Microarray analysis of Haemophilus parasuis gene expression

under in vitro growth conditions mimicking

the in vivo environment

Elena Melnikow a, Saffron Dornan c, Carole Sargent c, Michael Duszenko b,Gary Evans c, Nikolas Gunkel a, Paul M. Selzer a, Heinz J. Ullrich a,*

a Intervet Innovation, Drug Discovery, Zur Propstei, 55270 Schwabenheim, Germanyb Interfakultares Institut fur Biochemie, Universitat Tubingen, Hoppe-Seyler-Strasse 4, 72076 Tubingen, Germany

c Sygen International, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK

Received 22 February 2005; received in revised form 19 July 2005; accepted 1 August 2005

Abstract

Haemophilus parasuis is the causative agent of polyserositis in pigs, a mostly fatal disease on the rise especially in early-

weaned pigs and in pig herds with a high-health status. The mechanisms by which H. parasuis propagates through the body and

colonizes the serous membranes are unknown. We have used an H. parasuis microarray to identify virulence genes involved in

host adaptation. H. parasuis gene expression was analysed under in vitro growth conditions mimicking the environmental

conditions encountered during an infection. These included iron-limitation, acidic and temperature stress and growth under

microaerobic conditions. A kinetic impression of the gene regulation was obtained by analysing the transcription 10, 30 and

60 min after induction of the altered growth conditions. A total of 75 regulated H. parasuis genes were identified, most of which

coded for transporters of iron and sugar metabolites, metabolic enzymes, DNA metabolism and hypothetical proteins with

unknown functions. Furthermore, H. parasuis genes were identified that have homology to known virulence factors in other

pathogenic bacteria. Homologues of some of the identified H. parasuis genes are known to be expressed during natural and

experimental infections in pathogens of the Pasteurellaceae family.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Haemophilus parasuis; Virulence; Microarray; Gene expression

www.elsevier.com/locate/vetmic

Veterinary Microbiology 110 (2005) 255–263

* Corresponding author. Tel.: +49 6130 948267;

fax: +49 6130 948517.

E-mail address: joachim.ullrich@intervet.com (H.J. Ullrich).

0378-1135/$ – see front matter # 2005 Elsevier B.V. All rights reserved

doi:10.1016/j.vetmic.2005.08.007

1. Introduction

H. parasuis is a common opportunistic pathogen of

pigs which usually colonizes the upper respiratory

tract. Antibodies appear to play an important role in

protection of the piglet. Under some circumstances,

.

E. Melnikow et al. / Veterinary Microbiology 110 (2005) 255–263256

however, H. parasuis can cause Glasser’s disease,

which is characterized by a severe inflammation of the

serous membranes, including pleuritis, pericarditis

and peritonitis; it can also cause meningitis, arthritis

and pneumonia (Hoefling, 1991; Rapp-Gabrielson,

1999; Oliveira and Pijoan, 2004). This severe

presentation of the Glasser’s disease is commonly

observed in high health status farms in the America’s,

and is becoming increasingly important as multi-site

production systems expand (Rapp-Gabrielson, 1999).

H. parasuis infections are treated with penicillin or

ampicillin and vaccines are available, albeit with low

serotype cross-protection efficacy (Oliveira and

Pijoan, 2004). Despite the considerable loss suffered

by the pig breeding industry, not much is known about

the virulence determinants enabling H. parasuis to

successfully cause disease. Outer membrane proteins

and fimbriae have been implicated in colonization of

the lung epithelium (Munch et al., 1992). Transferrin-

binding proteins could play a role in scavenging iron

from porcine transferrin and a secreted neuraminidase

may allow the exploitation of host sialic acids as

carbon sources (Lichtensteiger and Vimr, 1997). An

involvement of LPS in septic disease progression has

been concluded from the presence of endotoxin in the

plasma and the appearance of clinical signs of

endotoxin shock in H. parasuis-infected pigs (Amano

et al., 1997).

A major obstacle to a better understanding of H.

parasuis virulence has been the paucity of studies

using genetic tools to identify virulence factors.

