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.

Fluidic Force Microscopy Demonstrates That Homophilic Adhesion by Candida albicans Als Proteins Is Mediated by Amyloid Bonds between Cells.

The fungal pathogen Candida albicans frequently forms drug-resistant biofilms in hospital settings and in chronic disease patients. Cell adhesion and biofilm formation involve a family of cell surface Als (agglutinin-like sequence) proteins. It is now well documented that amyloid-like clusters of laterally arranged Als proteins activate cell-cell adhesion under mechanical stress, but whether amyloid-like bonds form between aggregating cells is not known. To address this issue, we measure the forces driving Als5-mediated intercellular adhesion using an innovative fluidic force microscopy platform. Strong cell-cell adhesion is dependent on expression of amyloid-forming Als5 at high cell surface density and is inhibited by a short antiamyloid peptide. Furthermore, there is greatly attenuated binding between cells expressing amyloid-forming Als5 and cells with a nonamyloid form of Als5. Thus, homophilic bonding between Als5 proteins on adhering cells is the major mode of fungal aggregation, rather than protein-ligand interactions. These results point to a model whereby amyloid-like ß-sheet interactions play a dual role in cell-cell adhesion, that is, in formation of adhesin nanoclusters ( cis-interactions) and in homophilic bonding between amyloid sequences on opposing cells ( trans-interactions). Because potential amyloid-forming sequences are found in many microbial adhesins, we speculate that this novel mechanism of amyloid-based homophilic adhesion might be widespread and could represent an interesting target for treating biofilm-associated infections.

The Surface Protein SdrF Mediates Staphylococcus epidermidis Adherence to Keratin.

Background: Staphylococcus epidermidis, a major component of skin flora, is an opportunist, often causing prosthetic device infections. A family of structurally related proteins mediates staphylococcal attachment to host tissues, contributing to the success of S. epidermidis as a pathogen. We examined the ability of the surface protein SdrF to adhere to keratin, a major molecule expressed on the skin surface. Methods: A heterologous Lactococcus lactis expression system was used to express SdrF and its ligand-binding domains. Adherence to keratin types 1 and 10, human foreskin keratinocytes, and nasal epithelial cells was examined. Results: SdrF bound human keratins 1 and 10 and adhered to keratinocytes and epithelial cells. Binding involved both the A and B domains. Anti-SdrF antibodies reduced adherence of S. epidermidis to keratin and keratinocytes. RNA interference reduced keratin synthesis in keratinocytes and, as a result, SdrF adherence. Direct force measurements using atomic force microscopy showed that SdrF mediates bacterial adhesion to keratin 10 through strong and weak bonds involving the A and B regions; strong adhesion was primarily mediated by the A region. Conclusions: These studies demonstrate that SdrF mediates adherence to human keratin and suggest that SdrF may facilitate S. epidermidis colonization of the skin.

Forces guiding staphylococcal adhesion.

Staphylococcus epidermidis and Staphylococcus aureus are two important nosocomial pathogens that form biofilms on indwelling medical devices. Biofilm infections are difficult to fight as cells within the biofilm show increased resistance to antibiotics. Our understanding of the molecular interactions driving bacterial adhesion, the first stage of biofilm formation, has long been hampered by the paucity of appropriate force-measuring techniques. In this minireview, we discuss how atomic force microscopy techniques have enabled to shed light on the molecular forces at play during staphylococcal adhesion. Specific highlights include the study of the binding mechanisms of adhesion molecules by means of single-molecule force spectroscopy, the measurement of the forces involved in whole cell interactions using single-cell force spectroscopy, and the probing of the nanobiophysical properties of living bacteria via multiparametric imaging. Collectively, these findings emphasize the notion that force and function are tightly connected in staphylococcal adhesion.

Atomic force microscopy reveals a dual collagen-binding activity for the staphylococcal surface protein SdrF.

Staphylococcus epidermidis causes nosocomial infections by colonizing and forming biofilms on indwelling medical devices. This process involves specific interactions between cell wall-anchored (CWA) proteins and host proteins adsorbed onto the biomaterial. Here, we have explored the molecular forces by which the S. epidermidis CWA protein serine-aspartate repeat protein F (SdrF) binds to type I collagen, by means of advanced atomic force microscopy (AFM) techniques. Using single-cell force spectroscopy, we found that SdrF mediates bacterial adhesion to collagen-coated substrates through both weak and strong bonds. Single-molecule force spectroscopy demonstrated that these bonds involve the A and B regions of SdrF, thus revealing that the protein is capable of dual ligand-binding activity. Both weak and strong bonds showed high dissociation rates, indicating they are much less stable than those formed by the well-characterized 'dock, lock and latch' mechanism. Collectively, our results show that CWA proteins can bind to ligands by novel mechanisms. We anticipate that AFM will greatly contribute to the identification of novel binding partners and binding mechanisms in staphylococcal CWA proteins.

