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The Role of Acanthamoeba culbertsoni SerineProteases in Abating Microglial-Like CellCytokines and ChemokinesJenica HarrisonVirginia Commonwealth University

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© Jenica Ledah Harrison 2009

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THE ROLE OF ACANTHAMOEBA CULBERTSONI SERINE PROTEASES IN

ABATING MICROGLIAL-LIKE CELL CYTOKINES AND CHEMOKINES

A dissertation submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy at Virginia Commonwealth University.

by

JENICA LEDAH HARRISON B.S., Western Carolina University, 2000

Director: GUY A. CABRAL, PHD PROFESSOR, DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY

Virginia Commonwealth University

Richmond, Virginia

April, 2009

ii

Acknowledgements

I would like to thank the following individuals for their support during my

graduate career. Without your help I would not have been able to complete this program.

A special thanks to my Lord and Savior Jesus Christ who has walked with me

every step of my journey.

I am grateful to my advisor, Dr. Guy A. Cabral for allowing me to pursue my

research in his laboratory. Over the years, he has taught me the importance of “allowing

the data to speak” to the direction of the research. Most importantly, he has also taught

me how to be an upstanding research scientist. I would also like to thank Dr. Francine

Marciano-Cabral for all of her guidance and assistance with my research project.

I would like to thank my other graduate committee members; Dr. Kathleen

McCoy, Dr. Michael McVoy, and Dr. Sandra Welch for their advice and interest in my

research. I would also like to thank Dr. Joy Ware and Dr. Dan Huang for teaching me the

two-dimensional (iso-dalt) gel electrophoresis technique.

I am thankful to the National Institutes of Health for their financial support while

pursuing my graduate degree.

I would also like to thank Daniel Southern and Dr. Christine Stevens of the

Clinical Laboratory Sciences Program at Western Carolina University for introducing me

to clinical Microbiology and Immunology and encouraging my interest in research.

While pursuing this graduate degree I have had the honor of working with several

people in the Cabral and Marciano-Cabral laboratories that I would like to thank: Dr.

iii

Tammy Ferguson, Dr. Angela Fritzinger, Dr. Andrea Staab, Dr. Rebecca MacLean, Dr.

Erinn Raborn, Dr. Gabriela de Almeida Ferreira, Dr. Bruno da Rocha Azevedo, Dr.

Daniel Fraga, Dr. LaToya Griffin-Thomas, Christina Hartman, Melissa Jamerson, Alex

Mensah, and Olga Tavares-Sanchez.

Last, but definitely not least, I could like to thank my family; my mother Jeanette

Harrison, my father Jerry Harrison (deceased), my grandmother Viola M. Harrison

(deceased), Joyce Jefferson, Jasmin Bhalodia, Ronald Saunders, William Day, and

Deborah Day. Thanks for your prayers, advice, guidance, and love. You all have been

an integral part of my life and for that I am forever grateful.

iv

Table of Contents

Page

Acknowledgements ............................................................................................................. ii

List of Tables ..................................................................................................................... vi

List of Figures .................................................................................................................. vii

Abstract ...............................................................................................................................x

Introduction..........................................................................................................................1

Materials and Methods .......................................................................................................17

Reagents ......................................................................................................17

Amoebae ......................................................................................................17

Microglia .....................................................................................................18

Conditioned Medium ...................................................................................18

Two-Dimensional (Iso-Dalt) Gel Electrophoresis (2D-PAGE) ..................20

Gel Zymography ..........................................................................................21

Light Microscopy ........................................................................................22

Multiprobe Ribonuclease Protection Assay (RPA) .....................................22

Cytokine/Chemokine Protein Microarray ...................................................23

Enzyme-Linked Immunosorbent Assay (ELISA) .......................................24

Data Analysis ..............................................................................................25

Results...............................................................................................................................26

v

Discussion…………………………………………………………………....................57

Literature Cited……………………………………………………………....................69

vi

List of Tables

Page

Table 1: Genotypes of Acanthamoeba Associated with Disease in Humans...................4

vii

List of Figures

Page

Figure 1: Life Cycle Forms of Acanthamoeba ....................................................................2

Figure 2: Characteristic Granuloma Associated with Granulomatous Amoebic

Encephalitis (GAE) ..............................................................................................8

Figure 3: Visualization of Complete Neurobasal™-A Medium Proteins ..........................27

Figure 4: Visualization of A. culbertsoni-Conditioned Medium Proteins .........................28

Figure 5: A Comparison of the Effect of Propagation Medium on A. culbertsoni

Protease Activity ................................................................................................30

Figure 6: Titration of Protease Activity in 109

A. culbertsoni-Conditioned Medium ........31

Figure 7: Acanthamoeba culbertsoni Secrete Proteases that Are Inhibited by the

Serine Protease Inhibitor Phenylmethylsulphonylfluoride (PMSF) ...................33

Figure 8: Acanthamoeba culbertsoni Secrete Proteases that Are Not Inhibited by the

Cysteine Protease Inhibitor trans-Epoxysuccinyl-L-leucylamido

(4-guanidino)butane (E-64) ................................................................................34

Figure 9: A Comparison of the Proteolytic Activity in Three Different Types

of A. culbertsoni-Conditioned Media .................................................................35

Figure 10: Acanthamoeba culbertsoni-Conditioned Medium Proteolytic Activity

Increases in a Time-Related Manner and Is Augmented During Co-Culture

With BV-2 Cells ...............................................................................................37

viii

Figure 11: Acanthamoeba culbertsoni Induces Chemokine mRNA Expression by

BV-2 Cells ........................................................................................................38

Figure 12: Cytokine and Chemokine Protein Expression by BV-2 Cells following

9 h Co-culture with A. culbertsoni. ...................................................................40


24 h Co-culture with A. culbertsoni ..................................................................41

Figure 14: Light Microscopy Images of BV-2 cells, A. culbertsoni, and BV-2 cells

Plus A. culbertsoni Co-cultures ........................................................................43

Figure 15: Conditioned Medium from A. culbertsoni Degrades Constitutively

Expressed Cytokines and Chemokines Elicited by BV-2 Cells........................45

Figure 16: Effect of 109 A. culbertsoni-Conditioned Medium (ACM-HI) on BV-2

Cell Morphology ...............................................................................................47

Figure 17: Conditioned Medium from A. culbertsoni Degrades Cytokines and

Chemokines Elicited from Lipopolysaccharide (LPS) Stimulated BV-2

Cells ..................................................................................................................48

Figure 18: Acanthamoeba culbertsoni Proteases Degrade A. culbertsoni Induced

Microglial Chemokines from BV-2 cells ..........................................................50

Figure 19: Acanthamoeba culbertsoni Protease Activity Persists in Co-culture

Supernatants Following Incubation for 72 h .....................................................52

Figure 20: Acanthamoeba culbertsoni Serine Proteases Degrade TNF-α .........................53

Figure 21: Acanthamoeba culbertsoni Serine Proteases Degrade MIP-1α ........................54

ix

Figure 22: Acanthamoeba culbertsoni Serine Proteases Degrade MIP-2 ..........................55

x

Abstract

THE ROLE OF ACANTHAMOEBA CULBERTSONI SERINE PROTEASES IN

ABATING MICROGLIAL-LIKE CELL CYTOKINES AND CHEMOKINES

By Jenica L. Harrison, Ph.D.

A dissertation submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy at Virginia Commonwealth University.

Virginia Commonwealth University, 2009

Major Director: Guy A. Cabral, Ph.D.

Professor, Department of Microbiology and Immunology

Acanthamoeba culbertsoni is an opportunistic free-living amoeba that is causative

of granulomatous amoebic encephalitis (GAE), a chronic and often fatal central nervous

system (CNS) disease that is most prevalent in immune compromised individuals. One

hallmark of this disease is the formation of granulomas within the CNS, which are

commonly absent in immune compromised individuals. Granulomas are usually

composed of amoebae, microglia (CNS macrophages), macrophages, T cells, B cells, and

neutrophils. Previous studies have demonstrated that microglia respond to

Acanthamoeba by producing pro-inflammatory cytokines such as tumor necrosis factor

xi

alpha (TNF)-α, interleukin (IL)-1α, and IL-1β. In addition, activated microglia and

macrophages have been demonstrated to be cytolytic (i.e., amoebicidal) to

Acanthamoeba. Furthermore, previous studies also indicated that Acanthamoeba secrete

a myriad of factors including proteases. The role of these proteases during GAE has not

been fully elucidated; however, it is thought that these factors may aid in the chronic

persistence of Acanthamoeba within the CNS by modulating the host immune response.

Using two-dimensional (iso-dalt) gel electrophoresis, we demonstrated that A.

culbertsoni secrete factors that degrade culture medium proteins. Initial gelatin

zymography studies demonstrated that propagation of A. culbertsoni in medium with high

iron content leads to augmentation of protease activity. Gelatin zymography in concert

with protease inhibitors demonstrated that A. culbertsoni secrete proteases predominantly

of the serine protease class. Using an in vitro co-culture model, we demonstrated that

co-culture of A. culbertsoni with mouse microglial-like cells (BV-2 cells) results in the

augmentation of A. culbertsoni serine protease activity and stimulation of pro-

inflammatory cytokine and chemokine protein expression by microglial-like cells.

However, the A. culbertsoni-elicited proteases were shown to degrade microglial-like cell

elicited cytokines and chemokines. Collectively, our results suggest that A. culbertsoni-

secreted serine proteases may play a role in A. culbertsoni CNS immune evasion by

increasing A. culbertsoni CNS dissemination via the diminution of granuloma formation

and by dampening microglial-dependent cytokine response.

1

Introduction

Acanthamoeba belong to a group of opportunistic free-living amoebae, including

Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea, which have the

ability to cause disease in humans [Visvesvara et al. 2007]. Acanthamoeba are classified

in the kingdom Protista, phylum Rhizopodia, class Lobosa, order Amoebida, family

Acanthamoebidae, genus Acanthamoeba. Acanthamoeba are found worldwide and have

been isolated from a variety of environmental sources including air, soil, dust, tap water,

freshwater, seawater, swimming pools, air conditioning units, and contaminated contact

lens cases [Marciano-Cabral and Cabral 2003]. Acanthamoeba reproduce by binary

fission and have two life cycle stages; the actively feeding trophozoite and the dormant

cyst (Figure 1). The trophozoite stage represents the infective form of Acanthamoeba.

Morphologically, trophozoites average 12-35 μm in size, have a nucleus with a large

central nucleolus, and express spine-like projections called acanthapodia [Marciano-

Cabral and Cabral 2003; Khan 2006]. In the environment the trophozoite feeds on

bacteria (with a preference for gram-negative bacteria), algae, and yeast [Rodriquez-

Zaragoza et al. 1994; Visvesvara et al. 2007]. A double-walled round cyst, which ranges

in size from 5-20 μm, is formed during unfavorable environmental conditions such as

extreme temperature or pH changes [Marciano-Cabral and Cabral 2003; Khan 2006].

2

Figure 1: Life Cycle Forms of Acanthamoeba. Scanning electron micrographs of

Acanthamoeba (A) trophozoites displaying characteristic acanthapodia (arrows) and (B)

cyst. The scale bars designate 10 µm and 1 µm in A and B, respectively. Courtesy of Dr.

Francine Marciano-Cabral, Ph.D.

A

B

3

Grouping of Acanthamoeba species was originally established using cyst

morphology [Page 1967; Pussard and Pons 1977]. Using this criterion, species of

Acanthamoeba were divided into three groups based on having large cysts (Group I),

wrinkled ectocyts and endocysts with a triangular, polygonal, or oval morphology (Group

II), or smooth ectocytes and round endocyts (Group III). This classification scheme

proved to be challenging as cyst morphology may vary depending on conditions [Sawyer

and Griffin 1975]. Today, Acanthamoeba are grouped based on nuclear 18S ribosomal

rRNA sequence differences. Using this classification scheme, 12 different genotypes

(T1-T12) have been described [Stothard et al. 1998]. Most of the pathogenic

Acanthamoeba species belong to the T4 genotype [Khan 2006]. Table 1 lists the

classification of Acanthamoeba that have been linked to disease in humans.

