Force spectroscopy of the interaction between mycobacterial adhesins and heparan sulphate proteoglycan receptors.

Understanding the molecular interactions between bacterial adhesion proteins (adhesins) and their receptors is essential for elucidating the molecular mechanisms of bacterial pathogenesis. Here, atomic force microscopy (AFM) is used to explore the specific interactions between the heparin-binding hemagglutinin (HBHA) from Mycobacterium tuberculosis, and heparan sulphate proteoglycan (HSPG) receptors on live A549 pneumocytes. First, we show that the specific binding forces between single HBHA-HSPG pairs, 57+/-16 pN, are similar to the forces measured earlier between HBHA and heparin molecules. Second, we mapped the distribution of single HSPG receptors on the surface of A549 cells, revealing that the proteins are widely and homogeneously exposed. Third, we observed force curves with constant force plateaus at large pulling velocities, reflecting the extraction of membrane tethers or nanotubes. These single-molecule measurements provide new avenues in pathogenesis research, particularly for elucidating the molecular basis of pathogen-host interactions.

Interaction of the mycobacterial heparin-binding hemagglutinin with actin, as evidenced by single-molecule force spectroscopy.

Although Mycobacterium tuberculosis and related species are considered to be typical endosomal pathogens, recent studies have suggested that mycobacteria can be present in the cytoplasm of infected cells and cause cytoskeleton rearrangements, the mechanisms of which remain unknown. Here, we used single-molecule force spectroscopy to demonstrate that the heparin-binding hemagglutinin (HBHA), a surface adhesin from Mycobacterium tuberculosis displaying sequence similarities with actin-binding proteins, is able to bind to actin. Force curves recorded between actin and the coiled-coil, N-terminal domain of HBHA showed a bimodal distribution of binding forces reflecting the detection of single and double HBHA-actin interactions. Force curves obtained between actin and the lysine-rich C-terminal domain of HBHA showed a broader distribution of binding events, suggesting they originate primarily from intermolecular electrostatic bridges between cationic HBHA domains and anionic actin residues. We also explored the dynamics of the HBHA-actin interaction, showing that the binding force and binding frequency increased with the pulling speed and contact time, respectively. Taken together, our data indicate that HBHA is able to specifically bind actin, via both its N-terminal and C-terminal domains, strongly suggesting a role of the HBHA-actin interaction in the pathogenesis of mycobacterial diseases.

Single-molecule force spectroscopy of mycobacterial adhesin-adhesin interactions.

The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule. Here, we used single-molecule atomic-force microscopy to measure individual homophilic HBHA-HBHA interaction forces. Force curves recorded between tips and supports derivatized with HBHA proteins exposing their N-terminal domains showed a bimodal distribution of binding forces reflecting the formation of dimers or multimers. Moreover, the binding peaks showed elongation forces that were consistent with the unfolding of alpha-helical coiled-coil structures. By contrast, force curves obtained for proteins exposing their lysine-rich C-terminal domains showed a broader distribution of binding events, suggesting that they originate primarily from intermolecular electrostatic bridges between cationic and anionic residues rather than from specific coiled-coil interactions. Notably, similar homophilic HBHA-HBHA interactions were demonstrated on live mycobacteria producing HBHA, while they were not observed on an HBHA-deficient mutant. Together with the fact that HBHA mediates bacterial aggregation, these observations suggest that the single homophilic HBHA interactions measured here reflect the formation of multimers that may promote mycobacterial aggregation.

Probing molecular recognition sites on biosurfaces using AFM.

Knowledge of the molecular forces that drive receptor-ligand interactions is a key to gain a detailed understanding of cell adhesion events and to develop novel applications in biomaterials science. Until recently, there was no tool available for analyzing and mapping these forces on complex biosurfaces like cell surfaces. During the past decade, however, single-molecule atomic force microscopy (AFM) has opened exciting new opportunities for detecting and localizing molecular recognition forces on artificial biosurfaces and on living cells. In this review, we describe the general principles of the AFM technique, present procedures commonly used to prepare samples and tips, and discuss a number of applications that are relevant to the field of biomaterials.

Exploring the molecular forces within and between CbsA S-layer proteins using single molecule force spectroscopy.

We used single molecule atomic force microscopy (AFM) to gain insight into the molecular forces driving the folding and assembly of the S-layer protein CbsA. Force curves recorded between tips and supports modified with CbsA proteins showed sawtooth patterns with multiple force peaks of 58+/-26pN that we attribute to the unfolding of alpha-helices, in agreement with earlier secondary structure predictions. The average unfolding force increased with the pulling speed but was independent on the interaction time. Force curves obtained for CbsA peptides truncated in their C-terminal region showed similar periodic features, except that fewer force peaks were seen. Furthermore, the average unfolding force was 83+/-45pN, suggesting the domains were more stable. By contrast, cationic peptides truncated in their N-terminal region showed single force peaks of 366+/-149pN, presumably reflecting intermolecular electrostatic bridges rather than unfolding events. Interestingly, these large intermolecular forces increased not only with pulling speed but also with interaction time. We expect that the intra- and intermolecular forces measured here may play a significant role in controlling the stability and assembly of the CbsA protein.