Role of Ionic Strength in Staphylococcal Cell Aggregation.

Cell aggregation plays a key role in biofilm formation and pathogenesis of Staphylococcus species. Although the molecular basis of aggregation in Staphylococci has already been extensively investigated, the influence of environmental factors, such as ionic strength, remains poorly understood. In this paper, we report a new type of cellular aggregation of Staphylococci that depends solely on ionic strength. Seven strains out of 14, all belonging to staphylococcal species, formed large cell clusters within minutes in buffers of ionic strength ranging from 1.5 to 50 mM, whereas isolates belonging to other Gram-positive species did not display this phenotype. Atomic force microscopy (AFM) with chemically functionalized tips provided direct evidence that ionic strength modulates cell surface adhesive properties through changes in cell surface charge. The optimal ionic strength for aggregation was found to be strain dependent, but in all cases, bacterial aggregates formed at an ionic strength of 1.5-50 mM were rapidly dispersed in a solution of higher ionic strength, indicating a reversibility of the cell aggregation process. These findings suggest that some staphylococcal isolates can respond to ionic strength as an external stimulus to trigger rapid cell aggregation in a way that has not yet been reported.

Staphylococcus epidermidis Affinity for Fibrinogen-Coated Surfaces Correlates with the Abundance of the SdrG Adhesin on the Cell Surface.

Staphylococcus epidermidis is a world-leading pathogen in healthcare facilities, mainly causing medical device-associated infections. These nosocomial diseases often result in complications such as bacteremia, fibrosis, or peritonitis. The virulence of S. epidermidis relies on its ability to colonize surfaces and develop thereupon in the form of biofilms. Bacterial adherence on biomaterials, usually covered with plasma proteins after implantation, is a critical step leading to biofilm infections. The cell surface protein SdrG mediates adhesion of S. epidermidis to fibrinogen (Fg) through a specific "dock, lock, and latch" mechanism, which results in greatly stabilized protein-ligand complexes. Here, we combine single-molecule, single-cell, and whole population assays to investigate the extent to which the surface density of SdrG determines the ability of S. epidermidis clinical strains HB, ATCC 35984, and ATCC 12228 to bind to Fg-coated surfaces. Strains that showed enhanced adhesion on Fg-coated polydimethylsiloxane (PDMS) were characterized by increased amounts of SdrG proteins on the cell surface, as observed by single-molecule analysis. Consistent with previous reports showing increased expression of SdrG following in vivo exposure, this work provides direct evidence that abundance of SdrG on the cell surface of S. epidermidis strains dramatically improves their ability to bind to Fg-coated implanted medical devices.

An Amyloid Core Sequence in the Major Candida albicans Adhesin Als1p Mediates Cell-Cell Adhesion.

The human fungal commensal Candida albicans can become a serious opportunistic pathogen in immunocompromised hosts. The C. albicans cell adhesion protein Als1p is a highly expressed member of a large family of paralogous adhesins. Als1p can mediate binding to epithelial and endothelial cells, is upregulated in infections, and is important for biofilm formation. Als1p includes an amyloid-forming sequence at amino acids 325 to 331, identical to the sequence in the paralogs Als5p and Als3p. Therefore, we mutated Val326 to test whether this sequence is important for activity. Wild-type Als1p (Als1pWT) and Als1p with the V326N mutation (Als1pV326N) were expressed at similar levels in a Saccharomyces cerevisiae surface display model. Als1pV326N cells adhered to bovine serum albumin (BSA)-coated beads similarly to Als1pWT cells. However, cells displaying Als1pV326N showed visibly smaller aggregates and did not fluoresce in the presence of the amyloid-binding dye Thioflavin-T. A new analysis tool for single-molecule force spectroscopy-derived surface mapping showed that statistically significant force-dependent Als1p clustering occurred in Als1pWT cells but was absent in Als1pV326N cells. In single-cell force spectroscopy experiments, strong cell-cell adhesion was dependent on an intact amyloid core sequence on both interacting cells. Thus, the major adhesin Als1p interacts through amyloid-like ß-aggregation to cluster adhesin molecules in cis on the cell surface as well as in trans to form cell-cell bonds.IMPORTANCE Microbial cell surface adhesins control essential processes such as adhesion, colonization, and biofilm formation. In the opportunistic fungal pathogen Candida albicans, the agglutinin-like sequence (ALS) gene family encodes eight cell surface glycoproteins that mediate adherence to biotic and abiotic surfaces and cell-cell aggregation. Als proteins are critical for commensalism and virulence. Their activities include attachment and invasion of endothelial and epithelial cells, morphogenesis, and formation of biofilms on host tissue and indwelling medical catheters. At the molecular level, Als5p-mediated cell-cell aggregation is dependent on the formation of amyloid-like nanodomains between Als5p-expressing cells. A single-site mutation to valine 326 abolishes cellular aggregation and amyloid formation. Our results show that the binding characteristics of Als1p follow a mechanistic model similar to Als5p, despite its differential expression and biological roles.

