R E S E A R C H A R T I C L E

Comparative invivoand invitro analysesof putativevirulencefactorsofBurkholderia pseudomallei using lipopolysaccharide,capsuleand£agellinmutantsChanthiwa Wikraiphat1, Jaruek Charoensap1, Pongsak Utaisincharoen1, Surasak Wongratanacheewin2,Suwimol Taweechaisupapong3, Donald E. Woods4, Jan G.M. Bolscher5 & Stitaya Sirisinha1

1Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand; 2Department of Microbiology, Faculty of Medicine, Khon Kaen

University, Khon Kaen, Thailand; 3Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen, Thailand; 4Department of

Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Calgary, Alberta, Canada; and 5Department of Oral Biochemistry,

Academic Centre for Dentistry, VU Medical Center, Amsterdam, The Netherlands

Correspondence: Stitaya Sirisinha,

Department of Microbiology, Faculty of

Science, Mahidol University, Bangkok,

Thailand. Tel.: 1662 201 5675; fax: 1662

644 5411; e-mail: scssr@mahidol.ac.th

Received 19 February 2009; revised 14 May

2009; accepted 23 May 2009.

DOI:10.1111/j.1574-695X.2009.00574.x

Editor: Richard Marconi

Keywords

Burkholderia pseudomallei; polymorphonuclear

cell; macrophage; capsule; lipopolysaccharide;

flagella.

Abstract

Burkholderia pseudomallei is a gram-negative bacillus that is the causative agent of

melioidosis. We evaluated host–pathogen interaction at different levels using three

separate B. pseudomallei mutants generated by insertional inactivation. One of

these mutants is defective in the production of the polysaccharide side chains

associated with lipopolysaccharide; one does not produce the capsular polysac-

charide with the structure -3)-2-O-acetyl-6-deoxy-b-D-manno-heptopyranose-(1-;

and the third mutant does not produce flagellin. We compared the in vivo

virulence in BALB/c mice, the in vitro fate of intracellular survival inside human

polymorphonuclear cells (PMNs) and macrophages (Mfs) and the susceptibility

to killing by 30% normal human serum, reactive nitrogen and oxygen intermedi-

ates and antimicrobial peptides with that of their wild-type counterpart. The

lipopolysaccharide and capsule mutants demonstrated a marked reduction in

virulence for BALB/c mice, but the flagellin mutant was only slightly less virulent

than the parent strain. The results from the BALB/c mice experiments correlated

with survival in Mfs. The lipopolysaccharide and capsule mutants were also more

susceptible to killing by antimicrobial agents. All bacteria were equally susceptible

to killing by PMNs. Altogether, the data suggest that lipopolysaccharide and

capsule and, to a much lesser extent, flagella, are most likely associated with the

virulence of this bacterium and highlight the importance of intracellular killing by

PMNs and Mfs in disease pathogenesis.

Introduction

Burkholderia pseudomallei is a facultative gram-negative

bacillus that is the causative agent of a potentially fatal

disease known as melioidosis (White, 2003; Wiersinga et al.,

2006). Clinical manifestations vary from asymptomatic

infection to septic shock. The latter can have a mortality

rate as high as 80% despite appropriate antibiotic treatment.

The pathogenesis of melioidosis remains to be clarified,

but different lines of evidence suggest that innate immunity

is important in determining the outcome of infection

(Wiersinga et al., 2006). It was shown, for example, that

interferon-g (IFN-g) played an essential role in resistance in

a murine model of infection (Santanirand et al., 1999).

Enhancing IFN-g production before infection via CpG

oligodeoxynucleotide administration protected animals

against fatal challenge (Wongratanacheewin et al., 2004).

Macrophages (Mfs) preactivated with IFN-g could readily

kill B. pseudomallei (Miyagi et al., 1997). More recently,

the role of polymorphonuclear cells (PMNs) has attracted

the attention of many groups of investigators (Easton

et al., 2007; Wiersinga et al., 2007, 2008a, b), but attempts

to activate these cells with granulocyte colony-stimulating

factor in patients with severe sepsis due to melioidosis have

given rise to contradictory and questionable results (Cheng

et al., 2007).

