The Construction Of FliC And FimH Gene Deletion Mutants From Adherent-invasive Escherichia Coli And Related Function Analysis

Adherent-invasive Escherichia coli(AIEC)is related to the onset and development of intestinal infection.The virulence factors of AIEC influence its survival and replication in epithelia and macrophages.AIEC is one of the pathogenic bacteria involving in triggering IBD,so it attracts more attention in recent years.Although the pathogenesis of AIEC triggering IBD still remains to be known,flagella and fimbriae have been considered as two major pathogenic factors in the pathogen.F1iC encoded by fliC gene is the major structural subunit of filament,and playing a key role in pathogenic process.Meanwhile,FimH acts as the major part of type I fimbriae helping bacteria adhere to host cells,so it creates good conditions for bacteria invading the host.This research aims at AIEC FliC and FimH,and exploring their biological functions to better understand the pathogenesis of AIEC.According to NCBI GenBank database,we compared fliC and fimH gene sequences from different E.coli strains,designing the corresponding PCR primers to amplify the fliC and fimH gene of Adherent-invasive Escherichia Coli LF82(Wild-type,083:H1).After that,the PCR amplification products were purified,cloned and sequenced.Then,we designed the primers which contain a homologies 5' terminal to the target region and a homologies 3' terminal to the chloromycin-resistance cassette for Red recombination based one step PCR inactivating the target gene.According to the ?-Red recombination system,we used plasmid pKD3 as the template DNA to amplify the chloromycin-resistance cassette,then the purified PCR products were electroporated into LF82 carrying a heat-sensitive plasmid pKD46.With the assistance of three Red recombination enzymes,fliC or fimH was replaced by the cat gene,and the successful recombinant bacteria was selected by LB plates containing ampicilin and chloramphenicol.After that,the plasmid pCP20 was electroporated into the the first recombination bacteria,and the Flp recombinase expressed by pCP20 could excise the chloromycin-resistant cassette.Finally,the isogenic gene deletion LF82?fliC,LF82?fimH and LF82?fliC?fimH double mutants were constructed and confirmed by PCR detection and DNA sequencing data.On the base of mutant strains,we electroporated plasmid pBR322 which could express FliC or FimH protein into the second recombination mutants to construct the complemental strains LF82?fliC/pfliC and LF82?fimH/pfimH.Bacterial growth test indicats that there were no difference among wild type,mutants and complemental strains.Motility test shows that fliC mutants lost their motility on semisolid medium,but the wild type,fimH mutant and complemental strains still had strong motility.Meanwhile,biofilm formation test proves that flagella and type ? fimbriae could promote bacterial biofilm formation.Notably,the biofilm formation ability of fimH mutant decreased more significantly(p<0.01).In vitro,the adhesion test of Caco-2 cells shows that the adhesion ability of fliC mutant,fimH mutant and double mutant decreased by 74%,72%and 85%,respectively(p<0.01).Moreover,the invasion test indicates that the invasion ability of fliC mutant,fimH mutant and double mutant decreased by 59%(p<0.05),58%(p<0.05)and 86%(p<0.01),respectively.Sequentially we used Real-time PCR to detect the transcriptional changes of the major virulence factors in mutants and complemental strains.The result shows that the transcriptional level of fimH,papC,lpf,ibeA and hlyA in fliC mutant decreased by 43%,46%,74%,57%and 94%,respectively(p<0.05).The transcriptional level of fliC,papC,lpf,ibeA and hlyA in fimH mutant decreased by 43%,52%,78%,62%and 63%,respectively(p<0.05).The double mutant did not transcript fliC gene and fimH gene,and the transcriptional level of papC and hlyA decreased by 28%(p>0.05)and 57%(p<0.05).However,the transcriptional level of lpf,ibeA and vat increased by 6%,29%and 10%,respectively(p>0.05).The results suggest that fliC and fimH could be involved in the biological function of LF82.In addition,these two genes could cooperate with each other to involve in the pathogenesis of LF82,and there are some synergistic effects existing in different virulence factors.The adherence inhibition test shows that the process of AIEC adhering to Caco-2 cells could be inhibited by polyclonal antibody FliC and D-mannose.This suggests that FliC and FimH may be the potential therapeutic targets for blocking the initial attachment of the infection.The gut colonization test in C57BL/6 mice shows that AIEC LF82 mainly colonize in cecum and ileum.Compared to wild type,the colonization ability of fliC mutant,fimH mutant and double mutants decreased by 33%,25%and 26%,but with no significant differences(p>0.05).