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Single-molecule atomic force microscopy studies of microbial pathogens
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Single-molecule atomic force microscopy studies of microbial pathogens Marion Mathelié-Guinlet1, Felipe Viela1, Albertus Viljoen1, Jérôme Dehullu1 and Yves F. Dufrêne1,2 Abstract
Atomic force microscopy (AFM) has become a powerful multifunctional platform for probing microbial cell surfaces, one molecule at a time, thereby uncovering biophysical properties and interactions that are otherwise not accessible. Single-cell force spectroscopy has been widely used to quantify the adhesion forces of pathogens, including viruses, bacteria, and fungi, whereas single-molecule force spectroscopy has enabled the functional analysis and imaging of individual receptors on cell surfaces. In addition, AFM has been instrumental in assessing the inhibition of adhesion of pathogens by carbohydrates, antibodies, and peptides, thus showing promise for antiadhesion therapy. Addresses 1 Louvain Institute of Biomolecular Science and Technology, Université catholique de Louvain, Croix du Sud, 4-5, bte L7.07.06., B-1348, Louvain-la-Neuve, Belgium 2 Walloon Excellence in Life Sciences and Biotechnology (WELBIO), B-1300, Wavre, Belgium Corresponding author: Dufrêne, Yves F (Yves.Dufrene@uclouvain. be)
Current Opinion in Biomedical Engineering 2019, 12:1–7 This review comes from a themed issue on Molecular & Cellular Engineering: single molecule technology Edited by Deborah Leckband Received 9 July 2019, revised 12 July 2019, accepted 6 August 2019
https://doi.org/10.1016/j.cobme.2019.08.001 2468-4511/© 2019 Elsevier Inc. All rights reserved.
Keywords Atomic force microscopy, Single-molecule force spectroscopy, Singlecell force spectroscopy, Microbial pathogens, Infection, Cell adhesion.
Introduction Microbial infections often involve the adhesion of pathogens (fungi, bacteria, and viruses), to surfaces (biomaterials, host cells). For instance, human rhinoviruses infect their host by adhering to cell surface receptors such as the transmembrane glycoprotein InterCellular Adhesion Molecule (ICAM)-1 [1]. Being the major causative agent of the common cold, they have strong medicinal and socioeconomical impacts and, therefore, are the subject of intensive antiviral research efforts [1]. Other prototypical pathogens are the gramwww.sciencedirect.com
negative Escherichia coli bacterium, which causes urinary tract infections, and the gram-positive Staphylococcus aureus, involved in skin diseases and bloodstream infections. Both species trigger nosocomial infections that are difficult to control and prevent, therefore representing an economic burden [2]. Besides, pathogenicity is re-enforced by the ability of the cells to stick to biomedical devices (implants, catheters) and to develop surface-associated communities called biofilms, which are resistant to antibiotics [2,3]. Adhesion, the crucial initiating step leading to biofilms, needs to be fully understood to devise efficient antimicrobial strategies (antiadhesive coatings, antiadhesion molecules) that are complementary to antibiotic treatments [4]. Unlike ensemble techniques, single-molecule experiments probe individual molecules, thereby revealing events and properties that would otherwise be inaccessible [5]. Among these, atomic force microscopy (AFM) is the only technique which can detect, localize, and manipulate single molecules on live cells, while providing high-resolution topographic images of the cell surface [6,7]. Scanning a sharp tip across microbial samples provides novel insights into the structure of cell surfaces. In single-molecule force spectroscopy (SMFS), AFM tips labeled with cognate ligands are used to detect, localize, and manipulate individual receptors, whereas in single-cell force spectroscopy (SCFS), the tip is replaced by a living cell to measure single-cell adhesion forces. Both approaches provide a wealth of information on the strength, specificity, and dynamics of microbial cell surface interactions. Here, we survey recent progress in the use of these methods to study pathogen adhesion down to single-molecule resolution, and we discuss how this knowledge may be used to better understand and potentially fight microbial infections.
