DNA adenine methylation regulates SPI-5 expression in Salmonella enterica serovar Typhimurium.

SARNACKI SH, NOTO LLANA M, CERQUETTI MC, GIACOMODONATO MN.

Boston

Conferencia; 4th ASM Conference on Salmonella: The Bacterium, the Host and the Environment; 2013

ASM

DNA adenine methylation regulates virulence gene expression in certain bacteria, including Salmonella Typhimurium. The aim of this study was to investigate the involvement of DNA adenine methylase (Dam) in the in vivo expression and translocation of SopB (SPI-5-encoded effector) in SalmonellaTyphimurium. For this purpose, SopB-FLAG-tagged wild-type and damstrains of Salmonella Typhimurium were constructed by epitope tagging. The expression and secretion of SopB was determined in vitro and in vivo. Tagged proteins were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting with anti-FLAG antibodies. Bacteria were cultured under conditions that mimic the intestinal niche and the intracellular environment. Western blot and quantitative reverse transcriptase PCR analysis showed that dam mutant expresses lower levels of SopB/sopB(p < 0.05) than the wild-type strain under both cultured conditions. As well, SopB secretion was considerably impaired in this mutant. For in vivo experiments, cell monolayers and BALB/c mice were inoculated with tagged strains of Salmonella Typhimurium. Infecting bacteria and infected cells were recovered from HEp-2 cells and mesenteric lymph nodes (MLN). In agreement with in vitro experiments, the synthesis of SopB in dam mutants recovered from infected epithelial cells and from murine MLN 24 h post inoculation was reduced by 40 % respect to the wild type strain (p < 0.05). No SopB translocation by the dam mutant was detected in the cytosol of cells isolated from epithelial cells and murine MLN. Taken together, our results demonstrate that in Salmonella Typhimurium, Dam methylation modulates the expression and translocation of a SPI-5-encoded effector, like SopB.