CHARACTERISATION OF PROTEASES

INVOLVED IN PROTEOLYTIC

DEGRADATION OF HAEMOGLOBIN

IN THE HUMAN HOOKWORM

NECATOR AMERICANUS

Najju Ranjit

BBiotech (Hons)

Queensland Institute of Medical Research

Queensland University of Technology

A thesis submitted for the degree of Doctor of Philosophy

2008

ii

LIST OF KEYWORDS Necator americanus

Hookworm

Laser microscopy microdissection

Haemoglobin degradation

Haemoglobinases

Cysteine protease

Aspartic protease

Metalloprotease

Intestinal proteases

Vaccine candidates

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ABSTRACT

With over a billion people infected world wide, hookworms are considered as

important human pathogens, particularly in developing countries which have the

highest rates of infections. Hookworms reside in the gastrointestinal tract of the host

where they continuously feed on blood, leading to conditions such as chronic iron-

deficiency anaemia. The majority of blood-feeding parasites rely on proteins found

in blood to provide many of their nutritional requirements for growth, reproduction

and survival. Of the numerous proteins found in blood, haemoglobin (Hb) is one of

the most abundant. In order to acquire amino acids for protein synthesis, it is thought

that haematophagous parasites degrade Hb using various classes of endo- and exo-

proteases, in a manner similar to that which occurs in catabolism of proteins in

mammalian cellular lysosomes. This study identified and characterised proteases

involved in the Hb degradation process in the human hookworm, Necator

americanus, in order to identify potential candidate antigens for a vaccine that

interrupts blood-feeding.

Red blood cells ingested by hookworms are lysed to release Hb, which is

cleaved by various proteases into dipeptides or free amino acids and these are taken

up through the gut membrane by amino acid transporters. Proteases expressed in the

intestinal tract of hookworms are thought to play a major role in this process and

would therefore make good targets for vaccine candidates aimed at interrupting

blood-feeding. To identify these proteases, adult hookworms (both N. americanus

and Ancylostoma caninum) were sectioned and intestinal tissue was dissected via

laser microdissection microscopy. RNA extracted from the dissected tissue was used

to generate gut-specific cDNA, which then was used to create plasmid libraries. Each

library was subjected to shotgun sequencing, and of the 480 expressed sequence tags

(ESTs) sequenced from each species, 268 and 276 contigs were assembled from the

N. americanus and A. caninum libraries, respectively. Nine percent of N. americanus

and 6.5% of A. caninum contigs were considered novel as no homologues were

identified in any published/accessible database. The gene ontology (GO)

classification system was used to categorise the contigs to predicted biological

functions. Only 17% and 38% of N. americanus and A. caninum contigs,

respectively, were assigned GO categories, while the rest were classified as being of

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unknown function. The most highly represented GO categories were molecular

functions such as protein binding and catalytic activity. The most abundant

transcripts encoded fatty acid binding proteins, C-type lectins and activation

associated secreted proteins, indicative of the diversity of functions that occur in this

complex organ. Of particular interest to this study were the contigs that encoded for

cysteine and metalloproteases, expanding the list of potential N. americanus

haemoglobinases. In the N. americanus cDNA library, four contigs encoding for

cathepsin B cysteine proteases were identified. Three contigs from the A. caninum

and one contig from the N. americanus cDNA libraries encoded for metalloproteases,

including astacin-like and O-sialoglycoprotein endopeptidases, neither of which had

previously been reported from adult hookworms. Apart from haemoglobinases, other

mRNAs encoding potential vaccine candidate molecules were identified, including

anti-clotting factors, defensins and membrane proteins. This study confirmed that the

gut of hookworms encodes a diverse range of proteases, some of which are likely to

be involved in Hb digestion and have the potential to be hidden (cryptic) vaccine

antigens.

Four cysteine proteases (Na-CP-2, -3, -4 and -5) were identified from the gut

cDNA library of N. americanus. All four proteases belong to the clan CA, family C1,

share homology with human cathepsin B and possess a modified occluding loop.

Real-time PCR indicated that all transcripts were up-regulated in the adult stage of

the hookworm parasite with high levels of mRNA expression detected in gut cDNA.

All four proteases were expressed in recombinant form, but only Na-CP-3 was

successfully expressed in soluble form in the yeast Pichia pastoris. Proteolytic

activity for Na-CP-3 was detected on a gelatin zymogen gel, however no catalytic

activity was detected against the class-specific fluorogenic peptides Z-Phe-Arg-AMC

and Z-Arg-Arg-AMC. Mass spectrometry analysis of the purified protein suggested

that the pro-region had not been processed in trans when the protein was secreted by

yeast. Incubation of Na-CP-3 in salt buffers containing dextran sulfate resulted in

autoprocessing of the pro-region as detected by Western blot and catalytic activity

was detected against Z-Phe-Arg-AMC. Activated Na-CP-3 did not digest intact

tetrameric human Hb. The other three cysteine proteases (Na-CP-2, -4, and -5) were

expressed in insoluble form in Escherichia coli. Antibodies to all four proteins (Na-

CP-2 to 5) immunolocalised to the gut region of the adult worm, supporting mRNA

v

amplification results and strongly indicated that they might play a role in nutrient

acquisition.

Hb digestion in blood feeding parasites such as schistosomes and

Plasmodium spp. occurs via a semi-ordered cascade of proteolysis involving

numerous enzymes. In Plasmodium falciparum, at least three distinct mechanistic

classes of endopeptidases have been implicated in this process, and at least two

classes have been implicated in schistosomes. A similar process is thought to occur

in hookworms. An aspartic protease, Na-APR-1, was expressed in P. pastoris and

purified protein was shown to cleave the class-specific fluorogenic peptide 7-

Methoxycoumarin-4-Acetyl-GKPILFFRLK(DNP)-D-Arg-Amide. Recombinant Na-

APR-1 was able to cleave intact human Hb and completely degrade the 16 kDa

monomer and 32 kDa dimer within one hour. Recombinant Na-CP-3 was not able to

cleave intact Hb, but was able to further digest globin fragments that had previously

been digested with Na-APR-1. A clan MA metalloprotease, Na-MEP-1, was

identified in gut tissue of N. americanus and was expressed in recombinant form in

Hi5 insect cells using the baculovirus expression system. Recombinant Na-MEP-1

displayed proteolytic activity when assessed by gelatin zymography, but was

incapable of cleaving intact Hb. However, Na-MEP-1 did cleave globin fragments

which had previously been incubated with Na-APR-1 and Na-CP-3. Hb digested

with all three proteases was subjected to reverse phase HPLC and peptides were

analysed using Liquid Chromatography-Mass Spectrometry (LC-MS). A total of 74

cleavage sites were identified within Hb α and β chains. Na-APR-1 was responsible

for cleavage of Hb at the hinge region, probably unravelling the molecule so that Na-

CP-3 and Na-MEP-1 could gain access to globin peptides. All three proteases were

promiscuous in their subsite specificities, but the most common P1-P1′ residues were

hydrophobic and/or bulky in nature, such as Phe, Leu and Ala. Antibodies to all three

proteins (Na-APR-1, -CP-3, -MEP-1) immunolocalised to the gut region of the

worm, further supporting their roles in Hb degradation. These results suggest that Hb

degradation in N. americanus follows a similar pattern to that which has been

described in Plasomdium falciparum.

Studies conducted in this project have identified a number of potential

haemoglobinases and have demonstrated that the gut region of the hookworm

contains a multitude of proteases which could be targeted for production of new

chemotherapies or as vaccine candidates. Results presented here also suggest that the

vi

Hb degradation process occurs in an ordered cascade, similar to those which have

been reported in other haematophagous parasites. More importantly, it has been

confirmed that Na-APR-1 plays a crucial role in the initiation of the Hb degradation

process and therefore targeting this molecule as a vaccine candidate could provide

high levels of protection against hookworm infection.

vii

LIST OF PUBLICATIONS

Publication by candidate relevant to thesis:

N. Ranjit, M.K. Jones, D.J Stenzel, R.B Gasser, A. Loukas (2006). A survey of the

intestinal transcriptome of the hookworms, Necator americanus and Ancylostoma

caninum using tissue isolated by laser microdissection microscopy.

International Journal for Parasitology 36: 701-710 (Impact factor: 3.337)

N. Ranjit, B. Zhan, D. Stenzel, J. Mulvenna, R. Fujiwara, P. Hotez, A. Loukas

(2008). A family of cathepsin B cysteine proteases expressed in the gut of the human

hookworm, Necator americanus.

Molecular and Biochemical Parasitology 160: 90-9 (Impact factor: 2.641)

N. Ranjit, B. Zhan, B. Hamilton, D. Stenzel, J Lowther, M. Pearson, J. Gorman, P.

Hotez, A. Loukas (2008). Digestion of hemoglobin via an ordered cascade of

proteolysis in the intestine of the human hookworm Necator americanus

Submitted to Journal of Infectious Diseases (under review) (Impact factor:

6.035)

Additional publication by candidate relevant to the thesis but not forming part

of it:

A. Loukas, J. M.Bethony, S. Mendez, R. T. Fujiwara, G. N. Goud, N. Ranjit, B.

Zhan, K. Jones, M. E. Bottazzi, P. J. Hotez (2005). Vaccination with recombinant

aspartic hemoglobinase reduces parasite load and blood loss after hookworm

infection in dogs.

PLoS Medicine 2 (10): e295 (Impact factor: 13.750)

viii

TABLE OF CONTENTS LIST OF KEYWORDS ................................................................................................ ii ABSTRACT................................................................................................................. iii LIST OF PUBLICATIONS ........................................................................................ vii TABLE OF CONTENTS........................................................................................... viii LIST OF FIGURES AND TABLES.............................................................................xi LIST OF ABBREVATIONS ..................................................................................... xiii LIST OF ABBREVATIONS ..................................................................................... xiii STATEMENT OF ORIGINALITY............................................................................xvi STATEMENT BY SUPERVISOR.............................................................................xvi ACKNOWLEDGEMENTS ...................................................................................... xvii CHAPTER 1: INTRODUCTION ..................................................................................1

1.1 DESCRIPTION OF THE SCIENTIFIC PROBLEM INVESTIGATED ......2 1.2 OVERALL OBJECTIVE OF THE STUDY .................................................3 1.3 SPECIFIC AIMS OF THE STUDY ..............................................................3 1.4 ACCOUNT OF SCIENTIFIC PROGRESS LINKING THE SCIENTIFIC

PAPERS .........................................................................................................5 CHAPTER 2: LITERATURE REVIEW .......................................................................7

2.1 HOOKWORMS .............................................................................................8 2.1.1 Introduction............................................................................................8 2.1.2 Biology...................................................................................................8 2.1.3 Global Distribution ..............................................................................13 2.1.4 Clinical Aspects ...................................................................................14

2.1.4.1 Pathogenicity....................................................................................14 2.1.4.2 Immunology .....................................................................................17 2.1.4.3 Treatment .........................................................................................19 2.1.4.4 Vaccines ...........................................................................................20

2.2 PROTEASES ...............................................................................................21 2.2.1 Hookworm Larval Proteases ................................................................22 2.2.2 Adult Hookworm Proteases .................................................................26

2.2.2.1 Aspartic proteases ............................................................................26 2.2.2.2 Cysteine proteases............................................................................28 2.2.2.3 Metalloproteases ..............................................................................31 2.2.2.4 Exopeptidase and aminopeptidases..................................................33

2.3 HAEMOGLOBIN DIGESTION CASCADE..............................................34 2.4 SUMMARY .................................................................................................37 2.5 THESIS HYPOTHESIS...............................................................................38

CHAPTER 3: A SURVERY OF THE INTESTINAL TRANSCRIPTOMES OF THE HOOKWORMS, NECATOR AMERICANUS AND ANCYLOSTOMA CANINUM,USING TISSUES ISOLATED BY LASER MICRODISSECTION MICROSCOPY............................................................................................................40

3.1 CONTRIBUTIONS .....................................................................................41 3.2 ABSTRACT.................................................................................................42 3.3 INTRODUCTION .......................................................................................43 3.4 MATERIALS AND METHODS.................................................................44

3.4.1 Parasite material ...................................................................................44 3.4.2 Laser microdissection microscopy (LMM)..........................................45 3.4.3 RNA extraction, cDNA synthesis and detection of known gut

transcripts .............................................................................................45

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3.4.4 Construction of cDNA libraries ...........................................................46 3.4.5 Bioinformatic analyses.........................................................................46 3.4.6 Phylogenetic tree..................................................................................47

3.5 RESULTS AND DISCUSSION ..................................................................47 3.5.1 Extraction of gut tissues from hookworms ..........................................47 3.5.2 Tissue specificity of cDNA populations ..............................................48 3.5.3 Characteristics of the EST dataset........................................................49 3.5.4 Sequence analysis and gene ontology ..................................................49 3.5.5 Transcript abundance and highly represented genes............................53 3.5.6 Molecules involved in feeding.............................................................54 3.5.7 Immunomodulation..............................................................................58 3.5.8 Known and Potential Vaccine Antigens ..............................................59

3.6 CONCLUSION............................................................................................61 CHAPTER 4: A FAMILY OF CATHEPSIN B CYSTEINE PROTEASES EXPRESSED IN THE GUT OF THE HUMAN HOOKWORM, NECATOR AMERICANUS .............................................................................................................62

4.1 CONTRIBUTIONS .....................................................................................63 4.2 ABSTRACT.................................................................................................64 4.3 INTRODUCTION .......................................................................................65 4.4 MATERIALS AND METHODS.................................................................67

4.4.1 Phylogenetic tree..................................................................................67 4.4.2 Amplification of cysteine proteases genes from gut cDNA.................67 4.4.3 Quantitation of cysteine protease gene expression in different

developmental stages ...........................................................................68 4.4.4 Expression and purification of recombinant cysteine proteases ..........68 4.4.5 Autoactivation, catalytic activity assays. .............................................69 4.4.6 Identification of the pro-mature Na-CP-3 junction..............................71 4.4.7 Antibody production ............................................................................71 4.4.8 Immunolocalization .............................................................................71

4.5 RESULTS ....................................................................................................72 4.5.1 Sequence analysis of the cysteine proteases identified in N.

americanus ...........................................................................................72 4.5.2 Phylogenetic analysis of cathepsin B-like proteases............................73 4.5.3 Amplification of cysteine protease mRNAs from N. americanus gut

cDNA ...................................................................................................73 4.5.4 Developmental expression of cysteine protease genes ........................73 4.5.5 Expression of recombinant cysteine proteases.....................................77 4.5.6 Catalytic activity of Na-CP-3...............................................................78 4.5.7 Antibody production and immunolocalization of proteins ..................80

4.6 DISCUSSION ..............................................................................................81 CHAPTER 5: DIGESTION OF HEMOGLOBIN VIA AN ORDERED CASCADE OF PROTEOLYSIS IN THE INTESTINE OF THE HUMAN HOOKWORM, NECATOR AMERICANUS ..........................................................................................87

5.1 CONTRIBUTIONS .....................................................................................88 5.2 ABSTRACT.................................................................................................89 5.3 INTRODUCTION .......................................................................................90 5.4 MATERIALS AND METHODS.................................................................92

5.4.1 cDNA cloning ......................................................................................92 5.4.2 Protein expression and purification......................................................92 5.4.3 Catalytic activity of recombinant hemoglobinases ..............................93

x

5.4.4 Antibody production and immunolocalization.....................................94 5.4.5 Proteolysis of Hb by Na-APR-1, Na-CP-3 and Na-MEP-1 .................95 5.4.6 LC-MS and MS-MS analysis of Hb hydrolysates ...............................95

5.5 RESULTS ....................................................................................................96 5.5.1 Cloning of cDNAs encoding N. americanus hemoglobinases.............96 5.5.2 Expression and purification of Na-APR-1 and Na-MEP-1..................98 5.5.3 Catalytic activity assays .......................................................................99 5.5.4 Immunolocalization .............................................................................99 5.5.5 Hemoglobin degradation and LC-MS analysis of hemoglobin

hydrolysates. ......................................................................................100 5.6 DISCUSSION ............................................................................................105

CHAPTER 6: GENERAL DISCUSSION, CONCLUSION AND FUTURE DIRECTIONS............................................................................................................109

6.1 GENERAL DISCUSSION ........................................................................110 6.2 CONCLUSION AND FUTURE DIRECTIONS.......................................122

REFERENCES...........................................................................................................124 APPENDICES ...........................................................................................................138

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LIST OF FIGURES AND TABLES Figure 2.1. Scanning electron microgrpahs of the buccal cavities of human

hookworm species..................................................................................................9 Figure 2.2. Lifecycle of N. americanus .......................................................................11 Figure 2.3. Micrograph showing sectioned adult hookworm attached to intestinal

microvilli ..............................................................................................................11 Figure 2.4. Micrographs of N. americanus adult and egg............................................12 Figure 2.5. Global distribution of human hookworm infection (both N. americanus

and A. duodenale). ...............................................................................................14 Figure 2.6. Relationship between hookworm burden and anaemia and amount of

blood loss with different hookworm loads...........................................................17 Figure 2.7. Comparison of hookworm burden to other soil transmitted helminths. ....19 Figure 2.8. Percent reduction of A. caninum L3 that penetrated skin in an in vitro

model of tissue migration by hookworm larvae...................................................24 Figure 2.9. Schematic of the effects of anti-ASP-2 antibodies on host hookworm

burdens. ................................................................................................................26 Figure 2.10. Schematic diagram of the cysteine protease superfamily from parasitic

helminths. .............................................................................................................29 Figure 2.11. Families of zinc metalloproteases............................................................32 Figure 2.12. Tertiary structure of the haemoglobin molecule and the two subunits....34 Figure 2.13. Proposed haemoglobin degradation pathway in P. falciparum and S.

mansoni. ...............................................................................................................36 Figure 2.14. Schematic of proposed haemoglobinase cascade in the intestine of

blood-feeding nematodes. ....................................................................................37 Figure 2.15 Schematic for the development of a bivalent human hookworm vaccine.38 Figure 3.1. Light micrographs of adult hookworm section before and after laser

microdissection. ...................................................................................................48 Figure 3.2. Detection in gut but not ovary cDNA of transcripts corresponding to

proteins that were previously shown to be expressed in the intestine using immunolocalisation. .............................................................................................49

Table 3.1. Summary of the gut EST datasets for A. caninum and N. americanus contigs with ORFs containing ≥ 30 amino acids. ................................................51

Table 3.2. Novel clones with no homologues in any datasets that contain ORFs with predicted signal peptides. Double-ended arrows denote the predicted cleavage site of the signal peptide.......................................................................................52

Figure 3.3. Pie charts depicting gene ontology classifications of Ancylostoma caninum and Necator americanus gut ESTs identified in this study. ..................53

Table 3.3. The 10 most abundant contigs from the A. caninum and N. americanus gut expressed sequence tag (EST) datasets ................................................................55

Table 3.4. Hookoworm (Anyclostoma spp. plus Haemonchus and Caenorhabditis, of comparison) gut ESTs encoding proteolytic enzymes .........................................57

Figure 3.4. Multiple sequence alignment of contig Ac129 with homologous members of the C-type lectin family. ..................................................................................59

Figure 3.5. Neighbour joining phylogenetic tree showing the relationships of Ac173 and Na91 with other members of the Activation Associated Secretory Protein family. ..................................................................................................................60

Table 3.5. Contigs identified in this study with potential as vaccine antigens. ...........61 Table 4.1. General properties of N. americanus cathepsin B-like proteases ...............73

xii

Figure 4.1 Multiple sequence alignment of N. americanus cysteine proteases and human cathepsin B. ..............................................................................................75

Figure 4.2. Neighbour joining phylogenetic tree depicting the relationships of N. americanus cysteine proteases with homologues from other nematodes and other phyla............................................................................................................76

Figure 4.3. Amplification of cysteine protease mRNAs from N. americanus gut cDNA. ..................................................................................................................77

Figure 4.4. Developmental expression profiles of N. americanus cysteine protease mRNAs.................................................................................................................77

Figure 4.5. Expression and purification of recombinant N. americanus CatBs in yeast P. pastoris and E. coli. .........................................................................................78

Figure 4.6. Gelatin zymogram showing catalytic activity of purified recombinant Na-CP-3. ....................................................................................................................79

Figure 4.7. SDS-PAGE gel of purified recombinant pro-Na-CP-3 incubated with Pro-Q Emerald 300 glycoprotein stain (A). Activation of pro-Na-CP-3 (B)..............80

Figure 4.8. pH profile of the catalytic activity of recombinant Na-CP-3 after auto-processing.............................................................................................................80

Figure 4.9. Western blot showing recognition of recombinant Na-CP-2, CP-3, CP-4 and CP-5...............................................................................................................81

Figure 4.10. Immunolocalization of Na-CP-2, -3, -4 and -5 in transverse sections of adult N. americanus. ............................................................................................82

Figure 5.1. Multiple sequence alignment of Na-MEP-1 with Ac-MEP-1 from Ancylostoma caninum and human neprilysin 1....................................................97

Figure 5.2. Expression and purification of N. americanus hemoglobinases................98 Figure 5.3. Catalytic activity of recombinant Na-APR-1 expressed in yeast and Na-

MEP-1 expressed in insect cells...........................................................................99 Figure 5.4. Immunolocalization of Na-APR-1, Na-CP-3 and Na-MEP-1. ................100 Figure 5.5. Hemoglobin digestion with recombinant Na-APR-1, Na-CP-3 and Na-

MEP-1. ...............................................................................................................101 Figure 5.6. LC trace of hemoglobin incubated with various recombinant proteins for

18 hours at 37oC at pH 4.5 .................................................................................102 Figure 5.7. Map of hemoglobin α and β chains highlighting the cleavages made by N.

americanus recombinant haemoglobinases........................................................103 Figure 5.8. P4-P4′ subsite specificities of Na-APR-1, Na-CP-3 and Na-MEP-1. .....104

xiii

LIST OF ABBREVATIONS α alpha

aa amino acid

Asn Asparagine

APR aspartic protease

ASP activation associated secreted protein

β beta

BLAST Basic Local Alignment Search Tool

bp base pair

BSA bovine serum albumin

CAP cap analysis program

cDNA complementary deoxyribonucleic acid

CP cysteine protease

CRD carbohydrate recognition domain

C-TL C-type lectin

Cys Cysteine

Da Dalton

DALY disability-adjusted life year

DEPC diethyl pyrocarbonate

DNA deoxyribonucleic acid

dNTP dideoxyribonucleoside triphosphates

E-64 trans-Epoxysuccinyl-L-leucylamido-(4 guanidino) butane

EDTA ethylenediaminetetraacetic acid

ELISA enzyme-linked immunosorbent assay

epg eggs per gram

ES excretory/secretory

EST expressed sequence tag

FPLC fast protein liquid chromatography

g gram

GAG glycosaminoglycans

Gln Glutamine

Glu Glutamatic acid

GST Glutathione S-tranferase

xiv

h hour(s)

HAP histoaspartic protease

Hb haemoglobin

HCl hydrochloric acid

Hi5 Trichoplusia ni

His Histidine

HPLC high performance liquid chromatography

Igs immunoglobulins

IL interleukin

IPTG isopropyl β-D-thiogalactopyranoside

kb kilobase

kcat catalytic constant

kDa kilo dalton

Km Michaelis constant

L litre

L3 third-stage larvae

LB Luria-Bertani media

LC liquid chromatography

M molar

MEP metalloprotease

mg milligram

min minute(s)

ml millilitre

mM millimolar

MQ Milli-Q-purified water

mRNA messenger ribonucleic acid

MS mass spectrophotometry

MW molecular weight

ng nanograms

NIF neutrophil inhibitory factor

nm nanometres

NMS normal mouse serum

nr non redundant

nt nucleotide

xv

ORF open reading frame

PBS phosphate buffered saline

PBS/T phosphate buffered saline/Tween 20

PCR polymerase chain reaction

PM plasmepsin

PRP pathogenesis-related protein

PSP polysaccharides

RBC red blood cell

RNA ribonucleic acid

RNAi RNA interference

RP-HPLC reverse-phase high-performance liquid chromatography

RT room temperature

RT-PCR reverse transcription PCR

SDS sodium dodecyl sulfate

SDS-PAGE SDS-polyacrylamide gel electrophoresis

TFA trifluoroacetic acid

TSBP thiol sepharose binding protein

μg microgram

μl microlitre

μM micromolar

μm micrometer

WHO World Health Organization

YPD yeast peptone dextrose

xvi

STATEMENT OF ORIGINALITY

The work contained in this thesis has not been previously submitted to meet

requirements for an award at this or any other higher education institution. To the

best of my knowledge and belief, the thesis contains no material previously

published or written by another person except where due reference is made.

Najju Ranjit

2008

STATEMENT BY SUPERVISOR

All co-authors have provided their consent for the inclusion of the papers presented

in this thesis. The co-authors accept the student’s contribution to each manuscript

and description of co-authors’ contribution.

Alex Loukas

2008

xvii

ACKNOWLEDGEMENTS First and foremost I would like to sincerely thank my principal supervisor Dr Alex

Loukas for all his help, support and guidance throughout my PhD. This project

would not have been possible without all his input. Thank you for allowing me to

join your group and letting me work on this project, I am extremely grateful for

having been given this opportunity to work in such a great lab.

Thanks to my associate supervisor Dr Deb Stenzel, who made sure that all the correct

forms were filled and took time out of her busy schedule to make sure that my thesis

was up to scratch. Thank you for your words of encouragement.

Many thanks to the past and present members of the Loukas lab who helped me

enormously throughout the years. Special thanks to Mai Tran, Ben Datu, Soraya

Gaze and Leanne Cooper who were always there to lend a helping hand and gave me

plenty of great advice. Thank you for making my lab life so much more enjoyable.

To all my friends, thank you for being there for me and listening to me whenever I

needed to vent or escape from the lab. To my PhD buddies, Meru, Melina and Louise

thanks for keeping me company and always bringing a smile to my face. To my BoP

buddies, you guys are the best bunch of people I have ever worked with and I hope

we can all meet up again one day.

To everyone else who I met along this journey and who provided me with valuable

guidance and assistance, thank you!

And last but not least a great big thanks to my family, especially my parents to whom

I will forever be indebted to. Thank you for all your love and support throughout the

years. Both of you are my source of inspiration and I dedicate this thesis to you.

CHAPTER 1: INTRODUCTION

2

1.1 DESCRIPTION OF THE SCIENTIFIC PROBLEM

INVESTIGATED Hookworms are parasitic nematodes that reside in the upper region of the

small intestine of their mammalian hosts, where they attach to the mucosa and ingest

extravasated blood from ruptured intestinal capillaries and arterioles. In many

developing countries, hookworm infections are widespread, due to poor sanitation

conditions and a lack of comprehensive chemotherapy control programs (Hotez,

2007). The two major species infecting people are Necator americanus and

Ancylostoma duodenale, the former being more prevalent and widespread. The main

pathology associated with hookworm infection is iron deficiency anaemia, which is a

direct result of intestinal blood loss that occurs in heavy infections. Although the

overall prevalence and intensity of hookworm infections are higher in males than in

females, in part because males have greater occupational exposure to infection, iron

deficiency anaemia causes more adverse effects in young children and women of

child bearing age, due to their low iron stores (Brooker et al., 2004).

One of the main problems associated with the available chemotherapy options for

hookworms is the high rate of re-infection after treatment. Although drugs used for

treatment are highly effective in eliminating existing infections, they do not protect

against rapid re-infection, which is a common occurrence in many endemic

communities. Another concern of mass drug administration programs is the potential

risk of drug resistance occurring, as is the case with parasitic nematodes of sheep and

cattle where benzimidazole drug resistance is now well documented (Jabbar et al.,

2006). As yet there is no solid evidence of drug resistance in human helminths but

there have been reports of drug failure and diminished efficacy with treatments in a

number of African countries (Albonico et al., 2003). Also in stark contrast to

infection with other soil transmitted helminths, immunity against hookworms does

not develop in most people - in fact the oldest people living in an endemic

community sometimes have the heaviest worm burdens (Loukas et al., 2006).

A highly desirable goal to combat hookworm infection would be the production

of a prophylactic vaccine, one which reduces worm burden and leads to the decrease

of intestinal blood loss, the main cause of the pathology associated with this

infection. It has been suggested that a hookworm vaccine could be integrated into

current chemotherapy control programs, with chemotherapy given first to treat

3

existing infections, followed by a vaccine administered to prevent or significantly

delay reinfection (Bethony et al., 2005). Using this approach, vaccine-linked

chemotherapy would not only diminish the requirement for frequent and periodic

anthelmintic chemotherapy, but would also reduce the likelihood of the emergence of

drug resistance.

1.2 OVERALL OBJECTIVE OF THE STUDY As hookworms are complex multicellular parasites, it has been suggested that

an efficacious vaccine against this infection should consist of two antigens, one

which targets the infective larval stage and inhibits migration through the host, and

the second which targets defined physiological functions in the adult stage, such as

blood feeding. An antigen from the third-stage larvae, Na-ASP-2 (Bethony et al.,

2005, Goud et al., 2005) has already been identified and has completed phase one

clinical trials (Bethony et al., 2008). Therefore, the main objective of this study was

to identify and investigate mRNAs encoding proteases from the gut of adult N.

americanus and determine which of these enzymes are involved in haemoglobin

(Hb) degradation, and could therefore be considered haemoglobinases. By including

haemoglobinases in a cocktail hookworm vaccine, it is envisaged that the parasite’s

ability to digest and therefore absorb nutrients would be compromised, therefore

dramatically decreasing the viability of the parasite.

1.3 SPECIFIC AIMS OF THE STUDY There were three specific aims in this study. The first aim was to identify

mRNAs encoding potential vaccine antigens, particularly haemoglobinases, from the

intestinal cells of hookworms. The second aim was to express recombinant versions

of potential haemoglobinases in catalytically active form. The third aim was to assess

the abilities of these recombinant proteases to participate in a multi-enzyme cascade

of haemoglobinolysis and determine whether this occurs via an ordered pathway.

Finally, using tandem mass spectrometry, a Hb cleavage map was developed,

allowing inferences to be made on the subsite specificities of each of the recombinant

haemoglobinases.

In order to address the aims of this study the following experiments were

performed.

4

• Aim 1: Identification of the cDNAs encoding the major haemoglobinolytic

proteases from the intestine of adult N. americanus and the common dog

hookworm, Ancylostoma caninum. N. americanus and A. caninum gut tissues were

dissected using laser microdissection microscopy in order to prepare gut specific

mRNA for cDNA library construction and shot gun sequencing. The PALM

microbeam microdissector plus laser catapult microscope were used to dissect

intestinal tissue from adult hookworms. RNA was isolated from the extracted tissue

for subsequent cDNA synthesis followed by construction of gut cDNA plasmid

libraries. Four hundred and eighty expressed sequence tags (ESTs) were generated

from each library and these were subjected to extensive bioinformatics analyses

including filtering, clustering, gene ontology analyses and characterisation of

mRNAs encoding for proteases in particular.

• Aim 2: Expression and characterisation of potential haemoglobinases. Proteases

expressed by N. americanus that are orthologous to known/potential

haemoglobinases from other haematophagous parasites were selected, expressed in

recombinant form and purified using various expression systems. Recombinant

cysteine proteases were expressed in the yeast Pichia pastoris and bacterium

Escherichia coli. Recombinant aspartic protease was expressed in P. pastoris.

Metalloproteases were expressed in Trichoplusia ni Hi5 insect cells using the

baculovirus expression system. All recombinant proteins were purified via affinity

chromatography using the C-terminal hexa-histidine tags encoded by the expression

vectors. Polyclonal antibodies were generated in mice against N. americanus

proteases and these were used to localize the anatomic sites of expression in tissue

sections of adult N. americanus. Catalytic assays were conducted with purified

recombinant proteases using either zymogram gels or class-specific fluorogenic

peptides.

• Aim 3: Assessment of the ability of recombinant proteases to digest Hb or globin

peptides, development a cleavage map of human Hb and determine whether

digestion occurs in an ordered pathway. Necator americanus haemoglobinases

(both alone and in various combinations) were incubated with human Hb in vitro and

assessed using SDS-PAGE and liquid chromatography-mass spectrometry (LC-MS).

5

1.4 ACCOUNT OF SCIENTIFIC PROGRESS LINKING THE

SCIENTIFIC PAPERS It has been demonstrated in the blood-feeding nematode of ruminants,

Haemonchus contortus, that the intestine contains a myriad of hidden antigens, many

of which are presumed to be involved in the feeding process and are therefore good

targets for vaccine candidates. Following this concept the first paper presented in this

thesis (A survey of the intestinal transcriptomes of the hookworms, Necator

americanus and Ancylostoma caninum, using tissue isolated by laser

microdissection microscopy) conducts a survey of the intestinal transcriptomes of

the human and canine hookworms, N. americanus and A. caninum. This provided a

snapshot of the genes that are highly expressed in this tissue and enabled me to

identify intestinal proteases and assess their potential roles as haemoglobinases. At

least eleven potential haemoglobinases were detected from both the N. americanus

and A. caninum libraries, with cysteine and metalloproteases being the most

abundantly represented.

The second paper presented in this thesis (A family of cathepsin B cysteine

proteases expressed in the gut of the human hookworm, Necator americanus)

describes cysteine proteases (Na-CP-2, -3, -4 and -5) which were initially amplified

from a third-stage larval N. americanus cDNA phage library by colleagues from

George Washington University, USA. Gut cDNA (generated for paper #1) was used

to verify that these cysteine proteases were expressed in the intestinal tissue of the

adult worm, implying potential roles in blood-feeding. Immunolocalisation results

supported mRNA amplification data, and specific antibodies to each recombinant

cysteine protease bound to the gut microvillar surface, further verifying their

involvement in digestion of the blood meal. Recombinant Na-CP-3 was successfully

expressed in soluble form as a pro-enzyme and underwent auto-activation to a

mature protease in the presence of dextran sulfate. Catalytic activity was detected

using the fluorogenic peptide Z-Phe-Arg-aminomethylcoumarin, however activated

Na-CP-3 was incapable of digesting intact Hb. Nonetheless, it was suggested that

Na-CP-3 might have a role in nutrient acquisition by cleaving globin fragments after

other haemoglobinases had made initial cleavages of the Hb tetramer.

In the third paper (Digestion of hemoglobin via an ordered cascade of

proteolysis in the intestine of the human hookworm Necator americanus), an

6

ordered pathway of Hb digestion in N. americanus was determined. The aspartic

protease, Na-APR-1, was shown to cleave intact Hb. LC-MS was used to identify the

sites where APR-1 cleaved Hb - one of these sites was the hinge region of Hb,

implying that APR-1 unravels the tetrameric Hb protein, making it more susceptible

to further cleavage by other proteases. The cysteine protease, Na-CP-3, was shown to

cleave globin fragments after Hb had been digested with Na-APR-1, suggesting that

cleavage of Hb is initiated by the aspartic protease, and then cysteine proteases (CP-3

at least) act to further digest the globin fragments. In similar fashion to Na-CP-3, the

metalloprotease, Na-MEP-1, was also incapable of cleaving intact Hb but was able to

further cleave globin fragments following incubation of Hb with Na-APR-1 and Na-

CP-3. This study demonstrated that Hb digestion in N. americanus occurs in an

ordered cascade, thus validating the targeting of the proteases involved in this

process for vaccine development.

CHAPTER 2: LITERATURE REVIEW

8

2.1 HOOKWORMS 2.1.1 Introduction

More than a dozen different species of soil-transmitted helminths infect

humans in developing countries. Of these parasites, the two hookworm species

Necator americanus and Ancylostoma duodenale, stand out because of their

widespread prevalence and distribution, together contributing to hundreds of millions

of infections (Hotez et al., 2003c). These parasites have evolved complex life

histories with each stage expressing unique genes and gene families to promote its

survival in distinct niches. Helminth parasites utilise a large and diverse range of

proteases in order to infect, feed and reproduce in the host (Tort et al., 1999). Adult

hookworms are voracious blood feeders and rely on the acquisition of proteins found

in blood in order to meet their nutritional requirements. As with a number of other

haematophagous parasites, it has been suggested that hookworms employ a cascade

of proteolytic enzymes to break down proteins such as haemoglobin and serum

albumin to release amino acids needed for protein synthesis (Williamson et al.,

2003b). Unlike many mammalian acidic proteases which act in lysosomes,

homologous enzymes in helminths are released into the gut lumen (or outside of the

parasite altogether) where they digest their substrates extracellularly (Tort et al.,

1999). As such, these enzymes are accessible to antibodies when the parasite takes a

blood meal, making them viable targets for new therapies against hookworms and

other blood-feeding worms.

2.1.2 Biology

Hookworms are dioecious parasitic nematodes, with adult forms that

generally reside in the intestinal tract of their host (Roche and Layrisse, 1966). They

belong to the family Ancylostomatidae and are part of the superfamily

Strongyloidea. The two main genera that affect humans, Necator and Ancylostoma,

are characterised by the presence of either blunt cutting plates or sharp “teeth” that

line the adult parasite buccal capsule (Fig. 2.1). Necator americanus and

Ancylostoma duodenale are responsible for most human infections with hookworms,

although infection with the feline and canine hookworm Ancylostoma ceylancium

also occurs in parts of Asia as a consequence of zoonotic transmission (Hotez and

Pritchard, 1995). Ancylostoma ceylancium does not reach maturity in human hosts

9

and hence it is not associated with the substantial blood loss, seen in humans infected

with the other two hookworm species (Carroll and Grove, 1986). In north-eastern

Australia, the canine hookworm, A. caninum, has been shown to cause occasional

human intestinal infections, sometimes resulting in eosinophilic gastroenteritis

(Prociv and Croese, 1990). Another canine and feline hookworm (Ancylostoma

braziliense) can also infect humans and is the main cause of cutaneous larva

migrans, a self-limiting dermatologic condition characterized by serpiginous burrows

of 1 to 5 cm in length (Brooker et al., 2004, Hotez et al., 2004).

