| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 91 === Salmonella enteritidis is a facultative intracellular pathogen that can cause disease in humans and animals. Poultry meat and eggs are considered to be the major source of infection for infant, elderly, and those with impaired immune system. Egg associated salmonellosis have been raised because many strains of salmonella have become resistant to several of the antibiotics traditionally used to treat it. Today, modern chemotherapy must consider the treatment of antibiotics resistance and target factors that are required for the pathogenesis of bacterial infection. The purpose of this research is to characterize the virulence-associate factors and elucidate their roles in the pathogenesis of S.enteritidis.
In our previous study, two hemolytic activity genes have been cloned from genomic library of S.enteritidis (Sal-5). One gene encodes outer membrane protein P1 (OMPP1), and the other encodes hemolysin like protein (HLY). OMP are exposed on the surface of the bacterial cell and they can serve as phage receptors and react with antibodies. In this study, the results of cell fraction experiment indicated that OMPP1 is an outer membrane associated protein. HLY seems to have a cleavable N-terminal signal peptide and may be anchored in the membrane or secreted to the medium by prediction of computer. With their physical and biological characteristics, OMPP1 and HLY may be used successfully as vaccine antigens and good immunogens.
To evaluate whether OMPP1 and HLY could confer protective immunity against S.enteritidis infection or not, active and passive immunization experiments were performed. Mice immunized with OMPP1 showed 60％ protection and immunized with HLY showed 20％protection against challenged with 373 c.f.u. of S.enteritidis, compared to a 90％ mortality in mice immunized with BSA. For passive immunization experiment, mice challenged intra-peritoneally (I.P.) with 175 c.f.u. of S.enteritidis were intra-venous (I.V.) treated with 200ul anti-OMPP1 or anti-HLY antibody one times a day for five consecutive days. Antibody treatment resulted in a survival rate of 80％ for using anti-OMPP1 antibody and 10％ for using anti-HLY antibody, compared to a 90％ mortality in mice treated with pre-immune antibody. These results indicated that OMPP1 is a good immunogen and can provide the protective effects in both active and passive immunization.
In order to study the role of OMPP1 in the pathogenesis of Salmonella infection, ompp1-deficient strain was constructed by using integrated plasmid to disrupt the original ompp1 gene. In vitro studies of bacterial adhesion and invasion on intestinal epithelial cell lines and epithelial monolayer were performed. Compared to wild-type strain, the ompp1 mutant exhibited a 70％ decrease in adhesion ability and a 73％decrease in invasion ability. These results indicated that OMPP1 is involved in cellular adhesion and invasion, and plays an important role of Salmonella in natural infection route.
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