Attenuated virulence and protective efficacy of a Burkholderia pseudomallei bsa type III secretion mutant in murine models of melioidosis

Mark P. Stevens1,{dagger}, Ashraful Haque2,{dagger}, Timothy Atkins3,{dagger}, Jim Hill3,{dagger}, Michael W. Wood1, Anna Easton2, Michelle Nelson3, Cindy Underwood-Fowler3, Richard W. Titball2,3, Gregory J. Bancroft2 and Edouard E. Galyov1

1 Division of Microbiology, Institute for Animal Health, Compton Laboratory, Berkshire RG20 7NN, UK
2 Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel St, London WC1E 7HT, UK
3 Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK

Correspondence
Mark P. Stevens
mark-p.stevens{at}bbsrc.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Melioidosis is a severe infectious disease of animals and humans caused by the Gram-negative intracellular pathogen Burkholderia pseudomallei. An Inv/Mxi-Spa-like type III protein secretion apparatus, encoded by the B. pseudomallei bsa locus, facilitates bacterial invasion of epithelial cells, escape from endocytic vesicles and intracellular survival. This study investigated the role of the Bsa type III secretion system in the pathogenesis of melioidosis in murine models. B. pseudomallei bipD mutants, lacking a component of the translocation apparatus, were found to be significantly attenuated following intraperitoneal or intranasal challenge of BALB/c mice. Furthermore, a bipD mutant was attenuated in C57BL/6 IL-12 p40–/– mice, which are highly susceptible to B. pseudomallei infection. Mutation of bipD impaired bacterial replication in the liver and spleen of BALB/c mice in the early stages of infection. B. pseudomallei mutants lacking either the type III secreted guanine nucleotide exchange factor BopE or the putative effectors BopA or BopB exhibited varying degrees of attenuation, with mutations in bopA and bopB causing a significant delay in median time to death. This indicates that bsa-encoded type III secreted proteins may act in concert to determine the outcome of B. pseudomallei infection in mice. Mice inoculated with the B. pseudomallei bipD mutant were partially protected against subsequent challenge with wild-type B. pseudomallei. However, immunization of mice with purified BipD protein was not protective.


Abbreviations: GST, glutathione-S-transferase; HRP, horseradish peroxidase; MLD, median lethal dose; TTSS, type III secretion system

{dagger}These authors contributed equally to the work.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Burkholderia pseudomallei is the aetiological agent of melioidosis, a severe invasive infection of humans and animals that is endemic in tropical and subtropical areas. Clinical signs of melioidosis can vary from inapparent or subacute infections to localized or chronic suppurative infections, which may progress to acute septicaemia and pneumonia (reviewed by White, 2003). Latency and relapse are common even in melioidosis patients treated with appropriate antibiotics (Chaowagul et al., 1993). This may result in part from the ability of B. pseudomallei to invade non-phagocytic host cells, and to survive and replicate within phagocytes, where antibiotics may be less effective (Jones et al., 1996; Kespichayawattana et al., 2000; Pruksachartvuthi et al., 1990). The severe course of infection, aerosol infectivity and worldwide availability of B. pseudomallei has raised concerns that it may be used as a bioterror agent. No vaccine against melioidosis exists, and the molecular mechanisms underlying B. pseudomallei–host cell interactions and virulence are incompletely understood (reviewed by Stevens & Galyov, 2004).

Recently, a type III protein secretion apparatus was identified in B. pseudomallei (Bsa) that is similar to the Salmonella Inv/Spa/Prg and Shigella Ipa/Mxi/Spa systems (Attree & Attree, 2001; Rainbow et al., 2002; Stevens et al., 2002). Type III secretion systems (TTSSs) are key virulence determinants of Salmonella, Shigella and other Gram-negative facultative intracellular pathogens, and they serve to inject bacterial proteins into target cells (reviewed by Cornelis & van Gijsegem, 2000; Hueck, 1998; Galán, 2001; Sansonetti, 2001). A subset of type III secreted proteins (‘translocators’) is believed to interact with the eukaryotic cell membrane and mediate the delivery of secreted ‘effector’ proteins, which inhibit or subvert cellular processes. Studies in our laboratory and elsewhere have shown that mutations that disrupt the Salmonella Inv/Spa/Prg apparatus, the Sip translocator proteins and selected Sop effector proteins, reduce bacterial invasion and persistence in vivo and inhibit Salmonella-induced enteritis (reviewed by Wallis & Galyov, 2000; Zhang et al., 2003).