Single-cell force spectroscopy In SCFS experiments, cells are attached to the AFM cantilever to probe adhesion forces toward surfaces (cells, solid substrates). Among various immobilization strategies, the most straightforward consists in immersing the tip in a bacterial suspension and either leaving it to dry or fixing it. Although this strategy does not require any time-consuming functionalization steps, it may alter cell metabolism and properties. To cope with this issue, one may take advantage of electrostatic interactions between positively charged coatings, such Current Opinion in Biomedical Engineering 2019, 12:1–7
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as polyethylenimine or poly-L-lysine and the negatively charged surface of pathogens. However, this might lead to the attachment of multiple cells, making data interpretation very challenging. A versatile and reliable method was developed in which a glass bead is first attached to a tipless cantilever and then coated with a wet adhesive, thus allowing the capture of single viable cells [8]. In the past years, novel insights into the role of nonspecific forces in microbial cell adhesion have been gained through the use of SCFS. Applied to the adhesion of S. aureus to restorative dental biomaterials [9], the method showed that topological substrate characteristics such as roughness and hydrophobicity favor bacterial adhesion, while it was decreased in the presence of saliva proteins. Combining SCFS and Monte Carlo simulations, Thewes et al. [10] showed that an extended DLVO theory is needed when investigating bacterial adhesion to hydrophobic substrates and highlighted that protein density and structure are key to promoting strong adhesion, up to 8 nN. In the case of Streptococcus thermophilus, temperature-dependent adhesion to hydrophobic surfaces was found to be inhibited by the transcriptional regulator Rgg0182 which is associated with the synthesis of hydrophilic biopolymers [11]. The role of hydrophobic forces was also demonstrated for the fungal pathogens Candida albicans [12] and Candida glabrata [13]. Given that these fungi colonize biomedical devices and cause serious diseases in immunocompromised patients, it is of great importance to unravel and tentatively block their adhesion mechanisms. Als and Epa adhesins, which play major roles in infection in the two species, respectively, were found to mediate large adhesion forces and extended rupture lengths because of macromolecular unfolding of protein domains. In addition, adhesion of C. albicans was also probed against dendritic cells [14,15]. Riet et al. [14,15] found that host infection was promoted by the strong molecular recognition between mannan patterns in the cell wall of the yeast and specific immune cell receptors. SCFS has also enabled to capture specific forces between cell adhesion proteins (adhesins) and their ligands. Staphylococcal adhesins ClfA, ClfB, and SdrG were found to promote extremely strong adhesion toward host proteins such as fibrinogen (Fg) [16e18], resulting from the so-called ‘dock, lock, and latch’ (DLL) high-affinity mechanism [19]. The strength and dynamics of these remarkable bonds were further dissected by means of SMFS (see below; Figure 2). During biofilm accumulation, the S. aureus surface protein SasG was shown to be engaged in specific Zn2þ-dependent homophilic bonds, rather than in receptor-ligand bonds [20]. The force required to unfold individual SasG domains is remarkably strong, up to w500 pN, helping bacteria to resist physiological shear forces and maintain cellecell contacts. Deeper Current Opinion in Biomedical Engineering 2019, 12:1–7
insights were also obtained concerning the role of S. aureus adhesins in favoring attachment to host cells and tissues. In the context of skin colonization and disorder [21,22], strong adhesion forces (500 pN) were recorded on corneocyte surfaces, originating from multiple bonds between S. aureus adhesins and specific target ligands [21]. ClfB mediates skin adhesion that strengthens with mechanical stress, up to 1e2 nN, which plays important roles in skin-related disorders such as atopic dermatitis [22]. Also, Prystopiuk et al. [23] unraveled the mechanostability of the FnBPA-Fnintegrin ternary complex in guiding cellular invasion within minutes. Biofilm-related infections are difficult to eradicate because, in such communities, cells are protected from external challenges, especially from antibiotic treatments. This issue, together with the increasing occurrence of multiresistant strains, has urged the need for innovative antimicrobial strategies. An important branch of efforts has been the testing of antifouling and antiadhesion strategies to block the initiating step of biofilm formation [24]. The antifouling approach mostly concerns implanted biomaterials and aims to target a large spectrum of pathogens. Rodriguez-Emmenegger et al. [25] found that polyzwitterionic polymer brushes reduce by 99% the force and work needed to detach Yersinia pseudotuberculosis bacteria from a surface. Cationic nanoclusters dispersed in a controlled manner in such a polymer brush, also enhance the easy removal of attached S. aureus [26]. Sophorolipid-coated surfaces were shown to prevent the attachment of both grampositive and gram-negative bacteria [27]. The use of antiadhesion agents is especially relevant in the case of human infections and is generally specific to a given pathogen. SCFS showed that receptor analogs, that is, fullerene-based mannoconjugates, strongly block the adhesion of E. coli sugar-specific adhesin, FimH, to mannose surfaces [28]. These compounds may thus find applications for inhibiting intestinal colonization. Single-cell studies have also revealed that antibodies and peptides show strong antiadhesion potential, acting as competitive inhibitors for either adhesineligand interactions, such as the affinity of S. aureus Cna adhesion to collagen [29], or cellecell accumulation, such as SdrC-mediated homophilic bonds [30]. Conventional SCFS assays are time consuming, thus limiting their application for high throughput applications. One way to solve this problem is Fluidic force microscopy (FluidFM), which enables attaching reversibly single cells onto the aperture of microchanneled cantilevers via a pressure controller [31e33]. The hollow cantilever may thus be used as a forcerecording pipette, in liquid and under optical control [34], allowing single-cell adhesion measurements but also single-cell injection and extraction [31,35]. www.sciencedirect.com
Single-molecule atomic force microscopy Mathelié-Guinlet et al.
FluidFM has been used to quantify the forces between either yeasts, mammalian cells, or bacteria and abiotic surfaces [32,33,36]. Recently, the technology enabled researchers to quantify interaction forces between individual fungal cells, at increased throughput and without the need of chemical fixation. Cellecell experiments directly address the molecular interactions of functional adhesins in their native cellular environment. These experiments enabled the discovery of a novel function for C. albicans Als proteins, that is, driving homophilic cellecell adhesion through amyloid bonds [37] (Figure 1). In the future, FluidFM may contribute to the identification of small peptide inhibitors for antiadhesion therapy.