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FEMS Immunol Med Microbiol ]] (2009) 1–7 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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Although considerable attention has been paid to identi-

fying and characterizing the virulence factors of B. pseudo-

mallei, no definitive conclusions have been reached. Several

putative virulence factors have been implicated in disease

due to B. pseudomallei including lipopolysaccharide, the

capsular polysaccharide with the structure -3)-2-O-acetyl-6-

deoxy-b-D-manno-heptopyranose-(1- (hereafter referred to

as the mannoheptan capsule), flagella, the type III secretion

system, phospholipase C and several other toxins (Cheng &

Currie, 2005; Wiersinga et al., 2006). In the present study, we

used three B. pseudomallei mutants that we have developed

to compare their virulence in murine infection models, their

interaction with human PMNs and Mfs, as well as their

susceptibility to killing by antimicrobial agents in order to

obtain more informative data regarding virulence and

pathogenesis. The results suggest that lipopolysaccharide

and the mannoheptan capsule, but not flagella, are relevant

to the virulence of this bacterium.

Materials and methods

Bacterial cultivation and characterization

The lipopolysaccharide mutant SRM117 (DeShazer et al.,

1998), the mannoheptan capsule mutant SR1015 (mutated

in the wchB gene, which encodes for a protein with

homology to a glycosyltransferase, Reckseidler et al., 2001)

and flagellin mutant MM35 (DeShazer et al., 1997) were

generated from wild-type B. pseudomallei strain 1026b by

insertional mutagenesis. With regard to the insertional

mutagenesis, all the mutant strains contained a tetracycline

resistance cassette (Tcr) to indicate their insertional inacti-

vation. All strains were cultured at 37 1C in Luria–Bertani

(LB) medium for 16–18 h to obtain bacteria in the late

logarithmic phase of growth before harvesting and use in the

study. If not indicated otherwise, the medium for culturing

the mutants was always supplemented with tetracycline at a

concentration of 50mg mL�1 to prevent possible reversion

to the wild type. Under these conditions of growth, the

doubling times for all strains were calculated and found to

be c. 48 min. The number of viable bacteria, expressed as

CFU, was determined by serial dilution and the pour-plating

technique performed in duplicate using trypticase soy agar

(TSA) in the absence of an antibiotic. The presence or

absence of lipopolysaccharide, capsule or flagella in the

stock cultures (Table 1) was confirmed as described

(DeShazer et al., 1997; Anuntagool et al., 2000; Sirisinha

et al., 2000). Biofilm production was also determined (Tawee-

chaisupapong et al., 2005). Lethal dose 50% (LD50) determi-

nations in BALB/c mice for all four strains were performed as

described earlier (Wongratanacheewin et al., 2004).

M/ and PMN preparations

Mfs and PMNs were isolated from heparinized whole blood

of healthy human volunteers by Ficoll–Hypaque density

gradient separation (ethical clearance was provided by the

Ethical Committee of the Ramathibodi Hospital, Mahidol

University, Bangkok, Thailand, clearance number 2549/

452). In brief, peripheral blood mononuclear cells were

harvested from the fluid at the interface of the separation

tube, and monocytes were purified by positive selection of

CD141 cells using a magnetic cell sorter system (MACS@

Miltenyi Biotec, GmbH, Germany). The Mfs were gener-

ated from the purified monocytes by culturing the latter in

Iscove’s modified Dulbecco’s medium supplemented with

1% L-glutamine and 10% heat-inactivated human AB serum

(GemCellTM, West Sacramento, CA) at 37 1C for 5–7 days in

a humidified 5% CO2 incubator. The fluid in the lower layer

from the Ficoll–Hypaque separation tube was used for PMN

preparation (Watson et al., 1992). The PMNs were then

purified using 3% Dextran T500 (GE Healthcare, BioS-

ciences, Uppsala, Sweden). Residual contaminating red

blood cells were lysed in a hypotonic solution. The PMNs

were resuspended in DMEM culture medium supplemented

with 10% heat-inactivated fetal bovine serum (HyClone,

Logan, UT) and 1% L-glutamine and used immediately.

Survival and replication of B. pseudomallei wildtype and mutants in PMNs and M/s

PMNs (2� 106 cells) and Mfs (1� 105 cells) were infected

with the bacteria as described previously (Kespichayawatta-

na et al., 2000), using a multiplicity of infection of 2 : 1 and

1 : 1, respectively. Briefly, after 2 h of infection, extracellular

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Table 1. Phenotypic characteristics of Burkholderia pseudomallei wild type and mutants

Bacterial

strains

LD50 (CFU)

(BALB/c, IP)

Biofilm formation

(OD630 nm) 200 kDa

Reactive

with mAb to

lipopolysaccharide Motile

Wild type 104 0.807 1 1 1

Capsule mutant 4 109 1.296 � 1 1

Lipopolysaccharide mutant 108 2.461 1 � 1

Flagellin mutant 3.16�104 0.756 1 1 �

1, produces capsule or lipopolysaccharide, motile; � , does not produce capsule or lipopolysaccharide, nonmotile; IP, intraperitoneal challenge.

