RESUMO
A recombinant cosmid containing genes involved in Klebsiella pneumoniae C3 core lipopolysaccharide biosynthesis was identified by its ability to confer bacteriocin 28b resistance to Escherichia coli K-12. The recombinant cosmid contains 12 genes, the whole waa gene cluster, flanked by kbl and coaD genes, as was found in E. coli K-12. PCR amplification analysis showed that this cluster is conserved in representative K. pneumoniae strains. Partial nucleotide sequence determination showed that the same genes and gene order are found in K. pneumoniae subsp. ozaenae, for which the core chemical structure is known. Complementation analysis of known waa mutants from E. coli K-12 and/or Salmonella enterica led to the identification of genes involved in biosynthesis of the inner core backbone that are shared by these three members of the Enterobacteriaceae. K. pneumoniae orf10 mutants showed a two-log-fold reduction in a mice virulence assay and a strong decrease in capsule amount. Analysis of a constructed K. pneumoniae waaE deletion mutant suggests that the WaaE protein is involved in the transfer of the branch beta-D-Glc to the O-4 position of L-glycero-D-manno-heptose I, a feature shared by K. pneumoniae, Proteus mirabilis, and Yersinia enterocolitica.
Assuntos
Proteínas de Bactérias/genética , Bacteriocinas/metabolismo , Glucosiltransferases , Klebsiella pneumoniae/genética , Lipopolissacarídeos/biossíntese , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Southern Blotting , Cosmídeos , Eletroforese em Gel de Poliacrilamida , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Escherichia coli/genética , Teste de Complementação Genética , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/patogenicidade , Lipopolissacarídeos/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mapeamento Físico do Cromossomo , Análise de Sequência de DNA , Deleção de Sequência , VirulênciaRESUMO
The ability of Aeromonas hydrophila serogroup O:34 strains grown under different conditions (capsulated and non-capsulated) to adhere to and invade two fish cell lines was compared. The level of adherence was slightly higher when the strains were grown under conditions promoting capsule formation than when the same strains were grown under conditions which did not promote capsule formation. However, the most significant difference among the wild-type strains grown under conditions promoting capsule formation was the ability to invade the fish cell lines, which was significantly higher than when the same strains were grown under conditions which did not promote capsule formation. Isogenic unencapsulated mutants grown under conditions promoting capsule formation showed a lower ability to invade the fish cell lines than the parental capsulated strains. From these results, we concluded that the capsular polysaccharide is an important factor in intracellular invasion.