The B. pseudomallei bsa locus encodes homologues of Salmonella Sip translocator proteins (BipB, BipC and BipD) (Stevens et al., 2002). Salmonella SipB, SipC and SipD proteins are required for injection of effector proteins and invasion of epithelial cells in vitro (Kaniga et al., 1995; Collazo & Galán, 1997). In addition, SipD plays an important role in invasion of intestinal epithelia by Salmonella enterica serovar Dublin and the induction of enteritis (Bispham et al., 2001). Consistent with a role in the injection of effectors, mutation of the B. pseudomallei bipD gene impairs invasion of epithelial cells in vitro (Stevens et al., 2003). In addition, BipD is required for replication of B. pseudomallei in murine macrophage-like cells and for bacterial escape from endocytic vesicles and subsequent actin tail formation (Stevens et al., 2002). A protein encoded within the bsa locus (BopE) is secreted via the Bsa apparatus and influences invasion of HeLa cells, most likely by acting as a guanine nucleotide exchange factor for RhoGTPases that regulate the actin network (Stevens et al., 2003). Other putative bsa-encoded effector proteins have been identified, including BopA and BopB (Stevens et al., 2002). BopA is a homologue of the Shigella type III secreted protein IscB, which mediates cell-to-cell spread of Shigella by lysing the double membrane surrounding actin-based protrusions that project the bacteria into adjacent cells (Allaoui et al., 1992). BopB is predicted to be encoded at one end of the bsa locus by the seventh predicted gene downstream of bopA, and it contains an amino acid motif (CX5R) that is conserved in the catalytic domains of numerous phosphatases. A type III secreted protein of Salmonella containing a similar motif (SopB) influences inositol phosphate signalling pathways in eukaryotic cells, bacterial invasion and Salmonella-induced enteritis (Norris et al., 1998; Zhou et al., 2001).

The bsa locus is conserved in the glanders pathogen Burkholderia mallei, and two putative structural components of the type III secretion apparatus (BsaQ and BsaZ) were recently reported to be required for full virulence in rodent models of infection (Ulrich & DeShazer, 2004). The authors of that study did not examine the contribution of individual bsa-encoded translocator or effector proteins in pathogenesis, or the potential use of purified type III secreted proteins as subunit vaccines. TTSS components of other Gram-negative bacterial pathogens are protective antigens (Leary et al., 1995; Sawa et al., 1999). It is known that sera from convalescent melioidosis patients recognize the B. pseudomallei BipB, BipC and BipD proteins (Stevens et al., 2002); however, the protection offered by such responses has not been studied.

In this study, we investigated the role of BipD and known, or putative, bsa-encoded type III secreted effectors in B. pseudomallei virulence in murine models of melioidosis. In addition we examined the protective efficacy of the immune responses elicited by a B. pseudomallei bipD mutant strain and purified BipD protein.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and culture conditions.
B. pseudomallei strains 576 and 10276 were isolated from fatal cases of human melioidosis in Thailand and Bangladesh, respectively, and they were obtained from Dr Ty Pitt, Health Protection Agency, Colindale, London, UK. K96243 is the genome-sequenced strain of B. pseudomallei originally isolated from a meliodosis patient in Thailand, and it was supplied by Dr S. Songsivilai of Mahidol University. Defined mutants of B. pseudomallei strain 10276 with insertions in bsaZ, bipD and bopE have been described previously (Stevens et al., 2002). Escherichia coli S17-1{lambda}pir (Simon et al., 1983) was used as a conjugative donor of the pir-dependent suicide replicon pDM4 (oriR6K, mobRP4, sacBR, cat) and its derivatives (Milton et al., 1996). E. coli BL21(DE3) was used for expression of a BipD fusion protein from a pGEX-4T-1-based vector. Bacterial strains were amplified using Luria–Bertani (LB) broth, LB agar or tryptone soy agar containing, as appropriate, 100 µg ampicillin ml–1, 50 µg kanamycin ml–1 or 50 µg chloramphenicol ml–1. Bacterial strains were grown to stationary phase at 37 °C for 18–24 h, collected by centrifugation, then resuspended in phosphate-buffered saline (PBS) containing 20 % (v/v) glycerol, and frozen in 0·4 ml aliquots at a concentration of 5x108 c.f.u. ml–1 at –80 °C.

