Sofiane El-Kirat-Chatel

Quantifying the forces guiding microbial cell adhesion using single-cell force spectroscopy

During the past decades, several methods (e.g., electron microscopy, flow chamber experiments, surface chemical analysis, surface charge and surface hydrophobicity measurements) have been developed to investigate the mechanisms controlling the adhesion of microbial cells to other cells and to various other substrates. However, none of the traditional approaches are capable of looking at adhesion forces at the single-cell level. In recent years, atomic force microscopy (AFM) has been instrumental in measuring the forces driving microbial adhesion on a single-cell basis. The method, known as single-cell force spectroscopy (SCFS), consists of immobilizing a single living cell on an AFM cantilever and measuring the interaction forces between the cellular probe and a solid substrate or another cell. Here we present SCFS protocols that we have developed for quantifying the cell adhesion forces of medically important microbes. Although we focus mainly on the probiotic bacterium Lactobacillus plantarum, we also show that our procedures are applicable to pathogens, such as the bacterium Staphylococcus epidermidis and the yeast Candida albicans. For well-trained microscopists, the entire protocol can be mastered in 1 week.

Single-molecule imaging and functional analysis of Als adhesins and mannans during Candida albicans morphogenesis.

Cellular morphogenesis in the fungal pathogen Candida albicans is associated with changes in cell wall composition that play important roles in biofilm formation and immune responses. Yet, how fungal morphogenesis modulates the biophysical properties and interactions of the cell surface molecules is poorly understood, mainly owing to the paucity of high-resolution imaging techniques. Here, we use single-molecule atomic force microscopy to localize and analyze the key components of the surface of living C. albicans cells during morphogenesis. We show that the yeast-to-hypha transition leads to a major increase in the distribution, adhesion, unfolding, and extension of Als adhesins and their associated mannans on the cell surface. We also find that morphogenesis dramatically increases cell surface hydrophobicity. These molecular changes are critical for microbe-host interactions, including adhesion, colonization, and biofilm formation. The single-molecule experiments presented here offer promising prospects for understanding how microbial pathogens use cell surface molecules to modulate biofilm and immune interactions.

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

ABSTRACT 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. 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.

The binding force of the staphylococcal adhesin SdrG is remarkably strong

SdrG is a cell surface adhesin from Staphylococcus epidermidis which binds to the blood plasma protein fibrinogen (Fg). Ligand binding follows a ‘dock, lock and latch’ model involving dynamic conformational changes of the adhesin that result in a greatly stabilized adhesin–ligand complex. To date, the force and dynamics of this multistep interaction are poorly understood. Here we use atomic force microscopy (AFM) to unravel the binding strength and cell surface localization of SdrG at molecular resolution. Single‐cell force spectroscopy shows that SdrG mediates time‐dependent attachment to Fg‐coated surfaces. Single‐molecule force spectroscopy with Fg‐coated AFM tips demonstrates that the adhesin forms nanoscale domains on the cell surface, which we believe contribute to strengthen cell adhesion. Notably, we find that the rupture force of single SdrG–Fg bonds is very large, ∼ 2 nN, equivalent to the strength of a covalent bond, and shows a low dissociation rate, suggesting that the bond is very stable. The strong binding force, slow dissociation and clustering of SdrG provide a molecular foundation for the ability of S. epidermidis to colonize implanted biomaterials and to withstand physiological shear forces.

Single-cell force spectroscopy of the medically important Staphylococcus epidermidis–Candida albicans interaction

Despite the clinical importance of bacterial-fungal interactions, their molecular details are poorly understood. A hallmark of such medically important interspecies associations is the interaction between the two nosocomial pathogens Staphylococcus aureus and Candida albicans, which can lead to mixed biofilm-associated infections with enhanced antibiotic resistance. Here, we use single-cell force spectroscopy (SCFS) to quantify the forces engaged in bacterial-fungal co-adhesion, focusing on the poorly investigated S. epidermidis-C. albicans interaction. Force curves recorded between single bacterial and fungal germ tubes showed large adhesion forces (~5 nN) with extended rupture lengths (up to 500 nm). By contrast, bacteria poorly adhered to yeast cells, emphasizing the important role of the yeast-to-hyphae transition in mediating adhesion to bacterial cells. Analysis of mutant strains altered in cell wall composition allowed us to distinguish the main fungal components involved in adhesion, i.e. Als proteins and O-mannosylations. We suggest that the measured co-adhesion forces are involved in the formation of mixed biofilms, thus possibly as well in promoting polymicrobial infections. In the future, we anticipate that this SCFS platform will be used in nanomedicine to decipher the molecular mechanisms of a wide variety of pathogen-pathogen interactions and may help in designing novel anti-adhesion agents.

