RESUMEN
Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease of humans and animals. Gene clusters which encode capsular polysaccharide (type I O-PS) and LPS (type II O-PS), both of which play roles in virulence, have previously been identified. Here, the identification of two further putative clusters, type III O-PS and type IV O-PS, is reported. Mice challenged with type III O-PS or type IV O-PS mutants showed increased mean times to death (7.8 and 11.6 days) compared to those challenged with wild-type B. pseudomallei (3 days). To investigate the possible roles of polysaccharides in protection, mice were immunized with killed cells of wild-type B. pseudomallei or killed cells of B. pseudomallei with mutations in the O antigen, capsular polysaccharide, type III O-PS or type IV O-PS gene clusters. Immunization with all polysaccharide mutant strains resulted in delayed time to death compared to the naïve controls, following challenge with wild-type B. pseudomallei strain K96243. However, immunization with killed polysaccharide mutant strains conferred different degrees of protection, demonstrating the immunological importance of the polysaccharide clusters on the surface of B. pseudomallei.
Asunto(s)
Infecciones por Burkholderia/microbiología , Burkholderia pseudomallei/patogenicidad , Islas Genómicas/fisiología , Polisacáridos Bacterianos/fisiología , Vacunación , Factores de Virulencia/fisiología , Animales , Cápsulas Bacterianas/química , Cápsulas Bacterianas/genética , Vacunas Bacterianas/administración & dosificación , Infecciones por Burkholderia/prevención & control , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/inmunología , Femenino , Genes Bacterianos/genética , Inmunización Secundaria , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos BALB C , Familia de Multigenes/genética , Antígenos O/genética , Operón , Mutación Puntual , VirulenciaRESUMEN
Yersinia pestis, a Gram negative bacterium, causes bubonic and pneumonic plague. Emerging antibiotic resistance in clinical isolates is driving a need to develop novel antibiotics to treat infection by this transmissible and highly virulent pathogen. Proteins required for viability, so called essential genes, are attractive potential therapeutic targets, however, confirmation of essentiality is problematic. For the first time, we report the development of a system that allows the rapid determination of Y. pestis gene essentiality through mutagenesis and inducible expression of a plasmid borne copy of the target gene. Using this approach, we have confirmed the uridine monophosphate kinase PyrH as an essential protein in Y. pestis. This methodology and the tools we have developed will allow the confirmation of other putative essential genes in this dangerous pathogen, and facilitate the identification of novel targets for antimicrobial development.