Figure 2.1. Scanning electron microgrpahs of the buccal cavities of human hookworm species Left to right: Necator americanus and Ancylostoma duodenale. N. americanus has two cutting plates along the anterior margin while A. duodenale has two pairs of teeth. (http://www. mercksource.com and http://www.nematode.net)

There are significant pathobiological differences between the two major

human hookworms: N. americanus is smaller than A. duodenale, produces fewer

eggs, and causes less blood loss from the host (Albonico et al., 1998). N. americanus

is considered by some researchers to be better adapted to human parasitism because

of its diminished virulence relative to A. duodenale (Brooker et al., 2004). It is also

believed that N. americanus may be more adept at immune evasion (Pritchard and

Brown, 2001). In contrast, A. duodenale is considered to be the more “opportunistic

species” because of its ability to survive in more extreme environmental conditions,

its ability to cause infections via the oral route and its greater fecundity (Hotez et al.,

2003a). Ancylostoma duodenale L3 also have the unique ability to undergo arrested

development in humans and may enter human mammary glands prior to lactogenic

transmission (Yu et al., 1995).

The life cycle of hookworms is direct. Infection of humans is by the infective

third larval stage known as the L3. Generally, hookworms infect a host by larvae

penetrating the skin, although A. duodenale is also orally infective (Brooker et al.,

10

2004). Necator americanus is an obligate skin-penetrating parasite, and larvae of this

species cannot establish in the intestine when inoculated orally unless they first

penetrate the oral mucosa to enter the blood-stream and then migrate in the same

fashion as skin-penetrating larvae. The L3 attach to host skin on contact and

penetrate through hair follicles, into the dermis, where they enter blood or lymphatic

capillaries (Fig. 2.2). Skin penetration by L3 is facilitated by secretion of proteolytic

enzymes that degrade macromolecules of the host tissue (Vetter and Leegwater-vd

Linden, 1977, Williamson et al., 2006). It has been speculated that migration through

the skin is required to reactivate developmental pathways and trigger expression of

genes of the parasitic stage (Hawdon and Hotez, 1996). Following host entry, the L3

receive a signal present in mammalian serum and tissue that causes them to continue

development (Hawdon and Datu, 2003, Hawdon and Hotez, 1996). The host-

activated L3 then migrate through the vasculature and are swept via the afferent

circulation to the heart and then the pulmonary vasculature (Fig. 2.2). The larvae

then undergo tracheal migration by penetrating into the alveoli, to be coughed up into

the airways and then swallowed into the gut. After the larvae enter the

gastrointestinal tract they moult twice and differentiate into male and female adults.

Approximately 5-8 weeks pass from the time L3 larvae penetrate human skin until

the adult worms reach sexual maturity and mate (Hotez et al., 2004). The dioecious

adult hookworms live in the upper small intestine where they attach to the mucosa

via teeth (Ancylostoma species) or cutting plates (N. americanus) (Fig. 2.1), by

sucking clumps of villi into their buccal capsules, effectively burying their anterior

ends into the host intestinal mucosa or deeper (Loukas et al., 2005b) (Fig. 2.3).

11

Figure 2.2. Lifecycle of N. americanus Eggs are passed in the stool , and under favorable conditions (moisture, warmth, shade), larvae hatch in 1 to 2 days. The released rhabditiform larvae grow in the feces and/or the soil , and after 5 to 10 days (and two molts) they become become filariform (third-stage) larvae that are infective . On contact with the human host, the larvae penetrate the skin and are carried through the veins to the heart and then to the lungs. They penetrate into the pulmonary alveoli, ascend the bronchial tree to the pharynx, and are swallowed . The larvae reach the small intestine, where they reside and mature into adults. Adult worms live in the lumen of the small intestine, where they attach to the intestinal wall with resultant blood loss by the host. (http://www.dpd.cdc.gov/dpdx/HTML/Hookworm.htm)

Figure 2.3. Micrograph showing sectioned adult hookworm attached to intestinal microvilli The muscular pharynx is used to suck host tissue into the alimentary canal which is under hydrostatic pressure. (Loukas et al., 2005b)

12

The adult worms feed on host blood, and using radioactive tracers it has been

estimated that approximately 30 μl of blood/day is lost to a single N. americanus and

260 μl to A. duodenale (Pritchard et al., 1991). Ingestion of blood by the worm, and

associated intestinal blood loss from the host, begins just prior to egg production and

continues for the life of the hookworm. The large, anterior cephalic glands of the

adult hookworms secrete various products, including proteases and anti-coagulant

peptides, into the worm’s buccal cavity and oesophagus, as well as into the

attachment site in the host intestine to “predigest” host mucosal tissues (Loukas et

al., 2005b). Adult hookworms are thought to rely mostly on blood for nutrition, and

it has recently been shown that intact erythrocytes are ingested and lysed in the

parasite’s intestine by haemolytic proteins (Don et al., 2004).

Forty-five to sixty days after penetration of the host, female hookworms

begin laying eggs (9,000-11,000/day). The eggs, measuring approximately 60 μm x

40 μm, are transparent, thin-shelled and ovoid, with blunt, rounded ends (Fig. 2.4).

Eggs are passed in the host faeces, and embryonate in moist soil (25-28oC) before

developing to L1 stage larvae (Bethony et al., 2006a, Brooker et al., 2004).

Figure 2.4. Micrographs of N. americanus adult and egg. Left to right: female adult worm, male adult worm and egg. Female worms are approximately 9 to 11 mm, male worms are approx. 7 to 9 mm and eggs measure approx. 60 μm x 40 μm. The posterior end of the male worm is equipped with a characteristic copulatory bursa (arrow). The eggs are transparent, thin shelled and ovoid with blunt rounded ends. (Bethony et al., 2006a)

The hatched, first-stage larvae feed on organic debris and bacteria in the soil.

The larvae undergo two moults to become the infective third-stage larvae, which are

enveloped in the loose outer cuticular sheath left over from the second moult and are

approximately 600 μM in length (Bethony et al., 2006a, Brooker et al., 2004). At this

stage, the larvae may undergo developmental arrest and can live in the soil for weeks

if there is appropriate warmth, shade and moisture. The non-feeding, infective L3

migrate to the soil surface or low vegetation to maximize their chances of contacting

13

a new host (Hotez et al., 2004). The L3, in response to either an increase in

temperature or other undetermined cues on contact with the host, emerge from

developmental arrest, quickly shed their protective sheath and penetrate the host skin

(Fig 2.2).

2.1.3 Global Distribution

Hookworm infections predominantly occur in tropical and sub-tropical areas

and are most prevalent in regions where humidity is high and there is a lack of proper

sanitation (Bethony et al., 2006a). A major problem in these areas is the continued

use of human faeces as a crop fertilizer, which provides ideal conditions for the

perpetuation of the hookworm lifecycle. Adequate soil moisture and temperature are

the primary environmental requirements for maintaining endemic infections

(Brooker et al., 2004). Eggs that are shed in faeces can remain viable in moist soil

provided with the necessary environmental conditions for development.

The current global hookworm prevalence is shown in Figure 2.5. Hookworm

infections are particularly prevalent throughout much of sub-Saharan Africa, China,

Southeast Asia and the Pacific. Worldwide, N. americanus is the predominant agent

of human hookworm infection, while A. duodenale occurs in more scattered focal

environments where N. americanus cannot survive e.g. in colder and drier areas

(Hotez et al., 2004). Necator americanus is widely spread throughout the Caribbean

and Latin America and is a major pathogen in sub-Saharan Africa, Southeast Asia,

the Indian subcontinent and the Pacific islands. Coastal areas of these regions are

especially associated with high Necator transmission (Brooker et al., 2004). The

predominant regions for A. duodenale include northerly latitudes of south and west

China and India. Ancylostoma duodenale may survive in these harsher climates

because of its ability to undergo arrested development in host tissues (Schad et al.,

1973).

Throughout the world, mixed infections with both the major hookworm

species are common. It is estimated that nearly 1 billion people world wide harbour

these parasites, with recent estimates indicating that there are over 500 million

known clinically significant cases (Bethony et al., 2006a). Of these 500 million

people, approximately 44 million are pregnant women (Bundy et al., 1995). In sub-

Saharan Africa alone there are close to 200 million people infected with hookworms

(Crompton, 2000). Not surprisingly, and like many other neglected tropical diseases,

14

there is a striking global relationship between hookworm prevalence and low

socioeconomic status (de Silva et al., 2003).

Figure 2.5. Global distribution of human hookworm infection (both N. americanus and A. duodenale). The highest prevalence of hookworm occurs in sub-Saharan Africa and eastern Asia. High transmission also occurs in other areas of rural poverty in the tropics and southern China. (Hotez et al., 2005)

2.1.4 Clinical Aspects

2.1.4.1 Pathogenicity

The morbidity associated with hookworm infection is varied and ranges from

mild, transient clinical signs and symptoms to severe clinical disease. Moreover,

chronic infections are associated with effects on the physical growth, cognition and

productivity of individuals (Brooker et al., 2004). The overall impact of a hookworm

infection ultimately depends on the underlying health status of the patient (Brooker

et al., 2004). Although the adult hookworm elicits most of the pathological effects of

hookworm disease, the infective larvae may also contribute to disease during host

entry: the larvae release immunogenic and bioactive macromolecules, including

allergens and tissue invasive enzymes (Brown et al., 1999, Zhan et al., 2002). In

areas of high transmission, repeated L3 entry through the skin can result in a

cutaneous syndrome known as ground itch (Hotez et al., 2004). This comprises a

pruritic erythematous rash and appears mostly on the hands and feet where the

parasite penetrates the host. Zoonotic infection with A. braziliense L3 results in

cutaneous larva migrans which is characterised by serpiginous burrows appearing

15

most frequently on the feet, buttocks and abdomen (Blackwell and Vega-Lopez,

2001). Following entry into the human host, L3 undergo pulmonary migration which

can be accompanied by cough, sore throat and fever (Hotez et al., 2004). This usually

resolves when the L3 leave the lungs for the gastrointestinal tract. When A.

duodenale infection occurs via the oral route, the L3 migration can sometimes

produce a syndrome known as Wakana disease which is characterised by nausea,

vomiting, pharyngeal irritation and cough (Hotez et al., 2004).

The main pathogenic effect of hookworm infection is linked to the intestinal

blood loss that occurs during adult worm attachment and feeding in the host small

intestine (Hotez et al., 2004). Hookworms induce blood loss directly through

mechanical rupture of host capillaries and arterioles, followed by the release of a

battery of pharmacologically active polypeptides such as anticoagulants, anti-platelet

agents and antioxidants (Furmidge et al., 1996). In many developing countries, where

nutrition is inadequate, infection with these parasites is a leading cause of iron

deficiency anaemia, which results from the chronic blood loss associated with the

feeding of multiple adult worms (Loukas et al., 2006). In heavy infections, the blood

loss brought about by feeding activity can cause shortness of breath, lassitude and

angina, which can lead to congestive heart failure and, in the most severe cases,

death (Brooker et al., 2004). Children are especially vulnerable to hookworm

infection, and the effects of blood loss are exacerbated in children with nutrient

deprivation. Hookworm-induced iron deficiency anaemia has been shown to cause

developmental and mental retardation in children, and adverse maternal-foetal

outcomes in pregnant women (Crompton, 2000). In many developing countries,

anaemia in pregnancy is an important contributor to maternal mortality, especially

around the time of delivery. Severe maternal anaemia is also associated with reduced

birth weight which, in turn, is an important risk factor for infant mortality (Brooker

et al., 2004).

Morbidity is highest among patients that harbour large numbers of adult

parasites (Hotez et al., 2004). As hookworms do not replicate in the human host, the

number of adult worms present in the intestine of an infected person is directly

related to the level of exposure to infective larvae in the environment. Estimates of

the intensity of hookworm infections are typically obtained by using quantitative

faecal egg counts as a surrogate marker for worm burden (Hotez et al., 2005). The

World Health Organization (WHO) defines moderate-intensity infections as those

16

with 2,000-3,999 eggs per gram of faeces (epg) and heavy intensity as those with

≥4,000 epg (Hotez et al., 2005). Hookworm-induced blood loss is estimated to be as

high as 9 mL of blood per day in heavy infections and hookworm burdens of 40–160

worms are usually sufficient to cause anaemia (Fig. 2.6). Even light hookworm

infections (~300 epg) can contribute significantly to low haemoglobin and serum

ferritin levels in nutritionally-compromised hosts (Hotez et al., 2004).

Because hookworm infections cause more disability than death, the burden of

disease is typically assessed by using a metric known as the DALY (disability-

adjusted life year, i.e., the numbers of life years lost from premature death or

disability) (Bethony et al., 2006a). For hookworm infection, DALYs are determined

mainly on the basis of disability weights assigned to anaemia and cognition, with

estimates varying widely depending on the level of severity assigned to each

component (Hotez et al., 2003c). According to the 2002 global burden of disease

study conducted by WHO, hookworms caused the loss of 1.8 million DALYs

worldwide (Bethony et al., 2006a). However, estimates of the number of people

infected do not translate into the disease burden caused by hookworm, because not

everyone infected will develop clinically significant symptoms - morbidity is

typically related to the intensity of infection. Therefore, there is a need to revise

estimates of DALYs by combining data from attributable burdens of anaemia,

retarded childhood development, and pregnancy related morbidities resulting from

hookworm infection (Loukas et al., 2006).

17

A Hookworm infection Eggs per gram of

faeces Mean blood loss (mL/day) (SD)

Negative 0 1.24 (1.85) Light 1-999 1.46 (1.07) Moderate 1000-4999 2.96 (3.03) Heavy >5000 8.79 (1.10)

Figure 2.6. Relationship between hookworm burden and anaemia and amount of blood loss with different hookworm loads. Quantitative egg counts serve as an indirect measure of the adult-hookworm burden. The heavier the infection, the higher the blood loss, thus haemoglobin levels drop in proportion to infection. (A) (Hotez et al., 2004) (B) Adapted from (Loukas et al., 2006) 2.1.4.2 Immunology

The most studied aspect of the human immune response to hookworm

infection is antibody levels to crude larval and adult soluble extracts or adult

excretory/secretory (ES) products (Loukas and Prociv, 2001). As with most

helminths, the antibody response to hookworm consists predominantly of the Th2

antibody isotypes IgG1, IgG4 and IgE as well as the production of Th2 cytokines,

interleukin (IL)-4, -5, -9, -10, and -13 (Brooker et al., 2004). Adult hookworms also

induce the production of secretory IgE, IgG and IgM, but not IgA, and the levels of

these immunoglobulins (Igs) return to normal after successful anthelmintic treatment

(Brooker et al., 2004). Despite the extensive antibody response to infection, there is

limited evidence that these antibodies offer any protection by significantly reducing

either larval or adult hookworm numbers. Another hallmark feature of the immune

response to helminth infection is peripheral blood eosinophilia (Loukas and Prociv,

2001). Eosinophils also predominate in the inflammatory response to hookworm L3

in tissues (Maxwell et al., 1987). Hookworms appear to induce less intestinal

inflammation than most other intestinal nematodes, perhaps reflecting their

attachment and feeding strategies. Eosinophilia, mastocytosis and IgE stimulation are

the three main immune alterations observed during a hookworm infection in humans

(Brooker et al., 2004).

B

18

Hookworms excrete/secrete a myriad of products with the potential for

immunomodulation (Loukas et al., 2005b). In the case of several other nematodes,

injection of excretory/secretory products alone was shown to induce immune

responses similar to those observed during infection with live parasites in laboratory

animals (Allen and MacDonald, 1998). Characterisation of the composition of

hookworm and other nematode ES products have demonstrated the presence of many

different types of proteins, including proteases, protease inhibitors, C-type lectins,

anti-oxidants and anti-inflammatory proteins that might contribute to the cellular

hypo-responsiveness that is characteristic of chronic hookworm infections (Loukas

and Prociv, 2001). Other secreted proteins from hookworms have also been shown to

modulate the immune response, at least in vitro.

An anti-inflammatory polypeptide, termed neutrophil inhibitory factor (NIF),

that binds to the Mac-1 ligand has been identified in A. caninum (Moyle et al., 1994).

This molecule was demonstrated to inhibit neutrophil adhesion to endothelial cells

through blocking of the CD11/CD18 integrin on the neutrophil surface, as well as to

inhibit peroxide release from active neutrophils (Moyle et al., 1994). NIF belongs to

the pathogenesis-related protein (PRP) superfamily, a class of cysteine rich proteins

that is abundantly expressed by all parasitic nematodes investigated to date (Datu et

al., 2008). Published data suggest that the PRPs play diverse roles in nematode

parasitism, perhaps via ligand-receptor interactions courtesy of a large “binding”

groove that appears to accommodate peptide ligands (Asojo et al., 2005)

Unlike most other human helminthiases, such as Trichuris trichiura and

Ascaris lumbricoides, host resistance to infection and reinfection (after treatment)

with N. americanus and A. duodenale is not clear cut (reviewed in (Loukas et al.,

2005b)). In fact, a positive correlation between intensity of infection with N.

americanus and age has been demonstrated in human populations (Fig. 2.7) (Bethony

et al., 2002), which is in stark contrast to canine hookworm infections where age and

exposure related immunity occurs (Loukas and Prociv, 2001). While human

hookworm infections exhibit the hallmark features of Th2 responses, these immune

responses clearly fail to protect most infected people. The reason of the observed

failure of Th2 cells to mount effective anti-hookworm response remains unknown

(Loukas et al., 2005b).

19

Figure 2.7. Comparison of hookworm burden to other soil transmitted helminths. The hookworm burden increases with age, in contrast to the burden of other soil-transmitted helminths (e.g. Ascaris lumbricoides and Trichuris trichiura), which is highest in childhood. (Hotez et al., 2004) 2.1.4.3 Treatment

Because of the high transmission potential, hookworm infections have proven

to be extremely difficult to eliminate or eradicate in areas of poverty and poor

sanitation (Hotez, 2007). However, hookworm disease is easily treatable: oral doses

of the anthelmintic drug, albendazole can significantly reduce or completely cure

infection (Bethony et al., 2006a). Unfortunately, most hookworm infections occur in

developing and economically poor countries, where access to these medications is

limited or non-existent (Hotez, 2007). Additionally, anthelmintics do not provide

lasting protection, and there is no naturally acquired immunity (in most people) to

hookworms. Reinfection occurs rapidly, particularly in rural areas where activities

such as farming often provide ample opportunity for re-exposure to infective

hookworm larvae (Bethony et al., 2006a, Hotez, 2007).

Because infection with hookworm generally relies on the penetration of host

skin by infective larvae, simply wearing shoes can often provide an effective barrier

to exposure. However, as larvae can penetrate other areas of exposed skin this

practice is not 100% effective and, in the case of A. duodenale, infection can occur

by ingestion of infective larvae (Hotez, 2007). Individual predispositions to

hookworm infection appear to vary: some individuals have consistently higher (or

lower) infection levels than others in the same population and, moreover, these

individuals will re-acquire infection to previous levels after cure (Hotez et al., 2004).

A study conducted in an Iranian village revealed that 1-3% of the population

harboured the majority of the parasites (Croll and Ghadirain, 1981). It is well known

20

that host genetic and socio behavioural facts can contribute tremendously to

hookworm epidemiology.

The current widespread use of anthelmintics for treating hookworm and other

soil transmitted helminths has raised concerns of the potential of drug resistance

(Albonico, 2003). In some nematode species that parasitise livestock, benzimidazole

resistance occurs due to a single point mutation in nematode beta-tubulin alleles

(Schwenkenbecher et al., 2007, Albonico et al., 2004), and it is speculated that this

might partially account for an observed failure of mebendazole chemotherapy for

human hookworm in southern Mali (De Clercq et al., 1997). Studies have also shown

diminished efficacy with repeated targeted treatments of mebendazole in Zanzibar

(Albonico et al., 2003) and pyrantel pamoate failure against A. duodenale in Western

Australia (Reynoldson et al., 1997). However, as yet, there is not any conclusive

evidence of drug resistance in the hookworm species that infect humans.

2.1.4.4 Vaccines

Potential anthelmintic resistance in nematode populations and concerns about

the effects of drug residues on consumer health and the environment have focused

attention on developing effective anti-nematode vaccines. However, one of the major

issues that has been raised is whether it is feasible to develop a vaccine for an

infection in which natural exposure to the pathogen does not confer immunity (Hotez

et al., 2003c). This is especially highlighted by the fact that the cornerstone for the

development of first generation attenuated vaccines was based on the concept of

natural acquisition of immunity (Hotez et al., 2003c). A central challenge for

hookworm vaccine development will be to stimulate an artificial immune response

that is unique and results in disease burden reduction. Despite the lack of naturally

acquired immunity to hookworm infections in humans, there are two major lines of

evidence that support the feasibility of developing a hookworm vaccine.

Firstly, dogs immunised with radiation-attenuated larvae of A. caninum were

protected from challenge infection, with up to 90% reduction in worm burdens

compared to control dogs that did not receive the vaccine (Miller, 1967).

Interestingly, immunisation with dead larvae or non-irradiated larvae did not confer

similar levels of protection (Miller, 1978), inferring that protection was induced by

excretory/secretory antigens released by live larvae. Moreover, attenuation of L3

stops their development to adulthood and likely interferes with their implementation

21

of immunomodulatory strategies, accounting for the protection generated by

attenuated but not live larvae. What this vaccine provides is “proof of principle” that

antigens secreted by L3 upon migration through the host are capable of inducing

protective immune responses against healthy larvae during challenge infection

(Loukas et al., 2005b).

The second line of evidence comes from research conducted on the sheep

nematode, Haemonchus contortus (the “barber’s pole worm”), which is

phylogenetically related to hookworms. Sheep immunised with parasite extracts rich

in proteases derived from the intestine of adult worms showed high levels of

protection against infection (Knox and Smith, 2001). Among the antigens identified

in these extracts were parasite gut-derived glycoproteins that comprise a complex of

proteases and other components (designated H-gal-GP) (Smith et al., 1994).

Although the H-gal-GP complex is not ordinarily recognised during natural infection

and is considered a hidden antigen, vaccination with the complex provided high

levels of protection with respect to adult H. contortus worm burdens and fecundity

(Knox and Smith, 2001). In similar fashion, dogs vaccinated with extracts of the

oesophagus of adult A. caninum acquired immunity to this hookworm (Loukas and

Prociv, 2001). Antibodies from the vaccinated dogs had the capacity to neutralise

parasite protease activity, indicating that anti-enzyme antibodies have importance in

mediating protective immunity against hookworm infections (Loukas and Prociv,

2001).

Unlike vaccines developed against viruses and bacteria, where pathogens

reproduce within the host, and sterile immunity is imperative, the major goal of a

hookworm vaccine program is to decrease worm burdens to a level that minimizes

pathology (Loukas et al., 2005b). It is envisaged that the ultimate hookworm vaccine

will consist of a cocktail that elicits an immune response to at least two hookworm

proteins - probably one from the L3 larval stage to target penetrating and migrating

larvae, and another from the adult stage to minimize blood loss and anaemia (Loukas

et al., 2006).

2.2 PROTEASES Proteases encompass a broad class of hydrolytic enzymes that play essential roles in

cellular development and digestive processes, blood coagulation, inflammation,

22

wound healing and hormone processing. Recent studies have indicated that numerous

proteolytic enzymes are essential molecules in a wide range of processes that

determine parasitism, such as tissue penetration, feeding and immune evasion (Tort

et al., 1999). Proteases are now the current focus of drug and vaccine based control

programs for protistan and multicellular parasites (Dalton et al., 2003). Proteolytic

enzymes are classified into five major catalytic categories based on the structure of

their active site (serine, cysteine, threonine, aspartic) and dependency on co-factors

(metallopeptidases) (Rawlings et al., 2008).

Proteases produced by larval and adult hookworms play an important role in

assuring parasite invasiveness and completion of the hookworm lifecycle in the

appropriate host (Williamson et al., 2003b). Generally, proteases produced by

hookworm larval forms have been implicated in tissue penetration, immune evasion

and moulting, while proteases from adult forms are primarily implicated in tissue and

blood meal digestion (Williamson et al., 2003b). Until recently, not much was known

about the proteases of nematodes and their roles in haemoglobin proteolysis and

nutrient acquisition. It has been suggested that as proteins are abundant components

of blood, the major proteases found in hematophagous parasites are most likely to be

proteolytic digestive enzymes (Williamson et al., 2004). It also has been noted that

stage specific developmental regulation of these molecules often occurs (Hotez et al.,

2004).

2.2.1 Hookworm Larval Proteases

Molecules associated with hookworm invasion of skin and tissue have been

identified in all hookworm species, but several detected in dog hookworm, A.

caninum, have been the most extensively studied (Hotez et al., 2003b, Zhan et al.,

2002, Zhan et al., 2003). Secreted enzymes that have been identified in A. caninum

hookworm larvae include a hyaluronidase, zinc metalloproteases and cysteine-rich

secretory proteins (Hawdon et al., 1999, Hotez et al., 1992, Zhan et al., 1999).

Hyaluronic acid is a major component of the extracellular matrix and is also

associated with cell adhesion by ligand binding with the CD44 cell surface receptor

(Miyake et al., 1990). The release of hyaluronidase by invading hookworm larvae

would presumably facilitate migration of the larvae through the host dermal layers by

degrading cellular adhesion mediated by hyaluronic acid (Hotez et al., 1992). The

inability of A. braziliense to penetrate humans seems to be associated with the failure

23

of larvae to produce adequate digestive enzymes which could allow penetration

(Hotez et al., 1992). On the other hand, larvae of A. duodenale produce digestive

hydrolases that allow complete penetration of human skin and, therefore, completion

of the parasite lifecycle (Hotez et al., 1992). Datu recently identified a mRNA

encoding for a hyaluronidase from A. caninum that were activated with serum (Datu

et al., 2008).

The larval metalloprotease identified by (Hotez et al., 1990), termed Ac-

MTP-1, has similar effects on hyaluronic acid, and aids skin penetration by migrating

larvae. This molecule was shown to degrade fibronectin and tissue elastin in vitro

(Hotez et al., 1990). Williamson et al. then demonstrated that recombinant Ac-MTP-1

was capable of cleaving connective tissue proteins (Williamson et al., 2006).

Because this secreted enzyme displays activity against components of the

extracellular matrix, it was hypothesised that MTP-1 is important in parasite larval

invasion and aids in attachment of adult worms to the host intestinal wall (Zhan et

al., 2002). Secretion of zinc metalloproteases during the hookworm lifecycle may

indicate a key role for these enzymes in maintaining a parasitic relationship. Dogs

vaccinated with Ac-MTP-1, followed by challenge infection with A. caninum L3

larvae, revealed a statistically significant inverse association between anti-Ac-MTP-1

IgG2 antibody titers and the canine intestinal adult hookworm burden and

quantitative egg counts at necropsy (Hotez et al., 2003b). A recent vaccine trial

conducted with hamsters indicated that recombinant Ac-MTP-1 reduced worm

burden by up to 29% compared to a the control group which received adjuvant alone

(Xiao et al., 2008), suggesting that this molecule offers promise as a recombinant

vaccine. A similar metalloprotease was identified in the blood feeding nematode H.

contortus, and is involved in digestion of the larval cuticle, thus allowing the anterior

cap to open permitting larval escape (exsheathment) (Gamble et al., 1989).

The most abundant molecules released by penetrating hookworm larvae are

cysteine rich secretory proteins which belong to the pathogenesis related protein

(PRP) superfamily, and are termed Ancylostoma Secreted Proteins (ASPs) (Hawdon

and Hotez, 1996, Hotez et al., 2003c). ASP-1 is a 45 kDa protein with a double PRP

domain and is the major secreted protein in larval ES products (Hawdon et al., 1996).

Immunisation of mice with recombinant ASP-1 inhibited migration of A. caninum L3

larvae to the lungs, the endpoint of migration in this non-permissive host (Ghosh et

al., 1996). Although the biological functions of the ASPs are unknown, it has been

24

speculated that ASP-1 may modulate the host immune system in a manner which

may benefit the parasite (Zhan et al., 1999). Such an action would not be entirely

novel, as neutrophil inhibitory factor (a PRP family member) from A. caninum has

been hypothesized to modulate the host immune system (Moyle et al., 1994), and a

secreted hookworm zinc metalloprotease has been shown to specifically cleave the

eosinophil chemoattractant eotaxin and is hypothesised to aid in immune evasion

(Culley et al., 2000). ASPs share sequence identity with wasp venom allergens, and

adopt a fold that is similar to that displayed by some chemokines (Asojo et al., 2005).

A second ASP molecule, termed ASP-2, has also been identified in the ES

products of L3 that have been activated by the addition of serum in vitro (Hawdon et

al., 1999) ASP-2 is a 21 kDa protein with a single PRP domain. The molecule has

been immunolocalised to the glandular oesophagus of A. caninum L3, the basal

lamina of the body cavity, the channels that connect the glandular oesophagus to the

L3 surface, and on the L3 cuticle and epicuticle (Bethony et al., 2005). Vaccination

of hamsters with recombinant Ay-ASP-2 protein (from A. ceylanicum) resulted in

reduced egg output by female worms after challenge infection with A. ceylanicum L3

(Goud et al., 2004). Sera recovered from Ac-ASP-2 vaccinated dogs inhibited tissue

penetration by A. caninum L3 by 60% percent compared with control sera (Fig. 2.8)

(Bethony et al., 2005). A vaccine trial conducted in hamsters indicated that

vaccination with recombinant Na-ASP-2 protein (from N. americanus) reduced the

worm burden by 39% compared to controls that received adjuvant alone (Xiao et al.,

2008).

Figure 2.8. Percent reduction of A. caninum L3 that penetrated skin in an in vitro model of tissue migration by hookworm larvae. Na-ASP-2 recombinant protein was shown to inhibit larval invasion in vitro at similar levels to that of irradiated A. caninum L3 larvae. (Loukas et al., 2006)

25

Evidence to date indicates that anti-ASP-2 antibodies interact primarily with

the tissue-invading L3 stage of hookworms (Fujiwara et al., 2006). There are three

independent lines of evidence indicating that ASP-2 is a leading vaccine candidate

for human hookworm infection: 1) increased levels of IgE antibodies against ASP-2

appear to protect against heavy hookworm infection in humans; 2) anti-ASP-2

antibodies immunoprecipitate native ASP-2 from larval extracts and inhibit

hookworm larval invasion through tissue in vitro; and 3) vaccination with ASP-2

results in lower worm burdens and fecal egg counts in dogs (Bethony et al., 2005,

Fujiwara et al., 2006). The mechanism by which antibodies against ASP-2 reduce

host hookworm burdens and faecal egg counts is not known, but based on the

reported crystal structure of ASP-2, it has been proposed that the molecule could

function as an immunomodulator by mimicking chemokines (Asojo et al., 2005). As

it is inferred that ASP-2 vaccination interferes with the early stages of parasite

invasion of the host, vaccination with this molecule would decrease the number of

L3 larvae that reach the gastrointestinal tract, leading to reduced adult hookworm

burdens and, hence, reduced host blood loss (Bethony et al., 2006b) (Fig. 2.9). A

phase 1 study evaluating the safety and immunogenicity of Na-ASP-2/Alhydrogel in

healthy adults without evidence of hookworm infection and living in the United

States was conducted from 2005 through 2006. The vaccine was safe and well-

tolerated and induced significant anti–Na-ASP-2 IgG and cellular immune responses

(Bethony et al., 2008; Diemert et al., 2008).

In addition to the L3 secreted proteins, another surface protein from A.

caninum, Ac-16, also shows promise as a potential vaccine antigen (Diemert et al.,

2008; Fujiwara et al., 2007). Although Ac-16 is expressed during all stages of

hookworm development, it is an immunodominant larval surface protein that has

been shown to reduce faecal egg counts and blood loss in vaccinated dogs (Fujiwara

et al., 2007).

26

Figure 2.9. Schematic of the effects of anti-ASP-2 antibodies on host hookworm burdens. Anti-ASP-2 antibodies inhibit early host entry of hookworm L3 through tissues, resulting in reduced numbers of L3 that gain entry into the host gastrointestinal tract. (Bethony et al., 2005)

2.2.2 Adult Hookworm Proteases

2.2.2.1 Aspartic proteases

Aspartic proteases belong to clan AA and have two aspartic acid residues in

the active site cleft which are utilised for catalysis of peptide substrates (Rawlings et

al., 2008). They are optimally active at acidic pH, have endopeptidase activity and

nearly all known aspartic proteases are inhibited by pepstatin (Umezawa et al.,

1970). Aspartic proteases have several functions in mammals, including the

processing of hormones, growth factors and proteolytic enzymes (Tang and Wong,

1987). Aspartic proteases include pepsins, cathepsins D and E, and renins, and are

considered to be the most conserved group of the five classes of proteases (Tang and

Wong, 1987). One of the most well known aspartic proteases belongs to the A1

family of enzymes, which is typified by the mammalian gastric enzymes pepsin and

gastricsin. The A1 family also includes the lysosomal processing enzyme cathepsin

D (Rawlings et al., 2008).

Cathepsin D-like aspartic protease genes have been reported from human

blood flukes (Schistosoma japonicum and Schistosoma mansoni), which are known

to digest haemoglobin (Brindley et al., 2001, Tort et al., 1999). Gene knockout of

SmCatD demonstrated significant growth reduction of S. mansoni in vitro,

27

suppression of aspartic protease enzyme activity and an absence of black-pigment

heme in the gut, suggesting that this protein plays an important role in nutrient

absorption (Morales et al., 2008). Cathepsin D-like aspartic proteases have also been

identified from adult A. caninum and N. americanus. The A. caninum cathepsin-D-

like aspartic protease, termed Ac-APR-1, is 47% identical to SmCatD from S.

mansoni, and is expressed in the intestinal microvilli of adult hookworms

(Williamson et al., 2002). Dogs vaccinated with recombinant Ac-APR-1 had

significant reductions in hookworm burdens (33%) and fecal egg counts (70%), in

comparison to control dogs (Loukas et al., 2005a). More importantly, vaccinated

dogs had reduced blood loss and most did not develop anaemia, which is the main

pathology of hookworm disease (Loukas et al., 2005a). In addition, IgG from

vaccinated animals decreased the catalytic activity of the recombinant enzyme in

vitro and the antibody bound in situ to the intestines of worms recovered from

vaccinated dogs, implying that the antibody interferes with the ability of the parasite

to digest blood (Loukas et al., 2005a). A vaccine trial conducted with Ac-ARP-1 in

hamsters resulted in a 44% reduction in worm burden compared to control hamsters

that received adjuvant only (Xiao et al., 2008), further supporting the development of

this molecule as a recombinant vaccine for hookworm infection.

A second family of aspartic proteases, termed nemepsins, has been identified

from the intestines of blood-feeding strongyle nematodes (Williamson et al., 2003a).

This group of proteases resemble mammalian pepsin more closely than cathepsin D

(Williamson et al., 2003b). In N. americanus, a nemepsin termed Na-APR-2 was

characterised and localised primarily to the intestinal microvillar surface in adult

worms (Bethony et al., 2005). Mouse antisera to Na-APR-2 inhibited 50% of

infective hookworm larvae from penetrating mouse skin in vitro and inhibited the

ability of the protease to cleave peptide substrates in vitro (Williamson et al., 2003a).

A protease from H. contortus (termed Pep1) which is similar to mammalian

pepsinogen has been localized to the gut of H. contortus adult worms, where it is

believed to be involved in digestion of the blood meal (Longbottom et al., 1997).

Pep1 is a component of the highly host-protective integral membrane protein

complex, H-gal-GP, which reduced faecal egg count by 93% and worm burden by

75% when used as a vaccine in sheep (Smith et al., 2000). The contribution of Pep1

to the vaccine efficacy of H-gal-GP has yet to be determined.

28

Four aspartic proteases termed plasmepsin I, II, IV and histoaspartic protease

(HAP) have been identified in the erythrocytic stage of the malaria parasite,

Plasmodium falciparum, and have been localised to the food vacuole indicating that

they are involved in the blood digestion process (Banerjee et al., 2002). Studies

aimed at chemically inhibiting the plasmepsins, as well as gene knockout

experiments, showed that this group of proteases is important for parasite viability

and replication (Liu et al., 2005).

2.2.2.2 Cysteine proteases

Cysteine proteases play numerous roles in the biology of parasitic organisms,

which can range from general catabolic functions and protein processing to parasite

immune evasion, excystment/encystment, exsheathing, and cell and tissue invasion

(Tort et al., 1999). Parasite cysteine proteases are usually very immunogenic and

have been exploited as serodiagnostic markers and vaccine targets (Dalton et al.,

2003). Although host homologues exist, parasite cysteine proteases have distinct

structural and biochemical properties, including pH optima and stability, alterations

in peptide loops or domain extensions, diverse substrate specificities and cellular

locations (Sajid and McKerrow, 2002). Cysteine proteases of parasitic organisms are

divided into two main groups referred to as clans CA and CD (Barrett, 1994). The

most widely reported class of cysteine proteases from parasitic nematodes is clan CA

(Fig. 2.10). The clan CA proteases are further divided into families: C1, which

comprises cathepsins B and L-like proteases, and C2, which comprises calpain-like

proteases (Fig. 2.10) (Sajid and McKerrow, 2002). Cysteine proteases possess an

essential cysteine residue that forms a covalent intermediate complex with substrates.

The papain superfamily has a catalytic triad comprising of Cys, His and Asn residues

and a highly conserved Gln that forms the oxyanion hole (Sajid and McKerrow,

2002).

29

Figure 2.10. Schematic diagram of the cysteine protease superfamily from parasitic helminths. Cysteine proteases of parasitic organisms are divided into two main groups referred to as clans, CA and CD. Papain-like, or Clan CA proteases, are further divided into family C1 (cathepsin B and cathepsin L-like) and family C2 (calpain-like) (Sajid and McKerrow, 2002).