Of the approximately 20 species of Acanthamoeba that have been identified, only

twelve (A. astronyxis, A. castellanii, A. culbertsoni, A. divionesis, A. griffini, A. hatchetti,

A. healyi, A. lenticulata, A. lugdunensis, A. polyphaga, A. quina, and A. rhysodes) have

been linked to human disease [Marciano-Cabral and Cabral 2003; Visvesvara et al. 2007;

Centers for Disease Control]. Acanthamoeba are associated with an eye infection known

as amoebic keratitis (AK), a chronic and slow progressing central nervous (CNS) system

disease called granulomatous amoebic encephalitis (GAE), and a skin infection called

cutaneous acanthamoebiasis [Marciano-Cabral and Cabral 2003; Khan 2006; Visvesvara

et al. 2007]. It has been reported that approximately 80% of the normal human

population has serum antibodies against Acanthamoeba; however, Acanthamoeba

infections are generally rare and usually are found in individuals who have a

4

Table 1 – Genotypes of Acanthamoeba Associated with Disease

in Humans

Species Sequence Type Morphological

Group A. astronyxis T7 I

A. castellanii T4 II

A. culbertsoni T10 III

A. divionensis T4 II

A. griffini T3 II

A. hatchetti T11 II

A. healyi T12 III

A. lenticulata T5 III

A. lugdunensis T4 II

A. polyphaga T4 II

A. quina unknown II

A. rhysodes T4 II

Adapted from Stothard et al. 1998; Marciano-Cabral and Cabral 2003; Kilic et al. 2004.

5

predisposition for Acanthamoeba infection (i.e., contact lens wearers and immune

compromised individuals; discussed below) [Chappell et al. 2001].

AK is potentially a blinding eye infection and represents the most common

disease caused by Acanthamoeba in humans [Clarke and Niederkorn 2006a].

Acanthamoeba that have been linked to AK include A. castellanii, A. polyphaga, A.

hatchetti, A. culbertsoni, A. rhysodes, A. griffini, A. quina, and A. lugdunensis [Marciano-

Cabral and Cabral 2003]. This disease is most commonly associated with contact lens

wearers and those who have experienced corneal trauma [Niederkorn et al. 1999]. The

prevalence of this disease in contact lens wearers is thought to be associated with

exposure to contaminated water and/or contact lens solutions. Additionally, used contact

lenses are thought to provide unique environments that facilitate Acanthamoeba biofilm

formation on their surface, thus increasing the likelihood of infection [Khan 2006].

Adhesion of Acanthamoeba trophozoites to the corneal epithelium via a trophozoite

expressed mannose binding protein (MBP) is the first step in AK infection [Clarke and

Niederkorn 2006b]. Invasion of the cornea is facilitated by Acanthamoeba factors such

as extracellular matrix specific binding proteins and proteases (discussed below).

Symptoms of AK include photophobia, excessive tearing, and ocular pain [Marciano-

Cabral and Cabral 2003]. One hallmark of this disease is the development of an ocular

ring which results from immune cell infiltration. AK is diagnosed via corneal biopsy and

confocal microscopy [Visvesvara et al. 2007]. This infection is most successfully treated

when caught in its earliest stages. Chemotherapeutics used to treat AK include

6

polyhexamethylene biguanide (PHMB) or chlorhexidine digluconate (CHX) in

combination with propamidine isethionate (Brolene) [Khan 2006; Visvesvara et al. 2007].

The innate immune system is thought to be the primary host resistance element

involved in AK immunity. Diminution of macrophage and neutrophil responses in in

vitro and in vivo models of AK have been demonstrated to result in an increase in disease

severity; thus, these immunocytes are thought to represent the primary cells that act

against corneal invading Acanthamoeba [van Klink et al. 1996; Hurt et al. 2003c; Clarke

and Niederkorn 2006a]. Although the innate immune system is primarily involved in

controlling AK, secretory IgA (sIgA), a component of the adaptive immune system that is

normally found in tears, also is thought to play a role in resistance to AK infection.

Secretory IgA is thought to prevent the adhesion of trophozoites to corneal epithelium, to

augment the action of neutrophils, and to inhibit the deleterious actions of Acanthamoeba

secreted proteases [van Klink et al. 1997; Leher et al. 1998; Hurt et al. 2003b; Said et al.

2004; Clarke and Niederkorn et al. 2006a]

Acanthamoeba also can cause a cutaneous or disseminated infection known as

cutaneous acanthamoebiasis. Cutaneous acanthamoebiasis can occur in immune

competent individuals; however, it is associated more frequently with immune

compromised individuals [Marciano-Cabral and Cabral 2003]. It is thought that

cutaneous Acanthamoeba infections originate from direct contact of amoeba trophozoites

with broken skin or from hematogenous dissemination derived from the respiratory tract

or CNS [Marciano-Cabral and Cabral 2003]. Mortality from this infection has been

reported to be 73% among individuals without Acanthamoeba CNS infection and 100%

7

among those with CNS involvement [Torno et al. 2000]. One symptom of this disease is

the presence of draining non-healing skin ulcers. Skin biopsy in concert with

polymerase chain reaction (PCR), indirect immunofluorescence (IIF), and/or direct

isolation of amoebae from tissue are current diagnostic tools used for diagnosing this

infection. Oral itraconazole, pentamidine, and 5-fluocytosine in concert with topical

chlorhexidine gluconate and ketoconazole cream can be used to treat cutaneous

acanthamoebiasis [Helton et al. 1993; Slater et al. 1994].

GAE is a chronic and often fatal CNS disease. GAE is most prevalent in immune

compromised individuals [Marciano-Cabral and Cabral 2003]. Acanthamoeba that are

known to cause GAE include A. culbertsoni, A. castellanii, A. astronyxis, A. polyphaga,

A. healyi, and A. divionesis [Visvesvara et al. 2007]. Acanthamoeba trophozoites gain

access to the CNS via two distinct routes: through the olfactory neuroepithelium or

through hematogenous spread. However, it is believed that in human GAE trophozoites

most often gain access to the CNS via hematogenous spread originating from the lungs or

cutaneous infection [Marciano-Cabral and Cabral 2003; Khan 2008]. Specifically, it is

believed that trophozoites from the blood enter the brain parenchyma via the capillary

endothelium or the cerebral spinal fluid (CSF) via the endothelial cells of the choroid

plexus [Khan 2007]. Once in the CNS, microglia and invading peripheral macrophages

are the primary immune cells that interact with Acanthamoeba causing the release of pro-

inflammatory mediators [Marciano-Cabral and Cabral 2003]. GAE is characterized by

the formation of granulomas (Figure 2); however, granulomas are rarely observed in

8

Figure 2: Characteristic Granuloma Associated with Granulomatous Amoebic

Encephalitis (GAE). Hematoxylin and Eosin (H & E) stain of a GAE-associated

granuloma illustrating hallmark infiltration of microglia and macrophages surrounding

Acanthamoeba (arrow); bar designates 200 µm. Courtesy of Dr. Francine Marciano-

Cabral, Ph.D.

9

immune compromised individuals [Martinez 1982]. Granulomas formed during GAE are

composed of amoebae, microglia, macrophages, polymorphonuclear cells, T cells, and B

cells [Marciano-Cabral and Cabral 2003; Khan 2008]. Granulomas have been

demonstrated to be instrumental to slowing the progression of GAE as evidenced by the

finding that immune suppression of Acanthamoeba infected mice, that is induced by

treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ9-THC), results in an

increased mortality rate as compared to control mice [Marciano-Cabral et al. 2001]. The

increase in mortality rate of Δ9-THC-mediated immune suppressed mice infected with

Acanthamoeba was subsequently linked to a decreased presence of macrophages at focal

sites of Acanthamoeba in the brain [Cabral and Marciano-Cabral, 2004]. Symptoms of

GAE include nausea, vomiting, headache, stiff neck, seizures and lethargy [Marciano-

Cabral and Cabral 2003]. GAE is usually diagnosed upon autopsy. The observation of

brain lesions by Magnetic Resonance Imagery (MRI), observation of cysts by brain

biopsy, and use of PCR have been the principal modes for diagnosis of GAE. GAE is

treated with a combination chemotherapeutics including oral itraconazole, pentamidine,

sulfadiazine, fluconazole, and fluocytosine [Visvesvara et al. 2007]. Treatment can be

successful if GAE is diagnosed early; however, in most cases prognosis is poor even in

the presence of chemotherapeutics.

Microglia and CNS invading peripheral macrophages are thought to be the

primary cells that are responsible for mounting the immune response to invading

Acanthamoeba. Microglia, also known as “resident brain macrophages”, are one of four

types of glial cells in the CNS; other glial cells include astrocytes, oligodendrocytes, and

A

10

ependymal cells. Microglia exist in various states of activation, as do macrophages.

While in a resting state, microglia are in a ramified form in close association with

astrocytes and are not capable of phagocytosis. Microglia respond to a variety of stimuli

including cytokines such as interferon gamma (IFN)γ and TNF-α; and pathogen-derived

components such as lipopolysaccharide (LPS). Once activated, microglia are capable of

responding to sites of brain injury and can be characterized by their amoeboid shape,

surface receptor expression, and chemokine/cytokine production. Additionally, reactive

microglia are capable of migration to sites of damage via the chemokine/chemokine

receptor network, phagocytosis that is promoted by TNF-α, and antigen presentation that

is activated by IFNγ.

Reactive microglia express a plethora of cell surface receptors including: pattern

recognition receptors for LPS (CD14/Toll receptor 4 (TLR4)) and mannose (mannose

receptors); chemokine receptors CCR2, CCR3, CCR5, CXCR4, and CX3CR1; Fc

receptors Fc RI, RII, RIII; cytokine receptors for IFNγ, TNF (TNF RI and RII),

interleukin (IL)-1, and IL-12; and purinergic receptors [Vass and Lassmann et al. 1990;

Peress et al. 1993; Ulvestad et al. 1994; Peterson et al. 1995; Becher and Antel 1996;

Dopp et al. 1997; Harrison et al. 1998; Suzumura et al. 1998; Marzolo et al. 1999;

McManus et al. 2000; Simpson et al. 2000; Aloisi 2001; Bsibsi et al. 2002; Ogata et al.

2003; Rock et al. 2004]. Reactive microglia also are capable of producing and releasing a

host of immune mediators. Some of these include the chemokines IL-8, regulated on

activation, normal T-cell expressed and secreted (RANTES), macrophage inflammatory

protein (MIP)-1 α//, MIP-2, interferon gamma-inducible 10kDa protein (IP-10), and

11

monocyte chemoattractant protein (MCP)-1; and pro-inflammatory cytokines IL-1α, IL-

1β, IL-6, IL-12, and TNF-α [Aloisi 2001; Ambrosini and Aloisi 2004; Rock et al., 2004].

Studies have highlighted the role of microglia and CNS-invading peripheral

macrophages as instrumental immune effector cells during GAE, contributing to the

direct control of Acanthamoeba spread [Marciano-Cabral et al. 1998; Benedetto et al.

2002; Benedetto et al. 2003; Marciano-Cabral et al. 2004]. It has been demonstrated that

microglia elicit pro-inflammatory cytokines such as IL-1α, IL-1β, and TNF-α in response

to Acanthamoeba [Marciano-Cabral et al. 2004, Shin et al. 2001]. Moreover, Marciano-

Cabral et al. [2004] demonstrated that highly pathogenic A. culbertsoni stimulates the

secretion of higher levels of IL-1α, IL-1β, and TNF-α from primary rat microglia as

compared to when these cells are stimulated with weakly pathogenic A. castellanii.

However, reports indicate that such cytokines are not cytolytic to Acanthamoeba and,

rather, may serve to activate microglial/macrophage cells for contact-dependent mediated

killing of Acanthamoeba [Marciano-Cabral et al. 1998]. Activated macrophages have

been reported to have an enhanced ability to phagocytose and kill Acanthamoeba

[Marciano-Cabral et al. 1998; Alizadeh 2007]. In addition, microglia primed with IFN-γ

plus IL-1β or TNF-α have been shown to be amoebastatic or amoebicidal against A.

castellanii, respectively [Benedetto et al. 2002; Benedetto et al. 2003].

Acanthamoeba use both contact-dependent and contact-independent pathogenesis

mechanisms [Khan 2006]. Contact-dependent pathogenesis mechanisms include

adhesion to extracellular matrix proteins, binding to mannose sugars on host cells via

mannose binding protein (MBP) expressed by Acanthamoeba trophozoites, and

12

phagocytosis of host cells [Khan 2006; Rocha-Azevedo et al. 2009]. Contact-independent

pathogenesis mechanisms include the hydrolysis of ATP via ecto-ATPase expression and

secretion of phospholipases and proteases [Khan 2006].

The first step to Acanthamoeba host invasion involves recognition of, and

adhesion to, host tissue-expressed mannose and extracellular matrix proteins. Recently, it

has been demonstrated that highly pathogenic Acanthamoeba display differential

adherence to extracellular matrix proteins with a preference for laminin-1 [Rocha-

Azevedo et al. 2009]. This adherence is thought to be attributed to an uncharacterized 55

kDa membrane protein expressed by highly pathogenic Acanthamoeba [Rocha-Azevedo

et al. 2009]. Studies have highlighted the role of Acanthamoeba MBP in binding corneal

epithelial and human brain microvascular endothelial cells (HBMECs) [Morton et al.

