Study On Fima Mediated F18ab+Escherichi Coli Pathogenicity

Edema disease (ED) and post-weaning postweaning diarrhea (PWD) are both frequently fatal disease that usually occurs in post-weaning piglets. Affected pigs die quickly once they developing symptoms and surviving pigs may grow slowly. There is no effective vaccine or reliable therapy of this disease which has caused great economic loss in the whole world. F18ab+STEC (Shiga Toxin producing Escherichia coli) is the principal pathogenic bacteria leading to diarrhoea, edema, and mortality. As we all known, adhesions including F18ab fimbriae, type Ⅰ fimbriae, flagella, AIDA (adhesin involved in diffuse adherence) and toxins are two major pathogenic factors of F18ab+STEC causing infection, although the mechanism is remains to be elucidated. Given the proven role of type Ⅰ fimbriae in urinary tract infections (UTIs), it is reasonable hypothesise that it plays a direct role in enteric pathogenesis, which is not to be fully defined. This study target at the major structure subunit FimA of type Ⅰ fimbriae, exploring the role of type Ⅰ fimbriae in pathogenicity of F18ab+STEC.Based on the original sequences of fimA gene with different resources in Genebank, we used primers to detect and amplify the major structure subunit of type Ⅰ fimbriae in F18ab(Wild-type, O139:H1:F18ab, Stx2e). The purified PCR product was cloned into T vector and confirmed by DNA sequencing. Then we design primers which contain a homologies5’ terminal to the target region and a homologies3’terminal to the chloromycin-resistance cassette. First, template plasmid pKD3was used to amplify the chloromycin-resistant cassette for the replacement of the fimA gene. According to the λ-Red recombination system method, the purified PCR products were electroporated into the strain F18ab+E.coli carrying a heat-sensitive plasmid pKD46. Within the help of Red recombination enzyme, fimA gene was replaced by the Cat gene, and the successful recombinant bacteria was selected by LB plates containing chloramphenicol. The second recombination aimed at excision of the chloromycin-resistant cassette was performed by transforming another heat-sensitive vector pCP20expressing FLP recombinase into the recombinants. Finally, the isogenicF18ab△fimA mutants was confirmed by PCR and subsequent DNA sequencing.In the phenotypic test, we used yeast cell agglutination assays and MSHA (mannose sensitive hemagglutination) assays to further confirming the successful construction of fimA mutants. The AfimA mutants showed about68%reduction in adherence and61%reduction in invasion to IPEC-J2in vitro, and this is the first time to find that type Ⅰfimbriae was an important invasin for STEC in this study which provide more evidence for type Ⅰ fimbriae pathogenicity mechanism research. Biofilm formation qualification and quantitative assays demonstrated that type Ⅰ fimbriae is necessary to this process which significantly reduced the biofilm formation ability (p<0.01). Through realtime PCR test, we found that deletion of fimA in F18ab+STEC almost has no effect on fimE or fiml which is located just at its up-stream and down-stream in fim operon. In addition, deletion of fimA down-regulate fliC and fedF to73%and71%, respectively, which has no significant difference in comparison with the parent strains. On the contrary, the level of AIDA-I transcripts in AfimA mutants was3.3-fold more than in WT (0.01<p<0.05). However, loss of fimA did not affect transcription of stx2e, ecpA, sepA and kpsD. The result indicate that the bcteria virulence was directly and indirectly affected by the type Ⅰ fimbriae by interacting other adhesins and toxins. We further observe weather the type Ⅰ fimbriae is essencial in colonization of small intestinal in animal by adoption of adult mouse model. It showed that AfimA mutants defect in the ability of colonization which is consistent with the experiment in vitro. Compared with the wild type strains, deletion of fimA caused53%,69%,51%decrease respectively in duodenum, jejunum, ileum. In summary, the data above all suggest that type Ⅰ fimbriae is a key factor in F18ab+STEC pathogenicity which may offer us a new point to prevent from F18ab+STEC caused infections.