LuxR Mediated Quorum-sensing Regulation Mechanisms In Vibrio Alginolyticus

Vibrio alginolyticus is one of the opportunistic pathogens mediating high-mortality vibriosis in marine animals.Its host mainly includes various fishes,which has brought huge losses to the aquatic industry.It can respond to changes in seawater temperature and cause coral bleaching desease,and infect patients with low immunity and so on.The quorum sensing(QS)is an important regulatory system for the expression of virulence-related genes in vibrios.A variety of cell density-dependent and non-cell density-dependent virulence regulation mechanisms in Vibrio alginolyticus have been identified.The related signal transduction pathways regulate virulence factors by regulating the expression of LuxR protein,,which is the master quorum sensing regulator(MQSR).These virulence factors include exotoxin Asp,hemolysin,extracellular products,lipopolysaccharide,siderophore,secretion systems and biofilm,and so on.However,in Vibrio alginolyticus,the regulon of LuxR and the molecular mechanism of LuxR regulating target genes remain to be further studied.In addition,how Vibrio alginolyticus uses non-cell density-dependent environmental signal transduction systems and integrates them into complex quorum sensing systems to precisely regulate virulence expression remain to be further explored.LuxR homologs are TetR family master quorum sensing regulators that activate or represse expression of hundreds of genes that control collective behaviors in vibrios with underlying mechanism unknown.To illuminate how this regulator controls expression of various target genes,we applied ChIP-seq and DNase I-seq technologies.Vibrio alginolyticus LuxR controls expression of?280 genes that contain either symmetric palindrome(repDNA)or asymmetric(actDNA)binding motifs with different binding profiles.The median number of LuxR binding sites for activated genes are nearly double for that of repressed genes.Crystal structures of LuxR in complex with the respective repDNA and actDNA motifs revealed a new mode of LuxR DNA binding that involves contacts of its N-terminal extension to the minor groove.The N-terminal contacts mediated by Arg9 and Argll differ when LuxR binds to repDNA vs actDNA,leading to higher binding affinity at repressed targets.Moreover,modification of LuxR binding sites,binding profiles,and N-terminal extension have important consequences on QS-regulated phenotypes.These results facilitate fundamental understanding of the high flexibility of mechanisms of LuxR control of gene activation and repression in Vibrio QS,which may facilitate to design QS inhibiting chemicals that interfere with LuxR regulation to effectively control pathogens.In our previous work,it was found that temperature regulates the expression of LuxR through signal transduction pathways such as the controlled regulated intramembrane proteolysis(RIP),but its mechanism is not clear.In this study,we screened and identified most of the OMP-YxF of V.alginolyticus,which are potential heat-sensitive elements in the RIP pathway,such as OmpC and OmpU,through the transposon insertion sequencing(TIS)assay.The incorrect folding of the proteins may trigger the RIP signaling pathway and then activate DegS,DegP and other periplasmic/innermembrane proteases to cleave the anti-sigma factor,RseA,of RpoE,thereby releasing RpoE into the cytoplasm,which can activate the expression of temperature response-related genes and luxR.We also found that RpoE regulates a new alternative ? factor,RpoX,in Vibrio,which participates in the pathogenic mechanism and responds to environmental stresses such as heat and low osmotic stress.Besides that,RpoX also has some regulatory functions independent of RpoE,such as stress responses motility,biofilm formation and hemolytic activity.In addition,it was found that the expression of LuxR is also regulated by another master quorum sensing regulator VqsA.VqsA also directly binds to Pasp to regulate the expression and activity of Asp,not through QS.Its regulon includes virulence-related genes,such as exotoxin Asp,siderophore uptake,biotin synthesis,motility and chemotaxis,and T6SS,as well as many factors related to acid stress and oxygen stress,including AphB.LuxR,VqsA,and AphA form an important quorum sensing regulatory network.Collectively,these discoveries will enrich our knowledge how V.alginolyticus and other related bacteria fine-tune virulence gene expression and become infectious to marine animals when they respond to cell-density signals through QS signaling transduction system,in the changing marine environment.