INVESTIGADORES
SARNACKI Sebastian Hernan
congresos y reuniones científicas
Título:
Attenuated Dam Mutants Of Salmonella Enterica Has A Defect In The O Antigen Lipopolysaccharide (LPS) Synthesis.
Autor/es:
S. H. SARNACKI, M. N. GIACOMODONATO, M. NOTO LLANA, M. A. VALVANO M. C., CERQUETTI.
Lugar:
Victoria, BC, Canadá,
Reunión:
Conferencia; 2nd American Society for Microbiology Conference on Salmonella: From Pathogenesis to therapeutics.; 2006
Institución organizadora:
American Society for Microbiology (ASM)
Resumen:
The DNA adenine methylase (Dam) enzyme modulates a variety of processes such as DNA replication, mismatch repair and gene transcription. Salmonella enterica dam mutants have decreased infectivity in the murine model and have been recently suggested as promising vaccine candidates. We obtained Salmonella enterica serovar Enteritidis dam mutants by targeted and transposition mutagenesis. Using a murine model of salmonellosis, we observed that the S. Enteritidis dam mutants were attenuated when inoculated by intraperitoneal, oral, and intragastrical routes. Protection rates conferred by these mutants in immunized mice challenged with the parental strain of S. Enteritidis, varied between 40 to 60 %. We also found that dam mutants induced lower levels (p < 0.05) of intestinal IFN-ã than the parental strain (509 + 62 vs 798 + 82 pg/ ìg of protein, respectively), measured by ELISA. Western blot analysis showed that dam mutants induced a delayed activation of NF-kB. We also observed by silver-stained SDS-PAGE that dam mutants of S. Enteritidis produce a defective LPS, which was characterized by a reduced length in the O antigen polysaccharide. This LPS pattern was also observed in our laboratory in S. Typhimurium dam mutant, a strain proposed as a vaccine candidate, but no evident defect was observed in E. coli dam mutant. These observations suggest that the Dam protein in S. enterica may be involved in modulating LPS synthesis, more specifically the length distribution of O antigen polysaccharide, a function encoded by the wzz gene. In order to test whether the methylation by Dam might be involved in wzz regulation the transcriptional expression of the wzz gene, was studied with a wzz::lacZ fusion contained in a low copy number plasmid, pFZY1. The activity of Beta-galactosidase was assessed in wild type and dam mutant strains of S. Enteritidis. Analysis of Beta-galactosidase production by cells carrying plasmids with the fusion indicated that lack of Dam enzyme, increased transcription of the wzz promoter. Therefore, our results demonstrate that the dam gene modulates LPS synthesis and this defective LPS could explain, at least in part, the avirulent phenotype of S. Enteritidis dam mutants.