Cysteine proteases have been identified in whole worm extracts from A.

caninum and N. americanus, using the synthetic fluorogenic substrates such as Z-

Phe-Arg-AMC (Harrop et al., 1995, Loukas et al., 2000). Cysteine proteases have

also been identified in ES products obtained from adult hookworms, with a five fold

increase compared to whole worm extracts (Loukas et al., 2000). It is hypothesized

that secreted cysteine proteases may be involved in causing eosinophilic enteritis in

humans - an allergic response after zoonotic infection with A. caninum (Loukas et

al., 2000). Two A. caninum cysteine proteases, termed Ac-CP-1 and Ac-CP-2,

identified from the adult stage, are closely related to human and bovine cathepsin B

(Harrop et al., 1995). Molecular models of the active site of Ac-CP-1 indicate that,

although it is structurally similar to the cathepsin B gene, it may have cathepsin L-

30

like specificity (Harrop et al., 1995). Ac-CP-1 immunolocalises to the oesophageal,

amphidal and excretory glands of adult worms, which suggests that Ac-CP-1 is

available for secretion at the site of attachment to the host (Harrop et al., 1995). In

contrast, Ac-CP-2 immunolocalised to the brush border membrane of the intestine of

adult hookworms, suggesting that it is involved in blood meal digestion (Loukas et

al., 2004, Williamson et al., 2004).

Vaccination of dogs with Ac-CP-2 resulted in a marked decrease in faecal egg

count, and the number of female hookworms present in the intestine was

significantly reduced relative to control dogs (Loukas et al., 2004). Adult worms

recovered from the intestine of dogs vaccinated with Ac-CP-2 were significantly

smaller than hookworms from control dogs (Loukas et al., 2004). It was also shown

that anti–Ac-CP-2 antibodies bound to the gut of hookworms retrived from

vaccinated dogs, which suggests that these antibodies were ingested by the parasites

with their blood meal. IgG from vaccinated dogs decreased proteolytic activity of the

recombinant protein against a peptide substrate by 73%, which implies that

neutralizing antibodies were induced by vaccination (Loukas et al., 2004).

Cysteine proteases have also been identified in H. contortus. Five distinct

cysteine protease cDNAs have been cloned and termed AC1 to AC5 (Pratt, 1992).

Members of this gene family appear to be expressed only in the adult stage of this

parasite, and, hence, it has been hypothesized that these proteases are involved in

digestion of the blood meal (Pratt, 1990). These cysteine proteases have been

demonstrated to inhibit blood clot formation and to degrade haemoglobin,

fibrinogen, collagen and IgG, suggesting a role for the enzymes in attachment, blood

feeding and immune evasion by the adult worm (Roads and Fetterer, 1995). A

cysteine protease enriched fraction (TSBP), prepared from membrane extracts of

adult H. contortus, was shown to localise to the microvillar surface of intestinal cells

of the worm. Lambs immunised with TSBP were substantially protected against a

single challenge infection with H. contortus, with reductions in daily faecal egg

outputs of 77% and final worm burdens of 47%, compared to the control group

(Knox et al., 1999). The microvillar surface of worms that survived in, and were

recovered from, vaccinated lambs was found to be coated with sheep

immunoglobulin, and antibody harvested from vaccinated lambs functionally

inhibited the cysteine protease components of TSBP (Knox et al., 2005).

31

Several classes of cysteine protease have also been identified from adult stage

Schistosoma species: these include proteins with cathepsin B, C and L like functions

(Caffrey et al., 2004). Antisera against the cathepsin L-like proteases SmCL1 and

SmCL2 (from S. mansoni) recognize schistosome gut tissue, suggesting a role for

these proteases in blood meal digestion (Bogitsh et al., 2001). Similarly, cathepsin B-

like protease SmCB1 also immunolocalised to the gut region of adult schistosomes,

and was shown to be able to cleave haemoglobin, thus is thought to play role in

nutrient absorption (Delcroix et al., 2006, Lipps et al., 1996).

Cysteine protease activity has also been detected in P. falciparum, with three

proteases, termed falcipains 1-3, that are localised to the food vacuole and thought to

have a role in haemoglobin digestion (Shenai et al., 2000, Sijwali et al., 2001).

Experiments conducted with gene knockout of falcipain-2, showed that trophozoites

developed swollen, haemoglobin filled food vacuoles indicative of a block in

haemoglobin digestion. Gene disruption of falcipain-3 was unsuccessful, suggesting

that this protein is essential for survival of the erythrocytic parasite (Sijwali et al.,

2006).

2.2.2.3 Metalloproteases

Zinc metalloproteases are enzymes that rely on the presence of a zinc atom

coordinated by nucleophilic amino acids at the active site for catalytic activity

(Hooper, 1994). This enables the polarisation of the target scissile peptide bond

before nucleophilic attack and subsequent cleavage (Hooper, 1994). The majority of

the metalloproteases identified to date belong to the clan MA (Fig. 2.11) (Rawlings

et al., 2008) and are recognised by the presence of a HEXXH motif in which the two

His residues are zinc ligands and the Glu has a catalytic function (Hooper, 1994).

A major component of the H. contortus highly protective antigen complex,

H-gal-GP, is a family of four zinc metalloendopeptidases, designated MEPs 1–4.

These enzymes belong to the M13 zinc metalloendopeptidase family (EC 3.4.24.11),

which are also known as neutral endopeptidases or neprilysins (Newlands et al.,

2006). Vaccination of sheep with a combination of all four MEPs, separated

chromatographically from the rest of the complex, reduced H. contortus egg counts

by 45 to 50%, compared to the control group (Smith et al., 2003). Similarly, MEP3

alone or MEPs 1, 2 and 4 in combination each reduced egg counts by 33% (Smith et

al., 2003). It was therefore suggested that the MEPs are the host-protective

32

components of the H-gal-GP complex, and that MEP3 is the most effective member

of this metalloendopeptidase family (Smith et al., 2003). Jones and Hotez (2002)

cloned a metalloprotease from A. caninum: termed Ac-MEP-1, this protease is a

neprilysin-like zinc dependent enzyme and was shown to be expressed in the

intestinal lumen of adult stage hookworms (Jones and Hotez, 2002). Ac-MEP-1

exhibits significant similarity to the H. contortus developmentally regulated

metalloprotease, MEP1, which is expressed in L4 larvae and adult stages of the

parasite, and immunolocalises to the gut microvilli of the adult worm, where it is

hypothesized to play a role in blood meal digestion (Redmond et al., 1997).

Metallopeptidases have also been detected in P. falciparum, but unlike the

nematode metalloproteases, these proteases belong to clan ME, family M16, which

includes an “inverted” HXXEH active site motif (Fig. 2.11) (Eggleson et al., 1999).

P. falciparum falcilysin shares primary structural features with M16 family members

such as insulysin, mitochondrial processing peptidase, nardilysin, and pitrilysin, and

has been localised to the food vacuole where it is thought to play a role in

haemoglobin degradation (Eggleson et al., 1999).

Figure 2.11. Families of zinc metalloproteases. This schematic shows the families of the zinc metalloproteases and their inter-relationships, based on the sequence around the zinc binding residues. (Hooper, 1994)

33

2.2.2.4 Exopeptidase and aminopeptidases

In addition to the proteases mentioned above, which mainly have

endopeptidase and oligopeptidase activities, it has been suggested that

aminopeptidases and proteases with exopeptidase functions play a vital role in the

blood digestion process in hematophagous parasites (Williamson et al., 2003b).

Aminopeptidase activity was recently described in P. falciparum trophozoites,

suggesting that this activity was responsible for generating free amino acids from

haemoglobin peptides after prior digestion with plasmepsins, falcipains and

falycilysin (Stack et al., 2007, Gavigan et al., 2001).

Aminopeptidase activity has been detected in the intestine of N. americanus

(McLaren et al., 1974). AcDNA encoding an aminopeptidase has been identified

from A. caninum and is thought to be involved in the hookworm feeding process,

given that it is highly similar to the aminopeptidase from P. falciparum 1(Williamson

et al., 2004). A glycosylated gut membrane protein with aminopeptidase activity

known as H11 has been identified from H. contortus (Smith and Smith, 1993).

Sequence analysis of the H11 clone revealed that it is similar to mammalian

microsomal aminopeptidases which mediate the terminal events of digestion by

digesting small peptides (Smith et al., 1997). H11 is the most effective immunogen

isolated from a parasitic nematode to date, inducing high levels of protection, with

>90% reductions in H. contortus worm burdens compared to the control group

(Smith et al., 1993). H11 has been immunolocalised exclusively to the intestinal

brush-border of adult H. contortus and enzyme activity is inhibited by H11 antisera

in vitro (Smith et al., 1997). Similarly, sheep vaccinated with a leucine

aminopeptidase, a metalloprotease from Fasciola hepatica (the sheep liver fluke),

induced the production of neutralising antibodies and elicited 89% protection against

fascioliasis compared to the control group (Piacenza et al., 1999). It has also been

shown that both S. mansoni and S. japonicum express a gene encoding a member of

the M17 family of leucine aminopeptidases, and immunolocalisation studies showed

that this protein is synthesised in the gastrodermal cells surrounding the gut lumen

(McCarthy et al., 2004). Accordingly, it was proposed that peptides generated by

protein degradation in the lumen of the schistosome gut are absorbed into the

gastrodermal cells and are cleaved intracellularly by the aminopeptidase to free

1 T. Don and A. Loukas unpublished observation.

34

amino acids before being distributed to the internal tissues of the parasite (McCarthy

et al., 2004).

2.3 HAEMOGLOBIN DIGESTION CASCADE The main source of nutrient for haemotophagous parasites are proteins found

in the blood that they ingest. Blood is a specialized fluid that is composed of cells

suspended in plasma. The most abundant cells in blood are red blood cells, which

contain haemoglobin, an iron-containing protein. Haemoglobin is a tetramer

consisting of two α and two β subunits, non-covalently bound to each other and made

of 141 and 146 amino acid residues, respectively (Fig. 2.12). Each subunit has a

molecular weight of about 17 kDa, for a total molecular weight of the tetramer of

about 68 kDa. Each subunit is composed of a protein chain tightly associated with a

non-protein heme group. Each protein chain arranges into a set of alpha-helical

structural segments connected together in a globin fold arrangement, this folding

pattern contains a pocket which strongly binds the heme group.

A B Figure 2.12. Tertiary structure of the haemoglobin molecule and the two subunits Haemoglobin is a tetramer (A) consisting of two α and two β subunits (B) non-covalently bound to each other. (A) (sourced from http://chemistry.ewu.edu/jcorkill/biochem/HemoglobinMOM.jpg. (B) www.science.org.au/sats2004/images/mackay9.jpg)

In mammals, gastric digestion of proteins derived from food involves a

cascade of mechanistically distinct proteolytic enzymes including pepsin, trypsin and

gastricsin. Similarly, haemoglobin the major source of protein for blood-feeding

parasites, is thought to be degraded by a cascade of proteases found in the intestine

(or food vacuole in Plasmodium) (Williamson et al., 2003b). A proteolytic cascade of

haemoglobin digestion has been identified in the malaria parasite P. falciparum,

35

where a semi-ordered catalytic pathway exists and includes members of at least three

different mechanistic classes of endoproteases (Fig. 2.13A) (Goldberg, 2005). It is

believed that plasmepsin I and II initiate the degradation process by cleaving

haemoglobin at the hinge region. The process is then followed by cleavage of globin

fragments by other plasmepsins, falcipains and falcilysins (Goldberg, 2005). Lastly

aminopeptidases are thought to release free amino acids which can then be used for

protein synthesis. Aspartic and cysteine proteases present in the lumen and

surrounding gastrodermal cells of blood feeding adult schistosomes are also thought

to play a role in the degradation of haemoglobin through an ordered pathway (Fig.

2.13B) (Brindley et al., 1997, Delcroix et al., 2006). However, unlike the digestive

pathway that has been described in P. falciparum, there appears to be less

redundancy in schistosomes, as only one aspartic protease (catD) has been implicated

in initiating the process (Brindley et al., 2001). In schistosomes, both cathepsin B and

L-like cysteine proteases are capable of digesting haemoglobin fragments and in

addition, an asparaginyl endopeptidase has also been implicated in the process but

cannot cleave haemoglobin and is thought to be necessary for processing some of the

SmCatBs (Fig. 2.13B) (Bogitsh et al., 2001, Caffrey and Ruppel, 1997, Dalton et al.,

1997, Sajid et al., 2003). As yet, no metalloendopeptidase has been identified from

the gut of adult schistosomes, however a metallo leucine aminopeptidase has been

identified and is hypothesised to be involved in release of free amino acids

(McCarthy et al., 2004).

In similar fashion to P. falciparum and S. mansoni, it has been suggested that

hookworms also employ a cascade of haemoglobinolysis using multiple proteases of

distinct mechanistic classes (Fig. 2.14) (Williamson et al., 2003b). Don et al.

demonstrated that erythrocytes ingested by A. caninum are lysed in the gut using a

pore-forming membrane-bound hemolysin, which releases the red cell contents into

the intestinal lumen for proteolytic degradation (Don et al., 2004). Similarly to P.

falciparum plasmepsins (I-II) and falcipains (2-3), the A. caninum aspartic protease

Ac-APR-1 and the cathepsin B-like cysteine protease Ac-CP-2 were both shown to be

capable of digesting haemoglobin in vitro (Williamson et al., 2004). While

experiments conducted with Ac-MEP-1, indicated that like falcilysin, it was

incapable of digesting native haemoglobin or heat-denatured globin, it could further

digest globin fragments generated by initial cleavage with aspartic and cysteine

proteases (Williamson et al., 2004).

36

Figure 2.13. Proposed haemoglobin degradation pathway in P. falciparum and S. mansoni. In P. falciparum (A) it is proposed that the degradative enzymes function in a semi-ordered pathway, with plasmepsins making the initial cleavage in intact hemoglobin, followed by secondary cleavages by falcipains, falcilysin, while the dipeptidylpeptidases and aminopeptidases are presumed to function most efficiently in terminal degradation/amino acid release. (Goldberg, 2005) In S. mansoni (B) the primary cleavage of hemoglobin is facilitated by cathepsin D, followed by the endopeptidases cathepsins B1, L1. Peptides are then further broken down by exopeptidase cathepsin B1 and aminopeptidases. (Delcroix et al., 2006)

Hydrostatic pressure and peristalsis of the nematode pseudocoelom ensures

that blood ingested by adult hookworms has a rapid passage time through the

intestine and out of the anus (Roche and Layrisse, 1966). It is therefore important for

the parasite to quickly lyse ingested erythrocytes and employ fast acting

haemoglobinases in the intestinal lumen. Complete digestion of the haemoglobin

tetramer by Ac-APR-1 and Ac-CP-2 was evident after just 15 minutes in vitro

(Williamson et al., 2004), however it is difficult to extrapolate these findings to those

occurring in the gut of a worm in vivo; suffice to say that haemoglobinolysis occurs

rapidly with hookworm haemoglobinases.

A

B

37

Figure 2.14. Schematic of proposed haemoglobinase cascade in the intestine of blood-feeding nematodes. It is postulated that host Hb is initially cleaved by aspartic proteases and degraded to smaller peptides by cysteine proteases and then metalloproteases. Finally exopeptidases complete the digestion to constituent amino acids. (Williamson et al., 2003b)

2.4 SUMMARY Hookworms are one of the most debilitating intestinal pathogens in the

developing world. New control measures are required, the most important perhaps

being the development of a prophylactic vaccine. Proteases expressed by the various

life stages of blood-feeding parasites appear to be viable candidates for development

of novel therapeutics, so a more comprehensive understanding of their roles in host-

parasite interactions is warranted (Dalton et al., 2003). Unlike vaccines produced

against viruses and bacteria which reproduce asexually in the host, sterile immunity

against helminth parasites, which do not reproduce asexually, is not essential for an

effective vaccine (Loukas et al., 2005b). Therefore, an efficacious vaccine would be

one which decreases worm burden and egg output, thereby minimising pathology as

well as lowering transmission rates (Fig. 2.15). Elucidation of the molecular

mechanisms by which haematophagous worms digest blood should lead to the

production of new generation control strategies. By exploiting the absolute

requirement of hookworms for blood, a recombinant vaccine targeting the digestion

of the bloodmeal of these complex multicellular pathogens is a realistic near term

goal.

38

Figure 2.15 Schematic for the development of a bivalent human hookworm vaccine. A bivalent recombinant human hookworm vaccine should consist of a protein that targets (1) invasion and migration of the L3 (eg, Na-ASP-2) and (2) blood-feeding by the adult hookworm (eg haemoglobinase) in order to be efficacious. (Loukas et al., 2005b) 2.5 THESIS HYPOTHESIS

The main underlying hypothesis of this study is that human hookworm, N.

americanus, digests haemoglobin using a semi-ordered cascade of mechanistically

distinct proteases, termed haemoglobinases, which are expressed in the gut region of

the adult worm. Furthermore, it is hypothesised that haemoglobinases are excellent

targets for an anti-hookworm vaccine, as interfering with the parasite’s ability to

digest blood, will decrease their growth, fecundity and survival. It has previously

been demonstrated that proteases expressed in the gut of other parasitic helminths,

such as H. contortus and S. mansoni, are viable vaccine candidates, providing

significant levels of protection by reducing both worm burden and faecal egg output.

It is proposed that the ideal hookworm vaccine will consist of a cocktail of two

recombinant proteins - one targeting penetration and migration of infective larvae,

and a second protein (such as a haemoglobinase) that will interrupt blood-feeding by

adult worms, thereby reducing blood loss and anaemia.

39

This thesis has been submitted by publication. As such, each

research chapter (Chapters 3 to 5) presents a manuscript

which has been re-formatted to suit the style of the thesis.

Published versions of the papers are attached as

appendices.

CHAPTER 3: A SURVERY OF THE INTESTINAL

TRANSCRIPTOMES OF THE HOOKWORMS,

NECATOR AMERICANUS AND ANCYLOSTOMA

CANINUM,USING TISSUES ISOLATED BY

LASER MICRODISSECTION MICROSCOPY (International Journal for Parasitology 36: 701-710)

Najju Ranjita,b, Malcolm Jonesa,c, Deborah Stenzelb, Robin Gasserd, Alex

Loukasa,e aDivision of Infectious Diseases and Immunology, Queensland Institute of Medical Research,

Brisbane, QLD, Australia, bSchool of Life Sciences, Queensland University of Technology, Brisbane,

QLD, Australia, cSchool of Molecular and Microbial Sciences, The University of Queensland,

Brisbane, QLD, Australia, dDepartment of Veterinary Science, The University of Melbourne,

Werribee, VIC, Australia, eAustralian Centre for International and Tropical Health and Nutrition, The

University of Queensland, Brisbane, QLD, Australia.

41

3.1 CONTRIBUTIONS Contributor Statement of contribution

Najju Ranjit

-Designed all the experiments -Conducted all the experiments -Interpreted and analysed all the data -Drafted manuscript

Malcolm Jones -Aided in experimental design – LMM experiments -Provided feedback on manuscript

Deborah Stenzel -Discussed experimental design -Provided feedback on manuscript

Robin Gasser -Investigator on the grant that funded this project -Arranged for sequencing of ESTs -Provided feedback on manuscript

Alex Loukas -Aided in experimental design -Aided in data analysis -Aided in drafting manuscript

Principal Supervisor Confirmation I have sighted email or other correspondence from all Co-authors confirming their certifying authorship.

Alex Loukas 27-5-08 Name Signature Date

42

3.2 ABSTRACT The gastrointestinal tracts of multi-cellular blood-feeding parasites are targets

for vaccines and drugs. Recently, recombinant vaccines that interrupt the digestion of

blood in the hookworm gut have shown efficacy, so we explored the intestinal

transcriptomes of the human and canine hookworms, Necator americanus and

Ancylostoma caninum, respectively. We used Laser Microdissection Microscopy

(LMM) to dissect gut tissue from the parasites, extracted the RNA and generated

cDNA libraries. A total of 480 expressed sequence tags (ESTs) were sequenced from

each library and assembled into contigs, accounting for 268 N. americanus genes and

276 A. caninum genes. Only 17% of N. americanus and 36% of A. caninum contigs

were assigned Gene Ontology classifications. Twenty-six (9.8%) N. americanus and

18 (6.5%) A. caninum contigs did not have homologues in any databases including

dbEST – of these novel clones, seven N. americanus and three A. caninum contigs

had Open Reading Frames (ORFs) with predicted secretory signal peptides. The

most abundant transcripts corresponded to mRNAs encoding cholesterol- and fatty

acid-binding proteins, C-type lectins, Activation-Associated Secretory Proteins, and

proteases of different mechanistic classes, particularly astacin-like metallopeptidases.

ESTs corresponding to known and potential recombinant vaccines were identified

and these included homologues of proteases, anti-clotting factors, defensins and

integral membrane proteins involved in cell adhesion.

Keywords: Laser microdissection microscopy; Hookworm; Expressed sequence tag;

Intestine; Protease; Vaccine.

43

3.3 INTRODUCTION Hookworms are blood-feeding nematodes which inhabit the small intestines

of their definitive mammalian hosts. Infective, third-stage larvae (L3) penetrate the

host’s skin and migrate via the circulatory system to reside in the duodenum as adult

stage worms (1-1.5 cm in length). Adult parasites bury their anterior ends beneath the

mucosa of the bowel, rupture capillaries and feed on the extravasated blood. The

pathogenesis of hookworm infection is a direct consequence of the blood loss which

occurs during attachment and feeding. In developing countries, hookworms are the

leading cause of iron deficiency anaemia, which, in heavy infections, can cause

developmental and mental retardation in children as well as adverse maternal-foetal

outcomes in pregnant women (Hotez et al., 2004).

Current control strategies for hookworm are limited mainly to the treatment

of infected patients with anthelmintic drugs. However, due to increasing drug

resistance in parasitic nematodes of livestock and the perception that this may occur

in helminths of humans, as well as the absence of naturally acquired immunity in

most exposed people (Loukas et al., 2005b), the major focus of research has shifted

towards developing an effective hookworm vaccine. Through the auspices of the

Human Hookworm Vaccine Initiative, an antigen (Na-ASP-2) derived from the L3 of

Necator americanus, a major hookworm species of humans, was selected for

progression to clinical trials (Bethony et al., 2005, Goud et al., 2005). Na-ASP-2 is

expressed exclusively by the L3 and is only partially efficacious at reducing worm

burdens in vaccinated animals. Therefore, a useful human hookworm vaccine will

likely require a second antigen, preferably one derived from the adult blood-feeding

stage of the parasite (Loukas et al., 2005b, Hotez et al., 2003c).

Hookworms ingest red blood cells, lyse the cells in their intestines via pore-

forming proteins (Don et al., 2004) and digest the liberated haemoglobin using a

semi-ordered cascade of proteases, some of which have been characterised in vitro

(Hsieh et al., 2004). Vaccine trials in dogs with some of the recombinant

haemoglobin-degrading proteases (haemoglobinases) have shown encouraging levels

of efficacy (Don et al., 2004, Asojo et al., 2005). Moreover, a related nematode that

parasitizes livestock, Haemonchus contortus, can be successfully vaccinated against

using extracts enriched for haemoglobinases (Knox et al., 2005, Knox et al., 2001),

and a recombinant vaccine against cattle tick targets a gut membrane glycoprotein

44

(de la Fuente et al., 1999), lending support to the targeting of gut proteins for the

development of vaccines against blood-feeding parasites.

Expressed sequence tags (ESTs) have been characterised from hookworms

(Mitreva et al., 2005, Daub et al., 2000), but the majority of these sequences are

derived from the L3 stage for Ancylostoma sp. (approximately 20,000 -

www.nematode.net). Less than 5,000 ESTs from N. americanus have been described

(see www.nematodes.org) and are deposited in dbEST (www.ncbi.nlm.gov/dbEST)

(Parkinson et al., 2004). With a view to identifying mRNAs encoding potential new

vaccine antigens from the hookworm gut, we characterised gut-specific transcripts of

the two major hookworms of humans and canines, N. americanus and Ancylostoma

caninum, respectively.

Adult hookworms are small, which makes it very difficult to accurately

dissect individual tissues, unlike the situation for larger nematodes such as H.

contortus (Jasmer et al., 2001). However, with the development of Laser

Microdissection Microscopy (LMM), a technique which allows dissection of tissues

and even individual cells (Jones et al., 2004), it is now possible to dissect defined

organs and cells from small parasites for subsequent isolation of tissue-specific

proteins and nucleic acids. The potential of this application for extracting individual

cells/tissues from histological sections of helminths has been proposed (Jones et al.,

2004). Here, we describe the first application of LMM to the dissection of gut tissue

from adult N. americanus and A. caninum, extraction of RNA for production of

cDNA libraries, and comparative analyses of ESTs from each library.

3.4 MATERIALS AND METHODS 3.4.1 Parasite material

A Shanghai strain of N. americanus was maintained in hamsters at The

George Washington University, and worms were a kind gift from Drs Bin Zhan and

Peter Hotez. Adult A. caninum were collected from dogs in Brisbane, Queensland, as

described previously (Don et al., 2004). The recovered worms were washed 3× in

PBS, individually positioned in Optimal Cutting Temperature (OCT, Tissue-tek)

compound and snap-frozen on dry ice. Frozen blocks were kept at -80oC until

sectioned onto glass slides which were coated with poly-ethylene naphthalene

membrane. Frozen blocks were sectioned at a thickness of 7 microns.

45

3.4.2 Laser microdissection microscopy (LMM)

After sectioning, each slide was individually wrapped in plastic wrapping

pretreated with RNAase Zap (Ambion) and stored at -20oC until needed. Slides were

washed with diethylpyrocarbonate (DEPC) water to remove OCT, fixed in 100%

methanol, stained with hematoxylin to allow visualisation of intestinal tissue and

dehydrated with 70% ethanol, 100% ethanol and xylene respectively. Slides were

dried in a fume hood for ~ 2 hours. Intestinal or gonadal tissues were selected and

catapulted separately into 0.6 ml microfuge tube lids containing 30 μl of Trizol

(Invitrogen) using a PALM MicroBeam Laser Catapult Microscope (P.A.L.M.

Microlaser Technologies, Bernried, Germany).

3.4.3 RNA extraction, cDNA synthesis and detection of known gut transcripts

Total RNA was isolated from catapulted sections using Trizol (according to

the manufacturer’s instructions), with a yield of 200 ng for A. caninum and 130 ng

for N. americanus. The cDNA was generated using a SuperSMART PCR cDNA kit

(BD Clontech) where full-length cDNAs are generated by oligocapping. Second-

strand synthesis and PCR amplification were conducted according to the

manufacturer’s instructions. cDNA fragments >1 kb were size selected on a 0.8%

agarose gel, excised and gel extracted with a kit (Qiagen). Gut cDNA was used as a

template for PCR amplification of transcripts which were already known to be

expressed in the gut (via immunlocalisation) using gene specific oligonucleotide

primers that corresponded to the following cDNAs - Ac-mep-1 5’

CCGAAAAGGGACCACTTCCTG 3’ (forward primer), 5’

AGTCGCTAAGGCTTCCGTCG 3’ (reverse primer); Na-mep-1 5’

CGCGCCGGATCCGACAACGATAACCCACCA 3’ (forward primer), 5’

CGCGCCCTCGAGCTCTTGAACCCAAACTGA 3’ (reverse primer) (Jones and

Hotez, 2002) (N. Ranjit and A. Loukas, unpublished); Na-apr-1 5’

CGCGCCGAGCTCAGCGTTCATCGACGACTC 3’ (forward primer), 5’

CGCGCCAAGCTTAAAAAAGTAA 3’ (reverse primer) (Hotez et al., 2002); Na-

apr-2 5’ CGCGCCGAGCTCGGTGTATATAAAATCCCA 3’ (forward primer), 5’

CGCGCCAAGCTTAAATGTTTTACAGCTGCAAAACC 3’ (reverse primer)

(Bethony et al., 2005). Primers corresponding to a gene with a presumed ubiquitous

expression pattern, N. americanus β−tubulin (AF453524) - 5’

CGAATCTCGTGCCATATCGT 3’ (forward primer), 5’

46

TTCCTCCATACCCTCACCGA 3’ (reverse primer) - were used to amplify

transcripts from both cDNA populations, and cDNA recovered from whole adult

worms. Primers corresponding to mRNAs for proteins that localise to sites other than

the gut were also used in the PCR, employing as a template cDNA from gut, gonad

or whole adults. Non-gut derived cDNAs included Ac-asp-4 5

‘AAGCCAGTGTCTCACAGGAGGGT 3’ (forward primer), 5’

TCGGGTCTTGGTCATAGATGGGG 3’ (reverse primer) and Ac-asp-6 5’

TTTGTGGACCATAACAGTGCGGC 3’ (forward primer), 5’

TTTTGGGGAGTAGGGCAGACGA 3’ (reverse primer) (Zhan et al., 2003).

3.4.4 Construction of cDNA libraries

Non-directional cDNA libraries were constructed from gut cDNA in the

plasmid vector pGEM-T (Promega) using T-ended cloning. One hundred and fifty ng

of size-selected cDNA was added to 50 ng of vector. Colonies were screened using

blue-white selection. Five hundred clones from each library were randomly picked

and patched onto grided Luria Bertani (LB) agarose plates containing 100 μg/ml

ampicillin. Sequencing reactions were performed at AgGenomics (Melbourne,

Australia), utilising ABI BigDye terminator v3.1 and an ABI 3730xl DNA analyser.

Single-pass sequencing was performed on each template using the T7 primer.

3.4.5 Bioinformatic analyses

Sequence corresponding to vector was trimmed from the raw sequence data

using BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The

sequence of each EST was also manually edited in order to remove poly A tails and

poor quality sequence before further analyses. All edited sequences were condensed

into contigs or singletons using the contig analysis program (CAP) in BioEdit with

parameters of 100 bp overlap and a minimum of 95% identity at the nucleotide level.

Sequences were compared to other sequences in GenBank (nr), Wormbase

(http://blast.wormbase.org/db/searches/blat) and nematode.net

(http://www.nematode.net/index.php) using BLASTx and BLASTn

(http://www.ncbi.nlm.nih.gov/BLAST/), and WU-BLAST

(http://www.ebi.ac.uk/blast2) respectively. BLAST alignments with an E-value of ≤

1 × 10-5 were reported. Clusters were functionally categorised using InterProScan

(http://www.ebi.ac.uk/InterProScan) and clusters were mapped to the three

47

organising principles of gene ontology (http://www.geneontology.org). Files were

submitted as batch sequences at the Goblet web browser

(www.goblet.molgen.mpg.de/cgi-bin/goblet-batch.cgi), sequences were queried

against Wormbase (http://www.wormbase.org/) and alignments with an E-value of ≤

1 × 10-20 were reported. Multiple sequence alignments were conducted using

ClustalW at the BCM Search Launcher (http://searchlauncher.bcm.tmc.edu/multi-

align/multi-align.html). Predictions of signal peptides were conducted using SignalP

3.0 (http://www.cbs.dtu.dk/services/SignalP/) incorporating both neural networks

and hidden Markov models. Predictions of transmembrane domains were conducted

using TMPred (http://www.ch.embnet.org/software/TMPRED_form.html). The clan

and family assignments of proteolytic enzymes were analysed via the MEROPS

protease database (Rawlings et al., 2008).

3.4.6 Phylogenetic tree

Phylogenetic relationships were assessed and trees were generated by the

neighbour joining method using PAUP 4.0 beta version (Swofford, 1993).

Robustness was assessed by bootstrap analysis using 100 replicates; clades with

more than 50% support are denoted with bootstrap values on the branches.

3.5 RESULTS AND DISCUSSION 3.5.1 Extraction of gut tissues from hookworms

Intestinal and gonad tissues were readily identified by light microscopy in

both longitudinal and transverse sections of both A. caninum and N. americanus (Fig.

3.1). Each slide contained sections of 5-7 worms and tissue was extracted from 12

slides per species, corresponding to a total of ~ 720,000 μm2 tissue. Two hundred ng

and 130 ng of total RNA were extracted from A. caninum and N. americanus gut

tissue, generating 2.5 μg and 1.7 μg of double stranded cDNA respectively.

48

Figure 3.1. Light micrographs of adult hookworm section before and after laser microdissection. Longitudinal sections of A. caninum before (A) and after (B) removal of highlighted tissues by laser microdissection. Transverse sections of N. americanus before (C) and after (D) removal of highlighted tissues by laser microdissection. lu – gut lumen; in – intestine; ov – ovary. 3.5.2 Tissue specificity of cDNA populations

To verify that extracted tissue catapulted from intestinal tissues was gut-

derived and did not contain control tissue dissected from gonad, we used cDNA

populations extracted from gut and gonad as templates in the PCR employing

oligonucleotide primers corresponding to mRNAs for which the anatomic expression

sites of the corresponding proteins have previously been demonstrated using

immunofluorescence with specific antibodies against the recombinant proteins

(Hotez et al., 2002, Bethony et al., 2005, Jones and Hotez, 2002). Partial transcripts

corresponding to these mRNAs were successfully amplified from gut-derived but not

gonad-derived tissues (Fig. 3.2). An mRNA for a protein presumed to be

ubiquitously expressed throughout all tissues, β−tubulin (AF45352), was amplified

from both gut and gonad cDNAs. Two cDNAs encoding the activation associated

secreted proteins – Ac-ASP-4 which was immunolocalised to the cuticle surface and

Ac-ASP-6 which was immunolocalised to cephalic and excretory glands (Zhan et al.,

A

C

B

D

49

2003) - were amplified from gonad (and whole worm cDNA – not shown) but not

from gut cDNA (Fig. 3.2).

Figure 3.2. Detection in gut but not ovary cDNA of transcripts corresponding to proteins that were previously shown to be expressed in the intestine using immunolocalisation. Agarose gels stained with ethidium bromide showing PCR products amplified from N. americanus gut cDNA (A) or gonad cDNA (B). Panel A - lanes 1-6: Na-apr-2, Ac-mep-1, Na-apr-1, Na-β- tubulin, Ac-asp-6, Ac-asp-4. Panel B – lanes 1-5: Na-mep-1, Na-apr-1, Na- β -tubulin, Ac-asp-6, Ac-asp-4.

3.5.3 Characteristics of the EST dataset

ESTs were derived from the cDNA libraries representing the intestinal tissues

from each of N. americanus and A. caninum. Four hundred and eighty ESTs were

generated from each library and assembled into contigs to remove redundant

sequences and establish the quality and length of the sequences. Contigs were further

grouped into clusters to provide a non-redundant catalogue of partial genes

represented. Clusters ranged in size from a single EST to 20 and 30 ESTs for N.

americanus and A. caninum respectively, but generally most clusters consisted of

five or less ESTs. The average sizes of inserts were 400 bp for N. americanus and

490 bp for A. caninum. After the contig assembly, there were 268 N. americanus

cDNAs and 276 A. caninum cDNAs, achieving cDNA discovery rates of 55% and

57%, respectively.

3.5.4 Sequence analysis and gene ontology

In addition to the data provided in Table 3.1, 230 N. americanus contigs (86% of the

total) and 249 A. caninum contigs (90%) had ORFs ≥ 30 amino acids. Of these

contigs with ORFs of ≥ 30 amino acids, 136 N. americanus and 120 A. caninum

contigs had significant homologues in dbEST only, and did not have homologues in

GenBank nr; the vast majority of these homologues were derived from parasitic

nematodes. Of those N. americanus contigs with homologues in dbEST only, 41

corresponded to previously identified N. americanus ESTs in dbEST (≥ 95% identity

1200

300 300

A B

1 2 3 4 5 6 1 2 3 4 5

50

at the nt level over at least 100 nt). Of those A. caninum contigs with homologues in

dbEST only, 34 corresponded to previously identified A. caninum ESTs in dbEST.

Gene ontology (GO) and BLAST analyses revealed some interesting features

for the two datasets, particularly the relatively large number of contigs with no

homology to molecules with known functions (in any of the public databases

interrogated – Table 3.1). Of these, 7 N. americanus and 3 A. caninum contigs had

ORFs with putative N-terminal signal peptides (Table 3.2), indicating that the

corresponding mRNAs encoded secreted and/or transmembrane proteins with

unknown functions and no known homologues thus far identified in any organism.

Fig. 3.3 provides a summary of representatives categorized by major Gene

Ontology (GO). Level II GO hierarchy was chosen for the classification of ESTs. GO

categories revealed numerous transcripts encoding kinase activity, heat shock

proteins, and metabolic enzymes. Interestingly, only 36% of A. caninum and 17% of

N. americanus contigs mapped to GO categories, with the most common GO

category for both species being molecular functions, in particular binding and

catalytic activity. The two main predicted binding functions were protein- and ion-

binding, whereas the main catalytic activity was that of hydrolases. These GO

assignments were similar to those reported by Mitreva et al. (Mitreva et al., 2005) for

different life history stages of A. caninum and A. ceylanicum, where major GO

categories represented binding, catalytic activity, transporter activity and structural

molecular activity.

51

Table 3.1. Summary of the gut EST datasets for A. caninum and N. americanus contigs with ORFs containing ≥ 30 amino acids.

Species No. of contigsa

No. identical to

existing ESTsb

No. similar to

existing ESTsc

No. similar to

GenBank nr

No.

novel

No. novel with signal

peptide

A. caninum 249 (276) 52 (18.8%) 206 (74.6%) 122 (44.2%)

18

(6.5%) 3 (1.6%)

N. americanus 230 (268) 80 (29.8%) 184 (68.8%) 84 (31.3%)

26

(9.8%) 7 (2.6%)

a Numbers in brackets are total numbers of contigs irrespective of length of ORF. b Identity determined as ≥95% identity at the nucleotide level over at least 100 nt. c Similarity determined as E-value of ≤1.0x105.

52

Table 3.2. Novel clones with no homologues in any datasets that contain ORFs with predicted signal peptides. Double-ended arrows denote the predicted

cleavage site of the signal peptide.