Single-molecule force spectroscopy While SCFS probes adhesive forces of whole cells, SMFS captures the binding force and dynamics of
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single adhesins [38,39]. Perhaps the most striking discovery of the past years is that staphylococcal adhesins SdrG, ClfA, and ClfB bind to their protein ligand via extremely strong forces, w2 nN, equivalent to that of covalent bonds. Although the prototypical streptavidinebiotin bond had long been considered to be the strongest in nature (w0.2 nN), we now realize that pathogens have developed much stronger bonds to sustain pathogenicity. In early work, Herman et al. [16] found using both SCFS and SMFS that SdrG adhesin binds Fg with a force of 2 nN, reflecting the DLL mechanism (Figure 2). Recently, Milles et al. [40] combined SMFS with all-atom steered molecular dynamics simulations to show that the N2 and N3 domains of SdrG stabilize the Fg through a H bond network between a Fg peptide and the backbone of the adhesion (Figure 2). They also found that this mechanostability depends on the presence of calcium
Figure 1
Role of force-dependent amyloids in Candida albicans cell–cell adhesion. (a) Optical micrograph of a yeast aggregate. (b) In FluidFM experiments, a single yeast cell is immobilized on the cantilever aperture by applying a pressure difference. Labeling of the attached cell (in green) demonstrates that cell integrity is preserved, thus that the method is nondestructive. The cell probe is moved toward a target cell immobilized in a porous membrane and forces between interacting cells are measured. (c) Upon pulling cell–cell pairs apart by FluidFM-based SCFS, remarkable force curves are observed with multiple adhesion peaks separated by 2 nm. (d) Mechanism of amyloid-based adhesion. Force-induced unfolding of Als proteins lead to the exposure of hidden amyloid sequences, which trigger the lateral assembly (cis interactions) of the proteins on the cell surface. Strong homophilic adhesion (trans interactions) between adhesins on opposing cells is mediated by amyloid bonds. Shown in red is the amyloid sequence of the forceinduced unfolded T region, in orange. Reproduced with permission from (37).
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Figure 2
Ultrastrong interaction between the staphylococcal adhesin SdrG and fibrinogen. (a) The N2N3 subdomains of SdrG bind to fibrinogen (Fg) by the high-affinity dock, lock, and latch (DLL) mechanism involving sequential conformational changes in the subdomains. The binding site is a cleft between the N2 and N3 subdomains of SdrG. Once the ligand peptide is docked and stabilized by hydrophobic interactions and hydrogen bonds, a Cterminus extension of N3 subdomain folds over the ligand to insert and complement a b-sheet in the N2 subdomain. This mechanism thus greatly stabilizes the conformation of the SdrG-ligand complex. (b) SMFS reveals that the mechanical stability of the SdrG–Fg complex is remarkably high, equivalent to that of a covalent bond (~2 nN) [16,40]. (c) Simulations show that this extreme mechanostability results from an intricate hydrogen bond network (purple) between the ligand peptide backbone (orange) and the N2N3 subdomains (light and dark blue). Under load, the screw-like arrangement of hydrogen bonds maintains the peptide in a perfect shear geometry [40]. Adapted with permission from (40).
ions that stabilize B domains of SdrG, Ca2þ depletion leading to much smaller force [41]. A key finding is that the binding forces of CfA, ClfB, and surface protein A (SpA) are dramatically strengthened by mechanical tension, which is reminiscent of a catch bond mechanism, and highlight the role of physical forces in activating the adhesion of bacterial pathogens to host proteins [16,17,42]. Binding of SpA to von Willebrand factor (vWF) under shear stress, which does not occur through a DLL mechanism, is important for adherence to platelets and endothelial cells. AFM Current Opinion in Biomedical Engineering 2019, 12:1–7
experiments showed that the SpA-vWF interaction is regulated by a new force-dependent mechanism, in which mechanical extension of vWF leads to the exposure of a high affinity cryptic SpA-binding site, consistent with the shear force-controlled functions of vWF. This force-sensitive mechanism may help bacteria to resist shear stress of flowing blood during infection. Accordingly, these reports emphasize the importance of mechanobiology in controlling pathogen adhesion. Another area of research is the role of adhesion forces of bacterial appendages such as pili, which are implicated www.sciencedirect.com
Single-molecule atomic force microscopy Mathelié-Guinlet et al.
in pathogen virulence [43,44]. These structures show a unique ability to resist mechanical stress produced by mucus flow or the bloodstream. Becke et al. [45] reported the binding mechanism of RrgA and RrgB adhesins present in pili of Streptococcus pneumoniae to fibronectin (Fn) and collagen (Cn), respectively. They found that the weak interaction between RrgA and Fn occurs through a two-domain binding mechanism with short lifetime that allows the bacterium to first ‘gently’ scan the surface and then establish a strong adhesion. On the other hand, RrgB interacts with collagen, through the ‘collagen hug’ mechanism in a force-dependent manner which provides strong anchorage to the surface [46]. Finally, Rivas-Pardo et al. [47] linked the aforementioned high mechanostability of several adhesins to the Spy0128 pilus-mediated adhesion of Streptococcus pyogenes. Indeed, Spy0128 remains folded under mechanical stress, thanks to the formation of intramolecular isopeptide bonds, which, if blocked, leads to the instability of Spy0128 and its unfolding at low forces.
