Chapter 24 : Allelic Variation of the FimH Lectin of Escherichia coli Type 1 Fimbriae and Uropathogenesis

Schematic illustration of the genetic organization of the fim genes. The location of the genes within the fim gene cluster and their roles in regulation or biogenesis are indicated. Switching from ON to OFF phases is controlled by the inversion of a segment of DNA located between the fimE and fimA genes that contains the promoter for the fimA gene. Inversion is affected by FimB and FimE, as described in the text. In the ON orientation, fimAICDFGH is successfully transcribed, but in the OFF orientation, the message is aborted. Integration host factor (IHF) and Lrp bind to elements within the switch and also affect rates of inversion. Modified from reference 116 with permission.

Electron micrograph of type 1 fimbriated E. coli strain illustrating the typical numbers, lengths, and general morphology of the hair-like surface appendages. Type 1 fimbriae, and others of its class, are peritrichously arranged, can number in the hundreds per cell, and are rigid-appearing, straight structures. There are 1,000 or more FimA subunits polymerized into a 7-nm-diameter helical structure. This subunit makes up the majority of the fimbrial structure. The mannose-binding lectin, FimH, is located at the distal tips of the fimbriae (see Fig. 4 and 5 ). Reprinted from reference 52 with permission from Elsevier.

A 3-D reconstruction of a type 1 fimbria. The segment displayed in this model comprises 40 FimA subunits and covers 1.5 helical repeats. It has been surface rendered to include 100% of the nominal mass. The model shows the type 1 fimbria to be a hollow tube with walls that are formed by a helical string of elongated subunits associated in a head-to-tail orientation. Adjacent turns of the helix are connected via three binding sites, making the fimbriae relatively rigid structures. Reprinted from reference 52 with permission from Elsevier.

Schematic model for biogenesis of type 1 fimbriae. Nascent polypeptides of fimbrial subunits are transported across the inner membrane by the general secretion pathway. The periplasmic chaperone, FimC, binds to the polypeptides as they are being transported through the inner membrane. Polypeptides not protected by the chaperone are thought to be degraded by periplasmic proteases. The FimH subunit is held by FimC in a mannose-binding, nonpolymerizing form in the periplasm until delivered to the FimD assembly complex, or usher. The chaperone-subunit complexes arrive at the outer membrane usher, where they bind to previously delivered subunits, traverse the outer membrane as a ~2-nm-diameter linear filament, and then coil into a helical form at the external face of the usher. In this pathway, the translocation of subunits is highly ordered, with translocation of a FimH subunit being followed by that of FimF, FimG, and, finally, hundreds to thousands of copies of FimA. The precise number of FimF, FimG, and FimH subunits in this illustration is not known with absolute certainty. Reprinted from reference 116 , with permission.

Electron micrographs of type 1 fimbriae. (a and b) Isolated type 1 fimbriae negatively stained with uranyl formate. The main fimbrial shaft appears to be rather rigid and contains a central cavity, indicated by the dark thread of stain running parallel to the fimbrial axis. The 7-nm shafts end in a flexible, loosely coiled tip fibrillum (arrowheads) roughly half the diameter of the main shaft. (c) Immunolocalization of FimH at the fimbrial tips. Colloidal gold particles 8 nm in diameter were coated with polyclonal rabbit antibody against FimH. Gold particles are found exclusively at the fimbrial tips. Reprinted from reference 52 with permission from Elsevier.

Schematic diagram of typical N-linked glycans. Saccharide structures 1 and 2 are examples of high-mannose oligosaccharide chains. Saccharide 3 is an example of a typical hybrid-type glycan unit, one arm of which bears a trisaccharide. Saccharide 4 is an example of a complex-type glycan unit, both arms of which are terminally substituted with saccharides other than mannose (i.e., there is no terminal mannose). Prior to 1992, the primary type 1 fimbrial receptor was expected to have the structure of saccharide 1 or 3. Modified from reference 55 with permission.

Schematic diagram of the recombinant strains constructed to test the phenotypes conferred by different alleles of fimH. The host strain used was the ΔfimBEAICDFGH E. coli K-12 strain AAEC191A (11). Plasmid pPKL114 contains the entire fim gene cluster in a pBR322 replicon, with a stop linker inserted into the fimH gene. Because fimH is the last gene in the fim cluster, no polar effects would be expected. Plasmid pGB2-24 and subsequent derivatives contain fimH genes in a pACYC184 replicon, and these plasmids complement the fimH defect of pPKL114.

