Regulation Of Type Ⅵ Secretion System By QsvR In Vibrio Parahaemolyticus

Bacground:Vibrio parahaemolyticus,a Gram-negative halophilic bacterium,widely distributes in oceans and estuaries.After occurrence in bloodstream infection,it can cause septicemia,and even death.The type VI secretion system(T6SS)is a bacteriophage like protein injection machinery that injects effector proteins into prokaryotic or eukaryotic cells,contributing impotantly to the pathogenicity of V.parahaemolyticus.V.parahaemolyticus strain RIMD2210633 contains two distinct T6SS gene clusters in chromosomes I and II,termed T6SS1(VP1386-1420)and T6SS2(VPA1024-1046),respectively.T6SS1 is highly induced under warm marine condition(M broth,30℃),whereas T6SS2 is highly induced under imitated host condition(HI broth,37℃).The AraC-type protein QsvR was originally found to be involved in the regulation of biofilm formation in V.parahaemolyticus.A recently study demonstrated that QsvR integrates into quorum sensing(QS)circuit to control virulence gene expression.AphA and OpaR are the master QS regulators in V.parahaemolyticus,which play key regulatory roles at low cell density(LCD)and high cell density(HCD),respectively.At LCD,AphA can inhibit the expression of T6SS1 and T6SS2.At HCD,OpaR can inhibit and activate the expression of T6SS1 and T6SS2,respectively.Moreover,QsvR binds to the promoter DNA regions of aphA and opaR to repress and activate their transcription,respectively.Therefore,this study speculated that QsvR maybe have a regulatory effect on the expression of T6SS1 and T6SS2.Objective:The aim of study was to explore the regulation mechanism of QsvR on the transcription of T6SS1 and T6SS2 genes in V.parahaemolyticus.Methods:In the present study,the regulatory activities of QsvR on T6SS1 and T6SS2 were analyzed by combined utilization of cell adhesion test,primer extension,qRT-PCR,LacZ fusion,EMSA,and DNase I footprinting.Results:Under warm marine condition(M broth,30℃),the primer extenssion assay detected a single transcription start site for each T6SS1 related gene,which was located respectively at 64 bp,62 bp,93 bp and 232 bp upstream of the translation start site of VP 1388,hcp1,VP1400 and VP1409,and demonstated that QsvR inhibited the promoter activities of T6SS1 genes;the results of qRT-PCR showed that QsvR negatively regulated the transcription of T6SS1 genes;the results of EMSA showed that QsvR was able to bind derectly to the promoter DNA regions of VP1388,hcp1,VP1409 and VP1400.Under imitated host condition(HI broth,37℃),the primer extenssion assay detected a single transcription start site for each T6SS2 related gene,which was located respectively at 104 bp,119 bp and 72 bp upstream of the translation start site of VPA1027,VPA1043 and VPA1044,and demonstated that QsvR acticated the promoter activities of T6SS2 genes with LacZ fusion;the results of qRT-PCR showed that QsvR positively regulated the transcription of T6SS2 related genes;the results of EMSA showed that QsvR was able to bind directly to the promoter DNA regions of VPA1027,VPA1043 and VPA1044;the results of DNase I footprinting found that QsvR protected a single DNA region located from-347 to-51 bp,from-179 to+16 bp,and from-118 to-5 bp within the upstream regulatory region of VPA1027,VPA1043 and VPA1044,respectively;the results of cell adhesion test demonstrated that QsvR activated the cell adhesion activity of V.parahaemolyticus.Conclusion:Under imitated marine condition(M broth,30℃),QsvR directly inhibited the transcription of T6SS1 genes.Under imitated host condition(HI broth,37℃),QsvR directly activated the transcription of T6SS2 genes and enhance the cell adhesion activity of V.parahaemolyticus.