Abstract
Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were recognised by seropositive human sera from the endemic area. To conclude, several predicted autotransporters contribute to B. pseudomallei virulence and BpaC may do so by conferring resistance against complement-mediated killing.
Highlights
Burkholderia pseudomallei is a motile Gram-negative bacillus which is a saprophyte of tropical and sub-tropical soils and water
Our data indicate that most of the putative ATs play a role in the virulence of B. pseudomallei 10276 in BALB/c mice when infection is delivered by the intra-peritoneal route (BcaA, BoaA, BoaB, BpaA, BpaC, B. pseudomallei replication (BpaE), BbfA and BimA)
For LPS, overnight cultures of the B. pseudomallei strains were pelleted by centrifugation and the bacterial cells lysed by boiling for 10 minutes in 300 μl 1x SDS-PAGE sample buffer prior to Proteinase K (0.5 mg/ml) treatment for 1 h at 60°C
Summary
Burkholderia pseudomallei is a motile Gram-negative bacillus which is a saprophyte of tropical and sub-tropical soils and water. Autotransporters (ATs) comprise a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play roles in numerous environmental and virulence-associated interactions including persistence, adhesion, serum resistance and biofilm formation. A B. pseudomallei 1026b mutant in the gene encoding this same TAA, independently named bpaF, was found to display wild-type virulence via the intra-nasal route [15] This result was part of a study of eight predicted TAAs (excluding BimA) which found that only BapC played a role in virulence via the intra-nasal route in the BALB/c mouse model. One study has focused on the classical ATs; a mutant lacking the putative protease BcaA was found to have a reduced invasion and plaque formation in A549 cell lines but no virulence phenotype [17]. We have identified that one predicted TAA, BpaC, plays a role in resistance to complement-mediated killing by 45% (v/v) NHS via the classical and/or lectin arm of the complement pathway
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