Study On The Function And Regulation Of Accessory Colonization Factor AcfA Of Vibrio Alginolyticus To SOD,Flg And Dct

V.alginolyticus is a globally distributed condition pathogenic bacteria,which can cause fish,shrimp,shellfish and other marine creatures disease inordinately.Especially in recent years,there are frequent environment pollution in aquaculture water with eutrophication phenomenon.Vibriosis caused by V.alginolyticus shows a trend of wide epidemic range and frequent outbreaks,which leads to great damage and affect the healthy development of aquaculture.The successful colonization and propagation of Vibrio in the intestinal tract of the host is a critical first step for Vibrio to infect the host and cause disease.Acf A is an important virulence protein of Vibrio,which helps pathogenic bacteria adhere to the host mucosa effectively.However,the related function of Acf A in V.alginolyticus is still poorly studied.In this study,V.alginolyticus HY9901 was selected as the wild type strain,and mutant strain ?acf A was constructed by homologous recombination technology.The colonization status of strain WT and strain ?acf A was studied by using 16 S r DNA high-throughput sequencing technology.Then the regulation of Acf A in flagellum assembly,energy metabolism and other related genes in V.alginolyticus was studied by transcriptome sequencing.According to results of transcriptome sequencing and RT-PCR analysis,the sod B gene of V.alginolyticus was knocked out respectively to study whether the deletion of sod B affects the physiological characteristics and pathogenicity of V.alginolyticus.?-galactosidase reporter gene and bacterial single hybridization experiment were utilized to test the regulation of Acf A to SOD?Flg and Dct genes.The main results are as follows:1.Construction and biological characteristics of ?acf AV.alginolyticus HY9901 was used as the wild type strain,and strain ?acf A was constructed by homologous recombination technology.According to the analysis of biological characteristics,the knockout of acf A reduced the swarming ability,adhesion ability and virulence without affecting the genetic stability,external morphology,growth,extracellular enzyme activity and biofilm formation ability of V.alginolyticus.2.Analysis of intestinal bacteria of strain WT and strain ?acf A in grouperThe results of Alpha diversity index of intestinal mucosal bacteria of pearl gentian grouper showed that the sequencing depth of all samples was above 0.99.Chao1 and ACE index of the acf A group and WT group showed a declining trend over time.Moreover,the Chao1 and ACE index of the acf A group on the 1st,2nd and 3rd days were higher than that of the WT group on the 1st,2nd and 3rd days,respectively,indicating that the abundance of intestinal mucosal bacteria of grouper decreased after feeding strain WT.There was no significant difference in Simpson and Shannon indexes between acf A group and WT group.The Vibrio abundance in the WT group remained almost unchanged,while that of the acf A group decreased from 0.07% to 0.01%.Intestinal colonization experiment also showed that the number of ?acf A colonized grouper intestines was only 8.40% of that of strain WT after feeding.3.Transcriptome sequencing analysis of strain WT and strain ?acf AA total of 329 differentially expressed genes were screened by transcriptome sequencing analysis of strain WT and strain ?acf A,among which 157 genes were up-regulated and 172 genes were down-regulated.KEGG analysis showed that the differentially expressed genes of strain WT and strain ?acf A were mainly located in two-component system,microbial metabolism in diverse environments and carbon metabolism.20 genes related with flagellum assembly,energy metabolism,thiamine metabolism,carbohydrate and amino acid metabolism,signal transduction were significantly down-regulated.The results indicated that Acf A was involved in the regulation of flagellum assembly,energy metabolism and other related gene expression.4.Construction and biological characteristics of ?sod BBased on results of transcriptome sequencing and RT-PCR analysis,V.alginolyticus HY9901 was used as the wild type strain,and strain ?sod B was constructed by homologous recombination technology.According to the analysis of biological characteristics,the knockout of sod B enhanced the biofilm formation ability and extracellular enzyme activity,but reduced the swarming ability,adhesion ability,virulence,SOD activity and antioxidant ability without affecting the genetic stability,external morphology and growth of V.alginolyticus.5.Functional verification of regulation of Acf A to SOD,Flg and DctThe interaction between Acf A and the promoter regions of sod B,flg F,flg G,flg I and dct Q was tested by ?-galactosidase reporter gene and bacterial single hybridization experiment.The results showed that Acf A can positively regulate sod B,flg G and dct Q,and negatively regulate flg F and flg I.Acf A can be directly combind with flg F and flg I promoter regions,but not with sod B,flg G and dct Q promoter regions,indicating that Acf A is indirect in the regulation of sod B,flg G and dct Q.