Construction Of Salmonella Enterica Serovar Enteritidis C50336 SipA Mutant And Preliminary Study On Inflammatory Effect Induced By SipA

Salmonella enterica serovar Enteritidis (S. Enteritidis, SE) is a gram-negative and facultatively intracellular bacterium, parasitic in both human and animal intestines. As an important zoonotic foodborne pathogen, it can cause acute gastroenteritis and even severe systemic infection. Type three secretion system (T3SS) encoded by SPI-1 or SPI-2 is an important virulence factor for Salmonella. T3SS effectors play different roles in pathogenesis of Salmonella. Currently, the function of T3SS effectors has been investigated in S.Typhimurium, however, there are little such researches about their functions in S.Enteritidis (SE). S.Typhimurium SipA contributes to invasion of epithelial cells by modulating actin assembly through its C-terminal. While the N-terminal 425 amino acids of SipA trigger epithelial cell signaling. In addition, SipA contributes to intestinal inflammation in vivo, but the underlying mechanism has remained unclear. In this study, we constructed the sipA mutant of SE by homologous recombination system and investigated the relationship between SipA and inflammation. This study lays a foundation for further study on the molecular mechanism of sipA in the process of SE infection.1 The prokaryotic expression of SE T3SS effector SipA and construction of the mutant strain C50336△ipAWe constructed the prokaryotic expression plasmid pET30a(+)-sipA, and then transformed it into E. coli BL21 (DE3).The SipA protein was expressed efficiently after induction by IPTG SDS-PAGE and Western blotting analysis showed that the SipA protein was expressed efficiently in the soluble form and in predicted sizes in vitro. The purified protein SipA was injected into BALB/c mice to produce polyclonal antibodies.The sipA gene mutant of SE was constructed by X-Red recombination system, named as C50336△sipA. The corresponding complementary strain C50336△sipApsipA was also developed in this study. The expression of the protein SipA was further identified by Western-blot assy. Then the biological characteristics and virulence were determined. Among the three strains, there are no obviously differences in growth property, biochemical properties and virulence. And the pass-generation test showed that the hereditary property of mutant strain was stable. Construction of sipA mutant of SE provides the corresponding biological material for studying the role of SipA in the process of SE infection.2 Preliminary study on inflammatory effect induced by SipA in SEThe adhesion and invasion to Caco-2 by C50336△sipA was determined. The results showed the ability of adhesion to Caco-2 by C50336AsipA was similar to wild type strain C50336, but the ability of invasion was significantly decreased compared with the wild type strain and complementary strain. Caco-2 BBE cells were then infected by SE for detection of Bax associated with apoptosis. The apoptosis rate of Caco-2 BBE cells infected by C50336△ sipA was significantly decreased with comparison to wild type and complementary strain. Laser scanning confocal microscope and Western blot analysis identified that the expression of Bax protein associated with apoptosis was significnantly decreased in Caco-2 BBE cells infected by C50336△ sipA compared to wild type and complementary strain. The recombinant eukaryotic expression plasmid pcDNA3.1flag-sipA was constructed and then transfected transiently to HEK293T cells. Indirect immunofluorescent assay demonstrated the expression of SipA in HEK293T cells. After transfection of plasmid pcDNA3.1flag-sipA to HEK293T and HeLa cells respectively, cells were harvested to measure NF-κB transcriptional activity efficiency by luciferase reporter assay. HeLa cells stably transfected with an NF-icB--luciferase reporter was infected by SE, NF-κB activation was assessed using the NF-κB luciferase reporter system. The result showed expression of SipA in HEK293T and HeLa cells resulted in NF-κB activation. Activation of NF-κB in HEK293 cells induced by C50336△sipA was significantly decreased compared with wild type and complementary strain. These results indicated that SipA could activate NF-κB at the cellular level. The streptomycin pre-treated C57BL/6 mice were infected by C50336, C50336AsipA and C50336△sipApsipA. The pathological section results demonstrated that inflammatory infiltration and lesion of liver, colon and cecum lighten on the C50336△sipA group compared with C50336 and C50336△sipApsipA groups. The expression levels of inflammation cytokines from C57BL/6 mice serum showed that IL-6, TNF-a, MCP-1, IFN-y of C50336△sipA group decreased significantly compared with C50336 and C50336△sipApsipA groups. In conclusion, the effector protein SipA could induce host inflammation response during SE infection, and it will be helpful for us to understand the function of SipA in the process of SE infection.