Quorum Sensing-dependent Regulation Of Type Ⅲ Secretion System1and Type Ⅵ Secretion System2in Vibrio Parahaemolyticus

Vibrio parahaemolyticus (VP) is a halophilic bacterium that thrives inmarine, estuarine environments or commensally associated with differentshellfish species. Disease caused by V. parahaemolyticus is generallyassociated with the consumption of raw or undercooked seafood. At present,V. parahaemolyticus has become a leading cause of food poisoningthroughout the world. Quorum sensing (QS) systems are widely distributedin bacteria and act through complex signal transduction cascades involvingcell density-dependent synthesis, release, and detection of signal moleculescalled autoinducers.QS systems can respond to the change of living environment, which inturn regulate gene expression in response to fluctuations in cell density. QSsystems affect many bacteria biological functions in V. parahaemolyticus,such as the formation of biofilm, the change of the colony transparency,bacteria exercise capacity, and so on. AphA and OpaR are the two majorregulators of QS in V. parahaemolyticus, and they are abundantlyexpressed at low cell density (LCD) and high cell density (HCD),respectively, and also mediate the transition of bacteria between LCD andHCD.The type III secretion (T3SS) is a protein secretion machinery thatallows bacteria to deliver protein effectors across eukaryotic cellular membranes and induce cell damaged. T3SS1-induced cell death occursrapidly, within several hours after the inoculation of V. parahaemolyticus,which is associated with apoptosis or autophagy. During infection, theinitial adhesion of bacteria to a host cell is essential for the activation ofpathopoiesis. The type Ⅵ secretionⅡ(T6SS2)contribute to adhesion of V.parahaemolyticus to host cells. T3SS1and T6SS2are the key virulencedeterminants for Vibrio parahaemolyticus which are contribute to variousbiological functions during pathogenic process. This study focuses on thespecific transcriptional regulation mechanism of T3SS1and T6SS2by thecenteral QS system regulator AphA and OpaR.The transcription of the T3SS1genes of V. parahaemolyticus isregulated by the exsACDE regulatory cascade. Expression of the T3SS iscontrolled by QS system through repression of exsA in Vibrio harveyiwhich is close relatives with V. parahaemolyticus. Regulation mechanismof T3SS1by QS system is still not reported in V. parahaemolyticus.According to the bioinformatics, we predict that the transcription of theT3SS regulated by QS system may also through act with exsACDEregulatory cascade in V. parahaemolyticus. And based on the operonexperiment, four target genes were confirmed, which were exsC, exsB,exsD and VP1687(T3SS1effector protein).OpaR repress transcription of T6SS2of V. parahaemolyticus, howeverthe details of the regulatory mechanism have not reported. Based onbioinformatics and related literature, we speculate that QS system maydirectly act with the gene cluster of T6SS2to regulate its transcription andexpression in V. parahaemolyticus. T6SS2gene cluster is composed ofthree operons, so the first gene of each operon was chose as a target genefor further study, VPA1027, VPA1043and VPA1044.In this study, aphA and opaR mutation were constructed by using suicide plasmid pDS132and corresponding complementation strains wereestablished by using the plasmid pBAD33. His-AphA and His-OpaRrecombinant proteins were obtained using pET protein expression systemsin E.coli BL21(λDE3). After that, the relationships between regulator andpromoter region of the target genes were studied by electrophoresismobility shift assay (EMSA) and DNaseI footprint assay. The furtherregulation relationships were studied with the primer extension and LacZfusion β-galactosidase assay, then the regulatory mechanism was reportedin details.The results show that positive regulation of T3SS1(VP1687) by AphAthrough activating the gene of exsC, exsB and exsD, then promotecytotoxicity of bacteria to host cell. Negative regulation of T3SS1(VP1687)by OpaR through inhibiting the transcription of exsC, exsB and exsD, theninhibits cytotoxicity of bacteria to host cell. AphA and OpaR can directlycombine on promoter region of the exsB gene to regulate its transcriptionand expression. AphA and OpaR interact with exsC and exsD in a mannerof indirect. The results suggested that abundant of AphA can promoteexpression of T3SS1, so that a small amount of bacteria can cause diseaseat LCD. On the other hand, OpaR inhibits expression of T3SS1, whichresults in the low pathogenicity of bacteria and promote the spread ofbacteria at HCD.T6SS2was negatively regulated by AphA through inhibiting thethanscroption of VPA1027, VPA1043and VPA1044. T6SS2was positivelyregulated by OpaR through combining on promoter region of the geneVPA1027, VPA1043and VPA1044directly. These observations stronglysupported the notion that T6SS2might play important roles at themiddle/late stages of bacteria growth/infection.This research elaborated on QS system dependent T3SS1and T6SS2 transcriptional regulation mechanism in V. parahaemolyticus usingbioinformatics and classical molecular biology experiment at the first time,and provided a theoretical basis for reshape the gene regulatory networksand revealed pathogenic mechanism of V. parahaemolyticus.