Biochemical And Cellular Mechanisms Of SipA Induced Enteritis

Salmonella spp. are gram-negative bateria that cause diseases ranging from self-limiting enterocolitis to typhoid fever. Virulence determinants to salmonallae are the Salmonella pathogenicity islands in the chromosome. Now it has been confirmed there are five SPIs in the Salmonella chromosome from SPI1to SPI5.SPI1and SPI2encode type Ⅲ secretion system1and2, respectively. TTSS-1is associated with S. typhimurium’s ability to envade host cell cells.TTSS-2has the important role in the survival and replication of salmonella in the host cell.SipA, one of these effector proteins, binds to F-actin and polymerizes G-actin, which facilitates bacterial entry into epithelial cells. SipA is not only responsible for promoting actin polymerization, but also plays a role in the induction inflammation in the bovine and mice model. So our subject is planning to confirm the amino acids residues of SipA interaction with actin. Then we will construct sipA mutant strains that fail to interact with actin. At last, we investigate sipA mutant strains by mice animal model to elucidate the molecular mechanisms of SipA in serovar Typhimurium colitis.Researchers found SipA497-669is the domain to interact with actin. Based on this, we made a series of SipA truncation and tested their function by cosedimentation assay. We also constructed a set of SipA point mutation by Multi Site-Directed Mutagenesis Kit and check their function by cosedimentation assay. We show here that SipA514-651is the domain of interaction with actin and SipA635,637is the amino acids residues of interaction with actin. Then we investigated weather SipA514-651and SipA635,637have an effect on the critical concentration of G-actin by pyrene-actin assay. We found SipA514-651and SipA635,637can lower the critical concentration to promote actin polymerization.To further analyze the function of SipA, we constructed a set of sipA mutant strains including SipAL551A S.Typhimurium, SipAK635A5A S.Typhimurium, SipAE637W S. Typhimurium, SipAK635AE637W S. Typhimurium, SipA1580SK581A S. Typhimurium. We examined the function of these stains by using them to infect HeLa cells and mice. We found SipAK635AE637W S. Typhimurium was significantly less invasive than the wild-type strain. But Mice infected with SipAK635AE637W S. Typhimurium had severe inflammation similar to that of WT. We might get the conclusion that the key amino acids residue of SipA635,637to polymerize G-actin has no effect on murine colitis.Our discovery may help to elucidate pathogenesis of Salmonella and provide vital insights into basic host cell biology.