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bacteria were killed by adding kanamycin to the cell culture

to a final concentration of 250 mg mL�1. Two hours later, the

antibiotic concentration was changed to 20 mg mL�1. In

addition to the kanamycin, for experiments using the

mutants, tetracycline at a concentration of 50mg mL�1 was

also present. The number of viable intracellular bacteria

at different time intervals was determined by lysing the

infected cells with 0.1% Triton X-100, and the number of

bacteria in the lysate was quantified as described above. At

the end of all experiments, the viability of the infected cells

was determined by trypan blue staining, and experiments

with viability o 90% were discarded. Data were analyzed

using a paired-samples t-test. P-values o 0.05 were consid-

ered statistically significant.

Host-cell free killing assays

The sensitivity of the three mutants and the wild type to

killing by 30% fresh pooled normal human serum (NHS)

and of human neutrophil peptide-2 (HNP-2) was deter-

mined as described, respectively, by DeShazer et al. (1998)

and West et al. (2005). In brief, 106 CFU mL�1 of late log-

phase bacteria were incubated in LB broth containing 30%

fresh or heat-inactivated pooled human serum (obtained

from donors living in a nonendemic area of infection),

10 mg mL�1 of HNP-2 or in phosphate-buffered saline for 2 h

before residual bacteria were determined. In addition to

HNP-2, the killing activities of other antimicrobial peptides,

namely, cathelicidin (LL-37), histatins (histatin5 and its

variant, dhvar5) and lactoferrin peptides (LFcin 17-30

and LFampin 268-284), were also investigated. These pep-

tides were synthesized as described (Den Hertog et al., 2004;

van der Kraan et al., 2004). Bacteria (1� 107 CFU mL�1) in

1 mM potassium phosphate buffer (PPB), pH 7.0, were

added to equal volumes of the antimicrobial peptide solu-

tions. The final concentration of all the peptides was

100 mM. A bacterial suspension in PPB without a peptide

served as a control. After incubation at 37 1C for 1 h, the

number of residual bacteria was determined and the percen-

tage of killing was calculated using the formula [1� (CFU

sample/CFU control)]� 100%. Differences in susceptibility

to each antimicrobial peptide of each mutant compared

with that of the wild type were analyzed using Student’s t-

test. Statistical significance was considered when the P-value

was o 0.5.

Susceptibility to killing by reactive nitrogen(RNI) and oxygen (ROI) intermediates

The sensitivity of the bacteria to RNI killing was assessed by

culturing them in different concentrations of nitric oxide

(NO)-generating compounds, S-nitrosoglutathione (GSNO)

or 3-morpholinosydnonimine hydrochloride (SIN-1) (Chak-

ravortty et al., 2002). After 4 h of incubation, residual bacteria

were determined by serial dilution and plating on TSA. The

concentrations that killed 50% of the bacteria were then

calculated and expressed as lethal concentration 50%. H2O2

was used to test their sensitivity to ROI. For this test, the

bacteria were similarly cultured in LB broth that contained

different concentrations of H2O2, and the number of residual

bacteria was determined after 2 h of incubation.

Results

Virulence of B. pseudomallei wild type andmutants for BALB/c mice

The LD50 values of the wild type and mutants in BALB/c

mice are shown in Table 1. The virulence of lipopolysacchar-

ide and capsule mutants was markedly reduced compared

with that of the wild-type counterpart or the flagellin

mutant. Judging from these values, the capsule appeared to

be the most important virulence factor in this animal model,

as its LD50 value decreased by 4 5 log10 compared with that

of the wild type, and this was followed by that of the

lipopolysaccharide mutant.

