Mechanobiology: Shaping the future of cellular form and function.

Mechanobiology-the field studying how cells produce, sense, and respond to mechanical forces-is pivotal in the analysis of how cells and tissues take shape in development and disease. As we venture into the future of this field, pioneers share their insights, shaping the trajectory of future research and applications.

Mechanobiology: How pathogens use mechanics to modulate host interactions.

Mechanobiology explores how cells sense and respond to mechanical cues and how mechanics guide cell function, physiology, and disease. In this issue of Cell, Thacker and colleagues reveal how the tuberculosis-causing pathogen exploits the mechanical behavior of cord-like structures to promote infection, impacting immune response, antibiotic susceptibility, and treatment strategies.

Tight-packing of large pilin subunits provides distinct structural and mechanical properties for the Myxococcus xanthus type IVa pilus.

Type IVa pili (T4aP) are ubiquitous cell surface filaments important for surface motility, adhesion to surfaces, DNA uptake, biofilm formation, and virulence. T4aP are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While major pilins of structurally characterized T4aP have lengths of <165 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a conserved N-terminal domain and a variable C-terminal domain, and the additional residues of PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4aP (T4aPMx) at a resolution of 3.0 Å using cryo-EM. The T4aPMx follows the structural blueprint of other T4aP with the pilus core comprised of the interacting N-terminal α1-helices, while the globular domains decorate the T4aP surface. The atomic model of PilA built into this map shows that the large C-terminal domain has more extensive intersubunit contacts than major pilins in other T4aP. As expected from these greater contacts, the bending and axial stiffness of the T4aPMx is significantly higher than that of other T4aP and supports T4aP-dependent motility on surfaces of different stiffnesses. Notably, T4aPMx variants with interrupted intersubunit interfaces had decreased bending stiffness, pilus length, and strongly reduced motility. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4aP that expands the environmental conditions in which the T4aP system functions.

Nanomechanics of CCN1-Mediated Staphylococcus aureus Phagocytosis.

Phagocytosis is an essential mechanism of the human immune system where pathogens are eliminated by immune cells. The CCN1 protein plays an important role in the phagocytosis of Staphylococcus aureus by favoring the bridging of the αVß3 integrin to the bacterial peptidoglycan (PG), through mechanical forces that remain unknown. Here, we employ single-molecule experiments to unravel the nanomechanics of the PG-CCN1-αVß3 ternary complex. While CCN1 binds αVß3 integrins with moderate force (∼60 pN), much higher binding strengths (up to ∼800 pN) are observed between CCN1 and PG. Notably, the strength of both CCN1-αVß3 and CCN1-PG bonds is dramatically enhanced by tensile loading, favoring a model in which mechanical stress induces the exposure of cryptic integrin binding sites in CCN1 and multivalent binding between CCN1 lectin sites and monosaccharides along the PG glycan chains.

Staphylococcus aureus binds to the N-terminal region of corneodesmosin to adhere to the stratum corneum in atopic dermatitis.

Staphylococcus aureus colonizes the skin of the majority of patients with atopic dermatitis (AD), and its presence increases disease severity. Adhesion of S. aureus to corneocytes in the stratum corneum is a key initial event in colonization, but the bacterial and host factors contributing to this process have not been defined. Here, we show that S. aureus interacts with the host protein corneodesmosin. Corneodesmosin is aberrantly displayed on the tips of villus-like projections that occur on the surface of AD corneocytes as a result of low levels of skin humectants known as natural moisturizing factor (NMF). An S. aureus mutant deficient in fibronectin binding protein B (FnBPB) and clumping factor B (ClfB) did not bind to corneodesmosin in vitro. Using surface plasmon resonance, we found that FnBPB and ClfB proteins bound with similar affinities. The S. aureus binding site was localized to the N-terminal glycine-serine-rich region of corneodesmosin. Atomic force microscopy showed that the N-terminal region was present on corneocytes containing low levels of NMF and that blocking it with an antibody inhibited binding of individual S. aureus cells to corneocytes. Finally, we found that S. aureus mutants deficient in FnBPB or ClfB have a reduced ability to adhere to low-NMF corneocytes from patients. In summary, we show that FnBPB and ClfB interact with the accessible N-terminal region of corneodesmosin on AD corneocytes, allowing S. aureus to take advantage of the aberrant display of corneodesmosin that accompanies low NMF in AD. This interaction facilitates the characteristic strong binding of S. aureus to AD corneocytes.

