| Vibrio parahaemolyticus is a gram-negative bacterium,usually shaped,rod-shaped,with no spore.The bacteria generally grow in the ocean,and estuary environment.V.parahaemolyticus can infect fish,shrimp,crab,etc.,causing a large number of fish,shrimp,and crab to death in aquaculture,bringing economic losses to aquaculture.If people accidentally eat seafood with V.parahaemolyticus,it will cause abdominal pain,diarrhea,vomiting,and other clinical symptoms.EnvZ/OmpR is a two-component system widely present in bacteria,consisting of the transmembrane signal sensor EnvZ and the effector OmpR.The sensor EnvZ converts external stimuli into signal molecules,and after phosphorylation,the phosphoric groups are transferred to the effector OmpR to promote phosphorylation.The phosphorylated OmpR can regulate the expression of downstream related genes,thus enabling bacteria to adapt to the harsh environment.At present,EnvZ/OmpR has been reported in a variety of bacteria,and the system plays an important role in many bacteria.However,it is not clear whether this two-component system exists in V.parahaemolyticus(strain RMID2210633),whether it regulates the physiological functions of V.parahaemolyticus,and how it functions.However,studies have shown that EnvZ/OmpR can improve the survival rate of E.coli under high osmotic pressure;in addition,EnvZ/OmpR can also regulate the alkali tolerance of Vibrio cholerae.1.Identification of two-component system EnvZ/OmpR in Vibrio parahaemolyticusTo identify whether EnvZ/OmpR exists in V.parahaemolyticus,Blast homology analysis was performed on V.parahaemolyticus and other strains that have been identified to have EnvZ/OmpR.The results showed that the coding protein of VP0154 in V.parahaemolyticus genome had 92%homology with OmpR protein in Vibrio cholerae MS6 strain.The homology of OmpR with Escherichia coli MG1665 reached 84%.The coding protein of VP0155 was 78%homologous to EnvZ protein of V.cholerae MS6 strain,and 50%homologous to EnvZ protein of E.coli MG1665 strain.Therefore,the homologous protein of VP0155/VP0154 encoding Envz/OmpR in V.parahaemolyticus was preliminarily identified.The prediction of protein structure and function showed that the protein encoded by VP0154 has a REC Domain(which contains phosphorylated phosphoric acid receptor sites by histidine kinase)and a Trans_reg C Domain(which is associated with the REC Domain and has DNA binding ability).These two domains belong to conserved domains of response regulatory proteins.The protein encoded by VP0155 has a HAMP domain(containing transmembrane domain),HisKA domain(containing dimerization of histidine kinase and phosphate receptor domain),and HATPase c domain(containing histidine kinase activity),all of which belong to the conserved domain of histidine protein kinase.The predicted coding protein of vp0155/vp0154 has a typical two-component system domain,which further proves that vp0155/vp0154 encodes EnvZ/OmpR homologous protein.2.Investigation about the effect of EnvZ/OmpR on the physiological function of Vibrio p arahaemolyticu sAfter the deletion strain of envZ/ompR(vp0154/vp0155)was constructed in the V.parahaemolyticus genome,we determined the related phenotypes of the deletion strain.Firstly,the growth of V.parahaemolyticus under normal culture conditions was observed.It was found that neither envZ nor ompR deletion had any effect on the growth of V.parahaemolyticus under normal MLB conditions.Then we determined whether the growth of the deficient strains under high osmotic pressure would be affected.The experimental results showed that the deletion of envZ had no effect on the growth of V.parahaemolyticus under high osmotic pressure,but ompR played an important role in the growth of V.parahaemolyticus under high osmotic pressure.Similarly,when we detected the growth of the missing strain under acid and alkali conditions,we found that EnvZ did not participate in the acid and alkali resistance of V.parahaemolyticus,but OmpR played an important role in the alkali stress resistance of V.parahaemolyticus.To detect the effects of envZ/ompR deletion strains on the biofilm and motility of V.parahaemolyticus,biofilm formation and motility of the deletion strains were detected.The results showed that compared with the wild strain,the biofilm formation of the strain without envZ was significantly reduced.In addition,we measured the effect of envZ/ompR deletion strains on the motor ability of V.parahaemolyticus,and the results showed that envZ had significant positive regulation on the clustering and swimming movement of V.parahaemolyticus,while ompR had no significant regulation on the motor ability of V.parahaemolyticus.3.Study about the influence of EnvZ/OmpR on the alkali resistance mechanism of Vibrio p arahaemolyticu sTo further explore the molecular mechanism of EnvZ/OmpR,we screened some outer membrane protein genes that may be regulated by OmpR,and analyzed them by qRT-PCR using the whole RNA sequences of wild-type and OmpR deletion strains.The results showed that the expression of vp0493,vp1218,vpa0085,vpa1308 and vpa1745 outer membrane protein genes was inhibited in OmpR-deficient strains,indicating that OmpR positively regulates the expression of these genes.Then we constructed deletion strains of these genes and measured their growth curves under alkaline conditions.We found that the growth of vp0493,vp1218,vpa0085and vpa1745 deletion strains make no difference from that of the wild type under normal growth conditions,but their growth was weaker than that of the wild type under alkaline conditions.This phenotype is consistent with OmpR deletion strains,indirectly confirming OmpR regulation of these outer membrane protein genes.However,the growth of vpa1308 under normal MLB conditions is also weaker.The reason remains to be studied.It has been reported that OmpR of V.cholerae can maintain virulence and survival by inhibiting the expression of AphB and regulating downstream gene expression under alkaline conditions.To explore whether the regulation of OmpR on outer membrane proteins in V.parahaemolyticus could be mediated by AphB,we designed a fluorescence experiment to explore whether OmpR could regulate the expression of AphB and whether AphB could regulate the expression of those outer membrane proteins.The results showed that OmpR could inhibit the expression of AphB,and AphB could inhibit the expression of vp0493,vp1218 and vpa0085.AphB inhibited the expression of vp0493,vp1218 and vpa0085,while OmpR inhibited the expression of AphB,thus promoting the expression of these outer membrane proteins.As a result,the adaptability of V.parahaemolyticus under alkaline conditions was improved.In conclusion,EnvZ/OmpR can regulate physiological functions of V.parahaemolyticus including high osmotic pressure tolerance,acid,and alkaline resistance,biofilm resistance,and motor ability,and can indirectly and positively regulate the expressions of vp0493,vp1218 and vpa0085 to improve the alkali-resistance of V.parahaemolyticus in the alkaline environment by inhibiting the expression of AphB.In this experiment,the effect of EnvZ/OmpR on the physiological function of V.parahaemolyticus was investigated.And the alkali resistance mechanism of V.parahaemolyticus was studied by fluorescence quantitative PCR and fluorescence detection system.This paper is used to explore the adaptation mechanism of V.parahaemolyticus to harsh environments. |
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