Recently, Hill et al. (2003) have used a differential

display-based approach for the identification of seven

H. parasuis genes up-regulated during heat stress in

the presence and absence of swine serum. Heat-

induced genes coded for proteins involved in

transport, metabolism, and in the biosynthesis of fatty

acids and amino acids. The study by Hill et al.

provided a first glance at the complexity ofH. parasuis

host adaptation, but the results were limited by the

relatively small number of primers. Here we report on

the first effort to use a H. parasuis microarray in

unravelling host adaptation on a genome-wide scale.

We placed particular emphasis on the adaptation of H.

parasuis to the extremely low free iron availability in

the host, a major barrier to colonization for all

bacterial pathogens which is overcome by the

expression of specific iron-uptake systems (Ratledge

and Dover, 2000). Likewise, we investigated the

adaptation of H. parasuis to a temperature increase, as

experienced during fever; decreases in oxygen-

availability, as experienced during systemic propaga-

tion and cyanosis, and the response of H. parasuis to

acidic stress. Dozens of previously unknown H.

parasuis genes with homology to virulence factors in

closely related organisms like H. influenzae or P.

multocida were identified, pointing to striking

similarities in host adaptation within the Pasteurella-

ceae family.

2. Materials and methods

2.1. Bacterial strains and culture conditions

The Haemophilus parasuis strain 29775, serovar 5

was used. This strain has been shown to be virulent in

vivo in pigs (Oliveira et al., 2003; Blanco et al., 2004).

The strain originated from Dr. Ross’s Laboratory at

Iowa State University, USA. H. parasuis serovar 5 was

grown on chocolate-agar plates in a 5% CO2

atmosphere at 37 8C. Liquid growth medium consisted

of 6.0 g/l casein hydrolysate, 25 g/l yeast extract,

0.5 g/l KH2PO4, 0.1 g/l MgCl2�6H2O, 2.5 g/l NaCl,

1 ml/l of 1% CaCl2�2H2O, 0.5 ml/l of 0.5% FeSO4

�7H2O, 9.0 ml/l of 50% (w/v) glucose, 0.8 ml/l of 1%

L-cysteine, 4.0 ml/l of 10% (w/v) NAD, pH 7.4.

Exponential growing cultures (OD578� 0.5) were split

into separate Erlenmeyer flasks containing 200 ml

medium in each. One culture continued to grow under

standard conditions (37 8C, 250 rpm), whilst the other

was subjected to the experimental conditions. For

acidification, 0.5 M Na-acetate was added to decrease

the pH from 7.4 to 5.5. Iron was depleted by adding

2,2’-dipyridyl (Sigma, USA) to the growth medium to

give a final concentration of 200 mM. For induction of

heat stress, cultures were incubated at 40 8C.Microaerobic conditions were obtained by resuspend-

ing exponentially growing bacteria into medium that

was oxygen-deprived by 2 h aeration with N2. Control

bacteria were also subjected to harvesting but

resuspended into aerated medium. Growth under

oxygen-deprivation was carried out in sealed Erlen-

meyer flasks. For kinetic purposes, 50 ml control and

experimental culture samples were taken 10, 30 and

60 min after induction. Bacteria were centrifuged for

E. Melnikow et al. / Veterinary Microbiology 110 (2005) 255–263 257

5 min at 5000 g and 4 8C followed by immediate RNA

isolation. Each experiment was done in triplicate to

allow statistical analysis.

2.2. Construction of DNA microarrays

A genomic library of H. parasuis serovar 5 served

as a source for the H. parasuis microarray. Six

thousands inserts from the genomic library were

amplified by PCR using the M 13 forward and reverse

primers. PCR products of approximately 1.2 kbp were

purified using the Quiagen PCR purification kit

(Quiagen, Germany) and spotted in duplicate on

GAPS-II glass slides (Corning, USA) using a

Biorobotics (Genomic Solutions, USA) spotting

device. Negative control DNA consisted of eukaryotic

DNA from pig and pBluescript.