Clone Predicted Signal Sequence Ca S Y s D HMM internal TM

domainsb

Ac92 MRRRSVLLKPTTTNLSIVMLGFILGSPAVS↔VI No Yes Yes Yes Yes SP (0.905) 1

Ac94 MIQNIYLMLILNPHILFL↔YK No No Yes Yes Yes NS 0

Ac198 MAPGSRTSLLLAFALLCLPWLQEAGA↔VQ Yes Yes Yes Yes Yes SP (1.000) 0

Na60 MVRSAVCCSLLFLAPSTTT↔TI No Yes Yes Yes Yes SP (0.998) 1

Na130 MLTFIELLIGVVVIVGVA↔RY Yes Yes Yes Yes Yes SA (0.688) 1

Na138 MLFIYSVNSKTCLLLRFFHPEVVA↔SC Yes Yes No Yes No NS 0

Na144 MEIQKGVEGIGKVKDFLRIFQRFKFFNNCFGW

VFFWLFMFFCWCES↔FF

Yes Yes Yes No No SA (0.841) 0

Na152 MRQRMFLVLMRASYGGL↔ED No No No Yes No NS 0

Na157 MFFCSLFLFSLVFA↔WY Yes No Yes Yes Yes SP (0.850) 0

Na239 MFAYPPVYPLCTLCLGGIRGKSA↔GT Yes No Yes No No SP (0.857) 0

a The SignalP algorithm incorporates both neural networks and hidden Markov models. Output scores are predicted as being above (Yes) or below (No) a defined cut-off. C, ‘cleavage site’; S, ‘signal peptide’ score, Y, the combined C and S scores, s is the mean S score between the N-terminus and the cleavage site and D is the average of the s and Y scores.HMMis the hidden Markov model prediction with the prediction probability in parentheses. SP, signal peptide; SA, signal anchor; NS, non-secretory. b Number of predicted transmembrane domains C-terminal to the predicted signal sequence.

53

Figure 3.3. Pie charts depicting gene ontology classifications of Ancylostoma caninum and Necator americanus gut ESTs identified in this study. 3.5.5 Transcript abundance and highly represented genes

The 10 most abundant clusters from both libraries are presented in Table 3.3 and

account for 17% and 24% of ESTs for N. americanus and A. caninum, respectively.

Transcripts abundantly represented in both of the libraries included genes predicted

to encode proteins which carry out (and facilitate) key energetic and metabolic

processes, such as feeding.

In the A. caninum dataset, transcripts encoding proteases and proteins which bind

to both fatty acids and cholesterol were predominant. Vitellogenin was the most

abundant transcript accounting for 6% of ESTs sequenced and was also one of the

most abundant gut transcripts from N. americanus (Table 3.3). Vitellogenin

transcripts are also abundant in the intestine of H. contortus (Jasmer et al., 2001) and

Pristionchus pacificus (http://www.nematode.net/index.php). A fatty acid-binding

protein with invertebrate (including nematodes) and vertebrate homologues was also

represented by an abundant A. caninum transcript. A homologous hookworm protein

has been suggested to be involved in the scavenging, transport and metabolism of

fatty acids and sterols (Basavaraju et al., 2003), necessary for metabolic and

developmental processes, such as embryogenesis, glycoprotein synthesis, growth and

cellular differentiation (Kennedy, 2000).

A. caninum

20%

13%

3% 64%

N. americanus

7.49% 5.62%

1.12% 1.12%

1.50%

83.15%

Binding Catalytic activity Structural molecular activityTransporter activity Antioxidant activity Unknown

54

3.5.6 Molecules involved in feeding

cDNAs encoding known and new potential anticoagulants were identified in

the gut ESTs. Contig Ac72 was 99% identical to anticoagulant peptide 3 (AcAP3),

while contig Ac166 was 97% identical to AcAP4. Immunolocalisation of AcAP3

showed exclusive expression in the oesophagus, and it has been hypothesised that

these anticoagulants are critical during the blood meal as well as assisting in

digestion by keeping the blood in a liquid state once it has entered the digestive tract

(Mieszczanek et al., 2004).

To avoid the formation of clots at the site of attachment, hookworms secrete a

platelet inhibitor (HPI) which inhibits platelet aggregation and adhesion (Chadderdon

and Cappello, 1999, Hotez et al., 2003c, Del Valle et al., 2003). HPI has been

identified from the cephalic glands of adult worms (Del Valle et al., 2003), and the

identification herein of contig Ac93 (the ninth most abundant transcript in the A.

caninum dataset) suggests that HPI is expressed in the gut as well as the cephalic

glands. The presence of these transcripts in the gut is unlikely to be accounted for by

contamination of dissected gut tissue with oesophagus, because none of the tissue

sections used for laser microdissection contained oesophagus or pharynx. Moreover,

Ac93 was abundantly represented in the gut ESTs yet another mRNA from the

oesophagus, Ac-cp-1 (Sawangjaroen et al., 1995), was not detected in the gut ESTs.

55

Table 3.3. The 10 most abundant contigs from the A. caninum and N. americanus gut expressed sequence tag (EST) datasets

Hookworms are thought to digest the proteinaceous contents of lysed red

cells with a semi-ordered cascade of proteolysis, consisting of aspartic, cysteine and

metalloproteases, some of which have been characterised in vitro (Hsieh et al.,

2004). Three haemoglobinases expressed in the gut of A. caninum were all detected

by PCR in cDNA derived from the gut (Fig. 3.2). Two of these (Ac-APR-1 and Ac-

CP-2) were also detected as ESTs in the present study. At least twelve contigs

encoded for proteases from all four major mechanistic classes between the datasets

of the two species, and nine contigs corresponded to newly identified enzymes

Contig

no.

Closest homologue

Accession no.

(GenBank)

E-value

ESTs

per

contig

Ac153 A. ceylanicum Vitellogenin CB176085 6.10E-130 30

Ac81 A. ceylanicum Excretory/Secretory protein AY046590 4.10E-14 26

Ac201 A. ceylanicum Heat Shock Protein 20 CB338919 7.30E-63 16

Ac242 C. elegans Hypothetical protein CB037581 2.4E-31 16

Ac196 N. americanus 18s small subunit AY295811 3.70E-123 14

Ac129 C. elegans C-type lectin CB176035 2.70E-107 10

Ac197 C. elegans Astacin metalloprotease BM130887 7.60E-17 6

Ac8 A. ceylanicum Fatty acid binding protein CA341320 1.80E-78 5

Ac93 A. caninum Platelet inhibitor AF399709 1.40E-124 5

Ac155 A. caninum Cytochrome c oxidase subunit 1 AW627047 9.40E-45 5

Na205 N. americanus 18s ribosomal RNA AY295811 8.80E-132 20

Na85 N. americanus Heat shock protein 20 BG734476 1.60E-57 12

Na155 A. ceylanicum Lysozyme protein 8 CB190629 8.10E-41 12

Na160 A. ceylanicum Vitellogenin CB176264 7.70E-39 12

Na96 S. ratti MDR protein (ATP-binding cassette) CB098543 4.60E-23 10

Na265 X. index Non-functional folate binding protein CV579873 9.80E-31 9

Na220 N. americanus Amyloid precursor protein BG467555 1.30E-11 6

Na182 A. caninum NADH-ubiquinone oxidoreductase BM077443 1.20E-46 4

Na230 N. americanus Cathepsin B cysteine proteinase BI744492 4.00E-14 4

Na10 N. americanus Cytochrome c oxidase subunit 1 BU088443 1.20E-66 4

56

(Table 3.4). Three new metalloproteases of the astacin family were identified. An

astacin-like enzyme, Ac-MTP-1 is secreted by L3 during the invasion process (Zhan

et al., 2002) and digests connective tissue substrates (Williamson et al., 2006), but

this class of enzyme has not been described in adult hookworms. Compared with

other metalloprotease families, astacin-like enzymes (MEROPS – family M12A) are

the most abundantly expressed metalloproteases in C. elegans, and subgroup I of the

M12A family are secreted by pharyngeal cells into the lumen of the alimentary tract

of the nematode, where the protease is thought to be involved in the digestion of food

(Mohrlen et al., 2003). The M12A proteases identified amongst the A. caninum ESTs

might also be involved in digestion of the blood meal. Contig Ac70 encoded an O-

sialoglycoprotein endopeptidase, a family of metallopeptidases which only cleave

proteins that are O-sialoglycosylated. This family of proteases has not been reported

previously for nematodes which parasitize animals.

Cathepsin B-like cysteine proteases were also abundantly represented in the

gut and were the ninth most abundant gene family in the N. americanus dataset.

Four cathepsins B were identified from N. americanus (Table 3.4), two of which

were not found in the existing N. americanus ESTs derived from entire adult worm

cDNA. The H. contortus intestinal transcriptome is dominated by cysteine proteases

(~16%) (Jasmer et al., 2004, Jasmer et al., 2001), and while abundantly expressed in

N. americanus, the diversity of this gene family appears not to be as extensive as it is

for H. contortus.

57

Table 3.4. Hookoworm (Anyclostoma spp. plus Haemonchus and Caenorhabditis, of comparison) gut ESTs encoding proteolytic enzymes

Clan and family assignments and generic family names from the MEROPS database are provided. BLAST E-values and GenBank accession numbers are provided for homologues. a EST, expressed sequence tags. b ORF, open reading frames. c Previously identified protease known to be expressed in the gut using immunlocalisation

Contig Ac78 encoded an S1A family of serine proteases with similarity to

Manduca sexta prophenoloxidase-activating endopeptidase and vertebrate

coagulation factors VIIa and complement factors C1. A text search of nematode.net

(http://www.nematode.net/index.php) using “serine protease” or “serine proteinase”

Protease class

(MEROPS i.d.)

Contig no. Closest nr homologue Closest EST a ORF

length

(aa) b

Aspartic

Clan AA, Family A1

Cathepsin D

Ac132 Ac-APR-1c

A. caninum

U34888 (6.00x10-66)

84

Clan AA, Family A1 Na48 Na-APR-2c

N. americanus

AJ245458 (1.90x10-36)

43

Metallo-

Clan MA, Family M12

Astacin

Ac110 C. elegans

CAB05814.2 (2x10-15)

Anyclostoma ceylanicum

BM130887 (3.10x10-13)

62

Clan MA, Family M12

Ac197 C. briggsae

CAE60270.1 (7x10-5)

A. caninum

AF397162 (7.60x10-17)

45

Clan MA, Family M12

Na250 C. briggsae

CAE60270.1 (7x10-5)

A. ceylanicum

BM130887 (7.60x10-17)

45

Clan MK, Family M22

O-sialoglycoprotein

endopeptidase

Ac70 C. elegans

AAK29978.4 (3ex10-57)

Pristionchus pacificus

AI989172 (0.019)

142

Cysteine

Clan CA, Family C1A

Cathepsin B

Na56 H. contortus

CAA93278.1 (3x10-18)

N. americanus

BG468101 (1.70x10-12)

53

Clan CA, Family C1A

Na105 Glossina morsitans

AAK07477.2 (3ex10-20)

H. contortus

BM873292 (3.70x10-11)

66

Clan CA, Family C1A

Na191 Fasciola hepatica

CAD32937.1 (7x10-8)

N. americanus

BG468129 (1.10x10-40)

52

Clan CA, Family C1A

Na230 Lonomia obliqua

AAV91452.1 (1x10-19)

A. caninum

BI744492 (7.60x10-17)

53

Clan CA, Family C1A

Ac193 Ac-CP-2

A.caninum

U18912 (4.30x10-80)

88

Serine

Clan PA, Family S1A Ac78 Manduca sexta AAV91012.1 (8.00x10-4)

A. ceylanicum CA341432 (2.5 x10-4)

81

58

did not reveal any serine protease-encoding clusters from hookworms or other

nematodes that parasitise humans; three clusters corresponding to trypsin-like serine

proteases were identified from H. contortus. (A. Loukas, unpublished observation).

3.5.7 Immunomodulation

Another highly-represented gene (sixth most abundant in the A. caninum dataset)

encoded a C-type lectin (C-TL). C-TLs are proteins with a carbohydrate recognition

domain (CRD) which bind to glycoprotein ligands in a calcium-dependent manner.

They are important in a multitude of physiological processes in animals, including

innate and acquired immunity, haemostasis and wound repair. C-TLs are an

abundant gene family in C. elegans (Drickamer and Dodd, 1999), and some are

expressed in the intestine where they are upregulated in response to bacterial

infection (Mallo et al., 2002). Together with cysteine proteases, C-TLs are the most

abundant gene family in the gut ESTs from H. contortus (Jasmer et al., 2001),

although the functions of these molecules have yet to be determined. Identified in

the present study was a full length cDNA (contig Ac129) encoding an N-terminal

secretory signal peptide followed by a long form C-TL domain that shared homology

with Na-CTL-2, C. elegans lectins and CD69, an early leukocyte activation molecule

expressed at sites of chronic inflammation where it is thought to down regulate the

immune response through the production of TGF-β (Sancho et al., 2005) (Fig. 3.4).

A total of six contigs encoded additional C-TLs in the two hookworm EST datasets.

Activation-associated Secreted Proteins (or ASPs), are a family of nematode-

specific cysteine-rich, secreted proteins belonging to the pathogenesis-related protein

superfamily (Hawdon and Hotez, 1996). ASPs expressed by hookworm L3 are

efficacious recombinant vaccines (Bethony et al., 2005, Ghosh and Hotez, 1999).

Adult hookworms also secrete at least four other ASPs, and each of them localises to

a unique organ in the parasite (Zhan et al., 2003). In the present study, two novel

ASPs were identified - contigs Na91 and Ac173 (Fig. 3.5). The functions of most of

the ASPs in adult hookworms are still unknown, however the recent determination of

the crystal structure of Na-ASP-2 from N. americanus revealed a protein

conformation similar to that adopted by some chemokines, prompting speculation

about an immunomodulatory function (Asojo et al., 2005).

59

Ac129 1 ----MFFKSSLLFCVLTTALSVTIN----------------------------------- Na-CTL-2 1 ----MLFISSLFFCVLSTVSSTIIN----------------------------------- CeCBG03358 1 ----MRFFRFLVFPVIAGLSSVLAAPITSNDTVDGSGEAPETLLQNSEEQPHQRLKF--- CD69 1 ------------------------------------------------------------ NKR-P1B 1 MDSTTLVYADLNLARIQEPKHDSPPSLSPDTCRCPRWHRLALKFGCAGLILLVLVVIGLC * * Ac129 22 ----------------------------TTELKCPTGWFEYRDSCYFIDNPLAEYDRAQA Na-CTL-2 22 ----------------------------TTELTCPPGWFGYRDSCYFFDNPLLEHDKAEI CeCBG03358 54 -YNWDYKDLGTTAFEDISFPARQPPVAVNQSEQCPDGWLRFADSCYWIETELMGFAKAER CD69 1 ------------------------------VSSCSEDWVGYQRKCYFISTVKRSWTSAQN NKR-P1B 61 VLVLSVQKSSVQKICADVQENRTHTTGCSAKLECPQDWLSHRDKCFHVSQVSNTWKECRI * Ac129 54 RCWEQGATFLVAETPEEYTYVTEHSKPS-TWSWAGITQED----ENHLPKWSNNGGVDPA Na-CTL-2 54 KCWEMGSTLLVAETLDEYELITDRAKES-AWSWVGLTQSDDL--EHHIPQWSTSGGVDP- CeCBG03358 113 KCFEKQSTLFVANSLEEWDSIRSHSKEA-YFSWIGLVRFTHYEKSEQLPRWQTEGAINP- CD69 31 ACSEHGATLAVIDSEKDMNFLKRYAGR--EEHWVGLKKEP-----GHPWKWSNGKEFNN- NKR-P1B 121 DCDKKGATLLLIQDQEELRFLLDSIKEKYNSFWIGLSYTLT----DMNWKWINGTAFNS- * * * Ac129 109 TMINWLVKPFTPVANGWSTTAKCAAHLNVPVS--FAAYTFFLPCNIQINSICEKNFTLFP Na-CTL-2 110 ILINWLVKPYLAVSNGWTTQAKCAAHLNVPAGP-SASYTFFLPCTVQTYSICEKNATLFP CeCBG03358 171 TKMNWLIKPYKPIVNGWTSFANCAASYKSPATLESASYTFFYPCTYLLYSICERNSTIVN CD69 83 ----WFNV---TGSD------KCVFLKNTEVS--------SMECEKNLYWICNKPYK--- NKR-P1B 176 ----DVLKITGDTENG-----SCASISGDKVT--------SESCSTDNRWICQKELNHET Ac129 167 RIWDHGLVNLK Na-CTL-2 169 RIWEHGLIGL- CeCBG03358 231 VMQ-------- CD69 ----------- NKR-P1B 219 PSNDS------ Figure 3.4. Multiple sequence alignment of contig Ac129 with homologous members of the C-type lectin family. Arrow denotes signal peptide and asterisks denote conserved cysteine residues involved in disulfide bond formation. The conserved WIG motif is boxed. Black boxes denote identical and grey boxes denote similar residues. GenBank accession numbers for homologues are as follows: Na-CTL-2 - AF388311; CeCBG03358 - CAAC01000013; CD69 - NP_001772; NKR-P1B - AAK39100. 3.5.8 Known and Potential Vaccine Antigens

Secreted and membrane-bound proteins are targets for the development of

vaccines because they are exposed to the host immune response. Clones identified in

this study encoded proteins with proven and predicted potential as vaccine antigens

(Table 3.5). For example, vaccination with recombinant ASPs (Bethony et al., 2005,

Goud et al., 2005) and astacin-like metalloproteases (Hotez et al., 2003b) partially

protect dogs against challenge infection with hookworm L3 by targeting invading

larvae, and contigs encoding members of these protein families were identified in

this study. Vaccination of dogs with recombinant haemoglobinases results in

reduced worm burdens, worm fecundity and blood loss (Asojo et al., 2005, Loukas et

al., 2004). ESTs corresponding to two known haemoglobinase vaccine antigens from

adult hookworms (Ac-CP-2 and Ac-APR-1) were identified from the A. caninum

dataset. In addition, ESTs corresponding to four new cathepsin B cysteine proteases

were identified, and some of these might be involved in digestion of the blood meal.

60

Other interesting molecules from a vaccine perspective include contigs with

sequence similarities to anti-microbial alpha-defensins, tissue factor inhibitors, and

integral membrane proteins involved in cell adhesion.

Figure 3.5. Neighbour joining phylogenetic tree showing the relationships of Ac173 and Na91 with other members of the Activation Associated Secretory Protein family. Numbers on branches refer to bootstrap values from 100 replicates. The names and the GenBank accession numbers of the sequences used in the phylogenetic tree are as follows: A. caninum Ac-ASP-4 - AY217005; A. caninum Ac-ASP-6 - AY217007; N. americanus Na-ASP-2 - AY288089; Caenorhabditis elegans F49E11.5 and F49E11.9 - Z70308 (cosmid entry); Ov-VAL, Onchocerca volvulus venom allergen homologue (VAL) - AAB97282; Brugia malayi Bm-VAL - AAK12274; H. contortus Hc24 - AAC47714; A. caninum neutrophil inhibitory factor (NIF) - L27427; A. caninum platelet inhibitor (HPI) - AAK81732; Homo sapiens cysteine-rich secretory protein 2 (CRISP2) - S68682.

61

Table 3.5. Contigs identified in this study with potential as vaccine antigens.

3.6 CONCLUSION Here, we show that LMM is a very useful technique for the dissection of

defined tissues from helminth parasites. Moreover, the quality of nucleic acids

recovered from these tissues is sufficient to yield at least some full-length cDNAs.

The ESTs presented in this study add to the expanding catalogue of hookworm

genes, but importantly, provide the first set of tissue/organ-specific cDNAs from

these important blood-feeding parasites.

Acknowledgements

We thank Mary Lee for technical assistance, Bennett Datu, David McMillan

and Geoff Gobert for helpful discussions and advice, and Bin Zhan and Peter Hotez

for providing N. americanus. We also thank Clare Hopkins from AgGenomics for

conducting sequencing and Ian Smith for his support. This research was funded by

QIMR, a grant from the Bill and Melinda Gates Foundation awarded to the Sabin

Vaccine Initiative, and ARC Linkage Grant LP0667795.

Contig Function

Ac8,-11,-39 Fatty acid-binding proteins

Ac110,-197

Na250

Astacin metalloproteases

Ac27,-41,-62 -129,

-158,-261

C-type lectins

Ac161,-173,-214

Na91

Activation associated secreted proteins

Na56,-105,-191,-230 Cathepsin B cysteine proteases

Ac247,-202 Alpha defensins

Ac78 Serine protease

Na58,-143,-220,-251 Tissue factor inhibitors

CHAPTER 4: A FAMILY OF CATHEPSIN B

CYSTEINE PROTEASES EXPRESSED IN THE

GUT OF THE HUMAN HOOKWORM, NECATOR

AMERICANUS (Molecular and Biochemical Parasitology In press)

Najju Ranjita,b, Bin Zhanc, Deborah J. Stenzela, Jason Mulvennab,

Ricardo Fujiwarad, Peter J. Hotezc, Alex Loukasb

aSchool of Life Sciences, Queensland University of Technology, Brisbane, QLD, Australia; bDivision

of Infectious Diseases, Queensland Institute of Medical Research, Brisbane, QLD 4006, Australia; cDepartment of Microbiology, Immunology and Tropical Medicine, George Washington University

Medical Center, Washington DC 20037, USA; dCellular and Molecular Immunology Laboratory,

Centro de Pesquisas René Rachou, FIOCRUZ, Minas Gerais, Brazil

63

4.1 CONTRIBUTIONS Contributor Statement of contribution*

Najju Ranjit

-Designed all the experiments -Conducted all experiments except of the ones mentioned below -Interpreted and analysed all the data -Drafted manuscript

Bin Zhan -Provided cDNA clones -Provided feedback on manuscript

Deborah Stenzel -Discussed experimental design -Provided feedback on manuscript

Jason Mulvenna -Conducted proteomic analyses -Provided feedback on manuscript

Ricardo Fujiwara -Provided L3 larvae -Provided feedback on manuscript

Peter Hotez -Investigator on the grant that funded the project -Provided feedback on manuscript

Alex Loukas -Aided in experimental design -Aided in data analysis -Aided in drafting manuscript

Principal Supervisor Confirmation I have sighted email or other correspondence from all Co-authors confirming their certifying authorship.

Alex Loukas 27-5-08 Name Signature Date

64

4.2 ABSTRACT mRNAs encoding cathepsin B-like cysteine proteases (CatBs) are abundantly

expressed in the genomes of blood-feeding nematodes. Recombinant CatBs have

been partially efficacious in vaccine trials in animal models of hookworm infection,

supporting further investigation of these enzymes as new control tools. We recently

described a family of four distinct CatBs (Na-CP-2,-3,-4,-5) from the human

hookworm, Necator americanus. Here we show that these N. americanus CatBs form

a robust clade with other hookworm CatBs and are most similar to intestinal CatBs

from Haemonchus contortus. All four mRNAs (Na-cp-2, -3, -4 and -5) are

upregulated during the transition from a free-living larva to a blood-feeding adult

worm and are also expressed in gut tissue of adult N. americanus that was dissected

using laser microdissection microscopy. Recombinant Na-CP-3 was expressed in

soluble, secreted form in the yeast, Pichia pastoris, while Na-CP-2, -4 and -5 were

expressed in insoluble inclusion bodies in Escherichia coli. Recombinant Na-CP-3

was not catalytically active when secreted by yeast but underwent auto-activation to

an active enzyme at low pH in the presence of dextran sulphate. Activated Na-CP-3

digested gelatin and cleaved the fluorogenic cysteine protease substrate Z-Phe-Arg-

aminomethylcoumarin (AMC) but not Z-Arg-Arg-AMC. Recombinant Na-CP-3 did

not digest intact hemoglobin, but digested globin fragments generated by prior

hydrolysis with N. americanus aspartic hemoglobinases. Antibodies raised in mice to

all four recombinant proteins showed minimal cross-reactivity with each other, and

each antiserum bound to the intestine of adult N. americanus, supporting the

intestinal expression of their mRNAs. These data show that N. americanus expresses

a family of intestinal CatBs, many of which are likely to be involved in nutrient

acquisition and therefore are potential targets for chemotherapies and vaccines.

Keywords: Necator americanus; Cysteine protease; Cathepsin B; Hookworm;

Hemoglobin

65

4.3 INTRODUCTION Proteolytic enzymes are essential components of biological systems, ranging

from viruses to vertebrates. They have been divided into groups on the basis of the

catalytic mechanisms used during their hydrolytic processes (Rawlings et al., 2008).

There are five major catalytic types: aspartic, cysteine, metallo, serine and threonine

proteases. In addition to these five classes, it is believed that there are others which

are as yet undefined. The most abundantly represented family of proteases in

parasitic nematodes, particularly the blood-feeding strongyle nematodes, is the

papain-like cysteine protease family (family C1, MEROPS classification) (Sajid and

McKerrow, 2002).

Cysteine proteases from parasitic helminths perform numerous functions,

including house-keeping roles such as protein processing and turnover, degradation

of host proteins such as the extracellular dermal matrix during skin penetration,

hydrolysis of hemoglobin (Hb) for nutritional uptake, and inhibition of host

protective immune responses, including cleavage of immunoglobulin and

complement components to evade host immunity (reviewed by (Beckham et al.,

2006, Knox et al., 1999, Bethony et al., 2005)). Cysteine proteases of parasitic

organisms are divided into two main groups; clan CA and clan CD. Clan CA or

‘papain like’ proteases are further divided into two families, C1 and C2. Most

parasitic helminth proteases belong to the C1 family, which contains the cathepsin B-

and cathepsin L-like enzymes (Sajid and McKerrow, 2002). In contrast, the C2, or

calpain-like family of proteases appear to be less well represented in parasitic

helminths. Clan CD contains the C13 family, which are referred to as legumain-like

or asparaginyl endopeptidases.

Cathepsin B-like cysteine proteases (CatBs) generally constitute large multi-

gene families in both parasitic and non-parasitic helminths, including Haemonchus

contortus, Schistosoma sp. and Caenorhabditis sp. In parasitic helminths, CatBs are

often expressed in the alimentary canal of the worm, where they play proven or

suspected roles in digestion of protein for nutrient acquisition. In blood-feeding

nematodes, the CatB gene family has undergone enormous expansion. CatB-

encoding genes account for 16-17% of intestinal transcripts from H. contortus

(Jasmer et al., 2001, Jasmer et al., 2004), representing the most abundant and diverse

protein family in the gut of this parasite. Schistosoma mansoni ingests and lyses red

66

blood cells then digests Hb with a multi-enzyme network of proteases including the

CatB, SmCB1 (Lipps et al., 1996, Delcroix et al., 2006, Caffrey et al., 2004, Brindley

et al., 1997).

CatBs are also highly expressed in the hookworm gut. In a previous study, we

used laser microdissection microscopy to dissect gut tissue from adult hookworms

and showed that mRNAs encoding for C1 cysteine proteases (and other classes of

proteases) were abundantly represented in mRNAs from the gastrointestinal tract of

the blood-feeding stage (Ranjit et al., 2006).

Intestinal cysteine proteases are showing particular promise as efficacious

vaccine antigens against numerous helminth parasites (reviewed in (Bethony et al.,

2005, Dalton et al., 2003)). Cattle and sheep can be protected against infection with

the liver fluke, Fasciola hepatica, by vaccination with the major secreted cysteine

proteases (Dalton et al., 2003). Vaccination with protease-rich gut membrane extracts

of H. contortus results in upwards of 90% reduction in adult worms and fecal egg

burdens in lambs (reviewed in (Knox et al., 2001, Dalton et al., 2003)).

The adult stage of the dog hookworm, Ancylostoma caninum, digests Hb

using a semi-ordered pathway of proteolysis that involves the CatB, Ac-CP-2

(Williamson et al., 2004). Vaccination of dogs with Ac-CP-2 provided proof-of-

concept that hemoglobinolytic proteases (hemoglobinases) are efficacious vaccine

antigens against hookworm infection (Loukas et al., 2004). Catalytically active

recombinant Ac-CP-2 conferred partial protection to laboratory beagles, resulting in a

significant decrease in fecal egg counts and worm sizes. Moreover, the worms that

were recovered from CP-2-vaccinated dogs at necropsy had anti-CP-2 IgG adhered

to the parasite gut, and IgG from vaccinated dogs neutralized the hemoglobinolytic

activity of recombinant Ac-CP-2 in vitro (Loukas et al., 2004).

Here, we characterize the transcriptional profile, anatomical expression sites

and recombinant expression of four CatBs from adults of the human hookworm,

Necator americanus, and discuss their potential as vaccine antigens for human

necatoriasis.

67

4.4 MATERIALS AND METHODS 4.4.1 Phylogenetic tree

The phylogenetic relationships between N. americanus CatBs and other C1 family

peptidases were assessed based on amino acid sequence. The full length ORFs of Na-

CP-2, CP-3, CP-4 and CP-5 were aligned with homologues from other species using

ClustalW. A phylogenetic tree was constructed with distance matrix using the

neighbor-joining method with 1,000 bootstrap samplings in PAUP beta version 8.0

for Macintosh.

4.4.2 Amplification of cysteine proteases genes from gut cDNA

Cloning of Na-cp-2, -3, -4 and -5 cDNAs has been reported previously (Xiao et al.,

2008). We began our terminology from “Na-cp-2” and avoided the term “Na-cp-1”,

because the first N. americanus cysteine protease cDNA from N. americanus

deposited in GenBank is called “necpain” (GenBank - AJ132421) - there is no

corresponding publication in the literature describing necpain, but to avoid

confusion, we did not use the term “Na-cp-1” when naming the cDNAs described

herein (Xiao et al., 2008). To confirm that Na-cp-2, -3, -4, -5 mRNAs were

expressed in the gut of adult N. americanus, gut tissue extracted by laser

microdissection microscopy (LMM) (Ranjit et al., 2006) was used as template for

PCR. The following primers were used: Na-cp-2F: 5’-

TCTGTTTCGCTGGTTGAGCC (nt 61); Na-cp-2R: 5’-

TCCTGTCGGTCATCACCTCTGC (nt 435); Na-cp-3F: 5’-

GAGGAGGCAGAGAATCTTTC (nt 82);

Na-cp-3R: 5’- GCAACAGGCAAGGATGTCCG (nt 456); Na-cp-4F: 5’-

CGTCGTCCTTCTGGCAATAAAC (nt 123); Na-cp-4R: 5’-

GCGAATGAGACCGATGGAGG (nt 423); Na-cp-5F: 5’-

TTCCCCGCTGGTTGAACAG (nt 300); Na-cp-5R: 5’ –

CCATCGCATCCCATTCCG (nt 617).

Necator americanus alpha tubulin mRNA (GenBank accession no. BG467527) was

amplified as a constitutively expressed control using the primers Na-α tubulinF: 5’ –

CGAATCTCGTGCCATATCCT (nt 157), Na-α tubulinR: 5’ –

TTCCTCCATACCCTCACCGA (nt 628). Gonadal tissue cDNA which was

68

extracted by LMM in similar fashion as that of intestinal tissue (Ranjit et al., 2006)

was used as a template in PCR as a negative control.

4.4.3 Quantitation of cysteine protease gene expression in different

developmental stages

Real-time PCR reactions were conducted simultaneously for all candidate

genes using a Rotor-Gene 6000 thermal cycler (Corbett). Amplified products were

detected with SYBR Green I DNA binding dye. Single-stranded cDNA was prepared

from infective third stage larvae (L3) and adult worms of N. americanus, as reported

elsewhere (Datu et al., 2008), quantified spectrophotometrically, diluted to 50 ng/μl

with water and used as template for PCR. In each 20 μl reaction, 10 μl of SYBR

green super-mix (Applied Biosystems) was added to 100 nM of each primer with 250

ng of cDNA. All experiments were repeated twice with three replicates in each run

using the following cycle conditions: 3 min at 95°C, 45 cycles of 1 min at 95°C, 30

secs at 55°C and 30 secs at 72°C. A melt curve analysis step, included at the end of

each run, verified the absence of primer–dimers and non-specific products. Changes

in the expression of transcripts between L3 and adult worms were normalized to the

60S acidic ribosomal protein gene (GenBank accession no. BG734493). The

following primers were used: Na-cp-2F: 5’- GCTCAAGAACGCATGAAATC (nt

205); Na-cp-2R: 5’- GAAGGACATTCTGGCCATT (nt 359); Na-cp-3F: 5’-

CCGACGACAAATACTACGC (nt 680);

Na-cp-3R: 5’- GCCTGAAGTCACATAAACTCC (nt 834); Na-cp-4F: 5’-

CGTCCTTCTGGCAATAAACC (nt 126); Na-cp-4R: 5’-

TGTTCATTGGTTGGCGAATA (nt 272); Na-cp-5F: 5’-

CGATAGACAATGGTGTATGC (nt 647); Na-cp-5R: 5’ –

CTATTTCTGGTTTTGGTGGC (nt 747), Na-60SF: 5’ –

GTCGGAATCGTCGGAAAGTA (nt 39); Na-60SR: 5’ –

GTCTTGTTGCACTTCGAGCA (nt 205).

4.4.4 Expression and purification of recombinant cysteine proteases

We attempted to express all four proteases in the yeast, Pichia pastoris,

however only Na-CP-3 was produced in sufficient yields for use in this study. The

entire ORF encoding the pro-enzyme of Na-CP-3 (GenBank accession no.

ABL825237), excluding the predicted signal peptide, was cloned into the expression

69

vector pPICZαA (Invitrogen) according to the manufacturer’s instructions. The

correct reading frame was confirmed by sequencing the plasmid using the α-factor

and 3’AOX1 vector-derived primers. The recombinant plasmid was linearized with

the enzyme PmeI and transformed into P. pastoris X-33 strain by electroporation.

The transformants were selected on Zeocin (Invitrogen) Yeast Peptone Dextrose

(YPD) plates. Colonies were screened for cDNA insertion by PCR using gene

specific primers. Positive colonies were tested for protein expression as

recommended by the manufacturer using Western blot with an anti-hexa His

monoclonal antibody (Invitrogen). The clone exhibiting the highest expression was

scaled-up to 1.5 L suspension culture. Cells were harvested 96 hours post-induction

and supernatant was collected, concentrated to 200 ml by ultrafiltration using a 10

kDa cut off membrane (Pall Scientific) and buffer exchanged into binding buffer

(50mM NaH2PO4, 300mM NaCl and 10 mM imidazole). The recombinant protein

was purified by affinity chromatography on a Ni-NTA agarose column (Qiagen), and

purified protein was buffer exchanged into phosphate buffered saline (PBS) and

protein concentration determined using the bicinchoninic acid assay kit (Pierce).

For expression of Na-CP-2 (GenBank accession no. ABL85236), Na-CP-4

(GenBank accession no. ABL85238) and Na-CP-5 (GenBank accession no.

ABL85239), the ORFs without the signal peptides were cloned in frame into the

pET41a vector (Novagen) and the recombinant products were transformed into E.

coli strain BL21-DE2 (Invitrogen). Cultures were induced with isopropyl β-D-

thiogalactopyranoside (IPTG) at a final concentration of 1mM. Four hours post-

induction, the culture media were centrifuged at 20,000 g for 20 mins and cell pellets

were collected. The cell pellets were resuspended in lysis buffer (20 mM NaH2PO4,

500 mM NaCl) and subjected to two cycles of disruption in a French press (SLM

Instruments). Lysates were centrifuged at 20,000 g for 15 mins and pellets were

incubated in denaturing solubilization buffer (6 M GuHCl, 0.5 M NaCl, 50 mM Tris,

10 mM imidazole) for 2-4 hrs at RT. Recombinant proteins were purified by affinity

chromatography using Ni-NTA agarose (Qiagen) according to the manufacturer’s

instructions.

4.4.5 Autoactivation, catalytic activity assays.

To trans activate recombinant Na-CP-3 from its pro-form (as secreted by P.

pastoris) to its mature form, 100 μg of recombinant protein was incubated for 24

70

hours in AMT buffer (100mM sodium acetate, 100mM MES, 200 mM Tris, 4 mM

EDTA, 200 mM NaCl), 50 μg/ml dextran sulfate (DS 500K) and 10 mM DTT at one

pH unit increments from pH 4-7. The processed Na-CP-3 protein was then assayed

for a shift in molecular weight by western blot analysis using an anti-hexa His

antibody and catalytic activity against the fluorogenic peptide substrates

benzyloxycarbonyl-L-phenylalanine-L-arginine-7-amido-4-methyl-coumarin (Z-Phe-

Arg-AMC; Bachem) or Z-Arginine-ArginineAMC (Z-Arg-Arg; Bachem). Briefly, 10

μg of protein was added to the assay buffer containing 100 mM sodium acetate, 100

mM NaCl, 500 μM DTT to which either fluorogenic peptide was added to a final

concentration of 10 μM. The cysteine protease inhibitor E64 (Sigma) was included in

some reactions at a final concentration of 5 μM. Papain (Sigma) at a final

concentration of 1 μM and adult hookworm Excretory/Secretory (ES) products were

used as positive controls. Reactions were incubated at 37oC for up to 6 hrs and

cleavage of AMC was measured on a Fluostar Galaxy microtitre plate reader (BMG

Labtech) using excitation and emission wavelengths of 370 nm and 440 nm

respectively.

To further assess the catalytic activity of Na-CP-3, recombinant protein was

electrophoresed on precast gelatin zymogram gels (Invitrogen) as recommended by

the manufacturer with a slight modification - protease assay buffer (100 mM sodium

acetate, 100 mM NaCl and 10 mM DTT) was added to the zymogram developing

reagent. Adult hookworm ES products were used as a positive control for the

zymogram, and E64 was included in some assays to assess inhibition of enzymatic

activity.

To verify if recombinant Na-CP-3 was being glycosylated by P. pastoris, the

purified protein was electrophoresed on a 10% SDS-PAGE gel and was incubated

with Pro-Q Emerald 300 glycoprotein gel stain (Molecular Probes), as per the

manufacturer’s instructions.

To determine whether activated recombinant Na-CP-3 cleaved intact Hb or

globin peptides, 100 μg of Hb tetramer or Hb that had been pre-digested with

recombinant N. americanus aspartic protease Na-APR-1 (GenBank accession

number. CAC00543) (Acosta et al.2008) was incubated with activated Na-CP-3

protein (20 μg) in 0.1 M sodium acetate at pH 4.5 at 37oC. Samples were assessed

visually for cleavage of Hb by SDS-PAGE under native conditions, or using LC-MS

to assess cleavage of globin peptides (as described in section 4.4.5).

71

4.4.6 Identification of the pro-mature Na-CP-3 junction

To identify the cleavage site between the pro-region and mature protease of

Na-CP-3, protein was reduced and alkylated with DTT and ioadoacetamide and

digested with trypsin. Briefly, the peptides were injected onto a 150 μm C18 reverse

phase capillary column (Alltech) attached to an Ultimate 3000 nanoLC system

(Dionex) and eluted using a 50 min gradient from 5 to 55% acetonitrile. The mass

spectrometer (MicrOTOF-Q, Bruker) used an autoMSn methodology that collected

MS2 spectra for the two most intense ions in each full scan spectrum. Post-

acquisition spectral analysis was conducted using Biotools (Bruker).

