Bacterial fimbrial adhesins such as FimH are critical for host colonization and persistence under the mechanical forces encountered at sites of infection such as the urinary tract. The molecular mechanisms by which FimH, a key virulence factor of uropathogenic Escherichia coli, regulates its binding to host cell surface mannose moieties through conformational switching remain incompletely understood. FimH operates across a range of conformations that includes low- (LAS), intermediate-, and high-affinity (HAS) states and forms catch bonds that paradoxically strengthen under force. The allosteric pathways governing these transitions remain poorly defined due to experimental limitations that restrict understanding of key dynamic phenomena that underlie ligand-triggered conformational shifts and force-induced long-lived interactions. Such understanding is central to drug discovery efforts to target bacterial adhesion. Here, we present a model system that fully recapitulates the conformational repertoire of FimH in the absence of its pilin domain. Our findings demonstrate that a single mutation in the lectin domain stabilizes the LAS while allowing for ligand-binding-induced transition to a HAS-like conformation and catch bond formation, mirroring the behavior of the native FimH adhesin. We propose a dynamic allosteric mechanism that involves ultraslow, low-frequency dynamics for the ability of FimH to sustain long-lived interactions with mannose, under both static and force conditions.

Introduction: Streptococcus gordonii (SG), a commensal bacterium of the oral cavity, is a significant cause of native-valve (without prior valvular defect) and subacute infective endocarditis (IE), often attributed to its extraordinary ability to form biofilms. IE pathogenesis requires bacterial adhesion to the valve, which may involve binding to platelet (PLT) glycoprotein (GP) Ibα through SG serine-rich repeat (SRR) adhesins. In addition, the SG receptor PadA binds platelet integrin αIIbβ3. Binding to von Willebrand factor (VWF) has been shown for staphylococci but not for SG. The SG strain expressing the SRR adhesin GspB binds core 1 (C1) sialoglycans on the mechanosensory domain of GPIbα, 10712BR adhesin binds core 2 (C2) sialoglycans on the mucin stalk. Hsa adhesin binds both C1 and C2. Pathogenicity is at least partially determined by glycan-binding specificity. In a rat model, selective C1 binding results in enhanced virulence, contributing to infection initiation and progression. Here, we investigate the impact of glycan ligand specificity and the role of shear in SG binding to GPIbα and VWF, which also has C1 sialoglycans. Methods: Whole blood (WB), platelet-rich plasma (PRP), or washed PLTs were obtained from healthy donors (n = 29, 55% females). SG-PLT binding and PLT activation following a static 10 min incubation were measured by flow cytometry and under shear conditions using microfluidics, in isogenic SG strains (C1, C2, C1&2, and no (C0)-sialoglycan binding). Results: SG binding to blood cells, static. In WB, the C2 binder showed the highest binding to PLT (~70%), followed by C1&2 (~40%), with C1 and C0 showing minimal binding. WBC binding showed a different pattern; RBC binding was minimal. Between 15 and 40% of each strain bound blood cells with C2 and C1&2 binders showing a higher bound-fraction due to PLT binding. Previously reported plasma protein ligands of SG adhesins may account for the remainder of the binding. The same pattern of binding was seen in PRP as in WB. However, in washed PLTs, binding was ~3x higher, indicating that plasma contains inhibitory factors. C2 and C1&2-binders bound similarly. Glycocalicin or WM23 (mAb to the mucin stalk) can partially inhibit this binding, while SZ2 (mAb to GPIbα sulfo-tyrosine region) promotes binding. Effect of SG on platelet activation, static. In washed PLTs, C2 and C1&2 binders induced higher P-selectin expression (p<0.0001 and p=0.0001, respectively) and phosphatidylserine exposure compared to baseline (p = 0.03). In PRP, there were no apparent differences in PLT activation between strains. In PRP lumi-aggregometry, SG did not cause spontaneous PLT aggregation or ATP secretion. However, in the presence of minimal concentrations of ADP (1 µM), arachidonic acid (0.1 mM), or collagen (0.2 μg/mL), SG strains similarly induced maximal PLT aggregation. Studies under shear. We evaluated by microfluidics whether the binding of SG to PLT or VWF is shear-dependent. We first monitored adhesion of SG and PLT in WB perfused into VWF-coated channels. The C1-selective binder adhered more extensively to immobilized VWF than the other strains, independently of PLT adhesion, at both 500 and 2,000 s-1 shear rates. At 2,000 s-1, addition of SG induced greater PLT adhesion than without SG. We also studied the effect of SG on platelet thrombus formation in WB by perfusing the mixture into collagen-coated block-and-post channels, which provides high turbulent shear (24,000 s-1). C1 and C1&2 binding strains induced much larger thrombi, with greater SG incorporation. To evaluate SG-VWF binding, we perfused the bacteria over self-associated purified VWF (mono-pillar channels) and endothelium-secreted VWF (flow chambers with cultured HUVECs). We observed higher binding of the C1-binder to VWF strings around the pillar, possibly enhancing VWF self-association. On HUVECs, all SG strains except C0 formed beads-on-a-string structures on VWF, without colocalization with PLTs. Conclusion: The study shows a previously undescribed shear-dependent interaction between SG and VWF via SG's sialoglycan-binding SRR adhesin with C1 specificity. This provides a likely explanation for how the bacteria attach to heart valves at high shear rates to initiate infection and also suggests a reason for the increased virulence of the C1-binding strains. The C2-binding strains bind platelets through GPIbα, which may enhance bacterial clearance and reduce virulence.