2.3. RNA isolation and cDNA synthesis

RNA extractions were performed using the RNeasy

Maxi system from Quiagen (Germany) and quantified

spectrophotometrically (DU1 640, Beckman, USA).

Random primers were added (2.5 ml, 0.5 mg/ml,

Promega, USA) to 50 mg total RNA, and H2O was

added to give a final volume of 15.5 ml. The mixture

was incubated at 70 8C for 10 min, and snap cooled on

ice for 2 min. After cooling, 12.6 ml of an amino-allyl

master mix containing 6 ml five-fold first strand buffer

(Invitrogen, USA), 3 ml 0.1 M DTT (Invitrogen,

USA), 3 ml H2O, 0.6 ml 50-fold dNTPmix (dATP,

dCTP, dGTP 25 mM each, 10 mM dTTP (Promega,

USA), 5 mM aminoallyl dUTP (Sigma, USA)), and 40

U RNasin (Promega, USA), were added and incubated

at 42 8C for 2 min. A 200 U Superscript II reverse

transcriptase (Invitrogen, USA) was added and the

reaction was continued at 42 8C for 60 min. An

additional 200 U SuperScript II reverse transcriptase

was added and the reaction was incubated for another

1 h at 42 8C. The reaction was terminated by the

addition of 10 ml 0.5 M EDTA pH 8.0 and the RNA

was hydrolyzed by the addition of 10 ml 1 N NaOH

followed by incubation at 65 8C for 15 min. The

solution was neutralized with 25 ml 1 M Tris–HCl pH

8.0 and cooled on ice for 2 min. The cDNAs were

washed three times with 400 ml H2O using a

microcon-30 filter column (Millipore, UK). The

residual 100 ml volume was recovered, dried and

the cDNAwas solubilized in 4.5 ml H2O. The cDNAs

from cultures grown under altered environmental

conditions were labelled by adding 4.5 ml Cy3 NHS-

ester (20 nmol) in 100 mM sodium bicarbonate pH 9.3

(Amersham, USA), whilst cDNAs from control

cultures were labelled with Cy5 NHS-ester (20 nmol)

in the dark at RT for 60 min. The labelling reactions

were stopped with 4.5 ml of 4 M hydroxylamine for

15 min followed by addition of 35 ml of 0.1 M Na-

acetate pH 5.2.

2.4. Hybridizations and data analysis

For hybridizations, equal amounts of the Cy3- and

Cy5-labelled cDNA were combined and purified by

using a PCR purification system (Qiagen) according to

the manufacturer’s protocol. To the labelled cDNA,

1 ml yeast tRNA (4 mg/ml), 1 ml of polyA and 1 ml of

2 mg/ml pCRTopo (Invitrogen, USA) were added,

precipitated and resuspended in 50 ml of hybridization

buffer, consisting of Denhardt’s solution in 40%

formamide. Slides were incubated in a hybridization

chamber (Corning, USA) submerged in a water bath at

42 8C for 16 h. Microarray slides were washed for

5 min each in 1 � SSC and 0.1 � SSC followed by

five inverts in H2O and spin dried. A GenePix 4100A

scanner (Axon Instruments, USA) was used for

scanning of the microarrays. Signals were quality

checked and background-corrected using the Gene-

PixPro software (Axon Instruments, USA). Back-

ground-corrected expression values for each spot

(referred to as clones from the genomic library) were

imported into the VectorNTI Xpression 3.0 software

(Invitogen, USA). Only clones for which both

duplicates produced positive background-corrected

signal values were included in the data analysis. Mean

signal intensities for each duplicate spot were

normalized using lowess normalization which gen-

erates normalized log2 ratios of control and experi-

mental signal intensities (Quackenbush, 2002).

Control and experimental signal values for each time

point were compared using the Student’s t-test. Only

clones with a p-value <0.05 and at least 1.5-fold

difference between control and induced mean signal

values were called regulated. Reciprocal hybridiza-

tions did not reveal differences in the number or

identity of regulated clones (data not shown).

Clustering was performed using the unweighted

E. Melnikow et al. / Veterinary Microbiology 110 (2005) 255–263258

Fig. 1. Functional classification of 75 H. parasuis regulated genes.

