Allosteric catch bond properties of the FimH adhesin from Salmonella enterica serovar Typhimurium

Despite sharing the name and the ability to mediate mannose-sensitive adhesion, the type 1 fimbrial FimH adhesins of Salmonella Typhimurium and Escherichia coli share only 15% sequence identity. In the present study, we demonstrate that even with this limited identity in primary sequence, these two proteins share remarkable similarity of complex receptor binding and structural properties. In silico simulations suggest that, like E. coli FimH, Salmonella FimH has a two-domain tertiary structure topology, with a mannose-binding pocket located on the apex of a lectin domain. Structural analysis of mutations that enhance S. Typhimurium FimH binding to eukaryotic cells and mannose-BSA demonstrated that they are not located proximal to the predicted mannose-binding pocket but rather occur in the vicinity of the predicted interface between the lectin and pilin domains of the adhesin. This implies that the functional effect of such mutations is indirect and probably allosteric in nature. By analogy with E. coli FimH, we suggest that Salmonella FimH functions as an allosteric catch bond adhesin, where shear-induced separation of the lectin and pilin domains results in a shift from a low affinity to a high affinity binding conformation of the lectin domain. Indeed, we observed shear-enhanced binding of whole bacteria expressing S. Typhimurium type 1 fimbriae. In addition, we observed that anti-FimH antibodies activate rather than inhibit S. Typhimurium FimH mannose binding, consistent with the allosteric catch bond properties of this adhesin.     

FimH-mediated Escherichia coli K1 invasion of human brain microvascular endothelial cells

Adhesion to brain microvascular endothelial cells, which constitute the blood-brain barrier is considered important in Escherichia coli K1 bacterial penetration into the central nervous system. Type 1 fimbriae are known to mediate bacterial interactions with human brain microvascular endothelial cells (HBMEC). Here, we demonstrate that type 1 fimbriae, specifically FimH adhesin is not only an adhesive organelle that provides bacteria with a foothold on brain endothelial cells but also triggers signalling events that promote E. coli K1 invasion in HBMEC. This is shown by our demonstrations that exogenous FimH increases cytosolic-free-calcium levels as well as activates RhoA. Using purified recombinant mannose-recognition domain of FimH, we identified a glycosylphosphatidylinositol-anchored receptor, CD48, as a putative HBMEC receptor for FimH. Furthermore, E. coli K1 binding to and invasion of HBMEC were blocked by CD48 antibody. Taken together, these findings indicate that FimH induces host cell signalling cascades that are involved in E. coli K1 invasion of HBMEC and CD48 is a putative HBMEC receptor for FimH.     

FimH-mediated autoaggregation of Escherichia coli

Autoaggregation is a phenomenon thought to contribute to colonization of mammalian hosts by pathogenic bacteria. Type 1 fimbriae are surface organelles of Escherichia coli that mediate d-mannose-sensitive binding to various host surfaces. This binding is conferred by the minor fimbrial component FimH. In this study, we have used random mutagenesis to identify variants of the FimH adhesin that confer the ability of E. coli to autoaggregate and settle from liquid cultures. Three separate autoaggregating clones were identified, all of which contained multiple amino acid changes located within the N-terminal receptor-binding domain of FimH. Autoaggregation could not be inhibited by mannose, but was inhibited by growth at temperatures at or below 30 degrees C. Using green fluorescent protein (GFP) as a reporter, we show that the autoaggregating clones do not mix with wild-type fimbriated cells. Electron microscopy shows that autoaggregating cells produce fimbriae with a twisted and entangled appearance. We present evidence that autoaggregating versions of FimH also occur in nature. Our results stress the highly adaptive nature of the ubiquitous FimH adhesin.     