Catch Bond-Mediated Adhesion Drives Staphylococcus aureus Host Cell Invasion.

Various viruses and pathogenic bacteria interact with annexin A2 to invade mammalian cells. Here, we show that Staphylococcus aureus engages in extremely strong catch bonds for host cell invasion. By means of single-molecule atomic force microscopy, we find that bacterial surface-located clumping factors bind annexin A2 with extraordinary strength, indicating that these bonds are extremely resilient to mechanical tension. By determining the lifetimes of the complexes under increasing mechanical stress, we demonstrate that the adhesins form catch bonds with their ligand that are capable to sustain forces of 1500-1700 pN. The force-dependent adhesion mechanism identified here provides a molecular framework to explain how S. aureus pathogens tightly attach to host cells during invasion and shows promise for the design of new therapeutics against intracellular S. aureus.

Localized incorporation of outer membrane components in the pathogen Brucella abortus.

The zoonotic pathogen Brucella abortus is part of the Rhizobiales, which are alpha-proteobacteria displaying unipolar growth. Here, we show that this bacterium exhibits heterogeneity in its outer membrane composition, with clusters of rough lipopolysaccharide co-localizing with the essential outer membrane porin Omp2b, which is proposed to allow facilitated diffusion of solutes through the porin. We also show that the major outer membrane protein Omp25 and peptidoglycan are incorporated at the new pole and the division site, the expected growth sites. Interestingly, lipopolysaccharide is also inserted at the same growth sites. The absence of long-range diffusion of main components of the outer membrane could explain the apparent immobility of the Omp2b clusters, as well as unipolar and mid-cell localizations of newly incorporated outer membrane proteins and lipopolysaccharide. Unipolar growth and limited mobility of surface structures also suggest that new surface variants could arise in a few generations without the need of diluting pre-existing surface antigens.

Atomic Force Microscopy-Based Force Spectroscopy and Multiparametric Imaging of Biomolecular and Cellular Systems.

During the last three decades, a series of key technological improvements turned atomic force microscopy (AFM) into a nanoscopic laboratory to directly observe and chemically characterize molecular and cell biological systems under physiological conditions. Here, we review key technological improvements that have established AFM as an analytical tool to observe and quantify native biological systems from the micro- to the nanoscale. Native biological systems include living tissues, cells, and cellular components such as single or complexed proteins, nucleic acids, lipids, or sugars. We showcase the procedures to customize nanoscopic chemical laboratories by functionalizing AFM tips and outline the advantages and limitations in applying different AFM modes to chemically image, sense, and manipulate biosystems at (sub)nanometer spatial and millisecond temporal resolution. We further discuss theoretical approaches to extract the kinetic and thermodynamic parameters of specific biomolecular interactions detected by AFM for single bonds and extend the discussion to multiple bonds. Finally, we highlight the potential of combining AFM with optical microscopy and spectroscopy to address the full complexity of biological systems and to tackle fundamental challenges in life sciences.

Coevolution of the bacterial pheromone ComS and sensor ComR fine-tunes natural transformation in streptococci.

Competence for natural transformation extensively contributes to genome evolution and the rapid adaptability of bacteria dwelling in challenging environments. In most streptococci, this process is tightly controlled by the ComRS signaling system, which is activated through the direct interaction between the (R)RNPP-type ComR sensor and XIP pheromone (mature ComS). The overall mechanism of activation and the basis of pheromone selectivity have been previously reported in Gram-positive salivarius streptococci; however, detailed 3D-remodeling of ComR leading up to its activation remains only partially understood. Here, we identified using a semirational mutagenesis approach two residues in the pheromone XIP that bolster ComR sensor activation by interacting with two aromatic residues of its XIP-binding pocket. Random and targeted mutagenesis of ComR revealed that the interplay between these four residues remodels a network of aromatic-aromatic interactions involved in relaxing the sequestration of the DNA-binding domain. Based on these data, we propose a comprehensive model for ComR activation based on two major conformational changes of the XIP-binding domain. Notably, the stimulation of this newly identified trigger point by a single XIP substitution resulted in higher competence and enhanced transformability, suggesting that pheromone-sensor coevolution counter-selects for hyperactive systems in order to maintain a trade-off between competence and bacterial fitness. Overall, this study sheds new light on the ComRS activation mechanism and how it could be exploited for biotechnological and biomedical purposes.

