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
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ABSTRACT |
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These authors contributed equally to the work.
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INTRODUCTION |
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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.
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METHODS |
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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
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 S17-1pir 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 ml1.
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
-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 79 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 ml1 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.
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RESULTS |
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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 8 March 2004;
revised 7 May 2004;
accepted 11 May 2004.
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