Multiparametric imaging In the past 20 years, SMFS-based force-volume (FV) imaging has been used for mapping the distribution of single proteins, including staphylococcal adhesins [17,18,21,22,42,48]. Although valuable, FV is severely limited by its low spatial and temporal resolutions. Improvement of the scanning and detection mechanisms of force mapping has led to the development of multiparametric imaging methods, that is, quantitative imaging and peak force tapping. These modes have allowed researchers to simultaneously image the structure, adhesion, and elasticity of biological samples at high resolution and with higher speed than previously accessible in classical modes [49]. The technique may be used with functionalized tips to probe specific chemical and biological sites [49]. Currently, an exciting goal is to apply such biospecific multiparametric imaging technology to map and quantify the specific adhesion forces of pathogens. Alsteens et al. probed the interactions between mammalian cells and viruses at unprecedented resolution [50e52]. Using tips functionalized with single viruses, they quantified the adhesion force, kinetics, and the free energy landscape of the first binding steps of rabies virus to animal cells and demonstrated that initial binding of the virus is not driven by unspecific interactions [50]. Following the same strategy, Delguste et al. [52] showed that the herpesvirus envelope glycoproteins regulate attachment and release steps affecting the binding, diffusion and release potential of the virus on the cell surface. The role of viral glycoproteins was further balanced by the quantification of the decrease in viral mobility on the cell surface by viral envelope mucin-like regions [51]. In the biofilm context, multiparametric imaging of the S. aureus surface protein SasG showed that Zn2þ strongly alters the properties of the cell surface, which www.sciencedirect.com
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exhibited smoother, stiffer, and stickier characteristics than in the absence of these ions [20]. Together with SCFS data, these results pointed to a complex role of zinc in SasG-mediated adhesion: adsorption of zinc ions to cell wall components increases the cohesion of the cell surface, thus promoting the extension of SasG proteins beyond other surface components and making them fully available for zinc-dependent homophilic interactions. The adhesion forces of S. aureus onto human corneocytes were mapped, revealing the occurrence of specific interactions between pathogen adhesins and target ligands well spread on corneocytes [53]. Finally, amyloid nanodomains were mapped on C. albicans cells and found to exhibit different adhesiveness related to their stiffness and hydrophobic state [21].
Conclusions The studies reviewed here highlight the power of AFM techniques to understand the forces driving the adhesion of pathogens with unprecedented spatiotemporal resolution. With its unique capability to probe a wide range of forces, AFM has become an important multifunctional tool in microbiology and nanomedicine. Combining SMFS and SCFS allows us to quantify the forces between pathogens and cognate ligands or solid surfaces. Unique to these experiments is that they provide details of microbial adhesion that could not be obtained from ensemble averaged measurements. As most pathogens are subjected to physical stress, dynamic AFM studies suggest that force measurements, out-of equilibrium, might be much more relevant than traditional affinity values, obtained at equilibrium, to describe cell adhesion. Major breakthroughs from the past few years include (i) unveiling the role of hydrophobic forces in pathogen adhesion to solid surfaces, which is of particular importance in biofilm formation on biomedical implants, (ii) uncovering the extreme mechanostability of molecular complexes formed by bacterial adhesins, thus explaining how pathogens can resist high mechanical stresses in physiological environments such as the bloodstream, and (iii) understanding the dynamic steps in which viruses invade human cells. AFM has also proven to be a sensitive tool to assess the activity of antiadhesion compounds. Antiadhesive agents offer promise in preventing the initiating step of infection without killing the pathogens and thus decreasing their ability to mutate and adapt. An important challenge in developing adhesion inhibitors will be to enhance their blocking efficiency against multiple adhesin-ligand complexes and multivalent interactions.
Acknowledgements Work at the UCLouvain was supported by the Excellence of Science-EOS programme (Grant #30550343), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant #693630), the FNRS-WELBIO (Grant #WELBIO-CR2015A-05), the National Fund for Scientific Research (FNRS), and the ´ franc¸aise de Belgique (Concerted Research Department of the Communaute Research Action). Y.F.D. is a Research Director at the FRS-FNRS. Current Opinion in Biomedical Engineering 2019, 12:1–7
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Conflict of interest statement Nothing declared.
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45. Becke TD, Ness S, Gürster R, Schilling AF, di Guilmi A-M, Sudhop S, Hilleringmann M, Clausen-Schaumann H: Single molecule force spectroscopy reveals two-domain binding mode of pilus-1 tip protein RrgA of Streptococcus pneumoniae to fibronectin. ACS Nano 2018, 12: 549 – 558. 46. Becke TD, Ness S, Kaufmann BK, Hartmann B, Schilling AF, * Sudhop S, Hilleringmann M, Clausen-Schaumann H: Pilus-1 backbone protein RrgB of Streptococcus pneumoniae binds collagen I in a force-dependent way. ACS Nano 2019. https:// doi.org/10.1021/acsnano.9b02587. This work demonstrates the strong interaction between pilus protein RrgB and collagen, further strengthened by force loading and shear rate, similar to a catch bond mechanism. 47. Rivas-Pardo JA, Badilla CL, Tapia-Rojo R, Alonso-Caballero Á, * Fernández JM: Molecular strategy for blocking isopeptide bond formation in nascent pilin proteins. Proc Natl Acad Sci 2018, 115:9222–9227. Combining protein engineering and AFM, the team demonstrates that the anchorage of bacteria through pili might be counteracted by peptides compromising their mechanics and blocking the formation of isopeptide bonds. 48. Grandbois M, Dettmann W, Benoit M, Gaub HE: Affinity imaging of red blood cells using an atomic force microscope. J Histochem Cytochem 2000, 48:719–724. 49. Dufrêne YF, Martínez-Martín D, Medalsy I, Alsteens D, Müller DJ: Multiparametric imaging of biological systems by forcedistance curve–based AFM. Nat Methods 2013, 10:847–854. 50. Alsteens D, Newton R, Schubert R, Martinez-Martin D, * Delguste M, Roska B, Müller DJ: Nanomechanical mapping of first binding steps of a virus to animal cells. Nat Nanotechnol 2017, 12:177–183. Using multiparametric imaging with virus-functionalized tips, the authors show the specificity, strength and energy landscape of the initial interaction between viruses and mammalian cells. 51. Delguste M, Peerboom N, Le Brun G, Trybala E, Olofsson S, Bergström T, Alsteens D, Bally M: Regulatory mechanisms of the mucin-like region on herpes simplex virus during cellular attachment. ACS Chem Biol 2019, 14:534–542. 52. Delguste M, Zeippen C, Machiels B, Mast J, Gillet L, Alsteens D: Multivalent binding of herpesvirus to living cells is tightly regulated during infection. Sci Adv 2018, 4:eaat1273. 53. Formosa C, Schiavone M, Boisrame A, Richard ML, Duval RE, Dague E: Multiparametric imaging of adhesive nanodomains at the surface of Candida albicans by atomic force microscopy. Nanomed Nanotechnol Biol Med 2015, 11: 57 – 65.
