Construction And Characterization Of Compylobacter Jejuni FlhA Mutant

Campylobacter jejuni,a microaerophilic, asporous, Gram-negative bacterium, is the most frequently gastrointestinal food-borne pathogen worldwide.Flagellum can not only provid motility to the cell,but also is an important virulence factor,which plays an important role in adhesion, colonization, and the secretory of invasion protein of C. jejuni. Flagellar export apparatus is a very important structure of flagellar synthesis, assembly and work.A lot of researches consider that the flagellar export apparatus is homologous with the type III secretion systerm of the gram-negative bacterium in evolution.Genome analysis had indicated that the flagellar export apparatus were also the transportation apparatus of type III virulence proteins of C. jejuni.In order to search for the connections among the flagellar export apparatus, flagellar synthesis and pathogenesis, we construct a flhA deletion strain of C. jejuni flagellar export apparatus (HXW001),and study the function of flhA in virulence and molecule regulation.1. Construction of Campylobacter jejuni flhA mutant strainwe constructed a flhA deletion strain of C.jejuni. Firstly,the recombinant plasmid pMD19-T-flhA-kanr was constructed in E.coil JM109. Secondly,the plasmid was transformated into C. jejuni by electroporate,than we got the mutant strain HXW001 by homologus recombination.2. Virulence associated phenotypes research in HXW001The virulence associated phenotypes inculd cell growth,motility,cell struct, autoagglutin-ation,biofilm formation and so on. Compared with the wild type, the flhA mutant cell could grow well but lacks flagella, and the HXW001 lost the motility, and the ability of HXW001 for the formation of biofilm and autoagglutination was also decreased.These findings indicate that flhA is the molecule basise of flagellar struct and pathogenesis.3. Characterization of flhA in regulation of flagella synthesiswe investigated the role of flhA in regulation of intranscription of flaA, fliA and rpoN by Real-time PCR.We found that the transcription of the flaA and fliA were reduced, but the rpoN was not affected by flhA gene.These findings indicated that the flhA gene not noly plays a role in flagellin transportation but also work in regulating the transcription of the flagellin genes and regulatory factor genes.4. The role of flhA in transcription and secretion of Typeâ…¢virulence proteinsAt first, we studied the role of flhA in transcription by Real-time PCR.We found that the ciaB was not affected by flhA gene.For studying of the effects on the secretion of the type III virulence proteins by flhA,we construct the prokaryotic expression system of ciaB,and purificate the CiaB protein,for making the polyclonal antibodies of CiaB,in order to study the CiaB secretion difference between the wild type and HXW001 by Western blot.