Analysis Of The Pathogenic Role For SEF21and SEF14Fimbriae In Salmonella Enteritidis Infection

Salmonella enteritidis, a member of the family Enterobacteriaceae, which is gram-negative facultative bacteria, widely spread in the world. They cause substantial economic loss directly through mortality and poor growth after clinical disease, and also indirectly from animal carriage leading to cases of human salmonella infections with symptoms such as diarrhea, fever. It is an important pathogen for both human and animals. Several factors have been investigated in relation to their roles in pathogenicity of S.enteritidis, they interact well with each other and damage the host cells together. Among them, fimbriae play an important role. In this study, S.enteritidis SEF21and SEF14fimbriae were selected to study their relation with the pathogenicity. S.enteritidis express many kinds of fimbriae, they influence the initial attachment and colonization of the bacteria, also they can bind to the specific receptor on the surface, then induce systematic reaction. To study how SEF21or SEF14fimbriae act as single or group factor in the process of S.enteritidis infection, we constructed SEF21fimbriae deletion mutants combined with SEF14fimbriae, and studied their differences in in vitro and mice infection test.1To Construct fimA deletion mutants of S. enteritidis using Red recombination systemon the base of the reported gene sequence of fimA, the major subunit of SEF21, summitted in the GeneBank, we knocked out the fimA gene of two reference strains S.enteritidis using the Red recombination system. First, the primes were designed with the5’-end of homology to the flanking regions of fimA gene and the3’-end of homology to the cat gene, which is the chloramphenicol resistance gene of pKD3plasmid. Then the PCR products that has the chloramphenicol resistance and homologous sequences were introduced into strain50336、994、50336△sxf4、994△sefA by electroporation,。 With the help of pKD46, the target gene was replaced by homology extensions, the strains were selected according to their ampenicillin and chloramphenicol resistance. The chloramphenicol resistance gene was then eliminated by using the pCP20plasmid, which expresses Flp recombinase, generated the second-mutate-strain.50336△fimA,994△fimA,50336△sefA△fimA,994△sefA△fimA were confirmed by PCR amplication and sequencing. The successfully constructed mutants in this study will be the base to study the molecular pathogenesis mechanism of interaction between the SEF21and SEF14fimbriae and the host cells, prevention and control of the S.enteritidis-caused disease.2Studies into the role of the SEF21and SEF14fimbriae in the pathogenesis of S.enteritidisFimbriae play an important role in the adhesion of the pathogen to host cells in the initial binding and colonization. Microphages also involve in many reactions induced by S. enteritidis infection. To further investigate the role of the SEF21and SEF14fimbriae in pathogenesis, we transformed the pBR322-sefA plasmid and pACYC184-fimA recombinant plasmid to the mutants respectively, then obtained the complementary strains. With the series of wild type, deletion mutants and complementary strains, the function of both SEF21and SEF14was tested in the human intestinal Caco-2cell line and mouse peritoneal microphages in in vitro infection models. There was no significant difference between the wild-type strains and the△sefA,△fimA mutants for the adhesion ability to Caco-2cells, but the knock out of both fimA and sefA gene significantly decreased the adhesion ability. The efficacy of opsonized bacteria ingestion by mouse peritoneal macrophages were compared, and both the mutants were able to internalized in mouse peritoneal macrophages with much higher numbers compared to wild types, total bacterial numbers after phagosytosis in0.5h,1.5,4.5h suggested AfimA mutants cannot survival in murine macrophages. SEF21fimbriae may provide an advantage to the bacterium in avoiding killing by mouse peritoneal macrophages.3The effect of LD50to Balb/c mice by S.enteritidis and its mutantsThe LD50of S.enteritidis50336and mutant strains was determined in6-week-old Balb/c mice. Groups of Balb/c mice were infected by intraperitoneal injection with dose10to103cfu bacteriae separately. Mice were observed daily and following death their spleens, livers, lungs, kineys, small intestinals and caecae were aseptically taken out for the bacteriological detection. Salmonella were isolated and examined by PCR amplification. LD50of50336,50336△sefA,50336△fimA and their complemented strains were10.50cfu,8.06cfu,14.38cfu,16.77cfu,11.90cfu,11.90cfu,10cfu, respectively. It indicated the different effect of SEF21and SEF14fimbriae on the pathogenicity of S.enteritidis. The virulence of S.enteritidis in6-week-old Balb/c mice increased when injected with mutant strains50336△sefA, but decreased with50336△fimA and50336△sefA△fimA.50336△sefA△fimA not only performed poor adhesion ability, but also could be eliminated more easily by macrophages. When the mice were infected with S.enteritidis, the bacteria widely spread into multiple organs, bacteria were isolated from spleens, livers, lungs, kineys, small intestinals and caecae successfully, and PCR amplification of sdfl gene confirmed the symptoms were resulted form S.enteritidis infection. The test also indicated with SEF21and SEF14fimbriae, S.enteritidis can interacted with susceptible host cells more effectively, and live better in macrophages, that would be an advantage for recessive effection and long-term explusion of pathogen.