Adhesion of representative wild-type (A) and recombinant (B) M, MF, and MFP class strains to mannan (panel 1), Fn (panel 2), periodate-treated Fn (panel 3), and a synthetic peptide (panel 4). Strain designations are given in panels 5. The recombinant strains are constructed as indicated in the legend to Fig. 7 and the text. Open columns indicate bacteria incubated without D-mannose; solid columns indicate bacteria incubated with D-mannose. Values are the means and standard errors of the mean (n = 4) for each column. ND, not determined. O.D., optical density. Reprinted from reference 136 with permission.

Deduced amino acid sequences of several FimH variants. The polymorphic sites (sites in which there has been a nonsynonymous mutation in the codon) within the 300-residue FimH sequence are indicated. The positions are numbered vertically above each polymorphic amino acid, compared to the original FimH sequence published by Klemm and Christiansen ( 82 ). Positions that do not vary among the FimH alelles sequenced thus far are not present in the figure. Δ indicates a deleted residue. Substitutions that affect the adhesion phenotype are indicated in boldface type. The sequences are divided into two groups that differ from each other at residues 70 and 78, where Asn-to-Ser and Ser-to-Asn substitutions occur. Reprinted from reference 137 with permission.

Adhesion of fimB-transformed wild-type strains to mannan. Extra copies of fimB result in a more uniform expression of type 1 fimbriae, eliminating variable expression levels as one explanation for differences in adhesion. Open columns indicate bacteria incubated without α-methylmannoside; solid columns indicate bacteria incubated with 50 mM α-methylmannoside. Values are the means and standard errors of the mean (n = 4). Reprinted from reference 137 with permission.

Adhesion of recombinant strains constructed using fimH genes cloned from the wild-type strains shown in Fig. 10 . Columns and values are as in Fig. 10 . Reprinted from reference 137 with permission.

Adhesion of wild-type fecal isolates and isolates from patients with UTIs to mannan. All binding was inhibited by >80% by α-methylmannoside. To simplify the graphic presentation, data are arranged in groups of 0.25 × 106 bacteria bound per well. Since the data are plotted in this way, the actual numbers for circles placed behind the two reference lines fall below the line values, whereas those placed in front of the lines fall above the line values. Reprinted from reference 137 with permission.

Correlation of the abilities of seven recombinant strains to bind to mannan(MN)with their abilities to adhere to the J82 bladder epithelial cell line. Strain numbers are shown. Statistical analysis is given in the text. Reprinted from reference 138 with permission from the publisher.

Scatchard plot analyses of the binding of strains KB54 and KB91 to mannan at equilibrium. Data from a single experiment are presented, but the experiment was repeated several times and the results were essentially the same. Reprinted from reference 138 with permission from the publisher.

Adhesion of strains KB54 and KB91 to various glycoproteins. Abbreviations: Cas, bovine milk casein; αaGP, human α-acid glycoprotein; aTr, human serum apo-transferrin; Mn, yeast mannan; mIgAλ, mouse immunoglobulin Aλ; Lm, human laminin; OvAl, chicken egg albumin; mIgAκ, mouse immunoglobulin Aκ; TG, porcine thyroglobulin; POx, horseradish peroxidase; hIgA human immunoglobulin A; RNB, bovine RNase B. Values are means and standard errors of the mean (n = 3). Reprinted from reference 138 with permission from the publisher.

Schematic diagram of the N-linked glycan units of bovine RNase B. Data from reference 42. Reprinted from reference 55 with permission.

Scatchard plot analyses of binding of strains KB54 and KB91 to bovine RNase B at equilibrium. Data from a single experiment are presented, but the experiment was repeated several times and the results were essentially the same. Reprinted from reference 138 with permission from the publisher.

Correlation of the adhesion of seven recombinant strains ( Fig. 13 ) to Man3-BSA with their adhesion to bovine RNase B (bRB). Strain numbers are shown. Reprinted from reference 138 with permission from the publisher.

Correlation of the levels of adhesion of the same recombinant strains shown in Fig. 18 to Man3-BSA with their adhesion to mannan (MN). Strain numbers are shown. Reprinted from reference 138 with permission from the publisher.

Adhesion of 11 wild-type strains to high-Man moieties of bovine RNase B (HIGH-Man), monomannosylated BSA (Man1) and trimannosylated BSA (Man3). Reprinted from reference 55 with permission from the publisher.