Ethambutol-induced alterations in Mycobacterium bovis BCG imaged by atomic force microscopy.

Progress in understanding the structure-function relationships of the mycobacterial cell wall has been hampered by its complex architecture as well as by the lack of sensitive, high-resolution probing techniques. For the first time, we used atomic force microscopy (AFM) to image the surface topography of hydrated Mycobacterium bovis bacillus Calmette Guérin cells and to investigate the influence of the antimycobacterial drug ethambutol on the cell wall architecture. While untreated cells showed a very smooth and homogeneous surface morphology, incubation of cells in the presence of ethambutol caused dramatic changes of the fine surface structure. At 4 micro g mL(-1), the drug created concentric striations at the cell surface and disrupted a approximately 8 nm thick cell wall layer, attributed to the outer electron-opaque layer usually seen by electron microscopy, while at 10 micro g mL(-1) an underlying approximately 12 nm thick layer reflecting the thick electron-transparent layer was also altered. These noninvasive ultrastructural investigations provide novel information on the macromolecular architecture of the mycobacterial envelope as well as into the destructuring effects of ethambutol.

Direct measurement of Mycobacterium-fibronectin interactions.

Bacterial surface-associated proteins play essential roles in mediating pathogen-host interactions and represent privileged targets for anti-adhesion therapy. We used atomic force microscopy (AFM) to investigate, in vivo, the binding strength and surface distribution of fibronectin attachment proteins (FAPs) in Mycobacterium bovis bacillus Calmette-Guérin (BCG). We measured the specific binding forces of FAPs ( approximately 50 pN) and found that they increased with the loading rate, as observed earlier for other receptor-ligand systems. We also mapped the distribution of FAPs, revealing that the proteins are widely exposed on the mycobacterial surface. To demonstrate that the proteins are surface-associated, we showed that treatment of the cells with pullulanase, an enzyme possessing carbohydrate-degrading activities, or with protease, an enzyme that conducts proteolysis, led to a substantial reduction of the FAP surface density. A similar trend was also noted following treatment with ethambutol, an antibiotic which inhibits the synthesis of cell wall polysaccharides. The nanoscale analyses presented here complement traditional proteomic and molecular biology approaches for the functional analysis of surface-associated proteins, and may help in the search for novel anti-adhesive drugs.

Molecular mapping of lipoarabinomannans on mycobacteria.

Although the chemical composition of mycobacterial cell walls is well known, the 3D organization of the various constituents is not fully understood. In particular, it is unclear whether the major wall component lipoarabinomannan (LAM) is exposed on the outermost surface or hindered by other constituents such as mycolic acids. To address this pertinent question, we used atomic force microscopy (AFM) with tips bearing anti-LAM antibodies to detect single LAM molecules on Mycobacterium bovis BCG cells. First, we showed the ability of anti-LAM tips to detect isolated, purified LAM molecules. We then mapped the distribution of LAM on mycobacteria, prior to and after treatment with the drug isoniazid. We found that LAM was not exposed on the surface of native cells, pointing to the presence of a homogeneous layer of mycolic acids, whereas it was greatly exposed on isoniazid-treated cells, in agreement with the action mode of the drug. This single-molecule study provides novel insight into the architecture of mycobacterial cell walls and offers promising perspectives for understanding the action modes of antimycobacterial drugs.

Nanoscale imaging of microbial pathogens using atomic force microscopy.

The nanoscale exploration of microbes using atomic force microscopy (AFM) is an exciting research field that has expanded rapidly in the past years. Using AFM topographic imaging, investigators can visualize the surface structure of live cells under physiological conditions and with unprecedented resolution. In doing so, the effect of drugs and chemicals on the fine cell surface architecture can be monitored. Real-time imaging offers a means to follow dynamic events such as cell growth and division. In parallel, chemical force microscopy (CFM), in which AFM tips are modified with specific functional groups, allows researchers to measure interaction forces, such as hydrophobic forces, and to resolve nanoscale chemical heterogeneities on cells, on a scale of only approximately 25 functional groups. Lastly, molecular recognition imaging using spatially resolved force spectroscopy, dynamic recognition imaging or immunogold detection, enables microscopists to localize specific receptors, such as cell adhesion proteins or antibiotic binding sites. These noninvasive nanoscale analyses provide new avenues in pathogenesis research, particularly for investigating the action mode of antimicrobial drugs, and for elucidating the molecular basis of pathogen-host interactions.

