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 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.

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.

FimH is a mannose-specific adhesin located on the tip of type 1 fimbriae of Escherichia coli that is capable of mediating shearenhanced 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-ligand specific adhesion is among the most fundamental of biological phenomena in nature. This phenomenon underlies eukaryotic cell-cell or cell-surface attachment, initiates recognition and signaling events, binds bacteria or viruses to target cells, and mediates biofilm formation on medical implants. Usually, the adhesion is mediated by protein-protein, protein-lipid, or often, protein-saccharide interactions (1). Most of these adhesive interactions occur under a certain level of tensile force that arises from shear stress. Whereas a number of receptor-ligand binding interactions are weakened by tensile force, recent studies have shown that certain physiologically important biomolecular interactions can be enhanced by mechanical force (2-4). Although these studies have exposed a novel, so-called catch bond, mode of receptor-ligand adhesion, the precise structural mechanism for it remains elusive.One type of receptor-ligand interaction that is enhanced by mechanical force is that of the most common family of bacterial adhesins, mannose-specific type 1 fimbriae. In Escherichia coli, mannose-specific adhesion is mediated by the FimH adhesin at the tip of type 1 fimbriae. FimH has two domains, the mannosebinding lectin domain (Ld) 4 (1-156 aa) and the fimbria-incorporating pilin domain (Pd) (160 -273 aa), which are connected via a 3-aa interdomain linker peptide chain (5). The Ld is a ␤-barrel fold with the monomannose (1M)-bindin...

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 subunits that constitute the bacterial adhesive complex located at the tip of the fimbria form a hook-chain that acts as a rapid force-sensitive anchor at high flow.

Pyroptosis is an inflammatory form of programmed cell death following cellular damage or infection. It is a lytic process driven by gasdermin D-mediated cellular permeabilization and presumed osmotic forces thought to induce swelling and rupture. We found that pyroptotic cells do not spontaneously rupture in culture but lose mechanical resilience. As a result, cells were susceptible to rupture by extrinsic forces, such as shear stress or compression. Cell analyses revealed that all major cytoskeleton components were disrupted during pyroptosis and that sensitivity to rupture was calpaindependent and linked with cleavage of vimentin and loss of intermediate filaments. Moreover, while release of lactate dehydrogenase (LDH), HMGB1, and IL-1β occurred without rupture, rupture was required for release of large inflammatory stimuli-ASC specks, mitochondria, nuclei, and bacteria. Importantly, supernatants from ruptured cells were more immunostimulatory than those from nonruptured cells. These observations reveal undiscovered cellular events occurring during pyroptosis, define the mechanisms driving pyroptotic rupture, and highlight the immunologic importance of this event.

FimH, the adhesive subunit of type 1 fimbriae expressed by many enterobacteria, mediates mannosesensitive binding to target host cells. At the same time, fine receptor-structural specificities of FimH from different species can be substantially different, affecting bacterial tissue tropism and, as a result, the role of the particular fimbriae in pathogenesis. In this study, we compared functional properties of the FimH proteins from Escherichia coli and Klebsiella pneumoniae, which are both 279 amino acids in length but differ by some ϳ15% of residues. We show that K. pneumoniae FimH is unable to mediate adhesion in a monomannose-specific manner via terminally exposed Man␣(1-2) residues in N-linked oligosaccharides, which are the structural basis of the tropism of E. coli FimH for uroepithelial cells. However, K. pneumoniae FimH can bind to the terminally exposed Man␣(1-3)Man␤(1-4)GlcNAc␤1 trisaccharide, though only in a shear-dependent manner, wherein the binding is marginal at low shear force but enhanced sevenfold under increased shear. A single mutation in the K. pneumoniae FimH, S62A, converts the mode of binding from shear dependent to shear independent. This mutation has occurred naturally in the course of endemic circulation of a nosocomial uropathogenic clone and is identical to a pathogenicity-adaptive mutation found in highly virulent uropathogenic strains of E. coli, in which it also eliminates the dependence of E. coli binding on shear. The sheardependent binding properties of the K. pneumoniae and E. coli FimH proteins are mediated via an allosteric catch bond mechanism. Thus, despite differences in FimH structure and fine receptor specificity, the sheardependent nature of FimH-mediated adhesion is highly conserved between bacterial species, supporting its remarkable physiological significance.