Current Opinion in Biomedical Engineering 2019, 12:1–7
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Current Opinion in
Biomedical Engineering
Single-molecule atomic force microscopy studies of microbial pathogens Marion Mathelié-Guinlet1, Felipe Viela1, Albertus Viljoen1, Jérôme Dehullu1 and Yves F. Dufrêne1,2 Abstract
Atomic force microscopy (AFM) has become a powerful multifunctional platform for probing microbial cell surfaces, one molecule at a time, thereby uncovering biophysical properties and interactions that are otherwise not accessible. Single-cell force spectroscopy has been widely used to quantify the adhesion forces of pathogens, including viruses, bacteria, and fungi, whereas single-molecule force spectroscopy has enabled the functional analysis and imaging of individual receptors on cell surfaces. In addition, AFM has been instrumental in assessing the inhibition of adhesion of pathogens by carbohydrates, antibodies, and peptides, thus showing promise for antiadhesion therapy. Addresses 1 Louvain Institute of Biomolecular Science and Technology, Université catholique de Louvain, Croix du Sud, 4-5, bte L7.07.06., B-1348, Louvain-la-Neuve, Belgium 2 Walloon Excellence in Life Sciences and Biotechnology (WELBIO), B-1300, Wavre, Belgium Corresponding author: Dufrêne, Yves F (Yves.Dufrene@uclouvain. be)
Current Opinion in Biomedical Engineering 2019, 12:1–7 This review comes from a themed issue on Molecular & Cellular Engineering: single molecule technology Edited by Deborah Leckband Received 9 July 2019, revised 12 July 2019, accepted 6 August 2019
https://doi.org/10.1016/j.cobme.2019.08.001 2468-4511/© 2019 Elsevier Inc. All rights reserved.
Keywords Atomic force microscopy, Single-molecule force spectroscopy, Singlecell force spectroscopy, Microbial pathogens, Infection, Cell adhesion.
Introduction Microbial infections often involve the adhesion of pathogens (fungi, bacteria, and viruses), to surfaces (biomaterials, host cells). For instance, human rhinoviruses infect their host by adhering to cell surface receptors such as the transmembrane glycoprotein InterCellular Adhesion Molecule (ICAM)-1 [1]. Being the major causative agent of the common cold, they have strong medicinal and socioeconomical impacts and, therefore, are the subject of intensive antiviral research efforts [1]. Other prototypical pathogens are the gramwww.sciencedirect.com
negative Escherichia coli bacterium, which causes urinary tract infections, and the gram-positive Staphylococcus aureus, involved in skin diseases and bloodstream infections. Both species trigger nosocomial infections that are difficult to control and prevent, therefore representing an economic burden [2]. Besides, pathogenicity is re-enforced by the ability of the cells to stick to biomedical devices (implants, catheters) and to develop surface-associated communities called biofilms, which are resistant to antibiotics [2,3]. Adhesion, the crucial initiating step leading to biofilms, needs to be fully understood to devise efficient antimicrobial strategies (antiadhesive coatings, antiadhesion molecules) that are complementary to antibiotic treatments [4]. Unlike ensemble techniques, single-molecule experiments probe individual molecules, thereby revealing events and properties that would otherwise be inaccessible [5]. Among these, atomic force microscopy (AFM) is the only technique which can detect, localize, and manipulate single molecules on live cells, while providing high-resolution topographic images of the cell surface [6,7]. Scanning a sharp tip across microbial samples provides novel insights into the structure of cell surfaces. In single-molecule force spectroscopy (SMFS), AFM tips labeled with cognate ligands are used to detect, localize, and manipulate individual receptors, whereas in single-cell force spectroscopy (SCFS), the tip is replaced by a living cell to measure single-cell adhesion forces. Both approaches provide a wealth of information on the strength, specificity, and dynamics of microbial cell surface interactions. Here, we survey recent progress in the use of these methods to study pathogen adhesion down to single-molecule resolution, and we discuss how this knowledge may be used to better understand and potentially fight microbial infections.
