Studies On The Regulation Of SRNA In The Adhesion Process Of Vibrio Alginolyticus

Adhesion capability to fish mucus,which could be affected by environment conditions while the molecular mechanism is still unclear,is considered to be a key virulence factor of Vibrio alginolyticus.Other research in our group showed that stress conditions such as Cu,Pb,Hg and low pH were capable of reducing the adhesion ability of V.alginolyticus.Bacterial sRNAs are an emerging class of small regulatory RNAs Non-coding RNAs,which play a crucial role in the intricate regulation of bacterial gene expression,affecting the pathogenicity of bacteria.In order to investigate whether the sRNAs take part in the regulation of gene expression underlying decline in the adhesion ability of V.alginolyticus caused by stress conditions including Cu²⁺?50mg/L?,Pb²⁺?100mg/L?,Hg²⁺?50mg/L?and low pH?pH=5?,we combined high through-put sequencing with computational techniques to detect sRNAs dynamics in the stress samples,and through the RT-PCR technique to authenticate our sequencing results.There were a total of 976 sRNAs received from 5 samples through high throughput sequencing.The number of sRNAs from the sample Cu²⁺?50mg/L?,Pb²⁺?100mg/L?,Hg²⁺?50mg/L?,low pH?pH=5?and the control was 859,859,892,892 and 908,respectively.The length range of most of the sRNAs was 100-240 nt.By comparing the the expression of sRNAs between the stress strains and the wild strain,among 1083 sRNAs,we found that there are 32sRNAs showing a consistent changing trend either decreasingly or increasingly among 4 tress samples.30 sRNAs were up-regulated while 2 sRNAs were down-regulated by all stress treatments.The expression of randomly selected novel sRNAs was confirmed by QPCR,which reinforced the reliability of the sequencing data.GO and COG analysis of targe genes showed that the biological processes and functions such as“cell motility”,“response to chemical stimulus”,“establishment of localization”and“intracellular trafficking,secretion and vesicular transport”may be related to the decreased adhesion caused by the stess factors.Target prediction and KEGG pathway analysis indicated that among the significantly up-regulated target genes,type IV pilus assembly protein PilQ?K02666?,type IV pilus assembly protein PilM?K02662?,flagellar motor switch protein FliM?K02416?,chemotaxis protein MotB?K02557?and methyl-accepting chemotaxis protein MCP?K03406?may be closely related to bacterial adhesion.The sRNAs which palyed a key role in regulating these genes were Candidate 103?Candidate 635?Candidate 424?Candidate 283 and Candidate 907,respectively.The binding free energy of these sRNAs was-97.50,-83.00,-186.10,-86.65,-93.80 kcal/mol,respectively,which showed that the structure of the 5 sRNAs is relatively stable.Then the gene silencing experiment was conducted by the design of artificial bacterial sRNA on chemotaxis pathway related genes such as CheR?CheB?CheV?Aer and MCP.Our results indicated that although the inhibition effect of the sRNAs to the target genes was limited within 6 h,the inhibitory effect disappearing after 6 h,the inhibition was obvious.In conclusion,Candidate431,Candidate 103 and Candidate 907 could inhibit the expression of related genes in chemotaxis pathway in a short time to reduce bacterial adhesion.That is to say,these sRNAs may play a key role in the adhesion process of V.alginolyticus,and bacterial chemotaxis pathway related genes suppressed would lead to a reduced adhesion ability,indicating bacterial chemotaxis pathway was closely related to bacterial adhesion,which could lay a good foundation for further functional analysis of these sRNAs in the process of other pathways related to bacterial adhesion.Our results indicated that the perturbation of sRNAs expression may be involved in the chemical stress-induced reduction of the adhesion ability in V.alginolyticus.Our findings provided important information for further functional characterisation of sRNAs during the adhesion process of V.alginolyticus.