Molecular Mechanism Underlying The Novel Non-Coding RNA Vvrr2 Involved In The Regulation Of Vibrio Alginolyticus Virulence And Biofilm Formation

Vibrio alginolyticus is a common Marine conditioned pathogen,widely distributed in sea water and estuaries all over the world.It can cause disease and even death to a variety of Marine organisms.It is one of the main conditioned pathogenic pathogens of fish,shrimp,abalone,sea cucumber and other mariculture animals in recent years.At the same time,in addition to being pathogenic to aquatic animals,V.alginolyticus is one of the major opportunistic pathogens for people who eat raw or undercooked infected seafood or engage in aquatic activities.At present,the research on the pathogenic factors of V.alginolyticus mainly focuses on flagella,quorum sensing,lipopolysaccharide,biofilm and outer membrane proteins.However,the current prevention and treatment of vibriosis caused by V.alginolyticus is still dominated by antibiotics,and frequent use of antibiotics can easily produce drug-resistant strains,and there are still hazards such as drug residues.Therefore,it is urgent to find a new green,safe,efficient and convenient control method.There are many non-coding RNAs in bacteria,and there are usually7-9 bp complementary sequences between nc RNA and target gene m RNA.Therefore,nc RNA can bind to the m RNA of the target gene,thereby preventing the translation of the protein,or promoting the degradation of the m RNA,thereby regulating the gene at the post-transcriptional level,and thus widely participating in the physiological processes of bacteria.At present,there are few studies on the identification and function of non-coding RNA related to V.alginolyticus biofilm.In this study,the biofilm bacteria of V.alginolyticus at 12 h,24 h,36 h,48 h and 60 h were used as the research objects.The high-throughput sequencing technology was used to analyze the s RNA expression at different stages(12 h,24 h,36 h,48 h and 60 h)of V.alginolyticus biofilm formation.The s RNA with the most significant differential expression during biofilm formation was selected and named Vvrr2(Vibrio virulence regulatory RNA 2).By constructing Vvrr2 overexpression strains and further exploring their effects on virulence regulation and biofilm formation of V.alginolyticus,it provides a theoretical basis for the development of safe and effective green and efficient control methods.The main results are as follows :(1)The differential s RNAs of V.alginolyticus biofilm at different time periods were analyzed by RNA-seq technology.The results showed that a total of 81 differentially expressed s RNAs were found throughout the biofilm formation process compared with 12 h.We performed bioinformatics analysis on 81 differentially expressed s RNAs.Firstly,we concluded that the most significantly down-regulated s RNA in the whole biofilm formation process(four comparison groups)was AP023187＿pred RNA121,and the most significantly up-regulated s RNA was AP023187＿pred RNA145.Through further screening of s RNAs,we found that 80 s RNAs were involved in the regulation of biofilm formation.The top ten s RNAs related to biofilm formation in the predicted target genes of s RNAs were selected for KEGG enrichment analysis.The predicted target genes were mainly enriched in biofilm,bacterial secretion system,two-component system,quorum sensing system and other pathways.At the same time,trend analysis of all differentially expressed s RNAs showed that there were 26 up-regulated s RNAs and 30 down-regulated s RNAs during biofilm formation.After these series of bioinformatics analysis,we learned that the biofilm formation of V.alginolyticus was regulated by multiple s RNAs.We selected the s RNA most significantly related to the biofilm formation of V.alginolyticus and named it Vvrr2 for subsequent research.(2)The expression level of Vvrr2 under different stress conditions was detected by qRT-PCR.It was concluded that Vvrr2 was mainly involved in environmental regulation through negative regulation mode when V.alginolyticus was at a disadvantage.Subsequently,we constructed Vvrr2 overexpressing strains,and performed virulence phenotypic experiments such as growth curve,motility,adhesion,film formation,drug resistance,and extracellular polymeric enzyme activity.The results showed that Vvrr2 had significant effects on various virulence phenotypes of V.alginolyticus.The GFP reporter gene showed that fli G,lps,and che V may be important target genes of Vvrr2 and play an important regulatory role in the virulence regulation mechanism of V.alginolyticus.(3)In order to further study the effect of Vvrr2 on the biofilm of V.alginolyticus,we observed and analyzed the biofilm of V.alginolyticus by laser confocal microscopy and scanning electron microscopy.The results showed that overexpression of Vvrr2 delayed the time of three-dimensional structure of V.alginolyticus biofilm,and the biofilm formation ability was significantly weakened.Raman spectroscopy was used to evaluate the dynamic heterogeneity of the effect of Vvrr2 overexpression on the extracellular products during the biofilm formation of V.alginolyticus.It was shown that Vvrr2 affects the types and contents of polysaccharides,proteins,nucleic acids and lipids,and plays an important role in the adhesion,colony aggregation,biofilm maturation and degradation of V.alginolyticus.In summary,this study will further strengthen the understanding of the virulence regulation mechanism of V.alginolyticus.The nc RNA is involved in the regulation of the virulence mechanism and biofilm formation of V.alginolyticus to provide a theoretical basis,and also provide more possibilities for the treatment of V.alginolyticus.