Animals.
Female 7- to 10-week-old BALB/c and C57BL/6 IL-12 p40–/– mice were housed under specific pathogen-free conditions on a 12 h light/12 h dark cycle with free access to food and water. All animal experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986, and were approved by the local Ethical Review Committee.

Mutagenesis of the B. pseudomallei bipD, bopA, bopB and bopE genes.
The genes encoding BipD and BopE and the putative type III secreted proteins BopA and BopB were disrupted by homologous recombination using pir-dependent suicide replicons. The pDM4-based constructs for disruption of bipD and bopE have been described (Stevens et al., 2002). They were introduced into B. pseudomallei strain 576 by conjugation from E. coli S17-1{lambda}pir. Insertion mutants (576 bipD : : pDM4 and 576 bopE : : pDM4) were selected by plating on medium containing kanamycin and chloramphenicol, and verified by PCR using pDM4- and gene-specific primers as described previously (Stevens et al., 2002).

To mutate bopA, an internal fragment of the gene was amplified by PCR using the oligonucleotides 5'-CGAACACCTCGAGCGAGCGGCGGTTTTCC-3' and 5'-CGATGCAGATCTGCACCGACGCGCGGTTCGC-3' with Advantage GC2 DNA polymerase (Clontech) under optimal conditions for the amplification of GC-rich templates. The product was cloned into XhoI- and BglII-digested pDM4 via sites incorporated in the primers, and the construct was introduced into B. pseudomallei 576 from E. coli S17-1{lambda}pir as described above. A similar approach was used to mutate bopB. An internal fragment of the gene was amplified using the oligonucleotides 5'-CGCGCTCGAGGCCGCGGCGATTTTCGCCCAT-3' and 5'-CCAAGATCTGGTCATCATCCGCAGTATCGCT-3', the product was cloned into pDM4 opened with XhoI and BglII, and the construct was introduced into strain 576. Insertion mutants (576 bopA : : pDM4 and 576 bopB : : pDM4) were selected and verified as described above.

Infection of animals.
For each infection, aliquots of B. pseudomallei wild-type or mutant strains were thawed from frozen stocks. Bacterial cells were diluted in PBS to the required concentration and administered either via the intraperitoneal route (0·2 ml) or via the intranasal route (0·05 ml), and mice were then monitored twice daily for symptoms of infection. Viable count determinations were performed to confirm the inoculation dose. Median lethal doses (MLDs) were determined by the method of Reed & Muench (1938). To enumerate bacteria in the liver and spleen, the organs were aseptically removed and homogenized in sterile PBS by passing them through a 100 µm mesh cell-strainer. Serial tenfold dilutions of tissue homogenates were plated onto tryptone soy agar, and colonies were enumerated after 24 h. Stability of pDM4 insertions in bsa-encoded genes was verified by plating of the recovered bacteria on LB medium, either with or without 50 µg chloramphenicol ml–1.

