Mutational analysis of receptor binding mediated by the Dr family of Escherichia coli adhesins.
The fimbrial and afimbrial adhesins of the Dr family mediate the adherence of uropathogenic and diarrhoea-associated Escherichia coli to decay-accelerating factor (DAF) present on erythrocytes and other cell types. The Dr haemagglutinin binds type IV collagen and, unlike other members of the Dr family, mediates an adherence inhibited in the presence of chloramphenicol. We examined the ability of other members of the Dr family-AFAI, AFAIII, and F1845-to bind to type IV collagen, and demonstrated that the collagen-binding phenotype was unique to the Dr haemagglutinin. We employed site-directed mutagenesis to demonstrate the requirement of a negatively charged amino-acid at position 54 of the Dr haemagglutinin subunit for chloramphenicol sensitivity of binding. Mutations at position 32, 40, 54, 90, and 113 differently affected type IV collagen binding and chloramphenicol sensitivity of binding, while retaining DAF-binding capability. These results suggest the existence of a conformational receptor-binding domain in the major structural subunit of Dr family adhesins and demonstrate that chloramphenicol sensitivity of binding and adherence to type IV collagen were independent and separable phenotypes. Finally, we showed that the two conserved cysteine residues of Dr family structural subunits form a disulphide bond and that mutations of these residues abolish haemagglutination and binding to type IV collagen.
Carnoy
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Department of Microbiology, University of Washington, Seattle 98195-7242, USA.
Moseley
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SL
D018830:Adhesins, Escherichia coli / Q000502:physiology*; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D003094:Collagen / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006388:Hemagglutinins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D018830:Adhesins, Escherichia coli; D006388:Hemagglutinins; D003094:Collagen