Structural basis of tropism of Escherichia coli to the bladder during urinary tract infection

The first step in the colonization of the human urinary tract by pathogenic Escherichia coli is the mannose-sensitive binding of FimH, the adhesin present at the tip of type 1 pili, to the bladder epithelium. We elucidated crystallographically the interactions of FimH with D-mannose. The unique site binding pocket occupied by D-mannose was probed using site-directed mutagenesis. All but one of the mutants examined had greatly diminished mannose-binding activity and had also lost the ability to bind human bladder cells. The binding activity of the mono-saccharide D-mannose was delineated from this of mannotriose (Man(alpha 1-3)[Man(alpha 1-6)]Man) by generating mutants that abolished D-mannose binding but retained mannotriose binding activity. Our structure/function analysis demonstrated that the binding of the monosaccharide alpha-D-mannose is the primary bladder cell receptor for uropathogenic E. coli and that this event requires a highly conserved FimH binding pocket. The residues in the FimH mannose-binding pocket were sequenced and found to be invariant in over 200 uropathogenic strains of E. coli. Only enterohaemorrhagic E. coli (EHEC) possess a sequence variation within the mannose-binding pocket of FimH, suggesting a naturally occurring mechanism of attenuation in EHEC bacteria that would prevent them from being targeted to the urinary tract.     

Specific residues in the N-terminal domain of FimH stimulate type 1 fimbriae assembly in Escherichia coli following the initial binding of the adhesin to FimD usher

Type 1 fimbriae are assembled by the chaperone–usher pathway where periplasmic protein complexes formed between fimbrial subunits and the FimC chaperone are recruited by the outer membrane protein FimD (the usher) for their ordered polymerization and export. FimH adhesin initiates and stimulates type 1 fimbriae polymerization by interacting with FimD. Previously we showed that the N-terminal lectin domain of FimH (N-FimH) is necessary for binding of the adhesin to FimD. In this work, we have selected mutants in N-FimH that reduce the levels of adhesin and type 1 fimbriae displayed in Escherichia coli without altering the levels of FimH in the periplasm. The selected mutations are mostly concentrated in residues G15, N46 and D47. In contrast to other mutations isolated that simply affect binding of FimH to FimD (e.g. C3Y), these variants associate to FimD and alter its susceptibility to trypsin digestion similarly to wild-type FimH. Importantly, their mutant phenotype is rescued when FimD is activated in vivo by the coexpression of wild-type FimH. Altogether, these data indicate that residues G15, N46 and D47 play an important role following initial binding of FimH to FimD for efficient type 1 fimbriae polymerization by this outer membrane usher.     

Interdomain interaction in the FimH adhesin of Escherichia coli regulates the affinity to mannose

FimH is a mannose-specific adhesin located on the tip of type 1 fimbriae of Escherichia coli that is capable of mediating shear-enhanced bacterial adhesion. FimH consists of a fimbria-associated pilin domain and a mannose-binding lectin domain, with the binding pocket positioned opposite the interdomain interface. By using the yeast two-hybrid system, purified lectin and pilin domains, and docking simulations, we show here that the FimH domains interact with one another. The affinity for mannose is greatly enhanced (up to 300-fold) in FimH variants in which the interdomain interaction is disrupted by structural mutations in either the pilin or lectin domains. Also, affinity to mannose is dramatically enhanced in isolated lectin domains or in FimH complexed with the chaperone molecule that is wedged between the domains. Furthermore, FimH with native structure mediates weak binding at low shear stress but shifts to strong binding at high shear, whereas FimH with disrupted interdomain contacts (or the isolated lectin domain) mediates strong binding to mannose-coated surfaces even under low shear. We propose that interactions between lectin and pilin domains decrease the affinity of the mannose-binding pocket via an allosteric mechanism. We further suggest that mechanical force at high shear stress separates the two domains, allowing the lectin domain to switch from a low affinity to a high affinity state. This shift provides a mechanism for FimH-mediated shear-enhanced adhesion by enabling the adhesin to form catch bond-like interactions that are longer lived at high tensile force.     