The adhesin FimH is expressed by commensal Escherichia coli and is implicated in urinary tract infections, where it mediates adhesion to mannosylated glycoproteins on urinary and intestinal epithelial cells in the presence of a high-shear fluid environment. The FimH-mannose bond exhibits catch behavior in which bond lifetime increases with force, because tensile force induces a transition in FimH from a compact native to an elongated activated conformation with a higher affinity to mannose. However, the lifetime of the activated state of FimH has not been measured under force. Here we apply multiplexed magnetic tweezers to apply a preload force to activate FimH bonds with yeast mannan, then we measure the lifetime of these activated bonds under a wide range of forces above and below the preload force. A higher fraction of FimH-mannan bonds were activated above than below a critical preload force, confirming the FimH catch bond behavior. Once activated, FimH detached from mannose with multi-state kinetics, suggesting the existence of two bound states with a 20-fold difference in dissociation rates. The average lifetime of activated FimH-mannose bonds was 1000 to 10,000 s at forces of 30-70 pN. Structural explanations of the two bound states and the high force resistance provide insights into structural mechanisms for long-lived, force-resistant biomolecular interactions.

Diversity, equity, and inclusion (DEI) are interconnected with bioengineering, yet have historically been absent from accreditation standards and curricula. Toward educating DEI-competent bioengineers and meeting evolving accreditation requirements, we took a program-level approach to incorporate, catalog, and assess DEI content through the bioengineering undergraduate program. To support instructors in adding DEI content and inclusive pedagogy, our team developed a DEI planning worksheet and surveyed instructors pre- and post-course. Over the academic year, 74% of instructors provided a pre-term and/or post-term response. Of responding instructors, 91% described at least one DEI curricular content improvement, and 88% incorporated at least one new inclusive pedagogical approach. Based on the curricular adjustments reported by instructors, we grouped the bioengineering-related DEI content into five DEI competency categories: bioethics, inclusive design, inclusive scholarship, inclusive professionalism, and systemic inequality. To assess the DEI content incorporation, we employed direct assessment via course assignments, end-of-module student surveys, end-of-term course evaluations, and an end-of-year program review. When asked how much their experience in the program helped them develop specific DEI competencies, students reported a relatively high average of 3.79 (scale of 1 = “not at all” to 5 = “very much”). Additionally, based on student performance in course assignments and other student feedback, we found that instructors were able to effectively incorporate DEI content into a wide variety of courses. We offer this framework and lessons learned to be adopted by programs similarly motivated to train DEI-competent engineering professionals and provide an equitable, inclusive engineering education for all students.

