Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis. Virulence is determined by five Salmonella Pathogenicity Islands (SPI) encoded on the bacterial chromosome. SPI1 and SPI2 encode the structural components of two Type Three Secretion Sytems (TTSS) that translocate effectors into the host cell. The SPI1 effector SigD is an inositol phosphatase that induces the activation of Akt/PKB a mammalian serine threonine kinase. The role of Akt activation in pathogenesis remains unclear, however, in cultured epithelial cells it appears to be involved in preventing the onset of apoptosis in infected cells. More specifically we have found that a sigD deletion mutant induces Caspase 3 activation compared to wild type. Wild type Salmonella, but not the sigD mutant can block the induction of apoptosis by camptothecin. This may be important for the survival and replication of intracellular bacteria. In macrophages activation of Akt by Salmonella is more complex since lipopolysaccharide will activate the kinase in the absence of SigD. Comparison of the two pathways has revealed important differences. In particular, SigD-mediated activation is insensitive to Wortmannin a well described inhibitor of Akt activation via phosphatidylinositol-3-kinase. Thus SigD-dependent Akt activation appears to occur via a novel pathway. We have found that sigD also has effects up to 15 hours post invasion (p.i.). This is an important finding since it indicates that SPI1 effectors are not only involved in very early events. In this study we have investigated the role of SigD in iNOS induction in infected macrophages. We found that iNOS induction is affected by Sigd presumably in an Akt dependent process. Furthermore we found that SigD protein is detectable in infected cells for up to 8 hours p.i. Real time PCR confirmed that SigD continues to be expressed for at least 4 hours p.i. In order to further investigate the interaction between host cell and pathogen we are carrying out gene expression studies. Our initial experiments have concentrated on analyzing the expression of SPI1 and SPI2 genes by real time PCR. The results show that the expression is dependent on the mechansim of entry.
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