Screening For Invasion Related Genes Of Salmonella Enteritidis And Research On Characetristics Of Attenuated Strains

Salmonella enteritidis (SE) is a facultative intracellular pathogen. It causes poultry death followedby huge economic losses, and affects human health by entering the food chain through poultryproducts.Proper Salmonella attenuated strains may be vaccine candidates or act as live carriers toexpress exogenous gene and simultaneously can be used for further study on the infectious process invivo and invasion mechanism.We constructed a mutant library by the conjugal transfer method. Under the optimized conjugationconditions, the filter hybridization was proceeded between recipient bacteria SM6and donor bacteriaE.coli β2155carrying a suicide plasmid pUT-MiniTn5-Km2. After antibiotic resistance screening andPCR identification, a total of364mutants with mini-Tn5transposon inserting at different positions wereobtained. The invasion ability of them were investigated in epithelial cell Caco-2. With primaryscreening and repeated verification, eventually we obtained3stable attenuated strains2-33,2-25and2-19with poor invasion and proliferation in cells.Southern blotting was conducted to confirm the single insertion of transposon in attenuatedstrain.We then used genome walking to get unknown sequence upstream of insertion fragment andidentified the disrupted gene is hilA, which is known as regulated gene associated with invasion.With the help of Red recombination system, the chloramphenicol resistance gene flanked byhomologues of the hilA gene was amplified by PCR. The PCR products were electro-transformed intoSM6containing plasmid pKD46and the positive recombinants substituted the hilA gene forchloramphenicol gene were obtained. Then, the resistance gene was eliminated by importing plasmidpCP20and hilA gene was completely deleted in this procedure. After PCR identification andsequencing, we successfully constructed mutant SM6△hilA.We compared the growth curve, bacteria morphology, biochemical reaction and carbon sourcemetabolism of SM6and SM6△hilA and found little difference; The counting results of Caco-2cellinvasion test in vitro showed at3h post infection SM6△hilA decreased nearly100times than SM6, and1d about20times. That is to say, a significant reduction in invasion of SM6△hilA was obsereved.Pathogenicity in chicks was studied according to median lethal dose LD50and found the gene deletionstrain was56.23-fold weaker than that of the parent strain; After intraperitoneal injection with SM6andSM6△hilA respectively, SPF chicks were sacrificed at two day intervals to collect liver, spleen andcecum sterilely in order to determine the persistence and clearance of SM6and SM6△hilA in vivo andthe pathological damage. The amount of bacteria recovered from the tissues was taken down. In fact,the difference in colonization of liver is not obvious while in spleen and cecum the bacteria load of SM6△hilA is significantly more than wild strain SM6, especially in cecum. The result had difference with aprevious report. SM6caused obvious damage in the liver and spleen to a certain extent and the mutantmainly led to cecum pathological damage. We determined the antibody level in serum and found thegene deleted strain could induce apparent humoral immunity to some extent.This study used transposon mutagenesis to screen attenuated strains and constructed the hilA deleted mutant to research on related characteristics. The work laid theoretical foundation for thedevelopment of attenuated vaccines and live carriers. Most importantly the research provided clues andinformation for further study on the invasion mechanism of Salmonella.