4.4.7 Antibody production

Antibodies against all four purified recombinant proteins were raised in

female BALB/c mice (three mice per group). Pre-immune sera were collected by tail

bleed two days prior to the first injection. For the first immunization, mice were

injected in the tail subcutaneously with 25 μg of protein emulsified with an equal

volume of Freund’s complete adjuvant. Mice were boosted intraperitoneally, twice at

two weekly intervals, with 25 μg of protein emulsified with Freund’s incomplete

adjuvant. Two weeks after the final boost, mice were euthanized and blood was

collected via cardiac puncture. Blood from all 3 mice in each group was pooled. To

separate serum from cells, blood was incubated at 37oC for one hour and then

centrifuged at 10,000 g for 10 mins. Antibody endpoint titers were determined by

enzyme linked immunosorbent assay (ELISA), following the method of (Beckham et

al., 2006). To test for immunologic cross-reactivity between the different anti-CatB

sera, Western blot analyses were conducted by probing 1 μg of all four recombinant

cysteine proteases with mouse antisera to each enzyme, diluted 1:10,000 in antibody

dilution buffer (PBS/0.05% Tween-20/5% skimmed milk powder) for 1 hr at RT.

After washing 3 times for 5 mins each in PBS/0.05% Tween-20, blots were

incubated with goat anti-mouse IgG conjugated to horse radish peroxidase

(Chemicon) diluted 1:2,000 in antibody dilution buffer.

4.4.8 Immunolocalization

Adult N. americanus recovered from euthanized hamsters were fixed in 4%

formaldehyde and placed in OCT compound. OCT blocks were frozen on dry ice for

72

5 mins. Frozen blocks of fixed worms were cut by cryostat into 7 μm thick transverse

sections and mounted onto superfrosted slides and stored at -20oC until needed. To

rehydrate the sections, slides were incubated in PBS for 5 mins. Non-specific binding

was inhibited by blocking the sections with 5% fetal calf serum in PBS for one hour

at room temperature (RT). Slides were incubated with mouse antisera at various

dilutions (1:100, 1:250, 1:500, 1:1,000) with 1% Bovine Serum Albumin (BSA) in

PBST at RT for 1 hr. Anti-mouse IgG conjugated to Alexa Fluor 555 (Invitrogen)

was used as fluorescent secondary antibody at a dilution of 1:500 in 1% BSA/PBST

at RT for 1 hour. Slides were washed three times with PBST for 5 mins each, air

dried and coversliped with mounting media (Sigma). Slides were viewed and

photographed using a Leica IM100 fluorescent microscope.

4.5 RESULTS 4.5.1 Sequence analysis of the cysteine proteases identified in N. americanus

Four cDNAs encoding distinct CatBs were cloned from a N. americanus L3

cDNA library by Xiao et al. (2008); these were designated Na-cp-2, Na-cp-3, Na-cp-

4 and Na-cp-5, but their sequence features were not assessed in depth. All of the

ORFs consisted of a hydrophobic signal peptide at the N-terminus, followed by a

pro- region of between 72-77 amino acids (aa) and a mature protease sequence

(Table 4.1). All four proteases possessed the highly conserved residues of the

catalytic triad (Cys, His, Asn) as well as the oxyanion Gln. None of the four

proteases had the ERFNIN motif in their pro-regions (characteristic of non-cathepsin

B-like C1 proteases (Karrer et al., 1993)). All four proteases had the occluding loop

that is diagnostic of CatBs (Illy et al., 1997), however the loop was modified in Na-

CP-2 and CP-5, both of which contained just one of the conserved His doublet. Of

the four CatBs, only Na-CP-5 did not possess the haemoglobinase motif, as

described by (Baig et al., 2002). Interestingly, none of the CatBs had the Glu residue

at the base of the S2 pocket which is influential in determining substrate specificity

(Fig. 4.1) (Sajid and McKerrow, 2002). The four proteases shared 50-70% amino

acid identities with each other.

73

4.5.2 Phylogenetic analysis of cathepsin B-like proteases

A neighbour joining tree was constructed to compare the phylogenetic

relationships between the CatBs of N. americanus and other blood-feeding parasitic

and non-parasitic nematodes (Fig. 4.2). The outgroup for the tree was human

cathepsin F. All of the nematode CatBs formed one clade but it did not receive

greater than 50% bootstrap support. Within the nematode clade, the proteins were

further divided into more robust clades based on the species from which they were

derived. The five N. americanus proteases grouped with CatBs from Ancylostoma

spp. in a clade that obtained 69% bootstrap support. The hookworm CatBs grouped

with a clade of H. contortus CatBs to form a blood-feeding nematode clade with 61%

bootstrap support.

Table 4.1. General properties of N. americanus cathepsin B-like proteases

cDNA Size (bp) ORF size (aa)

Mature protein properties

(aa, MW, pI)

Predicted N-glycoslyation

sites

Na-cp-2 1134 347 262aa, 27.9 kDa, pI 6.36 140 (NGT)

Na-cp-3 1174 360 270aa, 30.1 kDa, pI 8.8 32 (NLS) 136 (NGT) 242 (NET) 298 (NGT)

Na-cp-4 1181 339 252aa, 28.3 kDa, pI 5.56 134 (NGT) 296 (NGT)

Na-cp-5 1236 342 254aa, 28.4 kDa, pI 8.15 101 (NCT) 135 (NGT) 245 (NET)

4.5.3 Amplification of cysteine protease mRNAs from N. americanus gut

cDNA

All four mRNAs were amplified by PCR from cDNA which had been

synthesised from tissue dissected from the gut of adult N. americanus (Fig. 4.3).

None of the mRNAs were amplified from dissected gonad tissue (not shown) which

served as a control.

4.5.4 Developmental expression of cysteine protease genes

Expression patterns of Na-cp-2-, -3, -4 and -5 mRNAs were determined from

N. americanus L3, whole adults and dissected adult gut using real-time PCR. In L3

cDNA, comparatively low expression levels were detected for Na-cp-3, -4 and -5,

74

and Na-cp-2 could not be detected above the negative control signal (Fig. 4.4). This

is somewhat surprising given that all four cDNAs were cloned from an L3 cDNA

library. All four mRNAs were highly upregulated in the adult stage, with increases of

229-, 60- and 70-fold for Na-cp-3, cp-4 and cp-5 respectively (Fig. 4.4). All four

mRNAs were more highly expressed in gut tissue than whole worm - both Na-cp-2

and cp-5 had 4-fold increases, while Na-cp-3 and -cp-4 underwent 20- and 70-fold

increases respectively (Fig. 4.4).

75

Na-CP-2 MLTLAALLISVSLVEPTGIGEFLAQPAPAYARRLTGQALVDYVNSHHSLYKAKYSPDAQE Na-CP-4 MKANFALVVVLLAINQLYADELLHKQESEHG--LSGQALVDYVNSHQSLFKTEYSPTNEQ Na-CP-5 MITIITLLLIASTVKSLTVEEYLARPVPEYATKLTGQAYVDYVNQHQSFYKAEYSPLVEQ Na-CP-3 -LILIALVVTALAQQPLSLKEYLEQPIPEEAENLSGEAFAEFLNKRQSFFTAKYTPNALN Necpain MLLFLTLFVAILAAD----EKILQDAVKKESKALTGHALAEFLRTLQSLFEVKKSEEVPV Human_CatB MWQLWASLCC-LLVLAN------ARSR-PSFHPLSDEL-VNYVNKRNTTWQAGHNFYNVD Na-CP-2 RMKSRIMDLSFMVDAEVMMEEMDQQEDIDLAVSLPESFDAREKWPECPSIG-LIRDQSAG Na-CP-4 FVKARIMDIKYMTEASHKYPRK----GINLNVELPERFDAREKWPHCASIG-LIRDQSAC Na-CP-5 YAKAVMRSEFMTKPNQNYVVKD-----VDLNINLPETFDAREKWPNCTSIR-TIRDQSNC Na-CP-3 ILKMRVMESRFLDNEEGEMLKE---EDMDFSEEIPVSFDARDKWPKCTSIG-FIRDQSHC Necpain RMKYLLPKHFMVKPKEEDRTKIQ------LDKEPPEKFDARDAWPYCREIIGHVRDQSRC Human_CatB MSYLKRLCGTFLGGPKPPQRVMFT-----EDLKLPASFDAREQWPQCPTIK-EIRDQGSC Na-CP-2 GGCWAVSSAEVMTDRICIQSNGTKQVYVSETDILSCCGQRCGSGCTSGVPRQAFNYAIRK Na-CP-4 GSCWAVSAASVMSDRLCIQTNGTNQKILSSADILACCGEDCGSGCEGGYPIQAYFYLENT Na-CP-5 GSCWAVSAASVMSDRLCIQSNGTIQSWASDTDILSCCWN-CGMGCDGGRPFAAFFFAIDN Na-CP-3 GSCWAVSSAETMSDRLCVQSNGTIKVLLSDTDILACCPN-CGAGCGGGHTIRAWEYFKNT Necpain GSCWAVSAASVMSDRLCVQSNGKIKLHVSDTDILACCGEFCGDGCSGGWPFQAWEWVRKY Human_CatB GSCWAFGAVEAISDRICIHTNAHVSVEVSAEDLLTCCGSMCGDGCNGGYPAEAWNFWTRK Na-CP-2 GVCSGGPYGTKGVCKPYPFYPCGYHAHLPYYGPCP-DGMWPTPTCEKACQSDYTVPYNDD Na-CP-4 GVCSGGEYREKNVCKPYPFYPCDG-----NYGPCPKEGAFDTPKCRKICQFRYPVPYEED Na-CP-5 GVCTGGPFREPNVCKPYAFYPCGRHQNQKYFGPCP-KELWPTPKCRKMCQLKYNVAYKDD Na-CP-3 GVCTGGLYGTKDSCKPYAFYPCKD----ESYGKCP-KDSFPTPKCRKICQYKYSKKYADD Necpain GVCTGGDYRAKGVCKPYAFHPCGNHENQVYYGVCP-KGSWPTPRCEKFCQRGYIKPYKKD Human_CatB GLVSGGLYESHVGCRPYSIPPCEHHVN---GSRPPCTGEGDTPKCSKICEPGYSPTYKQD Na-CP-2 RIFG--SKTIVLTGEEKIKREIFNNGPLVATYTVYEDFAYYKNGIYMTGLGRATGAHAVK Na-CP-4 KVFGKNSHILLQDNEARIRQEIFINGPVGANFYVFEDFIHYKEGIYKQTYGKWIGVHAIK Na-CP-5 KIYG-NDAYSLPNNETRIMQEIFTNGPVVGSFSVFADFAIYKKGVYVSNGIQQNGAHAVK Na-CP-3 KYYA-NSAYRIPQNETWIKLEIMRNGPVTASFRIYPDFGFYEKGVYVTSGGRELGGHAIK Necpain KFYA-KKSYWLPNDEKEIRLDIMKNGPVQAAFDVYEDFKLYKRGIYKHKEGIQTGGHAVK Human_CatB KHYG-YNSYSVSNSEKDIMAEIYKNGPVEGAFSVYSDFLLYKSGVYQHVTGEMMGGHAIR Na-CP-2 IIGWGEENG----VKYWLIANSWNTDWGEN-GFFRMLRGTNLCDIELSATGGTFKV---- Na-CP-4 LIGWGTENG----TDYWLVANSWNYDWGEN-GTFRILRGTNHCLIESQVIATEMIV---- Na-CP-5 IIGWGVQDG----LKYWLIANSWNNDWGDE-GYVRFLRGDNHCGIESRVVTGTMKV---- Na-CP-3 IIGWGTEKVNGTDLPYWLIANSWGTDWGENNGYFRILRGQNHCQIEQKVIAGMIKVPQPK Necpain IIGWGKDNG----TDYWLIANSWSKDWGES-GFFRMVRGENDCEIEDMITAGIMMV---- Human_CatB ILGWGVENG----TPYWLVANSWNTDWGDN-GFFKILRGQDHCGIESEVVAGIPRTDQYW Na-CP-2 ------------ Na-CP-4 ------------ Na-CP-5 ------------ Na-CP-3 SAGPPLQPNPSS Necpain ------------ Human_CatB EKI---------

Figure 4.1 Multiple sequence alignment of N. americanus cysteine proteases and human cathepsin B.

Blue font: Predicted signal peptide cleavage points, Aqua shading: Predicted cleavage points of mature and pro-domains, Red shading: Glutamine oxyanion hole, Pink shading: Catalytic triad - Cysteine, Histidine, Asparagine, Green shading: Occluding loop (Histidine doublet is underlined), Yellow shading: Haemoglobinase motif, Grey shading: Protease S2 pocket (arrow denoting Gln residue) Necpain GenBank accession number: CAB53364, Human CatB GenBank accession number: AAH10240

76

Figure 4.2. Neighbour joining phylogenetic tree depicting the relationships of N. americanus cysteine proteases with homologues from other nematodes and other phyla.

GenBank accession numbers are listed next to the protein names. Sm: S. mansoni, Ce: C. elegans, Hc: H. contortus

77

. Figure 4.3. Amplification of cysteine protease mRNAs from N. americanus gut cDNA. Lane 1: Na-cp-2, Lane 2: Na-cp-3, Lane 3: Na-cp-4, Lane 4: Na-cp-5. Figure 4.4. Developmental expression profiles of N. americanus cysteine protease mRNAs. Quantitation of mRNA levels for Na-cp-2, cp-3, cp-4 and cp-5 by real time PCR from infective larvae (L3), adult worms and adult worm gut tissue. Expression profiles are depicted as mRNA copy numbers using N. americanus 60S ribosomal RNA as a constitutively expressed control. Star denotes statistically significant difference of expression between L3 and adult cDNA (p≤0.05). Diamond denotes statistically significant difference of expression between adult and gut cDNA (p≤0.05). 4.5.5 Expression of recombinant cysteine proteases

Na-CP-3 was expressed in soluble form in P. pastoris X-33 strain. Protein

was detected by Western blot analysis using anti-hexa-His and anti-c-myc

monoclonal antibodies (Invitrogen). The protein was secreted into the culture

supernatant at ~3.5 mg/L and was purified via the nickel-NTA affinity

chromatography under native conditions. Na-CP-2, -4 and -5 were expressed as

recombinant proteins in E. coli strain BL21-DE2 in the vector pET41a. They were all

expressed in insoluble inclusion bodies and purified by nickel-NTA affinity

chromatography under denaturing conditions. Na-CP-2, CP-4 and CP-5 expressed at

4 mg/L, 5.5 mg/L and 7.5 mg/L respectively (Fig. 4.5). Attempts to remove urea and

refold the proteins into a soluble form were unsuccessful (not shown).

0101

Na-cp-2 Na-cp-3 Na-cp-4 Na-cp-5 Na-60s

N. americanus cysteine proteases

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Figure 4.5. Expression and purification of recombinant N. americanus CatBs in yeast P. pastoris and E. coli. Na-CP-2 (A), CP-3 (B), CP-4 (C) and CP-5 (D). Na-CP-2, CP-4 and CP-5 were expressed in E. coli; Na-CP-3 was expressed in P. pastoris. Lanes show protein purification steps on Ni-NTA resin. Lanes 1: Before binding, Lanes 2: Flow through, Lanes 3: Wash 1, Lanes 4: Wash 2, Lanes 5: Elution. 4.5.6 Catalytic activity of Na-CP-3

Purified recombinant Na-CP-3 did not cleave either Z-Phe-Arg-AMC or Z-

Arg-Arg-AMC, and the molecular weight of the purified protein (55kDa) indicated

that the enzyme had not undergone post-translational processing from its pro- form.

LC-MS analysis of the purified protein revealed that the 77 amino acid pro-domain

was still present. When the same recombinant protein was electrophoresed on a

gelatin gel, however, a zone of hydrolysis was visible at the predicted size of the pro-

enzyme, indicating that the renaturation process employed in gelatin zymography

was at least partially activating the pro-enzyme (Fig. 4.6). The gelatinolytic activity

was inhibited by E64 (cysteine protease inhibitor), confirming that this activity was

due to recombinant Na-CP-3. The recombinant CP-3 bound Pro-Q Emerald 300

79

glycoprotein stain, indicating that the protein had been glycosylated (Fig. 4.7a). In

order to facilitate trans processing of the pro- domain of Na-CP-3, the purified

protein was incubated in AMT buffer (containing dextran sulphate) at a range of pH

values. Western blotting of the processed protein samples indicated a shift in

molecular weight of the expected size from 55 kDa to ~30 kDa at pH 4 (Fig 4.7b).

Catalysis assays with the various samples indicated that auto-activated Na-CP-3

cleaved Z-Phe-Arg-AMC but not Z-Arg-Arg-AMC. Pro-enzyme that was activated

at pH 4 yielded the highest catalytic activity, followed by pro-enzymes activated at

pH 5 and pH 6 respectively (Fig. 4.8). No activity was detected from samples which

had been trans activated at pH 3, 7 or 8 (data not shown). Activated proteases

displayed catalytic activity against Z-Phe-Arg-AMC from pH 4 to 7, with highest

activity between pH 6 and 7 (Fig 4.8).

Na-CP-3 did not digest intact Hb tetramer but did digest globin peptides that

were generated by hydrolysis of Hb with the aspartic protease, Na-APR-1 (data not

shown), indicating a downstream role in the proposed hemoglobinolysis cascade

(Williamson et al., 2003b). The nature/identities of the peptides that were generated

by digestion of globin with Na-CP-3 are not shown here and have been submitted

elsewhere for publication as just one component of a multi-enzyme hemoglobinolytic

cascade (Ranjit et al., manuscript submitted).

Figure 4.6. Gelatin zymogram showing catalytic activity of purified recombinant Na-CP-3. Lane 1: 10 ng Na-CP-3, Lane 2: 1 ng Na-CP-3, Lane 3: 10 ng Na-CP-3 + E64

80

Figure 4.7. SDS-PAGE gel of purified recombinant pro-Na-CP-3 incubated with Pro-Q Emerald 300 glycoprotein stain (A). Activation of pro-Na-CP-3 (B). Western blot of purified recombinant Na-CP-3 activated in AMT buffer. Lane 1: no incubation, Lane 2: incubated O/N in AMT buffer, pH 4, Lane 3: incubated O/N in AMT buffer, pH 5, Lane 4: incubated O/N in AMT buffer pH 6.

Figure 4.8. pH profile of the catalytic activity of recombinant Na-CP-3 after auto-processing. Autoprocessing was facilitated in AMT buffer and conducted at pH 4, 5 or 6. The processed proteases were then assessed for catalytic activity at different pH values (x axis) against the fluorogenic substrate Z-Phe-Arg-aminomethylcoumarin. RFU – relative fluorescence units.

4.5.7 Antibody production and immunolocalization of proteins

Antisera were raised in BALB/c mice to all four proteases. Mice generated

antibody endpoint titers of 1:30,000 to 1:45,000 as determined by ELISA. Western

blot analyses conducted with all four antisera indicated specificity of each antibody

for its homologous enzyme, but there was slight cross reactivity between anti-Na-

CP-3 serum which strongly recognized Na-CP-3 and weakly bound to Na-CP-4, -CP-

81

5 and –CP-2. Anti-Na-CP-4 serum strongly bound to Na-CP-4 and very weakly

bound to Na-CP-5 (Fig. 4.9). Immunolocalization of each protease within adult N.

americanus tissue sections demonstrated binding of all four antibodies to the gut of

the parasite (Fig. 4.10), supporting the intestinal expression of the corresponding

mRNAs. Antisera to Na-CP-2 and Na-CP-4 bound strongly to the gut but also bound

weakly to the cuticle and/or hypodermis. We routinely see weak binding with some

antibodies to the cuticle/hypodermis region, and believe that this represents non-

specific antibody binding. Anti-Na-CP-3 showed the strongest immunofluorescent

labelling and this was clearly localised to the microvillar surface of the gut (Fig

4.10). The pre-immune serum did not bind to any structures (Fig. 4.10).

Figure 4.9. Western blot showing recognition of recombinant Na-CP-2, CP-3, CP-4 and CP-5. (listed under each panel) by antisera raised to each recombinant enzyme (listed above each panel). 4.6 DISCUSSION

The intestine of blood-feeding helminths is a protease-rich site (Ranjit et al.,

2006, Jasmer et al., 2001, Caffrey et al., 2004) and at least some of these proteins

play defined roles in nutrient acquisition. Cysteine proteases have been shown to be

one of the most abundantly expressed protease families in the gastrointestinal tracts

of parasitic helminths. These proteases are often developmentally regulated

throughout the complex parasitic life cycles, are frequently up-regulated in actively

feeding stages and have been shown to be commonly expressed in organs or

organelles involved in feeding (Jasmer et al., 2004). Here, we examined the

transcriptional expression, anatomical localisation and characterisation of four

different N. americanus cathepsin B- like proteases in order to gain an understanding

of what roles they might play and whether they would be effective as chemotherapy

or vaccine targets.

.

82

Figure 4.10. Immunolocalization of Na-CP-2, -3, -4 and -5 in transverse sections of adult N. americanus. Fluorescence images are on the left and corresponding bright field images are on the right for each antibody. Arrows indicate the intestine (int), intestinal microvillar surface (mv), cephalic gland (ceph) and cuticle (cut). A: α-Na-CP-2, B: α-Na-CP-3, C: α-Na-CP-4, D: α-Na-CP-5, E: Pre-vaccination serum.

A B

C D

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50 µm

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All four N. americanus proteinases have been classified as CatBs due to their

structure and sequence homology to cathepsin B proteins. They all have the essential

catalytic triad residues of cysteine, histidine and asparagine as well the highly

conserved glutamine that forms the oxyanion hole. In addition the conserved

glutamine is a crucial element in forming an electrophilic centre to stabilise the

tetrahedral intermediate during hydrolysis (Sajid and McKerrow, 2002).

In our study, three out of the four N. americanus CatBs, Na-CP-2, CP-3 and

CP-4, contained the so called ‘hemoglobinase motif’ described by (Baig et al., 2002).

This motif is located around the catalytic Asn and is thought to be diagnostic of

hemoglobinase activity. These authors suggested that CatBs which lack this motif

would not be able to degrade Hb readily and may play more generalised

housekeeping functions. Although there is sufficient evidence to indicate that CatBs

which lack this motif have limited abilities to degrading Hb, the reverse scenario

does not apply, i.e. not all CatBs which possess the motif will necessarily be

involved in Hb degradation. We found numerous CatBs from non-parasitic

organisms in GenBank which possessed the “hemoglobinase motif” (not shown),

including the free living protozoan Tetrahymena thermophila, the free living

nematode Caenorhabditis briggsae, the neuropathogenic bird schistosome

Trichobilharzia regenti (which feeds on nerve tissue) and the liver fluke Clonorchis

sinensis (which generally feeds on bile and epithelial cells rather than blood).

Although possession of this motif does not necessarily confirm that the protein will

have Hb degrading function, it is still a valuable tool for identifying potential

hemoglobinases. Na-CP-3 possesses the motif but seems incapable of cleaving intact

Hb, although it does digest globin fragments i.e. Hb that has been pre-digested with

other N. americanus proteases, indicating that Na-CP-3 plays a downstream role in

the Hb digestion cascade, functioning as a “globinase” rather than a hemoglobinase.

In addition to containing the “hemoglobinase motif”, all four N. americanus

CatBs were localized to the gut of adult worms and, moreover, mRNAs for all four

proteases were up-regulated in the transition from infective L3 to blood-feeding adult

worms. This strongly suggests that these enzymes are involved in blood-feeding, and

that vaccines that target this family of molecules might be efficacious against human

necatoriasis. Two CatBs, Ac-CP-1 and Ac-CP-2, have previously been characterised

from the adult stage of the dog hookworm, A. caninum (Harrop et al., 1995).

Although these two proteases share 86% amino acid sequence identity with each

84

other, they are expressed at distinct sites and are thought to have different functions -

Ac-CP-1 is expressed in the cephalic and excretory glands (Loukas et al., 2004) and

is detected in ES products (J. Mulvenna, A. Loukas, J. Gorman, unpublished),

suggesting an extracorporeal digestive function at the site of attachment. Ac-CP-2 is

localized to the brush border membrane of the intestine (Loukas et al., 2004) and is

involved in Hb digestion (Williamson et al., 2004). Vaccine trials with recombinant

Ac-CP-2 in the canine model resulted in a decrease in the number and fecundity of

female worms, stunted growth of adult worms and production of antibodies that

bound to the parasite gut in vivo and neutralised the activity of the enzyme in vitro

(Loukas et al., 2004). A recent study conducted by (Xiao et al., 2008) showed that

immunising with Na-CP-2, expressed as a denatured protein in E. coli, provided 29%

reduction in adult worm burdens (P < 0.05) in hamsters that were experimentally

challenged with N. americanus. Significant levels of protection against H. contortus

have been achieved in sheep by vaccination with a cysteine proteinase-enriched

fraction, TSBP (thiol sepharose binding protein) isolated from the gut of adult

parasites. This protection is associated with three CatBs (hmcp 1, 4 & 6). Sheep

immunized with a cocktail of these proteins expressed in bacteria had reduced faecal

egg counts and worm burdens compared to controls. Sera from immunized animals

also bound to the microvillar surface of the gut of adult H. contortus (Redmond and

Knox, 2006). The results of this haemonchosis vaccine trial, when coupled with the

protective efficacies in the canine hookworm model of Ac-CP-2 (Loukas et al., 2004)

and the cathepsin D aspartic protease Ac-APR-1 (Asojo et al., 2005), present a strong

case for exploitation of intestinal proteases as vaccine targets for hematophagous

nematodes.

The occluding loop is a diagnostic feature of CatBs, and modifications of this

loop were present in all four N. americanus proteases. In addition to its role in

conferring exopeptidase activity to CatBs, the occluding loop also governs the pH

dependence of auto-activation. In a study conducted on Fasciola hepatica,

recombinant FhCatB1 did not auto-activate upon secretion by yeast, but could be

auto-activated in a low pH buffer containing glycosaminoglycans (GAGs) or

polysulfated polysaccharides (PSPs) (Beckham et al., 2006). In similar fashion to

FhCatB1, we found that recombinant Na-CP-3 was secreted from Pichia as a

proenzyme rather than a processed mature protease, implying that the enzyme was

not auto-processed during its secretion from yeast, however in the presence of

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dextran sulphate and acidic pH, it underwent auto-processing and became

catalytically active. A study conducted with human procathepsin B suggested that

GAGs and PSPs bind to positively charged residues in the pro- peptide as well as the

His residues in the occluding loop, inducing a conformational change which unmasks

the active site and enables the access of a substrate molecule, which can then lead to

the intermolecular cleavage of the pro-domain (Caglic et al., 2007). Activation of

Na-CP-3 via this method supports the concept that processing of the proenzyme to

the mature form could be linked to the occluding loop. In another study, parasite

CatBs which were unable to undergo auto-processing required the presence of

another cysteine protease, asparaginyl endopeptidase (which cleaves on the C-

terminal side of Asn residues), to initiate the activation process (Sajid et al., 2003).

Na-CP-4 and Na-CP-5 are likely candidates for asparaginyl endopeptidase

processing, as both contain an Asn residue in the vicinity of the predicted pro-mature

junction (Fig. 4.1), however Na-CP-2 and Na-CP-3 do not contain an equivalent Asn.

It is well established that mammalian cathepsins B hydrolyse small peptides

at the P2 position, but cathepsins L are not efficient at cleaving peptides with a P2

Arg and, instead, prefer a bulky residue at P2 (Musil et al., 1991). Many CatBs of

parasitic nematodes display cathepsin B-like primary sequences, but their substrate

specificities are more reminiscent of cathepsins L in that they do not cleave

substrates with a P2 Arg. Indeed, ES products of A. caninum show a strong

preference for peptides with a P2 Phe, and CatBs predominate in ES products

(Mulvenna, Loukas, Gorman, personal communication) and in gene survey studies

(Mitreva et al., 2005, Ranjit et al., 2006, Datu et al., 2008). The substrate specificity

of mammalian cysteine proteases is thought to be determined by interactions in the

S2 pocket, particularly residue Glu-205 in human cathepsin B and the equivalent Ala-

205 in cathepsin L (reviewed in (Sajid and McKerrow, 2002)). The Glu residue can

accommodate and stabilise the polar group of Arg in the peptide, but Ala at this

position cannot bind to Arg. While many parasite proteases share sequence identities

with mammalian cathepsins B and L, many do not possess either an acidic or

hydrophobic residue at this site of the S2 pocket, making it difficult to classify these

proteases based solely on substrate specificity or sequence identity. For example, all

four N. americanus CatBs described in this study are structurally similar to

cathepsins B but all of them have substituted the S2 Glu for Gln, Ser, Arg or Lys,

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none of which are acidic or hydrophobic. This possibly accounts for the inability of

Na-CP-3, at least, to cleave Z-Arg-Arg.

In this study, we have investigated four cathepsin B-like cysteine proteases

expressed by the human hookworm N. americanus. The roles of CP-2, -4 and -5 in

blood-feeding have yet to be determined, but their developmental expression

patterns, anatomic sites of expression and sequence features suggest that, like CP-3,

they are involved in digestion of the blood meal. These enzymes therefore present as

attractive targets for development of an anti-blood-feeding vaccine that will reduce

worm viability and fecundity, which in turn could lessen the transmission of and

morbidity associated with hookworm infection.

Acknowledgements

This research was supported by grants from the National Health and Medical

Research Council (NHMRC, Australia) and Bill and Melinda Gates Foundation. NR

was supported by a QUTBLU award and funding from the ARC/NHMRC Research

Network for Parasitology. AL was supported by a Senior Research Fellowship from

NHMRC.

CHAPTER 5: DIGESTION OF HEMOGLOBIN

VIA AN ORDERED CASCADE OF PROTEOLYSIS

IN THE INTESTINE OF THE HUMAN

HOOKWORM, NECATOR AMERICANUS

Najju Ranjita,d, Bin Zhanc, Brett Hamiltonb, Deborah Stenzeld, Jonathan

Lowthere, Mark Pearsona, Jeffrey Gormanb, Peter Hotezc, Alex Loukasa

aHelminth Biology Laboratory and bProtein Discovery Centre, Division of Infectious Diseases,

Queensland Institute of Medical Research, Brisbane, Australia; cDepartment of Microbiology,

Immunology and Tropical Medicine, George Washington University, Washington DC, USA; dSchool

of Life Sciences, Queensland University of Technology, Brisbane, Australia; eInstitute for the

Biotechnology of Infectious Diseases, University of Technology, Sydney, Australia.

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5.1 CONTRIBUTIONS Contributor Statement of contribution*

Najju Ranjit

-Designed all the experiments expect for the ones mentioned below -Conducted all the experiments expect for the ones mentioned below -Analysed all the data -Drafted manuscript,

Bin Zhan -Provided Na-APR-1 recombinant protein -Submitted Na-MEP-1 sequence to GenBank -Provided feedback on manuscript

Brett Hamilton -Ran hemoglobin samples on mass spectrometer

Deborah Stenzel -Provided feedback on manuscript -Discussed experimental design

Jonathan Lowter -Designed Na-APR-1 kinetic activity assay -Calculated kinetics for Na-APR-1 activity -Provided feedback on manuscript

Mark Pearson -Designed Na-APR-1 activity assay -Helped conduct Na-APR-1 activity assay -Provided feedback on manuscript

Jeffery Gorman -Aided in proteomics aspects of experimental design for Figure 5.6 -Provided feedback on manuscript

Peter Hotez -Investigator on the grant that funded this project -Provided feedback on manuscript

Alex Loukas -Aided in experimental design -Aided in data analyses -Aided in drafting manuscript

Principal Supervisor Confirmation I have sighted email or other correspondence from all Co-authors confirming their certifying authorship.

Alex Loukas 27-5-08 Name Signature Date

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5.2 ABSTRACT Blood-feeding parasites utilize mechanistically distinct proteases in a vast

array of biological processes. Whilst the majority of these proteases have specialised

roles and generally function independently, many of them also work in a synergistic

fashion as components of multi-protease networks or cascades. In this study, we

investigate the roles of three distinct proteases, all of which are expressed in the gut

of adult Necator americanus hookworms. The A1 family aspartic protease, Na-APR-

1, and the C1 family cysteine protease, Na-CP-3, were expressed in recombinant

form in yeast and shown to be catalytically active towards synthetic peptide

substrates. A cDNA encoding an M13 family metalloprotease called Na-mep-1 was

cloned. Recombinant Na-MEP-1 was expressed in insect cells and displayed catalytic

activity in gelatin gels that was inhibited by 1,10-phenanthroline. Antibodies raised

to all three recombinant proteins were used to localize each native enzyme to the

intestine of adult N. americanus using immunofluorescence microscopy.

Recombinant Na-APR-1 cleaved intact Hb. In contrast, recombinant Na-CP-3 and

Na-MEP-1 could not cleave Hb but, instead, cleaved globin fragments after Hb

hydrolysis by Na-APR-1, implying an ordered process of hemoglobinolysis. Using

tandem mass spectrometry, 74 cleavage sites within Hb α and β chains were

characterised after digestion with all three proteases. All of the proteases

demonstrated a promiscuous subsite specificity within Hb – noteworthy subsite

preferences included bulky aromatic (Phe) and hydrophobic P1′ residues for Na-

APR-1, and hydrophobic (Ala and Leu) P1′ residues for Na-MEP-1. We conclude

that Hb digestion in N. americanus occurs in an ordered fashion, similar to that

described in the digestive vacuole of Plasmodium falciparum, and this provides a

potential mechanism by which these proteases exert their efficacy as recombinant

vaccines against hookworm infection.

Keywords: Hemoglobin digestion cascade; Hemoglobinase; Necator americanus;

aspartic protease; cysteine protease; metalloprotease

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5.3 INTRODUCTION Over 700 million people in developing countries are infected with the human

hookworms, Necator americanus and Ancylostoma duodenale (Hotez et al., 2004).

The main pathology associated with hookworm infection stems from intestinal blood

loss which can lead to iron deficiency anaemia in heavy infections. Hookworms are

voracious blood feeders, causing an estimated loss of up to 9 ml of blood per day in

heavily infected subjects (Lwambo et al., 1999). While anthelmintic chemotherapies

such as benzimidazoles are generally effective at removing adult worms from the

gut, reinfection occurs rapidly and often to equal or even higher infection intensities,

leading to concerns about the long-term viability of such practices (Hotez et al.,

2006). It has also been reported that, unlike other human helminthiases, clear-cut

protective immunity does not occur in most individuals exposed to hookworms

(Loukas et al., 2005b). This has culminated in efforts to develop a prophylactic

vaccine against hookworm infection as a sustainable solution for long-term control

(Bungiro and Cappello, 2004, Loukas et al., 2006).

Hematophagous parasites depend on the catabolism of blood proteins such as

hemoglobin (Hb) for survival. Schistosome blood flukes and the malaria parasite,

Plasmodium falciparum, digest Hb in the gastrodermis and digestive vacuole

respectively, using synergistic cascades consisting of enzymes belonging to distinct

mechanistic classes. In schistosomes, the intact Hb tetramer is cleaved initially by

aspartic proteases (Brindley et al., 2001, Delcroix et al., 2006) and to a lesser degree

by papain-like cysteine proteases; the cysteine proteases are thought to exert most of

their activity downstream of the aspartic protease by further catabolising globin

fragments (Delcroix et al., 2006). On the other hand, treatment of schistosomes with

double stranded RNA for the gastrodermal cathepsin B cysteine protease SmCB1

interrupted the ability of worms to digest serum albumin (Delcroix et al., 2006),

suggesting that the order in which the different hemoglobinases act differs for each

distinct protein substrate.

There are varying degrees of redundancy in hemoglobinolysis in blood-

feeding parasites. The roles and hierarchical positions of various Schistosoma and

Plasmodium proteases in the hemoglobinolytic processes have been debated, but the

application of gene knockout (Plasmodium) and gene silencing (Schistosoma)

technologies have resolved some of these issues. For example, hemoglobinase

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knockout and double knockout P. falciparum have been used to show that Hb

degradation is partially redundant and relies on dual protease families with

overlapping function (Liu et al., 2006). In S. mansoni, silencing of the mRNAs for

different hemoglobinases has shown an ordered and somewhat less redundant

pathway of Hb degradation (Delcroix et al., 2006, Morales et al., 2008).

Gene silencing techniques have not yet been successfully utilized with

hookworms and, indeed, some have questioned whether parasitic nematodes have the

required enzymes to process double stranded RNA (Knox et al., 2007, Viney and

Thompson, 2008). We have therefore taken a different approach to explore

hemoglobinolysis in blood-feeding hookworms. We previously identified proteases

involved in digestion of Hb (hemoglobinases) in the canine hookworm, Ancylostoma

caninum, and used recombinant enzymes to reveal a semi-ordered cascade of

proteolysis in vitro, whereby an aspartic and a cathepsin B-like cysteine protease

both digested intact Hb followed by further digestion of globin fragments into small

peptides by a metalloprotease (Williamson et al., 2004). Two of the enzymes

involved in this semi-ordered cascade were then shown to confer protection as

recombinant proteins against hookworm infection in dogs (Loukas et al., 2005a,

Loukas et al., 2004).

To characterize the hemoglobinolysis pathways used by human hookworms,

we used laser capture microdissection to isolate intestinal tissue from the adult stage

of N. americanus and identified the most highly expressed mRNAs encoding

proteases (Ranjit et al., 2006), including homologues of some of the A. caninum

hemoglobinases. Here, we describe the cDNA cloning and recombinant expression

of intestinal aspartic, cysteine and metalloproteases from N. americanus. We then

provide evidence for an ordered cascade of hemoglobinolysis whereby aspartic (Na-

APR-1), cysteine (Na-CP-3) and metalloproteases (Na-MEP-1), in that order, digest

Hb and globin fragments, and provide a map of the cleavage sites using liquid

chromatography – mass spectrometry (LC-MS). Our findings shed light on the

molecular mechanisms used by hookworms to obtain nutrition from human blood,

and further support the pursuit of hemoglobinases as targets for the development of

new drugs and vaccines against blood-feeding nematode parasites of humans and

livestock.