Modulation of plant plasma membrane structure by exogenous fatty acid hydroperoxide is a potential perception mechanism for their eliciting activity.

Oxylipins are lipid-derived molecules that are ubiquitous in eukaryotes and whose functions in plant physiology have been widely reported. They appear to play a major role in plant immunity by orchestrating reactive oxygen species (ROS) and hormone-dependent signalling pathways. The present work focuses on the specific case of fatty acid hydroperoxides (HPOs). Although some studies report their potential use as exogenous biocontrol agents for plant protection, evaluation of their efficiency in planta is lacking and no information is available about their mechanism of action. In this study, the potential of 13(S)-hydroperoxy-(9Z, 11E)-octadecadienoic acid (13-HPOD) and 13(S)-hydroperoxy-(9Z, 11E, 15Z)-octadecatrienoic acid (13-HPOT), as plant defence elicitors and the underlying mechanism of action is investigated. Arabidopsis thaliana leaf resistance to Botrytis cinerea was observed after root application with HPOs. They also activate early immunity-related defence responses, like ROS. As previous studies have demonstrated their ability to interact with plant plasma membranes (PPM), we have further investigated the effects of HPOs on biomimetic PPM structure using complementary biophysics tools. Results show that HPO insertion into PPM impacts its global structure without solubilizing it. The relationship between biological assays and biophysical analysis suggests that lipid amphiphilic elicitors that directly act on membrane lipids might trigger early plant defence events.

AFM in cellular and molecular microbiology.

The unique capabilities of the atomic force microscope (AFM), including super-resolution imaging, piconewton force-sensitivity, nanomanipulation and ability to work under physiological conditions, have offered exciting avenues for cellular and molecular biology research. AFM imaging has helped unravel the fine architectures of microbial cell envelopes at the nanoscale, and how these are altered by antimicrobial treatment. Nanomechanical measurements have shed new light on the elasticity, tensile strength and turgor pressure of single cells. Single-molecule and single-cell force spectroscopy experiments have revealed the forces and dynamics of receptor-ligand interactions, the nanoscale distribution of receptors on the cell surface and the elasticity and adhesiveness of bacterial pili. Importantly, recent force spectroscopy studies have demonstrated that extremely stable bonds are formed between bacterial adhesins and their cognate ligands, originating from a catch bond behaviour allowing the pathogen to reinforce adhesion under shear or tensile stress. Here, we survey how the versatility of AFM has enabled addressing crucial questions in microbiology, with emphasis on bacterial pathogens. TAKE AWAYS: AFM topographic imaging unravels the ultrastructure of bacterial envelopes. Nanomechanical mapping shows what makes cell envelopes stiff and resistant to drugs. Force spectroscopy characterises the molecular forces in pathogen adhesion. Stretching pili reveals a wealth of mechanical and adhesive responses.

AFM Identifies a Protein Complex Involved in Pathogen Adhesion Which Ruptures at Three Nanonewtons.

Staphylococci bind to the blood protein von Willebrand Factor (vWF), thereby causing endovascular infections. Whether and how this interaction occurs with the medically important pathogen Staphylococcus epidermidis is unknown. Using single-molecule experiments, we demonstrate that the S. epidermidis protein Aap binds vWF via an ultrastrong force, ∼3 nN, the strongest noncovalent biological bond ever reported, and we show that this interaction is activated by tensile loading, suggesting a catch-bond behavior. Aap-vWF binding involves exclusively the A1 domain of vWF but requires both the A and B domains of Aap, as revealed by inhibition assays using specific monoclonal antibodies. Collectively, our results point to a mechanism where force-induced unfolding of the B repeats activates the A domain of Aap, shifting it from a weak- to a strong-binding state, which then engages into an ultrastrong interaction with vWF A1. This shear-dependent function of Aap offers promise for innovative antistaphylococcal therapies.

AFM Unravels the Unique Adhesion Properties of the Caulobacter Type IVc Pilus Nanomachine.