Nanoscale Imaging of the Candida Macrophage Interaction Using Correlated Fluorescence-Atomic Force Microscopy

Knowledge of the molecular bases underlying the interaction of fungal pathogens with immune cells is critical to our understanding of fungal infections and offers exciting perspectives for controlling immune responses for therapy. Although fluorescence microscopy is a valuable tool to visualize pathogen-host interactions, the spatial resolution is low, meaning the fine structural details of the interacting cells cannot be observed. Here, we demonstrate the ability of correlated fluorescence-atomic force microscopy (AFM) to image the various steps of the interaction between fungal pathogens and macrophages with nanoscale resolution. We focus on Candida albicans, known to grow as two morphological forms (yeast cells, filamentous hyphae) that play important roles in modulating the interaction with macrophages. We observe the main steps of macrophage infection, including initial intercellular contact, phagocytosis by internalization of yeast cells, intracellular hyphal growth leading to mechanical stretching, and piercing of the macrophage membrane resulting in pathogen escape. While fluorescence imaging clearly distinguishes fungal cells from macrophages during the various steps of the process, AFM captures nanoscale structural features of the macrophage surface that are of high biological relevance, including ruffles, lamellipodia, filopodia, membrane remnants, and phagocytic cups. As fungal pathogenesis is mainly controlled by the ability of fungi to escape from immune cells, the nanoimaging platform established here has great potential in nanomedicine for understanding and controlling fungal infections.

Atomic force microscopy: a new look at pathogens

Microbial cells are highly complex and heterogeneous systems. In general, cell populations contain subgroups of cells which exhibit differences in growth rate as well as resistance to stress and drug treatment [1]. In addition, individual cells are spatially organized and heterogeneous, and this cellular heterogeneity is used to perform key functions [2]. This complexity emphasizes the need for single-cell analysis techniques in microbial research. Fluorescence imaging is a powerful tool to localize molecules in single cells [3], [4], but the resolution remains limited to the wavelength of the light source. On the other hand, high-resolution images of microbial structures can be obtained by electron microscopy techniques. In particular, cryo-electron tomography—or three-dimensional (3-D) electron microscopy—provides images of whole bacterial cells, at resolutions that are one to two orders of magnitude higher than those obtained with light microscopy [5]. In the past 20 years a new form of microscopy, atomic force microscopy (AFM), has revolutionized the way researchers probe the microbial cell surface. Instead of using an incident beam, AFM measures the minute forces acting between a sharp tip and the sample [6]–[8]. To generate a topographic image, the tip is attached to a cantilever that bends under force and is moved in three-dimensions using a piezoelectric scanner. While scanning the sample surface, cantilevers bending is measured by a laser beam focused on the free end of the cantilever and reflected into a photodiode. Unlike other microscopy techniques, 3-D images of cells and membranes are obtained at high resolution without staining, labelling or fixation, thus in physiological conditions. AFM is much more than a surface-imaging tool in that it also measures the localization and mechanical properties of the individual cell surface molecules. In this modality, known as single-molecule force spectroscopy, the cantilever deflection is recorded as a function of the vertical displacement of the scanner (as the sample is pushed towards the tip and it retracts) [6], [7]. This results in a cantilever deflection vs. scanner displacement curve, which is transformed into a force-distance curve using appropriate corrections. The characteristic adhesion force between tip and sample measured during retraction is used to probe the distribution and mechanics of single molecules, such as cell surface receptors. These novel AFM techniques complement traditional methods used to analyse microbial cell walls and provide new opportunities for understanding cell surface interactions. In this review, we provide a flavour of the various applications offered by AFM in microbiology, and we highlight some of the key breakthroughs the technique has enabled in pathogen research.