Immunization of BALB/c mice with purified BipD.
The pGEX-4T-1-based construct for expression of the translocator protein BipD as a glutathione-S-transferase (GST) fusion protein has been described previously (Stevens et al., 2002). Following expression in E. coli BL21(DE3) under isopropyl {beta}-D-thiogalactoside induction, BipD-GST was purified using glutathione Sepharose 4B resin according to the Amersham Pharmacia GST gene fusion protocol, and BipD was cleaved from GST by digestion with thrombin. Purified BipD protein, or GST or PBS as controls, were separately administered on days 1, 14 and 28 to ten female BALB/c mice aged 7–9 weeks. For each immunization, 10 µg protein was given in 100 µl of 50 % (v/v) Ribi adjuvant (Corixa) in PBS by the intraperitoneal route. On day 55, the mice received 3·3x104 c.f.u. B. pseudomallei strain K96243 by the intraperitoneal route, and they were monitored twice daily for the next 35 days. Expression of BipD by strain K96243 was confirmed by immunoblotting using BipD-specific rabbit polyclonal antiserum (data not shown).

Detection of BipD-specific antibodies.
A 96-well ELISA plate was coated overnight at 4 °C with either 100 µl of 1 µg ml–1 recombinant BipD in PBS or PBS control. Coated plates were washed three times with PBS plus 0·05 % Tween-20 (PBS-T). Plates were blocked with 5 % BSA in PBS, then washed with PBS-T as described above. Serum from animals immunized with BipD and PBS-treated controls (1/100 dilution in PBS) was added to an ELISA plate, serially diluted twofold in PBS, and then incubated at 37 °C for 60 min. After washing with PBS-T, HRP-conjugated secondary antibody, specific for mouse isotypes IgG1, IgG2a, IgG2b and IgG3 (Sigma), was added at a 1/2000 dilution to appropriate wells for 45 min. After washing, HRP activity was measured in a plate reader (414 nm absorbance) after 20 min incubation with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic) acid diammonium salt (ABTS) plus 0·02 % H2O2. The anti-BipD antibody titres for each IgG isotype were calculated as the maximum dilution of serum to give an A414 reading 0·1 U greater than background, and they are presented as the reciprocal of the dilution.

Statistical analysis.
Survival curves were compared using Log Rank tests, and P values <0·05 were taken to be significant.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The B. pseudomallei BipD protein is required for full virulence in murine models of melioidosis
To assess the importance of BipD in pathogenesis, a defined bipD mutant was constructed using B. pseudomallei strain 576, which has been extensively characterized in the BALB/c mouse model (Atkins et al., 2002a, b). Consistent with earlier findings (Stevens et al., 2002, 2003), mutation of the bipD gene in B. pseudomallei strain 576 impaired invasion of HeLa cells and intracellular survival in J774.2 murine macrophage-like cells, and prevented actin tail formation (data not shown). No effect of the bipD mutation on the growth rate of strain 576 in LB medium was detected (data not shown). In initial experiments to assess the virulence of the mutant strain, six groups of five 7- to 9-week-old female BALB/c mice were inoculated via the intraperitoneal route with increasing doses (101–106 c.f.u.) of either the 576 wild-type or the 576 bipD : : pDM4 mutant strain. At 35 days post-infection, the MLDs were calculated to be 80 c.f.u. for the B. pseudomallei 576 wild-type strain and 1·73x105 c.f.u. for the 576 bipD : : pDM4 mutant, indicating that the function of the Bsa TTSS is required for full virulence of B. pseudomallei.