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5.4 MATERIALS AND METHODS 5.4.1 cDNA cloning

Na-apr-1 was first reported by Williamson et al. (Williamson et al., 2002)

and is assigned GenBank accession number CAC00543. Na-cp-3 was first reported

by Xiao et al. (Xiao et al., 2008) and is assigned GenBank accession number

ABL85237. The Na-mep-1 cDNA was initially identified as a truncated EST

(BG467946) by using Ac-mep-1 as the query in a blast search of the Parasite

Genomes Database through Wu-Blast2 (http://www.ebi.ac.uk/blast2/parasites.html).

In order to clone the 5’ and 3’ ends of the cDNA, gene-specific primers (Na-MEPF2:

GAGCTTCAATCCACCGTACT; NaMEPF1: TAGTCAACTTCTCACCGACC);

NaMEPR4: CCAAGGAATTCCGCATCTTC; Na-MEPR7:

TCAAAGTGGGCAGATCGTAG; Na-MEPR8: ATAGCTCCGTAACGACTGAC)

were designed for 5′ and 3′ rapid amplification of cDNA ends (RACE) using adult N.

americanus RNA and a modified RNA ligase-mediated rapid amplification technique

(GeneRacer, Invitrogen) as described previously (Zhan et al., 2004). The full-length

cDNA sequence of Na-mep-1 was obtained by creating a consensus consisting of the

5′ and 3′ RACE products and the BG467946 EST sequence. The final sequence of

Na-mep-1 was confirmed by designing primers at either end of the ORF and

amplifying the full-length ORF by PCR. Multiple amplicons were generated and

sequenced. The cDNA sequence of Na-mep-1 has been deposited in GenBank with

accession number EU523699.

5.4.2 Protein expression and purification

We expressed Na-APR-1 in the yeast Pichia pastoris, as published earlier for

Ac-APR-1 from A. caninum (Loukas et al., 2005a). Expression of Na-CP-3 in P.

pastoris was reported by us recently (Ranjit et al., in press). Attempts to express Na-

MEP-1 in yeast were unsuccessful (not shown), so we expressed it in a

baculovirus/insect cell system. The entire open reading frame minus the predicted

signal peptide was amplified from adult N. americanus cDNA using PCR.

Amplicons were ligated into the baculovirus shuttle vector pHotWax, a modified

version of pMelBac (Invitrogen), where C-terminal V5 and 6×His epitopes were

inserted (Bethony et al., 2005). The plasmid contained an N-terminal melittin signal

peptide to direct secretion of the recombinant protein. Recombinant plasmids were

93

sequenced to verify their identities and transfected into Spodoptera frugiperda Sf9

insect cells following the Bac-N-Blue transfection protocol (Invitrogen). After

generation of a high titer viral stock, Trichoplusia ni Hi5 cells were infected with

recombinant virus. Cells were maintained at 27oC with constant shaking at 120 rpm

for 96 hours post infection. Supernatant from the baculovirus Hi5 cell culture was

collected by centrifugation at 20,000 g for 20 mins. The supernatant was then

concentrated to one-fifth original volume using a 10 kDa molecular weight cut off

membrane (Pall scientific) and was buffer exchanged by dialysis into binding buffer

(50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole). The concentrated supernatant

was applied onto a 2 ml Ni-NTA agarose column (Qiagen). The column was washed

with increasing concentrations of imidazole (10-60 mM) and protein was eluted with

250 mM imidazole. Eluate fractions were analysed by SDS-PAGE and recombinant

protein was detected by Western blot using an anti-V5 antibody (Invitrogen). Protein

concentration was determined using a bicinchoninic acid (BCA) assay kit (Pierce).

Na-MEP-1 was also expressed in E. coli to obtain sufficient protein for antibody

production. The entire open reading frame without the signal peptide was ligated into

pBAD/Thio-TOPO vector (Invitrogen) and transformed into E. coli strain BL21-DE2

(Invitrogen). To express recombinant protein, cultures were induced with arabinose

(0.2% final concentration) once they had reached an OD wavelength of 0.6 nm.

Culture medium was collected 4-6 hours post induction and centrifuged at 25,000 g

for 20 mins. Cell pellets were collected and resuspended in lysis buffer (20 mM

NaH2PO4, 500 mM NaCl). The cells were subjected to two cycles of disruption in a

French press (SLM Instruments). Lysates were centrifuged at 20,000 g for 15 mins,

supernatant was retained and pellets were incubated in solubilisation buffer (6 M

GuHCL, 50 mM Tris, 10 mM imidazole) for 2 hours at room temperature, and then

centrifuged again. Solubilised pellet was applied onto Ni-NTA agarose column.

Wash buffer (8 M Urea, 50 mM Tris, 10-60 mM imidazole gradient) was applied to

the column, as recommended by the manufacturer and protein was eluted with 250

mM imidazole. Purified protein was electrophoresed on SDS-PAGE gels and

detected by Western blot using an anti-His antibody (Invitrogen)

5.4.3 Catalytic activity of recombinant hemoglobinases

Recombinant Na-MEP-1 expressed in insect cells was electrophoresed on precast

gelatin zymogen gels (Invitrogen) as per the manufacturer’s instructions with slight

94

modifications – 10 mM CaCl2 or 10 mM ZnCl2 were added to the zymogram

developing buffer, and the pH of the developing buffer was titrated in single pH units

from pH 3-7. Excretory/secretory proteins of adult A. caninum were used as a

positive control for the gelatin gel and 1,10-phenanthroline (10 μM final

concentration) was included to inhibit metalloprotease activity. Gels were incubated

overnight at 37oC and stained with Coomassie Brilliant Blue (CBB) (BioRad)

followed by destaining. A zone of clearance in the gel after destaining was indicative

of proteolytic activity.

Catalytic activity of Na-APR-1 towards the flourogenic substrate 7-

Methoxycoumarin-4-Acetyl-GKPILFFRLK(DNP)-D-Arg-Amide (MoCAc-

GKPILFFRLK) (Sigma) was assayed in a Fluostar Optima microplate reader (BMG

Labtech) at 37°C. Rates of hydrolysis were recorded by monitoring the increase in

fluorescence measured in arbitrary units (relative fluorescence units – rfu) at

excitation and emission wavelengths of 330 nm and 390 nm, respectively. The pH

optimum for enzyme (1.0 μg) activity was determined by assaying in 50 mM sodium

acetate buffers at half-unit pH increments from pH 2-6. The final substrate

concentration was 1.0 μM and the final volume of each reaction was 100 µl.

Enzyme (0.105 nM) efficiency was assessed at pH 3.5 by measuring initial rates over

a range of substrate concentrations (0.2-25 μM). The catalytic constants kcat, Km and

kcat/Km were derived from the resulting Michaelis-Menten plot.

Catalytic activity of Na-CP-3 against the dipeptidyl substrate, Z-Phe-Arg-

aminomethylcoumarin (AMC), was described by us previously (Ranjit et al., in

press).

5.4.4 Antibody production and immunolocalization

Antibodies were raised in female BALB/c mice against the Na-MEP-1

pBAD/Thio-TOPO construct as previously described (Tran et al., 2006). Briefly, pre-

immune sera were collected two days prior to immunization and pooled. The mice

were injected with 25 μg of protein subcutaneously and boosted twice at two week

intervals. Whole blood was collected via cardiac puncture, sera were separated and

stored at -20oC. Production of a mouse antiserum raised to recombinant Na-CP-3 was

reported earlier (Ranjit et al., in press). A rabbit antiserum was produced to

recombinant Na-APR-1 as described for Ac-APR-1 (Loukas et al., 2005a).

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Immunolocalization was performed on 7 μm thick cryosections of formaldehyde

(4%) fixed adult N. americanus worms. Localization was performed as described

elsewhere (Don et al., 2007) with minor modifications. Briefly, slides were

rehydrated in PBS and non-specific binding was blocked by incubating the slides

with 5% fetal calf serum/PBS for one hour at room temperature (RT). Slides were

incubated with antisera at various concentrations (1:100 to 1:1,000) in 1% Bovine

Serum Albumin (BSA) for 1 hour at RT. Goat anti-mouse IgG Alexa Fluor 555

(Invitrogen) was used as fluorescent secondary antibody at a dilution of 1:500 in 1%

BSA/PBST; slides were incubated for 1 hour at RT. Slides were washed in PBST, air

dried and coverslips applied with mounting media (Sigma). Slides were viewed and

photographed using a Leica IM100 fluorescent microscope.

5.4.5 Proteolysis of Hb by Na-APR-1, Na-CP-3 and Na-MEP-1

Human Hb was prepared by lysing whole red blood cells in a hypotonic buffer

(1% PBS) (Brindley et al., 2001). Hb was incubated with individual recombinant

proteases at molar ratios of 72:1 Hb to Na-APR-1, 5:1 and 50:1 Hb to Na-CP-3, and

14:1 and 140:1 Hb to Na-MEP-1, in 0.1 M sodium acetate at 0.5 pH unit increments

from pH 3-7 for up to 18 hours at 37oC. Hb was also incubated with various

combinations of recombinant proteases at 37oC for 6 -18 hours. Each sample was

electrophoresed on a 15% SDS-PAGE gel under native conditions to observe the

amount of intact Hb that remained. Hb subsite preferences for each enzyme were

determined using the web logo program (http://weblogo.berkeley.edu/).

5.4.6 LC-MS and MS-MS analysis of Hb hydrolysates

Hb samples that had been incubated with various combinations of hookworm

proteases were subjected to reverse phase HPLC (RP-HPLC) and the Hb-derived

peptides were identified by liquid chromatography mass spectrometry (LC-MS). LC-

MS analysis was performed using an Ultimate 3000 nanoLC system (Dionex,

Germany) with a CAP-LC flow splitter and a variable wavelength UV-VIS

detector scanning at 214 nm coupled to a quadrupole time-of-flight mass

spectrometer (MicrOTOFq, Bruker Daltonics) operated with a low flow

electrospray needle. The column used was a Vydac monomeric C18 300 Å 3 μm 150

μm x 150 mm, at a flow rate of 1.2 μl.min-1. The mobile phase buffers used for the

gradient program were (A) water with 0.1% formic acid and (B) acetonitrile:water

96

(4:1) with 0.1% formic acid. The gradient program consisted of 5% B for 5 minutes,

linear ramping to 55% B over 29 min, linear ramping to 90% B over 1 min, holding

at 90% B for 9 min, ramping back to 5% B over 1 min, and holding at 5% B for 20

min. The mass spectrometer scanned 50-3000 m/z and acquired data for 50 mins of

each analysis. Data acquisition was facilitated using Hystar (Bruker) and data was

processed using Data Analysis (Bruker). The mass spectrometer used an autoMSn

methodology that collected MS2 spectra for the two most intense ions in each full

scan spectrum. The scan time was set to 0.5 seconds for the Survey Scan and the

MS2 spectra were recorded were the result of 2 microscans, giving an overall duty

cycle of 2.5 seconds. In addition, dynamic exclusion was used such that after 2 MS2

spectra, the precursor would be added to an exclusion list for 1 min. This allowed the

collection of the maximum number of MS2 spectra during the analysis. Calibration

was performed immediately prior to the analysis. The data was prepared into a

format suitable for Mascot database searching using Data Analysis, and Biotools

(Bruker, Germany) was used to store and further scrutinize the data and search

results. Mascot searches were performed using Swiss-Prot database and a 20 ppm

tolerance on the precursor, 0.2 Da tolerance on the product

ions, methionine oxidation as a variable modification, and charge states 1, 2 and

3. Searches were performed using no enzyme so that we could identify novel

protease cleavage sites generated by the hookworm hemoglobinases.

5.5 RESULTS 5.5.1 Cloning of cDNAs encoding N. americanus hemoglobinases

Cloning of Na-apr-1 (Williamson et al., 2002) and Na-cp-3 (Xiao et al., 2008) have

been reported elsewhere. Na-mep-1 was submitted to GenBank under accession

number EU523699. The ORF is comprised of 846 amino acids (excluding the signal

peptide of 26 amino acid residues) with a predicted molecular mass of 96.77 kDa and

pI of 6.69. It contains three predicted N-glycosylation sites (118-NRT, 251-NHT and

545-NMT), two zinc binding motifs and is a member of the M13 peptidase family

(Fig. 5.1). Closest homologues at the protein sequence level included predicted M13

family members from other hookworms (Ancylostoma sp.), the free-living nematode

Caenorhabditis elegans and the jewel wasp Nasonia vitripennis. Na-MEP-1 is 56%

identical at the amino acid level to Ac-MEP-1 from A. caninum.

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Na-MEP-1 MTKLLVSTAGLTGVVAALFITSLVFSILTWTRVKNDNDNPPRPKEPLSRPVVQLSSSIQT Ac-MEP-1 MAKLLEVTTGLVVLLGVLGVISVVFNVLTWLKLNENKDDSS-PAPKIWNVGEQDNTPVLT neprilysin ---------------------------MGKSESQMDITDINTPKPKKK----QRWTPLEI : . : : : * * :.: Na-MEP-1 TVTENVVTEPIVTVPTVSRTRVSAKTISPRSSATTSTRTLRTLTTPKFVATEAAP--RRN Ac-MEP-1 NLLVLEKEELAAKLKKTPYEEVDEQTVR-QSSVMKLRNIKNALFTPIEPVASALPPLRVN neprilysin SLSVLVLLLTIIAVTMIALYATYDDGICKSSDCIKS------------------------ .: : . . . : *. . Na-MEP-1 RTIMCPNYGVSDNSYAYQEAASFILSGLDERVNPCEDFYAFTCNKFLKDHKAEEHGVSRY Ac-MEP-1 DPKYCPSYGEPDKKYAYQEAASYLLSGLDQTVDPCEDLYAFTCNTYLRNHNATDIGVNRI neprilysin --------------------AARLIQNMDATTEPCTDFFKYACGGWLKRNVIPET-SSRY *: ::..:* .:** *:: ::*. :*: : : .* Na-MEP-1 GAIKELQDAVNTEIVDALFDVDVNDKKRSETERITKALLHDCVYHISPN-VPTETIINFL Ac-MEP-1 GTYKDAQDDVNAEIVEALEEVNVSDTKWSETERLVKATLFTCVHHTRAR-KPIDNSKNVL neprilysin GNFDILRDELEVVLKDVLQEPKTED---IVAVQKAKALYRSCINESAIDSRGGEPLLKLL * . :* ::. : :.* : ...* : : .** *: . : :.* Na-MEP-1 EEIARMFGGIPFLNHTLKEDFDVFAAMGEVEQNHAMGTLFSAMVSVDYKKIKQNSLFLSQ Ac-MEP-1 IEMRDLFGGIPFLNHTLKKDIDFFDIMGKFEQNHAMGTLLGAMVSVDFKNVNKHSLFLSQ neprilysin PDIYGWPVATENWEQKYGASWTAEKAIAQLNSKYGKKVLINLFVGTDDKNSVNHVIHIDQ :: . ::. . :.:.:.::. .*:. :*..* *: :: :.:.* Na-MEP-1 PRLPMP-REFYVLPQFTMKLKKRGLQIADVLKKFAEKILEEPDKYRDMIEKAAQDVVELE Ac-MEP-1 PYLPMA-RDFYVFPQHTKMVENRVSLINSVLRSFAEAVLDDPSPYLDLMSRSARDVVKLE neprilysin PRLGLPSRDYYECTGIYKEACTAYVDFMISVARLIRQEERLP-IDENQLALEMNKVMELE * * :. *::* . . : : : . * : : ..*::** Na-MEP-1 RRIALASWADAEMRNYAQQYNPYDLPTLKKAY------PSVKWESYLRSLLSTVGPVDFS Ac-MEP-1 MQIAMASWPESELRNYAQQHNPRTLNQLKAAY------PAIKWDSYFNALLSSVQGVDMN neprilysin KEIANATAKPEDRNDPMLLYNKMTLAQIQNNFSLEINGKPFSWLNFTNEIMSTVNISITN .** *: : .: :* * :: : ...* .: . ::*:* . Na-MEP-1 GPHKRLIISQPSYFGWLNALFNGNVVDENTIVNYIITHLIFEDAEFLGGIFKESAEDLNY Ac-MEP-1 --RQNIILTQPSYFGWLNALFNG-GADDKTIANYLLVHLILEEADFLGGALKTMVQKSDY neprilysin --EEDVVVYAPEYLTKLKPILTK--YSARDLQNLMSWRFIMDLVSSLSRTYKESRNAFRK .: ::: *.*: *:.::. . . : * : ::*:: .. *. * : Na-MEP-1 VRYAQRSGRGVARVGRQLMHQR-DTRGDPNIPCMNFIMTYMPYGPGYVYVRSKQQRNDVQ Ac-MEP-1 VPYALGRGKGVTRVGQQLTRSHDDTVEDANIQCLNSMMTYMPFGPGYVYVKSRKNRDDVV neprilysin ALYGTTSETATWRR------------------CANYVNGNMENAVGRLYVEAAFAG-ESK . *. .. * * * : * . * :**.: : Na-MEP-1 ADIRKQTELVIESFLNMTSGLKWMSSDSKEKARQKAKGMVRNYGWPQKLFGDFKSSEEID Ac-MEP-1 KDIEHQTELVFKNFVNMIGNLNWMTDASLELAMEKADTMVKNYGWPKDLFGNFRDSSKID neprilysin HVVEDLIAQIREVFIQTLDDLTWMDAETKKRAEEKALAIKERIGYPDDIVSNDNKLN--N :.. : : *:: ..*.** : : * :** : .. *:*..:..: .. . : Na-MEP-1 EYHKKDYAEILELTKTERSSLRYYRMRRVLIKGYSNRESLRLLLQDADRSNFLLSPALVS Ac-MEP-1 AYHKKDYGNIINLYK-ENITHNYYHIRRTMIKGYSNHESLRLLTEAPKRDHFLLSPALVN neprilysin EYLELNYK------------EDEYFENIIQNLKFSQSKQLKKLREKVDKDEWISGAAVVN * : :* * . :*: :.*: * : .:..:: ..*:*. Na-MEP-1 AWYQPERNSITFPYASFNPPYYSYEYPQAYNYGGQGGTAGHELVHGFDDQGVQFGPDGSL Ac-MEP-1 AWYIPERNSIAFPYAFWNPPYYNYEYPQACNYAGQGGTAGHELVHGFDDQGVQFAADGSL neprilysin AFYSSGRNQIVFPAGILQPPFFSAQQSNSLNYGGIGMVIGHEITHGFDDNGRNFNKDGDL *:* . **.*.** . :**::. : .:: **.* * . ***:.*****:* :* **.* Na-MEP-1 SRCTWYDCGWMDKRSKDGFNDMAQCVVTHYSTFCCPEQEGNIHCANGATTQGENIADIGG Ac-MEP-1 SDCTWIECGWLEEKSKKGFSDMAQCVVTQYSTQCCPQTGGVTHCANGATTQGENIADLGG neprilysin -------VDWWTQQSASNFKEQSQCMVYQYGNFSWDLAGG--QHLNGINTLGENIADNGG .* ::* ..*.: :**:* :*.. . * : ** .* ****** ** Na-MEP-1 EHAAYIAYREYIKS-LGHEEKRLPGLERYTPNQIFWITYGYSWCRSVTEEYLISQLLTDP Ac-MEP-1 QLAAYRAYREYITKERGEEEKRLPGLEQYTPNQIFWITYGYSWCMSQTDSSLIRQLLTDV neprilysin LGQAYRAYQNYIKK--NGEEKLLPGLDLN-HKQLFFLNFAQVWCGTYRPEYAVNSIKTDV ** **::**.. . *** ****: :*:*::.:. ** : . : .: ** Na-MEP-1 HAPSACRTNQVVQSIPAFGRDFGCSLGDRMYPAPEQRCSVWVQE Ac-MEP-1 HSPGSCRVNQVMQDIPEFALDFGCTMGQKMYPEPEQRCPVWVAE neprilysin HSPGNFRIIGTLQNSAEFSEAFHCRK--NSYMNPEKKCRVW--- *:*. * .:*. . *. * * . * **::* ** Figure 5.1. Multiple sequence alignment of Na-MEP-1 with Ac-MEP-1 from Ancylostoma caninum and human neprilysin 1. Signal peptides of the hookworm proteins are italicised. Putative N-linked glycosylation sites of Na-MEP-1 are shown in bold font. Black boxes surround the catalytic residues that bind to divalent metal ions (zinc binding motif). Asterisks denote conserved residues in all three sequences. Two dots denote residues conserved in two of the three sequences. Single dots denote similar amino acids in at least two of the sequences

98

5.5.2 Expression and purification of Na-APR-1 and Na-MEP-1

Na-APR-1 was secreted by yeast as a zymogen into culture medium. The

recombinant protein was purified on a nickel-NTA column and was produced at an

approximate concentration of 2.0 mg.L-1 of purified protein (Fig. 5.2A). Na-CP-3

was expressed in yeast and purified on nickel-NTA columns as described (Ranjit et

al., in press). Na-MEP-1 was expressed in Hi5 insect cells, secreted into culture

medium then purified by nickel-NTA chromatography at a yield of 0.2 mg.L-1 (Fig.

5.2B). The identities of the recombinant proteins were confirmed by western blot

analysis using anti-Hexa His and anti-cmyc for Na-APR-1 and anti-V5 for Na-MEP-

1 antibodies (not shown). Na-MEP-1 was also transformed into E. coli and protein

was expressed in insoluble inclusion bodies (Fig. 5.2C). The protein was purified

under denaturing conditions and verified by Western blot analysis using anti-hexa

His antibody. The yield of purified, denatured protein was 1.5mg.L-1. Attempts to

refold denatured Na-MEP-1 expressed in E. coli were unsuccessful (not shown). Na-

MEP-1 expressed in E.coli pBAD-Thio vector included a glutathione S-transferase

(GST) fusion thus the purified protein was approximately 129 kDa (33 kDa larger

than the protein purified from Hi5 insect cells).

Figure 5.2. Expression and purification of N. americanus hemoglobinases. Na-APR-1 expressed in the yeast Pichia pastoris X33 (A). Lane 1 – culture supernatant; 2 - flow through from nickel-NTA column; 3 - 20 mM imidazole wash; 4 – 60 mM imidazole wash; 5 -1 M imidazole eluate. Na-MEP-1 expressed in Hi5 insect cells (B). Lane 1 – culture supernatant; 2 – flow through from Ni-NTA column; 3 – 40 mM imidazole wash; 4 – 60 mM imidazole wash; 5 – 250mM imidazole eluate. Na-MEP-1 expressed in insoluble form in E. coli (C). Lane 1 – urea solubilised pellet; 2 – flow through from Ni-NTA column under denaturing conditions (6 M urea); 3 – 40 mM imidazole/6 M urea wash; 4 – 60 mM imidazole/6 M urea wash; 5 – 250 mM imidazole/6 M urea eluate. Arrows indicate purified protein.

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5.5.3 Catalytic activity assays

Na-APR-1 was secreted from P. pastoris in its pro-form and underwent auto-

activation at acid pH. Optimal cleavage of MoCAc-GKPILFFRLK was at pH 3.5

(not shown). MoCAc-GKPILFFRLK was hydrolysed by 0.105 nM recombinant Na-

APR-1 at pH 3.5 with a Km = 15.4 ± 0.7 μM, kcat = 32.2 ± 0.7 and kcat/Km =

2,090,909 M-1s-1 (Fig. 5.3A). As little as 5 ng of recombinant Na-MEP-1 displayed

gelatinolytic activity. The catalytic activity was dependent on the presence of

divalent cations (10 mM CaCl2) in the developing buffer; when CaCl2 was replaced

with ZnCl2, catalytic activity was no longer observed (Fig. 5.3B). Activity was

observed at pH 4.5-6.5 (not shown), and addition of 1,10- phenanthroline completely

inhibited catalytic activity.

Figure 5.3. Catalytic activity of recombinant Na-APR-1 expressed in yeast and Na-MEP-1 expressed in insect cells. Catalytic activity of Na-APR-1 was determined using the fluorogenic peptide MoCAc-GKPILFFRLK in 50 mM sodium acetate (A). A range of concentrations of peptide were hydrolysed by 0.105 nM of recombinant Na-APR-1 at pH 3.5. Gelatin zymogram showing catalytic activity of purified recombinant Na-MEP-1 (B). Lane 1: 10 ng Na-MEP-1+ CaCl2; 2: 5 ng Na-MEP-1 + CaCl2; Lane 3: 10 ng Na-MEP-1 + 1,10-phenanthroline

5.5.4 Immunolocalization

Immunofluorescence experiments were conducted with tissue sections of

adult N. americanus that had been removed from euthanised hamsters. Antibodies to

Na-APR-1, Na-CP-3 and Na-MEP-1 all localized to the intestine of the adult worm

(Fig. 5.4) and confirmed earlier reports of intestinal localization for Na-APR-1

(Williamson et al., 2002) and Na-CP-3 (Ranjit et al., in press). Pre-vaccination

control serum did not bind specifically to any structures (Fig 5.4D).

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Figure 5.4. Immunolocalization of Na-APR-1, Na-CP-3 and Na-MEP-1. Na-APR-1, Na-CP-3 and Na-MEP-1 in transverse (A, C, D) and longitudinal (B) sections of adult N. americanus. Arrows indicate the intestine. A: mouse α-Na-APR-1; B: mouse α-Na-CP-3; C: mouse α-Na-MEP-1; D: normal mouse serum. White bar denotes 50 μm.

5.5.5 Hemoglobin degradation and LC-MS analysis of hemoglobin

hydrolysates.

Human Hb was readily degraded by Na-APR-1, with digestion starting to

occur within 5 mins at pH 3.5 and pH 4.5; digestion was slower at pH 5.5 but still

occurred (Fig. 5.5A). In contrast, Na-CP-3 and Na-MEP-1 were not able to digest

intact Hb, even after 24 hour incubation (Fig. 5.5B). To observe whether Na-CP-3

and Na-MEP-1 acted downstream in the hemoglobinolysis pathway to digest globin

fragments after initial digestion of Hb with Na-APR-1, hydrolysates were observed

by LC-MS (Fig. 5.6). Nineteen cleavage sites were detected in the Hb α chain and a

further 18 in the Hb β chain after digestion with Na-APR-1. Supporting the SDS-

PAGE observations, neither Na-CP-3 nor Na-MEP-1 digested Hb when assessed by

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LC-MS (data not shown). However, when Na-APR-1 and Na-CP-3 were added to Hb

together, an additional 4 cleavage sites in the Hb α chain and 9 in the β chain were

detected. Na-MEP-1 did not digest Hb, but when added to Hb in the presence of Na-

APR-1 and Na-CP-3, a further 16 cleavage sites in the Hb α and 8 in the β chain

were detected (Fig. 5.7). None of the proteases displayed a distinct preference for

defined residues at the P4-P4′ subsites. Na-APR-1 preferred bulky aromatic and

hydrophobic residues at the P1 position, cleaving at 8 sites with a P1 Phe, 10 sites

with a P1 Leu and 5 sites with a P1 Ala. Na-CP-3 was also promiscuous in its

preferred P1 residues, mostly cleaving after hydrophobic and neutral amino acids.

Na-MEP-1 preferred P1′ Ala (5 sites), but also readily cleaved sites with P1′ Lys,

Ser, Asp and Leu (Fig. 5.8). Na-MEP-1 also showed a preference for hydrophobic

residues at P2 (Leu, Val and Ala).

Figure 5.5. Hemoglobin digestion with recombinant Na-APR-1, Na-CP-3 and Na-MEP-1. (A) One hundred μg of hemoglobin was incubated with 1 μg of recombinant Na-APR-1 various pHs at 37oC for 5 mins. Lane 1: 10 μg Hb; L2: Hb + Na-APR-1 pH 3.5; L3: Hb + Na-APR-1 pH 4.5; L4: Hb + Na-APR-1 pH 5.5 (B) One hundred μg of hemoglobin was incubated with various recombinant proteases at pH 4.5 at 37oC. Lane 1: 10 μg Hb; L2: Hb + Na-APR-1 (5 mins); L 3: Hb + Na-CP-3 (24 hrs); L4: Hb + Na-MEP-1 (24 hrs).

35

25

15

10

1 2 3 4

1 2 3 4

35

25

15

10

A B

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Figure 5.6. LC trace of hemoglobin incubated with various recombinant proteins for 18 hours at 37oC at pH 4.5 Colored lines represent the UV trace (214 nm) of hemoglobin. (A) Overlapping UV trace of all four samples; (B) Hb (C) Hb + Na-APR-1; (D) Hb + Na-APR-1 + Na-CP-3; (E) Hb + Na-APR-1 + Na-CP-3 + Na-MEP-1

0 1 0 2 0 3 0 4 0 5 0 6 0 T im e [m in ]

0

1 0

2 0

3 0

4 0

5 0

0 1 0 2 0 3 0 4 0 5 0 6 0 T im e [m in ]

0

1 0

2 0

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0 1 0 2 0 3 0 4 0 5 0 6 0 T im e [m in ]

0

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0

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0

1 0

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In te n s .[m A U ]

0 1 0 2 0 3 0 4 0 5 0 6 0 T im e [m in ]

0

1 0

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5 0

0 1 0 2 0 3 0 4 0 5 0 6 0 T im e [m in ]

0

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0

1 0

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4 0

5 0

0 1 0 2 0 3 0 4 0 5 0 6 0 T im e [m in ]

0

1 0

2 0

3 0

4 0

5 0

In te n s .[m A U ]

0 1 0 2 0 3 0 4 0 5 0 6 0 T i m e [ m i n ]

0

5

1 0

I n t e n s .[ m A U ]

0 1 0 2 0 3 0 4 0 5 0 6 0 T i m e [ m i n ]

0

5

1 0

I n t e n s .[ m A U ]

0 1 0 2 0 3 0 4 0 5 0 6 0 T i m e [ m i n ]

0

1 0

2 0

3 0

I n t e n s .[ m A U ]

0 1 0 2 0 3 0 4 0 5 0 6 0 T i m e [ m i n ]

0

1 0

2 0

3 0

I n t e n s .[ m A U ]

0 1 0 2 0 3 0 4 0 5 0 6 0 T i m e [ m i n ]

0

1 0

2 0

3 0

4 0

5 0

I n t e n s .[ m A U ]

0 1 0 2 0 3 0 4 0 5 0 6 0 T i m e [ m i n ]

0

1 0

2 0

3 0

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5 0

I n t e n s .[ m A U ]

0 1 0 2 0 3 0 4 0 5 0 6 0 T i m e [ m i n ]0

1 0

2 0

3 0

4 0

5 0

I n t e n s .[ m A U ]

0 1 0 2 0 3 0 4 0 5 0 6 0 T i m e [ m i n ]0

1 0

2 0

3 0

4 0

5 0

I n t e n s .[ m A U ]

A

B

C

D

E

103

Figure 5.7. Map of hemoglobin α and β chains highlighting the cleavages made by N. americanus recombinant haemoglobinases. All three enzymes were co-incubated with hemoglobin at pH 4.5. Black arrows – Na-APR-1 cleavage sites; green arrows – Na-CP-3 cleavage sites; red arrows- Na-MEP-1 cleavage sites. Stars denote cleavage in Hb hinge region.

104

Figure 5.8. P4-P4′ subsite specificities of Na-APR-1, Na-CP-3 and Na-MEP-1. (A) Na-APR-1, (B) Na-CP-3; (C) Na-MEP-1. MS/MS data compiled from Mascot searches was used to create an alignment of peptide cleavage sites which was submitted to the web logo program to generate the image. The letters denote each amino acid, the larger the letter the more frequently that residue occurred at each subsite.

A B C

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5.6 DISCUSSION To liberate Hb into the gut lumen, hookworms must first lyse the ingested

erythrocytes. They do this via a protein that creates pores in the erythrocyte

membrane (Don et al., 2004). Hookworms then, like other blood-feeding parasites,

digest blood via a cooperative and ordered cascade of hemoglobinolysis. Here, using

an in vitro approach we show that the human hookworm, N. americanus, can utilize a

pathway of mechanistically distinct proteases (hemoglobinases) to digest Hb and the

subsequent globin peptides, and that the pathway is similar to that used by the

malaria parasite, P. falciparum. It is ordered, consists of at least three distinct classes

of endopeptidases, aspartic, cysteine and metallo-enzymes, and can be a major target

for the development of new therapies.

Plasmodium spp. are no more closely related to nematodes than they are to

humans, inferring that convergent evolution has resulted in phylogenetically distant

pathogens adopting similar ordered cascades of Hb hydrolysis. P. falciparum makes

initial cleavages of Hb with aspartic proteases, called plasmepsins. Four distinct

plasmepsins (PM I, II, IV and HAP) cleave Hb in a semi-ordered fashion - the

process is initiated by PM I and II, allowing PM IV and HAP to then act downstream

in the cascade (Banerjee et al., 2002). However, P. falciparum clones with deletions

of each of these plasmepsins or combinations thereof remained viable, despite

prolonged doubling times, suggesting that at least the early phases of the

hemoglobinolysis process is redundant (Goldberg, 2005, Liu et al., 2006). This is in

contrast to the P. falciparum cysteine hemoglobinase, falcipain-2, which is essential

for Hb digestion and parasite growth (Sijwali and Rosenthal, 2004). Moreover,

falcipain-2 knockout parasites were over 1000-fold more sensitive to the aspartic

protease inhibitor, pepstatin, than were wild type controls, highlighting the

cooperative action of cysteine and aspartic hemoglobinases in this parasite (Sijwali et

al., 2006).

In contrast to Plasmodium, hemoglobinolysis is less redundant in the parasitic

flatworms. Current data suggests that only one aspartic protease and three clan CA

cysteine proteases are present in the gastrodermis of schistosomes, and only the

aspartic and two of the cysteine proteases (SmCB1 and SmCL1) have been shown to

digest the intact Hb tetramer (Brindley et al., 2001, Lipps et al., 1996). Further

evidence of an ordered cascade in schistosomes comes from studies employing RNA

106

interference (RNAi) (Delcroix et al., 2006). Silencing of the S. mansoni mRNA for

cathepsin D aspartic hemoglobinase resulted in an inability of larval worms to digest

Hb in vitro, and, more importantly, none of the worms treated with cathepsin D

double stranded RNA matured to adulthood when injected into mice (Morales et al.,

2008). Suppression of mRNA encoding the cathepsin B cysteine protease, SmCB1,

did not overtly affect Hb degradation, but slowed parasite growth considerably

(Correnti et al., 2005).

In the study described herein, as well as our earlier findings with A. caninum

(Williamson et al., 2004), we show that hookworms express intestinal homologues of

the major aspartic and cysteine hemoglobinases of schistosomes and Plasmodium

spp. Moreover, hookworms express M13 metalloprotease that digests globin

fragments but not intact Hb; only P. falciparum has an analogous

metalloendopeptidase (albeit an M16 peptidase), and a homologue has yet to be

reported from schistosomes.

Of the three hemoglobinases that we explored in this study, only one could

cleave intact Hb, the aspartic protease Na-APR-1. In our assays, Na-APR-1 was

responsible for making the initial cuts in the tetramer, thereby unravelling it and

making the globin fragments accessible to the other enzymes that act downstream in

the pathway. Na-APR-1 is by no means the only hemoglobinase that cleaves the

intact Hb molecule in hookworms. A pepsin-like aspartic protease, Na-APR-2, can

also cleave Hb, and does so at distinct sites to Na-APR-1 (Williamson et al., 2003a).

Moreover, the A. caninum cysteine protease, Ac-CP-2, cleaves intact Hb (Williamson

et al., 2004), implying at least some redundancy whereby distinct mechanistic classes

of enzyme can initiate the cascade, although they cleave Hb at distinct sites.

We recently reported a family of 5 cathepsin B cysteine proteases from the

gut of N. americanus (Ranjit et al., in press), most of which were identified from just

480 expressed sequence tags (ESTs). Only one of these enzymes, Na-CP-3, was

expressed in active form. Moreover, Na-CP-3, but not the other intestinal cathepsins

B, had a C-terminal insertion in the same region as the falcipain-2 Hb-interacting

motif (Pandey et al., 2005), suggesting that not all of the N. americanus intestinal

cathepsins B are involved in Hb digestion. Indeed, adult hookworms can be kept

alive for months in medium containing just serum in the absence of Hb or blood (D.

Smyth & A. Loukas, personal communication), implying that Hb is not the only food

source that hookworms can utilise for survival, at least in vitro. Although Na-CP-3

107

did not digest intact Hb and only digested globin fragments, it might be capable of

digesting other full-length serum proteins, such as albumin. In support of this, Hb

digestion in schistosomes is inhibited most effectively by aspartic protease inhibitors,

while digestion of serum albumin is most effectively inhibited by cysteine protease

inhibitors, implying that different classes of enzymes play distinct roles in the

digestion of different protein substrates in the parasitic helminths (Delcroix et al.,

2006).

Exopeptidases are involved in hemoglobinolysis in Plasmodium (Dalal and

Klemba, 2007, Stack et al., 2007) and schistosomes (Caffrey et al., 2004, McCarthy

et al., 2004). They are thought to exert their activities downstream of the

endopeptidases, removing terminal amino acids and dipeptides for transport into the

gastrodermal cells (schistosomes) or in the cytosol (Plasmodium). Hookworm ESTs

encoding amino- and carboxy-peptidases are present in public databases, and we

have actively expressed an A. caninum aminopeptidase and are currently exploring

its role in the Hb digestion process (T. Don, J. Lowther and A. Loukas, personal

communication).

The degree of redundancy, both in terms of numbers of proteases and

overlapping functions/substrate preferences, has not been addressed in any depth for

hookworms or other parasitic nematodes. Progress in this area has been hampered by

a number of roadblocks: the lack of a genome sequence and, until recently, a

substantive number of expressed sequence tags (ESTs); and absence of RNAi and

gene knockout technologies. As a result, studies on the functions of proteases of

parasitic nematodes have been restricted to in vitro observations only. For example,

the barber’s pole worm, Haemonchus contortus, is a blood-feeding nematode of

sheep that causes enormous economic losses worldwide. Like hookworms, the gut of

H. contortus contains a plethora of mechanistically distinct endo- and exopeptidases

(Jasmer et al., 2004, Knox et al., 2003, Williamson et al., 2003b). However, most

have not been expressed in catalytically active form and have yet to be unequivocally

assigned hemoglobinolytic functions, despite being expressed in the gut.

