Studies On Regulation Of Chemotaxis And Flagella Related Genes On Adhesion Of Vibrio Harveyi

Vibrio harveyi is a common pathogen in marine aquaculture environments,which can cause high mortality for aquatic fish,shrimp and shellfish,which results in serious damage in the aquaculture industry.The adhesion of bacteria to the host surface is one of the first steps in the infection process.There is a lot of mucus on the skin surface,the sputum and the inner wall of the intestine.Therefore,it is the first site of interaction between the pathogen and the host.Studies on the adhesion of pathogens to host mucus have attracted attention,especially for bacterial adhesion-related genes.V.harveyi has the ability to adhesion,however,there are few studies on the genes related to the adhesion regulation of V.harveyi.The genetic regulatory networks are still largely unknown.In this study,the key adhesion genes of V.harveyi were identified by the research on V.harveyi and the epithelial mucus of Larimichthys crocea,which strengthened the understanding of the adhesion mechanism of V.harveyi infection,in order to provide a theoretical reference for the prevention and treatment of fish disease.Mainly contents of this research are listed following:(1)The wild-type V.harveyi VH6110 was cultured in Luria-Bertani(LB)medium contain different concentrations of Cu2+(50 mg/L),Hg2+(25 mg/L),Zn2+(50 mg/L)and Pb2+(100 mg/L),gene expression level of adhesion-defective strain was confirmed by qRT-PCR to screening for genes that may be associated with adhesion.The results showed that the adhesion ability of V.harveyi after stress culture was significantly decreased.At the same time,the chemotactic genes cheA,cheB,cheR,cheV,cheY and flagella related genes fliA,fliR,and flrB were down-regulated.The results indicated that the cheA,cheB,cheR,che V,cheY,fliA,fliR and flrB genes may be associated with the adhesion of V.harveyi.The RNAi was used to silence the adhesion-related genes mentioned above,qRT-PCR and 16S rRNA sequencing were used to detect stably silenced strains.(2)After the stable silencing strains of V.harveyi were constructed,the effect of single gene silencing on V.harveyi was analyzed from an apparent level such as the adhesion ability,exercise capacity,biofilm formation ability,chemotaxis ability and flagellar synthesis ability.As a result,the adhesion ability and motility of cheA-RNAi,cheB-RNAi,cheR-RNAi,cheV-RNAi,cheY-RNAi,fliA-RNAi,fliR-RNAi and flrB-RNAi were significantly decreased.The result of biofilm formation ability experiments showed that the biofilm formation ability of cheA-RNAi,cheB-RNAi,cheR-RNAi and cheV-RNAi was significantly enhanced,while the biofilm formation ability of cheY-RNAi,fliA-RNAi,fliR-RNAiand flrB-RNAi were significantly decreased.The result of chemotaxis assay showed that the chemotaxis ability of cheA-RNAi,cheB-RNAi,cheR-RNAi,cheV-RNAi and cheY-RNAi decreased significantly.The flagella of V.harveyi were observed by transmission electron microscopy,the flagella of flrB-RNAi cells were significantly shorter than those of the control,what’s more,no flagella were observed on the cell surface of the fliA-RNAi and fliR-RNAi cells.The results suggested that the stable silencing of the target gene may reduce the adhesion of V.harveyi by affecting the motility of the bacteria.(3)In order to further understand the molecular mechanism of the stable silencing of the target gene on the adhesion regulation of V.harveyi,wild-type strains and the cheA-RNAi silencing strain were selected for transcriptome sequencing(RNA-seq)and a total of 134 significant differentially expressed genes(DEGs)were detected.The results of the GO analysis indicated that many differentially expressed genes are involved in establishing localization and response to stimuli,which are thought to be involved in bacterial adhesion.Among the 62 KEGG pathways corresponding to DEGs,5 pathways have been confirmed to be closely related to adhesion ability,such as:"biofilm formation-Vibrio cholerae","ABC transporter","oxidative phosphorylation","quorum sensing" and "Bacterial secretion system".All DEGs such as oppD and Hfq enriched in these KEGG pathways were significantly up-regulated.These results indicate that the DEGs found in this study may have an important effect on bacterial adhesion.V.harveyi may adapt to the decrease in adhesion caused by cheA by regulating adhesion-related pathways.(4)This study examined the trends in the adhesion and target gene expression of V.harveyi under different temperature,pH and salinity gradients.The results showed that all genes were sensitive to environmental conditions,but had different responses:under different temperature conditions,the expression levels of cheB,cheY,fliA and fliR had the same tendency of inverted U-shaped as bacterial adhesion;under different pH conditions,the expression levels of cheB,cheV,and cheY also had the same tendency of inverted U-shaped as bacterial adhesion;under different salinity conditions,there is no similar change in the expression level of the target gene and the adhesion of V.harveyi.The results indicate that cheB,cheV,cheY,fliA and fliR are more likely to be directly involved in the regulation of bacterial adhesion under different environmental conditions.In summary,chemotaxis and flagella related genes of V.harveyi regulate adhesion by affecting bacterial motility.In addition,they are sensitive to environmental conditions and participate in different degrees of adhesion regulation in different environments.RNA-seq and the further biological information analysis indicated that V.harveyi may adapt to the decrease in adhesion caused by cheA which could regulate adhesion-related pathways.This study initially explored the mechanism of chemotaxis and flagellar related genes on the adhesion regulation of V.harveyi,and provided a theoretical reference for the control of fish disease.