Adhesion is an important virulence factor of Vibrio alginolyticus. This factor may be affected by environment conditions; however, its molecular mechanism remains unclear. In our previous research, adhesion tender models were obtained by culturing V. alginolyticus under stresses such as Cu2+, Pb2+, Hg2+, and low pH. With high-throughput sequencing and bioinformatics analysis, we found that all of these stress treatments significantly affected the flagellar assembly pathway, which may play an important role in V. alginolyticus adhesion. Therefore, we hypothesized that the environmental stresses of the flagellar assembly pathway may be one way in which environment conditions affect adhesion. To verify our hypothesis, a bioinformatics analysis, QPCR, RNAi, in vitro adhesion assay and motility assay were performed. Our results indicated that 1) the flagellar assembly pathway was sensitive to environmental stresses, 2) the flagellar assembly pathway played an important role in V. alginolyticus adhesion, and 3) motility is not the only way in which the flagellar assembly pathway affects adhesion.

Adhesion capability to fish mucus is considered to be a key virulence factor of Vibrio alginolyticus, while the molecular mechanism is still unclear. Non-coding RNAs (ncRNAs) play a crucial role in the intricate regulation of bacterial gene expression, affecting the pathogenicity of bacteria. With high through-put sequencing and bioinformatics analysis, we identified three ncRNAs predicted to regulate the bacterial chemotaxis pathway in our previous study, which might play a key role in the adhesion process of V. alginolyticus. In the present study, we validated the effects of these three ncRNAs on their predicted target genes and the role of these three ncRNAs in the adhesion process of V. alginolyticus with RNAi, QPCR, capillary assay, and in vitro adhesion assay. According to our results, the expression of these ncRNAs and their predicted target genes were confirmed by QPCR, which reinforced the reliability of the sequencing data and the target prediction. We found that over-expression of these ncRNAs were capable of reducing the chemotactic and adhesion ability of V. alginolyticus, while the expression levels of their target genes were significantly reduced. Our results indicated that: 1) these three ncRNAs were really able to regulate the bacterial chemotaxis pathway; 2) these three ncRNAs played a key role in the adhesion process of V. alginolyticus.

Vibrio alginolyticus is a leading aquatic pathogen, causing huge losses to aquaculture. rpoS has been proven to play a variety of important roles in stress response and virulence in several bacteria. In our previous study, upon treatment with Cu²⁺, Pb²⁺, Hg²⁺ and low pH, the expression levels of rpoS were downregulated as assessed by RNA‐seq, while impaired adhesion ability was observed, indicating that rpoS might play roles in the regulation of adhesion. In the present study, the RNAi technology was used to knockdown rpoS in V. alginolyticus. In comparison with wild‐type V. alginolyticus, RNAi‐treated bacteria showed significantly impaired abilities of adhesion, growth, haemolytic, biofilm production, movement and virulence. Meanwhile, alterations of temperature, salinity, pH and starvation starkly affected rpoS expression. The present data suggested that rpoS is a critical regulator of virulence in V. alginolyticus; in addition, rpoS regulates bacterial adhesion in response to temperature, pH and nutrient content changes. These are helpful to explore its pathogenic mechanism and provide reference for disease control.