H. parasuis genes were classified according to the functional

classification of their homologues in the databases. Numbers are

indicated in parenthesis.

average hierarchical clustering algorithm and the

correlation coefficient matrix of the VectorNTI

Xpression 3.0 software. A total of 246 clones were

selected for up regulation under more than one in vitro

condition. Clones were sequenced by the Sanger

method with T3 and T7 primers using an ABI Prism

Sequencing System and reads of each clone were

aligned using the VectorNTI (Invitrogen, USA)

software. For annotation, H. parasuis sequences were

compared against public databases using the BLAST

algorithm. Since the microarray consisted of PCR

products from random clones of a genomic library,

some clones (133) contained sequences of more than

one ORF. The transcriptional signal of these clones

could not be traced back to a unique gene, therefore,

these clones were not included in further data analysis.

Of the 113 single ORF-containing clones several

duplicates were identified, reducing the total number

to 75 individual clones (Fig. 2).

3. Results and discussion

In order to identify the Haemophilus parasuis

virulence factors mediating the adaptation to the

distinct host microenvironments, a H. parasuis

microarray was produced from a genomic library of

a virulent serovar 5 strain. H. parasuis serovar 5 is the

most prevalent isolate in North America and is

reported to be highly pathogenic (Rapp-Gabrielson

et al., 1992; Amano et al., 1996). The H. parasuis

genome has not been sequenced; therefore 6000

inserts (referred to as clones) from a genomic library

averaging 1.2 kbp in length were PCR-amplified and

spotted in duplicate on glass slides. Provided the size

of the H. parasuis genome is within the range of the

closely related organisms, Haemophilus influenzae

and Haemophilus ducreyi (1.7–1.8 Mb), the micro-

array yielded an approximate four-fold coverage. H.

parasuis was grown under paucity of iron, oxygen

limitation, heat (40 8C) and acidic (pH 5.5) stress

conditions for 10, 30 and 60 min before the bacteria

were harvested and the RNAwas isolated. The cDNA

Table 1

Number of H. parasuis clones that were up- or down-regulated during all t

Expression value Iron limitation

�1.5-fold increase over control 65

�1.5-fold decrease over control 55

from bacteria grown under experimental conditions

was labelled with Cy3 and was co-hybridized on the

microarray with Cy5-labelled cDNA from RNA

derived from bacteria grown under control conditions.

Dependent on the environmental conditions, about

2–10% of the 6000 spotted clones showed signal

intensity changes of >1.5-fold over control intensities

(Table 1). Since the H. parasuis microarray consisted

of anonymous PCR products, selected clones had to be

sequenced and annotated. In order to restrict the

number of sequencing reactions, we placed a primary

focus on clones up-regulated under multiple condi-

tions and time points. This strategy selected for genes

that are likely to be induced in the varying host niches

populated by H. parasuis, and reduced the chance of

identifying false positive clones. A total of 75 H.

parasuis genes falling into 10 functional groups were

identified (Fig. 1). Hypothetical genes with unknown

functions formed the largest group, followed by genes

involved in DNA and energy metabolism, transport of

metabolites and amino acid biosynthesis. Relative

expression values of the 75 genes for all growth

conditions and time points are presented in Fig. 2.

The phosphotransferase system (PTS) appears to

play a prominent role in H. parasuis stress adaptation.

hree times points (10, 30, 60 min) under different growth conditions

Oxygen limitation pH 5.5 40 8C

246 329 380

173 138 281

E. Melnikow et al. / Veterinary Microbiology 110 (2005) 255–263 259

Fig. 2. Hierarchical clustering of regulated H. parasuis genes. Relative expression values for the three different time points are presented (left

panel). The intensity of the red and green colours symbolises the log2 expression values. The right panel shows the H. parasuis clone

identification numbers, the accession numbers and the description.

E. Melnikow et al. / Veterinary Microbiology 110 (2005) 255–263260

Fig. 2. (Continued ).