Mutation of bipD was also found to be strongly attenuating following inoculation of mice via the intranasal route and when using a different B. pseudomallei strain. Groups of ten 8-week-old BALB/c mice were inoculated intranasally either with 102 c.f.u. of strain 576 or 576 bipD : : pDM4, or with 104 c.f.u. of the previously described 10276 bipD : : pDM4 mutant or the 10276 parent strain. All mice inoculated intranasally with 576 died within 6 days, whilst 7/10 mice given the 576 bipD : : pDM4 mutant survived to day 47 (Fig. 1a). Bacteria were recovered from the spleens of 576 bipD : : pDM4-infected mice that died on days 15 and 40 post-inoculation, and they were found to be exclusively chloramphenicol resistant, indicating that the insertion is stable in vivo. All mice given the 10276 bipD : : pDM4 mutant survived, whilst mice given the 10276 strain died within 4 days (Fig. 1b). We also inoculated six mice intranasally with 104 c.f.u. strain 10276 bsaZ : : pDM4, which completely lacks the function of the TTSS (Stevens et al., 2003). All mice given this strain survived to 47 days post-inoculation (Fig. 1b). These observations further support our conclusion that a functional Bsa TTSS is required for full virulence of B. pseudomallei in mice. Mutation of bipD and bsaZ by pDM4 insertion is not expected to be polar, since the genes are predicted to be the last in their respective operons (Stevens et al., 2002).



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Fig. 1. Survival of BALB/c mice inoculated intranasally with (a) 1x102 c.f.u. B. pseudomallei 576 wild-type ({square}, 6 mice) or an isogenic 576 bipD : : pDM4 mutant ({lozenge}, 10 mice) or (b) 1x104 c.f.u. B. pseudomallei 10276 wild-type ({blacksquare}, 6 mice), an isogenic 10276 bipD : : pDM4 mutant ({blacklozenge}, 10 mice), or an isogenic 10276 bsaZ : : pDM4 mutant ({blacktriangleup}, 6 mice). Mice were observed daily and percentage survival was plotted against time.

 
Attenuation of the B. pseudomallei bipD mutant in BALB/c mice correlates with reduced bacterial replication in the liver and spleen in the early phase of infection
Groups of twelve 7- to 9-week-old BALB/c mice were inoculated via the intraperitoneal route with either 104 c.f.u. B. pseudomallei 576 wild-type or 104 c.f.u. 576 bipD : : pDM4. Three mice from each group were killed on days 1, 3, 5 and 7 post-inoculation, the liver and spleen were collected aseptically, and the number of viable bacteria per organ was enumerated by plating of serial dilutions of organ homogenates. No mice given the wild-type strain survived beyond 5 days post-inoculation. Identical numbers of the bipD mutant were recovered on medium with or without chloramphenicol, confirming the stability of the insertion. The spleens and livers of mice infected with the wild-type 576 strain were enlarged and contained multiple abscesses. Splenomegaly and abscess formation were less obvious with the 576 bipD : : pDM4 (data not shown). In mice given the wild-type strain, rapid replication of the bacteria in the liver and spleen was detected; however, the load of the 576 bipD : : pDM4 mutant in the spleen and liver was significantly lower at almost all time points (Fig. 2).



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Fig. 2. Course of bacterial replication in the spleen (a) and liver (b) of BALB/c mice after inoculation with 1x104 c.f.u. B. pseudomallei strains 576 ({blacksquare}) or 576 bipD : : pDM4 ({circ}) by the intraperitoneal route. Three mice from each group were killed on days 1, 3, 5 and 7 post-inoculation. Values are given as the mean±SEM.

 
B. pseudomallei mutants lacking known or putative type III secreted effector proteins are attenuated in mice to differing extents
The finding that a functional Bsa TTSS is required for full virulence of B. pseudomallei in mice implies that the injection of bacterial effector proteins into host cells influences the outcome of infection. We therefore assessed the contribution of known or putative bsa-encoded type III secreted proteins to B. pseudomallei virulence in mice. Insertion mutants of strain 576 lacking bopA, bopB and bopE were constructed using {lambda}pir-dependent suicide replicons. Mutation of bopA, bopB and bopE did not affect either the ability of B. pseudomallei to form actin tails following infection of J774.2 cells or the in vitro growth rate of the bacteria (data not shown). Groups of six 7- to 9-week-old BALB/c mice were inoculated via the intraperitoneal route with 5x104 c.f.u. 576 wild-type or one of the following mutant strains: 576 bipD : : pDM4, 576 bopA : : pDM4, 576 bopB : : pDM4 or 576 bopE : : pDM4. The median times to death for the wild-type and mutant strains were as follows: wild-type (20 days) < bopE (21 days) < bopB (30 days) < bopA (32 days) < bipD (50 days) (Fig. 3). Mutation of bipD significantly delayed time to death compared to the wild-type (P=0·0005). However, all six mice given the 576 bipD : : pDM4 mutant died by day 63, indicating that mutation of bipD slows, but does not abolish, the development of fatal melioidosis. Mutation of bopE did not cause a significant reduction in median time to death compared to the wild-type (P=0·59). Furthermore, we did not detect significant attenuation of either 576 bopE : : pDM4 or 10276 bopE : : pDM4 mutant following intranasal inoculation of groups of six BALB/c mice (data not shown). Mutation of bopA and bopB caused a significant delay in median time to death when compared to the bopE mutant (P=0·0155 for bopA, and 0·005 for bopB); however, the differences were not statistically significant when compared to the wild-type strain.



