Preliminary Study On The Regulation Of Type Ⅲ Secretion System And Its Effector Proteins By RpoE Protein Of Salmonella Typhimurium

Salmonella Typhimurium is an important zoonotic pathogen.When bacteria are in the biofilm conditions,their genes expression changes greatly compared with that in the eutrophication conditions.As an σ factor,RpoE can regulate biofilm formation,which is conductive to enhance bacterial resistance to adverse environment and affect the pathogenicity of Salmonella Typhimurium.Type Ⅲ secretion system(T3SS),encoded by both pathogenicity island 1(SPI-1)and pathogenicity Island 2(SPI-2),is the main virulence factor of Salmonella Typhimurium.T3SS encoded by different SPIs and their secreted effector proteins play different roles in each stage of bacterial pathogenicity.Therefore,rpoE may affect the virulence of Salmonella Typhimurium by regulating the expression of T3SS and its effector proteins.In this study,we used the Salmonella Typhimurium S025 and its rpoE gene mutant,which pocessses strong biofilm forming ability,to determine the regulatory effect of RpoE on T3SS under the condition of biofilm and non-biofilm.Our study will lay a foundation for further illuminate the pathogenic mechanism of Salmonella Typhimurium1.Transcription regulation of T3SS-related genes by rpoE gene of Salmonella TyphimuriumThe T3SS genes invA,iagB,iacP,and invC encoded by SPI-1 and their effector protein gene sopE2,and the T3SS gene sseB encoded by SPI-2 and its effector protein genes pipB,sseL,and sspH2 were selected.The primers of corresponding genes with better specificity were designed(including reference gene gyrA).The transcription level of wild strain S025 and its rpoE gene mutants were determined by real-time quantitative PCR under biofilm and non-biofilm condition(eutrophication condition and 28℃ planktonic condition).The results showed that the expression of invC,iacP,sseB,pipB,and sseL genes decreased significantly or extremely significantly in rpoE gene mutants compared with that in the wild strians,while the expression of invA and sopE2 genes showed completely different trends in different culture conditions,indicating that rpoE gene regulated transcription level of T3SS and its effector proteins,Finally,we screened four candidate genes with significant differences and consistent regulatory trends,including invA and sopE2 encoded by SPI-1,and sseB and sseL encoded by SPI-2,as the target genes for further study.2.Preparation for polyclonal antibody against partial T3SS genes of Salmonella TyphimuriumFour T3SS-related genes,including invA,sopE2,sseB,and sseL,were selected to construct the recombinant plasmids,including pET-30a-invA,pET-32a-sopE2,pGEX-6P-1-sseB,and pGEX-6P-1-sseL,respectively,by using prokaryotic expression vectors pET-30a/32a and pGEX-6P-1.The recombinant plasmids were transformed into BL21(DE3)cells,and the soluble proteins InvA(49 kDa),SopE2(47 kDa),SseB(48 kDa),and SseL(61 kDa)were induced.After purified by the commercial kit,the 6-week-old female BALB/c mice were immunized to obtain the polyclonal antiserum.The mutants of invA,sopE2,sseB,and sseL genes were constructed by,-red homologous recombination method.The polyclonal antiserum was verified by Western-blot.The results showed that the protein did not be detected in the mutants,suggesting that the preparation of polyclonal antiserumm was successful.However,the specificity of polyclonal antiserumm of different proteins was different.Polyclonal antiserum provide a tool for further exploring the regulatory effect of RpoE on T3SS and effector protein.3.Regulation of T3SS-related genes by RpoE of Salmonella Typhimurium in the protein levelBased on a strong biofilm forming ability of Salmmella Typhimurium strain S025 and its rpoE gene mutants,the rpoE gene complemented strain was constructed by using prokaryotic expression vector pGEX-6P-1.The biofilm forming abilities of wild strain,mutants,and complemented strain were determined by crystal violet staining.It was confirmed that rpoE gene expression in rpoE gene deletion mutan can rescue the biofilm formation.The prokaryotic expression vector pET-30a-rpoE was successfully constructed,and the purified fusion protein RpoE-His was successfully induced and purified.And the polyclonal antibodies against four proteins,including InvA,SopE2,SseB and SseL were used as the first antibody to detect the protein expression of wild strain S025,rpoE gene muatnts,and complemented strain under eutrophication condition,28℃ planktonic condition,and biofilm condition by Western-blot.The results showed that the expression of InvA and SopE2 protein was negatively regulated by RpoE,while the expression of SseL protein was positively regulated by RpoE.And the expression of SseB protein was only positively regulated by RpoE under eutrophication and biofilm condition.The results of protein expression were different from those of real-time quantitative PCR.Finally,the interaction between the expressed RpoE-His fusion protein and the four proteins in Salmonella Typhimurium S025 was determined by pulldown assay.The results showed that there was no direct interaction between RpoE and the four proteins,respectively.These results preliminarily elucidated that the regulation of T3SS-related proteins by RpoE under the condition of biofilm and non-biofilm.