Uncoiling mechanics of Escherichia coli type I fimbriae are optimized for catch bonds

We determined whether the molecular structures through which force is applied to receptor–ligand pairs are tuned to optimize cell adhesion under flow. The adhesive tethers of our model system, Escherichia coli, are type I fimbriae, which are anchored to the outer membrane of most E. coli strains. They consist of a fimbrial rod (0.3–1.5 μm in length) built from a helically coiled structural subunit, FimA, and an adhesive subunit, FimH, incorporated at the fimbrial tip. Previously reported data suggest that FimH binds to mannosylated ligands on the surfaces of host cells via catch bonds that are enhanced by the shear-originated tensile force. To understand whether the mechanical properties of the fimbrial rod regulate the stability of the FimH–mannose bond, we pulled the fimbriae via a mannosylated tip of an atomic force microscope. Individual fimbriae rapidly elongate for up to 10 μm at forces above 60 pN and rapidly contract again at forces below 25 pN. At intermediate forces, fimbriae change length more slowly, and discrete 5.0 ± 0.3–nm changes in length can be observed, consistent with uncoiling and coiling of the helical quaternary structure of one FimA subunit at a time. The force range at which fimbriae are relatively stable in length is the same as the optimal force range at which FimH–mannose bonds are longest lived. Higher or lower forces, which cause shorter bond lifetimes, cause rapid length changes in the fimbria that help maintain force at the optimal range for sustaining the FimH–mannose interaction. The modulation of force and the rate at which it is transmitted from the bacterial cell to the adhesive catch bond present a novel physiological role for the fimbrial rod in bacterial host cell adhesion. This suggests that the mechanical properties of the fimbrial shaft have codeveloped to optimize the stability of the terminal adhesive under flow.     

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.     

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.     

Shear-stabilized rolling behavior of E. coli examined with simulations

Escherichia coli exhibit both shear-stabilized rolling and a transition to stationary adhesion while adhering in fluid flow. Understanding the mechanism by which this shear-enhanced adhesion occurs is an important step in understanding bacterial pathogenesis. In this work, simulations are used to investigate the relative contributions of fimbrial deformation and bond transitions to the rolling and stationary adhesion of E. coli. Each E. coli body is surrounded by many long, thin fimbriae terminating in a single FimH receptor that is capable of forming a catch bond with mannose. As simulated cells progress along a mannosylated surface under flow, the fimbriae bend and buckle as they interact with the surface, and FimH-mannose bonds form and break according to a two-state, allosteric catch-bond model. In simulations, shear-stabilized rolling resulted from an increase in the low-affinity bond number due to increased fimbrial deformation with shear. Catch-bond formation did not occur during cell rolling, but instead led to the transition to stationary adhesion. In contrast, in leukocyte and platelet systems, catch bonds appear to be involved in the stabilization of rolling, and integrin activation is required for stationary adhesion.     

Three fim genes required for the regulation of length and mediation of adhesion of Escherichia coli type 1 fimbriae

Summary Three novel fim genes of Escherichia coli, fimF, fimG and fimH, were characterized. These genes were not necessary for the production of fimbriae but were shown to be involved in the adhesive property and longitudinal regulation of these structures. Complementation experiments indicated that both the major fimbrial subunit gene, fimA, and the fimH gene in combination with either the fimF or the fimG gene were required for mannose-specific adhesion. The fimF, fimG and fimH gene products were likewise shown to play a major role in the fimbrial morphology as longitudinal modulators. The amount of FimF, FimG and FimH proteins appeared to control the length and number of the fimbriae. The DNA sequence of a 2050 bp region containing the three genes was determined. The corresponding protein sequences all exhibited homology with the fimbrial subunit protein, FimA.     

