Claire Verbelen

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.

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.

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.

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. Copyright

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.

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.

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.

Imaging Chemical Groups and Molecular Recognition Sites on Live Cells Using AFM

Imaging the nanoscale distribution of specific chemical and biological sites on live cells is an important challenge in current life science research. In addition to imaging the surface topography of live cells, atomic force microscopy (AFM) is increasingly used to probe their chemical groups and biological receptors. In chemical force microscopy, AFM tips are modified with specific functional groups, thereby allowing investigators to probe chemical sites and their interactions on a scale of only ∼25 functional groups. In molecular recognition imaging, tips are functionalized with specific biomolecules, or samples labeled with immunogold particles, enabling researchers to localize specific receptors. Clearly, these nanoscale investigations provide new avenues in cellular biology and microbiology for elucidating the structure–function relationships of cell surfaces. In this chapter, we discuss the principles of these AFM modalities and their applications in life science research.

Towards a Nanoscale View of Microbial Surfaces Using the Atomic Force Microscope

In recent years, the atomic force microscope (AFM) has greatly improved our understanding of microbial surfaces. AFM imaging has proved to be a powerful tool for visualizing membrane proteins and live cells at high resolution and in physiological conditions. In addition, AFM force spectroscopy has enabled us to probe and map a variety of properties, including the elasticity of cell walls and cell surface molecules, and the unfolding forces of single proteins, and has allowed the detection and functional analysis of molecular recognition sites. These unique capabilities allow researchers to answer a number of questions that were inaccessible before, such as how does the surface architecture of microbes change as they grow or as they interact with an antibiotic, what are the conformational changes in single membrane proteins, and what are the molecular forces responsible for the interaction between host cells or specific molecules?