Receptor binding studies disclose a novel class of high-affinity inhibitors of the Escherichia coli FimH adhesin

Mannose-binding type 1 pili are important virulence factors for the establishment of Escherichia coli urinary tract infections (UTIs). These infections are initiated by adhesion of uropathogenic E. coli to uroplakin receptors in the uroepithelium via the FimH adhesin located at the tips of type 1 pili. Blocking of bacterial adhesion is able to prevent infection. Here, we provide for the first time binding data of the molecular events underlying type 1 fimbrial adherence, by crystallographic analyses of the FimH receptor binding domains from a uropathogenic and a K-12 strain, and affinity measurements with mannose, common mono- and disaccharides, and a series of alkyl and aryl mannosides. Our results illustrate that the lectin domain of the FimH adhesin is a stable and functional entity and that an exogenous butyl alpha-D-mannoside, bound in the crystal structures, exhibits a significantly better affinity for FimH (Kd = 0.15 microM) than mannose (Kd = 2.3 microM). Exploration of the binding affinities of alpha- d-mannosides with longer alkyl tails revealed affinities up to 5 nM. Aryl mannosides and fructose can also bind with high affinities to the FimH lectin domain, with a 100-fold improvement and 15-fold reduction in affinity, respectively, compared with mannose. Taken together, these relative FimH affinities correlate exceptionally well with the relative concentrations of the same glycans needed for the inhibition of adherence of type 1 piliated E. coli. We foresee that our findings will spark new ideas and initiatives for the development of UTI vaccines and anti-adhesive drugs to prevent anticipated and recurrent UTIs.     

Integrin-like allosteric properties of the catch bond-forming FimH adhesin of Escherichia coli

FimH is the adhesive subunit of type 1 fimbriae of the Escherichia coli that is composed of a mannose-binding lectin domain and a fimbria-incorporating pilin domain. FimH is able to interact with mannosylated surface via a shear-enhanced catch bond mechanism. We show that the FimH lectin domain possesses a ligand-induced binding site (LIBS), a type of allosterically regulated epitopes characterized in integrins. Analogous to integrins, in FimH the LIBS epitope becomes exposed in the presence of the ligand (or "activating" mutations) and is located far from the ligand-binding site, close to the interdomain interface. Also, the antibody binding to the LIBS shifts adhesin from the low to high affinity state. Binding of streptavidin to the biotinylated residue within the LIBS also locks FimH in the high affinity state, suggesting that the allosteric perturbations in FimH are sustained by the interdomain wedging. In the presence of antibodies, the strength of bacterial adhesion to mannose is increased similar to the increase observed under shear force, suggesting the same allosteric mechanism, a shift in the interdomain configuration. Thus, an integrin-like allosteric link between the binding pocket and the interdomain conformation can serve as the basis for the catch bond property of FimH and, possibly, other adhesive proteins.     

Recognition of the N-terminal lectin domain of FimH adhesin by the usher FimD is required for type 1 pilus biogenesis

In this work we discover that a specific recognition of the N-terminal lectin domain of FimH adhesin by the usher FimD is essential for the biogenesis of type 1 pili in Escherichia coli. These filamentous organelles are assembled by the chaperone-usher pathway, in which binary complexes between fimbrial subunits and the periplasmic chaperone FimC are recognized by the outer membrane protein FimD (the usher). FimH adhesin initiates fimbriae polymerization and is the first subunit incorporated in the filament. Accordingly, FimD shows higher affinity for the FimC/FimH complex although the structural basis of this specificity is unknown. We have analysed the assembly into fimbria, and the interaction with FimD in vivo, of FimH variants in which the N-terminal lectin domain of FimH was deleted or substituted by different immunoglobulin (Ig) domains, or in which these Ig domains were fused to the N-terminus of full-length FimH. From these data, along with the analysis of a FimH mutant with a single amino acid change (G16D) in the N-terminal lectin domain, we conclude that the lectin domain of FimH is recognized by FimD usher as an essential step for type 1 pilus biogenesis.     