Bacterial adhesion is the first step in the formation of surface biofilms. The number of bacteria that bind to a surface from the solution depends on how many bacteria can reach the surface (bacterial transport) and the strength of interactions between bacterial adhesins and surface receptors (adhesivity). By using microfluidic channels and video microscopy as well as computational simulations, we investigated how the interplay between bacterial transport and adhesivity affects the number of the common human pathogen Escherichia coli that bind to heterogeneous surfaces with different receptor densities. We determined that gravitational sedimentation causes bacteria to concentrate at the lower surface over time as fluid moves over a non-adhesive region, so bacteria preferentially adhere to adhesive regions on the lower, inflow-proximal areas that are downstream of non-adhesive regions within the entered compartments. Also, initial bacterial attachment to an adhesive region of a heterogeneous lower surface may be inhibited by shear due to mass transport effects alone rather than shear forces per se, because higher shear washes out the sedimented bacteria. We also provide a conceptual framework and theory that predict the impact of sedimentation on adhesion between and within adhesive regions in flow, where bacteria would likely bind both in vitro and in vivo, and how to normalize the bacterial binding level under experimental set-ups based on the flow compartment configuration.

Upon vascular injury, platelets form a hemostatic plug by binding to the subendothelium and to each other. Platelet-to-matrix binding is initially mediated by von Willebrand factor (VWF) and platelet-to-platelet binding is mediated mainly by fibrinogen and VWF. After binding, the actin cytoskeleton of a platelet drives its contraction, generating traction forces that are important to the cessation of bleeding. Our understanding of the relationship between adhesive environment, F-actin morphology, and traction forces is limited. Here, we examined F-actin morphology of platelets attached to surfaces coated with fibrinogen and VWF. We identified distinct F-actin patterns induced by these protein coatings and found that these patterns were identifiable into three classifications via machine learning: solid, nodular, and hollow. We observed that traction forces for platelets were significantly higher on VWF than on fibrinogen coatings and these forces varied by F-actin pattern. In addition, we analyzed the F-actin orientation in platelets and noted that their filaments were more circumferential when on fibrinogen coatings and having a hollow F-actin pattern, while they were more radial on VWF and having a solid F-actin pattern. Finally, we noted that subcellular localization of traction forces corresponded to protein coating and F-actin pattern: VWF-bound, solid platelets had higher forces at their central region while fibrinogen-bound, hollow platelets had higher forces at their periphery. These distinct F-actin patterns on fibrinogen and VWF and their differences in F-actin orientation, force magnitude, and force localization could have implications in hemostasis, thrombus architecture, and venous versus arterial thrombosis.

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.

In this issue of Blood, Bergal et al developed a new method to demonstrate that circulating von Willebrand Factor (VWF) multimers experience less tension and undergo less conformational elongation in shear flow than previously thought.(1) VWF is a blood protein that binds to and activates platelets to initiate hemostatic blood clots, particularly in arterial flow. This process must be precisely regulated, or thrombi would form spontaneously. The primary regulatory mechanism in VWF is mechanical tension; therefore, characterizing VWF tension and elongation in shear flow is critical for understanding the regulation of arterial hemostasis and thrombosis. Indeed, devices such as artificial heart valves and left ventricular assist devices, which introduce higher and different fluid stresses, are associated with the hemostatic and thrombotic dysregulation of VWF in many patients.

Allosteric proteins transition between 'inactive' and 'active' states. In general, such proteins assume distinct conformational states at the level of secondary, tertiary and/or quaternary structure. Different conformers of an allosteric protein can be antigenically dissimilar and induce antibodies with a highly distinctive specificities and neutralizing functional effects. Here we summarize studies on various functional types of monoclonal antibodies obtained against different allosteric conformers of the mannose-specific bacterial adhesin FimH - the most common cell attachment protein of Escherichia coli and other enterobacterial pathogens. Included are types of antibodies that activate the FimH function via interaction with ligand-induced binding sites or by wedging between domains as well as antibodies that inhibit FimH through orthosteric, parasteric, or novel dynasteric mechanisms. Understanding the molecular mechanism of antibody action against allosteric proteins provides insights on how to design antibodies with a desired functional effect, including those with neutralizing activity against bacterial and viral cell attachment proteins.