Mechanical Forces Guiding Staphylococcus aureus Cellular Invasion.

Staphylococcus aureus can invade various types of mammalian cells, thereby enabling it to evade host immune defenses and antibiotics. The current model for cellular invasion involves the interaction between the bacterial cell surface located fibronectin (Fn)-binding proteins (FnBPA and FnBPB) and the α5ß1 integrin in the host cell membrane. While it is believed that the extracellular matrix protein Fn serves as a bridging molecule between FnBPs and integrins, the fundamental forces involved are not known. Using single-cell and single-molecule experiments, we unravel the molecular forces guiding S. aureus cellular invasion, focusing on the prototypical three-component FnBPA-Fn-integrin interaction. We show that FnBPA mediates bacterial adhesion to soluble Fn via strong forces (∼1500 pN), consistent with a high-affinity tandem ß-zipper, and that the FnBPA-Fn complex further binds to immobilized α5ß1 integrins with a strength much higher than that of the classical Fn-integrin bond (∼100 pN). The high mechanical stability of the Fn bridge favors an invasion model in which Fn binding by FnBPA leads to the exposure of cryptic integrin-binding sites via allosteric activation, which in turn engage in a strong interaction with integrins. This activation mechanism emphasizes the importance of protein mechanobiology in regulating bacterial-host adhesion. We also find that Fn-dependent adhesion between S. aureus and endothelial cells strengthens with time, suggesting that internalization occurs within a few minutes. Collectively, our results provide a molecular foundation for the ability of FnBPA to trigger host cell invasion by S. aureus and offer promising prospects for the development of therapeutic approaches against intracellular pathogens.

Fibrinogen Activates the Capture of Human Plasminogen by Staphylococcal Fibronectin-Binding Proteins.

Invasive bacterial pathogens can capture host plasminogen (Plg) and allow it to form plasmin. This process is of medical importance as surface-bound plasmin promotes bacterial spread by cleaving tissue components and favors immune evasion by degrading opsonins. In Staphylococcus aureus, Plg binding is in part mediated by cell surface fibronectin-binding proteins (FnBPs), but the underlying molecular mechanism is not known. Here, we use single-cell and single-molecule techniques to demonstrate that FnBPs capture Plg by a sophisticated activation mechanism involving fibrinogen (Fg), another ligand found in the blood. We show that while FnBPs bind to Plg through weak (∼200-pN) molecular bonds, direct interaction of the adhesins with Fg through the high-affinity dock, lock, and latch mechanism dramatically increases the strength of the FnBP-Plg bond (up to ∼2,000 pN). Our results point to a new model in which the binding of Fg triggers major conformational changes in the FnBP protein, resulting in the buried Plg-binding domains being projected and exposed away from the cell surface, thereby promoting strong interactions with Plg. This study demonstrated a previously unidentified role for a ligand-binding interaction by a staphylococcal cell surface protein, i.e., changing the protein orientation to activate a cryptic biological function.IMPORTANCEStaphylococcus aureus captures human plasminogen (Plg) via cell wall fibronectin-binding proteins (FnBPs), but the underlying molecular mechanism is not known. Here we show that the forces involved in the interaction between Plg and FnBPs on the S. aureus surface are weak. However, we discovered that binding of fibrinogen to FnBPs dramatically strengthens the FnBP-Plg bond, therefore revealing an unanticipated role for Fg in the capture of Plg by S. aureus These experiments favor a model where Fg-induced conformational changes in FnBPs promote their interaction with Plg. This work uncovers a previously undescribed activation mechanism for a staphylococcal surface protein, whereby ligand-binding elicits a cryptic biological function.

