Although much progress has been made in the identification and characterization of adhesins borne by pathogenic bacteria, the molecular details underlying their interaction with host receptors remain largely unknown owing to the lack of appropriate probing techniques. Here we report a method, based on atomic force microscopy (AFM) with tips bearing biologically active molecules, for measuring the specific binding forces of individual adhesins and for mapping their distribution on the surface of living bacteria. First, we determined the adhesion forces between the heparin-binding haemagglutinin adhesin (HBHA) produced by Mycobacterium tuberculosis and heparin, used as a model sulphated glycoconjugate receptor. Both the adhesion frequency and adhesion force increased with contact time, indicating that the HBHA-heparin complex is formed via multiple intermolecular bridges. We then mapped the distribution of single HBHA molecules on the surface of living mycobacteria and found that the adhesin is not randomly distributed over the mycobacterial surface, but concentrated into nanodomains.

Knowledge of the mechanisms by which bacterial pili adhere to host cells and withstand external forces is critical to our understanding of their functional roles and offers exciting avenues in biomedicine for controlling the adhesion of bacterial pathogens and probiotics. While much progress has been made in the nanoscale characterization of pili from Gram-negative bacteria, the adhesive and mechanical properties of Gram-positive bacterial pili remain largely unknown. Here, we use single-molecule atomic force microscopy to unravel the binding mechanism of pili from the probiotic Gram-positive bacterium Lactobacillus rhamnosus GG (LGG). First, we show that SpaC, the key adhesion protein of the LGG pilus, is a multifunctional adhesin with broad specificity. SpaC forms homophilic trans-interactions engaged in bacterial aggregation and specifically binds mucin and collagen, two major extracellular components of host epithelial layers. Homophilic and heterophilic interactions display similar binding strengths and dissociation rates. Next, pulling experiments on living bacteria demonstrate that LGG pili exhibit two unique mechanical responses, that is, zipper-like adhesion involving multiple SpaC molecules distributed along the pilus length and nanospring properties enabling pili to resist high force. These mechanical properties may represent a generic mechanism among Gram-positive bacterial pili for strengthening adhesion and withstanding shear stresses in the natural environment. The single-molecule experiments presented here may help us to design molecules capable of promoting or inhibiting bacterial-host interactions.

Here we report on in vivo measurement of the mechanical behavior of a cell surface sensor using single-molecule atomic force microscopy. We focus on the yeast wall stress component sensor Wsc1, a plasma membrane protein that is thought to function as a rigid probe of the cell wall status. We first map the distribution of individual histidine-tagged sensors on living yeast cells by scanning the cell surface with atomic force microscopy tips carrying nitrilotriacetate groups. We then show that Wsc1 behaves like a linear nanospring that is capable of resisting high mechanical force and of responding to cell surface stress. Both a genomic pmt4 deletion and the insertion of a stretch of glycines in Wsc1 result in substantial alterations in protein spring properties, supporting the important role of glycosylation at the extracellular serine/threonine-rich region.

Elucidating the molecular mechanisms behind the strength and mechanics of cell adhesion proteins is of central importance in cell biology, and offers exciting avenues for the identification of potential drug targets. Here we use single-molecule force spectroscopy to investigate the adhesive and mechanical properties of the widely expressed Als5p cell adhesion protein from the opportunistic pathogen Candida albicans. We show that the forces required to unfold individual tandem repeats of the protein are in the 150–250 pN range, both on isolated molecules and on live cells. We also find that the unfolding probability increases with the number of tandem repeats and correlates with the level of cell adherence. We suggest that the modular and flexible nature of Als5p conveys both strength and toughness to the protein, making it ideally-suited for cell adhesion. The single molecule measurements presented here open new avenues for understanding the mechanical properties of adhesion molecules from mammalian and microbial cells, and may help us to elucidate their potential implications in diseases like inflammation, cancer and infection.

Currently, there is a growing need for methods that can quantify and map the molecular interactions of biological samples, both with high-force sensitivity and high spatial resolution. Force-volume imaging is a valuable atomic force microscopy (AFM) modality for probing specific sites on biosurfaces. However, the low speed and poor spatial resolution of this method have severely hampered its widespread use in life science research. We use a novel AFM mode (i.e., peak force tapping with chemically functionalized tips) to probe the localization and interactions of chemical and biological sites on living cells at high speed and high resolution (8 min for 1 μm × 1 μm images at 512 pixels × 512 pixels). First, we demonstrate the ability of the method to quantify and image hydrophobic forces on organic surfaces and on microbial pathogens. Next, we detect single sensor proteins on yeast cells, and we unravel their mechanical properties in relation to cellular function. Owing to its key capabilities (quantitative mapping, resolution of a few nanometers, and true correlation with topography), this novel biochemically sensitive imaging technique is a powerful complement to other advanced AFM modes for quantitative, high-resolution bioimaging.

