Study Relationship Cas9 Gene And Virulence In Campylobacter Jejuni

In recent years,there are more and more human and animal infected diseases by Campylobacter jejuni.To better prevent and control the disease,it is necessary to know more clearly the mechanism and cause of pathogenic bacteria produce virulence.In addition,the CRISPR-Cas system composed of CRISPR(Clustered regularly interspaced short palindromic repeat)and Cas gene(CRISPR-associated),as an adaptive immune system of bacteria,was found to be closely related to the virulence of bacteria.CRISPR-Cas system through the integration of exogenous DNA into the CRISPR sequence to identify secondary invasion of foreign DNA in order to degraded exogenous DNA,to resist the invasion of exogenous genetic elements.Since the CRISPR-Cas system can prevent foreign DNA damage itself,whether the CRISPR-Cas system can also allow bacteria to enter the other organisms faster or slower.For nearly a decade,the CRISPR-Cas9 system has been used as a novel gene editing technology in different organisms.Although the CRISPR-Cas9 system has been used in different organisms as a novel gene editing technology,it is role that in bacteria remains at the stage of adaptive immune defenses and its role in bacterial virulence.At present,There are still few related reports.Therefore,the purpose of this study is to understand the relationship between virulence and Cas9 gene in C.jejuni NCTC11168.To explore the relationship between Cas9 and virulence mainly from two aspects,on the one hand from the virulence phenotype,on the other hand from virulence genotypes.By combining these two aspects,we can fully understand the relationship between Cas9 and bacterial virulence,so as to provide more theoretical basis for solving the risk management and hazard control in Campylobacter jejuni.Homologous recombination vector was constructed by fusion PCR.The upstream and downstream of Cas9 gene were amplified about 1300 bp and the Kan gene was amplified with a size of about 1200 bp.The three fragments were then sequenced according to the fusion PCR method using the upstream fragment-Kan gene-downstream fragment And then ligated to pGEM-Teasy vector to construct homologous recombination vector p3HKan5 H.The p3HKan5 H vector was transformed into Campylobacter jejuni NCTC11168 by electrotransformation and natural transformation,and the mutants were selected and mutants were verified.The effect of Cas9 gene on the bacterial virulence phenotypes was investigated by measuring growth ability,morphology and migration of flagella,and biofilms.The effect of Cas9 gene on the bacterial virulence phenotypes was investigated by measuring growth ability,morphology and migration of flagella,and biofilms.Migration results show that: the migration of wild-type bacteria was significantly stronger than the mutant bacteria,there is a significant difference between the two strins.Migration mainly associated with bacterial flagella movement,since there are significant differences in migration force,it may that Cas9 gene have a certain role in bacterial flagella.Therefore,the morphology of flagella was observed by cryo-transmission electron microscopy to observe whether there was any significant change.However,the results showed that there were longer and complete flagella on both poles of wild-type and mutant,indicating that the flagella form did not change significantly.Biofilm results showed that the biofilm formation ability of the mutant was stronger than that of the wild.Cas9 can initially indicate that the virulence of Campylobacter jejuni has a certain impact.In addition to measuring the changes of the bacteria's own virulence,in vitro toxicity of the bacteria also needs to be tested,including the ability of the strains to infect and adhere to the cells and the viability of the strains in vitro.Mouse macrophages RAW264.7 and colon cancer cells Caco-2 were used in this experiment,and the wild-type and mutant strains were tested by the method of gentamicin protection.The experimental results showed that there was no significant difference in adhesive force and invasiveness after knocking out Cas9 gene,but there was a significant difference in the viability of cells,showing a tendency of weakening.The MTT assay was used to determine the virulence of the bacteria and the pathogenicity of the bacteria was tested by measuring the viability of the bacteria.The experimental results show that there is a significant difference between the two and the wild-type bacteria than the mutant toxin production ability,indicating that knock-out Cas9 gene,the bacterial toxin production decreased.In this study,Paired-end(PE)sequencing was performed on the constructed library using Next-Generation Sequencing(NGS)and Illumina NextSeq500 sequencing platform.The main purpose of RNA-Seq is to detect which virulence genes will change after Cas9 gene knockdown,so as to find out the relationship between Cas9 gene and these virulence genes.Overall,the results of the phenotypic analysis showed that ? Cas9 mutants showed weakened in four aspects of migration ability,biofilm formation ability,intracellular viability and toxin-producing ability compared with wild type bacteria.Virulence genotypes,RNA-Seq results showed that only a knockdown of Cas9 gene was down-regulated in all the differentially expressed genes,and other genes showed an up-regulation.In the up-regulated genes,no direct virulence-related genes were found.Up-regulated genes are some of the proteins involved in metabolism and hypothetical proteins of unknown function.Comprehensive analysis showed that knocking out the Cas9 gene,virulence phenotype showed a downward role in virulence genotypes did not appear to be a direct change,considering the role of negative regulation may be related to the regulation of changes in virulence.