Mechanical forces influence functions occurring inside and around cells. Forces have influenced some proteins to develop catch bond properties, in which the lifetime of the interaction between two molecules increases with force. Catch bonds have been observed in various proteins, many of which are associated with cellular adhesion. The maximum lifetime under force of most catch bonds is typically on the order of seconds. However, preliminary data suggests that the catch bond involving the bacterial adhesive, FimH, is much longer-lived. To understand what makes FimH so strong, we first sought to measure its lifetimes under different forces. To facilitate measuring long interactions, we utilized a novel magnetic tweezer apparatus that can apply constant stable forces to multiple magnetic beads simultaneously. Magnetic beads coated in ligand were manipulated via electromagnets to form bonds with immobilized FimH. After the catch bond was activated by a preload force, the tension on the bond was held constant until detachment.Single molecule interactions were ensured by diluting the concentration of FimH so that about 90% of observed interactions would only involve one FimH, based on Poisson statistics. We also implemented additional means of verifying single molecule behavior, such as examining the distance the FimH molecule and linker stretched during preload.At forces up to 70 pN, many FimH bonds outlasted the 15-minute collection period. In fact, we estimated the lifetime of the bond under 20-30 pN of force to be about 17 minutes. Like other catch bonds, once the FimH catch bond is activated, increasing force negatively affects the bond lifetime. However, when the change in unbinding rate due to force was estimated, we found that FimH exhibited a relatively force insensitive behavior. We believe this force insensitivity contributes to FimH's remarkably long lifetime under force.

Measuring the traction forces produced by cells provides insight into their behavior and physiological function. Here, we developed a technique (dubbed 'black dots') that microcontact prints a fluorescent micropattern onto a flexible substrate to measure cellular traction forces without constraining cell shape or needing to detach the cells. To demonstrate our technique, we assessed human platelets, which can generate a large range of forces within a population. We find platelets that exert more force have more spread area, are more circular, and have more uniformly distributed F-actin filaments. As a result of the high yield of data obtainable by this technique, we were able to evaluate multivariate mixed effects models with interaction terms and conduct a clustering analysis to identify clusters within our data. These statistical techniques demonstrated a complex relationship between spread area, circularity, F-actin dispersion, and platelet force, including cooperative effects that significantly associate with platelet traction forces. STATEMENT OF SIGNIFICANCE: Cells produce contractile forces during division, migration, or wound healing. Measuring cellular forces provides insight into their health, behavior, and function. We developed a technique that calculates cellular forces by seeding cells onto a pattern and quantifying how much each cell displaces the pattern. This technique is capable of measuring hundreds of cells without needing to detach them. Using this technique to evaluate human platelets, we find that platelets exerting more force tend to have more spread area, are more circular in shape, and have more uniformly distributed cytoskeletal filaments. Due to our high yield of data, we were able to apply statistical techniques that revealed combinatorial effects between these factors.

BACKGROUND:Recognition proteins are critical in many biotechnology applications and would be even more useful if their binding could be regulated. The current gold standard for recognition molecules, antibodies, lacks convenient regulation. Alternative scaffolds can be used to build recognition proteins with new functionalities, including regulated recognition molecules. Here we test the use of the bacterial adhesin FimH as a scaffold for regulated molecular recognition. FimH binds to its native small molecule target mannose in a conformation-dependent manner that can be regulated by two types of noncompetitive regulation: allosteric and parasteric.RESULTS:We demonstrate that conformational regulation of FimH can be maintained even after reengineering the binding site to recognize the non-mannosylated targets nickel or Penta-His antibody, resulting in an up to 7-fold difference in KD between the two conformations. Moreover, both the allosteric and parasteric regulatory mechanisms native to FimH can be used to regulate binding to its new target. In one mutant, addition of the native ligand mannose parasterically improves the mutant's affinity for Penta-His 4-fold, even as their epitopes overlap. In another mutant, the allosteric antibody mab21 reduces the mutant's affinity for Penta-His 7-fold. The advantage of noncompetitive regulation is further illustrated by the ability of this allosteric regulator to induce 98% detachment of Penta-His, even with modest differences in affinity.CONCLUSIONS:This illustrates the potential of FimH, with its deeply studied conformation-dependent binding, as a scaffold for conformationally regulated binding via multiple mechanisms.