1991; Alsam et al. 2003]. Acanthamoeba binding is thought to induce host cell cycle

arrest which may lead to apoptosis [Alizadeh et al. 1994; Shin et al. 2000; Sissons et al.

2004b; Sissons et al. 2005]. Moreover, it has been demonstrated that damage to neurons

and microglia occurs via contact-dependent mechanisms [Pettit et al. 1996; Marciano-

Cabral et al. 2000; Shin et al. 2000]. Acanthamoeba binding also has been linked to the

induction of Acanthamoeba signal transductional pathways leading to increased protease

secretion (discussed below) and activation of actin polymerization [Taylor et al. 1995;

Hurt 2003b; Clarke and Niederkorn 2006b; Khan 2006]. Actin polymerization is

instrumental to morphological changes that lead to phagocytosis of host cells.

Phagocytosis serves as a food acquiring process for amoebae as evidenced by the

presence of food cups during this event [Pettit et al. 1996].

13

One contact-independent pathogenesis mechanism used by Acanthamoeba is the

hydrolysis of ATP by membrane ecto-ATPases (ecto-nucleotidases) [Sissons et al.

2004a]. Ecto-ATPases belong to a group of enzymes known as ecto-enzymes that are

characterized by the extracellular expression of their active sites [Sissons et al. 2004a;

Goding 2000] Ecto-ATPases hydrolyse adenosine triphosphate (ATP) to adenosine

diphosphate (ADP) and adenosine monophosphate (AMP); and ADP to AMP [Goding

2000]. It has been demonstrated that clinical isolates of Acanthamoeba express higher

levels of ecto-ATPases than environmental isolates [Sissons et al. 2004a]. During

Acanthamoeba infection hydrolyzed ATP in the form of ADP acts on host cell expressed

P2Y2 purinergic receptors [Mattana et al. 2002]. This ADP activation has been

demonstrated to induce TNF-α secretion, caspase 3 activation, and apoptosis of

monocytic cells via P2Y2 receptors [Mattana et al. 2002]. Furthermore, ecto-ATPase

activity increases in the presence of mannose, indicating that receptor activity is

increased during host cell binding [Sissons et al. 2004a].

Other contact-independent pathogenesis mechanisms used by Acanthamoeba

include the production and secretion of phospholipases and proteases; of these, the

production of proteases has been most widely studied. It has been demonstrated that

pathogenic Acanthamoeba secrete more phospholipase A than non-pathogenic

Acanthamoeba [Cursons et al. 1978; Misra et al. 1983]. Pathogenic Acanthamoeba

produce serine, cysteine, and metallo- proteases [He et al. 1990; Hadás et al. 1993a;

Mitro et al. 1994; Mitra et al. 1995; Cao et al. 1998; Alfieri et al. 2000; Cho et al. 2000;

Hong et al. 2000; Khan et al. 2000; Kong et al. 2000; Na et al. 2001; Hong et al. 2002;

14

Na et al. 2002; Alsam et al. 2005; Kim et al. 2006; Sissons et al. 2006]. However, serine

proteases represent the major class of proteases secreted by Acanthamoeba [Khan 2006].

Serine proteases constitute a class of enzymes that have the amino acid serine

located in their active site and are able to cleave peptide bonds. Highly virulent

Acanthamoeba have been demonstrated to secrete higher levels of proteases than less

virulent Acanthamoeba [Hadás et al. 1993b; Khan et al. 2000]. In Acanthamoeba’s

natural environment, serine proteases are thought to play a key role in encystment and

excystment [Moon et al. 2008; Dudley et al. 2008]. Acanthamoeba serine proteases also

are believed to facilitate invasion and evasion within the infected host.

Many studies have linked Acanthamoeba serine proteases to host invasion.

Acanthamoeba serine proteases have been demonstrated to degrade ECM components

such as collagen, laminin, elastin [Kong et al. 2000; Na et al. 2001; Na et al. 2002; Hurt

et al. 2003a; Sissons et al. 2006]. In addition, Acanthamoeba binding to corneal

epithelial cells via MBP has been shown to increase the expression of a 133kDa

Acanthamoeba serine protease called mannose-induced protease (MIP-133); and,

subsequent studies found this protease to be cytolytic to these cells [Hurt et al. 2003a and

Hurt et al. 2003b]. Using an in vitro blood-brain barrier (BBB) model, Alsam et al.

[2003] demonstrated that, although Acanthamoeba binding to HBMECs can be blocked

in vitro with exogenous mannose, HBMEC cell cytotoxicity increased in the presence of

exogenous mannose. Subsequent studies demonstrated that conditioned medium from A.

castellanii increased BBB permeability and that this effect was prevented in the presence

of the serine protease inhibitor phenylmethylsulphonylfluoride (PMSF) [Alsam et al.

15

2005]. Moreover, it has been demonstrated that conditioned medium from

Acanthamoeba degrades zonula occludens 1 (ZO-1) and occludin tight junction proteins

of HBMEC monolayers; this action is believed to be mediated by Acanthamoeba serine

proteases [Khan 2007].

Acanthamoeba serine proteases also have been implicated as having a functional

role in evasion of host cell defenses. Previous studies have indicated that Acanthamoeba

serine proteases are able to degrade the immunoglobulins secretory IgA (sIgA), IgG,

IgM; the cytokines IL-1α and IL-1β; and other proteins such as plasminogen,

hemoglobin, fibrinogen, and albumin [Kong et al. 2000; Na et al. 2001; Na et al. 2002;

Sissons et al. 2006]. However, all of these studies were performed using exogenous

substrates (i.e., recombinant protein substrates) and, though they give important insight to

the potential functional role of serine proteases in host immune evasion, there is still a

paucity of information with regards to how Acanthamoeba specifically use their serine

proteases for immune evasion.

The goal of the present study was to determine the functional relevance of A.

culbertsoni-elicited serine proteases in CNS immune evasion. We hypothesized that

serine proteases provide an effective means of host immune evasion for Acanthamoeba

by dampening microglial immune response through the degradation of elicited cytokines

and chemokines. In order to test our hypothesis we proposed four specific aims: i) to

characterize proteases elicited by A. culbertsoni, ii) to determine the cytokine and

chemokine profile elicited by microglial-like cells in response to A. culbertsoni, iii) to

determine if A. culbertsoni-elicited factors could degrade constitutive and LPS-stimulated

16

cytokines and chemokines produced by microglial-like cells, and iv) to determine if pro-

inflammatory cytokines and chemokines elicited by microglial-like cells in the presence

A. culbertsoni could be degraded by A. culbertsoni serine proteases.

We demonstrate that A. culbertsoni secrete serine proteases, the levels of which

are augmented in the presence of microglial-like cells. A. culbertsoni induced the gene

expression of a plethora of pro-inflammatory cytokines and chemokines by microglial-

like cells as assessed by Multiprobe Ribonuclease Protection Assay (RPA) and

cytokine/chemokine protein microarrray. However, we demonstrate that the cytokines

and chemokines elicited from microglial-like cells were degraded by A. culbertsoni serine

proteases. While it is likely that A. culbertsoni use a multifactorial process to elicit

pathogenesis during GAE, these studies suggest that A. culbertsoni serine proteases aid in

A. culbertsoni CNS immune evasion by specifically targeting microglial facilitated

granuloma formation and anti-microbial activity.

17

Materials and Methods

Reagents. Dulbecco’s Modified Eagle’s Medium (DMEM; with phenol red, without L-

glutamine), 100X penicillin/streptomycin, 1M HEPES, 100X nonessential amino acids

(NEAA), 200 mM L-glutamine, and 100X Modified Eagle’s Medium (MEM) vitamins

were purchased from Cellgro/Mediatech, Inc. (Herndon, VA). Fetal Bovine Serum

(FBS) and heat-inactivated (HI) FBS were purchased from both Cellgro/Mediatech, Inc.

and KSE Scientific (Durham, NC). Neurobasal™-A medium (without phenol red), B-27

supplement, 1X phosphate buffered saline (PBS; without MgCl2 and CaCl2; pH 7.4), and

TRIzol®

reagent were purchased from Invitrogen (Carlsbad, CA). Lipopolysaccharide

(LPS), phenylmethylsulphonylfluoride (PMSF), trans-Epoxysuccinyl-L-leucylamido(4-

guanidino)butane (E-64), and glucose were purchased from Sigma-Aldrich (St. Louis,

MO). HyClone® 100X penicillin G/streptomycin/amphotericin B, Remel oxoid

neutralized liver digest, and Remel proteose peptone were purchased from Thermo Fisher

Scientific (Lenexa, KS). Bacto™ yeast extract was purchased from Becton, Dickinson

and Company (Franklin Lakes, NJ). Dextrose was purchased from J.T. Baker Chemicals

(Phillipsburg, NJ).

Amoebae. Acanthamoeba culbertsoni (ATCC # 30171) were acquired from the

American Type Culture Collection (ATCC, Manassas, VA). Axenic (i.e., culture free of

18

other contaminating microorganisms) amoebae were maintained either in oxoid medium

(0.55% w/v oxoid neutralized liver digest, 0.3% w/v dextrose, 0.5% w/v proteose

peptone, 0.25% w/v yeast extract, 1% FBS, and 0.1% hemin) or proteose peptone, yeast,

glucose (PYG) medium (2% w/v proteose peptone, 0.2% w/v yeast extract, and 0.1M

glucose) at 37ºC.

Microglia. Immortalized primary mouse microglial-like BV-2 cells were a gift from Dr.

Michael McKinney of the Mayo Clinic (Jacksonville, FL). BV-2 cells were maintained

in complete DMEM (i.e., DMEM supplemented with 10% HI-FBS, 100 U/ml penicillin

G, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, 0.01M HEPES, 1X NEAA,

2mM L-glutamine, and 1X MEM vitamins) at 37ºC with 5% CO2.

Conditioned Medium. Microglial conditioned medium (MCM) was generated by

culturing 106 BV-2 cells in T25 cm

2 (Greiner, Monroe, NC) tissue culture flasks in

complete DMEM overnight at 37ºC with 5% CO2. The following day, cultures were

washed with sterile PBS and 5ml complete Neurobasal™-A medium (serum free,

supplemented with 1X B-27 supplement, 100 U/ml penicillin, 100 µg/ml streptomycin,

and 0.5 mM L-glutamine) were added. Neurobasal™-A medium is specifically

formulated to mimic the serum-free environment of the brain. Cells were cultured an

additional 9-24 h at 37ºC with 5% CO2. The supernatant (i.e., conditioned medium) was

collected by centrifugation at 1700 x g for 10 min at 4°C. Conditioned medium from LPS

stimulated BV-2 cells was generated in a similar manner except that BV-2 cells were

initially cultured overnight in 60 mm x 15 mm petri dishes at 37ºC with 5% CO2. Cells

19

were washed with PBS, complete Neurobasal™-A medium (CNBA) containing 100

ng/ml LPS was added, and cultures were incubated 8 h at 37ºC with 5% CO2.

Supernatants (LPS/MCM) were collected by centrifugation (1700 x g, 10 min, 4°C) and

stored at -80°C until assayed for cytokines and chemokines.

Acanthamoeba culbertsoni conditioned medium (ACM) was generated from

cultures of 109 A. culbertsoni (ACM-HI) and 10

6 A. culbertsoni (ACM-LO). For

generation of ACM-HI, axenic A. culbertsoni were cultured in Erlenmeyer flasks

containing 1L oxoid or PYG medium at 37ºC with shaking for 4 days to yield

approximately 109 amoebae. The amoebae were pelleted by centrifugation at 6090 x g

for 20 min at 4°C, 5ml CNBA were added to the pellet and the pellet was incubated at

37ºC for 24 h. The amoebae were re-pelleted by centrifugation (1700 x g, 10 min, 4°C).

The supernatant (i.e., conditioned medium) was removed and centrifuged at (1700 x g, 10

min, 4°C) to remove debris, and the conditioned medium (i.e., supernatant) was stored at

-80ºC until used in experiments. The protein concentration of ACM-HI was determined

by the Bradford method [1976], dilutions of this conditioned medium were used in select

experiments. ACM-LO was prepared by incubating 106

axenic A. culbertsoni, which was

originally propagated in oxoid medium, in 5ml CNBA in T25 cm2

flasks at 37ºC with 5%

CO2 for 24 h. The supernatant was then removed, centrifuged (1700 x g, 10 min, 4°C)

and stored -80ºC.

Conditioned medium from co-cultures of BV-2 cells and A. culbertsoni

(MCM/ACM) was generated by first culturing 106 BV-2 cells in complete DMEM in T25

cm2 flasks overnight at 37ºC with 5% CO2. The next day, the cells were washed with

20

PBS, CNBA was added, and 106 A. culbertsoni (originally propagated in oxoid medium)

was added to the BV-2 cell cultures. Co-cultures were incubated at 37ºC with 5% CO2 for

9-24 h. Culture supernatants were then harvested by centrifugation at (1700 x g, 10 min,

4°C) and stored at -80ºC.