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Fig. 3. Survival of BALB/c mice inoculated via the intraperitoneal route with 5x104 c.f.u. B. pseudomallei 576 wild-type, isogenic 576 bipD : : pDM4, 576 bopA : : pDM4, 576 bopB : : pDM4, 576 bopE : : pDM4 mutant strains or PBS (6 mice per group).

 
In this experiment, BALB/c mice inoculated via the intraperitoneal route with 5x104 c.f.u. 576 wild-type exhibited a longer median time to death than that observed in the experiments to determine MLD and the kinetics of replication in liver and spleen. Whilst these studies used the same dose, inoculation route and host, the experiments were performed at two independent laboratories and the discrepancy is likely to reflect differences in the source of animals and the humane end-point criteria applied. Regardless of such considerations, experiments with internal controls performed at either laboratory show a statistically significant reduction in B. pseudomallei virulence caused by mutation of bipD and bsaZ.

To assess the residual virulence of the bipD mutant in greater detail we infected C57BL/6 IL-12 p40–/– mice with the 576 wild-type and the bipD : : pDM4 mutant strain. Mice in which interleukin (IL)-12 has been depleted by the administration of anti-IL-12 monoclonal antibody are acutely susceptible to B. pseudomallei infection (Santanirand et al., 1999). We inoculated groups of five C57BL/6 IL-12 p40–/– mice with 103 c.f.u. strain 576, 103 c.f.u. 576 bipD : : pDM4 or PBS via the intraperitoneal route. The median time to death of C57BL/6 IL-12 p40–/– mice inoculated with the 576 bipD : : pDM4 mutant was 13 days, compared to 6 days with the wild-type strain (P=0·0173) (Fig. 4).



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Fig. 4. Survival of C57BL/6 IL-12 p40–/– mice inoculated with 1x103 c.f.u. B. pseudomallei 576 wild-type ({blacksquare}), an isogenic 576 bipD : : pDM4 ({circ}) mutant or sterile PBS ({square}) by the intraperitoneal route (10 mice per group).

 
Infection of BALB/c mice with the B. pseudomallei bipD mutant partially protects against challenge with wild-type bacteria
Survivors of the initial intraperitoneal challenges with the B. pseudomallei 576 bipD : : pDM4 mutant were inoculated 5 weeks after the first inoculation with a matched dose of the 576 wild-type. Survival was monitored for 35 days after rechallenge. A delay in the expected median time to death was observed in all groups. A survival rate of 60% was observed for mice dosed with 104 c.f.u. 576 bipD : : pDM4 then rechallenged with 104 c.f.u. wild-type 576 (Fig. 5); therefore, prior infection with the 576 bipD : : pDM4 mutant partially protected mice from infection with the wild-type organism. Control mice (not age-matched) given 104 c.f.u. wild-type 576 at the same time all died.



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Fig. 5. Survival of mice inoculated via the intraperitoneal route with 104 c.f.u. B. pseudomallei 576 bipD : : pDM4 and then rechallenged at day 35 post-inoculation (arrow) with 104 c.f.u. wild-type 576 strain ({bullet}, 5 mice). Mice inoculated with 576 at day 0 ({blacksquare}) all died by day 4 post-inoculation. Control mice ({blacktriangleup}, not age-matched) infected on day 35 with the rechallenge dose of 576 all died.