Three-dimensional particle tracking is a routine experimental procedure for various biophysical applications including magnetic tweezers. A common method for tracking the axial position of particles involves the analysis of diffraction rings whose pattern depends sensitively on the axial position of the bead relative to the focal plane. To infer the axial position, the observed rings are compared with reference images of a bead at known axial positions. Often the precision or accuracy of these algorithms is measured on immobilized beads over a limited axial range, whereas many experiments are performed using freely mobile beads. This inconsistency raises the possibility of incorrect estimates of experimental uncertainty. By manipulating magnetic beads in a bidirectional magnetic tweezer setup, we evaluated the error associated with tracking mobile magnetic beads and found that the error of tracking a moving magnetic bead increases by almost an order of magnitude compared with the error of tracking a stationary bead. We found that this additional error can be ameliorated by excluding the center-most region of the diffraction ring pattern from tracking analysis. Evaluation of the limitations of a tracking algorithm is essential for understanding the error associated with a measurement. These findings promise to bring increased resolution to three-dimensional bead tracking of magnetic microspheres.

ABSTRACTMeasuring the traction forces produced by cells provides insight into their behavior and physiological function. Here, we developed a technique (dubbed ‘black dots’) that microcontact prints a fluorescent micropattern onto a flexible substrate to measure cellular traction forces without constraining cell shape or needing to detach the cells. To demonstrate our technique, we assessed human platelets, which can generate a large range of forces within a population. We find platelets that exert more force have more spread area, are more circular, and have more uniformly distributed F-actin filaments. As a result of the high yield of data obtainable by this technique, we were able to evaluate multivariate mixed effects models with interaction terms and conduct a clustering analysis to identify clusters within our data. These statistical techniques demonstrated a complex relationship between spread area, circularity, F-actin dispersion, and platelet force, including cooperative effects that significantly associate with platelet traction forces.GRAPHICAL ABSTRACT

Platelets bind, spread, and contract to stop bleeding and reinforce hemostatic plugs. Platelet contraction occurs when interactions between actin and myosin create cytoskeletal tension and force generation. In this study, single-cell platelet forces were measured using a novel microcontact-printed, reference-free traction force microscopy approach. This approach facilitates single-cell force measurement on a contiguous topology that enables unrestricted platelet spreading without necessitating platelet removal to calculate force. Microcontact printing is used to deposit a grid of fluorescent bovine serum albumin onto a flexible polydimethylsiloxane substrate. Platelets from healthy donors were washed and seeded onto von Willebrand Factor-coated substrates. Platelets were fixed, stained, and imaged via confocal microscopy to simultaneously visualize fluorescently labeled platelets and grid displacement (used to calculate single-cell force.) Because microcontact-printing facilitates printing the grid on large, scalable surfaces, platelet forces were able to be measured on >100 single-cells per donor (N = 6 donors). Donor-to-donor platelet force variability was examined, and some donors produce significantly more force per platelet than other donors (p<0.01). Even within each donor, ∼15-fold variability in single platelet forces exists. A well-established area-force relationship (more spread cells produce more force) accounts for some variability in force generation, but for cells spread equally, there is still ∼5-fold differences in force generation. After examining the relationship between cell shape and force, a relationship was observed wherein platelets that are more circular produce more force. Additionally, amongst spread platelets, platelets with more spread F-actin produce more force. This versatile method that facilitates measurement of single-cell forces over large areas without restricting cell spreading or requiring cell removal reveals donor variability, cell shape, and F-actin localization as contributors to the large variability of single-platelet forces.

Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an ‘inactive’ low-affinity state (LAS) to an ‘active’ high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH’s ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E . coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function.

Mechanical force exponentially weakens most protein-ligand interactions. However, some proteins possess catch bond properties in which the lifetime of the protein and ligand increases in the presence of force. Catch bonds have been observed in proteins including cell and bacterial adhesive proteins. One such bacterial adhesive, FimH, allows E. coli to resist strong shear forces found in high flow environments of the intestinal or urinary tract. In the presence of force, FimH has a remarkably long lifetime that has never been measured before. To fully characterize the profile of the FimH catch bond, we utilized a novel magnetic tweezer apparatus that can apply constant stable forces to multiple magnetic beads simultaneously. Magnetic beads coated in ligand were manipulated via electromagnets to form bonds with immobilized FimH. After the catch bond was activated by a preload force, the tension on the bond was held at a constant force until detachment. At forces up to 70 pN, many FimH bonds outlasted the 15-minute collection period. In fact, we estimated lifetime of the bond under 20-30 pN of force to be about 17 minutes. In contrast, other catch bond forming proteins, integrins, cadherins and P-selectins, last less than 10 seconds and the high affinity bond between biotin and streptavidin unbinds within a second within the same force range. Like other catch bonds, once the FimH catch bond is activated, increasing force negatively affects the bond lifetime. However, when the change in unbinding rate due to force was estimated, we found that FimH exhibited a relatively force insensitive behavior. We believe this relative force insensitivity contributes to FimH's remarkably long lifetime under force.

Adhesive interactions must withstand mechanical forces occurring in an organism. Forces have influenced some proteins such as integrins, clotting factors, and bacterial adhesive proteins to develop catch bond properties, in which the lifetime between a biomolecule and ligand increases with applied force. One of the most well studied catch bond behaviors is found between the E. coli adhesive protein, FimH, and its ligand, mannose. FimH has two distinct conformations with significantly different mannose affinities. The low affinity conformation predominates the initial FimH-mannose interactions, as this state, characterized with an open binding pocket, facilitates fast kinetics. A force pulling on the FimH-mannose interaction elongates FimH, inducing a transition into the high mannose affinity state, which includes a closed mannose binding pocket. While in this force-stabilized high affinity conformation, FimH has a remarkably long-lived lifetime that has never been fully measured before. We suspect the closed binding pocket is responsible for this lifetime, and mannose dissociation occurs when the pocket spontaneously opens. If the opening of this pocket occurs independent of the strength of the applied force, mannose unbinding would thus represent a force independent, or ideal bond, interaction. To explore this possibility, we measured the lifetimes of the mannose-FimH interaction at various forces with FimH in the high affinity state. We utilized a magnetic tweezer apparatus, to apply various constant forces to mannose-coated magnetic beads bound to immobilized FimH. While true ideal bond behavior cannot be concluded with certainty, the FimH-mannose bond often outlasted the 15-minute collection period, even at high forces relative to other catch bond forming proteins. Understanding the relationship between force and FimH's incredibly stable binding pocket, would grant further insight into the catch bond mechanism associated with FimH.

SignificancePyroptosis is a form of inflammatory cell death. It is driven by plasma membrane pore formation that is thought to rupture cells through osmotic influx, thereby releasing intracellular content that initiates an inflammatory response to fight acute infection and tissue damage. However, when chronic or unregulated, this process can drive autoimmune disease and cancer. Here, we show that pyroptotic cells do not undergo rupture in culture. Instead, the calcium-dependent protease calpain destroys intermediate filaments, which provide mechanical resilience to cells. As a result, cells are susceptible to rupture by mechanical disruption from shear stress or compression as occurs in the blood stream or within tissues. Thus, our data reveal potential targets for the therapeutic intervention of autoimmune diseases and cancer.

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.