Bacterial pili are proteinaceous motorized nanomachines that play various functional roles including surface adherence, bacterial motion, and virulence. The surface-contact sensor type IVc (or Tad) pilus is widely distributed in both Gram-positive and Gram-negative bacteria. In Caulobacter crescentus, this nanofilament, though crucial for surface colonization, has never been thoroughly investigated at the molecular level. As Caulobacter assembles several surface appendages at specific stages of the cell cycle, we designed a fluorescence-based screen to selectively study single piliated cells and combined it with atomic force microscopy and genetic manipulation to quantify the nanoscale adhesion of the type IVc pilus to hydrophobic substrates. We demonstrate that this nanofilament exhibits high stickiness compared to the canonical type IVa/b pili, resulting mostly from multiple hydrophobic interactions along the fiber length, and that it features nanospring mechanical properties. Our findings may be helpful to better understand the structure-function relationship of bacterial pilus nanomachines.

Seeing and Touching the Mycomembrane at the Nanoscale.

Mycobacteria have unique cell envelopes, surface properties, and growth dynamics, which all play a part in the ability of these important pathogens to infect, evade host immunity, disseminate, and resist antibiotic challenges. Recent atomic force microscopy (AFM) studies have brought new insights into the nanometer-scale ultrastructural, adhesive, and mechanical properties of mycobacteria. The molecular forces with which mycobacterial adhesins bind to host factors, like heparin and fibronectin, and the hydrophobic properties of the mycomembrane have been unraveled by AFM force spectroscopy studies. Real-time correlative AFM and fluorescence imaging have delineated a complex interplay between surface ultrastructure, tensile stresses within the cell envelope, and cellular processes leading to division. The unique capabilities of AFM, which include subdiffraction-limit topographic imaging and piconewton force sensitivity, have great potential to resolve important questions that remain unanswered on the molecular interactions, surface properties, and growth dynamics of this important class of pathogens.

What makes bacterial pathogens so sticky?

Pathogenic bacteria use a variety of cell surface adhesins to promote binding to host tissues and protein-coated biomaterials, as well as cell-cell aggregation. These cellular interactions represent the first essential step that leads to host colonization and infection. Atomic force microscopy (AFM) has greatly contributed to increase our understanding of the specific interactions at play during microbial adhesion, down to the single-molecule level. A key asset of AFM is that adhesive interactions are studied under mechanical force, which is highly relevant as surface-attached pathogens are often exposed to physical stresses in the human body. These studies have identified sophisticated binding mechanisms in adhesins, which represent promising new targets for antiadhesion therapy.

Host-specialized fibrinogen-binding by a bacterial surface protein promotes biofilm formation and innate immune evasion.

Fibrinogen is an essential part of the blood coagulation cascade and a major component of the extracellular matrix in mammals. The interface between fibrinogen and bacterial pathogens is an important determinant of the outcome of infection. Here, we demonstrate that a canine host-restricted skin pathogen, Staphylococcus pseudintermedius, produces a cell wall-associated protein (SpsL) that has evolved the capacity for high strength binding to canine fibrinogen, with reduced binding to fibrinogen of other mammalian species including humans. Binding occurs via the surface-expressed N2N3 subdomains, of the SpsL A-domain, to multiple sites in the fibrinogen α-chain C-domain by a mechanism analogous to the classical dock, lock, and latch binding model. Host-specific binding is dependent on a tandem repeat region of the fibrinogen α-chain, a region highly divergent between mammals. Of note, we discovered that the tandem repeat region is also polymorphic in different canine breeds suggesting a potential influence on canine host susceptibility to S. pseudintermedius infection. Importantly, the strong host-specific fibrinogen-binding interaction of SpsL to canine fibrinogen is essential for bacterial aggregation and biofilm formation, and promotes resistance to neutrophil phagocytosis, suggesting a key role for the interaction during pathogenesis. Taken together, we have dissected a bacterial surface protein-ligand interaction resulting from the co-evolution of host and pathogen that promotes host-specific innate immune evasion and may contribute to its host-restricted ecology.

Staphylococcus aureus adhesion in endovascular infections is controlled by the ArlRS-MgrA signaling cascade.