 
Immunization of BALB/c mice with purified BipD protein does not protect against challenge with wild-type B. pseudomallei
Groups of ten 7- to 9-week-old female BALB/c mice were immunized via the intraperitoneal route three times at 14 day intervals with 10 µg purified BipD protein with Ribi adjuvant in PBS. Control mice were immunized with 10 µg GST plus adjuvant or with PBS. A marked induction of BipD-specific IgG was detected by ELISA at day 54 after the first immunization. Titre units of IgG isotypes were as follows: total IgG, 1005; IgG1, 1510; IgG2a, 1242; IgG2b 845; IgG3, 7. On day 55 after the first immunization, mice were challenged with 3·3x104 c.f.u. B. pseudomallei strain K96243 by the intraperitoneal route and monitored for 35 days. A slight delay in median time to death was observed in the group of mice immunized with BipD compared to the control groups (Fig. 6) (median time to death BipD, 15·75±0·74 days; GST, 14·6±0·51 days; PBS, 14·3±1·55 days); however, the differences were not statistically significant. An overall survival rate of 20% was observed in the group immunized with BipD, whereas immunization with GST or PBS did not protect against lethal challenge (Fig. 6).



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Fig. 6. Survival of BALB/c mice immunized with purified BipD ({blacktriangleup}), GST ({bullet}) or PBS ({blacksquare}) (10 mice per group) and challenged via the intraperitoneal route with 3·3x104 c.f.u. B. pseudomallei strain K96243 on day 55 after the first immunization.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We assessed the role of components of the Bsa TTSS in B. pseudomallei virulence and immunity. Specifically, we assessed the role of BipD, a putative translocator protein that is required for invasion of epithelial cells, survival in murine macrophage-like cells, escape from endocytic vesicles and subsequent actin-tail formation (Stevens et al., 2002, 2003). We also investigated the effect of mutation of genes encoding a type III secreted guanine nucleotide exchange factor that facilitates invasion (BopE; Stevens et al., 2003) and the putative type III secreted effector proteins BopA and BopB.

A B. pseudomallei strain 576 bipD mutant was calculated to have a MLD in BALB/c mice of 1·73x105 c.f.u. compared to 80 c.f.u. for the parent strain (a greater than 2160-fold reduction in MLD). Significant attenuation was also observed following administration of the bipD mutant to BALB/c mice via the intranasal route, which is likely to represent a more natural route of infection. Mutation of bipD had the same effect on virulence following intranasal inoculation when a different B. pseudomallei strain was used. This suggests that the attenuation of the 576 bipD : : pDM4 mutant is unlikely to be explained by second-site mutations in the genome of the mutant strain. Furthermore, a bsaZ mutant of strain 10276 was attenuated to the same degree as the bipD mutant following intranasal infection; this confirms that the bsa-encoded Inv/Mxi-Spa-like TTSS plays an important role in the pathogenesis of melioidosis in mice. Our data are in agreement with the finding that structural components of the B. mallei strain ATCC 23344 Bsa apparatus are required for full virulence in rodents (Ulrich & DeShazer, 2004).

Splenomegaly and abscess formation were less pronounced in the liver and spleen of mice infected with the B. pseudomallei bipD mutant on days 1, 3, 5 and 7 post-infection, compared to mice infected with the wild-type strain. This is consistent with the reduced replication rate of the bipD mutant in the early phase of infection and the observations of Ulrich & DeShazer (2004). It is noteworthy that considerable animal-to-animal variation in bacterial load was detected with apparent clearance of the bipD mutant in some, but not all, animals. This reinforces the importance of examining groups of mice rather than single animals at each time interval, and it may explain why only some mice later succumbed to infection with the bipD mutant strain. Mice that died or reached a humane end-point following infection with the bipD mutant contained only mutant bacteria upon post-mortem examination, indicating that the insertion was stable in vivo and that the mice succumbed to the overall burden of the mutant strain and not to a revertant. Attenuation of the bipD mutant was not complete, and was significantly less than that caused by mutation of genes required for capsular polysaccharide or branched-chain amino acid biosynthesis (Atkins et al., 2002a, b). Nevertheless, mutation of bipD caused a significant delay in the median time to death compared to the wild-type in C57BL/6 IL-12 p40–/– mice, which are highly susceptible to B. pseudomallei infection.

