Research On QsvR Regulon And Its Regulation On C-di-GMP Metabolism In Vibrio Parahaemolyticus

Background: Vibrio parahaemolyticus,the leading cause of seafood-related bacterial gastroenteritis in humans,produces various virulence factors and has a strong ability to form biofilms on the surface.QsvR,an Ara C-type transcriptional regulator,controls the virulence,biofilm formation and motility of V.parahemolyticus,but whether it can regulate other cellular pathways needs to be further investigated.Cyclic c-di-GMP(cdi-GMP),a ubiquitous second messenger in bacteria,has regulatory effects on multiple cellular pathways including biofilm formation,virulence and mobility.However,the regulatory mechanism of QsvR on c-di-GMP metabolism in V.parahemolyticus is still not fully understood.Objective: The aims of this work were to investigate QsvR regulon in V.parahemolyticus RIMD2210633 by RNA-seq,and to explore the regulatory mechanism of QsvR on genes involved in c-di-GMP metabolism.Methods: Crystal violet(CV)staining was performed to investigate the effect of QsvR on the biofilm formation by V.parahemolyticus.RNA-seq was used to analyze the differentially expressed genes between WT and ΔqsvR under the biofilm formation conditions.Real time quantitative PCR(qPCR)and lux fusion and electrophoretic mobility shift assay(EMSA)were employed to verify the RNA-seq data and to analyze the regulatory relationships of QsvR on c-di-GMP metabolism associated genes.Several c-di-GMP metabolism associated genes were deleted from the WT genome,and then their roles were investigated by combined utilization of transparency test,CV staining,measurement of c-di-GMP levels,swimming and swarming.Result: The results of growth curves showed that WT and ΔqsvR manifested similar growth rates in all the current growth conditions,indicating that QsvR did not affect the growth of V.parahaemolyticus.The results of CV staining showed that ΔqsvR produced more biofilms than WT at all time points tested(24 and 48 h),suggesting that QsvR repressed biofilm formation by V.parahaemolyticus.The RNA-seq data showed that QsvR regulated 1735 genes,of which 855 were downregulated genes and 880 were upregulated.DEGs has been classified into 3 Ontologies by GOseq,including 30 GO terms.A total of 1735 differentially expressed genes were classified into 6 types of KEGG pathways,and1483 were annotated into 21 COG pathways.The differentially expressed genes include: 16 exopolysaccharide-associated genes(14 were upregulated and 2 were downregulated),19 capsular polysaccharide-associated genes(all were down-regulated),15 type IV pili-related genes(all were downregulated),35 polar flagellar genes(all were downregulated except for flg A),16 lateral flagellar genes(7were downregulated and 9 were upregulated),and 24 c-di-GMP metabolism-related genes(11 were downregulated and 13 were upregulated).The qPCR and lux fusion assays confirmed the reliability of RNA-seq.In addition,the c-di-GMP levels in WT were higher than those in ΔqsvR under the tested growth conditions,indicating that QsvR promoted the production of c-di-GMP in V.parahemolyticus.The EMSA results showed that QsvR was able to bind the promoter DNA regions of 19 c-di-GMP metabolism-related genes to regulate their transcription.Moreover,VP0117,VPA0198 and VP2979 are putative c-di-GMP metabolismassociated genes,and thus we constructed the vp0117 mutant(Δvp0117),VP0117-EAL domain deletion-mutant(Δvp0117-EAL),VP0117-GGDEF domain deletion-mutant(Δvp0117-GGDEF),vpa0198 mutant(Δvpa0198),and vp2979 mutant(Δvp2979).The results of biofilm-associated phenotypes showed that the biofilm formation abilities ofΔvp0117and Δvp0117-EAL were stronger than that of WT,suggesting that VP0117 inhibited biofilm formation by V.parahemolyticus.VP0117 also inhibited the production of c-di-GMP in V.parahemolyticus.The swarming capacities ofΔvp0117and Δvp0117-EAL were similar to that of WT,while their swimming abilities were inhibited.VP0117 did not affect the production of capsular polysaccharide.Furthermore,VPA0198 and VP2979 seemed to have no regulatory activities on the production of c-di-GMP,capsular polysaccharide synthesis and biofilm formation,but they inhibited swarming of V.parahaemolyticus;VP2979 inhibited swimming of V.parahaemolyticus.Conclusion: QsvR is a global regulator in V.parahaemolyticus,controlling the expression of a large number of genes involved multiple cellular pathways.QsvR directly regulated the transcription of many c-di-GMP metabolism-associated genes,thereby promoting the intracellular c-di-GMP levels of V.parahaemolyticus under the biofilm formation conditions.