Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance

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.

Fig 1. Net intracellular replication of the B. pseudomallei AT mutants in J774.2 macrophage-like cells.

Viable intracellular bacteria were enumerated (output divided by input CFU) for the duplicate experimental samples of each of the AT mutants and the data was normalised against the wild-type parent strain. Data from five experiments was expressed as the mean and standard error of the mean; statistical significance was analysed using Student’s t test.

Fig 2. The bpaC mutant is sensitive to complement-mediated killing.

The bpaC mutant demonstrates a 1.1-log₁₀ reduced survival in 45% (v/v) NHS after 3 h incubation relative to the parent strain (1.08 x 10³ CFU versus the 1.22 x 10⁴ CFU input, p = 0.0003). This sensitivity could be abrogated by the addition of 10 mM EDTA or 10 mM EGTA, 5 mM MgCl₂. Graphs show mean results with error bars displaying standard error of the mean; statistical significance was calculated using Student’s t test.

Fig 3. Complementation of the bpaC mutant serum sensitivity and intracellular survival phenotypes.

a. Complementation of the bpaC mutant with a full-length copy of the bpaC gene expressed in trans by the inducible pME6032 vector (pME-bpaC) abrogated the serum sensitive phenotype seen for the bpaC mutant harbouring empty pME6032 vector (pME). Graphs show mean results with error bars displaying standard error of the mean; statistical significance was calculated using Student’s t test. b. Complementation of the bpaC mutant with a full-length copy of the bpaC gene expressed in trans by the inducible pME6032 vector (pME-bpaC) partially complemented the defect in net intracellular replication seen for the bpaC mutant harbouring pME6032 vector with a full-length copy of the bbfA gene (pME-bbfA). Graphs show normalised mean results with error bars displaying standard error of the mean; statistical significance was calculated using Student’s t test.

Fig 4. Systematic assessment of ATs for three pathogenesis-associated phenotypes.

A Venn diagram of the phenotypic results of the complete repertoire of ATs for virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 macrophage-like cells and survival in 45% (v/v) normal human serum.

Fig 5. Immune recognition of recombinant proteins.

a) Recombinant N-terminal GST fusion proteins of the passenger domain of four ATs were used as antigens (1 μg/ml) to stimulate whole blood (WB) from human seropositive donors, alongside PHA and fixed B. pseudomallei strain K96243 positive controls. The IFN-γ response of WB was measured by ELISA. BpaE and BcaA (225.4 pg/ml ± 45.7 pg/ml and 214 pg/ml ± 56.7 pg/ml respectively; p value <0.001) relative to the medium-only control average (4.99 pg/ml ± 1.7 pg/ml) elicited a strong IFN-γ response from WB from seropositive donors (n = 2; black and grey bars represent the two individual donors) while BpaC and BatA did not induce a significant lymphocyte recall response. b) The four antigens were also tested for recognition by human seropositive sera from a melioidosis endemic area using an indirect sandwich ELISA. BpaE and BcaA (absorbance index of 7.96 ± 0.82 and 4.86 ± 0.55 respectively; p value <0.001) were both recognised by seropositive human sera from the endemic area while values for neither BpaC nor BatA were significantly different from the negative control.