Quorum Sensing-dependent Regulation Of Type ? Secretion System ? In Vibrio Parahaemolyticus

Vibrio parahaemolyticus is a gram-negative halophilic bacterium that thrives in warm –environments. It naturally occurs in coastal areasbottom sediments and marine animals. Disease caused by V. parahaemolyticus is generally associated with the consumption of raw or undercooked seafood,include fever?nausea?vomiting?abdominal cramping and watery diarrhea. Severe cases can develop into fatal septicemia and cause death without timely and effective treatment. Quorum-sensing(QS) is a regulation mechanism of bacterium group behavior,AphA and OpaR are the two core of QS regulators that are expressed at low cell density and high cell density,QS regulated a feature of gradient production of them with expressed between low cell density and high cell density.The T6SS1 gene cluster(VP1386-1414) is composed of 29 consecutive genes forming7 putative operons on the larger chromosome,so the first gene of each operon was chose as atarget gene for study, VP1388?VP1393?VP1400 and VP1409.In this study, aph A?ToxR and opa R mutation were constructed by using suicide plasmid p DS132. His-AphA?His-ToxR and His-OpaR recombinant proteins were obtained using p ET protein expression systems in E.coli BL21(?DE3). The primer extension, Real Time PCR, electrophoretic mobility shift assay, and DNase I footprinting assays will be then carried out to characterize the trancscriptional regulation of T6SS1 genes by the quorum-sensing(QS) core regulators OpaR and AphA; the final combined phenotpic and melecular dataset will promote us to gain the deeper understanding of distinct phenotpes and QS-mediated cell dencsity-dependent reciprocal regulation of T6SS1 expression in V. parahaemolyticus. The results show :1. T6SS1 was negatively regulated by AphA through inhibiting the thanscroption of VP1388?VP1393?VP1400 and VP1409.2. T6SS1 was negatively regulated by ToxR through combining on promoter region of the gene VP1393?VP1400 and VP1409 directly.3. The results suggested negatively regulation of T6SS1 by OpaR.Through combining on promoter region of the gene VP1388?VP1400 and VP1409 directly.4. These observations strongly supported the notion that T6SS1 might play important roles at the early stages of bacteria growth/infectionan and inhibited the action of other bacteria.