RESUMO
Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/genética , Proteoma/genética , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Células CACO-2 , Cromatografia Líquida , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/ultraestrutura , Humanos , Intestinos/citologia , Intestinos/microbiologia , Lactococcus lactis/metabolismo , Lactococcus lactis/ultraestrutura , Microscopia Eletrônica , Anotação de Sequência Molecular , Dados de Sequência Molecular , Família Multigênica , Fragmentos de Peptídeos/análise , Plasmídeos , Probióticos/química , Proteólise , Proteoma/metabolismo , Espectrometria de Massas em Tandem , Tripsina/químicaRESUMO
Homology-facilitated illegitimate recombination has been described in three naturally competent bacterial species. It permits integration of small linear DNA molecules into the chromosome by homologous recombination at one end of the linear DNA substrate, and illegitimate recombination at the other end. We report that homology-facilitated illegitimate recombination also occurs in Escherichia coli during conjugation with small non-replicative plasmids, but at a low frequency of 3×10(-10) per recipient cell. The fate of linear DNA in E. coli is either RecBCD-dependent degradation, or circularisation by ligation, and integration into the chromosome by single crossing-over. We also report that the observed single crossing-overs are recA-dependent, but essentially recBCD, and recFOR independent. This suggests that other, still unknown, proteins may act as mediator for the loading of RecA on DNA during single crossing-over recombination in E. coli.