Ribbon diagram illustrating the three-dimensional structure of the FimH lectin subunit complexed with the FimC chaperone and cocrystallized with cyclohexylbutanoyl-N-hydroxyethyl-D-glucamide (C-HEGA). FimH is folded into two domains connected by a short linker arm. The NH2-terminal lectin domain binds to mannosylated receptors, and the COOH-terminal pilin domain anchors FimH to the proximal subunits of the fimbrial superstructure. The lectin domain is an 11-strand elongated β-barrel that exhibits a mannose-size pocket at the tip of the domain distal from the linker arm connecting the two domains. The figure was generously provided by Stefan Knight.

β-Sheet topology diagram of the lectin domain of FimH. The lengths of the sheets and loops do not relect the actual size, but the relative positions of the labeled residues are accurately indicated. The filled circles indicate the positions of point mutations that induce a dual low-Man1-binding/high-Man1-binding phenotype (for details, see reference 132). The crosses indicate residues interacting with the receptor analog, C-HEGA. The open circle indicates the position of an E89K mutation (see reference 141). Reprinted from reference 141 with permission from Blackwell Publishing.

Luminal surface topography of the mouse urothelium. Quick-freeze, deep-etch, and rotary shadowed images of mouse bladder urothelium are shown. (a) An overview showing several crystalline plaques (P) interrupted by hinge areas (H). The upper right corner is an area where the apical surface membrane has been cross-fractured and the cytosol has been water etched, thus exposing the underlying cytoskeleton (Cy). (b) Higher-magnification image of the urothelial plaque and its fast Fourier transform (inset), showing the hexagonal symmetry of the packing and the twisted hexagonal symmetry of individual particles. Reprinted from reference 72 with permission from Elsevier.

Localization of UPIa receptor for FimH on the six inner domains of the mouse 16-nm AUM particle. (a) UPIa specificity of the FimH-FimC complex. The FimH-FimC complex was biotinylated and incubated with proteins from purified mouse urothelial plaques that had been resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Lane 1 shows total proteins of mouse urothelial plaques visualized by Coomassie blue staining, showing the separation of UPII (15 kDa), UPIa (24 kDa), UPIb (27 kDa), and UPIII 947 kDa). There is excellent separation between UPIa and UPIb in this figure. Lane 2 shows the selective binding of biotinylated FimH/FimC to UPIa. Lane 3 shows the selective binding of [35S] methionine-labeled type 1 fimbriated E. coli to UPIa. MW, molecular mass standards. (b) A 2-D difference map of the mouse urothelial plaque images collected in the presence and absence of FimH and FimC. (c) Localization of the FimH-binding site on the six inner domains of the 16-nm AUM particle when projected onto a 3-D model of the particle. Reprinted from reference 102 with permission from Elsevier.

Adhesion of low-Man1-binding and high-Man1-binding strains to AUMs. Reprinted from reference 55 with permission from the publisher.

Scanning electron micrograph of a high-Man1-binding phenotype recombinant E. coli strain binding to the surface of mouse bladder epithelial cells. The mosaic pattern of adhesion of this E. coli strain is striking. Cells bearing hundreds of bound E. coli bacteria are intermingled with cells bearing essentially none. Bar, 20 μm.

Colonization of mouse bladders by isogenic E. coli expressing nonfunctional (M−) FimH, low-Man1-binding (ML) FimH, or high-Man1-binding (MH) Fim H subunits. Bars indicate mean CFU per bladder; error bars indicate standard error of the mean. P values indicating level of significance between different groups are indicated. Reprinted from reference 139 with permission from the publisher.

Essentially equivalent binding of E. coli to buccal epithelial cells (0% α-methylmannoside) and inhibition of this interaction by increasing levels of α-methyl-D-mannopyrannoside. Reprinted from reference 139 with permission from the publisher.

Inhibition of the interaction of E. coli and buccal cells by whole, stimulated human saliva. Reprinted from reference 139 with permission from the publisher.

Phylogenetic tree (unrooted phylogram) of FimH protein variants. The protein tree was built by collapsing branch regions containing silent mutations, using a maximum-likelihood fimH gene tree. The nodes are separated into primary, secondary, and extended zones (see the text). The node labeled CONS corresponds to the consensus structure FimH. All other nodes are labeled with the replacement mutation by which they vary from the immediate ancestral node. Where the same replacement was acquired independently, they are distinguished by a lowercase letter. The grey circles represent FimH variants with intranodal synonymous variations, i.e., encoded by multiple gene alleles. The numbers within circles give the total numbers of strains with the indicated mutation. The small solid circles represent FimH variants found in a single strain in the collection. Reprinted from reference 142 with permission from the publisher.