Expression of SopA and SopB effector proteins in dam mutants of Salmonella Typhimurium during the epithelial cells infection

GIACOMODONATO MN, GARCÍA CATTANEO AS, SARNACKI SH, NOTO LLANA MA, UZZAU S, RUBINO S, CERQUETTI MC

Istanbul. Turkey.

Congreso; IUMS Congresses 2008: the XII. International Congress of Bacteriology and Applied Microbiology.; 2008

IUMS

Deoxyadenosine methyltransferase (Dam) protein is required for expression of virulence genes in certain bacteria included Salmonella Typhimurium but the mechanism of the Dam requirement for Salmonella spp. virulence is still a matter of discussion. The aim of this study was to analyze the involvement of Dam protein on the expression and secretion of SPI-1 proteins, such as SopA and SopB. We obtained dam mutants of Salmonella Typhimurium, named STD2795 Ddam sopA::3xFLAG and STD2796 Ddam sopB::3xFLAG by epitope tagging. The expression and secretion of SopA and SopB were determined in bacterial culture and in intracellular bacteria recovered from infected epithelial cells (HEp-2). Bacterial culture supernatants and pellets were used to investigate secreted-proteins and cell-associated proteins, respectively. Bacterial strains were grown under SPI-1 conditions. Western blot analyze showed that dam mutants express lower levels of SopA and SopB than the wild type strain (p<0.05). We observed that dam mutants synthesize SopA under conditions that are nonpermissive for synthesis in wild-type strains (28º C). However, these mutants were unable to secrete SopA under any culture condition. On the other hand, we found that SopB secretion in dam mutants, under SPI-1 conditions, is reduced compared to the virulent strain (p<0.05). Analysis of intracellularly grown bacteria in a 20 minutes and 24 hours gentamicina protection assay revealed the presence of SopA and SopB in both dam and wild type strains. The amount of SopA detected in dam mutants was lower than the wild type strain (p <0.05), whereas no differences were observed on SopB expression.  Complementation of the dam mutation fully restored the proteins profile observed in the wild type strain. Taken together, our results demonstrate that Dam methylation modulates the Salmonella Typhimurium SopA expression and secretion.