Comparative structure-function analysis of mannose-specific FimH adhesins from Klebsiella pneumoniae and Escherichia coli - PubMed (original) (raw)

Comparative structure-function analysis of mannose-specific FimH adhesins from Klebsiella pneumoniae and Escherichia coli

Steen G Stahlhut et al. J Bacteriol. 2009 Nov.

Abstract

FimH, the adhesive subunit of type 1 fimbriae expressed by many enterobacteria, mediates mannose-sensitive binding to target host cells. At the same time, fine receptor-structural specificities of FimH from different species can be substantially different, affecting bacterial tissue tropism and, as a result, the role of the particular fimbriae in pathogenesis. In this study, we compared functional properties of the FimH proteins from Escherichia coli and Klebsiella pneumoniae, which are both 279 amino acids in length but differ by some approximately 15% of residues. We show that K. pneumoniae FimH is unable to mediate adhesion in a monomannose-specific manner via terminally exposed Manalpha(1-2) residues in N-linked oligosaccharides, which are the structural basis of the tropism of E. coli FimH for uroepithelial cells. However, K. pneumoniae FimH can bind to the terminally exposed Manalpha(1-3)Manbeta(1-4)GlcNAcbeta1 trisaccharide, though only in a shear-dependent manner, wherein the binding is marginal at low shear force but enhanced sevenfold under increased shear. A single mutation in the K. pneumoniae FimH, S62A, converts the mode of binding from shear dependent to shear independent. This mutation has occurred naturally in the course of endemic circulation of a nosocomial uropathogenic clone and is identical to a pathogenicity-adaptive mutation found in highly virulent uropathogenic strains of E. coli, in which it also eliminates the dependence of E. coli binding on shear. The shear-dependent binding properties of the K. pneumoniae and E. coli FimH proteins are mediated via an allosteric catch bond mechanism. Thus, despite differences in FimH structure and fine receptor specificity, the shear-dependent nature of FimH-mediated adhesion is highly conserved between bacterial species, supporting its remarkable physiological significance.

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Figures

FIG. 1.

FIG. 1.

(A and B) FimH lectin (top) and pilin (bottom) domains shown from each side. The location of the carbohydrate binding pocket is indicated in light yellow (4). Polymorphisms distinguishing E. coli F18 and K. pneumoniae cas663 are shown in green. Within-species K. pneumoniae naturally occurring mutations are shown in blue; residues 94 and 105 (the latter is not visible in Fig. 1) have mutations both within K. pneumoniae and between E. coli and K. pneumoniae. Within-species E. coli naturally occurring mutations which increase binding to mannose are shown in red. Point mutations with pathoadaptive functional changes occurring both in E. coli and in K. pneumoniae (V27T and S62A) are shown in purple. Mutations in residues 105 (blue) and 106 (green) are hidden in the figures. (C) Binding pocket of FimH lectin domain. Polymorphisms distinguishing E. coli F18 and K. pneumoniae cas663 are shown in green. Mutations which functionally change the properties of FimH binding to 1M are shown in yellow. Mutations which functionally change the properties of FimH binding to 3M are shown in orange. (D) LIBS epitope (residues 29 and 152 to 157) shown in gray at bottom of the lectin domain of FimH. Polymorphisms distinguishing E. coli F18 and K. pneumoniae cas663 are shown in green.

FIG. 2.

FIG. 2.

(A) Accumulation of K. pneumoniae expressing K. pneumoniae FimH-S62 and FimH-A62 in PPFC on monomannosylated BSA (1M) and bovine RNase (3M) substrates. Accumulation experiments were carried out at low shear (light bars, 0.1 dyne/cm2) and high shear (dark bars, 1.0 dyne/cm2) in a 5-min period, in at least three independent experiments; error bars represent standard errors. (B) Accumulation of E. coli expressing E. coli FimH-S62 and FimH-A62 or K. pneumoniae FimH-S62 and FimH-A62 in PPFC on 1M substrate. Accumulation experiments were carried out at low shear (light bars, 0.1 dyne/cm2) and high shear (dark bars, 1.0 dyne/cm2) in a 5-min period, in at least three independent experiments; error bars represent standard errors. (C) Accumulation of E. coli expressing E. coli FimH-S62 and FimH-A62 or K. pneumoniae FimH-S62 and FimH-A62 in PPFC on 3M substrate. Accumulation experiments were carried out at low shear (light bars, 0.1 dyne/cm2) and high shear (dark bars, 1.0 dyne/cm2) in a 5-min period, in at least three independent experiments; error bars represent standard errors. (D) Accumulation of E. coli expressing E. coli FimH-S62 and FimH-A62 or K. pneumoniae FimH-S62 and FimH-A62 in PPFC on SBA (same kind of experiments). Accumulation experiments were carried out at low shear (light bars, 0.1 dyne/cm2) and high shear (dark bars, 1.0 dyne/cm2) in a 5-min period, in at least three independent experiments; error bars represent standard errors.

FIG. 3.

FIG. 3.

Isogenic recombinant E. coli strains harboring wild-type E. coli (E.c.) and K. pneumoniae (K.pn.) fimH genes express equal amounts of FimH. (A) Bacteria were immobilized on plastic. The amount of FimH on bacteria was evaluated by ELISA using purified polyclonal (Pab280) anti-FimH-Ld antibodies at a concentration of either 100 or 10 μg/ml. (B) Bacteria, in different concentrations, were bound to immobilized purified polyclonal anti-FimH-Ld antibodies. Bound bacteria were detected by growth assay.

FIG. 4.

FIG. 4.

(A) Polyclonal antibodies with affinity to the lectin domain of the K. pneumoniae FimH protein and specific monoclonal antibody (MAb21) recognizing the LIBS epitope of the lectin domain, bound to purified fimbriae of K. pneumoniae FimH-S62 (dark bars) and FimH-A62 (light bars). Data are shown as optical densities at 650 nm. (B) Polyclonal antibodies with affinity to the lectin domain of the E. coli FimH protein and specific monoclonal antibody (MAb21) recognizing the LIBS epitope of the lectin domain, bound to purified fimbriae of E. coli FimH-S62 (dark bars) and FimH-A62 (light bars). Data are shown as optical densities at 650 nm.

FIG. 5.

FIG. 5.

Schematic representation of high-mannose type N-linked oligosaccharide structures. (A) Structure of the Man5GlcNAc2Asn in 3M (11). (B) Structure of Man9GlcNAc2Asn in SBA (9). Filled shapes indicate residues interacting with FimH. Within the shaded oval is the Manα(1→3)Manβ(1→4)GlcNAc(1→) trisaccharide that has high-affinity interaction with FimH in terminal configuration (as in 3M but not SBA).

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