Construction and characterization of type I fimbriae fimH deletion mutant from avian pathogenic Escherichia coli

Abstract:

The wild type avian pathogenic Escherichia coli (APEC) isolate A2 (Serotype O2:K89) was selected as the prototype of the (APEC ) TypeⅠFimbriae. Based on the original sequences of TypeⅠFimbriae operon gene clusters in the GenBank,we generated PCR products by using primers with the homologies extension of fimH gene to be deleted and template plasmid pKD3 carrying selectable antibiotic chloramphenicol resistance(cat) gene that is flanked by FRT(FLP recognition target)sites. By using the PCR products, the A2 △fimH::Cat deletion mutant from A2 isolate was constructed by λRed-mediated recombination system in the flanking homologies. After selection, the resistance gene located in the A2△fimH::Cat mutant was eliminated in the second recombination, by using a helper plasmid pCP20, a tempera-ture-sensitive one encoding and expressing the FLP recombinase, which acts on the directly repeated FRT sites flanking the resistance gene. The A2△fimH mutant obtained was further confirmed by fimH PCR amplification and sequencing. The A2△fimH mutant 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 A2△fimH deletion mutant restored the agglutination ability of both binding guinea pig erythrocytes and yeast cells when transfected the compatible recombinant plasmid pBR322-fimH with fimH insert in the fimH complementation assay. Very similar to the wild type A2 isolate, The obtained binding activity from the A2△fimH mutant in the complementation assay was completely inhibited when pretreated with 0.5% mannose solution. Compared with the wild type isolate, the A2△fimH mutant grew slowly during all stages of growth. This work provides the basis for us to study the molecular pathogenesis mechanisms of interaction between the APEC TypeⅠ Fim-briae and susceptible host cells, extra-intestinal infection, prevention and control of the APEC-caused disease.