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.     

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.     

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.     

Mechanism of allosteric propagation across a β‐sheet structure investigated by molecular dynamics simulations

The bacterial adhesin FimH consists of an allosterically regulated mannose‐binding lectin domain and a covalently linked inhibitory pilin domain. Under normal conditions, the two domains are bound to each other, and FimH interacts weakly with mannose. However, under tensile force, the domains separate and the lectin domain undergoes conformational changes that strengthen its bond with mannose. Comparison of the crystallographic structures of the low and the high affinity state of the lectin domain reveals conformational changes mainly in the regulatory inter‐domain region, the mannose binding site and a large β sheet that connects the two distally located regions. Here, molecular dynamics simulations investigated how conformational changes are propagated within and between different regions of the lectin domain. It was found that the inter‐domain region moves towards the high affinity conformation as it becomes more compact and buries exposed hydrophobic surface after separation of the pilin domain. The mannose binding site was more rigid in the high affinity state, which prevented water penetration into the pocket. The large central β sheet demonstrated a soft spring‐like twisting. Its twisting motion was moderately correlated to fluctuations in both the regulatory and the binding region, whereas a weak correlation was seen in a direct comparison of these two distal sites. The results suggest a so called “population shift” model whereby binding of the lectin domain to either the pilin domain or mannose locks the β sheet in a rather twisted or flat conformation, stabilizing the low or the high affinity state, respectively. Proteins 2016; 84:990–1008. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

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.     

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.     

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.     

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.     

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.     

Allosteric Coupling in the Bacterial Adhesive Protein FimH

The protein FimH is expressed by the majority of commensal and uropathogenic strains of Escherichia coli on the tips of type 1 fimbriae and mediates adhesion via a catch bond to its ligand mannose. Crystal structures of FimH show an allosteric conformational change, but it remains unclear whether all of the observed structural differences are part of the allosteric mechanism. Here we use the protein structural analysis tool RosettaDesign combined with human insight to identify and synthesize 10 mutations in four regions that we predicted would stabilize one of the conformations of that region. The function of each variant was characterized by measuring binding to the ligand mannose, whereas the allosteric state was determined using a conformation-specific monoclonal antibody. These studies demonstrated that each region investigated was indeed part of the FimH allosteric mechanism. However, the studies strongly suggested that some regions were more tightly coupled to mannose binding and others to antibody binding. In addition, we identified many FimH variants that appear locked in the low affinity state. Knowledge of regulatory sites outside the active and effector sites as well as the ability to make FimH variants locked in the low affinity state may be crucial to the future development of novel antiadhesive and antimicrobial therapies using allosteric regulation to inhibit FimH.     

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.     

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.     

Catch-bond model derived from allostery explains force-activated bacterial adhesion

High shear enhances the adhesion of Escherichia coli bacteria binding to mannose coated surfaces via the adhesin FimH, raising the question as to whether FimH forms catch bonds that are stronger under tensile mechanical force. Here, we study the length of time that E. coli pause on mannosylated surfaces and report a double exponential decay in the duration of the pauses. This double exponential decay is unlike previous single molecule or whole cell data for other catch bonds, and indicates the existence of two distinct conformational states. We present a mathematical model, derived from the common notion of chemical allostery, which describes the lifetime of a catch bond in which mechanical force regulates the transitions between two conformational states that have different unbinding rates. The model explains these characteristics of the data: a double exponential decay, an increase in both the likelihood and lifetime of the high-binding state with shear stress, and a biphasic effect of force on detachment rates. The model parameters estimated from the data are consistent with the force-induced structural changes shown earlier in FimH. This strongly suggests that FimH forms allosteric catch bonds. The model advances our understanding of both catch bonds and the role of allostery in regulating protein activity.     

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-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 fimbrial adhesin F17-G of enterotoxigenic Escherichia coli has an immunoglobulin-like lectin domain that binds N-acetylglucosamine

The F17-G adhesin at the tip of flexible F17 fimbriae of enterotoxigenic Escherichia coli mediates binding to N-acetyl-beta-D-glucosamine-presenting receptors on the microvilli of the intestinal epithelium of ruminants. We report the 1.7 A resolution crystal structure of the lectin domain of F17-G, both free and in complex with N-acetylglucosamine. The monosaccharide is bound on the side of the ellipsoid-shaped protein in a conserved site around which all natural variations of F17-G are clustered. A model is proposed for the interaction between F17-fimbriated E. coli and microvilli with enhanced affinity compared with the binding constant we determined for F17-G binding to N-acetylglucosamine (0.85 mM-1). Unexpectedly, the F17-G structure reveals that the lectin domains of the F17-G, PapGII and FimH fimbrial adhesins all share the immunoglobulin-like fold of the structural components (pilins) of their fimbriae, despite lack of any sequence identity. Fold comparisons with pilin and chaperone structures of the chaperone/usher pathway highlight the central role of the C-terminal beta-strand G of the immunoglobulin-like fold and provides new insights into pilus assembly, function and adhesion.     

Catch bond-mediated adhesion without a shear threshold: trimannose versus monomannose interactions with the FimH adhesin of Escherichia coli

The FimH protein is the adhesive subunit of Escherichia coli type 1 fimbriae. It mediates shear-dependent bacterial binding to monomannose (1M)-coated surfaces manifested by the existence of a shear threshold for binding, below which bacteria do not adhere. The 1M-specific shear-dependent binding of FimH is consistent with so-called catch bond interactions, whose lifetime is increased by tensile force. We show here that the oligosaccharide-specific interaction of FimH with another of its ligands, trimannose (3M), lacks a shear threshold for binding, since the number of bacteria binding under static conditions is higher than under any flow. However, similar to 1M, the binding strength of surface-interacting bacteria is enhanced by shear. Bacteria transition from rolling into firm stationary surface adhesion as the shear increases. The shear-enhanced bacterial binding on 3M is mediated by catch bond properties of the 1M-binding subsite within the extended oligosaccharide-binding pocket of FimH, since structural mutations in the putative force-responsive region and in the binding site affect 1M- and 3M-specific binding in an identical manner. A shear-dependent conversion of the adhesion mode is also exhibited by P-fimbriated E. coli adhering to digalactose surfaces.     

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.     

The cysteine bond in the Escherichia coli FimH adhesin is critical for adhesion under flow conditions

Cysteine bonds are found near the ligand-binding sites of a wide range of microbial adhesive proteins, including the FimH adhesin of Escherichia coli. We show here that removal of the cysteine bond in the mannose-binding domain of FimH did not affect FimH-mannose binding under static or low shear conditions (< or = 0.2 dyne cm(-2)). However, the adhesion level was substantially decreased under increased fluid flow. Under intermediate shear (2 dynes cm(-2)), the ON-rate of bacterial attachment was significantly decreased for disulphide-free mutants. Molecular dynamics simulations demonstrated that the lower ON-rate of cysteine bond-free FimH could be due to destabilization of the mannose-free binding pocket of FimH. In contrast, mutant and wild-type FimH had similar conformation when bound to mannose, explaining their similar binding strength to mannose under intermediate shear. The stabilizing effect of mannose on disulphide-free FimH was also confirmed by protection of the FimH from thermal and chemical inactivation in the presence of mannose. However, this stabilizing effect could not protect the integrity of FimH structure under high shear (> 20 dynes cm(-2)), where lack of the disulphide significantly increased adhesion OFF-rates. Thus, the cysteine bonds in bacterial adhesins could be adapted to enable bacteria to bind target surfaces under increased shear conditions.     

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.