Study On Molecular Mechanism Of EnvZ/OmpR Sensing Ferric Iron And Regulating Vibrio Parahaemolyticus Virulence Factor

Vibrio parahaemolyticus?V.parahaemolyticus?is a Gram-negative bacterium that naturally lives in marine environments including sea water,sea bottom sediments,and seafood products including fish and shellfish.It would cause vomiting,fever,diarrhea even death after infected,so it is an important foodborne pathogen that seriously threaten the safety of aquatic foods.Exploring the pathogenic mechanism of V.parahaemolyticus is important for preventing and controlling foodborne diseases.Bacterial pathogenicity is closely related to bacterial virulence factors.The expression of virulence factors is strictly regulated by environmental factors such as osmotic pressure,metal ions,bacteria density,and two-component regulatory system.The two-component regulatory system?TCS?is an essential mechanism for bacteria to sense,respond and adapt to environmental stress and growth conditions.Therefore,identifying the virulence factors regulated by TCS and exploring the molecular mechanism of how TCS sense environment signal?s?,will lay a theoretical foundation for the control of V.parahaemolyticus infection and the development of novel drugs.However,we know little about the role and mechanism of the TCS in V.parahaemolyticus sensing environmental signals to regulate virulence factors.This project starts with the screening of total of 50 histidine kinase?HK?mutants that regulate the motility and biofilm formation of V.parahaemolyticus,and focus on a classic TCS protein—EnvZ/Omp R.From the perspective of gene transcription regulation,the regulation of EnvZ/Omp R in biofilm formation,motility,bacterial secretion system and other virulence factors,and also the molecular mechanism of them were thoroughly studied.Transcriptome sequencing?RNA-sequencing?was used to investigate the transcriptional regulation network of EnvZ and wild-type V.parahaemolyticus living under iron deplete environment.The phosphorylation assay reveals that EnvZ can be phosphorylated by sensing the ferric iron(Fe³⁺).Furthermore,surface plasmon resonance was used to determine the binding of EnvZ and Fe³⁺.The specific binding site is predicted and determined ulteriorly.This project reveals the pathogenic mechanism of V.parahaemolyticus and provides a theoretical basis for disease control.The main findings and conclusions are as follows:1. The function of TCS in V.parahaemolyticusThe motility agar plate methods were used to determine the swimming and swarming motility of 50 HK mutants of V.parahaemolyticus.The results showed that the swimming motility of 14 strains altered significantly,among them,that of 12 strains decreased and 2strains increased.The swarming motility of 19 strains significantly reduced compare to wild-type strain.The biofilm formation of 13 strains significantly reduced and 10 significantly enhanced.According to the above results,we focus on a classic TCS—EnvZ/Omp R,then the gene sequences,protein structures and functional domains were preliminarily analyzed.The gene deletion mutants and complemental strains were constructed.These results provide understanding and directions of the role of TCS in V.parahaemolyticus.2. EnvZ/Omp R regulate virulence factors of V.parahaemolyticusEnvZ/Omp R positively regulates the motility and biofilm formation of V.parahaemolyticus and the regulation is closely related to protein phosphorylation.EnvZ/Omp R has no significant effect on swimming motility.EnvZ/Omp R regulate the killing ability of V.parahaemolyticus to E.coli,which mainly depends on T6SS2.Compared with wild-type strain,?env Z mutant had stronger tolerance to 10%Na Cl and 0.039%hydrogen peroxide,but has no significant effect to 20%sucrose,which illustrate that EnvZ plays a role in salinity stress and oxygen stress.The colonization of?env Z mutant in the infant rabbit intestine was weaker than that in wild-type strain,indicating that EnvZ can promote the colonization of V.parahaemolyticus in the host.3. Molecular mechanism of EnvZ/Omp R regulating virulence of V.parahaemolyticusTranscriptome sequencing?RNA-seq?results showed that the KEGG pathway regulated by EnvZ mainly includes oxidative phosphorylation,biofilm,amino acid metabolism,bacterial secretion system,biosynthesis of secondary metabolites,two-component signal transduction system,etc.Among them,pathways related to motility,biofilm formation,type III secretion system?T3SS?and type VI secretion system?T6SS?,outer membrane proteins,iron uptake system and other virulence-related functions had prominent changes in transcription level,and the transcription level of the gene omp N showed extremely down-regulated.Omp R participates in the regulation of omp N,exs C,hcp2,omp N,vp2362,vpa0842,vpa0651,vpa1403,vpa1447,vpa0248 and Omp R-his directly bound to the promoter regions of these genes.The specific binding site was located between-327 and-302 bp upstream of the ATG start codon of omp N and this region harbors 5'-GATCACGAAACCTC-3'sequence,which is consistent with the motif predicted by MEME-suite.Furthermore,EnvZ regulate swarming motility and biofilm formation in V.parahaemolyticus by regulating the transcription of downstream genes vpa0842 and omp N,respectively.4. Molecular mechanism on EnvZ regulating virulence factor by sensing Fe³⁺EnvZ can regulate its protein phosphorylation by sensing changes in Fe³⁺concentration in the environment with concentration-dependent manner.Under iron deplete conditions,33%of the genes in the transcriptome of wild-type strain undergo more than 1.5-fold regulatory changes.The altered KEGG pathway included several physiological metabolic functions such as the metabolism of microorganisms in various environments,antibiotic biosynthesis,TCS,biosynthesis of secondary metabolites,synthesis of multiple amino acids,and amino acids metabolism,etc.Moreover,Fe³⁺can regulate the biofilm formation and swarm movement of V.parahaemolyticus.EnvZ plays an important role in sensing Fe³⁺to regulate the swarming motility but does not play a decisive role in biofilm formation of V.parahaemolyticus.In addition,EnvZ bound Fe³⁺directly in vitro,and the 103^rd amino acid?glutamine?plays a key role in this binding.