Min oscillations in bacteria as real-time reporter of environmental challenges at the single-cell level.

Min oscillations are a fascinating mechanism used by Escherichia coli to find their middle. Beyond their biological role, they provide a convenient and relatively unexplored method to monitor the effect of sublethal environmental challenges on bacterial physiology in real-time and at the single-cell level. In this review, we discuss the original papers that put forward the idea of using Min oscillations as a reporting tool to monitor the effect of extracellular cationic compounds, including antibiotics. More recent work from our laboratory explores this tool to follow bacterial response to other challenges such as weak mechanical interactions with nanomaterials or photodynamic treatment. We discuss the physiological meaning of the changes in Min oscillation period, likely related to membrane potential dynamics, as well as the benefits and limitations of using oscillations as a reporter in fluorescence microscopy. Overall, Min oscillations are a useful addition to the fluorescence microscopy toolbox in order to visualize stress responses in E. coli, and have the potential to provide full mechanistic understanding of the events that lead to bacterial cell death in different contexts.

A molecular device for the redox quality control of GroEL/ES substrates.

Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10 cofactor) to GroEL induces CnoX release. Cryoelectron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal α-helix. Furthermore, we identified complexes in which CnoX, bound to GroEL, forms mixed disulfides with GroEL substrates, indicating that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, suggesting that Hsp60 molecular plugins have been conserved through evolution.

Versatile Near-Infrared Super-Resolution Imaging of Amyloid Fibrils with the Fluorogenic Probe CRANAD-2.

CRANAD-2 is a fluorogenic curcumin derivative used for near-infrared detection and imaging in vivo of amyloid aggregates, which are involved in neurodegenerative diseases. We explore the performance of CRANAD-2 in two super-resolution imaging techniques, namely stimulated emission depletion (STED) and single-molecule localization microscopy (SMLM), with markedly different fluorophore requirements. By conveniently adapting the concentration of CRANAD-2, which transiently binds to amyloid fibrils, we show that it performs well in both techniques, achieving a resolution in the range of 45-55 nm. Correlation of SMLM with atomic force microscopy (AFM) validates the resolution of fine features in the reconstructed super-resolved image. The good performance and versatility of CRANAD-2 provides a powerful tool for near-infrared nanoscopic imaging of amyloids in vitro and in vivo.

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.

Staphylococcus aureus vWF-binding protein triggers a strong interaction between clumping factor A and host vWF.

The Staphylococcus aureus cell wall-anchored adhesin ClfA binds to the very large blood circulating protein, von Willebrand factor (vWF) via vWF-binding protein (vWbp), a secreted protein that does not bind the cell wall covalently. Here we perform force spectroscopy studies on living bacteria to unravel the molecular mechanism of this interaction. We discover that the presence of all three binding partners leads to very high binding forces (2000 pN), largely outperforming other known ternary complexes involving adhesins. Strikingly, our experiments indicate that a direct interaction involving features of the dock, lock and latch mechanism must occur between ClfA and vWF to sustain the extreme tensile strength of the ternary complex. Our results support a previously undescribed mechanism whereby vWbp activates a direct, ultra-strong interaction between ClfA and vWF. This intriguing interaction represents a potential target for therapeutic interventions, including synthetic peptides inhibiting the ultra-strong interactions between ClfA and its ligands.

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.

Single-cell fluidic force microscopy reveals stress-dependent molecular interactions in yeast mating.

Sexual agglutinins of the budding yeast Saccharomyces cerevisiae are proteins mediating cell aggregation during mating. Complementary agglutinins expressed by cells of opposite mating types "a" and "α" bind together to promote agglutination and facilitate fusion of haploid cells. By means of an innovative single-cell manipulation assay combining fluidic force microscopy with force spectroscopy, we unravel the strength of single specific bonds between a- and α-agglutinins (~100 pN) which require pheromone induction. Prolonged cell-cell contact strongly increases adhesion between mating cells, likely resulting from an increased expression of agglutinins. In addition, we highlight the critical role of disulfide bonds of the a-agglutinin and of histidine residue H273 of α-agglutinin. Most interestingly, we find that mechanical tension enhances the interaction strength, pointing to a model where physical stress induces conformational changes in the agglutinins, from a weak-binding folded state, to a strong-binding extended state. Our single-cell technology shows promises for understanding and controlling the complex mechanism of yeast sexuality.

Stress-Induced Catch-Bonds to Enhance Bacterial Adhesion.