Single-cell force spectroscopy In SCFS experiments, cells are attached to the AFM cantilever to probe adhesion forces toward surfaces (cells, solid substrates). Among various immobilization strategies, the most straightforward consists in immersing the tip in a bacterial suspension and either leaving it to dry or fixing it. Although this strategy does not require any time-consuming functionalization steps, it may alter cell metabolism and properties. To cope with this issue, one may take advantage of electrostatic interactions between positively charged coatings, such Current Opinion in Biomedical Engineering 2019, 12:1–7
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as polyethylenimine or poly-L-lysine and the negatively charged surface of pathogens. However, this might lead to the attachment of multiple cells, making data interpretation very challenging. A versatile and reliable method was developed in which a glass bead is first attached to a tipless cantilever and then coated with a wet adhesive, thus allowing the capture of single viable cells [8]. In the past years, novel insights into the role of nonspecific forces in microbial cell adhesion have been gained through the use of SCFS. Applied to the adhesion of S. aureus to restorative dental biomaterials [9], the method showed that topological substrate characteristics such as roughness and hydrophobicity favor bacterial adhesion, while it was decreased in the presence of saliva proteins. Combining SCFS and Monte Carlo simulations, Thewes et al. [10] showed that an extended DLVO theory is needed when investigating bacterial adhesion to hydrophobic substrates and highlighted that protein density and structure are key to promoting strong adhesion, up to 8 nN. In the case of Streptococcus thermophilus, temperature-dependent adhesion to hydrophobic surfaces was found to be inhibited by the transcriptional regulator Rgg0182 which is associated with the synthesis of hydrophilic biopolymers [11]. The role of hydrophobic forces was also demonstrated for the fungal pathogens Candida albicans [12] and Candida glabrata [13]. Given that these fungi colonize biomedical devices and cause serious diseases in immunocompromised patients, it is of great importance to unravel and tentatively block their adhesion mechanisms. Als and Epa adhesins, which play major roles in infection in the two species, respectively, were found to mediate large adhesion forces and extended rupture lengths because of macromolecular unfolding of protein domains. In addition, adhesion of C. albicans was also probed against dendritic cells [14,15]. Riet et al. [14,15] found that host infection was promoted by the strong molecular recognition between mannan patterns in the cell wall of the yeast and specific immune cell receptors. SCFS has also enabled to capture specific forces between cell adhesion proteins (adhesins) and their ligands. Staphylococcal adhesins ClfA, ClfB, and SdrG were found to promote extremely strong adhesion toward host proteins such as fibrinogen (Fg) [16e18], resulting from the so-called ‘dock, lock, and latch’ (DLL) high-affinity mechanism [19]. The strength and dynamics of these remarkable bonds were further dissected by means of SMFS (see below; Figure 2). During biofilm accumulation, the S. aureus surface protein SasG was shown to be engaged in specific Zn2þ-dependent homophilic bonds, rather than in receptor-ligand bonds [20]. The force required to unfold individual SasG domains is remarkably strong, up to w500 pN, helping bacteria to resist physiological shear forces and maintain cellecell contacts. Deeper Current Opinion in Biomedical Engineering 2019, 12:1–7
insights were also obtained concerning the role of S. aureus adhesins in favoring attachment to host cells and tissues. In the context of skin colonization and disorder [21,22], strong adhesion forces (500 pN) were recorded on corneocyte surfaces, originating from multiple bonds between S. aureus adhesins and specific target ligands [21]. ClfB mediates skin adhesion that strengthens with mechanical stress, up to 1e2 nN, which plays important roles in skin-related disorders such as atopic dermatitis [22]. Also, Prystopiuk et al. [23] unraveled the mechanostability of the FnBPA-Fnintegrin ternary complex in guiding cellular invasion within minutes. Biofilm-related infections are difficult to eradicate because, in such communities, cells are protected from external challenges, especially from antibiotic treatments. This issue, together with the increasing occurrence of multiresistant strains, has urged the need for innovative antimicrobial strategies. An important branch of efforts has been the testing of antifouling and antiadhesion strategies to block the initiating step of biofilm formation [24]. The antifouling approach mostly concerns implanted biomaterials and aims to target a large spectrum of pathogens. Rodriguez-Emmenegger et al. [25] found that polyzwitterionic polymer brushes reduce by 99% the force and work needed to detach Yersinia pseudotuberculosis bacteria from a surface. Cationic nanoclusters dispersed in a controlled manner in such a polymer brush, also enhance the easy removal of attached S. aureus [26]. Sophorolipid-coated surfaces were shown to prevent the attachment of both grampositive and gram-negative bacteria [27]. The use of antiadhesion agents is especially relevant in the case of human infections and is generally specific to a given pathogen. SCFS showed that receptor analogs, that is, fullerene-based mannoconjugates, strongly block the adhesion of E. coli sugar-specific adhesin, FimH, to mannose surfaces [28]. These compounds may thus find applications for inhibiting intestinal colonization. Single-cell studies have also revealed that antibodies and peptides show strong antiadhesion potential, acting as competitive inhibitors for either adhesineligand interactions, such as the affinity of S. aureus Cna adhesion to collagen [29], or cellecell accumulation, such as SdrC-mediated homophilic bonds [30]. Conventional SCFS assays are time consuming, thus limiting their application for high throughput applications. One way to solve this problem is Fluidic force microscopy (FluidFM), which enables attaching reversibly single cells onto the aperture of microchanneled cantilevers via a pressure controller [31e33]. The hollow cantilever may thus be used as a forcerecording pipette, in liquid and under optical control [34], allowing single-cell adhesion measurements but also single-cell injection and extraction [31,35]. www.sciencedirect.com
Single-molecule atomic force microscopy Mathelié-Guinlet et al.
