Mutational analysis of receptor binding mediated by the Dr family of Escherichia coli adhesins

Abstract

SummaryThe 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.

DAF- and collagen-binding properties of chimeric Dr fimbriae

“…Previously it has been shown that DraE mutations at positions T88 and I111 affect the type IV collagen binding of the DraE adhesin (Carnoy & Moseley, 1997). Recent research revealed that residues P40, P43, I114 and Y115 are also implicated in collagen binding by DraE fimbrial subunits.…”

“…Plasmid pCC90, corresponding to the dra operon with its promoter region and regulatory genes upstream of the draB gene deleted, and pCC90D54stop, with a mutated draE gene (the GAC triplet of the draE gene encoding Asp-54 was replaced with a stop codon), were provided by S. Moseley, University of Washington, Seattle (Carnoy & Moseley, 1997).…”

“…Additionally, the interactions of DraE adhesin with the 7s domain (tetramer) of type IV collagen localized at the basement membrane renal interstitium, which underlies all epithelia, may facilitate colonization and development of persistent renal infections caused by E. coli Dr + strains. Replacement of a single amino acid at the 113 position of DraE results in loss of the type IV collagenbinding property (Carnoy & Moseley, 1997;Selvarangan et al, 2004). Furthermore, previous investigations have shown that the Dr adhesins AfaE-I, AfaE-III, DraE and DaaE adhere to CEA and related cell adhesion molecules (CEACAM1 and CEACAM6).…”

“…Both epitopes were inserted into the DraE adhesin in place of the N-terminal region of the surface-exposed domain 2 (from V28 to A38) located between C19 and C51, conserved among the Dr family adhesins. Initially, the insertion position of heterologous epitopes was chosen based on mutagenesis studies (Carnoy & Moseley, 1997) and computer analysis of surface-exposed domains (Emini et al, 1985), but this was later confirmed by the atomic resolution structure of the AfaE-III afimbrial subunit, which is closely related to the DraE fimbrial subunit . The construction of potential chimeric fimbrial vaccines should be preceded by research focused on biological and physico-chemical properties of fimbrial subunits after insertion of immune-relevant peptides.…”

“…Members of this family bind to the Dr a blood-group antigen present on the decayaccelerating factor (DAF; CD55), a complement regulatory and signalling molecule (Nowicki et al, 2001). Dr haemagglutinin (a major structural subunit of Dr fimbriae) is the only member of the Dr family adhesins that has the unique ability to bind to two receptors -DAF and type IV collagen (Westerlund et al, 1989;Carnoy & Moseley, 1997). The binding of Dr fimbriae (expressed at the bacterial cell surface) to the short consensus repeat 3 domain (SCR-3) of the DAF receptor leads to the internalization of Dr + E. coli into epithelial cells (Nowicki et al, 1993).…”