Intracellular survival and replication ofB. pseudomallei in human PMNs and M/s

The fates of B. pseudomallei wild type and the three mutants

after being internalized by human PMNs and Mfs were

followed for c. 16–18 h when the experiments were termi-

nated and host cells were still viable. Inside the PMNs, both

the wild type and the mutants displayed similar survival

profiles (Fig. 1). The absolute numbers of intracellular

bacteria, expressed as CFU of viable count, gradually

declined during these time intervals, and at the end of the

experiments there was a reduction of between 1 and 2 log10

of viable intracellular bacterial counts for all strains com-

pared with their initial 3-h time points. However, unlike the

results with the PMNs, the Mfs could not suppress the

growth of the B. pseudomallei wild type and at the end of the

experiment the absolute numbers of intracellular bacteria

increased by at least 1 log10. Its doubling time in the Mfs

was about 1 h 50 min, a value similar to that obtained with

other wild-type strains that we have tested (Charoensap

et al., 2009). The profile of the intracellular lipopolysacchar-

ide mutant, on the other hand, was markedly different from

that of the wild type, and by the end of the experiment the

residual numbers of the lipopolysaccharide mutant were

significantly lower than those of the wild type by 4 2 log10

(P = 0.036). Hence, the oligosaccharide component of the

lipopolysaccharide appeared to be one of the components of

B. pseudomallei that conferred resistance against intracellu-

lar killing by human Mfs.

It is apparent from Fig. 1b that the numbers of the capsule

mutant at the 16-h time point were markedly different from

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FEMS Immunol Med Microbiol ]] (2009) 1–7 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

3Analysis of putative virulence factors of B. pseudomallei

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those of the wild type. The difference was more obvious

when these end-point values were transformed into the

percentage of their respective initial values at the 3-h time

point (Po 0.01). When calculated, the doubling time of the

capsule mutant was considerably prolonged, i.e., 4 5 h. The

survival profile of flagellin mutant, on the other hand, was

almost the same as that of the wild type; however, it

appeared to replicate slightly slower, having its doubling

time slightly prolonged from 1 h 50 min found with the wild

type to 2 h 16 min. Altogether, the survival data of

B. pseudomallei in the Mfs were in agreement with the

results obtained from the LD50 values in animals in that

both the lipopolysaccharide and the capsule appeared to be

more related to virulence than the flagella.

Susceptibility of B. pseudomallei wild type andmutants to killing by NHS

The contribution of lipopolysaccharide, capsule and flagella

to resistance to killing by NHS was analyzed by simulta-

neously testing all four strains in a cell-free system in the

presence or absence of 30% fresh NHS. The data summar-

ized in Table 2 showed that the capsule and flagellin mutants

exhibited similar degrees of resistance to killing as that of the

wild type. The level of resistance to killing of the lipopoly-

saccharide mutant, on the other hand, decreased signifi-

cantly (P = 0.006) compared with other strains. At the end of

the 2-h exposure, essentially all lipopolysaccharide mutant

organisms were killed. The bactericidal activity of NHS for

B. pseudomallei appeared to be associated with heat-labile

component(s), as its activity could be totally destroyed by

heating at 56 1C for 30 min (Table 2).

Susceptibility of B. pseudomallei wild type andmutants to killing by antimicrobial peptides

The susceptibility of B. pseudomallei wild type and the three

mutants to killing by different antimicrobial peptides was

investigated. While both the wild type and all three mutants

were found to be resistant to killing by 10 mg mL�1 of HNP-2

(data not shown), all strains were highly susceptible to

killing by 100 mM of cathelicidin LL-37 (Table 2). On the

other hand, when compared with their wild-type counter-

part, only the lipopolysaccharide and the capsule mutants

showed enhanced susceptibility to killing by histatins and

lactoferrin peptides (Table 2), whereas the flagellin mutant

appeared to be indistinguishable from the wild type with

regard to this susceptibility.

Susceptibility of B. pseudomallei wild type andmutants to killing by RNIs and ROI

Although these intermediates are known to be generated

intracellularly by some phagocytic cells, in order to avoid

misinterpretation of the data, the experiments were per-

formed in a cell-free system. The results presented in Table 2

showed that, compared with the wild type, the lipopolysac-

charide and, to a lesser extent, the flagellin mutant, appeared

to have enhanced resistance to killing by the NO-generating

compounds. With the ROI testing using H2O2, only the

capsule and, to a lesser extent, flagellin mutants appeared to

have enhanced resistance against killing. On the other hand,

there was a slight, if any, increase in susceptibility for the

lipopolysaccharide mutant compared with the wild type

(Table 2). These observations altogether suggested that the

lipopolysaccharide component may be one of the inherent

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Fig. 1. Intracellular survival kinetics of Burkholderia pseudomallei wild

type and mutants inside human PMNs (a) and Mfs (b). The results are

expressed as mean and SE of the mean of absolute number of

intracellular bacteria (CFU), each from five different donors. Survival data

of the wild type and capsule mutant inside Mfs were also expressed as

percent internalization (c).