We used atomic force microscopy (AFM) to explore the antigen binding forces of individual Fv fragments of antilysozyme antibodies (Fv). To detect single molecular recognition events, genetically engineered histidine-tagged Fv fragments were coupled onto AFM tips modified with mixed self-assembled monolayers (SAMs) of nitrilotriacetic acid- and tri(ethylene glycol)-terminated alkanethiols while lysozyme (Lyso) was covalently immobilized onto mixed SAMs of carboxyl- and hydroxyl-terminated alkanethiols. The quality of the functionalization procedure was validated using X-ray photoelectron spectroscopy (surface chemical composition), AFM imaging (surface morphology in aqueous solution), and surface plasmon resonance (SPR, specific binding in aqueous solution). AFM force-distance curves recorded at a loading rate of 5000 pN/s between Fv- and Lyso-modified surfaces yielded a distribution of unbinding forces composed of integer multiples of an elementary force quantum of approximately 50 pN that we attribute to the rupture of a single antibody-antigen pair. Injection of a solution containing free Lyso caused a dramatic reduction of adhesion probability, indicating that the measured 50 pN unbinding forces are due to the specific antibody-antigen interaction. To investigate the dynamics of the interaction, force-distance curves were recorded at various loading rates. Plots of unbinding force vs log(loading rate) revealed two distinct linear regimes with ascending slopes, indicating multiple barriers were present in the energy landscape. The kinetic off-rate constant of dissociation (k(off) approximately = 1 x 10(-3) s(-1)) obtained by extrapolating the data of the low-strength regime to zero force was in the range of the k(off) estimated by SPR.

The knowledge of molecular mechanisms underlying the adhesive and mechanical properties of cell surface-associated molecules is a key to understanding their functions. In this context, single-molecule force spectroscopy (SMFS) has recently offered new opportunities for probing the adhesion and mechanics of polysaccharides and proteins on live cells. Here we present a protocol that we have used to analyze polysaccharide chains of different nature on the bacterium Lactobacillus rhamnosus GG. We describe procedures (i) for functionalizing atomic force microscopy (AFM) tips with Pseudomonas aeruginosa-I or concanavalin A lectins, (ii) for stretching specific polysaccharide molecules on live bacteria using SMFS with lectin tips and (iii) for mapping the localization, adhesion and extension of individual polysaccharide chains. We also discuss data treatment, emphasizing how to gain insight into the elasticity of the stretched macromolecules using the extended freely jointed chain model. Even though the presented protocol is for L. rhamnosus, it can be easily modified for other cell types. For users having expertise in the field, the entire protocol can be completed in about 5 d.

Signalling is a key feature of living cells which frequently involves the local clustering of specific proteins in the plasma membrane. How such protein clustering is achieved within membrane microdomains (“rafts”) is an important, yet largely unsolved problem in cell biology. The plasma membrane of yeast cells represents a good model to address this issue, since it features protein domains that are sufficiently large and stable to be observed by fluorescence microscopy. Here, we demonstrate the ability of single-molecule atomic force microscopy to resolve lateral clustering of the cell integrity sensor Wsc1 in living Saccharomyces cerevisiae cells. We first localize individual wild-type sensors on the cell surface, revealing that they form clusters of ∼200 nm size. Analyses of three different mutants indicate that the cysteine-rich domain of Wsc1 has a crucial, not yet anticipated function in sensor clustering and signalling. Clustering of Wsc1 is strongly enhanced in deionized water or at elevated temperature, suggesting its relevance in proper stress response. Using in vivo GFP-localization, we also find that non-clustering mutant sensors accumulate in the vacuole, indicating that clustering may prevent endocytosis and sensor turnover. This study represents the first in vivo single-molecule demonstration for clustering of a transmembrane protein in S. cerevisiae. Our findings indicate that in yeast, like in higher eukaryotes, signalling is coupled to the localized enrichment of sensors and receptors within membrane patches.