Physical forces have a profound influence on bacterial cell physiology and disease. A striking example is the formation of catch-bonds that reinforce under mechanical stress. While mannose-binding by the Escherichia coli FimH adhesin has long been the only thoroughly studied microbial catch-bond, it has recently become clear that proteins from other species, such as staphylococci, are also engaged in such stress-dependent interactions.

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.

Force-clamp spectroscopy identifies a catch bond mechanism in a Gram-positive pathogen.

Physical forces have profound effects on cellular behavior, physiology, and disease. Perhaps the most intruiguing and fascinating example is the formation of catch-bonds that strengthen cellular adhesion under shear stresses. Today mannose-binding by the Escherichia coli FimH adhesin remains one of the rare microbial catch-bond thoroughly characterized at the molecular level. Here we provide a quantitative demonstration of a catch-bond in living Gram-positive pathogens using force-clamp spectroscopy. We show that the dock, lock, and latch interaction between staphylococcal surface protein SpsD and fibrinogen is strong, and exhibits an unusual catch-slip transition. The bond lifetime first grows with force, but ultimately decreases to behave as a slip bond beyond a critical force (~1 nN) that is orders of magnitude higher than for previously investigated complexes. This catch-bond, never reported for a staphylococcal adhesin, provides the pathogen with a mechanism to tightly control its adhesive function during colonization and infection.

The Molecular Complex between Staphylococcal Adhesin SpsD and Fibronectin Sustains Mechanical Forces in the Nanonewton Range.

The bacterial pathogen Staphylococcus pseudintermedius is involved in canine otitis externa and pyoderma as well as in surgical wound and urinary tract infections. Invasion of canine epithelial cells is promoted by S. pseudintermedius fibronectin (Fn)-binding proteins SpsD and SpsL through molecular interactions that are currently unknown. By means of single-molecule experiments, we discover that both adhesins have distinct molecular mechanisms for binding to Fn. We show that the SpsD-Fn interaction has a strength equivalent to that of a covalent bond (∼1.5 to 1.8 nN), which is an order of magnitude stronger than the binding force of classical receptor-ligand complexes. We suggest that this extreme mechanostability originates from the ß-sheet organization of a tandem ß-zipper. Upon binding to FnI modules, the intrinsically disordered binding sequences of SpsD would shift into an ordered structure by forming additional ß-strands along triple peptide ß-sheets in the Fn molecule. Dynamic force measurements reveal an unexpected behavior, i.e., that strong bonds are activated by mechanical tension as observed with catch bonds. By contrast, the SpsL-Fn interaction involves multiple weak bonds (∼0.2 nN) that rupture sequentially under force. Together with the recently described dock, lock, and latch complex, the ultrastrong interaction unraveled here is among the strongest noncovalent biological interaction measured to date. Our findings may find applications for the identification of inhibitory compounds to treat infections triggered by pathogens engaged in tandem ß-zipper interactions.IMPORTANCE Binding of Staphylococcus pseudintermedius surface proteins SpsD and SpsL to fibronectin (Fn) plays a critical role in the invasion of canine epithelial cells. Here, we discover that both adhesins have different mechanisms for binding to Fn. The force required to separate SpsD from Fn is extremely strong, consistent with the unusual ß-sheet organization of a high-affinity tandem ß-zipper. By contrast, unbinding of the SpsL-Fn complex involves the sequential rupture of single weak bonds. Our findings may be of biological relevance as SpsD and SpsL are likely to play complementary roles during invasion. While the SpsD ß-zipper supports strong bacterial adhesion and triggers invasion, the weak SpsL interaction would favor fast detachment, enabling the pathogen to colonize new sites.

Nanomechanics of the molecular complex between staphylococcal adhesin SpsD and elastin.

Staphylococcus pseudintermedius surface protein SpsD binds to extracellular matrix proteins to invade canine epithelial cells. Using single-molecule experiments, we show that SpsD engages in two modes of interaction with elastin that are tightly controlled by physical stress. Binding is weak (∼100 pN) at low tensile force (i.e. loading rate), but is dramatically enhanced (up to ∼1500 pN) by mechanical tension. Consistent with a "dock, lock, and latch" (DLL) mechanism, this force represents among the highest mechanical strengths known for a non-covalent biological interaction. The transition from weak to strong binding correlates with an increase in molecular stiffness but, surprisingly, with a decrease in molecular extension. This unanticipated mechanical behavior indicates that the adhesin is engaged in two distinct interaction mechanisms. Our results emphasize the crucial role of protein nanomechanics in the adhesion of staphylococci, and illustrate their wide diversity of force-dependent ligand-binding activities. These single-molecule mechanical experiments may contribute to the development of antiadhesion approaches to treat infections caused by S. pseudintermedius and other bacterial pathogens engaged in DLL interactions.

