title: 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.
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom.
authors.forename: Natalie R
authors.initials: NR
authors.lastname: Lazar Adler
authors.affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, United Kingdom.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Stevens
authors.affiliation: Biomedical Sciences, Defence Science and Technology Laboratory, Porton Down, United Kingdom.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Dean
authors.affiliation: Biomedical Sciences, Defence Science and Technology Laboratory, Porton Down, United Kingdom.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Saint
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom.
authors.forename: Depesh
authors.initials: D
authors.lastname: Pankhania
authors.affiliation: Biomedical Sciences, Defence Science and Technology Laboratory, Porton Down, United Kingdom.
authors.forename: Joann L
authors.initials: JL
authors.lastname: Prior
authors.affiliation: Biomedical Sciences, Defence Science and Technology Laboratory, Porton Down, United Kingdom; School of Biosciences, Geoffrey Pope Building, University of Exeter, Exeter, United Kingdom.
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Atkins
authors.affiliation: The Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Bianca
authors.initials: B
authors.lastname: Kessler
authors.affiliation: The Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Arnone
authors.initials: A
authors.lastname: Nithichanon
authors.affiliation: The Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand.
authors.forename: Ganjana
authors.initials: G
authors.lastname: Lertmemongkolchai
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, United Kingdom.
authors.forename: Edouard E
authors.initials: EE
authors.lastname: Galyov
mesh_terms: D056704:Adaptive Immunity; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D016957:Burkholderia pseudomallei / Q000235:genetics / Q000472:pathogenicity*; D002460:Cell Line; D004195:Disease Models, Animal; D006801:Humans; D007111:Immunity, Cellular; D037102:Lectins / Q000378:metabolism; D008554:Melioidosis / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D050296:Microbial Viability; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D044967:Serum / Q000737:chemistry; D000069320:Type V Secretion Systems / Q000235:genetics* / Q000378:metabolism; D014774:Virulence / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D037102:Lectins; D011994:Recombinant Proteins; D000069320:Type V Secretion Systems
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