Two-Dimensional (Iso-Dalt) Gel Electrophoresis (2D-PAGE). Two-dimensional (Iso-

Dalt) gel electrophoresis was used to separate proteins in CNBA (background control)

and ACM. Briefly, amoebae were cultured in Erlenmeyer flasks containing 1L PYG

medium at 37ºC with shaking for 4 days to yield approximately 109 amoebae. The

amoebae were pelleted by centrifugation at 6090 x g for 20 min at 4°C, 5ml CNBA were

added to the pellet and the pellet was incubated at 37ºC for 24 h. The amoebae were re-

pelleted by centrifugation (1700 x g, 10 min, 4°C). The supernatant (i.e., conditioned

medium) was removed and centrifuged at (1700 x g, 10 min, 4°C) to remove debris, and

the conditioned medium (i.e., supernatant) was stored at -80ºC until used in experiments.

In order to remove interfering salts before performing the isoelectric focusing first-

dimension, samples were dialyzed overnight against ultrapure deionized water using

Slide-A-Lyzer 3.5K molecular weight cutoff dialysis cassettes (Pierce Biotechnology,

Inc, Rockford, IL). Protein concentrations were determined by the Bradford method

[Bradford, 1976] and 500g of CNBA protein and 1000g of ACM protein were added

to ReadyPrepTM

rehydration/sample buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.2%

Bio-Lyte®

3/10 ampholyte, and trace bromophenol blue) (Bio-Rad, Hercules, CA) in a

total volume of 200 μl. A carbonic anhydrase carbamylated isoelectric point marker

(20l) (GE Healthcare, Piscataway, NJ) was added to the CNBA protein sample before

21

performing first dimension isoelectric focusing. The protein samples were applied to

ReadyStripTM

IPG pH 3-10 (Bio-Rad) strips that had been previously rehydrated with

ReadyPrepTM

rehydration/sample buffer overnight at room temperature. The samples

were allowed to passively rehydrate for 2 h at room temperature. The proteins were

isoeletrically focused for 45,900 and 59,000 volt hours for CNBA and ACM,

respectively, using a PROTEAN®

IEF cell (Bio-Rad). Electrophoresis in the second

dimension was performed in 10-20% gradient SDS-PAGE gels using the CriterionTM

gel

electrophoresis system (Bio-Rad). The gels were stained with Bio-SafeTM

Coomassie

Blue G-250 (Bio-Rad). Gels were scanned on a Microtek ScanMaker 9800XL/TMA

1600 flatbed scanner (Microtek, Santa Monica, CA) interfaced with a Compaq computer

(Hewlett-Packard Company, Houston, TX) using SilverFast scanning software.

Gel Zymography. Gel zymography was performed to measure protease activity in

conditioned media. Briefly, 10μl 2X Novex® tris-glycine SDS sample buffer (Invitrogen)

were added to each 10μl sample prior to loading. In select experiments, samples were

incubated (37ºC for 30 min.) with PMSF and E-64 prior to electrophoresis in order to

assess for serine and cysteine protease activity, respectively. Samples were loaded into

pre-cast Novex® 10% gelatin zymogram gels (Invitrogen) and gels were electrophoresed

(125V, 90 min) in Novex®

tris-glycine SDS buffer. Following electrophoresis gels were

incubated (30 min, room temperature) in 1X Novex®

zymogram

renaturing buffer

(Invitrogen) with gentle rocking, and then equilibrated (30 min, room temperature) in 1X

Novex® zymogram developing buffer, pH 7.5 (Invitrogen) with gentle rocking.

Following the addition of fresh 1X Novex® zymogram developing buffer, pH 7.5, gels

22

were incubated at 37ºC overnight. Protease digestion was viewed as areas of clearing on

a blue background following staining with SimplyBlue™ Safestain (Invitrogen). Gels

were scanned on a Microtek ScanMaker 9800XL/TMA 1600 flatbed scanner interfaced

with a Compaq computer using SilverFast scanning software.

Light Microscopy. Light microscopy images of BV-2 cell cultures, A. culbertsoni

cultures, and BV-2/ A. culbertsoni co-cultures were acquired using an Olympus CK2

inverted microscope (Opelco, Washington, DC) in concert with an attached XV-GP230

digital video camera (Panasonic, Yokohoma, Japan). A Dell Dimension XPS1450

computer (Dell, Inc., Round Rock, Texas) programmed with Videum 100 hardware and

Window NT software (Winnov, Sunnyvale, CA) was used to capture images.

Multiprobe Ribonuclease Protection Assay (RPA). BV-2 cells (1.5 x 106) were co-

cultured (6 h) with A. culbertsoni in 60 mm culture dishes at 37oC with 5% CO2. Total

RNA was prepared from cell cultures using TRIzol®

reagent (Invitrogen) according to the

manufacturer’s instructions, subjected to isopropanol precipitation, and dissolved directly

in 1X hybridization buffer (BD Biosciences/PharMingen, San Diego, CA). A Riboquant

RPA was used to assess for levels of mouse chemokine mRNA (mCK-5c probe template

set; BD Biosciences/PharMingen). The ribo-probes were labeled with 32

P [UTP] (MP

Biomedicals, Aurora, OH) to a specific activity of greater than 3,000 Ci/mmol. The

isolated RNA samples then were hybridized with the probe overnight at 56oC and the

protected fragments were resolved on a 6% polyacrylamide gel containing 6M urea.

Imaging of the protected fragments was performed using a 445 SI Phosphorimager

23

(Molecular Dynamics, Sunnyvale, CA). The pixel intensity of each band was quantified

using ImageQuant 4.1 software (Molecular Dynamics) and the amount of chemokine

mRNA was normalized for loading by dividing the pixel value for the chemokine

expression band by the sum of the pixel values for the mRNAs of the housekeeping

genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a ribosomal protein,

L32.

Cytokine/Chemokine Protein Microarray. The RayBio® Mouse Cytokine Antibody

Array III (RayBiotech, Inc., Norcross GA) was used as a semi-quantitative measure of

cytokines and chemokines in culture supernatants according to the manufacturer’s

instructions. Briefly, arrays were blocked (30 min, room temperature) in manufacturer

supplied blocking buffer. After blocking, 1ml of culture supernatant was added.

Following incubation overnight at 4°C, the membranes were washed with manufacturer

provided wash buffers and incubated (1.5 h) with biotin-conjugated anti-cytokine

antibodies. After washing to remove unbound biotin-conjugated anti-cytokine

antibodies, the membranes were incubated (2 h) with horseradish peroxidase (HRPO)-

conjugated strepavidin, washed, and protein was visualized by chemilumenescent

reaction in concert with a Kodak X-OMAT 2000A processing instrument (Kodak,

Rochester, NY) and Kodak BioMAX XAR film. Film was scanned on a Microtek

ScanMaker 9800XL/TMA1600 flatbed scanner and densitometry was performed using

Quantity One® software (Bio-Rad, Hercules, CA).

24

Enzyme-linked Immunosorbent Assay (ELISA). BV-2 cells were co-cultured with A.

culbertsoni for 9-24 h (37ºC with 5% CO2), culture supernatant (MCM/ACM) was

collected and incubated (37ºC with 5% CO2) an additional 12-48 h. DuoSet mouse

sandwich ELISA kits (R & D Systems, Inc., Minneapolis, MN) were used to quantify

levels of TNF-α, MIP-1α, and MIP-2 in MCM/ACM samples. For ELISA, Corning

Costar

EIA plates (Corning, Inc., Lowell, MA) were coated with 0.8 µg/ml goat anti-

mouse TNF-α, 0.4 µg/mL goat anti-mouse MIP-1α, or 2.0 g/ml rat anti-mouse MIP-2,

incubated overnight at room temperature, washed (0.05% Tween-20 in PBS, pH 7.4), and

blocked (1 h) with reagent diluent (1% BSA in PBS, pH 7.4, 0.2 m filtered). Following

addition of samples or standards (100 µl), plates were incubated (2 h) at room

temperature, washed, and incubated (2 h, room temperature) with 150 ng/ml biotinylated

goat anti-mouse TNF-α, 100 ng/ml biotinylated goat anti-mouse MIP-1α, or 75 ng/ml

biotinylated goat anti-mouse MIP-2. The plates then were washed, and streptavidin

conjugated HPRO was added. Following incubation (20 min, room temperature) a 1:1

mixture of manufacturer provided substrate reagent (H2O2 and Tetramethylbenzidine

(TMB)) was added (20 min, room temperature). After addition of stop solution (2 N

H2SO4), the optical density of each sample was determined using a SpectraMax 250

spectrophotometer (MDS Analytical Technologies, Sunnyvale, CA) in concert with

SoftMax Pro software (MDS Analytical Technologies). All wells were read at 450nm

with a correction wavelength of 570nm. Standards consisted of two-fold dilutions of

recombinant mouse TNF-α (2000 pg/ml to 31.25 pg/ml), MIP-1α (500 pg/ml to 7.81

pg/ml), or MIP-2 (1000 pg/ml to 15.63 pg/ml).

25

Data Analysis. For cytokine/chemokine protein microarrays, background was subtracted

and the density of duplicate cytokine and chemokine array spots (i.e., the sum of pixels

per array spot), which corresponded to the relative amount of cytokines or chemokines in

each supernatant sample, was normalized to the average density of the standards (i.e.,

internal positive controls consisting of biotin-conjugated IgG) included in each

membrane. Density values were graphed as the mean value intensity and a fold

difference of > 2.1 as compared to control was considered significant. Error bars

represent the + S.D. of duplicate cytokine or chemokine spots. For ELISA, the percent

maximum response was calculated by comparing the optical density of samples that were

incubated for an additional 12-48 h to optical density of the starting co-culture

supernatant (i.e., 9 h or 24 h co-culture supernatant); the optical density of the co-culture

supernatant at 9 h or 24 h was considered the 100% value. Samples were performed in

triplicate and the average optical density of each sample was used to calculate percent

maximum response.

26

Results

Acanthamoeba culbertsoni Secrete Degradative Enzymes.

Two-dimensional (iso-dalt) gel electrophoresis (2D-PAGE) was used to visualize

and compare proteins of CNBA versus ACM. For this study, supernatant form A.

culbertsoni (i.e., ACM) was generated from 109

amoebae which were propagated in PYG

medium, pelleted, and incubated in the presence of CNBA; the resulting supernatant was

designated as ACM-HI. The proteomic profile of CNBA revealed a large protein spot at

the approximate molecular weight (kDa) of 62, which is consistent with the approximate

molecular weight predicted for albumin (Figure 3). A carbonic anhydrase pI marker,

which was included as an internal standard within the CNBA two-dimensional gel, had a

molecular weight of 26 kDa and a pI range approximately 4.8-6.7 (Figure 3). ACM-HI

exhibited a proteomic profile that was distinct from that of CNBA (Figure 4). Notably,

the ACM-HI proteomic profile displayed a complete absence of the 62 kDa albumin spot

(Figure 4). These results are consistent with the presence of A. culbertsoni degradative

enzymes in ACM-HI.

27

Figure 3: Visualization of Complete Neurobasal-A Medium Proteins. Complete

Neurobasal-A Medium proteins (500 µg) were separated based on relative charge (pI)

(pH 3-10) in the first dimension and relative molecular weight in the second dimension

(10-20% SDS-PAGE). Proteins were visualized using Coomassie G-250 protein stain.

The top arrow indicates a major protein spot at 62 kDa. The bottom arrow indicates a

carbonic anhydrase pI marker at 26 kDa and pI range of ~ 4.8-6.7 that was included as an

internal standard. Complete Neurobasal-A Medium was used to generate conditioned

medium from BV-2 cell cultures, A. culbertsoni cultures, and BV-2 cell plus A.

culbertsoni co-cultures.

28

Figure 4: Visualization of A. culbertsoni-Conditioned Medium Proteins. Acanthamoeba culbertsoni conditioned medium (ACM) was generated from

approximately 109

amoebae (i.e., ACM-HI) which were propagated in proteose peptone,

yeast, glucose (PYG) medium. Following propagation, amoebae were pelleted and

incubated in the presence of complete Neurobasal™-A medium (24 h). Supernatant (i.e.,

conditioned medium) was harvested and proteins (1000 g) were separated based on

relative charge (pI) (pH 3-10) in the first dimension and relative molecular weight in the

second dimension (10-20% SDS-PAGE). Following electrophoresis, proteins were

visualized using Coomassie G-250 protein stain.

29

Acanthamoeba culbertsoni Secrete Serine Proteases.