Fast chemical force microscopy demonstrates that glycopeptidolipids define nanodomains of varying hydrophobicity on mycobacteria.

Mycobacterium abscessus is an emerging multidrug-resistant bacterial pathogen causing severe lung infections in cystic fibrosis patients. A remarkable trait of this mycobacterial species is its ability to form morphologically smooth (S) and rough (R) colonies. The S-to-R transition is caused by the loss of glycopeptidolipids (GPLs) in the outer layer of the cell envelope and correlates with an increase in cording and virulence. Despite the physiological and medical importance of this morphological transition, whether it involves changes in cell surface properties remains unknown. Herein, we combine recently developed quantitative imaging (QI) atomic force microscopy (AFM) with hydrophobic tips to quantitatively map the surface structure and hydrophobicity of M. abscessus at high spatiotemporal resolution, and to assess how these properties are modulated by the S-to-R transition and by treatment with an inhibitor of the mycolic acid transporter MmpL3. We discover that loss of GPLs leads to major modifications in surface hydrophobicity, without any apparent change in cell surface ultrastructure. While R bacilli are homogeneously hydrophobic, S bacilli feature unusual variations of nanoscale hydrophobic properties. These previously undescribed cell surface nanodomains are likely to play critical roles in bacterial adhesion, aggregation, phenotypic heterogeneity and transmission, and in turn in virulence and pathogenicity. Our study also suggests that MmpL3 inhibitors show promise in nanomedicine as chemotherapeutic agents to interfere with the highly hydrophobic nature of the mycobacterial cell wall. The advantages of QI-AFM with hydrophobic tips are the ability to map chemical and structural properties simultaneously and at high resolution, applicable to a wide range of biosystems.

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.

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.

Nanonewton forces between Staphylococcus aureus surface protein IsdB and vitronectin.

Single-molecule experiments have recently revealed that the interaction between staphylococcal surface proteins and their ligands can be extremely strong, equivalent to the strength of covalent bonds. Here, we report on the unusually high binding strength between Staphylococcus aureus iron-regulated surface determinant B (IsdB) and vitronectin (Vn), an essential human blood protein known to interact with bacterial pathogens. The IsdB-Vn interaction is dramatically strengthened by mechanical tension, with forces up to 2000 pN at a loading rate of 105 pN s-1. In line with this, flow experiments show that IsdB-mediated bacterial adhesion to Vn is enhanced by fluid shear stress. The stress-dependent binding of IsdB to Vn is likely to play a role in promoting bacterial adhesion to human cells under fluid shear stress conditions.

Adhesion of Staphylococcus aureus to Candida albicans During Co-Infection Promotes Bacterial Dissemination Through the Host Immune Response.

Interspecies interactions greatly influence the virulence, drug tolerance and ultimately the outcome of polymicrobial biofilm infections. A synergistic interaction is observed between the fungus Candida albicans and the bacterium Staphylococcus aureus. These species are both normal commensals of most healthy humans and co-exist in several niches of the host. However, under certain circumstances, they can cause hospital-acquired infections with high morbidity and mortality rates. Using a mouse model of oral co-infection, we previously showed that an oral infection with C. albicans predisposes to a secondary systemic infection with S. aureus. Here, we unraveled this intriguing mechanism of bacterial dissemination. Using static and dynamic adhesion assays in combination with single-cell force spectroscopy, we identified C. albicans Als1 and Als3 adhesins as the molecular players involved in the interaction with S. aureus and in subsequent bacterial dissemination. Remarkably, we identified the host immune response as a key element required for bacterial dissemination. We found that the level of immunosuppression of the host plays a critical yet paradoxical role in this process. In addition, secretion of candidalysin, the C. albicans peptide responsible for immune activation and cell damage, is required for C. albicans colonization and subsequent bacterial dissemination. The physical interaction with C. albicans enhances bacterial uptake by phagocytic immune cells, thereby enabling an opportunity to disseminate.

Mechanostability of the Fibrinogen Bridge between Staphylococcal Surface Protein ClfA and Endothelial Cell Integrin αVß3.