Clonal analysis reveals high rate of structural mutations in fimbrial adhesins of extraintestinal pathogenic Escherichia coli

Type 1 fimbriae of Escherichia coli mediate mannose-specific adhesion to host epithelial surfaces and consist of a major, antigenically variable pilin subunit, FimA, and a minor, structurally conserved adhesive subunit, FimH, located on the fimbrial tip. We have analysed the variability of fimA and fimH in strains of vaginal and other origin that belong to one of the most prominent clonal groups of extraintestinal pathogenic E. coli, comprised of O1:K1-, O2:K1- and O18:K1-based serotypes. Multiple locus sequence typing (MLST) of this group revealed that the strains have identical (at all but one nucleotide position) eight housekeeping loci around the genome and belong to the ST95 complex defined by the publicly available E. coli MLST database. Multiple highly diverse fimA alleles have been introduced into the ST95 clonal complex via horizontal transfer, at a frequency comparable to that of genes defining the major O- and H-antigens. However, no further significant FimA diversification has occurred via point mutation after the transfers. In contrast, while fimH alleles also move horizontally (along with the fimA loci), they acquire point amino acid replacements at a higher rate than either housekeeping genes or fimA. These FimH mutations enhance binding to monomannose receptors and bacterial tropism for human vaginal epithelium. A similar pattern of rapid within-clonal structural evolution of the adhesive, but not pilin, subunit is also seen, respectively, in papG and papA alleles of the di-galactose-specific P-fimbriae. Thus, while structurally diverse pilin subunits of E. coli fimbriae are under selective pressure for frequent horizontal transfer between clones, the adhesive subunits of extraintestinal E. coli are under strong positive selection (Dn/Ds > 1 for fimH and papG) for functionally adaptive amino acid replacements.     

Shear-dependent 'stick-and-roll' adhesion of type 1 fimbriated Escherichia coli

It is generally assumed that bacteria are washed off surfaces as fluid flow increases because they adhere through 'slip-bonds' that weaken under mechanical force. However, we show here that the opposite is true for Escherichia coli attachment to monomannose-coated surfaces via the type 1 fimbrial adhesive subunit, FimH. Raising the shear stress (within the physiologically relevant range) increased accumulation of type 1 fimbriated bacteria on monomannose surfaces by up to two orders of magnitude, and reducing the shear stress caused them to detach. In contrast, bacterial binding to anti-FimH antibody-coated surfaces showed essentially the opposite behaviour, detaching when the shear stress was increased. These results can be explained if FimH is force-activated; that is, that FimH mediates 'catch-bonds' with mannose that are strengthened by tensile mechanical force. As a result, on monomannose-coated surfaces, bacteria displayed a complex 'stick-and-roll' adhesion in which they tended to roll over the surface at low shear but increasingly halted to stick firmly as the shear was increased. Mutations in FimH that were predicted earlier to increase or decrease force-induced conformational changes in FimH were furthermore shown here to increase or decrease the probability that bacteria exhibited the stationary versus the rolling mode of adhesion. This 'stick-and-roll' adhesion could allow type 1 fimbriated bacteria to move along mannosylated surfaces under relatively low flow conditions and to accumulate preferentially in high shear regions.     

The bacterial fimbrial tip acts as a mechanical force sensor

There is increasing evidence that the catch bond mechanism, where binding becomes stronger under tensile force, is a common property among non-covalent interactions between biological molecules that are exposed to mechanical force in vivo. Here, by using the multi-protein tip complex of the mannose-binding type 1 fimbriae of Escherichia coli, we show how the entire quaternary structure of the adhesive organella is adapted to facilitate binding under mechanically dynamic conditions induced by flow. The fimbrial tip mediates shear-dependent adhesion of bacteria to uroepithelial cells and demonstrates force-enhanced interaction with mannose in single molecule force spectroscopy experiments. The mannose-binding, lectin domain of the apex-positioned adhesive protein FimH is docked to the anchoring pilin domain in a distinct hooked manner. The hooked conformation is highly stable in molecular dynamics simulations under no force conditions but permits an easy separation of the domains upon application of an external tensile force, allowing the lectin domain to switch from a low- to a high-affinity state. The conformation between the FimH pilin domain and the following FimG subunit of the tip is open and stable even when tensile force is applied, providing an extended lever arm for the hook unhinging under shear. Finally, the conformation between FimG and FimF subunits is highly flexible even in the absence of tensile force, conferring to the FimH adhesin an exploratory function and high binding rates. The fimbrial tip of type 1 Escherichia coli is optimized to have a dual functionality: flexible exploration and force sensing. Comparison to other structures suggests that this property is common in unrelated bacterial and eukaryotic adhesive complexes that must function in dynamic conditions.     

