Construction Of The Type Ⅰ Fimbriae FimH Deletion Mutant From Avian Pathogenic Escherichia Coli And Some Related Biological Function

Avian colibacillosis caused by avian pathogenic Escherichia coli (APEC) is one of the most common infectious diseases in birds, and is often complicated with combination of other pathogen infection such as bacteria or virus, and responsible for worldwide economic losses to the poultry industry.Several virulence factors associated with APEC, including type I fimbriae and P fimbriae, curli, aerobactin iron-sequestering system, K1capsular antigen, resistance to killing by the complement and so on were demonstrated. Among them, type I fimbriae were elucidated well, it may play an important role in pathogenicity. It can facilitate adherence of the bacteria to host cells during the initial infection stage, which is believed to be an important prerequisite for successful invasion and colonization in the pathogenesis of E.coli. Of all clinic isolates from APEC, more than 95% ones express type I fimbriae, type I fimbriae with heteropolymers protein are composed primarily of the structural subunit, FimA, and minor amounts of at least three ancillary subunits, FimF, FimG, and FimH. The minor component FimH is a mannose-specific adhesin located on the tip of type I fimbriae of Escherichia coli and responsible for the D-mannose-sensitive (MS) adhesion of E. coli to the target cells.In order to explore the role of type I fimbriae, especially the FimH in the pathogenicity. Based on the original sequences of type I fimbriae operon gene clusters in the GenBank.The wild type APEC isolates A2(Serotype O2:K89), CE128(Serotype O78:K89),CE129(Serotype O1:K89)and C1(from the intestinal tract of health chichen, serotype unidentified)were selected as the prototype of type I fimbriae and construct the fimH mutant isolate by using Red recombination system. First, the chloramphenicol resistance (cat) gene flanked by homology extensions of fimH gene was amplified by PCR. The PCR products were introduced into wild type strain A2,CE128,CE129,C1 by electroporation, with the help ofλRed-mediated recombination system, fimH gene was replace by homology extensions connected with cat gene, the strains expressing chloramphenicol resistance gene were selected by chloramphenicol agar, the chloramphenicol resistance gene was then eliminated by using a helper plasmid, pCP20, encoding the Flp recombinase. Using this system, fimH gene in chromosome of Escherichia coli was deleted. A2△fimH,CE128△fimH,CE129△fimH,C1△fimH mutants obtained and further confirmed by PCR amplification and sequencing. With the most part of fimH gene have been deleted, the△fimH mutants have little chance to revert back to the parent strains.Based on the 4△fimH mutants, some assays related to the adhesion assay and other some biological characteristics were carried out. In guinea pig erythrocytes and yeast agglutination assay, all of the 4 wild type strains have agglutination ability of both binding guinea pig erythrocytes and yeast cells, and the agglutination capability were completely inhibited when pretreated with 0.5% mannose solution, but all of the△fimH mutants with the deletion of fimH adhesin in the fim gene cluster lost the ability of agglutination reaction with both guinea pig erythrocytes and yeast cells. The△fimH mutants restored the agglutination ability of both binding guinea pig erythrocytes and yeast cells in the fimH complementation assay, very similar to the wild type. Guinea pig erythrocytes and yeast agglutination assay demonstrated the FimH is a mannose-specific adhesion and responsible for the D-mannose-sensitive (MS). Compared with the wild type isolate, the△fimH mutants grew slowly during all stages of growth. In the adhesion with chicken heterophils and A549 cells assay, wild type strains demonstrated a higher association with chicken heterophils and A549 cells than△fimH mutants.These△fimH mutants were constructed by usingλRed-mediated recombination system, and the most part of fimH gen has been deleted. Compared with other mutant strains constructed by conventional methods, these△fimH mutants without the fimH gen and carry no any selection marker, which can be easier manipulated to construct the mutants with the second gene or multi-gene deletion in the next turn of construction. This work provides the basis to study the the molecular pathogenesis mechanisms of interaction between the APEC type I fimbriae and susceptible host cells, and the mechanisms of interaction between FimH and cells in host defences.