Binding of the Staphylococcus aureus surface protein clumping factor A (ClfA) to endothelial cell integrin αVß3 plays a crucial role during sepsis, by causing endothelial cell apoptosis and loss of barrier integrity. ClfA uses the blood plasma protein fibrinogen (Fg) to bind to αVß3 but how this is achieved at the molecular level is not known. Here we investigate the mechanical strength of the three-component ClfA-Fg-αVß3 interaction on living bacteria, by means of single-molecule experiments. We find that the ClfA-Fg-αVß3 ternary complex is extremely stable, being able to sustain forces (∼800 pN) that are much stronger than those of classical bonds between integrins and the Arg-Gly-Asp (RGD) tripeptide sequence (∼100 pN). Adhesion forces between single bacteria and αVß3 are strongly inhibited by an anti-αVß3 antibody, the RGD peptide, and the cyclic RGD peptide cilengitide, showing that formation of the complex involves RGD-dependent binding sites and can be efficiently inhibited by αVß3 blockers. Collectively, our experiments favor a binding mechanism involving the extraordinary elasticity of Fg. In the absence of mechanical stress, RGD572-574 sequences in the Aα chains mediate weak binding to αVß3, whereas under high mechanical stress exposure of cryptic Aα chain RGD95-97 sequences leads to extremely strong binding to the integrin. Our results identify an unexpected and previously undescribed force-dependent binding mechanism between ClfA and αVß3 on endothelial cells, which could represent a potential target to fight staphylococcal bloodstream infections.

Bacterial pathogens under high-tension: Staphylococcus aureus adhesion to von Willebrand factor is activated by force.

Attachment of Staphylococcus aureus to platelets and endothelial cells involves binding of bacterial cell surface protein A (SpA) to the large plasma glycoprotein von Willebrand factor (vWF). SpA-mediated bacterial adhesion to vWF is controlled by fluid shear stress, yet little is currently known about the underlying molecular mechanism. In a recent publication, we showed that the SpA-vWF interaction is tightly regulated by mechanical force. By means of single-molecule pulling experiments, we found that the SpA-vWF bond is extremely strong, being able to resist forces which largely outperform the strength of typical receptor-ligand bonds. In line with flow experiments, strong adhesion is activated by mechanical tension. These results suggest that force induces conformational changes in the vWF molecule, from a globular to an extended state, leading to the exposure of cryptic binding sites to which SpA strongly binds. This force-sensitive mechanism may largely contribute to help S. aureus bacteria to resist shear stress of flowing blood during infection.

Binding of Staphylococcus aureus Protein A to von Willebrand Factor Is Regulated by Mechanical Force.

Binding of Staphylococcus aureus to the large plasma glycoprotein von Willebrand factor (vWF) is controlled by hydrodynamic flow conditions. Currently, we know little about the molecular details of this shear-stress-dependent interaction. Using single-molecule atomic force microscopy, we demonstrate that vWF binds to the S. aureus surface protein A (SpA) via a previously undescribed force-sensitive mechanism. We identify an extremely strong SpA-vWF interaction, capable of withstanding forces of ∼2 nN, both in laboratory and in clinically relevant methicillin-resistant S. aureus (MRSA) strains. Strong bonds are activated by mechanical stress, consistent with flow experiments revealing that bacteria adhere in larger amounts to vWF surfaces when the shear rate is increased. We suggest that force-enhanced adhesion may involve conformational changes in vWF. Under force, elongation of vWF may lead to the exposure of a high-affinity cryptic SpA-binding site to which bacteria firmly attach. In addition, force-induced structural changes in the SpA domains may also promote strong, high-affinity binding. This force-regulated interaction might be of medical importance as it may play a role in bacterial adherence to platelets and to damaged blood vessels.IMPORTANCEStaphylococcus aureus protein A (SpA) binds to von Willebrand factor (vWF) under flow. While vWF binding to SpA plays a role in S. aureus adherence to platelets and endothelial cells under shear stress, the molecular basis of this stress-dependent interaction has not yet been elucidated. Here we show that the SpA-vWF interaction is regulated by a new force-dependent mechanism. The results suggest that mechanical extension of vWF may lead to the exposure of a high-affinity cryptic SpA-binding site, consistent with the shear force-controlled functions of vWF. Moreover, strong binding may be promoted by force-induced structural changes in the SpA domains. This study highlights the role of mechanoregulation in controlling the adhesion of S. aureus and shows promise for the design of small inhibitors capable of blocking colonization under high shear stress.