Quantitative differences in adhesiveness of type 1 fimbriated Escherichia coli due to structural differences in fimH genes.

Type 1 fimbriae are heteropolymeric surface organelles responsible for the D-mannose-sensitive (MS) adhesion of Escherichia coli. We recently reported that variation of receptor specificity of type 1 fimbriae can result solely from minor alterations in the structure of the gene for the FimH adhesin subunit. To further study the relationship between allelic variation of the fimH gene and adhesive properties of type 1 fimbriae, the fimH genes from five additional strains were cloned and used to complement the FimH deletion in E. coli KB18. When the parental and recombinant strains were tested for adhesion to immobilized mannan, a wide quantitative range in the ability of bacteria to adhere was noted. The differences in adhesion do not appear to be due to differences in the levels of fimbriation or relative levels of incorporation of FimH, because these parameters were similar in low-adhesion and high-adhesion strains. The nucleotide sequence for each of the fimH genes was determined. Analysis of deduced FimH sequences allowed identification of two sequence homology groups, based on the presence of Asn-70 and Ser-78 or Ser-70 and Asn-78 residues. The consensus sequences for each group conferred very low adhesion activity, and this low-adhesion phenotype predominated among a group of 43 fecal isolates. Strains isolated from a different host niche, the urinary tract, expressed type 1 fimbriae that conferred an increased level of adhesion. The results presented here strongly suggest that the quantitative variations in MS adhesion are due primarily to structural differences in the FimH adhesin. The observed differences in MS adhesion among populations of E. coli isolated from different host niches call attention to the possibility that phenotypic variants of FimH may play a functional role in populations dynamics.     

Bacterial adhesion to target cells enhanced by shear force

Surface adhesion of bacteria generally occurs in the presence of shear stress, and the lifetime of receptor bonds is expected to be shortened in the presence of external force. However, by using Escherichia coli expressing the lectin-like adhesin FimH and guinea pig erythrocytes in flow chamber experiments, we show that bacterial attachment to target cells switches from loose to firm upon a 10-fold increase in shear stress applied. Steered molecular dynamics simulations of tertiary structure of the FimH receptor binding domain and subsequent site-directed mutagenesis studies indicate that shear-enhancement of the FimH-receptor interactions involves extension of the interdomain linker chain under mechanical force. The ability of FimH to function as a force sensor provides a molecular mechanism for discrimination between surface-exposed and soluble receptor molecules.     

Glycosylation changes as important factors for the susceptibility to urinary tract infection

FimH is the type?1 fimbrial tip adhesin and invasin of Escherichia coli. Its ligands are the glycans on specific proteins enriched in membrane microdomains. FimH binding shows high-affinity recognition of paucimannosidic glycans, which are shortened high-mannose glycans such as oligomannose-3 and -5. FimH can recognize equally the (single) high-mannose glycan on uroplakin Ia, on the urinary defence protein uromodulin or Tamm-Horsfall glycoprotein and on the intestinal GP2 glycoprotein present in Peyer's patches. E. coli bacteria may attach to epithelial cells via hundreds of fimbriae in a multivalent fashion. This binding is considered to provoke conformational changes in the glycoprotein receptor that translate into signalling in the cytoplasm of the infected epithelial cell. Bladder cell invasion by the uropathogenic bacterium is the prelude to recurrent and persistent urinary tract infections in humans. Patients suffering from diabetes mellitus are more prone to contract urinary tract infections. In a study of women, despite longer treatments with a more potent antibiotic, these patients also have more often recurrences of urinary tract infections compared with women without diabetes. Type?1 fimbriae are the most important virulence factors used not only for adhesion of E. coli in the urinary tract, but also for the colonization by E. coli in patients with Crohn's disease or ulcerative colitis. It appears that the increased prevalence of urinary tract infections in diabetic women is not the result of a difference in the bacteria, but is due to changes in the uroepithelial cells leading to an increased adherence of E. coli expressing type?1 fimbriae. Hypothetically, these changes are in the glycosylation of the infected cells. The present article focuses on possible underlying mechanisms for glycosylation changes in the uroepithelial cell receptors for FimH. Like diabetes, bacterial adhesion induces apoptosis that may bring the endoplasmic reticulum membrane with immature mannosylated glycoproteins to the surface. Indicatively, clathrin-mediated vesicle trafficking of glucose transporters is disturbed in diabetics, which would interfere further with the biosynthesis and localization of complex N-linked glycans.     

