QsvR And OpaR Work Coordinately To Regulate Biofilm Formation By Vibrio Parahaemolyticus

Background: Biofilm formation by Vibrio parahaemolyticus requires specific structures,including exopolysaccharide(EPS),type IV pili and capsular polysaccharide(CPS),and is tightly regulated by various regulatory pathways such as quorum sensing(QS)and c-di-GMP.Qsv R is an Ara C-type regulator that is integrated into the regulatory cascade of QS by directly regulating the transcription of the master QS regulators Aph A and Opa R.Deletion of qsv R in the wild-type(WT)or opa R mutant(Δopa R)backgrounds alters the biofilm formation ability of V.parahaemolyticus,suggesting that Qsv R may coordinate with Opa R to control biofilm formation.Objective: The aim of this study was to investigate the combined regulation of Qsv R and Opa R on the biofilm formation by V.parahaemolyticus.Methods: The combined regulation of Qsv R and Opa R on biofilm formation was investigated using crystal violet(CV)staining assay and colony morphology assay.The enzyme-linked immunosorbent assay was employed to analyze the regulatory effects of Qsv R and Opa R on c-di-GMP metabolism in V.parahaemolyticus.The transparency experiments were performed to explore the regulatory effects of Qsv R and Opa R on the changes in colony transparency.The EPS-associated genes,cps A and scv E,type Ⅳ pilirelated genes,pli A and msh B,c-di-GMP metabolism-related genes,scr A and scr G,as well as the CPS-associated genes,VP0218 and VP0219,were selected as the target genes to investigate the Qsv R-and Opa R-mediated gene regulation.The q PCR and Lac Z fusion assays were applied to analyze the regulatory relationship of Qsv R and Opa R on each target gene.Two-plasmid reporter assay and EMSA were performed to investigate whether Qsv R and Opa R had direct regulatory effects on the target genes in E.coli and in vitro.Results: The results of CV staining and colony morphology assays showed that Δqsv R/p BAD33,Δopa R/p BAD33 and Δqsv RΔopa R/p BAD33 formed more biofilms and produced more wrinkled colony than the other stains.Δqsv R/p BAD33-opa R and Δopa R/p BAD33-qsv R exhibited restored biofilm production and colony morphology,indicating that Qsv R and Opa R coordinately inhibited the biofilm formation by V.parahemolyticus.The results of enzyme-linked immunosorbent assay showed that the intracellular c-di-GMP levels in Δqsv R/p BAD33,Δopa R/p BAD33,and Δqsv RΔopa R/p BAD33 were significantly enhanced relative to WT/p BAD33,with restoration in C-Δqsv R,Δqsv R/p BAD33-opa R,C-Δopa R,and Δopa R/p BAD33-qsv R,indicating that Qsv R and Opa R coordinately inhibited the synthesis of c-di-GMP.Transparency experiments showed that Δqsv R/p BAD33,Δopa R/p BAD33 and Δqsv RΔopa R/p BAD33 produced translucent colonies,whereas WT/p BAD33,C-Δqsv R,Δqsv R/p BAD33-opa R,C-Δopa R and Δopa R/ p BAD33-qsv R produced opaque colonies,indicating that Qsv R and Opa R coordinately regulated the transformation of colony transparency from opaque to translucent.Additionally,Opa R directly inhibited cps A and scv E transcription,while Qsv R did not regulate their transcription.Qsv R and Opa R were able to directly and coordinately promote the transcription of msh A1,pil A,scr G,VP0218 and VP0219.Opa R directly but Qsv R indirectly inhibited scr A transcription.The results of competitive EMSA experiments showed that His-Qsv R and His-Opa R did not have competitive DNA binding activities to the target promoters.In brief,we showed that Qsv R and Opa R worked coordinately to repress biofilmassociated phenotypes and c-di-GMP metabolism,but to promote V.parahaemolyticus to form opaque(OP)colonies.Qsv R was able to restore the biofilm-associated phenotypic changes caused by Δopa R,and vice versa.Qsv R and Opa R coordinately regulated the transcription of EPS production-associated genes,type IV pili-related genes,CPS synthesis-associated genes as well as the c-di-GMP metabolism-related genes.Moreover,His-Qsv R and His-Opa R had no competitive binding activities to the target promoter DNA regions.Conclusion: Qsv R and Opa R coordinately inhibited the biofilm formation by V.parahaemolyticus.Qsv R restored the biofilm-associated phenotypic changes caused by Δopa R,and vice versa.However,they had no additive regulation activities on the target genes.QsvR and Opa R regulated biofilm formation by directly regulating the transcription of genes involved in biofilm formation.Therefore,Qsv R operated with Opa R to negatively regulate biofilm formation by precisely controlling the transcription of multiple biofilm formation-associated genes in V.parahaemolyticus...