Cloning and expression of an afimbrial adhesin (AFA-I) responsible for P blood group-independent, mannose-resistant hemagglutination from a pyelonephritic Escherichia coli strain

Abstract

The uropathogenic Escherichia coli KS52 strain expresses a mannose-resistant hemagglutinin involving an erythrocyte recognition site distinct from the at-digalactoside glycosphingolipid receptor identified for the uropathogenic E. coli strains specifying a P adhesin. The KS52 strain showed three major properties. (i) It agglutinated human erythrocytes of all tested blood groups. (ii) Hemagglutinin activity was found both in the supernatant fluid L-broth cultures and in cells grown on L-agar plates. (iii) No fimbriae in organisms grown on L-agar plates were detected by electron microscopy. Whole-cell DNA from the KS52 strain was size fractionated and cloned into the pHC79 cosmid vector. Three recombinant cosmids expressing a mannoseresistant hemagglutination (MRHA) phenotype were characterized and used to subclone the smallest DNA fragment able to confer the same MRHA properties as the parent strain. A 6.7-kilobase chromosomal DNA fragment cloned in pBR322 (pIL14) was shown to be necessary for host-cell MRHA expression and uroepithelial cell adherence. The insert encoded the production of a 16,000-dalton hemagglutinin. This polypeptide could be detected in culture supernatant fluids, in E. coli minicells harboring the pIL14 plasmid, and, by immunoblotting, in the KS52 strain and E. coli whole cells harboring the pIL14 plasmid. No homology was detected by Southern hybridization between the cloned insert and the DNA of the operon responsible for MRHA in the P-specifying, fimbriate strains (pap operon).

Preface

“…Afimbrial adhesins (AFA) are the first adhesin structures that are not associated with fimbriae. They were observed for the first time on a uropathogenic E. coli strain (Labigne-Roussel et al, 1984). At present at least eight different afa gene clusters are known.…”

“…The afaI gene cluster identified first from E. coli strains associated with urinary and intestinal infections encodes AfaABCDE proteins and it is involved in adhesion to epithelial cells and haemagglutination (Labigne-Roussel and Falkow, 1988). Three Afa proteins, AfaB, AfaC and AfaE, are required for the mannose-resistant haemagglutination (MRHA) and for adherence to uroepithel cells (Labigne-Roussel et al, 1984;Labigne-Roussel and Falkow, 1988). Among these three proteins AfaA and AfaF are transcriptional regulators, AfaB functions as a chaperone, AfaC is an outer membrane usher, AfaD is an invasin, and AfaE is the adhesin protein (Walz et al, 1985).…”

Pathogenesis of Human Diffusely Adhering Escherichia coli Expressing Afa/Dr Adhesins (Afa/Dr DAEC): Current Insights and Future Challenges

“…To detect daaC, daaE, afaB, and afaC sequences, probes and PCR primers have been developed (32,45,54,55). DNA probes included the following: drb (56), a 260-bp PstI fragment of the pIL14 plasmid (afa-1 operon) coding for the AfaE-I adhesin of uropathogenic Afa/Dr DAEC KS52 strain (25); daaC, a 300-bp PstI fragment of the plasmid pSSS1 daa operon (57); a 390-bp I fragment of the pSLM852 daa operon (58); a DNA fragment homologous to daaE (59); and a probe designed from the M030 sequence found to be specifically present in wild-type, diarrheaassociated Afa/Dr DAEC strain C1845 (60,61).…”

“…Agnès Labigne and Chantal Le Bouguénec have extensively described the pathogenicity mechanisms of E. coli strains bearing the afimbrial adhesins (Afa) encoded by the afa operons ( Table 1). The first Afa adhesin was isolated from the wild-type prototype UPEC strain KS52 by Labigne et al (25,37). The 6.7-kb chromosomal DNA fragment essential for a mannoseresistant hemagglutination (MRHA) phenotype in human erythrocytes and for adhesion onto uroepithelial cells contains five genes: afaA, afaE, afaD, afaB, and afaC (24).…”

“…In commensal E. coli from humans (in Europe, the United States, Australia, and Japan), B2 group E. coli strains are predominant (20), and it is noteworthy that these E. coli strains displayed a high capacity to colonize epithelia (21)(22)(23). The name "Afa/Dr DAEC" was proposed in 2005 to define a family of human UTI-or diarrhea-associated clinical E. coli isolates harboring ad-hesins encoded by the afa (24)(25)(26)(27)(28), dra (29,30), and daa (31,32) operons, having a similar genetic organization and displaying a similar receptor specificity for human decay-accelerating factor (hDAF) and members of the family of human carcinoembryonic antigen cell adhesion molecules (hCEACAMs) (11). It is important to note that the name "Dr family" has been used by Bogdan Nowicki and coworkers as dictated by the receptor specificity of Afa, Dr, and F1845 adhesins for the Dr blood group antigen (33,34).…”