A homologue of the membrane-bound EIIC compo-

nent of the PTS of Streptococcus pneumoniae was

found to be up-regulated under all four growth

conditions. Furthermore, another H. parasuis gene

with homology to the sucrose-specific PTS gene ptsB

of Pasteurella multocidawas up-regulated (>1.5-fold)

10 min after heat stress induction and 60 min after

oxygen limitation. Previously, Hill et al. (2003)

E. Melnikow et al. / Veterinary Microbiology 110 (2005) 255–263 261

observed the induction of enzyme I expression of the

H. parasuis PTS system under heat stress. Further-

more, the expression of crr, an enzyme II component

of the PTS system of P. multocida, is induced under

nutrient limitation (Paustian et al., 2002). These data

support the view of a major role of the PTS system in

host adaptation by members of the Pasteurellaceae

family. Regulation of the H. parasuis homologue of

the P. multocida spermidine/putrescine transporter

PotD under three out of four growth conditions

suggests that polyamine uptake via PotD is likely to

occur in vivo as well. Polyamines like spermidine and

putrescine are essential for cell growth by influencing

gene expression through interaction with mRNAs and

tRNAs (Igarashi and Kashiwagi, 1999). PotD is

expressed during the infection of calves by Mannhei-

mia haemolytica, another member of the Pasteur-

ellaceae family, and potD mutants in S. pneumoniae

are attenuated (Polissi et al., 1998; Pandher and

Murphy, 1996). Furthermore, the intracellular poly-

amine level influences the expression of genes

involved in oxidative stress adaptation of Escherichia

coli (Jung and Kim, 2003). Other H. parasuis

transporters, up-regulated under multiple conditions,

were homologues of GlpT and MalF in P. multocida

and Salmonella typhimurium, respectively. GlpT

mediates glycerol-3-phosphate uptake and is regulated

by FnR, a positive regulator of genes induced during

E. coli oxygen limitation (Wong and Kwan, 1992).

The induction of glpT expression during microaerobic

growth points to a similar mode of FnR-dependent

regulation in H. parasuis. MalF is part of the maltose

transport complex. The up-regulation of malF

expression in H. parasuis probably reflects the

necessity of maltose uptake for energy supply during

an infection, but may also be involved in production

and secretion of virulence factors, as in the case of

Vibrio cholerae (Lang et al., 1994). The importance of

adaptation to the nutritional host conditions for

successful colonization was further corroborated by

the induction of several H. parasuis genes involved in

the synthesis of amino acids, DNA, cofactors and

energy metabolism. For instance, genes involved in

the biosynthesis of valine and leucine, leuD, leuB and

ilvC, were up-regulated under microaerobic, acidic

and temperature stress conditions. LeuB expression is

positively influenced by the leucine-responsive pro-

tein Lrp, a major regulator of adaptive responses in E.

coli. Whether H. parasuis expresses lrp is not known,

but the closely related organisms H. influenzae and P.

multocida do express genes with homology to lrp. Lrp

also influences the expression of the small subunit of

the glutamate synthase, gltD, involved in the produc-

tion of glutamate (Ernsting et al., 1992). Indeed, a

homologue of GltD was induced in H. parasuis under

multiple stress conditions. The induction of gltD

expression under acidic growth conditions could

reflect the importance of glutamate as a source for

ammonia in pH homeostasis, as has been hypothesized

for Helicobacter pylori colonization of the acidic

stomach (Merrell et al., 2003).

Two putative proteases were found up-regulated

under acidic, temperature and heat stress conditions.

One showed high homology to a protease of H.

influenzae (HI0419), which clusters with collagenase-

degrading proteins characterized by PrtC, a collage-

nase-degrading protein of Porphyromonas gingivalis.

The other protease showed weak homology to a serine

protease of Fusobacterium nucleatum subsp. vincentii

with an unknown function. Another candidate

virulence factor was identified under oxygen-limiting

conditions with homology to the hemolysin HhdA of

H. ducreyi. The regulation of H. parasuis hhdA

expression in response to oxygen deprivation is

consistent with the detection of hhdA transcription

in the microaerobic environment of pustules in H.

ducreyi-infected human volunteers (Throm and

Spinola, 2001).