B. pseudomallei bopE mutants were not significantly attenuated in BALB/c mice compared to the wild-type following either intraperitoneal or intranasal inoculation. In contrast, mutation of the putative bsa-encoded effectors BopA and BopB caused a statistically significant increase in median time to death in BALB/c mice following intraperitoneal inoculation, at least when compared to the bopE mutant. BopA is a homologue of Shigella IscB, which is required for cell-to-cell spread of Shigella (Allaoui et al., 1992). B. pseudomallei is also capable of cell-to-cell spread and cell fusion (Kespichayawattana et al., 2000); the role of BopA in these processes is the subject of investigation in our laboratory. BopB contains a motif shared with the catalytic domains of numerous phosphatases and potentially may subvert or inhibit eukaryotic cell signalling pathways. The finding that mutations affecting known or putative effector proteins have more subtle effects on B. pseudomallei virulence than disruption of the translocator BipD implies that they may act in concert to influence the outcome of infection, as is the case with Salmonella type III secreted effector proteins (Wallis & Galyov, 2000; Zhang et al., 2003). We cannot preclude the possibility that pDM4 insertions in bopA and bopB may have disrupted the expression of nearby genes. However the observation that the bopA and bopB mutants exhibit normal actin tail formation in J774.2 cells implies that the in vivo phenotype is unlikely to be the result of indirect effects on the function of the Bsa type III secretion apparatus, since this is required for endosome escape (Stevens et al., 2002).

Mice infected with the B. pseudomallei bipD mutant were partially protected against a challenge with the wild-type organism and the nature and efficacy of antigen-specific humoral and cell-mediated immune responses elicited by the B. pseudomallei bipD mutant requires further study. Protection conferred by the bipD mutant was not complete and may be influenced by the activation of innate immunity by persistent organisms at the time of rechallenge.

Immunization with purified BipD did not confer significant protection against B. pseudomallei infection. Co-administration of BipD with purified BipC did not improve protection, and BipB alone was not protective (J. Hill, M. W. Wood & E. E. Galyov, unpublished observations). The V-antigen proteins involved in translocation of type III secreted effector proteins in Yersinia and Pseudomonas have proven to be effective protective antigens (Leary et al., 1995; Sawa et al., 1999). It remains to be determined if other components of the bsa-encoded TTSS can induce protective immune responses against B. pseudomallei.

We have demonstrated that the Bsa type III protein secretion is required for full virulence of B. pseudomallei in mice. B. pseudomallei contains two other putative type III secretion loci, which are similar to the hrp gene cluster of Ralstonia solanacearum and TTSS loci in other plant pathogens (Winstanley et al., 1999; Rainbow et al., 2002); the role played by these systems in virulence awaits investigation. A mutation in the plant pathogen-like type III secretion apparatus of Burkholderia cepacia genomovar III was recently reported to be attenuating in a murine model of respiratory infection (Tomich et al., 2003). Thus TTSSs appear to play key roles in the pathogenesis of Burkholderia infections in animals.


   ACKNOWLEDGEMENTS
 
This work was supported by the Biotechnology and Biological Sciences Research Council, UK (E. E. G. and M. P. S. 201/C20021), and the United Kingdom Ministry of Defence. We gratefully acknowledge the expert advice of Dr Debbie Smith and the staff of the Biological Services Facility at LSHTM.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Received 8 March 2004; revised 7 May 2004; accepted 11 May 2004.



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