Gelatin zymography was employed in order to determine whether differences in

the two-dimensional (iso-dalt) gel electrophoresis proteomic profiles of CNBA versus

ACM-HI were attributed to the presence of A. culbertsoni protease activity. Gelatin

zymography is a technique that allows for the detection of serine, cysteine, and metallo-

proteases. Initially, the proteolytic profiles of ACM-HI generated from amoebae

propagated in either oxoid or PYG medium were compared (Figure 5). Acanthamoeba

culbertsoni propagated in oxoid or PYG medium exhibited three common protease bands

of approximately 187, 97, and 58 kDa (Figure 5). Propagation of A. culbertsoni in oxoid

medium resulted in protease activity that was more robust as compared to amoebae

propagated in PYG medium. CNBA did not show evidence of protease activity (Figure

5). These results indicate that components of the oxoid medium influence A. culbertsoni

protease levels. Oxoid medium was used to propagate amoebae in all subsequent

experiments.

Titration of ACM-HI revealed protease activity at positions corresponding to

187, 97, 90, and 58 kDa (Figure 6). Protease activity located at positions corresponding

187 and 58 kDa were the most robust. Protease activity located at positions

corresponding to 97 and 90 kDa was significantly reduced at 0.5 µg as compared to the

starting concentration at 1.5 µg (Figure 6).

Protease inhibitors in concert with gelatin zymography were used to identify the

class of proteases secreted by A. culbertsoni. Initially, ACM-HI (1.0 µg) was incubated

with either the serine protease inhibitor PMSF (2.0-0.125 mM) or anhydrous ethanol

30

Figure 5: A Comparison of the Effect of Propagation Medium on A. culbertsoni

Protease Activity. A. culbertsoni (109) were propagated in either oxoid (OX) or PYG

media. Then amoebae were pelleted and cultured in the presence of complete

Neurobasal™-A medium (background control, C) for 24 h. Supernatants (i.e., A.

culbertsoni-conditioned medium; ACM) were collected and 1.0 µg of ACM protein was

assessed by gelatin zymography. Arrows indicate protease activity at positions

corresponding to 187, 97, and 58 kDa. Relative molecular weight was calculated on the

basis of the mean center point for each respective band on gelatin zymograms.

31

Figure 6: Titration of Protease Activity in 109 A. culbertsoni-Conditioned Medium.

The protease activity of 1.5-0.25 µg of 109 A. culbertsoni-conditioned medium (ACM-

HI) was assessed by gelatin zymography. Arrows indicate protease activity at positions

corresponding to 187, 97, 90, and 58 kDa. Relative molecular weight was calculated on

the basis of the mean center point for each respective band on gelatin zymograms.

32

(PMSF vehicle) (Figure 7). All ACM-HI protease activity was sensitive to PMSF

indicating that A. culbertsoni secrete serine proteases (Figure 7). The PMSF vehicle did

not have an effect on protease activity. Specifically, ACM-HI serine protease activity

was completely inhibited by PMSF at concentrations as low as 1.0 mM. As expected,

lower concentrations of PMSF (i.e., 0.500-0.125 mM) resulted in slight recovery of

protease activity at 187 kDa. Based on these results, 1.0 mM PMSF was used in all

subsequent experiments requiring the use of protease inhibitors. ACM-HI (0.75 µg) was

also incubated with the cysteine protease inhibitor E-64 (10-1 µM) at pH 7.5 (Figure 8).

Gelatin zymogram studies were also performed on ACM-HI (1.0 µg) which was

incubated in the presence of 10 µM E-64 at pH 5.0 (data not shown). Gelatin

zymography revealed that ACM-HI protease activity was not sensitive to E-64 (Figure 8

and data not shown). This result suggests that A. culbertsoni cysteine protease activity

was not detectable under the experimental conditions used.

Protease activity in ACM-HI, conditioned medium from 106 amoebae (ACM-

LO), and conditioned medium from co-cultures of BV-2 cells (mouse microglial-like

cells) plus A. culbertsoni (MCM/ACM) were compared using gelatin zymography.

ACM-HI, ACM-LO, and MCM/ACM exhibited three common protease bands

corresponding to 187, 97, and 58 kDa (Figure 9). In addition, all protease bands were

serine proteases as evidenced by their sensitivity to PMSF. The similarities in protease

activity from ACM-LO and MCM/ACM indicate that co-culture of A. culbertsoni with

BV-2 cells did not elicit differential patterns of protease expression. However, an overall

increase in protease activity was observed in MCM/ACM as compared to ACM-LO,

33

Figure 7: Acanthamoeba culbertsoni Secrete Proteases that Are Inhibited by the

Serine Protease Inhibitor Phenylmethylsulphonylfluoride (PMSF). Conditioned

medium (ACM-HI) (1.0 µg) from 109 A. culbertsoni was subjected to no treatment (0),

incubated (30 min, 37°C) in the presence of 5% anhydrous ethanol that served as the

vehicle (V) for PMSF, or incubated (30 min, 37°C) in the presence of the serine protease

inhibitor PMSF (2.0-0.125 mM). Arrows indicate protease activity at positions

corresponding to 187, 97, 90, and 58 kDa. Relative molecular weight was approximated

based on comparable gelatin zymograms.

34

Figure 8: Acanthamoeba culbertsoni Secrete Proteases that Are Not Inhibited by the

Cysteine Protease Inhibitor trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane

(E-64). 109 A. culbertsoni-conditioned medium (ACM-HI) (0.75 µg) was subjected to no

treatment (0) or incubated (30 min, 37°C) in the presence of the cysteine protease

inhibitor E-64 (10-1 µM). Arrows indicate protease activity at positions corresponding to

187, 97, 90, and 58 kDa. Relative molecular weight was calculated on the basis of the

mean center point for each respective band on gelatin zymograms.

35

Figure 9: A Comparison of the Proteolytic Activity in Three Different Types of A.

culbertsoni-Conditioned Media. Equal volumes of 109 A. culbertsoni-conditioned

medium (ACM-HI), 106 A. culbertsoni-conditioned medium (ACM-LO), and conditioned

medium from co-culture of 106 A. culbertsoni plus 10

6 BV-2 cells (MCM/ACM) were

subjected to no treatment (0), incubated (30 min, 37°C) in the presence of 5% anhydrous

ethanol that served as the vehicle (V) for PMSF, or incubated (30 min, 37°C) in the

presence of the serine protease inhibitor PMSF (P) (1.0 mM), and protease activity was

assessed using gelatin zymography. Arrows indicate protease activity at positions

corresponding to 187, 97, and 58 kDa. Relative molecular weight was calculated on the

basis of the mean center point for each respective band on gelatin zymograms.

36

indicating that co-culture of A. culbertsoni with microglial-like cells augments A.

culbertsoni protease activity.

Gelatin zymography also was used to compare protease activity in ACM-LO and

MCM/ACM from 9-24 h cultures (Figure 10). Protease activity in ACM-LO and

MCM/ACM from 9 h culture was less intense as compared to the protease activity at 24

h, indicating a time-related accumulation of A. culbertsoni proteases in culture

supernatant. Protease activity in MCM/ACM was more intense than that of ACM-LO at

all time points studied, indicating that co-culture with BV-2 cells augments A. culbertsoni

protease activity (Figure 10).

Acanthamoeba culbertsoni Stimulate Microglial-Like Cells to Produce and Secrete

Cytokines and Chemokines.

In order to determine whether BV-2 cells were inducibly responsive to A.

culbertsoni in terms of expressing chemokines, BV-2 cells were co-cultured (6 h) with A.

culbertsoni and the levels of select chemokine mRNAs were evaluated by RPA (Figure

11). BV-2 cells maintained in the absence of A. culbertsoni did not elicit discernable

levels of chemokine mRNAs. In contrast, A. culbertsoni stimulated BV-2 cells to

produce mRNAs for monocyte chemoattractant protein (MCP)-1, interferon gamma-

inducible 10kDa protein (IP-10), macrophage inflammatory protein (MIP)-2, MIP-1α,

and MIP-1β. Of these, the most robust induction levels were those for MIP-1α.

Acanthamoeba did not express mRNAs for cytokines nor internal controls included in the

RPA (data not shown).

37

Figure 10: Acanthamoeba culbertsoni-Conditioned Medium Proteolytic Activity

Increases in a Time-Related Manner and Is Augmented During Co-culture With

BV-2 Cells. The proteolytic activity of conditioned medium from 106 A. culbertsoni

(ACM-LO) and conditioned medium from co-culture of 106 A. culbertsoni plus 10

6 BV-2

cells (MCM/ACM) (9-24 h cultures) was assessed by gelatin zymography. ACM-LO is

labeled “A” and MCM/ACM is labeled “M/A”. Arrows indicate protease activity located

at positions corresponding to 187, 97, and 58 kDa. Relative molecular weight was

calculated on the basis of the mean center point for each respective band on gelatin

zymograms.

38

Figure 11: Acanthamoeba culbertsoni Induces Chemokine mRNA Expression by BV-

2 Cells. A Multiprobe Ribonuclease Protection (RPA) assay was used to measure

chemokine mRNA expression by BV-2 cells following co-culture (6 h) with A.

culbertsoni. (A) RPA: lanes labeled “P” represent the undigested probes for LTN,

RANTES, MIP-1α, MIP-1β, MIP-2, IP-10, MCP-1, TCA-3, Eotaxin and the

constitutively expressed internal controls L32 and GAPDH. Lanes labeled “M + A” and

“M” represent samples from BV-2 cells plus A. culbertsoni and BV-2 cells alone,

respectively. (B) Graphical depiction of RPA results. Courtesy of Dr. Erinn Raborn,

Ph.D.

A

B

39

In order to determine the profile of cytokines/chemokines at the protein level that

were elicited in response to A. culbertsoni, culture supernatants from BV-2 cell cultures

maintained in the absence (MCM) or presence of A. culbertsoni (MCM/ACM) (9-24 h)

were examined using cytokine/chemokine protein microarray (Figures 12 and 13). A 9

h period post co-culture was selected for assessment since it represents a 3 h differential

time interval post assessment of mRNA elicitation and thereby putatively allowed for the

production of a protein product (Figure 12). A 24 h period post co-culture time was

selected in order to assess time-related differences in cytokine and chemokine expression

as compared to 9 h culture supernatants (Figure 13). BV-2 cell cultures maintained (9

h) in the absence of A. culbertsoni constitutively expressed relatively high levels of MCP-

1, MIP-1α, and MIP-1γ as well as moderate levels of MIP-2, platelet factor 4 (PF-4), P-

Selectin, soluble tumor necrosis factor receptor (sTNF R) I and sTNF RII (Figure 12).

BV-2 cells maintained (9 h) in co-culture with A. culbertsoni expressed decreased levels

of MIP-1α (2.1 fold decrease), MIP-1γ (2.1 fold decrease), PF-4 (2.3 fold decrease),

sTNF RI (3.4 fold decrease), and sTNF RII (2.6 fold decrease) (Figure 12). There was

also a decrease in MCP-1 levels (2.0 fold decrease) observed in supernatants from co-

cultures of BV-2 cells and A. culbertsoni (Figure 12). However, augmented levels of

granulocyte-colony stimulating factor (G-CSF) (2.3 fold increase), IL-12p40/70 (2.5 fold

increase), TNF-α (2.6 fold increase), and MIP-3α (4.1 fold increase) were observed from

co-culture supernatants as compared to supernatants for BV-2 cell cultures maintained in

the absence of A. culbertsoni for 9 h (Figure 12). There was also an increase in IL-1α

levels (1.5 fold increase) observed in supernatants from co-cultures of BV-2 cells and

40

Figure 12: Cytokine and Chemokine Protein Expression by BV-2 Cells following 9 h

Co-culture with A. culbertsoni. The RayBio® Mouse Cytokine Antibody Array III

arrays were used to screen for cytokines and chemokines in supernatants from BV-2 cell

culture (9 h, MCM) and BV-2 cell plus A. culbertsoni co-culture (9 h, MCM/ACM). (A)

Membrane arrays comparing cytokine and chemokine proteins of MCM versus

MCM/ACM. (B) Graphical depiction of select cytokines and chemokines in MCM versus

MCM/ACM. The relative amount of each cytokine and chemokine was normalized using

the average density of the standards (Std.) represented in each membrane. Cytokines and

chemokines are labeled: (1) G-CSF, (2) IL-1α, (3) IL-1β, (4) IL-12p40/70, (5) MCP-1,

(6) MIP-1α, (7) MIP-1γ, (8) MIP-2, (9) MIP-3β, (10) MIP-3α, (11) PF-4, (12) P-Selectin,

(13) RANTES, (14) TNF-α, (15) sTNF RI, and (16) sTNF RII. * indicates a ≥ 2.1 fold

increase as compared to MCM, + indicates a ≥ 2.1 fold decrease as compared to MCM.