Mechanical Strength and Inhibition of the Staphylococcus aureus Collagen-Binding Protein Cna.

The bacterial pathogen Staphylococcus aureus expresses a variety of cell surface adhesion proteins that bind to host extracellular matrix proteins. Among these, the collagen (Cn)-binding protein Cna plays important roles in bacterium-host adherence and in immune evasion. While it is well established that the A region of Cna mediates ligand binding, whether the repetitive B region has a dedicated function is not known. Here, we report the direct measurement of the mechanical strength of Cna-Cn bonds on living bacteria, and we quantify the antiadhesion activity of monoclonal antibodies (MAbs) targeting this interaction. We demonstrate that the strength of Cna-Cn bonds in vivo is very strong (~1.2 nN), consistent with the high-affinity "collagen hug" mechanism. The B region is required for strong ligand binding and has been found to function as a spring capable of sustaining high forces. This previously undescribed mechanical response of the B region is of biological significance as it provides a means to project the A region away from the bacterial surface and to maintain bacterial adhesion under conditions of high forces. We further quantified the antiadhesion activity of MAbs raised against the A region of Cna directly on living bacteria without the need for labeling or purification. Some MAbs are more efficient in blocking single-cell adhesion, suggesting that they act as competitive inhibitors that bind Cna residues directly involved in ligand binding. This report highlights the role of protein mechanics in activating the function of staphylococcal adhesion proteins and emphasizes the potential of antibodies to prevent staphylococcal adhesion and biofilm formation. IMPORTANCE: Cna is a collagen (Cn)-binding protein from Staphylococcus aureus that is involved in pathogenesis. Currently, we know little about the functional role of the repetitive B region of the protein. Here, we unravel the mechanical strength of Cna in living bacteria. We show that single Cna-Cn bonds are very strong, reflecting high-affinity binding by the collagen hug mechanism. We discovered that the B region behaves as a nanospring capable of sustaining high forces. This unanticipated mechanical response, not previously described for any staphylococcal adhesin, favors a model in which the B region has a mechanical function that is essential for strong ligand binding. Finally, we assess the antiadhesion activity of monoclonal antibodies against Cna, suggesting that they could be used to inhibit S. aureus adhesion.

Staphylococcus aureus Fibronectin-Binding Protein A Mediates Cell-Cell Adhesion through Low-Affinity Homophilic Bonds.

UNLABELLED: Staphylococcus aureus is an important opportunistic pathogen which is a leading cause of biofilm-associated infections on indwelling medical devices. The cell surface-located fibronectin-binding protein A (FnBPA) plays an important role in the accumulation phase of biofilm formation by methicillin-resistant S. aureus (MRSA), but the underlying molecular interactions are not yet established. Here, we use single-cell and single-molecule atomic force microscopy to unravel the mechanism by which FnBPA mediates intercellular adhesion. We show that FnBPA is responsible for specific cell-cell interactions that involve the FnBPA A domain and cause microscale cell aggregation. We demonstrate that the strength of FnBPA-mediated adhesion originates from multiple low-affinity homophilic interactions between FnBPA A domains on neighboring cells. Low-affinity binding by means of FnBPA may be important for biofilm dynamics. These results provide a molecular basis for the ability of FnBPA to promote cell accumulation during S. aureus biofilm formation. We speculate that homophilic interactions may represent a generic strategy among staphylococcal cell surface proteins for guiding intercellular adhesion. As biofilm formation by MRSA strains depends on proteins rather than polysaccharides, our approach offers exciting prospects for the design of drugs or vaccines to inhibit protein-dependent intercellular interactions in MRSA biofilms. IMPORTANCE: Staphylococcus aureus is a human pathogen that forms biofilms on indwelling medical devices, such as central venous catheters and prosthetic joints. This leads to biofilm infections that are difficult to treat with antibiotics because many cells within the biofilm matrix are dormant. The fibronectin-binding proteins (FnBPs) FnBPA and FnBPB promote biofilm formation by clinically relevant methicillin-resistant S. aureus (MRSA) strains, but the molecular mechanisms involved remain poorly understood. We used atomic force microscopy techniques to demonstrate that FnBPA mediates cell-cell adhesion via multiple, low-affinity homophilic bonds between FnBPA A domains on adjacent cells. Therefore, FnBP-mediated homophilic interactions represent an interesting target to prevent MRSA biofilms. We propose that such homophilic mechanisms may be widespread among staphylococcal cell surface proteins, providing a means to guide intercellular adhesion and biofilm accumulation.