Construction Of The GspE Gene Mutant Strain Of Salmonella Typhimurium And Study On Its Biological Function

Salmonella Typhimurium is the main serotype causing salmonellosis in humans and animals,which resulting in gastroenteritis in humans and livestock,typhoid-like systemic infection in mice.The main virulence factors of Salmonella include adhesin,flagella,secretion system,and virulence plasmid.Salmonella outer membrane proteins(OMPs)can induce macrophage apoptosis,involved with drug resistance,and play an important role in the pathogenesis.T2SS is the main way for protein exportion,and GspE is a key protein of T2SS.As an energy pump,it provides energy for protein secretion.The secreted proteins are virulence factors,toxins or lyase that is required to infect the host.Focus on importance of GspE,we delete the gspE gene of T2SS and differentially expressed OMPs were screened by proteomics in order to further explore the pathogenic mechanism of Salmonella Typhimurium.1?Construction of gspE gene mutant strain of Salmonella Typhimurium and iTRAQ of outer membrane proteinsAccording to the full-length sequence of the USDA-ARS-USMARC-1808 gspE gene of Salmonella typhimurium published in GenBank(Accession Number:CP014969.1),a pair of identification primers was designed in its conserved region.The target fragment of gspE was amplified by PCR using S.Typhimurium W32 as template.The mutant strain W32?gspE was successfully constructed by recombinant technology,as well as the complemented strain W32?gspE/pgspE.When finished,the outer membrane proteins of S.Typhimurium W32 and the mutant strain W32?gspE were extracted by differential centrifugation,and quantified by Bradford and SDS-PAGE.With iTRAQ technology,we identified 1444 proteins(1199 proteins were identified as having at least two unique peptides).The proteins with significant differences was 393(W32:W32?gspE)(the number of significant differential proteins was correspondingly screened according to up_regulate?1.5 or down_regulate?0.67,P?0.05).271 up-regulated proteins and 122 down-regulated proteins were identified compared to W32AgspE.Functional annotations(including GO,COG,and Pathway analysis)were performed for all identified proteins and significant differential proteins.Hierarchical cluster analysis and functional enrichment analysis were performed for all significant differential proteins(GO enrichment,Pathway enrichment).The results showed that the expression of virulence proteins associated with pathogenic pathways such as adhesion and invasion changed in gspE mutant strain,which provided basis for further studies on the virulence-related mechanisms of S.Typhimurium.2?The regulatory function of GspE in pathogenicity of Salmonella TyphimuriumOn the basis of the above studies,the effects of GspE on S.Typhimurium pathogenesis were investigated through growth curve assay,qualitative and quantitative biofilm assay,motility assay,adhesion assay,invasion assay.The results indicate the absence of gspE reduced the biofilm formation ability,which was significantly lower than wild-type strain(P<0.05).Meanwhile,adhesion to DF1 and invasion to HD 11 cells reduced significantly when compared to wild-type strains(P<0.01).In the motility assay,the mutant strain was slightly increasedcompared to the wild-type strain.Subsequently,the relevant genes were examined at transcriptional level,including sefA(main subunits of sef21 pilus),fimA(main subunit of type I pilus),agfA(main subunit of thin-aggregated pili),f7iC(major subunit of flagellum),cpxR(two-component signal transduction system),phoP(two-component system),cobB(Salmonella acid resistance).The results showed that compare to W32,fimA,agfA,sefA,and cobB gene in the mutant strain were up-regulated,while fliC,phoP,and cpxR gene were down-regulated.The expression of fimA,phoP,and cpxR gene in the mutant strainwas consistent with the results of proteomics.In summary,GspE protein can regulate the expression of genes related to the two-component regulatory system,pilin-related and related proteins in S.Typhimurium W32,affect its adhesion and invasion ability,and further regulate the pathogenicity of S.Typhimurium.