Construction Of SEF14 Fimbrial Deletion Mutants From Salmonella Enteritidis And Analysis Of The Related Functions

Salmonella enteritidis is a kind of pantropic salmonella.It not only cause poultry diseases, costing serious economic losses, moreover it is one of the main foodborne pathogenic bacteria, and it has great significance in public health. Fimbriae have been shown to be involved in colonization and in adherence to specific host target tissues in the early stages of infection and also can serve as potential immunogens against many pathogens that colonize epithelial cell surfaces. There are many kinds of fimbrial structures existed on the surface of Salmonella enteritidis,SEF14 fimbriae have been separated since the 1990s, and only found in Salmonella enteritidis and Salmonella Dublin, suggesting that SEF14 fimbriae may affect serovar-specific virulence traits. Currently, the function of SEF14 fimbriae is still being analysised.In order to study the fuction of SEF14 fimbriae and its contribution to the pathogenesis mechanism of Salmonella entertidis, we knocked out sefA and sefD subunit genes of SEF14 fimbriae from three different animal or human origin of Salmonella enteritidis including domestic standard strain 50336,avian original isolate S.O9 and human original isolate 43 by using homologue recombination strategies from both the suicide vector system and Red recombination system, three kinds of isogenic mutants forâ–³sefA andâ–³sefD were successfully constructed, and based on theâ–³sefA mutants,â–³sefAâ–³sefD mutants were constructed by the Red recombination system. The recombination plasmid containing homologous DNA fragment should be constructed in the suicide vector system and then transformed into Salmonella enteritidis stains. The first recombination frequency was high, and these recombinant strains could easily lose their plasmids encoding antibiotics resistance genes by passaging positive colonies from first recombinant on plates containing 5% sucrose during the second recombination, with no scar left in the chromosome DNA; however, the second recombination rate was low. Compared with the suicide vector system, the PCR products in Red recombination system could be directly transformed into Salmonella enteritidis stains by one step, but both of the PCR products and competent cells needed high quality and the first recombination rate obtained was comparatively low; while in the second recombination the FRT sites would be left in the chromosome. Although both of the two systems can be used to delete the target genes in Salmonella enteritidis chromosome and without any resistant genes remained, Red recombination system is more convenient and efficient in the practical operation process.Based on the deletion mutants constructed above, the wild-type strains and the deletion mutantsâ–³sefA,â–³sefD, andâ–³sefAâ–³sefD of Salmonella enteritidis were tested in binding and invasion assays using porcine intestinal epithelial cell lines (IPEC-J2) and mouse peritoneal microphages as an in vitro infection model, and analysised the differences between the wild-type strains and the mutants based on the data. The results showed that there was no significant difference between the wild-type strains and the deletion mutants in their abilities to adhere to IPEC-J2 cells during 4 hours of the test, suggesting that SEF14 fimbriae may not mediate the adherence between Salmonella enteritidis and the intestinal epithelium cells in vitro tests.While the mouse peritoneal macrophages were able to internalize high numbers of Salmonella enteritidis during 4 hours. However, compared with the wild-type strains, the numbers of enterance and surviaval of the mutants in mouse peritoneal macrophages were greatly different, the mutants enhanced the phagocytosis ability of the macrophages, while the same time they also improved their survival abilities in macrophages. These results may indicate that both of SEFA and SEFD subunits can affect the fuction of SEF14 fimbriae in vitro tests, and SEF14 fimbriae is good for the enterance and survival of Salmonella enteritidis in macrophages, which really be beneficial to its pathogen-carrying state infection and long-term sheding bacteria.