Establishment Of FliC Deletion Mutant From Salmonella Enteritidis And Corresponding Function Analysis

Salmonella serotype Enteritidis (SE) is a group of Gram negative (G") bacillus which is spore negative and flagellar positive and motive. It’s one of the most significant zoonosis. Salmonella present a huge threat to the development of breeding industry, especially poultry husbandry. Apart from the high morality in young chicken and retarded growth in broiler, Salmonella may also cause a severely negative impact on laying chiken, from the drop of their layingcapacity to final death. Moreover, Salmonella is obviously the most important food-borne pathogen. Mistaken intake of chicken eggs or other chicken products contaminated by Salmonella may result in food poison of human beings, indicating significance in public health issues. The pathogenicity of Salmonella enteritidis is closely related to multiple virulence factors. Under various pathogenic mechanisms, diffenent Salmonella enteritidis virulence factors may co-act on the host, cause illness or even death of the host. Among the multiple virulence factors, flagellar is the most unignorable as the major motion organ of Salmonella, providing assistance in purposed chemotaxis motion so that bacteria are capable of getting access to nutritient in the outer environment while avoiding potential harmful substances. However, the unknown relationship of Salmonella flagellar with the formation of biofilm and its role in inducing immuno-reaction and inflammatory reaction in host body, especially the mechanism of how this kind of bacteria managed to attached to the intestine mucosa has aroused high attention among academic world. Therefore, Salmonella flagellar gene fliC was thoroughly studied in this research by establishing fliC deletion mutation strain of wildtype and by further delete this gene in sefA mutants obtained earlier in our lab to build flagellar-fimbria double mutants of Salmonella. Then in vitro cell tests were carried out to further analyze the potential function of Salmonella flagella in the process of adhersion, invasion and the possible synergistic effect between flagellar and fimbria, hoping to reveal the molecular mechanism valuable for clinical diagnosis.With the sequence acknowledgement of Salmonella flagelar gene fliC, we were able to adopt Red recombination system to perform fliC gene knock out on domestic standard Salmonella strain CMCC(B)50336and international standard strain994. PCR amplification products containing fliC gene sequence homologous arms and Cat expression box were firstly transferred into Salmonella50336and994with pKD46plasimid already imported. Then with the help of Red recombinase, recombination was happened at homologous sequence to achieve50336△fliC::Cat and994△fliC::Cat. During the second recombination step, pCP20of the thermosensitive recombinase Flp was adopted to eliminate the Cat resistance genes mark to obtain50336△fliC and994△fliC Same procedure was performed to achieve50336△sefA△fliC and994△se/A△fliC on the basis of50336△sefA and994△sefA. Transfer pBR322into50336△fliC and994△fliC to get their corresponding compensation strains50336△fliC/pfliC and994△JliC/pfliC. In phenotypic tests, these fliC mutants:lacked motibility on solid medium and flargella under electron microscope, and didn’t react to Salmonella Enteritidis flagellar McAb with no agglutination reaction visable to naked eyes. The establishment of fliC mutants and their corresponding compensation strains provide an opportunity to futher explore the potential function of flagella and SEF14fimbria in the pathogenic process of Salmonella.Different from planktonic living style, bacteria biofilm is a kind of mechanism adopted by some bacteria to better adapt to disadvantaged environmental conditions. The majority of bacteria can form this biofilm under certain conditions. In this study, we found that Salmonella Enteritidis50336and994both possess the capability to form biofilm. Flagella and mobility mediated by flagellar are of great importance in biofilm formation factors. In bio-film formation tests, results showed that the ability of50336△fliC and994△fliC to form biofilm was substancially compromised compared to their wildtypes while the compensation strains showed no such decreased ability in biofilm formation. The biofilm formation quantitative test results further indicated that biofilm formation ability of Salmonella declined greatly after the deletion of flagellar gene fliC.Adhersion and the after colonization are the initial steps in the pathogenic process againstthe host intestine system and flagellar somehow induced this process. Adhersion ability comparison of Salmonella flagellar mutant strains, fimbria SEF14mutant strains, flagellar-fimbria double deletion strains and their wildtypes was conducted on Caco-2cells and IPEC-J2cells. From the above results, we safely speculate that flagellar may play a significant role in the process of adhesion while it may also cooperate with fimbria in some mechanism still unknown.