B

A

41


24 h Co-culture with A. culbertsoni. The RayBio® Mouse Cytokine Antibody Array III

arrays were used to screen for cytokines and chemokines in supernatants from BV-2 cell

culture (24 h, MCM) and BV-2 cell plus A. culbertsoni co-culture (24 h, MCM/ACM).

(A) Membrane arrays comparing cytokine and chemokine proteins of MCM versus

MCM/ACM. (B) Graphical depiction of select cytokines and chemokines in MCM versus

MCM/ACM. The relative amount of each cytokine and chemokine was normalized using

the average density of the standards (Std.) represented in each membrane. Cytokines and

chemokines are labeled: (1) G-CSF, (2) IL-1α, (3) IL-1β, (4) IL-12p40/70, (5) MCP-1,

(6) MIP-1α, (7) MIP-1γ, (8) MIP-2, (9) MIP-3β, (10) MIP-3α, (11) PF-4, (12) P-Selectin,

(13) RANTES, (14) TNF-α, (15) sTNF RI, and (16) sTNF RII. * indicates a ≥ 2.1 fold

increase as compared to MCM, + indicates a ≥ 2.1 fold decrease as compared to MCM.

A

B

42

A. culbertsoni (Figure 12). Levels of MCP-1, MIP-1α, MIP-1γ, MIP-2, MIP-3β, MIP-

3α, P-Selectin, sTNF RI, and sTNF RII in 24 h supernatants from BV-2 cells maintained

in the absence of A. culbertsoni were similar to those of BV-2 cells co-cultured with A.

culbertsoni (Figure 13). Augmented levels of G-CSF (4.5 fold increase), IL-12p40/70

(3.7 fold increase), and TNF-α (4.7 fold increase) were observed from co-culture

supernatants as compared to supernatants from BV-2 cell cultures maintained in the

absence of A. culbertsoni for 24 h (Figure 13). An increase in IL-1α levels (1.7 fold

increase) was also observed in supernatants from co-cultures of BV-2 cells and A.

culbertsoni (Figure 13). In addition, a decrease in PF-4 (2.2 fold decrease) and normal T-

cell expressed and secreted (RANTES) (2.1 fold decrease) was observed from co-culture

supernatants as compared to supernatants for BV-2 cell cultures maintained in the

absence of A. culbertsoni for 24 h (Figure 13). Notably, there was an overall decrease in

MIP-3α (5.0 fold decrease) and TNF-α (3.8 fold decrease) levels in 24 h co-culture

supernatants as compared to 9 h co-culture supernatants (Figures 12 and 13).

Light microscopy was used to observe the morphology of BV-2 cells, A.

culbertsoni, and BV-2 cell plus A. culbertsoni co-cultures maintained in CNBA for 9-24

h (Figure 14). BV-2 cells maintained in the absence of A. culbertsoni (9-24 h) were

attached to the plastic substratum and displayed a ramified morphology, consistent with

these microglial-like cells being in a resting state (Figure 14 A-C). A. culbertsoni

maintained in the absence of BV-2 cells (9-24 h) were observed attached to the plastic

substratum and in their trophozoite form with multiple vacuoles (Figure 14 D-F). BV-2

cells co-cultured with A. culbertsoni (9-24 h) were observed in a rounded (i.e., amoeboid)

43

Figure 14: Light Microscopy Images of BV-2 Cells, A. culbertsoni, and BV-2 Cells

Plus A. culbertsoni Co-cultures. 106 BV-2 cells, 10

6 A. culbertsoni, and 10

6 BV-2 cells

plus 106 A. culbertsoni were cultured for 9 h (A, D, G), 15 h (B, E, H), and 24 h (C, F, I).

(A-C) BV-2 cell cultures, (D-F) A. culbertsoni cultures, and (G-I) BV-2 cell plus A.

culbertsoni co-cultures. Arrows in A-C indicate BV-2 cell processes associated with

ramified morphology. Arrows in D-F indicate vacuoles associated with A. culbertsoni

trophozoites. Arrows in G-H indicate BV-2 cells. 40x magnification.

A

B

C I

H

G D

E

F

44

form and detached from the plastic substratum, which is consistent with these cells being

in a reactive or cytotoxic state (Figure 14 G-I). Notably, BV-2 cells maintained 24 h in

the presence of A. culbertsoni were observed to be over 90% detached from the plastic

substratum (Figure 14 I). A. culbertsoni morphology in co-culture with BV-2 cells was

similar to that for cultures of A. culbertsoni maintained in the absence of BV-2 cells

(Figure 14 G-I).

Acanthamoeba culbertsoni Elicited Factors Degrade Constitutively-Expressed

Microglial-Like Cell Cytokines and Chemokines.

In order to determine further the effect of A. culbertsoni on cytokines and

chemokines elicited from BV-2 cells, BV-2 cells were cultured in the absence (8 and 18.5

h) or presence (4-18.5 h) of 0.70 mg/ml of ACM-HI and levels of cytokines and

chemokines were assessed by cytokine/chemokine protein microarray (Figure 15). A

concentration of 0.70 mg/ml ACM-HI represented a 1:2.5 dilution (dilution factor

calculated based on the average ACM-HI concentration in all experiments represented) of

the original ACM-HI medium. Supernatant from BV-2 cells maintained in the absence of

ACM-HI for 8 h elicited MCP-1, MIP-1α, MIP-1γ, MIP-2, PF-4, P-Selectin, regulated on

activation, RANTES, sTNF RI, and sTNF RII (Figure 15). The relative amount of all of

these immune factors was increased in supernatants from BV-2 cells maintained in the

absence of ACM-HI for 18.5 h (Figure 15). In addition, eotaxin-2 also was elicited by

BV-2 cells in the absence of ACM-HI following culture for 18.5 h (Figure 15). In

contrast, supernatant from BV-2 cells maintained in the presence of ACM-HI for 4-8 h

45

Figure 15: Conditioned Medium from A. culbertsoni Degrades Constitutively

Expressed Cytokines and Chemokines Elicited by BV-2 Cells. The RayBio® Mouse

Cytokine Antibody Array III arrays were used to screen for cytokines and chemokines in

supernatants from BV-2 cell cultures maintained in Neurobasal-A medium (control

medium; BV-2) for 8-18.5 h or in ACM-HI (BV-2 + ACM-HI) (0.70 mg/ml) for 4-18.5

h. Chemokines are labeled: (5) MCP-1, (6) MIP-1α, (7) MIP-1γ, (8) MIP-2, (11) PF-4,

(12) P-Selectin, (13) RANTES, (15) sTNF RI, (16) sTNF RII, and (17) Eotaxin-2.

Internal standards are labeled “Std”.

46

exhibited overall diminished levels of constitutively expressed cytokines and chemokines

(Figure 15). In addition, culturing BV-2 cells in the presence of ACM-HI for 18.5 h

resulted in the complete depletion of constitutively expressed cytokines and chemokines

(Figure 15).

Morphological studies using light microscopy revealed that BV-2 cells

maintained for 18.5 h in the absence of ACM-HI displayed a ramified morphology

(Figure 16). However, BV-2 cells exposed to ACM-HI for 4-8 h displayed an amoeboid

morphology consistent with BV-2 cells being in an active state or exhibiting incipient

cytotoxicity. Maintenance of BV-2 cells in the presence of ACM-HI for 18.5 h resulted

in an amoeboid morphology that exhibited membrane blebbing in > 50% of BV-2 cells,

ruptures in cell membranes, and extrusion of cytosol (Figure 16). These observations

are in agreement with factors elicited from A. culbertsoni, present in high concentration,

being cytotoxic to BV-2 cells.

In order to further confirm if factors elicited from A. culbertsoni could deplete

BV-2 cell pro-inflammatory cytokines and chemokines, BV-2 cells were exposed to 100

ng/ml LPS for 8 h, culture supernatant was collected (LPS/MCM), then incubated in the

absence or presence of ACM-HI (0.70 mg/ml) for an additional 8 h. The presence of

cytokine and chemokine proteins in resulting supernatants was assessed by

cytokine/chemokine protein microarray (Figure 17). A concentration of 0.70 mg/ml

ACM-HI represented a 1:2.5 dilution (dilution factor calculated based on the average

ACM-HI concentration in all experiments represented) of the original ACM-HI medium

to LPS/MCM. LPS treatment of BV-2 cells resulted in a robust induction of cytokines

47

Figure 16: Effect of 109 A. culbertsoni-Conditioned Medium (ACM-HI) on BV-2 Cell

Morphology. BV-2 cells were cultured in NeurobasalA medium (control medium) or

treated with ACM-HI (0.70 mg/ml) for 4-18.5 h. Arrows indicate areas of cytosol

extrusion at 18.5 h. 40x magnification.

48

Figure 17: Conditioned Medium from A. culbertsoni Degrades Cytokines and

Chemokines Elicited from Lipopolysaccharide (LPS) Stimulated BV-2 Cells. BV-2

cells were stimulated (8 h) with 100 ng/ml LPS and culture supernatant (LPS/MCM) was

collected. Then, LPS/MCM was incubated with ACM-HI (0.70 mg/ml) (B) or an equal

volume of complete Neurobasal™-A medium (control) for 8 h (A). Cytokines and

chemokines are labeled: (1) G-CSF, (4) IL-12p40/70, (5) MCP-1, (6) MIP-1α, (7) MIP-

1γ, (8) MIP-2, (13) RANTES, (14) TNF-α (15) sTNF RI, (16) sTNF RII, and (18) GM-

CSF, (19) IL-6.

49

and chemokines. BV-2 cell stimulation with LPS induced the production of G-CSF,

granulocyte macrophage-colony stimulating (GM-CSF), interleukin-6 (IL-6), IL-

12p40/70, MCP-1, MIP-1α, MIP-1γ, MIP-2, RANTES, TNF-α, sTNF RI, and sTNF RII

(Figure 17). Thus, the profile for LPS induction of cytokines and chemokines from BV-

2 cells differed from that resulting from induction with A. culbertsoni. In particular,

robust levels of IL-6 were elicited in the presence of LPS and not in the presence of A.

culbertsoni. Incubation of LPS/MCM with ACM-HI (0.70 mg/ml) for 8 h resulted in

nearly complete depletion of cytokines and chemokines (Figure 17).

Acanthamoeba culbertsoni Serine Proteases Are Associated with the Degradation of

A. culbertsoni-Stimulated Microglial-Like Cell Cytokines and Chemokines.

In order to determine whether A. culbertsoni proteases could exert a similar effect

on cytokines and chemokines elicited from BV-2 cells in the presence of A. culbertsoni,

BV-2 cells and A. culbertsoni were co-cultured for 24 h (1:1 ratio), the culture

supernatants (presumably containing amoebae-elicited proteases plus cytokines and

chemokines produced by BV-2 cells) were collected and incubated for an additional 72

h, and the presence of cytokine and chemokine proteins were assessed by protein

microarray (Figure 18). Using this approach, a decrease in MIP-1α (8.3 fold decrease),

PF-4 (2.1 fold decrease), and RANTES (2.1 fold decrease) was observed from co-culture

supernatants as compared to supernatants for BV-2 cell cultures maintained in the

absence of A. culbertsoni (Figure 18). Levels of TNF-α and sTNF RI were also

decreased (1.8 fold decrease and 1.7 fold decrease, respectively) (Figure 18).

50

Figure 18: Acanthamoeba culbertsoni Proteases Degrade A. culbertsoni Induced

Microglial Chemokines from BV-2 Cells. BV-2 cells (106) were cultured alone or co-

cultured with A. culbertsoni (106) for 24 h. The supernatant was harvested and incubated

an additional 72 h and the relative amount of cytokines and chemokines present was

measured using RayBio® Mouse Cytokine Antibody Array III assay. (A) Membrane

array for culture supernatant from BV-2 cells (MCM). (B) Membrane array for culture

supernatant from BV-2 cells co-cultured with A. culbertsoni (MCM/ACM). (C)

Graphical depiction comparing select chemokines. Chemokines are labeled: (5) MCP-1,

(6) MIP-1α, (7) MIP-1γ, (8) MIP-2, (11) PF-4, (13) RANTES, (14) TNF-α, and (15)

sTNF RI. + indicates a ≥ 2.1 fold decrease as compared to MCM.

51

Protease activity associated with culture supernatants from Figure 18 was

assessed by gelatin zymography. BV-2 cells maintained in the absence of A. culbertsoni

did not exhibit protease activity (Figure 19). In contrast, A. culbertsoni co-cultured with

BV-2 cells (24 h) exhibited protease activity at zymogram gel positions that corresponded

to 187, 97, and 58 kDa. This protease activity remained stable following an additional 72

h of incubation at 37ºC (Figure 19).

In order to determine if A. culbertsoni serine proteases were specifically linked to

A. culbertsoni stimulated BV-2 cell cytokine and chemokine degradation, BV-2 cells

were co-cultured with A. culbertsoni for 9-24 h, supernatants were collected and

incubated an additional 12-48 h in the presence or absence of PMSF. The levels of TNF-

α, MIP-1α, and MIP-2 proteins in resulting supernatants were assessed by ELISA.