FluidFM has been used to quantify the forces between either yeasts, mammalian cells, or bacteria and abiotic surfaces [32,33,36]. Recently, the technology enabled researchers to quantify interaction forces between individual fungal cells, at increased throughput and without the need of chemical fixation. Cellecell experiments directly address the molecular interactions of functional adhesins in their native cellular environment. These experiments enabled the discovery of a novel function for C. albicans Als proteins, that is, driving homophilic cellecell adhesion through amyloid bonds [37] (Figure 1). In the future, FluidFM may contribute to the identification of small peptide inhibitors for antiadhesion therapy.
Single-molecule force spectroscopy While SCFS probes adhesive forces of whole cells, SMFS captures the binding force and dynamics of
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single adhesins [38,39]. Perhaps the most striking discovery of the past years is that staphylococcal adhesins SdrG, ClfA, and ClfB bind to their protein ligand via extremely strong forces, w2 nN, equivalent to that of covalent bonds. Although the prototypical streptavidinebiotin bond had long been considered to be the strongest in nature (w0.2 nN), we now realize that pathogens have developed much stronger bonds to sustain pathogenicity. In early work, Herman et al. [16] found using both SCFS and SMFS that SdrG adhesin binds Fg with a force of 2 nN, reflecting the DLL mechanism (Figure 2). Recently, Milles et al. [40] combined SMFS with all-atom steered molecular dynamics simulations to show that the N2 and N3 domains of SdrG stabilize the Fg through a H bond network between a Fg peptide and the backbone of the adhesion (Figure 2). They also found that this mechanostability depends on the presence of calcium
Figure 1
Role of force-dependent amyloids in Candida albicans cell–cell adhesion. (a) Optical micrograph of a yeast aggregate. (b) In FluidFM experiments, a single yeast cell is immobilized on the cantilever aperture by applying a pressure difference. Labeling of the attached cell (in green) demonstrates that cell integrity is preserved, thus that the method is nondestructive. The cell probe is moved toward a target cell immobilized in a porous membrane and forces between interacting cells are measured. (c) Upon pulling cell–cell pairs apart by FluidFM-based SCFS, remarkable force curves are observed with multiple adhesion peaks separated by 2 nm. (d) Mechanism of amyloid-based adhesion. Force-induced unfolding of Als proteins lead to the exposure of hidden amyloid sequences, which trigger the lateral assembly (cis interactions) of the proteins on the cell surface. Strong homophilic adhesion (trans interactions) between adhesins on opposing cells is mediated by amyloid bonds. Shown in red is the amyloid sequence of the forceinduced unfolded T region, in orange. Reproduced with permission from (37).
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Current Opinion in Biomedical Engineering 2019, 12:1–7
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Figure 2
Ultrastrong interaction between the staphylococcal adhesin SdrG and fibrinogen. (a) The N2N3 subdomains of SdrG bind to fibrinogen (Fg) by the high-affinity dock, lock, and latch (DLL) mechanism involving sequential conformational changes in the subdomains. The binding site is a cleft between the N2 and N3 subdomains of SdrG. Once the ligand peptide is docked and stabilized by hydrophobic interactions and hydrogen bonds, a Cterminus extension of N3 subdomain folds over the ligand to insert and complement a b-sheet in the N2 subdomain. This mechanism thus greatly stabilizes the conformation of the SdrG-ligand complex. (b) SMFS reveals that the mechanical stability of the SdrG–Fg complex is remarkably high, equivalent to that of a covalent bond (~2 nN) [16,40]. (c) Simulations show that this extreme mechanostability results from an intricate hydrogen bond network (purple) between the ligand peptide backbone (orange) and the N2N3 subdomains (light and dark blue). Under load, the screw-like arrangement of hydrogen bonds maintains the peptide in a perfect shear geometry [40]. Adapted with permission from (40).
ions that stabilize B domains of SdrG, Ca2þ depletion leading to much smaller force [41]. A key finding is that the binding forces of CfA, ClfB, and surface protein A (SpA) are dramatically strengthened by mechanical tension, which is reminiscent of a catch bond mechanism, and highlight the role of physical forces in activating the adhesion of bacterial pathogens to host proteins [16,17,42]. Binding of SpA to von Willebrand factor (vWF) under shear stress, which does not occur through a DLL mechanism, is important for adherence to platelets and endothelial cells. AFM Current Opinion in Biomedical Engineering 2019, 12:1–7
experiments showed that the SpA-vWF interaction is regulated by a new force-dependent mechanism, in which mechanical extension of vWF leads to the exposure of a high affinity cryptic SpA-binding site, consistent with the shear force-controlled functions of vWF. This force-sensitive mechanism may help bacteria to resist shear stress of flowing blood during infection. Accordingly, these reports emphasize the importance of mechanobiology in controlling pathogen adhesion. Another area of research is the role of adhesion forces of bacterial appendages such as pili, which are implicated www.sciencedirect.com
Single-molecule atomic force microscopy Mathelié-Guinlet et al.
