Functional Flexibility of the FimH Adhesin: Insights from a Random Mutant Library

Abstract

Type 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces. This binding is conferred by the minor fimbrial component FimH. Naturally occurring variants of the FimH protein have been selected in nature for their ability to recognize specific receptor targets. In particular, variants that bind strongly to terminally exposed monomannose residues have been associated with a pathogenicity-adaptive phenotype that enhances E. coli colonization of extraintestinal locations such as the urinary bladder. In this study we have used random mutagenesis to specifically identify nonselective mutations in the FimH adhesin which modify its binding phenotype. Isogenic E. coli clones expressing FimH variants were tested for their ability to bind yeast cells and model glycoproteins that contain oligosaccharide moieties rich in either terminal monomannose, oligomannose, or nonmannose residues. Both the monomannose-and the oligomannose-binding capacity of type 1 fimbriae could be altered by minor amino acid changes in the FimH protein. The monomannose-binding phenotype was particularly sensitive to changes, with extensive differences in binding being observed in comparison to wild-type FimH levels. Different structural alterations were able to cause similar functional changes in FimH, suggesting a high degree of flexibility to target recognition by this adhesin. Alteration of residue P49 of the mature FimH protein, which occurs within the recently elucidated carbohydrate-binding pocket of FimH, completely abolished its function. Amino acid changes that increased the binding capacity of FimH were located outside receptor-interacting residues, indicating that functional changes relevant to pathogenicity are likely to be due to conformational changes of the adhesin.

Characterization of Escherichia coli Type 1 Pilus Mutants with Altered Binding Specificities

“…noted by Schembri et al (26). Unfortunately, the allele described by Schembri et al had an additional lesion.…”

Molecular Characterization of the Escherichia coli Asymptomatic Bacteriuria Strain 83972: the Taming of a Pathogen

“…4). By analogy with the FimH adhesin (22,25), variations that alter the con- formational stability of the protein loops that carry the receptor-interacting residues may also account for its lack of function. E. coli 83972 was carried by a young girl for 3 years without any symptoms.…”

Fimbrial surface display systems in bacteria: from vaccines to random libraries

“…We have shown that peptide library display can be combined with alterations in the natural receptor-binding region to independently modulate the binding of FimH to two ligands simultaneously (Schembri & Klemm, 1998 ;Schembri et al, 2000). An obvious advantage inherent in this binary system could be that the immobilization of bacteria using one adhesive domain may facilitate the use of the cells in detection systems for metals, or perhaps directly as biosorption agents for the removal of toxic or precious metals from the environment.…”

“…The initial translocation of organelle components across the cytoplasmic membrane is dependant on the normal type II export system. However, further export from the periplasm to the cell exterior is mediated by a specific two-component system consisting of a periplasmic chaperone and an usher, an outermembrane-located pore, which serves as assembly platform (Hultgren et al, 1996 ;Klemm & Schembri, 2000). A highly choreographed series of specific molecular interactions ultimately leads to the formation of the fimbrial organelle, a polymeric structure in which hundreds of subunits are held together by non-covalent subunit-subunit interactions.…”