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properties associated with the resistance and susceptibility

of B. pseudomallei to killing by NOI and ROI and, as a

consequence, may influence the outcome of the organism’s

intracellular fate in the phagocytes. The capsule and, to a

lesser extent, the flagellin mutants appeared to be more

resistant to ROI killing than the wild type. The latter also

had a considerable delay in stimulating respiratory burst in

human PMNs compared with the wild type and the other

two mutants, judging from a chemiluminescence assay using

200 mM luminol (data not shown).

Discussion

In the present study, we attempted to provide additional

evidence to further identify and verify the possible role of

three putative virulence factors, namely, lipopolysaccharide,

capsule and flagella, in disease caused by B. pseudomallei.

This was done by comparative analyses of the three respec-

tive mutants with their wild-type counterpart in inducing

disease in susceptible BALB/c mice, their intracellular fates

in human PMNs and Mfs and their susceptibility to killing

by serum, antimicrobial peptides, ROI and RNIs. The results

from our studies strongly suggest that the lipopolysacchar-

ide and capsule, and to a much lesser extent flagella, are

associated with the virulence of this bacterium. Moreover,

because both the lipopolysaccharide and the capsule mu-

tants demonstrated noticeably higher biofilm production

than either the flagellin mutant or the wild type (Table 1), it

appears that biofilms do not play a significant role in the

disease process. This conclusion is consistent with our

previous report using a B. pseudomallei wild type possessing

different biofilm contents and a biofilm mutant (Taweechai-

supapong et al., 2005).

The observation that the lipopolysaccharide represented

one of the virulence components of B. pseudomallei is not

really unexpected (Table 1 and Fig. 1) because it was

suggested previously by our own group (DeShazer et al.,

1997; Archaroen et al., 2007 Q1). In one of these reports, we

showed that compared with the wild type, this same

lipopolysaccharide mutant was more susceptible to killing

during the early phase of infection by a mouse Mf cell line

RAW264.7 and the data obtained suggested that this may be

related to its ability to activate IFN-b and iNOS, both of

which are known to upregulate NO production that killed

this intracellular bacterium (Arjcharoen et al., 2007). It

could be argued, however, that in that report a mouse cell

line was used and so the conclusion must be interpreted

with caution. Therefore, in the present study, we similarly

tested host–bacterial interactions using freshly isolated

human phagocytic cells infected with B. pseudomallei wild

type and three different mutants. With the human system,

we found again that the lipopolysaccharide mutant was

more susceptible to killing by the Mfs than its wild-type

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53 Tab

le2.

Leth

alco

nce

ntr

atio

n50%

(LC

50)va

lues

and

susc

eptibili

tyle

vels

(with�

SD)of

Burk

hold

eria

pse

udom

alle

iwild

type

and

muta

nts

toki

lling

by

seru

man

ddiffe

rent

antim

icro

bia

lagen

ts

Bac

teria

Res

idual

bac

teria

(CFU

)%

Kill

ed(n

=6)

LC50

30%

NH

S

30%

hea

t-in

activa

ted

NH

SPB

S

His

tatins

(100mM

)La

ctofe

rrin

s(1

00mM

)C

ather

icid

in

(100mM

)RN

Is(m

M)

RO

I(mM

)

(n=

4)

His

tatin

5D

hva

r5LF

cin

17-3

0

Lfam

pin

268-2

84

LL-3

7SI

N-1

GSN

OH

2O

2

Wild

type

1.1

4�

10

61.2

3�

10

61.5

8�

10

658.0�

6.9

39.3�

9.2

25.2�

7.9

20.7�

18.9

99.9�

0.1

00.6

52.7

536�

6

Cap

sule

muta

nt

1.1

1�

10

61.1

8�

10

61.4

5�

10

678.1�

6.1�

74.7�

5.9�

71.1�

9.4�

80.5�

7.5�

99.5�

0.0

6�

0.5

52.7

273�

14

Lipopoly

sacc

har

ide

muta

nt

41

1.2

2�

10

61.5

3�

10

658.1�

9.7

80.0�

9.5�

60.2�

18.1�

57.8�

19.9�

99.9�

0.0

33.0

53.5

22�

3

Flag

ellin

muta

nt

1.4

5�

10

61.1

9�

10

61.8

1�

10

640.7�

21.8

51.9�

11.1

38.9�

13.8

16.8�

19.3

99.7�

1.0

21.8

53.6

555�

13

� Po

0.0

1co

mpar

edw

ith

stra

in1026b.

PBS,

phosp

hat

e-buff

ered

salin

e.