Previous assessment of cytokines and chemokines using protein microarrays indicated

that TNF-α expression was optimal at 9 h and that MIP-1α and MIP-2 levels were stable

up to 24 h. Thus, 9 h and 24 h co-culture times were chosen for assessment of TNF-α

and for MIP-1α and MIP-2, respectively. TNF-α, MIP-1α and MIP-2 were chosen due to

their roles in: i) recruiting microglia/macrophages (MIP-1α), T cells (MIP-1α), and

neutrophils (MIP-2) and ii) activating microglia/macrophages for amoebicidal activity

(TNF-α). TNF-α, MIP-1α and MIP-2 present in co-culture supernatants in the absence

of PMSF were degraded in a time-related manner (Figures 20-22). Specifically, TNF-α

was decreased by approximately 80% in co-culture supernatants maintained for an

additional 12 h as compared to levels in initial co-culture supernatants (Figure 20).

52

Figure 19: Acanthamoeba culbertsoni Protease Activity Persists in Co-culture

Supernatants Following Incubation for 72 h. BV-2 cells (106) were cultured alone or

co-cultured with A. culbertsoni (106) for 24 h. The supernatant was harvested, incubated

an additional 72 h, and protease activity of an equal volume of each supernatant was

assessed using gelatin zymography. Supernatants from BV-2 cells alone and BV-2 cells

co-cultured with A. culbertsoni are designated as “M” and “M/A”, respectively.

Supernatants from 24 h cultures were considered as the starting time and are labeled 0 h.

53

Figure 20: Acanthamoeba culbertsoni Serine Proteases Degrade TNF-α. Enzyme

linked immunosorbent assay (ELISA) was used to measure TNF-α produced by BV-2

cells (106) co-cultured with A. culbertsoni (10

6) for 9 h. The culture supernatant was

collected and incubated an additional 12-48 h with or without the serine protease

inhibitor PMSF (1.0 mM). The percent maximum response was calculated by comparing

the optical density of samples that were incubated for an additional 12-48 h to optical

density of the starting co-culture supernatant (i.e., 9 h co-culture supernatant). The

optical density of the co-culture supernatant at 9 h was equivalent to 1695 pg/ml TNF-α

and was considered the 100% value. Samples were performed in triplicate and the

average optical density of each sample was used to calculate percent maximum response.

54

Figure 21: Acanthamoeba culbertsoni Serine Proteases Degrade MIP-1α. Enzyme

linked immunosorbent assay (ELISA) was used to measure MIP-1α produced by BV-2



collected and incubated an additional 12-48 h with or without the serine protease

inhibitor PMSF (1.0 mM). The percent maximum response was calculated by comparing

the optical density of samples that were incubated for an additional 12-48 h to optical

density of the starting co-culture supernatant (i.e., 24 h co-culture supernatant). The

optical density of the co-culture supernatant at 24 h was equivalent to 3983 pg/ml MIP-

1α and was considered the 100% value. Samples were performed in triplicate and the

average optical density of each sample was used to calculate percent maximum response.

55

Figure 22: Acanthamoeba culbertsoni Serine Proteases Degrade MIP-2. Enzyme

linked immunosorbent assay (ELISA) was used to measure MIP-2 produced by BV-2



collected and incubated an additional 12-48 h with or without the addition of the serine

protease inhibitor PMSF (1.0 mM). The percent maximum response was calculated by

comparing the optical density of samples that were incubated for an additional 12-48 h to

optical density of the starting co-culture supernatant (i.e., 24 h co-culture supernatant).

The optical density of the starting co-culture supernatant was equivalent to 5247 pg/ml

MIP-2 and was considered the 100% value. Samples were performed in triplicate and

the average optical density of each sample was used to calculate percent maximum

response.

56

Furthermore, TNF-α levels in co-culture supernatants were shown to be decreased by

approximately 97% following an additional 48 h incubation as compared to levels

observed in initial co-culture supernatants. An approximate 88% and 99% reduction in

MIP-1α levels was observed in co-culture supernatants maintained for an additional 12 h

and 48 h, respectively, as compared to levels in initial co-culture supernatants (Figure

21). In addition, MIP-2 levels were diminished by approximately 50% and 96% in co-

culture supernatants maintained for an additional 12 h and 48 h, respectively, as

compared to levels in initial co-culture supernatants (Figure 22). Degradation of TNF-α,

MIP-1α, and MIP-2 in co-culture supernatants was reversed in the presence of PMSF,

consistent with A. culbertsoni serine proteases as playing a role in their degradation.

Specifically, in the presence of PMSF an approximate 60% and 70% difference in TNF-α

and MIP-1α levels, respectively, was observed in co-culture supernatants incubated for an

additional 12 h as compared to co-culture supernatants maintained an additional 12 h in

the absence of PMSF (Figures 20 and 21). The presence of PMSF in co-culture

supernatant resulted in the full recovery of MIP-2 levels at all time points studied (Figure

22). Although there was an overall increase in TNF-α and MIP-1α levels in co-culture

supernatants maintained in the presence of PMSF as compared to co-culture supernatants

maintained in the absence of PMSF, a time-related decrease of TNF-α and MIP-1α also

was observed in co-culture supernatants maintained for an additional 12-48 h in the

presence of PMSF (Figures 20 and 21). These observations may represent a natural

breakdown of these factors within the extracellular milieu; or, they may represent the

degradation of these factors by other A. culbertsoni elicited enzymes.

57

Discussion

Acanthamoeba are free-living amoebae that are found worldwide and can cause

opportunistic infections in humans. Acanthamoeba have been isolated from a variety of

environmental sources including air, soil, dust, tap water, freshwater, seawater,

swimming pools, air conditioning units, and contaminated lens cases [Marciano-Cabral

and Cabral 2003]. Acanthamoeba have two life cycle stages; the trophozoite and the

cyst. The trophozoite stage represents the feeding as well as the infective form of

Acanthamoeba [Rodriquez-Zaragoza et al. 1994; Marciano-Cabral and Cabral 2003;

Visvesvara et al. 2007]. The cyst stage represents a dormant phase that is formed under

adverse environmental conditions such as extreme temperature or pH changes [Marciano-

Cabral and Cabral 2003].

Acanthamoeba secrete a plethora of enzymes including phospholipases and

proteases. It has been demonstrated that pathogenic Acanthamoeba secrete more

phospholipase A and proteases than non-pathogenic Acanthamoeba [Cursons et al. 1978;

Misra et al. 1983; Hadás et al. 1993b; Khan et al. 2000]. Additionally, it has been

demonstrated that pathogenic Acanthamoeba produce serine, cysteine, and metallo-

proteases [He et al. 1990; Hadás et al. 1993a; Mitro et al. 1994; Mitra et al. 1995; Cao et

al. 1998; Alfieri et al. 2000; Cho et al. 2000; Hong et al. 2000; Khan et al. 2000; Kong et

al. 2000; Na et al. 2001; Hong et al. 2002; Na et al. 2002; Alsam et al. 2005 ; Kim et al.

58

2006; Sissons et al. 2006]. In our studies a comparison of two-dimensional (iso-dalt) gel

electrophoresis proteomic profiles of complete Neurobasal™-A medium versus A.

culbertsoni-conditioned complete Neurobasal™-A medium (i.e., A. culbertsoni-

conditioned medium) suggested that A. culbertsoni secrete factors that are able to degrade

albumin and putatively other proteins during in vitro culture. Our findings are consistent

with studies by Kong et al. [2000] that demonstrated that a serine protease from A. healyi

degrades albumin. Using gelatin zymography, we detected A. culbertsoni-secreted serine

proteases corresponding to positions at 187, 97, 90, and 58 kDa. However, protease

activity located at the position corresponding to 90 kDa was not always observed in our

gelatin zymography assays of A. culbertsoni-conditioned medium. Thus, it is possible

that protease activity detected at this position could represent a breakdown product. Our

gelatin zymography results are consistent with those obtained from previous studies that

demonstrated that pathogenic T4 genotype Acanthamoeba secrete serine proteases at

positions corresponding to 188, 97, and 55 kDa [Cao et al. 1998; Serrano-Luna et al.

2006]. We propose that the A. culbertsoni serine proteases of the approximate molecular

weights of 187, 97, and 58 kDa identified in our studies likely represent serine proteases

similar to that of pathogenic Acanthamoeba T4 genotype identified at 188, 97, and 55

kDa, respectively. Differences between the relative molecular weights of serine

proteases identified in our studies and those of others are likely due to variations in the

construction of zymography gels. Thus, our results suggest that the T10 genotype A.

culbertsoni used in our studies may secrete similar serine proteases as pathogenic T4

genotype Acanthamoeba; suggesting that certain proteases may convey pathogenicity.

59

Using the cysteine protease inhibitor E-64 we did not observe cysteine protease activity

in A. culbertsoni-conditioned medium. Studies by Alferi et al. [2000] indicate that

Acanthamoeba cysteine proteases are most active at pH 4.0-5.0. Our gelatin zymogram

studies were performed at pH 5.0 and 7.5. Furthermore, Serrano-Luna et al. [2006] were

not able to detect cysteine protease activity in Acanthamoeba conditioned medium using

E-64 at pH 3.0 - 9.0; but, they were successful at detecting cysteine protease activity

using the cysteine protease inhibitor N-ethylmaleimide (NEM) [Serrano-Luna et al.

2006]. Thus, their results suggest that E-64 may not be the optimal inhibitor to use for

the detection of cysteine protease activity in Acanthamoeba-conditioned medium. Using

gelatin zymography, we also demonstrated that A. culbertsoni propagation medium does

not influence the proteolytic profile of A. culbertsoni; however, levels of protease activity

secreted from A. culbertsoni were affected. Our results suggest that the higher iron

content in oxoid medium, which contains neutralized liver digest, as compared to that in

PYG medium influences the level of protease activity elicited from A. culbertsoni. Our

results are consistent with those of Melo-Braga et al. [2003] that demonstrated that iron-

depletion reduces cysteine protease activity elicited from the bovine pathogen

Tritrichomonas foetus. Collectively, our results indicate that A. culbertsoni secrete

predominantly serine proteases and highlight the importance of the selection of medium

used for the growth of Acanthamoeba in various studies since these may affect levels of

secreted proteases. Acanthamoeba serine proteases are thought to serve a functional role

in encystment and excystment of amoeba while they inhabit the environment [Dudley et

60

al. 2008; Moon et al. 2008]. However, it is also thought that serine proteases may play a

role in Acanthamoeba host invasion and immune evasion.

Only a select group of Acanthamoeba species, including A. culbertsoni, have been

linked to disease in humans. Acanthamoeba are known to cause three types of infections

in humans; AK, cutaneous acanthamoebiasis, and GAE [Marciano-Cabral and Cabral

2003; Khan 2006; Visvesvara et al. 2007]. AK is a potentially blinding eye infection that

is most frequently associated with contact lens wearers and it the most common disease

caused by Acanthamoeba [Clarke and Niederkorn 2006a]. Cutaneous acanthamoebiasis

is an Acanthamoeba skin infection that can result in hematogenous spread of

Acanthamoeba throughout the host. Cutaneous acanthamoebiasis is commonly

associated with immune compromised individuals. Acanthamoeba can also cause a

chronic and slow progressing CNS disease called GAE.

GAE is most prevalent in immune compromised individuals [Marciano-Cabral

and Cabral 2003]. It is believed that in humans GAE is most often caused by the entry of

trophozoites into the CNS via hematogenous spread originating from the lungs or

cutaneous infection [Marciano-Cabral and Cabral 2003; Khan 2008]. However,

Acanthamoeba trophozoites may also gain access to the CNS via the olfactory

neuroepithelium. Studies by Alsam et al. [2005], using a human brain endothelial cell

model, implicated Acanthamoeba serine proteases in the induction of blood brain barrier

permeability; thus, demonstrating a potential role of Acanthamoeba serine proteases in

CNS invasion leading to GAE. GAE is characterized by the formation of granulomas

within the CNS; however, granulomas are rarely observed in immune compromised

61

individuals [Martinez 1982]. Granulomas are usually composed of amoebae, microglia,

macrophages, T cells, B cells, and neutrophils.

