| F18+Escherichia coli (F18+E. coli) widely spread and are a major cause of economic losses in the pig industry due to diarrhoea, growth retardation and mortality, however, no vaccine or good therapy exists to date. F18fimbriae and toxins are the known virulence factors responsible for F18+E. coli caused infections. After establishment of initial adherence by F18fimbriae, they release toxins that change the functions of enterocytes by increasing secrection and reducing absorption. Although flagella, bacterial locomotive organelles, contribute to some pathogen pathogenesis, neither previous nor current models of F18+E. coli pathogenesis have implicated the role of flagella. Since both F18ab and F18ac reference strains carry the flagella with good motility, we would explore the relationship between F18+E. coli flagella and its pathogenicity in this study.Based on the original sequences of the flagellin gene fliC, F18fimbriae gene fedA and the rotory motor motA gene in the GenBank, the two F18+E. coli reference strains, F18ab (Wild-type, O139:H1:F18ab, Stx2e) and F18ac (Wild-type, O157:HO19:F18ac,4P-, STa+, STb+) E. coli were chosen as the parent strains to construct the above isogenic mutants by using λ-Red recombination system method. First, we generated PCR products by using primers with the homologies extension of fliC, fedA and motA genes to be deleted and template plasmid pKD3carrying selectable antibiotic chloramphenicol resistance (Cat) gene that is flanked by FRT (FLP recognition target) sites. The F18ab/ac△fliC::Cat, F18ab/ac△fedA::Cat and F18ab/ac△motA::Cat for excision of the chloramphenicol cassette by introduction of the FLP recombinase-expressing vector pCP20. The final deletion of these genes in parent strains was confirmed by PCR and DNA sequencing. The isogenic F18△fliC mutants were non-motile on0.3% semi-solid agar, unlike the control parent strains, suggesting that the mutated flagella were rendered non-functional in these mutants. To further confirming this, mutants flagella were not observed by TEM, and flagellin protein expression was absent in Western blot analysis of the whole-cell lysates. Although the mot A mutants possess flagella, they were nonmotile on semi-solid agar. The agglutination assay indicated that the fedA mutant strain lost the ability to agglutinate with the F18fimbriae polyclonal antibody and no fimbriae expression was confirmed. Based on the single mutants of fliC, fedA and fliC double mutants were constructed in the same method, they could neither agglutinate with F18fimbriae polyclonal antibody nor exhibit motility on a semi-solid agar plate. The mutants obtained in this study will gain insight into the mechanism of F18+E. coli pathogenicity in the following experiments.In general, bacterial attachment to enterocytes, with subsequent colonization of the intestinal epithelium, is regarded as the initial and crucial step in the pathogenesis of enteric diseases. Although flagella have been reported to mediate some certain of pathogens adherence, whether they play a role in F18+E. coli adherence has not been elucidated. The△fliC mutants, on the other hand, showed a significant reduction in the ability to adhere to IPEC-J2and IPEC-1cells in the presence of4%mannose when compared to the wild-type strains, indicating that flagella played important role in the binding assay between F18+E. coli and the cells. In contrast, the adherence ability of the fimbriae-deficient F18△fedA mutants were not significantly different from wild-type F18+E. coli strains. Like the△fliC mutants, the adherence assay showed the F18△fliCfedA double mutants with the lower adherence capacity to adhere to IPEC-J2and IPEC-1cells. Furthermore, we tested the requirement of flagella for adherence by using blocking antibodies against H1or H19flagella to block adherence. A reduction in adherence was obtained with anti-H1or H19antibodies at1:10dilutions, respectively. Likewise, the ability of motA mutants adhere to these two cell lines was also decreased. Collectively, these data confirmed that flagella are necessary and sufficient for F18+E. coli adherence. In addition, the F18+E. coli fliC deletion mutants up-regulate about2-fold the expression of type I fimbriae produced by F18+E. coli strains. The levels of F18fimbriae and AIDA-1up-regulate about1.2-fold and1.3-fold after fliC deletion.respectively. Overall, these data suggested that F18+E. coli coordinately regulate flagella and other surface appendix organelles. The data described above demonstrated that flagella were involved in F18+E. coli initial adherence. We showed that flagella play roles in the process through two ways. One way is that flagella-mediated motility promoted and facilated interaction between F18+E. coli and host cells. The other one is that flagella could mediate adherence as adhesins.Flagella and flagellar motility are important invasins for many invasive pathogenic bacteria. In this study, we investigated the capacity of invasion ability of F18ab E. coli and F18ac E. coli to invade both IPEC-J2and IPEC-1cell monolays. We found that F18ac E. coli parent strain did not possess invasion ability like other ETEC did, while F18ab E. coli parent strain could invade both cell lines. The invasion ability of IPEC-J2cells and IPEC-1cells by F18ab△fliC mutants was significantly impaired when compared to the wild-type strains. The invasion ability of IPEC-J2cells by F18ab△fliC mutant decreased about91%and invasion of IPEC-1cells reduced approximatly63%. Flagella were also required for invasion, as the complemented strain (F18ab△fliC/p△fliC) restored the F18ab△fliC mutant’s capacity to invade both celllines. In addition, we found that F18ab E. coli invasion ability at37℃was stronger than at30℃, that due to more flagella expression at37℃. In couclusion, we first found that F18ab E. coli possess invasion ability, and flagellum was an important invasin for it in this study.Bacterial biofilms are community of microorganism attached to a surface in adverse environment, which is opposed to a single bacteriae in a plantonic state. Most pathogens can form a biofilm. In this study, we found that both F18ab E. coli and F18ac E. coli strains could form biofilm in vitro, and F18ab E. coli had stronger ability than F18ac E. coli. Flagella and flagella-mediated motility are one of the important factors that influence biofilm formation. While the F18ab△fliC mutant was not able to produce the typical large microcolonies displayed by the wild-type strain, the complemented strain could form microcolonies of bacterial clusters, although microcolonies were still smaller than those produced by the parent strain. Biofilm formation on the glass tube surface by the△fliC mutant was reduced when compared with the wild-type strain. However, even the wild-type F18ac E. coli strains could only form small microcolonies and weak biofilm. The biofilm formation by F18ac E. coli flagella mutants was hardly seen. In quantitative assay, the ability of both F18ab E. coli and F18ac E. coli to form biofilm was markly reduced when compared to the wild-type strains. Thus, flagella seem to play an important role in F18ab E. coli biofilm and microcolony formation in vitro.Bacterial monomer flagellin can induce production of inflammatory cytokines IL-8through Toll-like receptor5signaling pathway. In this study, we found that all the F18+E. coli fliC, fedA and fliCfedA mutants had a reduced ability to stimulate IL-8production by Caco-2cells infection when compared to the wild-type strains. The level of IL-8production by F18+E. coli fliC, fedA and fliCfedA mutants decreased about68%,43%and86%respectivly. These results not only indicate that flagellin is the main factor inducing IL-8production in vitro, but are also the first to suggest the novel finding that F18fimbriae can potentially induce IL-8.The results presented in this study strongly suggest a role for flagella in F18+E. coli adhering to IPEC-J2and IPEC-1cells, forming biofilm, and stimulating IL-8production from Caco-2cells in vitro. In addition, flagella of F18ab E. coli are required for its efficent invasion. Due to their good immnuogenicity and important roles in F18+E. coli pathogenesis, flagella are particularly attractive for the development of vaccine candidates to prevent from F18+E. coli caused infections. |
PDF Full Text Request