Nonetheless, parasite-derived H. contortus antigen complexes that are enriched for

intestinal proteases are highly efficacious vaccines (Knox et al., 2003, Knox and

Smith, 2001). This strongly supports the case for dissecting the molecular basis of

the haemoglobinolytic pathway in blood-feeding nematodes.

108

N. americanus intestinal cells produce an array of proteolytic enzymes; our

study has now attributed a hemoglobinolytic function to several of these. This is by

no means the complete set of enzymes involved in the pathway, but serves to

highlight the complexity of the process and shows that at least some level of order

exists. The pathway is similar to that described for the canine hookworm, A.

caninum, but several key differences exist; Na-CP-3 was incapable of cleaving intact

Hb and instead cleaved globin peptides generated by hydrolysis with Na-APR-1,

however, Ac-CP-2 is the only cysteine hemoglobinase identified thus far from A.

caninum, that cleaves intact Hb. Numerous cysteine proteases are present in the gut

of N. americanus (Ranjit et al., in press), so we cannot yet discount the presence of

an intestinal cathepsin B that digests intact Hb.

Unlike Plasmodium hemoglobinases, which are within the digestive vacuole

and inaccessible to antibodies, hookworm hemoglobinases are extracellular and

exposed to host antibodies when the worm ingests blood. Antibodies against Ac-

APR-1 and Ac-CP-2 bind to the intestine of feeding hookworms, neutralize the

catalytic activity of the target enzymes and damage the epithelial surface (Loukas et

al., 2005a, Loukas et al., 2004). By understanding the functions of these enzymes in

the intestine of the worm we can obtain a more in-depth understanding of blood-

feeding in parasitic nematodes and make more informed decisions on hemoglobinase

antigen selection and mechanisms for their delivery as recombinant vaccines.

Acknowledgements

We thank Tristan Wallis for his technical assistance and helpful suggestions.

This research was supported by grants from the National Health and Medical

Research Council (NHMRC, Australia), The Sabin Vaccine Institute and the Bill and

Melinda Gates Foundation. NR was supported by a QUTBLU award and funding

from the ARC/NHMRC Research Network for Parasitology. AL was supported by a

Senior Research Fellowship from NHMRC.

CHAPTER 6: GENERAL DISCUSSION,

CONCLUSION AND FUTURE DIRECTIONS

110

6.1 GENERAL DISCUSSION Hookworm infections are highly endemic in many developing countries. The

nature of the infection varies from person to person, ranging from no overt symptoms

(usually light infections) to lethal, depending on the intensity of infection as well as

the nutritional and physiological status of the patient. At present, the control of

hookworm infections is dependent on anthelmintic drugs, and while this mode of

treatment provides successful cure, there are growing concerns that continued use of

these therapies could potentially lead to the emergence of drug resistant parasites

(Hotez et al., 2006). Vaccines are an attractive alternative to anthelmintics and, while

they may not achieve the rapid parasite clearance obtainable by drug treatment, the

effects of vaccination are prolonged, offering long-term protection against infection

and reinfection.

In developing a hookworm vaccine, it is essential to identify one or a few out

of the numerous potential antigens, as it is not feasible to assess all possible

molecules for vaccine efficacy in animal models. Immunological studies conducted

with the sheep nematode H. contortus have suggested that parasitic nematode

antigens can be categorised into two groups - either natural antigens or hidden

antigens (Munn, 1997). ES products and exposed somatic antigens which induce an

immune response in the host during the course of infection are termed natural

antigens, whilst antigens that do not induce an immune response during infection

(because they are hidden from the afferent immune system) are referred to as hidden

antigens (Newton and Munn, 1999). Each type of antigen can induce a robust

immune response in isolation, however it has been suggested that by combining

members of the two groups, their effects may be synergistic and they could target

distinct physiological pathways and developmental stages (Munn, 1997).

It is believed that an efficacious hookworm vaccine should consist of a

combination of two antigens, one of which is expressed/secreted by the infective

larvae and is involved in skin penetration and migration, and a second one which

targets proteins expressed in the gut of the adult stage, such as those involved in

blood-feeding (Loukas et al., 2006). A secreted protein, termed Na-ASP-2, from L3

N. americanus larvae, has already been selected and tested in phase I clinical trials

(Diemert et al., 2008) on the basis of its ability to partially protect laboratory animals

against hookworm challenge infections (Bethony et al., 2005, Goud et al., 2004). In

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addition to Na-ASP-2, it has been proposed that a second antigen, one from the adult

blood-feeding stage, would also be needed for an optimal vaccine.

The process by which haemoglobin (Hb) is broken down into free amino

acids is a focus of investigation in a number of haematophagous parasites, as it has

long been thought that targeting this process could lead to identification of new

chemotherapeutic strategies or vaccines (Delcroix et al., 2006). In lysosomes of

mammalian cells, endopeptidases act cooperatively with exopeptidases in the

catabolism of proteins. There is now mounting evidence that haematophagous

parasites digest Hb using a similar cooperative cascade, whereby endopeptidases that

are similar to mammalian cathepsins B and D act together with exopeptidases that

are similar to mammalian dipeptidyl peptidases and aminopeptidases (Williamson et

al., 2003b). Studies conducted with hematophagous parasites such as P. falciparum

and S. mansoni have shown that there is a hierarchical system whereby defined

proteases initiate the cleavage of the intact Hb tetramer, which is subsequently

cleaved by other endo-proteases and then exo-proteases, with the endpoint being

small globin peptides and free amino acids that are readily absorbed to provide

nutrients for the parasite. A similar network of enzymes has been shown to digest Hb

in the canine hookworm, A. caninum, and this model of Hb digestion has been

loosely termed a ‘haemoglobin digestion cascade’ (Delcroix et al., 2006, Williamson

et al., 2004).

The main objective of this thesis was to identify and characterise potential

vaccine candidate molecules from the adult stage of N. americanus, in particular

seeking those proteins that are involved in feeding and nutrient acquisition. To date,

there has been a paucity of literature on the feeding process in human hookworms,

mostly due to the difficulty in maintaining them in laboratory animals. If the blood-

feeding process is to be targeted as a potential vaccine strategy for human

hookworms, it is imperative to gain a comprehensive understanding of the molecular

events in this process and to determine which proteases are involved in the enzymatic

digestion of the blood-meal.

It is thought that the main source of nutrition for hookworms comes from

proteins contained in the blood that they ingest from ruptured capillaries in the host

intestinal wall - one of most highly abundant proteins in blood is haemoglobin (Hb).

In this study, I have investigated the roles of various intestinal proteases which are

thought to play a role in haemoglobin degradation, aiming to establish which

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protease would be the most viable target as a vaccine candidate. The first manuscript

from this study (A survey of the intestinal transcriptome of the hookworms,

Necator americanus and Ancylostoma caninum using tissue isolated by laser

microdissection microscopy) details the investigation of the intestinal transcriptome

from both human and canine hookworm species. This study was conducted in order

to profile the proteins expressed in the intestine of the worms and gain a general

snapshot of the functions that occur in this specialised organ. As it is thought that Hb

digestion processes takes place in intestine of the worm, I determined which groups

of proteases were expressed in this region, and selected several of these for more

detailed characterisation. The aim here was to obtain better knowledge of which

proteins might be the most important to target in a vaccine development program.

The N. americanus gut cDNA generated in this study was not only used for the

identification of novel protease mRNAs, but was also valuable in verifying which of

the previously identified proteases such as Na-APR-2, Na-CP-2-5, and other proteins

with potential roles in blood-feeding were expressed in the gut of this hookworm,

further strengthening the claim that they are involved in digestion of the blood-meal.

The data I have presented in the following two manuscripts (A family of cathepsin

B cysteine proteases expressed in the gut of the human hookworm, Necator

americanus and Digestion of haemoglobin via an ordered cascade of proteolysis

in the intestine of the human hookworm, Necator americanus), provides the first

detailed evidence of the roles of several individual proteins, belonging to three

distinct classes of proteases, expressed by N. americanus. The majority of the

proteases I found are orthologs of proteases expressed by other hematophagous

helminths and protozoa, some of which have been already been shown to play a role

in Hb digestion process in these parasites (Delcroix et al., 2006, Goldberg, 2005). I

postulated that, due to their similarity in protein sequence and structure, the N.

americanus proteases identified in this study would also be involved in the Hb

degradation process.

Aspartic proteases

Studies conducted with several hematophagous parasites have implied that

aspartic proteases play a significant role in the initial degradation of tetrameric Hb by

cleaving it at the hinge region. In S. mansoni and P. falciparum, it has been shown

that cathepsin D-like enzymes make initial cuts in Hb (Banerjee et al., 2002,

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Delcroix et al., 2006). Moreover, Williamson et al. demonstrated that Ac-APR-1 and

Na-APR-1 cleave intact dog and human Hb, and suggested that this enzyme was

likely to be responsible for the initiation of the Hb digestion cascade in hookworms

(Williamson et al., 2002). My results confirm this hypothesis, as MS/MS data

showed that Na-APR-1 cleaved the Hb α chain at Phe-33 – Leu-34 and Hb β chain at

Phe-41 – Phe-42, which are critical sites for the maintenance of Hb conformation.

Recent published studies on cathepsin D-like proteases from S. mansoni and P.

falciparum demonstrated that suppression of these enzymes, either at the RNA

expression level or with chemical inhibitors of aspartic proteases, resulted in the

parasites being incapable of digesting haemoglobin properly, leading to retarded

growth and reduction in fecundity in S. mansoni (Delcroix et al., 2006, Morales et

al., 2008) and slower replication time in P. falciparum (Liu et al., 2005). Vaccination

of dogs with Ac-APR-1 resulted in significant reductions in worm burden and faecal

egg output, but most importantly, vaccination protected the host against substantial

blood loss (Loukas et al., 2005a). These results clearly indicate that this group of

proteases play a critical role in Hb degradation and therefore warrant targeting as

vaccine candidates.

Cysteine proteases

Although it has been shown that aspartic proteases play a principal role in the

Hb digestion process, studies investigating the effects of specific inhibitors on

proteolytic activity of hookworm extracts have revealed that the inhibition of aspartic

proteases alone does not completely ablate Hb degradation (Williamson personal

communication). In fact, complete inhibition of Hb degradation was only observed

when all four protease inhibitors were utilised, suggesting that other classes of

proteases are necessary for the Hb degradation process to occur efficiently. A

number of studies have now shown that cysteine proteases also play a substantial role

in Hb digestion process (Sajid et al., 2003, Sijwali et al., 2006).

Cathepsin B-like cysteine proteases have been found to be abundantly

expressed in a number of blood-feeding parasites, particularly in the gut of the

nematode H. contortus. Indeed, this gene family has undergone enormous expansion

in Haemonchus, and mRNAs encoding cathepsins account for approximately 16% of

all transcripts in the adult female worm intestine (Jasmer et al., 2004). For this

reason, I have proposed that cathepsins B also play a major role in

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haemoglobinolysis in the gut of hookworms, and they were therefore a major focus

of this study.

Cysteine proteases of parasites are often developmentally regulated, showing

highest expression levels in actively feeding stages (Jasmer et al., 2004). Moreover,

they are often primarily localised in the region where digestion of food occurs, such

as the intestine of helminths. Kumar et al. characterised a cysteine protease from the

exsheathing fluid of N. americanus larvae (Kumar and Pritchard, 1992), but this is

the only report in the literature on cysteine proteases from human hookworms. A N.

americanus cDNA sequence, encoding a cathepsin B protease called necpain, has

been deposited in GenBank, but a publication has not accompanied this submission.

In the second manuscript from this thesis (A family of cathepsin B cysteine

proteases expressed in the gut of the human hookworm, Necator americanus), I

characterised four additional mRNAs encoding cathepsins B and showed that all four

are expressed in the intestine of N. americanus. A comparison of the ORFs of the

four N. americanus cathepsins B (Na-CP-2, -3, -4, -5) showed that these proteins

shared the greatest sequence homology with the A. caninum cysteine proteases Ac-

CP-2 and Ac-CP-1. Despite these two A. caninum proteases sharing 86% amino acid

identity, they are expressed in two different locations and are thought to play distinct

roles: Ac-CP-1 has been localised to cephalic and excretory glands, which suggests

that it is secreted into host tissue by the adult worm during attachment and feeding,

while Ac-CP-2 has been localised to the brush border membrane of the worm

intestine and is involved in Hb digestion (Loukas et al., 2004, Williamson et al.,

2004). Therefore, to determine the roles of the N. americanus cysteine proteases

described earlier in my study, immunolocalisation studies and functional assays were

undertaken.

Published studies of cysteine proteases from a range of parasites have

demonstrated high levels of expression of secreted recombinant proteins from yeast,

in particular, Pichia pastoris (Beckham et al., 2006, Sajid et al., 2003). I therefore

adopted this strategy for the expression of all four N. americanus cysteine proteases,

initially transforming plasmids into the X-33 strain of P. pastoris. However, only Na-

CP-3 and CP-5 were expressed and secreted into the culture supernatant, and only

Na-CP-3 was expressed at sufficiently high yield to pursue further characterisation.

Numerous experimental parameters were modified in an attempt to obtain or improve

expression of Na-CP-2, -4 and -5. These included transforming into the KM71H

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strain of P. pastoris, addition of protease inhibitor cocktails (to prevent digestion of

the recombinant protein by itself or by yeast proteases), varying the pH of the culture

medium, and expression of the mature form (in the absence of the pro-region). None

of these variables affected the levels of protein produced (data not shown). Further

characterisation (in terms of catalytic activity) of the proteases Na-CP-2, -4 and -5

was therefore deemed unfeasible.

As Na-CP-3 was the only recombinant protein which expressed in yeast at

high yield, I restricted further analyses of enzymatic activity to this protein. When

the pro-form of the protease was electrophoresed in a gelatin gel with dithiothreitol

as the reducing agent, a zone of clearance was detected in the gel, indicating that cis-

processing was occurring in the gel, to yield an active mature enzyme. In contrast to

this result, catalytic activity could not be detected using clan CA-specific peptide

substrates bearing a fluorochrome, indicating an absence of catalytic activity. MS

analysis of the secreted recombinant protease revealed that the pro-region of the

protein had not been cleaved off during secretion from P. pastoris. Incubation of

recombinant Na-CP-3 for extended periods in low pH buffers with thiol agents did

not result in auto-activation (data not shown). Similar scenarios have been described

for F. hepatica FhCatB1 and S. mansoni SmCB1, where the pro-regions were not

cleaved during secretion, thereby inhibiting catalytic activity (Beckham et al., 2006,

Sajid et al., 2003). In the case of FhCatB1, the pro-region was removed by

incubating the protein with a large negatively charged molecule such as dextran

sulfate, while the SmCB1 pro-region was removed via a trans-processing event with

an asparaginyl endopeptidase, an enzyme that belongs to another family of cysteine

proteases and is also expressed in the schistosome gut (Beckham et al., 2006, Sajid et

al., 2003). An asparaginyl endopeptidase has been reported from A. caninum (D.

Smyth & A. Loukas, personal communication) and has also been shown to be

expressed in the gut of H. contortus (Oliver et al., 2006), so it is likely that this

enzyme is also expressed in the gut of N. americanus. Asparaginyl endopeptidases

cleave on the C-terminal side of Asn residues. It is unlikely that Na-CP-3 is activated

by asparaginyl endopeptidase due to the absence of an Asn residue in the vicinity of

the predicted pro-mature junction. However, Na-CP-4 and CP-5 do possess Asn

residues in this vicinity and may be candidates for trans-processing by asparaginyl

endopeptidase.

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To further address the activation status of pro-Na-CP-3, a similar strategy to

that used for activation of FhCatB1 was employed (Beckham et al., 2006). This

involved incubation of pro-Na-CP-3 with dextran sulfate, which resulted in cis-

processing of the protein and auto-catalytic removal of the pro-region. The processed

protease demonstrated catalytic activity against the diagnostic fluorogenic peptide Z-

Phe-Arg-AMC. This result verified that Na-CP-3 was catalytically activity, thus the

next step was to determine whether or not it was capable of cleaving intact Hb.

Previous studies on cathepsin B proteases from blood-feeding parasites, such as S.

mansoni SmCB1 and A. caninum Ac-CP-2, indicated that these proteins were capable

of cleaving native Hb. However, Na-CP-3 could not cleave intact Hb, although it was

capable of further digesting globin fragments which had initially been cleaved by the

aspartic protease, Na-APR-1. Na-CP-3 is expressed in the hookworm gut and the cp-

3 mRNA is more highly expressed in gut tissue than any of the other cathepsin B

mRNAs examined in this study, further suggesting that Na-CP-3 plays a pivitol role

in nutrient acquisition. Although not tested in this study, it is possible that Na-CP-3

might play a more upstream role in cleavage of other blood proteins such as serum

albumin, fibrinogen and immunoglobulins. Delcroix et al. reported that gene

knockdown of S. mansoni cysteine proteases did not result in a significant reduction

of haemoglobin digestion, but did substantially impact on digestion of albumin

(Delcroix et al., 2006). Similarly Correnti et al, demonstrated that schistosomes

treated with SmCB1-dsRNA were viable and developed intestinal heme

pigmentation indicative of haemoglobin digestion, but showed significant growth

retardation when compared to control parasites, indicating that SmCB1 function is

not essential for haemoglobin digestion but is necessary for normal parasite growth

(Correnti et al., 2005).

Although catalytic activity was not established for the other three cysteine

proteases (Na-CP-2, -4 and -5) because recombinant protein could not be expressed

in native form in my study, it is speculated that these proteases are also involved in

nutrient acquisition. Localisation studies conducted with antibodies produced against

the denatured proteins demonstrated that all three proteases are expressed in the gut

of the worm. Moreover, real time PCR results demonstrated that the mRNAs

encoding these proteases were upregulated in adult worms, and were most highly

expressed in the gut tissue. In addition, both Na-CP-2 and CP-4 possess the so called

“haemoglobinase” motif as described by (Baig et al., 2002) and, although this does

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not guarantee that these proteins are haemoglobinolytic, it is strongly suggestive of

this function.

Knox et al. demonstrated that vaccination of sheep with a thiol-sepharose

binding protein (TSBP) extract from adult H. contortus reduced worm burdens in

sheep intestines by 47% (Knox et al., 2005). Redmond et al. further demonstrated

that vaccination with a cocktail of three different cysteine proteases resulted in 38%

reduction in numbers of H. contortus in sheep compared to controls, indicating that

the majority of the protective immunity generated by TSBP was due to the combined

effects of a few cysteine proteases (Redmond and Knox, 2006). In similar fashion, all

four N. americanus cysteine proteases described in this study might work

synergistically, and present a better vaccine target when combined, rather than

focusing on each isolated enzyme. Similarly, their roles could be redundant, as seen

for the P. falciparum plasmepsins, and to a lesser extent, the falcipains (Liu et al.,

2006), so that if one mRNA was suppressed, others could compensate with minimal

or no effect on overall feeding of the worm. In like fashion, if redundancy exists

amongst the cathepsins B, antibodies targeting just one protease might not directly

interrupt blood feeding. A vaccine trial conducted with Ac-CP-2 did not result in

reduced worm burden, but did cause a significant decrease in the size of adult worms

and the fecundity of female worms (Loukas et al., 2004). Whilst the Ac-CP-2 vaccine

was not as efficacious (in terms of reductions in adult worms) as the Ac-APR-1

vaccine (which had a reduction of worm burden by 33%) (Loukas et al., 2005a), it

did have a significant effect on fecundity in particular, indicating that this group of

proteases is a valid target for new chemotherapies.

A recent study by Xiao et al showed that vaccination of hamsters with Na-

CP-2 resulted in a 28% reduction in worm burdens (Xiao et al., 2008). While the

level of protection was not particularly high, the Na-CP-2 immunogen that was used

was expressed in E. coli in denatured form and could not be refolded in soluble form.

As a result, the recombinant protein was not properly folded and would not have

displayed many of the potential conformational epitopes of the native protein. If this

vaccine trial was repeated with correctly folded protease, it is reasonable to assume

that the level of protection would increase.

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Metalloproteases

In addition to aspartic and cysteine proteases, a number of hematophagous

parasites, including the nematodes A. caninum and H. contortus, and the malaria

parasite P. falciparum, express a third mechanistic class of proteases - the

metalloproteases - that play a downstream role in haemoglobinolysis. Surprisingly, a

metalloendopeptidase involved in haemoglobinolysis has not yet been reported from

schistosomes, implying that the convergent evolution of this digestive process in

blood-feeding parasites is more obvious in Plasmodium spp. and the blood-feeding

nematodes than it is in the schistosomes. None of the metalloenzymes described so

far from haematophagous parasites can digest intact Hb, and all appear to act

downstream in the cascade after aspartic and/or cysteine proteases have generated

globin peptides (Eggleson et al., 1999, Williamson et al., 2004).

Presented here is the first report of identification and characterisation of a

metalloprotease from N. americanus. Previously, an eotaxin-cleaving

metalloprotease activity was described from N. americanus ES products, but neither

the protein nor the cDNA were isolated (Culley et al., 2000). In my study, it was

demonstrated that a metalloprotease (termed Na-MEP-1) is expressed in the gut of

adult N. americanus and, in similar fashion to other metallo-haemoglobinases, it

cannot digest intact Hb but instead digests globin fragments, providing further

support for the concept of an ordered digestive process in hookworms. A vaccine

trial has been conducted by Smith et al. with the four metalloproteases fractionated

from the H. contortus H-gal-GP complex, which provided up to 50% reduction in

egg count, however no significant protection was reported with bacterially expressed

forms of the metalloproteases indicating that conformational epitopes are required

for immunity (Smith et al., 2003). Unlike other proteases, little is know about what

level of effective immunity can be provided by individual metalloprotease antigens.

Nonetheless, vaccine trials with these molecules should be undertaken as they have

been implicated in Hb degradation and in immune evasion.

Hidden antigens

The strategy of identifying vaccine candidate molecules using hidden

antigens such as gut molecules has been successfully implemented in both H.

contortus and the cattle tick, Boophilus microplus (Munn, 1997). In the case of B.

microplus, a gut membrane structural protein termed Bm86 has been produced in

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recombinant form as a commercial vaccine and provides high levels of protection

against tick infestation (de la Fuente et al., 1999). While a commercial vaccine has

yet to be produced from H. contortus gut proteins, a number of semi-purified extracts

from the gut membrane have been shown to be highly protective in vaccine trials,

with worm burden reductions as high as 90% (Knox and Smith, 2001). Interestingly,

the protective gut antigen complexes from H. contortus comprise various classes of

proteases including aspartic, cysteine, metalloendopeptiodases and microsomal

aminopeptidases, indicating the complexity of the process involved in nutrient

acquisition. What has hampered progress in the development of a commercial

haemonchosis vaccine has been the inability to produce many of the

haemoglobinases in recombinant soluble form, with appropriate post-translational

modifications (Dalton et al., 2003), and at sufficient yields for viable scale-up.

Nonetheless, what these studies clearly establish is that targeting proteases which are

expressed in the gut and are involved in feeding is a valid approach to vaccine (and

drug) development. The distinct benefit of targeting hidden antigens for a vaccine is

that there has been no selective pressure placed upon these molecules by the host

immune system, so many of the polymorphisms that are found in exposed

extracellular antigens (Good et al., 2004), are not evident in gut proteins (Munn,

1997).

Hookworms are smaller than Haemonchus sp., making dissection of gut

tissue via standard methods (Jasmer et al., 2001) extremely difficult. Instead, in my

study laser microscopy microdissection (LMM) was used to remove the gut – this

was the first report of the use of LMM to dissect tissues from a parasitic helminth.

The approach was highly successful and proved to be an excellent method for

isolating specific tissue from defined hookworm organs – intestine and gonad. RNA

isolated from the catapulted gut sections was used to synthesise cDNA which was

then transformed into a plasmid library. Shotgun sequencing of clones from these gut

tissue specific libraries from both N. americanus and A. caninum provided a snapshot

of the intestinal transcriptome, particularly the cDNAs that encode proteases. Many

of the H. contortus gut proteases, particularly those that form the H-gal-GP complex,

are hidden from the host’s immune system (Newton and Munn, 1999), and are not

ES products. By analogy, the haemoglobinases in the hookworm gut are also likely

to be hidden antigens, but this has not been comprehensively addressed. In

unpublished data from our laboratory (J. Mulvenna & A. Loukas, personal

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communication), very few cysteine proteases were detected in the ES products of

adult A. caninum using a proteomics approach. In my study, the transcriptomes of

gut tissue from both N. americanus and A. caninum were investigated in order to

gain a better understanding of the nature, abundance and diversity of the major

proteases which are expressed in this organ and to identify potential vaccine

candidates, particularly those that shared sequence identities with H. contortus

proteases present in H-gal-GP.

Abundant hookworm gut transcripts

The gene ontology (GO) classification system was used to identify the

functional roles of some of the intestinal contigs. A large number of contigs could

not be placed into specific GO categories, but of the ~30% of contigs which did

receive a GO assignment, these had diverse predicted functions. The most frequently

predicted GO functions for cDNAs from the gut of both species of hookworms was

protein/ion binding and catalytic activity, indicative of nutrient digestion and uptake

processes that occur in this tissue. Some mRNAs were particularly abundant, such as

those encoding vitellogenin, fatty acid binding proteins and heat shock proteins.

Vitellogenin was one of the most highly abundant transcripts in both A. caninum and

N. americanus libraries, and while this protein is generally associated with

embryogenesis, Caenorhabditis elegans vitellogenin is expressed in the intestine

where it transports cholesterol from the gut to the oocytes (Matyash et al., 2001). As

nematodes are auxotrophic for sterols, uptake and transportation of cholesterol to the

gonads is essential for viable reproduction (Matyash et al., 2001). Heat shock protein

20 was another highly abundant gut transcript, which aligns with reports on the

importance of this family of proteins during the transition of nematodes from the

external environment into the mammalian host. In H. contortus, HSP-20 has been

localised to both the intestine and reproductive organs and is thought to play a role in

preparing parasites for the stress of moving from the pastures into a warm blooded

host – specifically, the sudden rise in temperature and exposure to the host immune

response (Hartman et al., 2003).

Whilst a large number of gut mRNAs with GO assignments encoded general

house-keeping proteins, numerous contigs that encoded proteins with suggested or

proven roles in feeding were identified in my study. These included anticoagulants

and platelet inhibitors, as well as all of the proteases described earlier. Hookworm

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anticoagulants and platelet inhibitors are expressed in the oesophageal and cephalic

glands from where they are secreted into host tissue at the attachment site (Del Valle

et al., 2003), but their expression in the gut has not been reported previously. It is

possible that they function in the parasite gut by inhibiting clot formation of ingested

blood.

Novel hookworm gut transcripts

Prior to this study, only one metalloprotease had been identified in the gut of

adult hookworms – the M13 family member Ac-MEP-1 from A. caninum (Jones and

Hotez, 2002). In my study, the N. americanus orthologue, Na-MEP-1, was identified.

However, an additional four novel metalloproteases were identified in the intestinal

EST datasets (Ranjit et al., 2006) - three of these belonged to the M12 family, also

known as astacins, and one belonged to the M22 family of O-sialoglycoprotein

endopeptidases. To date, M12 proteases have only been found in hookworm L3

larval stage ES products (Zhan et al., 2002) where they are thought to assist in tissue

migration during entry into the mammalian host (Williamson et al., 2006). In C.

elegans, M12 proteases are secreted into the alimentary tract where they are thought

to be involved in digestion of food (Mohrlen et al., 2003), so it is feasible that these

hookworm M12 astacin-like proteases are also involved in nutrient acquisition from

blood. The identification of an O-sialoglycoprotein endopeptidase represents the first

report of an M22 protease in parasitic nematodes. These enzymes are highly specific

for O-sialoglycosylated proteins such as glycophorin A which is a component of red

blood cells (Rawlings et al., 2008).

Serine proteases do not appear to be widely expressed in parasitic nematodes.

In my study an EST encoding a serine protease was detected from A. caninum gut

cDNA library, this protease belongs to the clan CA, S1A family which is classified

as chymotrypsin (Rawlings et al., 2008). In vertebrates, intestinal protein digestion is

largely the work of serine proteases, primarily members of the trypsin family Apart

from the serine protease activity which was detected in the H. contortus TBSP

complex (Knox et al., 1999), there have not been any other reports of serine

proteases involved in the feeding process in helminths, possibly indicating that this

group of proteases does not play a major role in the blood digestion process in

parasites. In contrast, serine proteases are integral to the digestion process in blood

feeding insects such as Anopheles species (Muller et al., 1993). It is believed that the

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evolutionary transition from cysteine/aspartic to serine proteases occurred in

arthropods or molluscs (Delcroix et al., 2006).

Other than APR-1 and APR-2, additional aspartic protease cDNAs were not

identified in gut tissue from either hookworm species in my study, suggesting that

only a small number of aspartic proteases are involved in Hb digestion, and this early

phase of Hb digestion might not be as redundant as it is in Plasmodium, where at

least four aspartic proteases are found in the digestive vacuole. My results further

highlight the validity of targeting these enzymes as vaccines and drug targets.

However it is important to note that only a small number of ESTs were sequenced

from the gut cDNA libraries – it is noteworthy that I did not identify intestinal ESTs

encoding aminopeptidases or haemolysins, two groups of proteins that are thought to

be important in blood-feeding parasites. However, using PCR, Don et al. amplified

two cDNAs encoding pore-forming saposins (Don et al., 2007) and an

aminopeptidase (T. Don & A. Loukas, personal communication) from the A.

caninum gut cDNA library, indicating the presence but not abundance of these

enzymes in hookworm intestinal tissue.

6.2 CONCLUSION AND FUTURE DIRECTIONS Results presented in this thesis have verified the complex nature of the Hb

degradation process in hookworms, as it was demonstrated that this process involves

numerous proteases from various mechanistic classes. I have shown that aspartic

proteases play a principal role in initiating the cleavage of the tetrameric Hb protein

in the gut of adult N. americanus, while cysteine proteases and metalloproteases also

play essential, but downstream, roles in this process after aspartic proteases have

made initial cuts. The data presented implies that Hb digestion in N. americanus

occurs in an ordered fashion, similar to that described in the digestive vacuole of

Plasmodium falciparum, although there appears to be less redundancy in

hookworms, at least in the early stages of the process. The conclusions drawn have

established that all three mechanistic classes of proteases are needed for Hb

degradation to occur efficiently. The aspartic protease Na-APR-1 plays the pivotal

role in initiating the process, and therefore might be considered a good target for

vaccine or drug development. Vaccine trials with Ac-APR-1 followed by a

heterologous challenge with N. americanus L3 have already been conducted in a

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hamster model of necatoriasis, and a 44% reduction in adult worms was obtained

(Xiao et al., 2008). My studies suggest that even greater reductions might be

achieved when hamsters are immunized with Na-APR-1 and receive a homologous

N. americanus larval challenge. These trials, including assessment of the vaccine

efficacy of Na-CP-3 and –MEP-1 are now underway at the Institute of Parastic

Diseases in China.

My investigation of the gut transcriptional profile of adult hookworms

indicated that proteases are, as expected, abundantly represented in this tissue. There

were numerous additional proteases identified from gut tissue ESTs, other than those

investigated in detail in this study. Many of these might have roles in

haemoglobinolysis process, thus further investigation of these enymes is necessary.

Moreover, there were numerous other transcripts identified which are predicted to be

involved in various other processes that could be targeted as vaccines, including

haemolysins, lipid/cholesterol binding proteins and amino acid transporters, some of

which are now under examination as vaccine candidates.

Further research into the up and downstream process of Hb degradation in

hookworms is required as it is still unknown how red blood cells are lysed to release

Hb or the process by which amino acids are transported across gut cells. As yet, no

exopeptidases (caboxy and amino) have been indentified in the Hb digestion process

in hookworms, however these enzymes have been implicated in a down stream role

in other haematophagous parasites such as S. manosoni and P. falciparum. Thus it is

highly probable that homologous proteases are also utilised by hookworms in this

process, and idenfication of such enzymes is warranted.

Due to the sheer number of people infected with hookworms worldwide, it is

imperative that an efficacious vaccine is produced in order to combat this insidious

disease. The ultimate hookworm vaccine would be one that targets both the larval

and adult stages, assaulting the parasite at both major developmental stages. As with

other haematophagous parasites, Hb degradation is essential for the survival of these

parasites and, therefore, further dissection of the molecular mechanisms of this

process is essential if we are to exploit the requirement of hookworms for blood

feeding. This will, and indeed already has, facilitated the identification and

development of target molecules that will form the basis of molecular vaccines

against this and other neglected diseases of humans and animals.

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APPENDICES

APPENDIX 1

A survey of the intestinal transcriptome of the hookworms, Necator

americanus and Ancylostoma caninum using tissue isolated by laser

microdissection microscopy.

International Journal for Parasitology 36: 701-710 N. Ranjit, M.K. Jones, D.J Stenzel, R.B Gasser, A. Loukas (2006).

APPENDIX 2

A family of cathepsin B cysteine proteases expressed in the gut of the

human hookworm, Necator americanus.

Molecular and Biochemical Parasitology 160: 90-9 N. Ranjit, B. Zhan, D. Stenzel, J. Mulvenna, R. Fujiwara, P. Hotez, A. Loukas

(2008).

APPENDIX 3

Vaccination with recombinant aspartic hemoglobinase reduces parasite

load and blood loss after hookworm infection in dogs.

PLoS Medicine 2 (10): e295 A. Loukas, J. M.Bethony, S. Mendez, R. T. Fujiwara, G. N. Goud, N. Ranjit, B.

Zhan, K. Jones, M. E. Bottazzi, P. J. Hotez (2005).

Vaccination with Recombinant AsparticHemoglobinase Reduces Parasite Load andBlood Loss after Hookworm Infection in DogsAlex Loukas

1*, Jeffrey M. Bethony

2, Susana Mendez

2, Ricardo T. Fujiwara

2, Gaddam Narsa Goud

2, Najju Ranjit

1,

Bin Zhan2, Karen Jones

2, Maria Elena Bottazzi

2, Peter J. Hotez

2*

1 Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Brisbane, Queensland, Australia, 2 Department of Microbiology and Tropical

Medicine, The George Washington University Medical Center, Washington, District of Columbia, United States of America

Competing Interests: The authorshave declared that no competinginterests exist.

Author Contributions: AL, JMB,MEB, and PJH designed the study.AL, RTF, GNG, NR, BZ, performedexperiments. AL, PJH, JMB, SM, andKJ analyzed the data. AL, PJH, JMB,and SM contributed to writing thepaper.

Academic Editor: MariaYazdanbakhsh, Leiden UniversityMedical Center, the Netherlands

Citation: Loukas A, Bethony JM,Mendez S, Fujiwara RT, Goud GN, etal. (2005) Vaccination withrecombinant aspartichemoglobinase reduces parasiteload and blood loss after hookworminfection. PLoS Med 2(10): e295.

Received: April 8, 2005Accepted: July 13, 2005Published: October 4, 2005

DOI:10.1371/journal.pmed.0020295

Copyright: � 2005 Loukas et al. Thisis an open-access article distributedunder the terms of the CreativeCommons Attribution License, whichpermits unrestricted use,distribution, and reproduction in anymedium, provided the originalauthor and source are credited.

Abbreviations: APR-1, Ancylostomacaninum aspartic protease 1; AS03,GlaxoSmithKline Adjuvant System01; ELISA, enzyme-linkedimmunosorbent assay; epg, eggs pergram of feces; Hb, hemoglobin; L3,third stage larvae

*To whom correspondence shouldbe addressed. E-mail: alex.loukas@qimr.edu.au (AL); mtmpjh@gwumc.edu (PJH)

A B S T R A C TBackground

Hookworms infect 730 million people in developing countries where they are a leading causeof intestinal blood loss and iron-deficiency anemia. At the site of attachment to the host, adulthookworms ingest blood and lyse the erythrocytes to release hemoglobin. The parasitessubsequently digest hemoglobin in their intestines using a cascade of proteolysis that beginswith the Ancylostoma caninum aspartic protease 1, APR-1.

Methods and Findings

We show that vaccination of dogs with recombinant Ac-APR-1 induced antibody and cellularresponses and resulted in significantly reduced hookworm burdens (p ¼ 0.056) and fecal eggcounts (p¼ 0.018) in vaccinated dogs compared to control dogs after challenge with infectivelarvae of A. caninum. Most importantly, vaccinated dogs were protected against blood loss (p¼0.049) and most did not develop anemia, the major pathologic sequela of hookworm disease.IgG from vaccinated animals decreased the catalytic activity of the recombinant enzyme in vitroand the antibody bound in situ to the intestines of worms recovered from vaccinated dogs,implying that the vaccine interferes with the parasite’s ability to digest blood.

Conclusion

To the best of our knowledge, this is the first report of a recombinant vaccine from ahematophagous parasite that significantly reduces both parasite load and blood loss, and itsupports the development of APR-1 as a human hookworm vaccine.

PLoS Medicine | www.plosmedicine.org October 2005 | Volume 2 | Issue 10 | e2951008

Open access, freely available online PLoSMEDICINE

Introduction

Hookworms infect more than 700 million people intropical and subtropical regions of the world. The majorspecies infecting humans are Necator americanus and Ancylos-toma duodenale. The parasites feed on blood, causing iron-deficiency anemia, and as such, are a major cause of diseaseburden in developing countries [1]. Unlike other humanhelminthiases, worm burdens do not generally decrease withage; in fact, recent findings revealed that the heaviest wormburdens are found among the elderly [2,3]. Whereasanthelminthic chemotherapy with benzimidazole drugs iseffective in eliminating existing adult parasites, re-infectionoccurs rapidly after treatment [4], making a vaccine againsthookworm disease a desirable goal.