RMSD analysis of structures of the bacterial protein FimH identifies five conformations of its lectin domain

FimH is a bacterial adhesin protein located at the tip of Escherichia coli fimbria that functions to adhere bacteria to host cells. Thus, FimH is a critical factor in bacterial infections such as urinary tract infections and is of interest in drug development. It is also involved in vaccine development and as a model for understanding shear-enhanced catch bond cell adhesion. To date, over 60 structures have been deposited in the Protein Data Bank showing interactions between FimH and mannose ligands, potential inhibitors, and other fimbrial proteins. In addition to providing insights about ligand recognition and fimbrial assembly, these structures provide insights into conformational changes in the two domains of FimH that are critical for its function. To gain further insights into these structural changes, we have superposed FimH's mannose binding lectin domain in all these structures and categorized the structures into five groups of lectin domain conformers using RMSD as a metric. Many structures also include the pilin domain, which anchors FimH to the fimbriae and regulates the conformation and function of the lectin domain. For these structures, we have also compared the relative orientations of the two domains. These structural analyses enhance our understanding of the conformational changes associated with FimH ligand binding and domain-domain interactions, including its catch bond behavior through allosteric action of force in bacterial adhesion.     

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.     

FimH forms catch bonds that are enhanced by mechanical force due to allosteric regulation

The bacterial adhesive protein, FimH, is the most common adhesin of Escherichia coli and mediates weak adhesion at low flow but strong adhesion at high flow. There is evidence that this occurs because FimH forms catch bonds, defined as bonds that are strengthened by tensile mechanical force. Here, we applied force to single isolated FimH bonds with an atomic force microscope in order to test this directly. If force was loaded slowly, most of the bonds broke up at low force (<60 piconewtons of rupture force). However, when force was loaded rapidly, all bonds survived until much higher force (140-180 piconewtons of rupture force), behavior that indicates a catch bond. Structural mutations or pretreatment with a monoclonal antibody, both of which allosterically stabilize a high affinity conformation of FimH, cause all bonds to survive until high forces regardless of the rate at which force is applied. Pretreatment of FimH bonds with intermediate force has the same strengthening effect on the bonds. This demonstrates that FimH forms catch bonds and that tensile force induces an allosteric switch to the high affinity, strong binding conformation of the adhesin. The catch bond behavior of FimH, the amount of force needed to regulate FimH, and the allosteric mechanism all provide insight into how bacteria bind and form biofilms in fluid flow. Additionally, these observations may provide a means for designing antiadhesive mechanisms.     

The major subunit of Escherichia coli type 1 fimbriae is not required for D-mannose-specific adhesion

Type 1 fimbriae are surface organelles on Escherichia coli, which mediate specific binding to D-mannose-containing structures. These fimbriae are heteropolymers composed of a major building element, the FimA protein, and small amounts of the FimF, FimG and FimH proteins. The FimH protein is uniquely responsible for the D-mannose receptor binding. In this work data are presented which indicate that the major subunit of type 1 fimbriae is dispensable for D-mannose-specific binding. A recombinant strain was studied which harboured an insertional deletion in the fimA gene, and was thereby unable to produce type 1 fimbriae; however, it was still able to express a D-mannose-binding phenotype. However, the deletion resulted in a 25-fold reduction of the adhesive potential, as measured by binding to D-mannose-coated Sepharose beads. Serological and specific receptor binding evidence is presented that suggests that the FimH adhesion is capable of being exposed on the bacterial surface without being an integral part of the fimbriae.     