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counterpart during the entire period of observation (Fig. 1).

We would like to suggest that the decreased virulence of the

lipopolysaccharide mutant in our in vivo mouse model

(Table 1) is a combined result of their intracellular fates in

Mfs, together with enhanced susceptibility to killing by

serum, antimicrobial agents and, to a lesser extent, by ROI

(Table 2). The data presented also allow us to conclude that

the capsule is another important virulence factor for this

bacterium. Its ability to confer some degree of resistance

against intracellular killing by human Mfs in particular

sheds additional light on its possible mechanism of patho-

genicity. The association of the capsule with virulence had

been reported previously in another animal model (Reck-

seidler et al., 2001), but it was then suggested that this is

probably attributable to the fact that the capsule is known to

interfere with complement activation and subsequently

phagocytosis of B. pseudomallei (Reckseidler et al., 2005).

In addition to these possible mechanisms, our data in Fig. 1,

showing that the capsule mutant survived rather poorly in

the Mfs when compared with the wild type, provide

another possible mechanism of resistance against killing. It

should be mentioned that the slight increase in the number

of viable capsule-mutant organisms in the lysate at the later

time points (Fig. 1) was unlikely to be caused by a premature

release of bacteria from dying infected cells into the super-

natant fluids because the viability of these infected cells was

still higher than 90% when the experiment was terminated.

The ability of the capsule to confer enhanced resistance

against intracellular killing may represent a novel mechan-

ism of capsule function, and this phenomenon has never

been reported before for B. pseudomallei. This observation

is, however, in accordance with the results reported recently

for Neisseria meningitidis (Spinosa et al., 2007). It was

demonstrated further in that model that the presence of the

capsule correlated with its enhanced resistance to killing by

defensins, cathelicidins, protegerins and polymyxin B, a

finding that is also similar to our data on the susceptibility

of Burkholderia to killing by the antimicrobial peptides used

in our study (Table 2). Moreover, the enhanced resistance of

the capsule mutant to ROI killing as noted in the table may

contribute to its prolonged survival in the Mfs. The

importance of Mfs in host defense against B. pseudomallei

infection was convincingly demonstrated with the data

showing that Mf depletion by dichloromethylene bipho-

sphonate-containing liposomes rendered mice more suscep-

tible to B. pseudomalleiQ2 infection (Breitbach et al., 2006).

Consistent with the animal study reported earlier (Miyagi

et al., 1997), we have recently demonstrated in the human

system that although B. pseudomallei was able to survive

intracellular killing inside the Mfs, the killing capacity

could be readily enhanced after activation by IFN-g (Char-

oensap et al., 2009). On the other hand, the role of PMNs

should not be overlooked as it was shown earlier that they

were needed in defense against B. pseudomallei infection in a

mouse model (Easton et al., 2007; Wiersinga et al., 2008a, b).

Our results (Fig. 1), demonstrating the efficacy of PMNs in

killing B. pseudomallei in vitro, supported its potential role

in host defense against B. pseudomallei infection.

Lastly, it should be mentioned that the role of flagella is

yet to be established. For example, although we recently

reported that flagella were needed for the invasion of

B. pseudomallei into nonphagocytic cells (Chuaygud et al.,

2008), it is inconsistent with the data in the present study

using primary human phagocytic cell cultures. It is therefore

possible to attribute the difference to the types of cells used

in the two studies. Our in vivo data using flagellin mutant

bacteria in BALB/c mice (Table 1) were also different from

what was reported earlier by Chua et al. (2003). The

discrepancy between our results and those of the previous

results may be explained by a difference in the route of

bacterial challenge. In their study, they clearly demonstrated

that flagella were a virulence determinant as their flagellin-

defective mutant was avirulent for BALB/c mice when

deposited intranasally. When challenged by the intraperito-

neal route, however, as shown in the current studies, flagella

are not required for virulence. In summary, while we

showed here, using in vitro and in vivo experiments,

that lipopolysaccharide and the capsular polysaccharide

with the structure -3)-2-O-acetyl-6-deoxy-b-D-manno-

heptopyranose-(1- are definitely important virulence factors

for B. pseudomallei, the role of flagellin remains unsettled

and needs to be investigated further.

Acknowledgements

This work was supported by grants from the National

Science and Technology Development Agency (Thailand),

the Royal Golden Jubilee PhD Program of the Thailand

Research Fund, the Institutional Strengthening Program,

Faculty of Science, and the RA Scholarship, Faculty of

Graduated Studies, Mahidol University.

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