Microglia, resident brain macrophages, and invading peripheral macrophages

have been indicated as the primary immune cells that elicit a direct response to CNS

invading Acanthamoeba [Marciano-Cabral and Cabral 2003]. It has been reported that

primary rat microglia release pro-inflammatory mediators, such as IL-1α, IL-1β, and

TNF-α in response to Acanthamoeba [Marciano-Cabral et al. 2004]. Reports also

indicate that such cytokines are not cytolytic to Acanthamoeba and, rather, may serve to

activate microglial/macrophage cells for contact-dependent mediated killing of

Acanthamoeba [Marciano-Cabral et al. 1998]. Indeed, activated microglia and

macrophages have been demonstrated to be cytolytic (i.e., amoebicidal) to Acanthamoeba

[Benedetto et al. 2002; Benedetto et al. 2003]. We demonstrated that BV-2 microglial-

like cells produce mRNAs for MCP-1, MIP-1α, MIP-1β, and MIP-2 in response to A.

culbertsoni. These observations suggest that exposure of the microglial-like cells to

Acanthamoeba resulted in de novo gene induction. The results were extended at the

protein level wherein MCP-1, MIP-1α, and MIP-1γ were constitutively produced by BV-

2 cells at relatively high levels at early time periods (i.e., 9 h). Furthermore, we

demonstrated that A. culbertsoni augmented the protein level of G-CSF, IL-1α, IL-

12p40/70, TNF-α, and MIP-3α secreted by BV-2 cells. It is important to note that in our

studies we did not observe an A. culbertsoni-dependent induction of BV-2 cell-elicited

IL-1β expression as demonstrated in previous studies [Shin et al. 2001; Marciano-Cabral

et al. 2004]. Thus, differences between our results and those of others are likely due to

62

the differences in microglial cell type and/or culture conditions used. G-CSF stimulates

the growth and activation of neutrophils [Fitzgerald et al. 2001]. IL-1α, IL-1β, and TNF-

α are microglial, macrophage, and T cell activating cytokines. Furthermore, TNF-α plays

an instrumental role in the activation of microglial and macrophage anti-microbial

activity via the activation of phagocytosis. IL-12 serves as an important signal for the

induction of the Th1 cell phenotype and also serves as a stimulatory signal for Th1

cytokine secretion; and microglia are thought to represent the main source of IL-12

within the CNS [Stalder et al. 1997; Aloisi 2001]. MIP-3α stimulates the recruitment of

activated and memory T cells, dendritic cells, and B cells [Dieu et al. 1998; Tanaka et al.

1999]. Overall, our results suggest that microglia elicit a variety of cytokines and

chemokines in response to A. culbertsoni which are capable of autocrine and paracrine

immune cell activation facilitating the formation of granulomas via the recruitment of

immune cells, including microglia, macrophages, T cells, B cells, and neutrophils, to

focal sites of Acanthamoeba within the CNS. It is important to note that we also

observed a decrease in sTNF RI and s TNF RII levels in supernatants from BV-2 cells

stimulated with A. culbertsoni for 9 h as compared to supernatants from un-stimulated

BV-2 cells. sTNF RI and sTNF RII represent soluble forms of TNF RI and TNF RII,

respectively, and their role in CNS immunity has not been fully elucidated; however,

studies have indicated a link between increased levels sTNF RI in cerebrospinal fluid to

the clinical onset of multiple sclerosis [Tsukada et al. 1993]. In contrast to the induction

of pro-inflammatory factors observed at 9 h co-culture, we observed a decrease in levels

of MCP-1, MIP-1α, MIP-1γ and PF-4 at 9 h. MCP-1 and MIP-1α stimulate the

63

chemotaxis of microglia, macrophages, dendritic cells, and T helper 1 (Th1) cells

[Peterson et al. 1997; Dieu et al. 1998; Aloisi 2001]. MIP-1γ serves as a chemotactic

signal for dendritic and memory T cells. PF-4 recently has been reported to induce the

migration of microglia via the CXCR3 chemokine receptor [de Jong et al. 2008]. In

addition, we detected a decrease in PF-4 and RANTES levels in supernatants from BV-2

cells stimulated with A. culbertsoni for 24 h as compared to supernatants from un-

stimulated BV-2 cells. RANTES induces the migration of microglia, macrophages, T

cells, and dendritic cells [Aloisi 2001]. We also observed an overall decrease in the

levels of TNF-α and MIP-3α protein at 24 h. The decreases in the aforementioned

cytokines and chemokines could represent changes in the transcriptional or translational

regulation of these BV-2 cell-elicited factors in the presence of A. culbertsoni or,

alternatively, could reflect the degradation of these factors within the extracellular milieu.

Our studies implicate proteases that are secreted by Acanthamoeba as playing a

major role in the decrease in levels of chemokines and cytokines in the extracellular

milieu. In this context, previous studies have demonstrated that of Acanthamoeba serine

proteases are able to degrade immune modulating factors such as sIgA, IgG, IgM, IL-1α,

and IL-1β, implicating these enzymes as playing a role in host immune evasion [Kong et

al. 2000; Na et al. 2001; Na et al. 2002]. However, in these studies recombinant

exogenous substrates were used. Our studies are novel in that they demonstrated that

proteases elicited by Acanthamoeba degrade cytokines and chemokines natively secreted

by microglial-like cells. That is, we observed that constitutively-expressed as well as

LPS-stimulated cytokines and chemokines from BV-2 cells were degraded in the

64

presence of conditioned medium from A. culbertsoni cultures. Constitutively expressed

BV-2 cell cytokines and chemokines which were degraded in the presence of A.

culbertsoni- elicited factors included eotaxin-2, MCP-1, MIP-1α, MIP-1γ, MIP-2, PF-4,

P-Selectin, RANTES, sTNF RI and sTNF RII. In addition, LPS-stimulated cytokines and

chemokines were degraded. LPS-stimulated BV-2 cell cytokines and chemokines which

were degraded in the presence of A. culbertsoni-elicited factors included G-CSF, GM-

CSF, IL-6, IL-12p40/p70, MCP-1, MIP-1α, MIP-1γ, MIP-2, RANTES, TNF-α, sTNF RI,

and sTNF RII. It is important to note that the pattern of cytokines and chemokines

elicited by LPS-stimulated BV-2 cells was different than that of A. culbertsoni-

stimulated BV-2 cells. In particular, LPS caused BV-2 cells to elicit a high level of IL-6

which was not detected when BV-2 cells were stimulated with A. culbertsoni. Moreover,

MIP-3α and MIP-3β were elicited by A. culbertsoni-stimulated BV-2 cells and not by

LPS-stimulated BV-2 cells. Furthermore, our studies suggest that A. culbertsoni serine

proteases are specifically linked to the degradation of microglial cytokines and

chemokines (e.g. TNF-α, MIP-1α, and MIP-2). Our results parallel findings of others

that demonstrate that the serine proteases cathepsin G, elastase, and proteinase 3 degrade

human MIP-1α; and that a Group A streptococcal serine protease degrades MIP-2 [Ryu et

al. 2005; Hidalgo-Grass et al. 2006]. Furthermore, studies by Ferrante and Bates [1988]

indicate that A. culbertsoni elicit an elastase; thus, it can be postulated that this serine

protease may also be linked to the degradation of microglial-secreted cytokines and

chemokines. Collectively, our results suggest that A. culbertsoni-elicited factors degrade

endogenously produced BV-2 cell-elicited cytokines and chemokines, indicating a mode

65

by which A. culbertsoni applies a contact-independent mechanism to dampen microglial

facilitated CNS immune cell responsiveness which, in turn, may lead to the downstream

diminution of granuloma formation and microglial/macrophage anti-microbial activity.

We also detected a time-related augmentation in the level of serine protease

activity from A. culbertsoni in the presence of BV-2 cells. The augmentation in A.

culbertsoni serine protease activity in conditioned medium may be linked to A.

culbertsoni binding to microglial-like cells via trophozoite expressed mannose-binding

protein (MBP), and is consistent with reports that binding of Acanthamoeba to mannose

on human brain microvascular endothelial cells via Acanthamoeba-expressed MBP

increases the amount of secreted protease [Alsam et al. 2003; Alsam et al. 2005]. One

mode by which A. culbertsoni serine proteases affect microglial responsiveness may be

through activation of microglial-expressed receptors called protease-activated receptors

(PARs), the activation of which results in the release of microglial cytokines and

chemokines. PARs are a group of G-protein coupled receptors that are activated by

serine and cysteine proteases. To date, four PARs have been described; PAR1, PAR2,

PAR3, and PAR4. PARs are expressed by a variety of cells including platelets,

endothelial cells, epithelial cells, monocytes, T cells, natural killer (NK) cells, astrocytes,

neurons, and microglia [Balcaitis et al. 2003; Steinhoff et al. 2005]. We have confirmed

published reports that BV-2 cells express mRNA for PAR1, PAR3, and PAR4 (data not

shown) [Balcaitis et al. 2003]. PARs are activated when proteases recognize and cleave a

specific amino acid sequence located at the N-terminus of the receptor; the resulting N-

terminus acts as a tethered ligand by binding back on the receptor’s extracellular loop

66

region. PAR1 can be activated by thrombin, Factor Xa, plasmin, and activated protein C

[Ossovskaya and Bunnett 2004; Steinhoff et al. 2005]. PAR2 can be activated by trypsin,

tryptase, Factor VIIa, Factor Xa, and exogenous proteases such as gingipain-R of

Porphyromonas gingivalis [Ossovskaya and Bunnett 2004; Steinhoff et al. 2005]. PAR3

is believed to act as a cofactor for PAR4; and, PAR3 and PAR4 can be activated by

thrombin and cathepsin G [Nakanishi-Matsui, 2000; Steinhoff et al. 2005]. Activation of

PARs has been linked to inflammation [Steinhoff et al. 2005]. Specifically, it has been

suggested that PAR1 activation leads to the production of IL-1, IL-6, and MCP-1 by

monocytes [Colotta et al. 1994; Naldini et al. 2000; Naldini et al. 2002; Steinhoff et al.

2005; Li et al. 2006]. PAR1 activation has also been linked to IL-8 production in

macrophages [Zheng et al. 2007]. Activation of monocyte expressed PAR2 has been

demonstrated to induce IL-1β, IL-6, and IL-8 production [Johansson et al. 2005]. PAR4

has been linked to IL-6 production by monocytes [Li et al, 2006]. Thus, we propose that

it is possible that A. culbertsoni serine proteases act through BV-2 microglial-like cell-

expressed PARs leading to the secretion of cytokines and chemokines observed in our

studies. However, further studies using specific PAR agonists and antagonists/blockers

would be required in order to establish a functional linkage between in A. culbertsoni-

elicited serine protease and microglial-cell cytokine/chemokine production.

Proteases acting through PAR receptors have been linked to cell apoptosis

[Steinhoff et al. 2005]. For example, PAR1 thrombin activation has been demonstrated

to induce neuronal cell apoptosis [Smirnova et al. 1998]. These observations are

consistent with our results from our light microscopy studies which demonstrated that

67

conditioned medium from A. culbertsoni is toxic to BV-2 cells suggestive of apoptosis.

Additionally, these findings are in agreement with those that demonstrated that

Acanthamoeba elicited factors induce morphological changes and lysis of epithelial cells

[Mattana et al. 1997].

In summary, while it is likely that Acanthamoeba uses a multiplicity of contact-

dependent and contact-independent mechanisms to elicit pathology within the CNS

during GAE, our results suggest that Acanthamoeba have developed strategies via the

elicitation of serine proteases to aid immune evasion in that compartment. Specifically,

we postulate that serine proteases released by Acanthamoeba initially act on microglia to

induce the production of a plethora of cytokines and chemokines, possibly mediated

through the activation of PAR receptors on microglia. These cytokines and chemokines

elicited by microglia in response to Acanthamoeba serve to activate and recruit additional

microglia, macrophages, T cells, and neutrophils to focal sites of infection leading to the

formation of granulomas. Within these granulomas it is postulated that microglia and

macrophages are activated by T cell-elicited IFN-γ in combination with T

cell/microglial/macrophage-elicited TNF-α, leading to microglial/macrophage-dependent

amoebicidal activity [Benedetto et al. 2002; Benedetto et al. 2003]. However, under

conditions of high Acanthamoeba burden when relatively high concentrations of

Acanthamoeba may be present, we suggest that the attendant higher concentration of

Acanthamoeba serine proteases leads to the degradation of microglial cytokines and

chemokines. This diminution, in turn, may lead to a decrease in microglial/macrophage

activation resulting in the decrease of granuloma formation and microglial/macrophage

68

anti-microbial activity. The resultant outcome is dissemination of Acanthamoeba within

the CNS and progressive neuropathology. Additionally, our results suggest that

Acanthamoeba proteases induce microglial cell apoptosis, thus, representing a means by

which these opportunistic pathogens may process microglia as a source of food. In

conclusion, our results suggest that Acanthamoeba serine proteases provide a multi-tailed

approach for Acanthamoeba to effectively evade host CNS immunity. Furthermore, it

can be postulated that appropriately designed therapeutics which block the potential

activation of microglial expressed PARs by Acanthamoeba elicited serine proteases used

in combination with current therapeutic approaches could prove to be an effective means

of benefiting the clinical outcome of patients with GAE.

69

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