Expression and Secretion of SopA Effector Protein in DNA Adenine Methylase (dam) Mutants of Salmonella Typhimurium

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

Rosario, Argentina

Congreso; SAIB 2006. XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2006

SAIB. Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

DNA adenine methylase (Dam) protein plays an important role in the pathogenesis of several bacterial species, including Salmonella Typhimurium. Here we examined the effect of Dam protein on the expression and secretion of Salmonella pathogenicity island 1 (SPI1) proteins, such as SopA. We obtained a SopA tagged Dam mutant of S. Typhimurium named STD2795. Bacteria were grown under SPI-1 condition. The expression and secretion of SopA was determined in bacterial pellets and culture supernatans, respectively. Invasion and proliferation were studied in Hep-2 cells. Analysis of methylation status showed that STD2795 has unmethylated DNA, this pattern was reverted in the complemented strain. It was found that the dam mutant invades epithelial cells 58 % less than the wild type (wt). The proliferation of the mutant within Hep-2 cells was also affected. Proliferation but not invasiveness was restored in the complemented strain. Western blot analysis showed STD2795 expresses lower levels of SopA (p< 0.001) than the wt strain under SPI-1 conditions. When cultured at 28 ºC the mutant expressed similar levels of SopA whereas wt and complemented strains showed no expression. On the other hand, SopA secretion was only observed in the wt strain under SPI-1 conditions. The partial inability of the dam mutant to invade epithelial cells could be related to a reduced secretion of invasion proteins encoded in the SPI-1, like SopA.