| Salmonella Enteritidis is an important zoonotic pathogen that has a broad host spectrum, which can cause intestinal or systemic disease in the human and animal. Salmonella pullorum is a host-adapted pathogen in avian species, which can infect chickens with different stages, as well as long-term colonization and growth retardation. This diseases lead to enormous economic loss in poultry industry and poses a serious threat to human and animal health.Salmonella infect hosts through the oral route where they adhere and invade in intestinal epithelial cells or lymphoid tissue localized mainly in the Peyer’s patch. Infected phagocytes move to lymphoid tissues where bacterial undertake multiplication. In this process, adhesion to the host cell is the prerequisite request of Salmonella pathogenicity. Although role of FimH in Urinary Tract Infections have been discussed thoroughly, its function in Salmonella Enteritids and Salmonella pullorum have been rarely defined. This study focus on function ofFimH in type I fimbriae, and explores its biological function in the pathogenicity of Salmonella Enteritidis and S.pullorums.The FimA prokaryotic expression vectors were constructed, through vaccinating BALB/c mice with purified FimA, specific polyclonal antibody was obtained. The expression of type Ⅰ fimbriae were detected in both Salmonella Enteritidis and S.pullorum in vitro by high specific mice anti-FimA antibody. Based on the original DNA sequences of the fimH gene for Salmonella in GenBank, specific primers were designed to amplify the fimH in Salmonella Enteritidis CMCC(B)50336 and S.pullorum 15218, which were chosen as the parent strains to construct the fimH isogenic mutants by λ-Red recombination system. The PCR products for the replacement the fimH of the parents strains above were first generated by using the template plasmid pKD3 and primers which contain a homologies at the 5’terminal to the target region and homologies at the 3’terminal to the chloromycin-resistance cassette (cat is flanked by FRT sites, which were recognition by FLP). Secondly, the PCR products were, respectively, introduced into Salmonella Enteritidis CMCC(B)50336 and S.pullorum 15218 carrying a heat-sensitive plasmid pKD46 by electroporation. With the functions of Exo, Beta and Gam of X Red recombination system, fimH was replaced by cat gene.50336 △fimH::cat and 15218 △fimH::cat mutants were selected by LB plates containing chloramphenicol. The chloromycin-resistant cassette from 50336:△fimH::cat and 15218△ fimH::cat were excised using the FLP recombinase, which were expressed by the vector pCP20. Finally, the isogenic 50336△fimH and 15218△fimH mutants were confirmed by PCR and subsequent DNA sequencing. Mannose sensitive heamoglutination and yeast cell agglutination assays indicate that the fimH mutants lost agglutination ability in Salmonella Enteritidis. S.pullorums and their fimH mutants also lost agglutination ability to chicken erythrocytes and guinea pig erythrocytes, as well as yeast cells. Bio film qualification and quantitative assays demonstrated that fimH mutants had a significant reduction in bio film formation ability compared with Salmonella Enteritidis (p<0.05). The △fimH mutants of Salmonella Enteritidis showed about 62% decrease in adherence to HD-11 in vitro compared with Salmonella Enteritidis, while S.pullorums with the low adhesion ability to HD-11. Through Real-Time PCR, we found deletion of fimH up-regulated csgD and ompD compared with wild type, respectively. In summary, FimH, the subunit of type I fimbriae, plays an indispensable role in the biofilm formation and aspects of HD-11 adhesion. All these factors proved that FimH of Salmonella Enteritidis involved in the ability to biofilm formation, and the adhesion ability to avian macrophage cells. The FimH of S.pullorums, however, losts the ability to biofilm formation, and the adhesion ability to avian macrophage cells. |
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