in pathogen virulence [43,44]. These structures show a unique ability to resist mechanical stress produced by mucus flow or the bloodstream. Becke et al. [45] reported the binding mechanism of RrgA and RrgB adhesins present in pili of Streptococcus pneumoniae to fibronectin (Fn) and collagen (Cn), respectively. They found that the weak interaction between RrgA and Fn occurs through a two-domain binding mechanism with short lifetime that allows the bacterium to first ‘gently’ scan the surface and then establish a strong adhesion. On the other hand, RrgB interacts with collagen, through the ‘collagen hug’ mechanism in a force-dependent manner which provides strong anchorage to the surface [46]. Finally, Rivas-Pardo et al. [47] linked the aforementioned high mechanostability of several adhesins to the Spy0128 pilus-mediated adhesion of Streptococcus pyogenes. Indeed, Spy0128 remains folded under mechanical stress, thanks to the formation of intramolecular isopeptide bonds, which, if blocked, leads to the instability of Spy0128 and its unfolding at low forces.
Multiparametric imaging In the past 20 years, SMFS-based force-volume (FV) imaging has been used for mapping the distribution of single proteins, including staphylococcal adhesins [17,18,21,22,42,48]. Although valuable, FV is severely limited by its low spatial and temporal resolutions. Improvement of the scanning and detection mechanisms of force mapping has led to the development of multiparametric imaging methods, that is, quantitative imaging and peak force tapping. These modes have allowed researchers to simultaneously image the structure, adhesion, and elasticity of biological samples at high resolution and with higher speed than previously accessible in classical modes [49]. The technique may be used with functionalized tips to probe specific chemical and biological sites [49]. Currently, an exciting goal is to apply such biospecific multiparametric imaging technology to map and quantify the specific adhesion forces of pathogens. Alsteens et al. probed the interactions between mammalian cells and viruses at unprecedented resolution [50e52]. Using tips functionalized with single viruses, they quantified the adhesion force, kinetics, and the free energy landscape of the first binding steps of rabies virus to animal cells and demonstrated that initial binding of the virus is not driven by unspecific interactions [50]. Following the same strategy, Delguste et al. [52] showed that the herpesvirus envelope glycoproteins regulate attachment and release steps affecting the binding, diffusion and release potential of the virus on the cell surface. The role of viral glycoproteins was further balanced by the quantification of the decrease in viral mobility on the cell surface by viral envelope mucin-like regions [51]. In the biofilm context, multiparametric imaging of the S. aureus surface protein SasG showed that Zn2þ strongly alters the properties of the cell surface, which www.sciencedirect.com
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exhibited smoother, stiffer, and stickier characteristics than in the absence of these ions [20]. Together with SCFS data, these results pointed to a complex role of zinc in SasG-mediated adhesion: adsorption of zinc ions to cell wall components increases the cohesion of the cell surface, thus promoting the extension of SasG proteins beyond other surface components and making them fully available for zinc-dependent homophilic interactions. The adhesion forces of S. aureus onto human corneocytes were mapped, revealing the occurrence of specific interactions between pathogen adhesins and target ligands well spread on corneocytes [53]. Finally, amyloid nanodomains were mapped on C. albicans cells and found to exhibit different adhesiveness related to their stiffness and hydrophobic state [21].
Conclusions The studies reviewed here highlight the power of AFM techniques to understand the forces driving the adhesion of pathogens with unprecedented spatiotemporal resolution. With its unique capability to probe a wide range of forces, AFM has become an important multifunctional tool in microbiology and nanomedicine. Combining SMFS and SCFS allows us to quantify the forces between pathogens and cognate ligands or solid surfaces. Unique to these experiments is that they provide details of microbial adhesion that could not be obtained from ensemble averaged measurements. As most pathogens are subjected to physical stress, dynamic AFM studies suggest that force measurements, out-of equilibrium, might be much more relevant than traditional affinity values, obtained at equilibrium, to describe cell adhesion. Major breakthroughs from the past few years include (i) unveiling the role of hydrophobic forces in pathogen adhesion to solid surfaces, which is of particular importance in biofilm formation on biomedical implants, (ii) uncovering the extreme mechanostability of molecular complexes formed by bacterial adhesins, thus explaining how pathogens can resist high mechanical stresses in physiological environments such as the bloodstream, and (iii) understanding the dynamic steps in which viruses invade human cells. AFM has also proven to be a sensitive tool to assess the activity of antiadhesion compounds. Antiadhesive agents offer promise in preventing the initiating step of infection without killing the pathogens and thus decreasing their ability to mutate and adapt. An important challenge in developing adhesion inhibitors will be to enhance their blocking efficiency against multiple adhesin-ligand complexes and multivalent interactions.
Acknowledgements Work at the UCLouvain was supported by the Excellence of Science-EOS programme (Grant #30550343), the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant #693630), the FNRS-WELBIO (Grant #WELBIO-CR2015A-05), the National Fund for Scientific Research (FNRS), and the ´ franc¸aise de Belgique (Concerted Research Department of the Communaute Research Action). Y.F.D. is a Research Director at the FRS-FNRS. Current Opinion in Biomedical Engineering 2019, 12:1–7
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Conflict of interest statement Nothing declared.
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