Sortase-catalyzed assembly of distinct heteromeric fimbriae in Actinomyces naeslundii

Two types of adhesive fimbriae are expressed by Actinomyces; however, the architecture and the mechanism of assembly of these structures remain poorly understood. In this study we characterized two fimbrial gene clusters present in the genome of Actinomyces naeslundii strain MG-1. By using immunoelectron microscopy and biochemical analysis, we showed that the fimQ-fimP-srtC1-fimR gene cluster encodes a fimbrial structure (designated type 1) that contains a major subunit, FimP, forming the shaft and a minor subunit, FimQ, located primarily at the tip. Similarly, the fimB-fimA-srtC2 gene cluster encodes a distinct fimbrial structure (designated type 2) composed of a shaft protein, FimA, and a tip protein, FimB. By using allelic exchange, we constructed an in-frame deletion mutant that lacks the SrtC2 sortase. This mutant produces abundant type 1 fimbriae and expresses the monomeric FimA and FimB proteins, but it does not assemble type 2 fimbriae. Thus, SrtC2 is a fimbria-specific sortase that is essential for assembly of the type 2 fimbriae. Together, our experiments pave the way for several lines of molecular investigation that are necessary to elucidate the fimbrial assembly pathways in Actinomyces and their function in the pathogenesis of different biofilm-related oral diseases.     

Two autonomous structural modules in the fimbrial shaft adhesin FimA mediate Actinomyces interactions with streptococci and host cells during oral biofilm development

By combining X-ray crystallography and modelling, we describe here the atomic structure of distinct adhesive moieties of FimA, the shaft fimbrillin of Actinomyces type 2 fimbriae, which uniquely mediates the receptor-dependent intercellular interactions between Actinomyces and oral streptococci as well as host cells during the development of oral biofilms. The FimA adhesin is built with three IgG-like domains, each of which harbours an intramolecular isopeptide bond, previously described in several Gram-positive pilins. Genetic and biochemical studies demonstrate that although these isopeptide bonds are dispensable for fimbrial assembly, cell-cell interactions and biofilm formation, they contribute significantly to the proteolytic stability of FimA. Remarkably, FimA harbours two autonomous adhesive modules, which structurally resemble the Staphylococcus aureus Cna B domain. Each isolated module can bind the plasma glycoprotein asialofetuin as well as the polysaccharide receptors present on the surface of oral streptococci and epithelial cells. Thus, FimA should serve as an excellent paradigm for the development of therapeutic strategies and elucidating the precise molecular mechanisms underlying the interactions between cellular receptors and Gram-positive fimbriae.     

Porphyromonas gingivalis FimA Fimbriae: Fimbrial Assembly by fimA Alone in the fim Gene Cluster and Differential Antigenicity among fimA Genotypes

The periodontal pathogen Porphyromonas gingivalis colonizes largely through FimA fimbriae, composed of polymerized FimA encoded by fimA. fimA exists as a single copy within the fim gene cluster (fim cluster), which consists of seven genes: fimX, pgmA and fimA-E. Using an expression vector, fimA alone was inserted into a mutant from which the whole fim cluster was deleted, and the resultant complement exhibited a fimbrial structure. Thus, the genes of the fim cluster other than fimA were not essential for the assembly of FimA fimbriae, although they were reported to influence FimA protein expression. It is known that there are various genotypes for fimA, and it was indicated that the genotype was related to the morphological features of FimA fimbriae, especially the length, and to the pathogenicity of the bacterium. We next complemented the fim cluster-deletion mutant with fimA genes cloned from P. gingivalis strains including genotypes I to V. All genotypes showed a long fimbrial structure, indicating that FimA itself had nothing to do with regulation of the fimbrial length. In FimA fimbriae purified from the complemented strains, types I, II, and III showed slightly higher thermostability than types IV and V. Antisera of mice immunized with each purified fimbria principally recognized the polymeric, structural conformation of the fimbriae, and showed low cross-reactivity among genotypes, indicating that FimA fimbriae of each genotype were antigenically different. Additionally, the activity of a macrophage cell line stimulated with the purified fimbriae was much lower than that induced by Escherichia coli lipopolysaccharide.     