Amongst the heat-shock proteins, a homologue of

the P. multocida stress protein ClpB was up-regulated

under heat, acidic and iron-limiting growth conditions.

ClpB belongs to the Clp/HSP100 family of molecular

chaperons, which rescue denatured proteins from the

aggregated state (Lee et al., 2003). ClpB mutants in

Listeria monocytogenes and S. typhimurium are

severely attenuated in virulence, demonstrating the

importance of ClpB in establishing a successful

infection (Turner et al., 1998; Chastanet et al., 2004).

The induction of clpB expression in H. parasuis under

temperature stress is consistent with previous reports

in gram-negative and -positive organisms (Ekaza

et al., 2001;Weiner et al., 2003). The induction of clpB

expression under iron-limitation has not been reported

previously and could reflect the participation of ClpB

in the disaggregation of proteins that require iron for

proper folding. In this context it is interesting to note

E. Melnikow et al. / Veterinary Microbiology 110 (2005) 255–263262

that in H. pylori iron-regulated genes and clpB are co-

regulated during transition into the stationary growth

phase, suggesting that depletion of iron in the medium

triggered the clpB expression (Thompson et al., 2003).

A number of H. parasuis genes were specifically

induced during iron-limitation. These included homo-

logues of the hxuCBA genes of H. influenzae and the

yfeA genes of P. multocida. The hxuCBA genes of H.

influenzae are required for the utilization of free heme

and heme-hemopexin and are organised in one operon

(Hanson et al., 1992; Cope et al., 1998). The

expression of the H. parasuis hxuCBA genes in

response to iron depletion did not differ significantly

during the 60 min time course, suggesting that

hxuCBA of H. parasuis are also organised in one

operon. The up-regulation of hxuC during iron-

limitation is in agreement with the induction of hemR,

the HxuC homologue of P. multocida, under iron-

limiting conditions (Paustian et al., 2001). Further-

more, hxuA and hemR (hxuC) expression has been

detected during otitis media caused by H. influenzae

and in the liver of P. multocida-infected chickens,

respectively, suggesting a prominent role of heme-

hemopexin uptake in host adaptation within the

Pasteurellaceae family (Boyce et al., 2004). YfeA

codes for a periplasmic binding protein first char-

acterized in Yersinia pestis as part of the iron uptake

system YfeABCD (Bearden et al., 1998; Bearden and

Perry, 1999). The yfe operon may play an important

part in iron supply in vivo. Deletions in yfeAB caused

strong attenuation of Y. pestis in experimental

infections of mice, and expression of yfeABCD has

been demonstrated in the blood and liver of P.

multocida-infected chickens (Boyce et al., 2004).

A large number of genes coded for proteins with

unknown functions, with several showing up-regula-

tion under multiple conditions. One of the most

intriguing hypothetical proteins (P14_A05) is a

homologue of a hypothetical protein of V. cholerae.

This protein exhibited a steady and strong rise after

induction of acidic and temperature stress, and showed

increased levels under microaerobic conditions.

Protein domain analysis in the NCBI domain database

revealed a weak homology to exopolyphosphatase-

related proteins involved in the stringent response.

In conclusion, by using microarray technology we

were able to identify many new factors potentially

involved in H. parasuis virulence and host adaptation.

Some of the H. parasuis genes identified in vitro were

homologues of known virulence factors or homo-

logues of genes, which are expressed in the context of

an infection in other members of the Pasteurellaceae

family. The latter is an important confirmation for the

relevance of the selected in vitro environmental stress

conditions. This study is a first step into a

comprehensive, global analysis of H. parasuis

virulence and must be followed by the development

of mutagenesis tools in H. parasuis to verify the

importance of potential virulence factors in experi-

mental animal infections.

Acknowledgements

This work was in part funded by the PathoCHIP

project of the European Community, EC contract

number QLK2-CT-2000-00726. The bacterial strain

was kindly provided by Drs. Carlos Pijoan and Simone

Oliveira from the University of Minnesota, USA.

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