Structural and Population Characterization of MrkD,the Adhesive Subunit of Type 3 Fimbriae

Type 3 fimbriae are adhesive organelles found in enterobacterial pathogens. The fimbriae promote biofilm formation on biotic and abiotic surfaces; however, the exact identity of the receptor for the type 3 fimbriae adhesin, MrkD, remains elusive. We analyzed naturally occurring structural and functional variabilities of the MrkD adhesin from Klebsiella pneumoniae and Escherichia coli isolates of diverse origins. We identified a total of 33 allelic variants of mrkD among 90 K. pneumoniae isolates and 10 allelic variants among 608 E. coli isolates, encoding 11 and 9 protein variants, respectively. Based on the level of accumulated silent variability between the alleles, mrkD was acquired a relatively long time ago in K. pneumoniae but recently in E. coli. However, unlike K. pneumoniae, mrkD in E. coli is actively evolving under a strong positive selection by accumulation of mutations, often targeting the same positions in the protein. Several naturally occurring MrkD protein variants from E. coli were found to be significantly less adherent when tested in a mannan-binding assay and showed reduced biofilm-forming capacity. Functional examination of the MrkD adhesin in flow chamber experiments determined that it interacts with Saccharomyces cerevisiae cells in a shear-dependent manner, i.e., the binding is catch-bond-like and enhanced under increasing shear conditions. Homology modeling strongly suggested that MrkD has a two-domain structure, comprising a pilin domain anchoring the adhesin to the fimbrial shaft and a lectin domain containing the binding pocket; this is similar to structures found in other catch-bond-forming fimbrial adhesins in enterobacteria.     

Molecular basis for the enterocyte tropism exhibited by Salmonella typhimurium type 1 fimbriae

Salmonella typhimurium exhibits a distinct tropism for mouse enterocytes that is linked to their expression of type 1 fimbriae. The distinct binding traits of Salmonella type 1 fimbriae is also reflected in their binding to selected mannosylated proteins and in their ability to promote secondary bacterial aggregation on enterocyte surfaces. The determinant of binding in Salmonella type 1 fimbriae is a 35-kDa structurally distinct fimbrial subunit, FimHS, because inactivation of fimHS abolished binding activity in the resulting mutant without any apparent effect on fimbrial expression. Surprisingly, when expressed in the absence of other fimbrial components and as a translational fusion protein with MalE, FimHS failed to demonstrate any specific binding tropism and bound equally to all cells and mannosylated proteins tested. To determine if the binding specificity of Salmonella type 1 fimbriae was determined by the fimbrial shaft that is intimately associated with FimHS, we replaced the amino-terminal half of FimHS with the corresponding sequence from Escherichia coli FimH (FimHE) that contains the receptor binding domain of FimHE. The resulting hybrid fimbriae bearing FimHES on a Salmonella fimbrial shaft exhibited binding traits that resembled that of Salmonella rather than E. coli fimbriae. Apparently, the quaternary constraints imposed by the fimbrial shaft on the adhesin determine the distinct binding traits of S. typhimurium type 1 fimbriae.     

Heterobinary Adhesins Based on the Escherichia coli FimH Fimbrial Protein

The FimH adhesin of Escherichia coli type 1 fimbriae confers the ability to bind to d-mannosides by virtue of a receptor-binding domain located in its N-terminal region. This protein was engineered into a heterobifunctional adhesin by introducing a secondary binding site in the C-terminal region. The insertion of histidine clusters into this site resulted in coordination of various metal ions by recombinant cells expressing chimeric FimH proteins. In addition, libraries consisting of random peptide sequences inserted into the FimH display system and screened by a “panning” technique were used to identify specific sequences conferring the ability to adhere to Ni²⁺ and Cu²⁺. Recombinant cells expressing heterobifunctional FimH adhesins could adhere simultaneously to both metals and saccharides. Finally, combining the metal-binding modifications with alterations in the natural receptor-binding region demonstrated the ability to independently modulate the binding of FimH to two ligands simultaneously.

Expression systems for the display of heterologous protein segments facilitate the presentation of both defined and random peptide sequences at exposed regions of surface proteins of filamentous bacteriophage virions, bacteria, and yeasts (2, 4). We are particularly interested in the display of heterologous peptides in type 1 fimbriae. Such surface organelles are found on the majority of Escherichia coli strains and confer the ability to bind to specific surfaces. A single type 1 fimbria is a heteropolymer that is 7 nm wide and approximately 1 μm long. It consists of approximately 1,000 subunits of the major element, FimA, that are polymerized in a right-handed helical structure that also contains low levels of the minor components FimF, FimG, and FimH (9). The FimH protein has been shown to be the actual receptor-binding molecule which recognizes α-d-mannose-containing structures (10). Because of this, type 1 fimbriated bacteria readily agglutinate yeast cells (a rich source of mannan).The FimH adhesin is located at the tip of each fimbria and also is interspersed along the fimbrial shaft (6, 10). The results of linker insertion mutagenesis (16) and analyses of naturally occurring variants (17–19) and hybrid proteins constructed by fusing FimH to FocH (8) and MalE (21) suggest that the FimH protein consists of two major domains, each constituting roughly one-half of the molecule; the N-terminal domain seems to contain the receptor-binding site, while the C-terminal domain seems to contain the recognition sequences for export and bioassembly.In previous studies we investigated the ability of FimH to display heterologous peptides in connection with the development of vaccine systems. Various heterologous sequences, representing immune-relevant sectors of foreign proteins, were authentically displayed on the bacterial surface in FimH (12). These observations led us to believe that the FimH protein is an ideal candidate for display of random peptide sequences and for construction of designer adhesins (i.e., proteins manipulated to bind to targets of choice). Here we describe simultaneous heterobifunctional binding of recombinant cells expressing chimeric FimH proteins to metal and d-mannose targets.     

The affinity of the FimH fimbrial adhesin is receptor-driven and quasi-independent of Escherichia coli pathotypes

Type-1 fimbriae are important virulence factors for the establishment of Escherichia coli urinary tract infections. Bacterial adhesion to the high-mannosylated uroplakin Ia glycoprotein receptors of bladder epithelium is mediated by the FimH adhesin. Previous studies have attributed differences in mannose-sensitive adhesion phenotypes between faecal and uropathogenic E. coli to sequence variation in the FimH receptor-binding domain. We find that FimH variants from uropathogenic, faecal and enterohaemorrhagic isolates express the same specificities and affinities for high-mannose structures. The only exceptions are FimHs from O157 strains that carry a mutation (Asn135Lys) in the mannose-binding pocket that abolishes all binding. A high-mannose microarray shows that all substructures are bound by FimH and that the largest oligomannose is not necessarily the best binder. Affinity measurements demonstrate a strong preference towards oligomannosides exposing Manalpha1-3Man at their non-reducing end. Binding is further enhanced by the beta1-4-linkage to GlcNAc, where binding is 100-fold better than that of alpha-d-mannose. Manalpha1-3Manbeta1-4GlcNAc, a major oligosaccharide present in the urine of alpha-mannosidosis patients, thus constitutes a well-defined FimH epitope. Differences in affinities for high-mannose structures are at least 10-fold larger than differences in numbers of adherent bacteria between faecal and uropathogenic strains. Our results imply that the carbohydrate expression profile of targeted host tissues and of natural inhibitors in urine, such as Tamm-Horsfall protein, are stronger determinants of adhesion than FimH variation.     