Differential mechanical stability of filamin A rod segments

Prompted by recent reports suggesting that interaction of filamin A (FLNa) with its binding partners is regulated by mechanical force, we examined mechanical properties of FLNa domains using magnetic tweezers. FLNa, an actin cross-linking protein, consists of two subunits that dimerize through a C-terminal self-association domain. Each subunit contains an N-terminal spectrin-related actin-binding domain followed by 24 immunoglobulinlike (Ig) repeats. The Ig repeats in the rod 1 segment (repeats 1–15) are arranged as a linear array, whereas rod 2 (repeats 16–23) is more compact due to interdomain interactions. In the rod 1 segment, repeats 9–15 augment F-actin binding to a much greater extent than do repeats 1–8. Here, we report that the three segments are unfolded at different forces under the same loading rate. Remarkably, we found that repeats 16–23 are susceptible to forces of ∼10 pN or even less, whereas the repeats in the rod 1 segment can withstand significantly higher forces. The differential force response of FLNa Ig domains has broad implications, since these domains not only support the tension of actin network but also interact with many transmembrane and signaling proteins, mostly in the rod 2 segment. In particular, our finding of unfolding of repeats 16–23 at ∼10 pN or less is consistent with the hypothesized force-sensing function of the rod 2 segment in FLNa.     

For catch bonds, it all hinges on the interdomain region

Tensile mechanical force was long assumed to increase the detachment rates of biological adhesive bonds (Bell, 1978). However, in the last few years, several receptor-ligand pairs were shown to form "catch bonds," whose lifetimes are enhanced by moderate amounts of force. These include the bacterial adhesive protein FimH binding to its ligand mannose (Thomas et al., 2002; Thomas et al., 2006), blood cell adhesion proteins P- and L-selectin binding to sialyl Lewis X (sLe(X))-containing ligands (Marshall et al., 2003; Evans et al., 2004; Sarangapani et al., 2004), and the myosin-actin motor protein interaction (Guo and Guilford, 2006). The structural mechanism behind this counterintuitive force-enhanced catch bond behavior is of great interest.     

Recombinant FimH Adhesin Demonstrates How the Allosteric Catch Bond Mechanism Can Support Fast and Strong Bacterial Attachment in the Absence of Shear

The FimH protein of Escherichia coli is a model two-domain adhesin that is able to mediate an allosteric catch bond mechanism of bacterial cell attachment, where the mannose-binding lectin domain switches from an ‘inactive’ conformation with fast binding to mannose to an ‘active’ conformation with slow detachment from mannose. Because mechanical tensile force favors separation of the domains and, thus, FimH activation, it has been thought that the catch bonds can only be manifested in a fluidic shear-dependent mode of adhesion. Here, we used recombinant FimH variants with a weakened inter-domain interaction and show that a fast and sustained allosteric activation of FimH can also occur under static, non-shear conditions. Moreover, it appears that lectin domain conformational activation happens intrinsically at a constant rate, independently from its ability to interact with the pilin domain or mannose. However, the latter two factors control the rate of FimH deactivation. Thus, the allosteric catch bond mechanism can be a much broader phenomenon involved in both fast and strong cell-pathogen attachments under a broad range of hydrodynamic conditions. This concept that allostery can enable more effective receptor-ligand interactions is fundamentally different from the conventional wisdom that allostery provides a mechanism to turn binding off under specific conditions.     

Beyond induced-fit receptor-ligand interactions: structural changes that can significantly extend bond lifetimes

While the lifetime of conventional receptor-ligand interactions is shortened by tensile mechanical force, some recently discovered interactions, termed catch bonds, can be strengthened by force. Motivated by the search for the underpinning structural mechanisms, we here explore the structural dynamics of the binding site of the bacterial adhesive protein FimH by molecular dynamics and steered molecular dynamics. While the crystal structure of only one FimH conformation has been reported so far, we describe two distinctively different conformations of the mannose-bound FimH binding site. Force-induced dissociation was slowed when the mannose ring rotated such that additional force-bearing hydrogen bonds formed with the base of the FimH binding pocket. The lifetime of the complex was further enhanced significantly by rigidifying this base. We finally show how even sub-angstrom spatial alterations of the hydrogen bonding pattern within the base can lead to significantly decreased bond lifetimes.     

Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities

Background  

Actinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galβ binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking.