FlgE Protein Expression And The Construction Of FlhA/flhB Gene Deletion Mutants In Salmonella Typhimurium

Salmonella Typhimurium is a pathogen of multiple host infections which can lead to diseases such as gastroenteritis.Its pathogenicity is closely related to many virulence factors,such as type III secretion system,virulence plasmids,flagella,pili.Among them,flagella are the one of the main virulence factors,not only conduct an important motor organ of bacteria,but also participate in important biological processes such as biofiom formation and adhesion on epithelial cell.Besides,as the main component of flagella,flagellin is also an important immune activator which can bind to TLR5 receptors.It can activate immune response and then stimulate the production of a series of cytokines.The flagella structure consists of three parts: filament,hook,basal body(motor,at least four circular structures,and a specialized flagella type III secretion system).Among them,the hook is composed of FlgE,which is responsible for connecting the filament and basal body.The type III secretion system of flagella which is also known as flagella export apparatus,is mainly used to synthesize and output flagellin.The presence of flagella hook and flagella export apparatus plays an important role in the flagella structure of the bacteria.In order to understand the characteristic and molecular mechanism of the related genes in the structure of flagella and explain the pathogenic mechanism of bacteria,we have carried out the research from the following three aspects:(1)The hook is encoded by the flgE gene.The flagellin was obtained by heterologous expression and polyclonal antibodies were prepared.By cloning the flgE gene and connecting the pET28 a plasmid,and the induced protein was about 42 kDa with soluble expression.The antiserum was obtained by using the protein to immunize mice.The indirect ELISA showed that the polyclonal antibodies have good titers and the western blot proved they can specifically bind to flagellin.(2)Through establishing an interaction model of flagellin and macrophages,the experiment analyzed the inflammatory effect of flagellin at different levels.The cell viability was determined by cell counting kit-8 and the relative mRNA expression of cytokine was analyzed by real-time fluorescence quantitative PCR.The production of reactive oxygen species was also observed by fluorescence microscope.It was showed that low concentration of flagellin has no effect on the viability of macrophage and promote the expression of a series of cytokine such as IL-1??TNF-?.At the same time,flagellin can promote the production of reactive oxygen species as well.(3)The flhA and flhB gene mutants of flagella export apparatus was constructed by ?-red homologous recombination technique and corresponding complementations were obtained.We analyzed the phenotypic changes in different aspects.The results showed that the growth performance of ?flhA and ?flhB was not affected,but its motility decreased compared with wild type.The ability of biofilm formation at different times was evaluated by crystal violet staining.It was clearly showed that the biofilm formation ability of flhA or flhB gene deletion mutants was lower than the wild type.Meanwhile,the autoaggregation ability associated with bacterial biofilm formation was also influenced obviously.In summary,the analysis of the two structure from protein and molecular level has deepened the cognition of flagella,which not only helps us understand the pathogenic mechanism of bacteria,but also lays the foundation for the subsequent work such as inhibiting bacterial infection.