The Roles Of Flagellar Assembly Pathway Andbacterial Chemotaxis Pathway In The Adherence Of Vibrio Alginolyticus

Vibrio alginolyticus is a halophilic Gram-negative bacterium,which is an important opportunist pathogen.Diseases caused by Vibrio alginolyticus cause serious economic loss to the aquaculture industry.Therefore,the pathogenicity of Vibrio alginolyticus has gained extensive research interest.Bacterial adhesion is an important step in the initial stage of infection.The bacterial adhesion is influenced remarkably by bacterial factors,adhesion substrate and environmental factors.The process of bacterial adhesion is regulated by genes.Although some genes have been proved to be associated with bacterial adhesion,the mechanisms of regulation is still unclear.Our research investigated Vibrio alginolyticus and the regulation mechanisms related to bacterial adhesion.Using RNA-seq and bioinformatics analysis,we found that flagellar assembly pathway and bacterial chemotaxis pathway may closely associate with bacterial adhesion.To verify the reliability of the RNA-seq data,we conducted quantitative real-time PCR(QPCR).The QPCR results matched those of the RNA-seq,which reinforced the reliability of the sequencing data.Transient gene silencing was conducted to the differential expression genes(DEGs)in flagellar assembly pathway and bacterial chemotaxis pathway with short interfering RNA(siRNA),followed by in vitro adhesion assay.The QPCR results indicated that the siRNAs could cause significant gene silencing at 1-6 h.Meanwhile,the results of in vitro adhesion assay proved that the adhesion ability of V.alginolyticus under RNAi conditions was significantly impaired.Stable gene silencing was conducted through recombinant plasmid,followed by QPCR,in vitro adhesion assay,soft agar plate motility assay and transmission electron microscope observation.The results showed that the adhesion ability of V.alginolyticus was significantly impaired after stable gene silencing.In flagellar assembly pathway,the results of soft agar plate motility assay and transmission electron microscope observation demonstrated that not all the strains after stable gene silencing decreased in motility,which showed that motility is not the only avenue through which the flagellar assembly pathway affected adhesion.In bacterial chemotaxis pathway,the results of soft agar plate motility assay displayed that the motility of all the stains after stable gene silencing decreased,which indicated that bacterial chemotaxis pathway may regulate bacterial adhesion through motility.Our research investigated the expression of the upstream and downstream genes of DEGs in bacterial chemotaxis pathway.The results demonstrated that the gene regulation of adhesion is complicated.The stable gene silencing of cheV could affect the upstream and downstream genes most significantly,indicating the important role of cheV in bacterial chemotaxis pathway.Finally,we investigated the expression change of DEGs under stress conditions(temperature,pH,salinity and starvation).The results showed that the same DEG responded to the stresses differently,and also different DEGs responded to the same stress differently,which indicated that the DEGs are sensitive to the environmental factors,especially pH and starvation.In conclusion,the flagellar assembly pathway and the bacterial chemotaxis pathway was sensitive to environmental stresses and played a key role in the adhesion process of V.alginolyticus.Motility is not the only avenue through which the flagellar assembly pathway affected adhesion,while the bacterial chemotaxis pathway may regulate bacterial adhesion through motility.