Canines can be successfully vaccinated against infectionwith the dog hookworm, Ancylostoma caninum, by immunizationwith third-stage infective larvae (L3) that have been attenu-ated with ionizing radiation [5–7]. Subsequently, varying levelsof vaccine efficacy have been reported for the major antigenssecreted by hookworm L3 using hamsters [8,9] and dogs [10].Despite obtaining encouraging levels of protection with larvalantigens, only partial reductions in parasite load (fecal eggcounts and adult worm burdens) were reported. Moreover,protective antigens from the larval stage are only expressed byL3, and not adult worms, rendering antibodies against theseL3 secretions useless against parasites that have successfullyreached adulthood in the gut and begun to feed on blood. Wetherefore suggest that an ideal hookworm vaccine wouldrequire a cocktail of two recombinant proteins, one targetingthe infective larva and the second targeting the blood-feedingadult stage of the parasite [11].

Of the different families of proteins expressed by blood-feeding parasitic helminths, proteolytic enzymes have shownpromise as intervention targets for vaccine development[12,13]. Proteases are pivotal for a parasitic existence,mediating fundamental physiologic processes such as molting,tissue invasion, feeding, embryogenesis, and evasion of hostimmune responses [12,14]. Parasite extracts enriched forproteases protect sheep against the blood-feeding nematodesHaemonchus contortus [15–18] and Ostertagia ostertagi [19]; how-ever, significant protective efficacy has not been shown with apurified recombinant protease from nematodes of livestock.

Hookworms feed by burying their anterior ends in theintestinal mucosa of the host, rupturing capillaries andingesting the liberated blood. Erythrocytes are lysed by poreformation [20], releasing hemoglobin (Hb) into the lumen ofthe parasite’s intestine, where it is degraded by a semi-ordered pathway of catalysis that involves aspartic, cysteine,and metalloproteases [21]. Vaccination of dogs with acatalytically active recombinant cysteine hemoglobinase, Ac-CP-2, induced antibodies that neutralized proteolytic activityand provided partial protection to vaccinees by reducing eggoutput (a measure of intestinal worm burden) and worm size,but significant reductions of adult worm burdens and/orblood loss were not observed [22]. Anemia is the primarypathology associated with hookworm infection, and anultimate human hookworm vaccine would limit the amountof blood loss caused by feeding worms and maintain normallevels of Hb. This is particularly important in young childrenas well as women of child-bearing age, in whom menstrual,

and particularly fetal, Hb demands are considerable, render-ing these populations most vulnerable to the parasite [1].Here we describe vaccination of dogs with the aspartic

hemoglobinase of A. caninum, Ac-APR-1 [21,23] and show thatvaccination resulted in the production of neutralizing anti-bodies, significantly reduced egg counts, and significantlyreduced adult worm burdens. Most importantly, Hb levels ofvaccinated dogs were significantly higher than those of dogsthat were vaccinated with adjuvant alone after parasitechallenge. These data show that aspartic hemoglobinases,particularly APR-1, are efficacious vaccines against caninehookworm disease, providing strong support for furtherinvestigation and development of APR-1 as a recombinantvaccine against human hookworm disease.

Methods

Expression of Recombinant Ac-APR-1 in Pichia pastorisThe entire open reading frame of Ac-APR-1 encoding the

zymogen (spanning Ser-17 to the C-terminal Phe-446) butexcluding the predicted signal peptide was cloned into theexpression vector pPIC-Za (Invitrogen, Carlsbad, California,United States) using the XbaI and EcoRI sites. Yeast, P. pastorisX 33, was transformed with the vector encoding the Ac-APR-1zymogen as recommended by the manufacturer (Invitrogen)with modifications. Protein disulfide isomerase (PDI) gene inthe vector pPIC3.5 (a gift from Mehmet Inan, University ofNebraska, Lincoln, Nebraska, United States) was cut with SacIand transformed into P. pastoris X 33 cells which were alreadytransformed with Ac-apr-1 following the manufacturer’sinstructions. Eight transformed colonies were picked fromYPD plates containing Geneticin (0.5–1.0 mg�ml�1) andZeocin (1.0 mg�ml�1) and tested for Ac-APR-1 expressionfollowing the manufacturer’s instructions. The highestexpressing colony was selected and transferred to suspensionculture in flasks containing BMG medium (buffered minimalglycerol: 1.34% yeast nitrogen base, 0.00004% d-biotin, 1% w/v glycerol, and 100 mM potassium phosphate, [pH 6.0]).Suspension cultures were then transferred to a Bioflo 3000fermentor (New Brunswick Scientific, Edison, New Jersey,United States) utilizing a 5-l vessel as described [8]. Therecombinant protein was secreted into culture medium andaffinity purified on nickel-agarose as described elsewhere [8].Progress of purification was monitored using SDS-PAGE gelsstained with Coomassie Brilliant Blue and immunoblots usingmonoclonal antibodies to the vector-derived myc epitope.Recombinant Ac-APR-1 was treated with PNGase F and O-glycosidase, according to the manufacturer’s instructions(Enzymatic CarboRelease kit; QA-Bio, San Mateo, California,United States), under denaturing conditions to remove anyN-linked and O-linked oligosaccharides. Deglycosylation wasperformed only to confirm the presence of N-linked sugarson the recombinant molecule. All remaining studies wereconducted with the glycoprotein.

Activation and Hemoglobinolytic Activity of RecombinantAPR-1The unactivated zymogen was used for vaccination. A small

amount of the purified protein, however, was bufferexchanged into 100 mM sodium formate (pH 3.6)/0.15 MNaCl using a PD10 desalting column (Amersham Biosciences,Little Chalfont, United Kingdom) to facilitate proteolytic

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activation and removal of the pro-region. One microgram ofpurified, activated protease was then added to 10 lg of dogHb in the same buffer and incubated at 37 8C for 2 h.Cleavage of Hb was assessed visually by staining SDS-PAGEgels with Coomassie Brilliant Blue.

Animal HusbandryPurpose-bred, parasite naive, male beagles aged 8 6 1 wk

were purchased from Marshall Farms (North Rose, New York,United States), identified by ear tattoo, and maintained in theGeorge Washington University Animal Research Facility aspreviously described [24]. The experiments were conductedaccording to a protocol approved by the University AnimalCare and Use Committee (IACUC 48–12,0 [12,1]E). Before thefirst vaccination and after each subsequent one, a bloodsample was obtained from each dog.

Vaccine Study Design and Antigen-Adjuvant FormulationThe vaccine trial was designed to test Ac-APR-1 zymogen

formulated with the adjuvant AS03 [25], obtained fromGlaxoSmithKline (a kind gift from Drs. Joe Cohen and SylvieCayphas; GSK Biologicals, Rixensart, Belgium). To make sixdoses of Ac-APR-1 formulated with AS03, 600 lg ofrecombinant protein (1.5 ml of Ac-APR-1 at a concentrationof 0.4 mg�ml�1) was mixed with 1.2 ml of 20 mM Tris-HCl, 0.5M NaCl (pH 7.9), and 1.5 ml of AS03; the contents of the tubewere vortex mixed for 30 sec then shaken at low speed for 10min. Dogs were immunized with 100 lg of formulated antigenin a final volume of 0.5 ml. AS03-only control was prepared asdescribed above, with PBS included instead of Ac-APR-1.

Canine Immunizations and Antibody MeasurementsFive beagles were immunized three times with AS03-

formulated Ac-APR-1 by intramuscular injection. The vaccinewas administered on days 0, 21, and 42, beginning when thedogs were 62 6 4 d of age. As negative controls, five beagleswere also injected intramuscularly with an equivalent amountof AS03 using the identical schedule. Blood was drawn at leastonce every 21 d and serum was separated from cells bycentrifugation. Enzyme-linked immunosorbent assays (ELI-SA) were performed as previously described [24]. Recombi-nant Ac-APR-1 was coated onto microtiter plates at aconcentration of 5.0 lg�ml�1. Dog sera were titrated between1:100 and 1:23 106 to determine endpoint titers (the highestdilution of test group [APR-1] sera that gave a mean O.D. of�33 the mean optical density (OD) of sera from the controlgroup). Anti-canine IgG1, IgG2, and IgE antibodies conju-gated to horseradish peroxidase (Bethyl Laboratories, Mont-gomery, Texas, United States) were used at a dilution of1:1,000. Blood was collected from dogs before immunizationsand 7 d after the third vaccination but before L3 challenge.

Stimulation of and Cytokine Measurements from CulturedWhole Blood

Lymphoproliferation assays were performed using a wholeblood microassay as previously described [26]. Briefly, 25 ll ofheparinized blood was diluted in 200 ll of RPMI 1640medium (Gibco, Invitrogen) supplemented with 3% anti-biotic/antimycotic solution (Gibco). All tests were performedin triplicate in 96-well flat-bottomed culture plates usingrecombinant APR-1 at a concentration of 25 lg�ml�1 andconcanavalin A (ConA; Sigma-Aldrich, St. Louis, Missouri,United States) at 80 lg�ml�1. Incubation was carried out in a

humidified 5% CO2 atmosphere at 37 8C for 2 d (ConA-stimulated cultures) and 5 d (APR-1). Cells were pulsed for 6 hwith 1.0 lCi of [3H] thymidine (PerkinElmer Life AndAnalytical Sciences, Boston, Massachusetts, United States)and harvested onto glass fiber filters. Radioactive incorpo-ration was determined by liquid scintillation spectrometry.Proliferation responses were expressed as stimulation indices,SI (where SI¼mean proliferation of stimulated cultures/meanproliferation of unstimulated cultures). For cytokine analyses,whole blood (collected as described above) was diluted 1:8 inRPMI supplemented with 3% antibiotic/antimycotic solutionin a 48-well flat-bottomed culture plate with a final volume of1.0 ml per well. Cells were stimulated by the addition of 25lg�ml�1 of recombinant APR-1. After 48 h of incubation at 378C, 700 ll of supernatant was removed from each well andstored at�20 8C until required for the cytokine assay. IL-4, IL-10, and IFN-c were measured using a capture ELISA assay fordogs (R & D Systems, Minneapolis, Minnesota, United States)following the manufacturer’s instructions. Biotin-labeleddetection antibodies were used (100 ng�ml�1), revealed withstreptavidin-HRP (Amersham Biosciences), and plates weredeveloped with OPD (O-Phenylenediamine) substrate system(Sigma-Aldrich).

Hb MeasurementsTo determine Hb concentrations of experimental dogs, 1–2

ml of blood were collected in EDTA and analyzed using aQBC VetAutoread Hematology System and VETTEST Soft-ware (IDEXX Laboratories, Westbrook, Maine, United States).

Hookworm Infections and Parasite RecoveryTwo weeks after the final immunization, dogs were

anaesthetized using a combination of ketamine and xylazine(20 mg�kg�1 and 10 mg�kg�1 respectively) and infected via thefootpad with 500 A. caninum L3 as described elsewhere [22].Quantitative hookworm egg counts (McMaster technique)were obtained for each dog 3 d per wk from days 12–26postinfection. Four weeks postinfection, the dogs were killedby intravenous injection of barbiturate, and adult hookwormswere recovered and counted from the small and largeintestines at necropsy [24]. The sex of each adult worm wasdetermined as described elsewhere [8]. Approximately 1–2 cmlengths of small intestine were removed and stored informalin for future histopathologic analysis.

Statistical MethodsIn most cases, the small size of the samples did not enable

us to determine if values were normally distributed, so thefollowing non-parametric tests were used: Mann-Whitney Uwas used to test whether two independent samples (groups)came from the same population, and the Kruskal Wallis H testwas used to determine if several independent samples camefrom the same population. Normally distributed variableswere tested in the following manner: The independent-samples t-test procedure was used to compare the means fortwo groups, and an analysis of variance was used to test thehypothesis that several means are equal, followed by a Dunnetpost hoc multiple comparison t-test to compare the vaccinetreatment groups against the control group. Differences wereconsidered statistically significant if the calculated p-valuewas equal to or less than 0.1 (two-sided). The percentagereduction or increase in adult hookworm burden in the

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vaccinated group was expressed relative to the control groupas described elsewhere [24].

ImmunohistochemistryAdult hookworms were recovered at necropsy from vacci-

nated dogs and control dogs, washed briefly, then fixed andsectioned as previously described [22]. To observe whether IgGfrom vaccinated but not control dogs, bound to APR-1 liningthe intestinal microvillar surface of worms in situ, sectionswere probed with Cy3-conjugated rabbit anti-dog IgG (JacksonImmunoresearch, West Grove, Pennsylvania, United States) ata dilution of 1:500 as described elsewhere [27]. Sections werevisualized using a Leica IM 100 inverted fluorescence micro-scope (Leica Microsystems, Wetzlar, Germany).

Effect of Anti–Ac-APR-1 IgG on Proteolytic ActivityCanine IgG was purified from sera of vaccinated dogs using

protein A-agarose (Amersham Biosciences) as previouslydescribed [23]. Purified IgG (0.2 lg) was incubated with 1.0lg of recombinant Ac-APR-1 for 45 mins prior to assessingcatalytic activity of APR-1 against the fluorogenic substrate o-aminobenzoyl-IEF-nFRL-NH2 as described previously [23].The aspartic protease inhibitor, pepstatin A, was included ata final concentration of 1.0 lM as a positive control forenzymatic inhibition. Data was recorded from triplicateexperiments and presented as relative fluorescence unitsusing a TD700 fluorometer (Turner Designs, Sunnyvale,California, United States).

Results

Secretion of Catalytically Active Ac-APR-1 by P. pastorisYeast secreted the APR-1 zymogen into culture medium at

an approximate concentration of 1.0 mg�l�1 (Figure 1A). In theabsence of co-expression with the PDI chaperone, the amountof APR-1 secreted by P. pastoris was approximately half thatobtained here (not shown). Ac-APR-1 has one potentialglycosylation site at Asn-29 of the zymogen (after removal ofthe signal peptide), and treatment with PNGase F decreasedthe size of the recombinant protein by the expected size (2–3kDa; not shown). The activated recombinant protease readilydigested canine Hb at acidic pH (Figure 1B), confirming thatAc-APR-1 expressed in yeast is catalytically active and digestedHb with similar efficiency to recombinant Ac-APR-1 producedin baculovirus (data not shown).

Recombinant Ac-APR-1 Is Immunogenic in DogsAS03 was used as an adjuvant based on its ability to induce a

higher IgG1 response and greater reduction in hookworm eggcounts when used to vaccinate dogs in a head-to-headcomparison of a cysteine hemoglobinase formulated withfour different adjuvants [22]. Dogs immunized with recombi-nant Ac-APR-1 formulated with AS03 produced IgG1 and IgG2antibody responses as measured by ELISA using the recombi-nant protein (Figure 2). IgE titers were low (,1:1,500) and

Figure 1. P. pastoris Secrete Ac-APR-1 Zymogen that Autoactivates at

Low pH and Degrades Canine Hb

SDS-PAGE gel stained with Coomassie Brilliant Blue showing purificationof recombinant APR-1 zymogen from P. pastoris culture supernatant.(A) Lane 1, molecular weight markers; lane 2, concentrated culturesupernatant; lane 3, flow-through from a nickel-IDA column; lane 4, 5mM imidazole wash; lane 5, 20 mM imidazole column eluate; lane 6, 60mM imidazole eluate; and lane 7, 1 M imidazole eluate. Purifiedrecombinant APR-1 zymogen was activated by buffer exchange into 0.1M sodium formate/0.1 M NaCl (pH 3.6).(B) Lane 1, molecular weight markers; lane 2, 5.0 lg of canine Hb (pH3.6); and lane 3, 5.0 lg of canine Hb (pH 3.6) incubated with 0.2 lg ofrecombinant APR-1.DOI: 10.1371/journal.pmed.0020295.g001

Figure 2. The Geometric Mean Titers of the IgG1 and IgG2 Antibody

Responses of Dogs Vaccinated with Recombinant Ac-APR-1 Formulated

with AS03 or AS03 Alone

LC, day on which dogs were challenged with hookworm L3; N, day ofnecropsy; V1, V2, and V3, days on which animals were vaccinated.DOI: 10.1371/journal.pmed.0020295.g002

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were not sustained past challenge. We did not adsorb IgG fromserum before measuring IgE in this study; however, in previoustrials IgG was removed and we did not see a difference inantigen-specific IgE titers. For vaccinated dogs, maximumIgG2 titers of 1:121,500 were attained by all five dogs after thesecond vaccination. High titers persisted through challengeand decreased to 1:26,098 by necropsy. IgG1 titers peaked at1:13,500 after the third vaccination in all four dogs anddropped to 1:3,600 by necropsy. Dogs immunized withadjuvant alone did not generate detectable immune responsesgreater than 1:500, even after larval challenge.

Dogs rapidly acquire resistance to hookworm with matur-ity. A single dog was therefore removed from the controlgroup (for all analyses) because its weight was greater than theacceptable range at all time points after the first vaccination(mean plus or minus three standard errors).

Vaccination Induces Antigen-Specific Cell Proliferationand Cytokine Production

Vaccination with APR-1 induced a high level of lympho-cyte/leukocyte proliferation compared with control dogswhen cells were stimulated with APR-1 (p , 0.01, t-test). Cellsfrom both vaccinated and control dogs proliferated equallywhen stimulated with mitogen (Figure 3A and 3B). Nosignificant proliferation to APR-1 was observed before theimmunization process. Immunization with APR-1 elicitedantigen-specific production of IFN-c (p ¼ 0.03, t-test) (Figure3C). In contrast, we did not detect significant production of

IL-4 or IL-10 after stimulation with APR-1 in eithervaccinated or control groups (not shown).

Vaccination with Ac-APR-1 Decreases Fecundity of FemaleHookwormsDogs develop age- and exposure-related immunity to A.

caninum [5], so we therefore observed egg counts fromvaccinated animals up to 26 d postchallenge, after which weoften observe a significant decrease in egg counts in somedogs. Because of daily variation in egg counts from infecteddogs (A. Loukas. S. Mendez, and P. Hotez, unpublished data),we analyzed the data in two ways. Firstly, the median eggcounts for days 21, 23, and 26 postinfection were used tocompare worm fecundity between vaccinated and controlgroups. A 70% decrease in median egg counts was observedin dogs vaccinated with Ac-APR-1 (2,650 eggs per gram offeces [epg]) compared with dogs that were vaccinated withadjuvant alone (8,725 epg) when median egg counts werecalculated for the three time points measured after larvalchallenge (Figure 4A). We then compared geometric meanvalues of egg counts between the two groups (Figure 4B), andshowed that mean egg counts of the vaccinated animalsremained lower than the control animals as worms becamefecund by day 21, implying that fecundity of female wormsdiminished significantly as they began to feed on bloodcontaining anti–APR-1 antibodies. By day 26 postchallenge,there was an 85% reduction in mean egg counts between thetwo groups. For statistical analyses, we transformed egg

Figure 3. Canine Cellular Immune Response to Vaccination with Recombinant Ac-APR-1

Cell proliferation of whole blood cells from vaccinated (APR-1) and control dogs (AS03) when stimulated with concanavalin A (A) or recombinant Ac-APR-1 (B) before (day 0) and after the final immunization (day 51). The p-value comparing the mean differences between the vaccinated group andcontrols is denoted. Detection of secreted IFN-c in whole blood cultures taken from vaccinated and control dogs before and after immunization (C).Mean cytokine concentrations are indicated in pg�ml�1 with standard error bars. Statistically significant differences are indicated above the bars by p-values. APR, stimulated with recombinant APR-1; NS, non-stimulated cultures.DOI: 10.1371/journal.pmed.0020295.g003

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counts into log values and ran the test in two ways: (1)comparing the log transformed epgs in the last three eggcounts by analysis of variance (Kruskall-Wallis) revealed nosignificant differences among the groups for the last three eggcounts when each time point was considered individually; and(2) comparing pooled data from the last three egg counts

using a Mann-Whitney test (APR-1 versus control), revealed astatistically significant difference (p ¼ 0.018).

Vaccination with Ac-APR-1 Significantly Reduces AdultHookworm BurdensA statistically significant difference at the p � 0.1 level (p¼

0.095; Mann-Whitney U test) was detected for a one-sided testbetween median adult worm burdens recovered fromvaccinated dogs (182) compared with control dogs (270) butnot for a two-sided test (p ¼ 0.190) (Figure 5). Percentagereduction of the median worm counts was 33% when datafrom both sexes of worms were combined, 30% for maleworms (p¼ 0.111 [2-sided] or p¼ 0.056 [1-sided]) and 40% forfemale worms (p ¼ 0.1905 [2-sided] or p ¼ 0.0952 [1-sided]),again supporting the enhanced effect of the vaccine onfemale worms given their increased nutritional requirementsfor egg production.

Vaccination with APR-1 Protects against AnemiaHb levels in four of the five dogs that were vaccinated with

APR-1 were significantly elevated when compared withcontrol dogs (adjuvant alone) after challenge infection(Figure 6). The median Hb concentration of vaccinated dogsfor the last two time points (0 and 7 d prior to necropsy) was12.45 g�dl�1 compared with 9.5 g�dl�1 for the control dogs thatwere immunized with adjuvant alone (p ¼ 0.049; Mann-Whitney U test). A decline in Hb levels was seen in all of thecontrol dogs after challenge infection; the decline wasmarked in three of the four dogs. Four of the five dogs thatwere vaccinated with APR-1 did not show a similar decline,and had Hb levels within (or very close to) the normal clinicalrange of 12–14 g�dl�1. One dog (C5) from the vaccinatedgroup did become anemic (Hb concentration was 9.6 g�dl�1),and this animal had more female worms (120 compared witha mean of 88 female worms for the group) and more maleworms (87 compared with a mean of 80 male worms for thegroup). However, using both Spearman and Pearson tests, wedid not detect a significant correlation between wormburdens (for either or both sexes) and Hb status of thevaccinated dogs.

Anti–APR-1 Antibodies Are Ingested by and Bind to theIntestine of Feeding HookwormsThe site of anatomical expression of Ac-APR-1 within adult

hookworms has been previously reported by us to be the

Figure 4. Vaccination with APR-1 Reduces Fecal Egg Counts of Dogs

after Challenge Infection with Hookworms

Statistically significant reduction (p¼ 0.018) in median fecal egg countssampled on days 21, 23, and 26 of dogs vaccinated with APR-1 comparedto dogs that received adjuvant alone.(A). Geometric mean values of fecal egg counts from vaccinated andcontrol dogs between challenge infection and necropsy.(B). Error bars refer to the standard error of the mean.DOI: 10.1371/journal.pmed.0020295.g004

Figure 5. Vaccination with APR-1 Reduces Adult Worm Burdens of Dogs after Challenge Infection with Hookworms

Statistically significant reduction at the p, 0.1 level (p¼0.065) in median adult worm (both sexes) burdens of dogs vaccinated with APR-1 compared todogs that received adjuvant alone (A). Reductions are also shown when only male (B) (p ¼ 0.111) and only female (C) (p ¼ 0.1905) worms wereconsidered; however, statistically significant reductions were not achieved for single sex analyses. Bars represent the median value for each group.DOI: 10.1371/journal.pmed.0020295.g005

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microvillar surface of the gut [21,23]. To determine whethervaccination of dogs induced circulating antibodies thatbound to the intestinal lumen during infection, parasiteswere removed from vaccinated dogs, fixed, sectioned, andprobed with anti-dog IgG conjugated to Cy3. Wormsrecovered from dogs immunized with Ac-APR-1 but not fromdogs immunized with adjuvant alone reacted with Cy3-conjugated anti-dog IgG (Figure 7), indicating that anti–

APR-1 antibodies were ingested with the blood-meal of theworm and subsequently bound specifically to the intestine ofthe parasite in situ.

IgG from Dogs Vaccinated with Ac-APR-1 NeutralizesProteolytic Activity In VitroPurified IgG from dogs that were immunized with Ac-APR-1

reduced the catalytic activity of the enzyme by 71%,compared with just 6% reduction when an equivalentamount of IgG from dogs immunized with adjuvant alonewas assessed (Table 1). The aspartic protease inhibitor,pepstatin A, inhibits catalytic activity of APR-1 [23] and wastherefore used as a positive control to obtain 100% inhibitionfor comparative purposes.

Discussion

Here we describe protective vaccination of dogs with arecombinant aspartic hemoglobinase, a pivotal enzyme in theinitiation of Hb digestion in the gut of canine hookworms[12,21]. We show that APR-1 provides the best efficacy thusfar reported for a recombinant vaccine aimed at reducinghookworm egg counts, intestinal worm burdens, and hook-worm-induced blood loss.The vaccine efficacy of recombinant Ac-APR-1 expressed in

baculovirus-infected insect cells was described earlier by us

Figure 6. Vaccination of Dogs with APR-1 Reduces Blood Loss and

Protects against Anemia

Hb concentrations of vaccinated dogs were significantly (p ¼ 0.049)greater than those of control dogs when blood was drawn after larvalchallenge (0 and 7 d before necropsy [post]) but not when blood wasdrawn 5 d before larval challenge (pre).DOI: 10.1371/journal.pmed.0020295.g006

Figure 7. Antibodies Bind In Situ to the Intestines of Hookworms that Feed on Vaccinated Dogs

Detection of antibodies that bound to the gut of worms recovered from vaccinated dogs (A and B) but not control dogs (C and D) byimmunofluorescence. Binding was detected using Cy3-conjugated rabbit anti-dog IgG, allowing only detection of antibodies that had bound in situwhile parasites were feeding on blood from vaccinated or control dogs. ic. intestinal contents; in, hookworm intestine; mv, intestinal microvillar surface;ro, reproductive organs.DOI: 10.1371/journal.pmed.0020295.g007

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[24]; however, this initial vaccine trial was hampered bylimited availability of the recombinant protein: Suboptimaldoses were used and antibody responses (titers ,10,000) werefirst observed just 1 wk following the third (and final)immunization, and only in some dogs. Despite the weakantibody responses, a statistically significant reduction inmean (18%, p , 0.05) and median (23%) hookworm burdenswere observed. In addition there was a shift of adulthookworms from the small intestine to the colon [24].However, no reduction in the mean fecal egg counts wereobserved, and hematologic parameters were not assessed. Theimproved immunogenicity of APR-1 observed in this studymight also be attributed to use of the adjuvant AS03compared with alhydrogel in the previous study. We haveshown in a head-to-head comparison of a hookworm cysteinehemoglobinase formulated with different adjuvants (includ-ing alhydrogel) that AS03-formulated protein generatedhigher antibody titers and afforded greater protection tovaccinated dogs [22]. In this study, we show that yeast-derivedAPR-1 provides the best efficacy thus far reported for arecombinant vaccine aimed at reducing hookworm load andpotential transmission. Moreover, we show that vaccinationprotects against the pathology associated with worm-inducedblood loss, or hookworm disease.

Hookworms bury their anterior ends into the intestinalmucosa to feed, secreting anticoagulants to promote bloodflow and stop clot formation at the site of attachment(reviewed in [28]). Numerous anticoagulant peptides havebeen reported from hookworms [29–31], and their combinedactivities result in ‘‘leakage’’ of blood around the attachmentsite and into the host intestine [32]. It is not known whetherthe majority of blood loss during a hookworm infection is dueto leakage around the feeding site or from ingested bloodthat enters the parasite’s alimentary canal for nutritionalpurposes. To address this, attempts have been made tomeasure blood lost from the anus of A. caninum (i.e., bloodthat has passed through the parasite’s alimentary canal);varying calculations have been proposed ranging from 0.14–0.8 ml blood expelled over 24 h per adult worm (reviewed in[32]). Whatever the true figure is, significant blood loss occursvia this route, supporting the hypothesis that vaccination withAPR-1 damages that parasite’s intestine and results indecreased blood intake (and blood loss) by feeding worms.

The immunological parameters required for vaccine-induced protection against hookworm infection were, untilrecently, poorly defined. Protection against A. caninum byvaccination of dogs with radiation-attenuated L3 wasreported many years ago [5]; however, it was not untilrecently that murine [8,33] and canine [34] studies revealedthe protective mechanisms of the irradiated larval vaccine ata cellular level. These studies suggested that a T-helper type-2response is induced by vaccination with irradiated L3;however the authors did not prove that a T-helper type-1response abrogates protection. In our study reported here,dogs vaccinated with APR-1 generated strong memoryresponses to the recombinant antigen and did not secreteTh-2 cytokines but instead secreted IFN-c in response tostimulation with recombinant APR-1. Moreover, the domi-nant antibody isotype induced by vaccination was IgG2,suggesting that a Th-1-like response was generated. Unlikethe clear association between IgG2 and type I cytokines suchas IFN-c in mice and humans, little is known about thisassociation in dogs. Experimental evidence using the caninemodel suggests that immune responses (Th1 versus Th2) are,however, linked to isotype production. For example, animalsinfected with and protected against visceral leishmaniasis(Th1 response) or Salmonella (also a Th1 response) mount ahigher IgG2 than IgG1 response [35,36]. Our data [34] showthat dogs immunized with irradiated hookworm larvaedemonstrated a stronger production of IgG1 (also supportedby [37]) which accompanied IL-4 production, implying a Th2cytokine response in dogs is accompanied by the sameimmunoglobulin isotypes seen in humans and mice. Based onthe current data, we cannot conclude that a Th-1 response toAPR-1 is required to obtain protection; however, it does notinhibit the development of a protective memory response. Itshould also be considered that successful immunity to thedifferent developmental stages of hookworms might requirevery different immune response phenotypes, not unlike thoseseen in schistosomiasis [38]. Further studies will explore theeffects of vaccination with APR-1 formulated with differentadjuvants and co-factors (e.g., cytokines) that will promote aTh2 response.Hematophagous helminths require blood as a source of

nutrients to mature and reproduce. Female schistosomesingest 13 times as many erythrocytes and ingest them aboutnine times faster than male worms [39]. Moreover, mRNAsencoding Hb-degrading proteases of schistosomes are over-expressed in female worms [40]. Although similar studies haveyet to be performed for hookworms, female hookworms arebigger than males and lay up to 10,000 eggs per day, implyingthat they have a greater metabolism and therefore greaterdemand for erythrocytes. Ac-APR-1 degrades Hb in the gutlumen of the worm, and it is therefore not surprising thatinterruption of the function of APR-1 via the action ofneutralizing antibodies has a deleterious effect on theestablishment of worms, particularly females and theirsubsequent egg production. We observed a similar (althoughnot as pronounced) phenomenon when dogs were vaccinatedwith the cysteine hemoglobinase, Ac-CP-2, followed bychallenge infection with A. caninum L3 [22]. Vaccination withCP-2, however, did not result in reduced adult worm burdensor reduced blood loss, essential attributes of an efficacioushookworm vaccine.Vaccination of livestock and laboratory animals with

Table 1. Reduction in Cleavage of the Fluorogenic Substrate o-Aminobenzoyl-IEF-nFRL-NH2 When 1.0 lg of Recombinant Ac-APR-1 Was Pre-Incubated with 0.2 lg of IgG Purified from Sera ofDogs Vaccinated with APR-1/AS03 or AS03 Alone (Control)

Protease and

Treatment

Corrected Relative

Fluorescence Units

Mean Percent

Reduction in Cleavage

of IEF-nFRL-NH2

APR-1 þ buffer 362 6 13 0

APR-1 þ a-APR-1 IgG 104 6 24 71

APR-1 þ control IgG 340 6 41 6

APR-1 þ pepstatin 0 100

Percent reductions caused by incubation of APR-1 with IgGs were determined using 1.0 lM pepstatin as positive

(100% reduction) control. Baseline was set at zero using the relative fluorescence of the positive control.

DOI: 10.1371/journal.pmed.0020295.t001

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aspartic proteases of other nematodes, as well as trematodehelminths, has resulted in antifecundity/antiembryonationeffects. Immunization of sheep with the intestinal brushborder complex, H-gal-GP, confers high levels of protection(both antiparasite and antifecundity) against H. contortus andat least three different protease activities, including asparticproteases, have been detected in this extract [16,41].Immunization of sheep with aspartic protease-enrichedfractions of H. contortus membranes resulted in 36%reduction in adult worms and 48% reduction in fecal eggoutput [17]. Vaccination of sheep with denatured H. contortusproteases or recombinant proteases expressed in bacteria,however, did not confer protection, suggesting that con-formational epitopes are important in protection [17].Vaccination of mice with recombinant aspartic protease ofthe human blood fluke, Schistosoma mansoni, resulted in 21%–38% reduction in adult parasites after challenge withinfective cercariae; however a reduction in eggs depositedin the liver (the cause of most pathology in schistosomiasis)was not detected [42]. Protective efficacy of aspartic proteaseshas been observed against fungal pathogens as well. Vacci-nation of mice with secreted aspartic proteases of Candidaalbicans, known virulence factors in candidiasis, protectedanimals against a lethal challenge infection and inhibitedcolonization of fungi in the kidneys [43]. Moreover, passivetransfer of serum from vaccinated animals conferred pro-tection, pointing towards an antibody-mediated protectivemechanism.

Almost all of the pathology and morbidity of humanhookworm infection results from intestinal blood loss causedby large numbers of adult hookworms. Depending on hostiron and protein stores, a range of hookworm intensities,equivalent to burdens of 40 to 160 worms, is associated withHb levels below 11 g�dl�1, the World Health Organizationthreshold for anemia. In Tanzania, Nepal, and Vietnam wherehost iron stores are generally depleted, there is a directcorrelation between the number of adult hookworms in theintestine and host blood loss [1,44]. Therefore the optimalhookworm vaccine will be one that either prevents L3 fromdeveloping into adult blood-feeding hookworms, or one thatblocks the establishment, survival, and fecundity of the adultparasites in the intestine [3,45]. Achieving both goals willlikely require a vaccine cocktail comprised of an L3 antigen,such as ASP-2 now under clinical development [46,47], and anadult gut protease, such as APR-1.

An effective hookworm vaccine need not attain 100%efficacy. Unlike many unicellular organisms that reproduceasexually within the host, nematodes need to sexuallyreproduce. Therefore, small numbers of adult worms willgenerate fewer eggs to contaminate the environment, andsubsequently reduce transmission. More importantly, becausehookworms are blood feeders, a partial reduction in adultworm burden equates to a decrease in pathology, notablyiron-deficiency anemia [44]. Mathematical modeling ofschistosomiasis in China showed that elimination of theparasite could be attained using an antifecundity vaccine thattargets egg output with 75% efficacy [48], and it is likely that asimilar scenario applies to long-term elimination of soil-transmitted helminths such as hookworms. An orthologue ofAc-APR-1 has been reported from the major human hook-worm, N. americanus [23]. Na-APR-1 is structurally andantigenically very similar to Ac-APR-1 and also functions as

a hemoglobinase [23]. For this reason, we believe that APR-1is now the major vaccine antigen from the adult stage of theparasite, and as such, Na-APR-1 should undergo processdevelopment and enter into Phase I clinical trials as a vaccinefor human hookworm infection. This vaccine strategy is nowbeing implemented for a larval hookworm antigen, withPhase 1 human trials using ASP-2 formulated with Alhydrogelalready underway [49]. Based on the data reported here, APR-1 may also be selected for downstream process development,manufactured under good clinical manufacturing processes,and tested in the clinic.

Supporting Information

Accession NumbersThe GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession num-bers for the gene products mentioned in this paper are Ac-APR-1(U34888) and Na-APR-1 (AJ245459).

Acknowledgments

This work was supported by a grant from the Bill and Melinda GatesFoundation awarded to the Sabin Vaccine Institute. AL is supportedby a Career Development Award from the National Health andMedical Research Council of Australia. JMB is supported by anInternational Research Scientist Development Award (1K01TW00009) from the Fogarty Center. For technical assistance and/orhelpful advice, we thank Yan Wang, Lilian Bueno, Azra Dobardzic,Reshad Dobardzic, Andre Samuel, Sonia Ahn, Aaron Witherspoon,Clay Winters, Estelle Schoch, John Hawdon, and Philip Russell. Wewould like to acknowledge Joe Cohen and Sylvie Cayphas ofGlaxoSmithKline Biologicals (Rixensart, Belgium) for providingAS03 and technical assistance with formulation.

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Patient Summary

Background Hookworms are parasites of the intestines. They can infectmany animals, including dogs, cats, and people. Worldwide, about oneperson in five has a hookworm infection. Most of these one billionpeople live in tropical countries. Hookworm is not spread from person toperson, because at one stage of its lifecycle, the parasite needs to be inthe soil. In areas where hookworm is common, people who have contactwith soil that contains human feces are at high risk of infection; becausechildren play on soil and often go barefoot, they have the greatest risk.Infection leads to blood loss and a decrease in the amount of iron, andthis causes anemia (i.e., because of a lack of iron, the blood cannot carryoxygen efficiently). There are effective drugs to treat the infection, butthey do not prevent the patient from becoming re-infected. Making avaccine against hookworm is therefore a priority. Some vaccines for usein animals have already been developed, but their effectiveness is limitedto one stage of the hookworm’s lifecycle. The aim is to find a vaccine thatworks against more than one of the stages that the parasite passesthrough in its lifecycle.

What Did the Researchers Do and Find? The researchers focused ontwo enzymes the parasite needs in order to live. Building on earlierresearch and using a species of hookworm that affects dogs, theresearchers aimed to make these enzymes the ‘‘target’’ of a vaccine.They first vaccinated dogs, then infected them with hookworm. Thesedogs had fewer parasites than dogs that had not been vaccinated. Mostimportantly, vaccinated dogs were protected against blood loss, andmost did not develop anemia. Laboratory tests confirmed that the targetenzymes had been damaged.

What Do These Findings Mean? This is the best result so far for ahookworm vaccine used in dogs. The authors believe that, as well asreducing parasite numbers, the vaccine reduces the ability of theparasite to take in blood, which would explain the reduction in anemia.The researchers have called for trials to begin with a vaccine targetedagainst similar enzymes in the species of hookworm that mostcommonly affects humans.

Where Can I Get More Information Online? The US Centers for DiseaseControl have a fact sheet on hookworm:http://www.cdc.gov/ncidod/dpd/parasites/hookworm/factsht_hookworm.htm.The Sabin Vaccine Institute has an overview of the Human HookwormVaccine Initiative:http://www.sabin.org/hookworm.htm.

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Vaccination with Hookworm Hemoglobinase