Wendy E. Thomas

Bacterial Adhesion to Target Cells Enhanced by Shear Force

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

Biophysics of Catch Bonds

Receptor-ligand bonds strengthened by tensile mechanical force are referred to as catch bonds. This review examines experimental data and biophysical theory to analyze why mechanical force prolongs the lifetime of these bonds rather than shortens the lifetime by pulling the ligand out of the binding pocket. Although many mathematical models can explain catch bonds, experiments using structural variants have been more helpful in determining how catch bonds work. The underlying mechanism has been worked out so far only for the bacterial adhesive protein FimH. This protein forms catch bonds because it is allosterically activated when mechanical force pulls an inhibitory domain away from the ligand-binding domain. Other catch bond-forming proteins, including blood cell adhesion proteins called selectins and the motor protein myosin, show evidence of allosteric regulation between two domains, but it remains unclear if this is related to their catch bond behavior.

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.

Structural Basis for Mechanical Force Regulation of the Adhesin FimH via Finger Trap-like β Sheet Twisting

The Escherichia coli fimbrial adhesive protein, FimH, mediates shear-dependent binding to mannosylated surfaces via force-enhanced allosteric catch bonds, but the underlying structural mechanism was previously unknown. Here we present the crystal structure of FimH incorporated into the multiprotein fimbrial tip, where the anchoring (pilin) domain of FimH interacts with the mannose-binding (lectin) domain and causes a twist in the beta sandwich fold of the latter. This loosens the mannose-binding pocket on the opposite end of the lectin domain, resulting in an inactive low-affinity state of the adhesin. The autoinhibition effect of the pilin domain is removed by application of tensile force across the bond, which separates the domains and causes the lectin domain to untwist and clamp tightly around the ligand like a finger-trap toy. Thus, beta sandwich domains, which are common in multidomain proteins exposed to tensile force in vivo, can undergo drastic allosteric changes and be subjected to mechanical regulation.

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.

A structural model for force regulated integrin binding to fibronectin's RGD-synergy site.

The synergy site on fibronectins FN-III(9) module, located approximately 32 A away from the RGD-loop on FN-III(10), greatly enhances integrin alpha(5)beta(1) mediated cell binding. Since fibronectin is exposed to mechanical forces acting on the extracellular matrix in vivo, we used steered molecular dynamics to study how mechanical stretching of FN-III(9-10) affects the relative distance between these two synergistic sites. Our simulations predict the existence of an intermediate state prior to unfolding. In this state, the synergy-RGD distance is increased from 32 A to approximately 55 A, while the conformations of both sites remain unperturbed. This distance is too large for both sites to co-bind the same receptor, as indicated by experiments that confirm that increasing the length of the linker chain between FN-III(9) and FN-III(10) reduces alpha(5)beta(1) binding. Our simulations thus suggest that increased alpha(5)beta(1)-binding attributed to the synergy site, along with the associated downstream cell-signaling events, can be turned off mechanically by stretching FN-III(9-10) into this intermediate state. The potential physiological implications are discussed.

Catch bonds in adhesion.

One of the most exciting discoveries in biological adhesion is the recent and counter-intuitive observation that the lifetimes of some biological adhesive bonds, called catch bonds, are enhanced by tensile mechanical force. At least two types of adhesive proteins have been shown to form catch bonds--blood proteins called selectins and a bacterial protein called FimH. Both mediate shear-enhanced adhesion, in which cells bind more strongly at high shear than at low shear. Single-molecule experiments and cell-free assays have now clearly demonstrated that catch bonds exist and mediate shear-enhanced adhesion. However, the mechanics of cellular organelles also contribute to shear-enhanced adhesion by modulating the force applied to catch bonds. This review examines how individual catch bond behavior contributes to shear-enhanced cellular adhesion for the two best-understood examples. The lessons from these systems offer design principles for understanding other types of shear-enhanced adhesion and for engineering nanostructured force-dependent adhesives out of catch bonds.

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.

Catch-Bond Mechanism of Force-Enhanced Adhesion: Counterintuitive, Elusive, but … Widespread?

Catch bonds are bonds between a ligand and its receptor that are enhanced by mechanical force pulling the ligand-receptor complex apart. To date, catch-bond formation has been documented for the most common Escherichia coli adhesin, FimH, and for P-/L-selectins, universally expressed by leukocytes, platelets, and blood vessel walls. One compelling explanation for catch bonds is that force-induced structural alterations in the receptor protein are allosterically linked to a high-affinity conformation of its ligand-binding pocket. Catch-bond properties are likely to be widespread among adhesive proteins, thus calling for a detailed understanding of their underlying mechanisms and physiological significance.