Staphylococcus aureus is a leading cause of endovascular infections. This bacterial pathogen uses a diverse array of surface adhesins to clump in blood and adhere to vessel walls, leading to endothelial damage, development of intravascular vegetations and secondary infectious foci, and overall disease progression. In this work, we describe a novel strategy used by S. aureus to control adhesion and clumping through activity of the ArlRS two-component regulatory system, and its downstream effector MgrA. Utilizing a combination of in vitro cellular assays, and single-cell atomic force microscopy, we demonstrated that inactivation of this ArlRS-MgrA cascade inhibits S. aureus adhesion to a vast array of relevant host molecules (fibrinogen, fibronectin, von Willebrand factor, collagen), its clumping with fibrinogen, and its attachment to human endothelial cells and vascular structures. This impact on S. aureus adhesion was apparent in low shear environments, and in physiological levels of shear stress, as well as in vivo in mouse models. These effects were likely mediated by the de-repression of giant surface proteins Ebh, SraP, and SasG, caused by inactivation of the ArlRS-MgrA cascade. In our in vitro assays, these giant proteins collectively shielded the function of other surface adhesins and impaired their binding to cognate ligands. Finally, we demonstrated that the ArlRS-MgrA regulatory cascade is a druggable target through the identification of a small-molecule inhibitor of ArlRS signaling. Our findings suggest a novel approach for the pharmacological treatment and prevention of S. aureus endovascular infections through targeting the ArlRS-MgrA regulatory system.

Staphylococcus aureus clumping factor A is a force-sensitive molecular switch that activates bacterial adhesion.

Clumping factor A (ClfA), a cell-wall-anchored protein from Staphylococcus aureus, is a virulence factor in various infections and facilitates the colonization of protein-coated biomaterials. ClfA promotes bacterial adhesion to the blood plasma protein fibrinogen (Fg) via molecular forces that have not been studied so far. A unique, yet poorly understood, feature of ClfA is its ability to favor adhesion to Fg at high shear stress. Unraveling the strength and dynamics of the ClfA-Fg interaction would help us better understand how S. aureus colonizes implanted devices and withstands physiological shear stress. By means of single-molecule experiments, we show that ClfA behaves as a force-sensitive molecular switch that potentiates staphylococcal adhesion under mechanical stress. The bond between ClfA and immobilized Fg is weak (∼0.1 nN) at low tensile force, but is dramatically enhanced (∼1.5 nN) by mechanical tension, as observed with catch bonds. Strong bonds, but not weak ones, are inhibited by a peptide mimicking the C-terminal segment of the Fg γ-chain. These results point to a model whereby ClfA interacts with Fg via two distinct binding sites, the adhesive function of which is regulated by mechanical tension. This force-activated mechanism is of biological significance because it explains at the molecular level the ability of ClfA to promote bacterial attachment under high physiological shear stress.

Single-Molecule Analysis Demonstrates Stress-Enhanced Binding between Staphylococcus aureus Surface Protein IsdB and Host Cell Integrins.

Binding of Staphylococcus aureus surface proteins to endothelial cell integrins plays essential roles in host cell adhesion and invasion, eventually leading to life-threatening diseases. The staphylococcal protein IsdB binds to ß3-containing integrins through a mechanism that has never been thoroughly investigated. Here, we identify and characterize at the nanoscale a previously undescribed stress-dependent adhesion between IsdB and integrin αVß3. The strength of single IsdB-αVß3 interactions is moderate (∼100 pN) under low stress, but it increases dramatically under high stress (∼1000-2000 pN) to exceed the forces traditionally reported for the binding between integrins and Arg-Gly-Asp (RGD) sequences. We suggest a mechanism where high mechanical stress induces conformational changes in the integrin from a low-affinity, weak binding state to a high-affinity, strong binding state. This single-molecule study highlights that direct adhesin-integrin interactions represent potential targets to fight staphylococcal infections.

How Microbes Use Force To Control Adhesion.

Microbial adhesion and biofilm formation are usually studied using molecular and cellular biology assays, optical and electron microscopy, or laminar flow chamber experiments. Today, atomic force microscopy (AFM) represents a valuable addition to these approaches, enabling the measurement of forces involved in microbial adhesion at the single-molecule level. In this minireview, we discuss recent discoveries made applying state-of-the-art AFM techniques to microbial specimens in order to understand the strength and dynamics of adhesive interactions. These studies shed new light on the molecular mechanisms of adhesion and demonstrate an intimate relationship between force and function in microbial adhesins.