Identification And Primary Functional Analysis Of Genes Mediating Intramarcophages Salmonella Enteritidis Exit

Salmonella spp.is an important zoonotic pathogen,which can easily bring serious harm to the livestock industry and human health,and so salmonellosis is one of the main diseases for priority prevention and control during livestock and poultry breeding in our country.Extra-intestinal systemic infection is one of pathogenic properties of Salmonella spp.,and macrophages are the main carrier of Salmonella systemic infection in vivo.The exit of intramacrophages Salmonella is one of key steps in Salmonella systemic infection,which accelerates the spread speed of in vivo and causes extra-intestinal systemic infection.At present,the genes which decide the ability of intramacrophages Salmonella exit are still unknown,and further study is needed.So signature-tagged mutagenesis was used to establish a transposon mutant library of SE.Then single mutant infected RAW264.7 cells,the exit ability of intramacrophages Salmonella is assessed by the number of Salmonella inside or outside macrophages one by one,and some weak/strong mutants were screened out,the functional genes were determined by the flanking sequence of the transposon in bacterial genome.The role of these genes in Salmonella infection,virulence and possible mechanism was preliminary analyzed.1 Identification of gene(s)mediating intramacrophages SE exitThe donor E.coli x7213(Tn5)and the recipient C50041 were mixed for conjugation,a transposon mutant library of SE was successfully constructed.Then single mutant infected RAW264.7 cells.The mutants were screened according to the number of Salmonella inside or outside macrophages at 8 hours post infection,and exit ability of intramacrophages Salmonella was assessed.In this study,12 mutants were screened out from 887 transposon mutants,and the genes with transposon inserted were located by PCR and sequencing.A total of 10 related genes were identified,which included 6 named and 4 hypothetical genes.2 Preliminary analysis of biological features of trmE,tgt,focA and purL mutantsBased on homologous recombination,C50041?trmE,C50041?tgt,C50041?focA and C50041?purL mutants were constructed by suicide plasmid pGMB152 and their biological characteristics were studied,their corresponding transposon mutants were also as a control.The results of growth and biochemical characteristics showed that the loss of tgt,focA and purL genes had no effect on the growth rate and biochemical characteristics of Salmonella,while the loss of trmE gene significantly decreased the growth rate,but it also did not affect the biochemical characteristics.In in vitro assays,compared with the wild-type C50041,the invasion and proliferation rates of C50041 ?trmE,C50041 ?tgt and C50041?purL on murine macrophages RAW264.7 and J774A.1 were significantly reduced,while that of C50041?focA did not change.In in vivo assays,compared to wild-type C50041,the LD50 of C50041?trmE and C50041?purL increased by more than 1,000 folds,and C50041?tgt increased by 15.87 folds,while that of C50041?focA decreased slightly.The dynamic analysis in vivo showed that the count of C50041?trmE,C50041?tgt,and C50041?purL isolated from liver and spleen of mice was less than that of wild-type during the same period,indicating that their spread ability in vivo became weaker,while C50041?focA could be isolated at 1 dpi but C50041 could not,which proved that it had an enhanced spread ability compared to wild-type.Pathological changes showed that C50041?tgt and C50041?focA could cause strong lesions in liver and spleen,while C50041?trmE and C50041?purL caused weak lesions.The result of immune protection showed that the protective efficacies of C50041?trmE and C50041?purL were up to 60%and 80%,respectively,indicating that trmE and purL genes could be used as live attenuated Salmonella vaccine candidate genes.Detection of cytokines by qRT-PCR showed that the mRNA level of TNF-?,IFN-y,IL-1?and IL-12 in the spleen of mice infected with each mutant changed significantly,indicating that the infection of mutants changed the immune response of mice.3 Preliminary study on the mechanism of SE exit from macrophagesDue to the loss of trmE or focA gene weakened or enhanced the intramacrophages SE exit,respectively,the related mechanism was further studied.After LDH test,C50041?trmE caused lower cytotoxicity to RAW264.7 and J774A.1 cells,while that of C50041?focA have significantly increased.The detection results of apoptosis and pyroptosis by flow cytometry and western-blot,respectively,showed that compared with wild-type C50041,apoptosis and pyroptosis levels induced by C50041?trmE were significantly reduced,indicating that its ability to induce programmed cell death of macrophages was decreased,while that of C50041?focA had no significant change.The result of genes expression by qRT-PCR showed that the mRNA level of SPI-1 genes of C50041?trmE did not change,while that of flagellar biosynthesis ? factor fliA,flagellar motor stator proteins motA,motB,and flagellar biosynthetic proteins flgB,flgK were all significantly down-regulated.It proved that loss of trmE gene significantly inhibited the synthesis of SE flagella.The motility analysis showed that the motility of C50041?trmE was significantly decreased,while that of C50041?focA had no significant change.In all,12 mutants were screened from 887 transposon mutants,10 related genes were identified.In in vitro and vivo assays,trmE,tgt,focA and purL genes had been further confirmed to participate in the exit from macrophages and the pathogenicity of SE.These results not only broaden our understanding to the mechanism of Salmonella exit from macrophages,but also provide a basis for the development of Salmonella vaccines or drugs.