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.