Organization of the mycobacterial cell wall: a nanoscale view.

The biosynthesis of the Mycobacterium tuberculosis cell wall is targeted by some of the most powerful antituberculous drugs. To date, the molecular mechanisms by which these antibiotics affect the cell wall characteristics are not well understood. Here, we used atomic force microscopy - in three different modes - to probe the nanoscale surface properties of live mycobacteria and their modifications upon incubation with four antimycobacterial drugs: isoniazid, ethionamide, ethambutol, and streptomycine. Topographic imaging, combined with quantitative surface roughness analysis, demonstrated that all drugs induce a substantial increase of surface roughness to an extent that correlates with the localization of the target (i.e., synthesis of mycolic acids, arabinogalactans, or proteins). Chemical force microscopy with hydrophobic tips revealed that the structural alterations induced by isoniazid and ethambutol were correlated with a dramatic decrease of cell surface hydrophobicity, reflecting the removal of the outermost mycolic acid layer. Consistent with this finding, tapping mode imaging, combined with immunogold labeling, showed that the two drugs lead to the massive exposure of hydrophilic lipoarabinomannans at the surface. Taken together, these structural, chemical, and immunological data provide novel insight into the action mode of antimycobacterial drugs, as well as into the spatial organization of the mycobacterial cell wall.

The NTA-His6 bond is strong enough for AFM single-molecular recognition studies.

There is a need in current atomic force microscopy (AFM) molecular recognition studies for generic methods for the stable, functional attachment of proteins on tips and solid supports. In the last few years, the site-directed nitrilotriacetic acid (NTA)-polyhistidine (Hisn) system has been increasingly used towards this goal. Yet, a crucial question in this context is whether the NTA-Hisn bond is sufficiently strong for ensuring stable protein immobilization during force spectroscopy measurements. Here, we measured the forces between AFM tips modified with NTA-terminated alkanethiols and solid supports functionalized with His6-Gly-Cys peptides in the presence of Ni2+. The force histogram obtained at a loading rate of 6600 pN s(-1) showed three maxima at rupture forces of 153 +/- 57 pN, 316 +/- 50 pN and 468 +/- 44 pN, that we attribute primarily to monovalent and multivalent interactions between a single His6 moiety and one, two and three NTA groups, respectively. The measured forces are well above the 50-100 pN unbinding forces typically observed by AFM for receptor-ligand pairs. The plot of adhesion force versus log (loading rate) revealed a linear regime, from which we deduced a kinetic off-rate constant of dissociation, k(off) approximately 0.07 s(-1). This value is in the range of that estimated for the multivalent interaction involving two NTA, using fluorescence measurements, and may account for an increased binding stability of the NTA-His6 bond. We conclude that the NTA-His6 system is a powerful, well-suited platform for the stable, oriented immobilization of proteins in AFM single-molecule studies.

Chemical force microscopy of single live cells.

Traditionally, cell surface properties have been difficult to study at the subcellular level, especially on hydrated, live cells. Here, we demonstrate the ability of chemical force microscopy to map the hydrophobicity of single live cells with nanoscale resolution. After validating the technique on reference surfaces with known chemistry, we probe the local hydrophobic character of two medically important microorganisms, Aspergillus fumigatus and Mycobacterium bovis, in relation with function. Applicable to a wide variety of cells, the chemically sensitive imaging method presented here provides new opportunities for studying the nanoscale surface properties of live cells and for understanding their roles in mediating cellular events.

Towards a nanoscale view of fungal surfaces.

In the past years, atomic force microscopy (AFM) has offered novel possibilities for exploring the nanoscale surface properties of fungal cells. For the first time, AFM imaging enables investigators to visualize fine surface structures, such as rodlets, directly on native hydrated cells. Moreover, real-time imaging can be used to follow cell surface dynamics during cell growth and to monitor the effect of molecules such as enzymes and drugs. In fact, AFM is much more than a microscope in that when used in the force spectroscopy mode, it allows measurement of physicochemical properties such as surface energy and surface charge, to probe the elasticity of cell wall components and macromolecules, and to analyse the force and localization of molecular recognition events.

Single-molecule force spectroscopy and imaging of the vancomycin/D-Ala-D-Ala interaction.

The clinically important vancomycin antibiotic inhibits the growth of pathogens such as Staphylococcus aureus by blocking cell wall synthesis through specific recognition of nascent peptidoglycan terminating in D-Ala-D-Ala. Here, we demonstrate the ability of single-molecule atomic force microscopy with antibiotic-modified tips to measure the specific binding forces of vancomycin and to map individual ligands on living bacteria. The single-molecule approach presented here provides new opportunities for understanding the binding mechanisms of antibiotics and for exploring the architecture of bacterial cell walls.