The distinct binding specificities exhibited by enterobacterial type 1 fimbriae are determined by their fimbrial shafts

Type 1 fimbriae of enterobacteria are heteropolymeric organelles of adhesion composed of FimH, a mannose-binding lectin, and a shaft composed primarily of FimA. We compared the binding activities of recombinant clones expressing type 1 fimbriae from Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium for gut and uroepithelial cells and for various soluble mannosylated proteins. Each fimbria was characterized by its capacity to bind particular epithelial cells and to aggregate mannoproteins. However, when each respective FimH subunit was cloned and expressed in the absence of its shaft as a fusion protein with MalE, each FimH bound a wide range of mannose-containing compounds. In addition, we found that expression of FimH on a heterologous fimbrial shaft, e.g. K. pneumoniae FimH on the E. coli fimbrial shaft or vice versa, altered the binding specificity of FimH such that it closely resembled that of the native heterologous type 1 fimbriae. Furthermore, attachment to and invasion of bladder epithelial cells, which were mediated much better by native E. coli type 1 fimbriae compared with native K. pneumoniae type 1 fimbriae, were found to be dependent on the background of the fimbrial shaft (E. coli versus K. pneumoniae) rather than the background of the FimH expressed. Thus, the distinct binding specificities of different enterobacterial type 1 fimbriae cannot be ascribed solely to the primary structure of their respective FimH subunits, but are also modulated by the fimbrial shaft on which each FimH subunit is presented, possibly through conformational constraints imposed on FimH by the fimbrial shaft. The capacity of type 1 fimbrial shafts to modulate the tissue tropism of different enterobacterial species represents a novel function for these highly organized structures.     

Cutting edge: FimH adhesin of type 1 fimbriae is a novel TLR4 ligand

Several TLR ligands of bacterial origin induce innate immune responses. Although FimH, the adhesin portion of type 1 fimbria, plays an important role in the pathogenicity of some gram-negative bacteria, its ability to stimulate the innate immune system via TLR signaling remains unclear. In this study we report that FimH induces potent innate responses in a MyD88-dependent fashion. The FimH-induced innate activity was restricted to cells expressing TLR4. In addition, FimH was able to bind directly to TLR4. More importantly, cells unresponsive to LPS were responsive to FimH and the presence or absence of MD-2 and CD14 had no effect on FimH activity. Our data suggest that TLR4 is a functional receptor for the adhesin portion of bacterial type 1 fimbria.     

Characterization of Escherichia coli type 1 pilus mutants with altered binding specificities

PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in fimH, the chromosomal gene that encodes the adhesin protein (FimH) of Escherichia coli type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket.     

The solution structure of PapGII from uropathogenic Escherichia coli and its recognition of glycolipid receptors

Uropathogenic Escherichia coli (UPEC) is the primary cause of symptomatic urinary tract infection. The P-pili, a bacterial surface organelle, mediates the bacterial host–cell adhesion. The PapG adhesin has generated much interest in recent years, not only because of its clinical value, i.e. in the prevention of microbial adherence, but also because of its ability to promote virulence. Using multidimensional nuclear magnetic resonance (NMR) and deuteration we have determined the solution structure of the adhesin domain from PapGII (PapGII-198). The novel structure of PapGII-198 is composed of a large elongated jellyroll motif. Despite an automated search of the structural database failing to reveal any similar proteins, PapGII adhesin shares some structural similarities with FimH. Furthermore, interpretation of NMR-titration data has enabled us to identify the putative binding site for the globoseries of oligosaccharides. This work provides insight into UPEC pathogenesis as well as aiding the development of preventative therapies and the guidance of future mutagenesis programmes.