Single-cell force spectroscopy of Als-mediated fungal adhesion

Macroscopic assays that are traditionally used to investigate the adhesion behaviour of microbial cells provide averaged information obtained on large populations of cells and do not measure the fundamental forces driving single-cell adhesion. Here, we use single-cell force spectroscopy (SCFS) to quantify the specific and non-specific forces engaged in the adhesion of the human fungal pathogen Candida albicans. Saccharomyces cerevisiae cells expressing the C. albicans adhesion protein Als5p were attached on atomic force microscopy tipless cantilevers using a bioinspired polydopamine wet polymer, and force-distance curves were recorded between the obtained cell probes and various solid surfaces. Force signatures obtained on hydrophobic substrates exhibited large adhesion forces (1.25 ± 0.2 nN) with extended rupture lengths (up to 400 nm), attributed to the binding and stretching of the hydrophobic tandem repeats of Als5p. Data collected on fibronectin (Fn) -coated substrates featured strong adhesion forces (2.8 ± 0.6 nN), reflecting specific binding between Fn and the N-terminal immunoglobulin-like regions of Als5p, followed by weakly adhesive macromolecular bonds. Both hydrophobic and Fn adhesion forces increased with contact time, emphasizing the important role that time plays in strengthening adhesion. Our SCFS methodology provides a versatile platform in biomedicine for understanding the fundamental forces driving adhesion and biofilm formation in fungal pathogens.

Single-molecule atomic force microscopy unravels the binding mechanism of a Burkholderia cenocepacia trimeric autotransporter adhesin

Trimeric autotransporter adhesins (TAAs) are bacterial surface proteins that fulfil important functions in pathogenic Gram‐negative bacteria. Prominent examples of TAAs are found in Burkholderia cepacia complex, a group of bacterial species causing severe infections in patients with cystic fibrosis. While there is strong evidence that Burkholderia cenocepacia TAAs mediate adhesion, aggregation and colonization of the respiratory epithelium, we still know very little about the molecular mechanisms behind these interactions. Here, we use single‐molecule atomic force microscopy to unravel the binding mechanism of BCAM0224, a prototype TAA from B. cenocepacia K56‐2. We show that the adhesin forms homophilic trans‐interactions engaged in bacterial aggregation, and that it behaves as a spring capable to withstand high forces. We also find that BCAM0224 binds collagen, a major extracellular component of host epithelia. Both homophilic and heterophilic interactions display low binding affinity, which could be important for epithelium colonization. We then demonstrate that BCAM0224 recognizes receptors on living pneumocytes, and leads to the formation of membrane tethers that may play a role in promoting adhesion. Collectively, our results show that BCAM0224 is a multifunctional adhesin endowed with remarkable binding properties, which may represent a general mechanism among TAAs for strengthening bacterial adhesion.

Force Nanoscopy as a Versatile Platform for Quantifying the Activity of Antiadhesion Compounds Targeting Bacterial Pathogens

The development of bacterial strains that are resistant to multiple antibiotics has urged the need for new antibacterial therapies. An exciting approach to fight bacterial diseases is the use of antiadhesive agents capable to block the adhesion of the pathogens to host tissues, the first step of infection. We report the use of a novel atomic force microscopy (AFM) platform for quantifying the activity of antiadhesion compounds directly on living bacteria, thus without labeling or purification. Novel fullerene-based mannoconjugates bearing 10 carbohydrate ligands and a thiol bond were efficiently prepared. The thiol functionality could be exploited as a convenient handle to graft the multimeric species onto AFM tips. Using a combination of single-molecule and single-cell AFM assays, we demonstrate that, unlike mannosidic monomers, multivalent glycofullerenes strongly block the adhesion of uropathogenic Escherichia coli bacteria to their carbohydrate receptors. We expect that the nanoscopy technique developed here will help designing new antiadhesion drugs to treat microbial infections, including those caused by multidrug resistant organisms.