Microbial Nanoscopy: Breakthroughs, Challenges, and Opportunities Yves F. Dufrêne*,†,‡ †
Institute of Life Sciences, Université Catholique de Louvain, Croix du Sud, 4-5, bte L7.07.06., Louvain-la-Neuve B-1348, Belgium Walloon Excellence in Life Sciences and Biotechnology (WELBIO), Avenue Pasteur, 6, Wavre 1300, Belgium
‡
ABSTRACT: Studying the structure, properties, and interactions of microbial cells is key to understanding the functions of the microbiome. Recent advances in nanotechnology have offered new tools to probe microbes at the single-molecule and single-cell levels. In this issue of ACS Nano, Kumar et al. present an atomic force microscopy method that is capable of imaging the nanoscale organization of bacterial proteins in native, curved membranes. This study represents an important step forward in the development of nanoscopy techniques for analyzing biological systems with large curvature and vertical dimensions, such as membrane vesicles and bacterial cells.
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force nanoscopy. Rather than using an incident beam, a sharp tip is moved across the sample while sensing the small forces acting on the probe tip. This technique enables surfaces to be imaged and manipulated with atomic (/molecular) resolution. Today, AFM modalities represent a powerful toolbox in microbiology, enabling researchers to image and to manipulate cell surface structures under physiological conditions and with nanoscale resolution (Figure 1).
nderstanding the functions of the microbiome will require new tools that can image and manipulate microbes at various scales, from individual molecules up to microbial communities.1 For years, microbiologists have been using microscopy to look at microbes.2 The invention of the light microscope in the 17th century enabled microbiologists to observe the morphological details of individual cells directly. Later, fluorescence microscopy techniques were developed to localize specific molecules in cells and to visualize the organization and heterogeneity of cells in microbial communities. Currently, confocal laser scanning microscopy is routinely used to examine the complexity of thick samples, like microbial biofilms. Yet, the spatial resolution of optical microscopy is limited by the diffraction limit of light (∼200 nm), meaning the arrangement and interactions of single molecules cannot be resolved. Electron microscopy provides higher resolution, but needs to be operated in a vacuum. So there is clearly a need for novel techniques that can probe microbes on the nanoscale and in physiological conditions.
LIVE-CELL NANOSCOPY Recent advances in nanoscopy techniques have offered new opportunities to study single cells and single molecules in microbiology.3 In 2014, Betzig, Hell, and Moerner received the Nobel Prize in Chemistry for their outstanding work in superresolution microscopy, also called optical nanoscopy. By breaking the optical diffraction limit, optical nanoscopy can study cellular structures and machineries to a resolution of 20 nm. Scanning probe techniques represent another way to look at cells at high resolution. Binnig, Gerber, and Quate were awarded the 2016 Kavli prize in nanoscience for the invention and development of atomic force microscopy (AFM), also called © 2017 American Chemical Society
Figure 1. AFM for microbiology. While AFM imaging captures the supramolecular structure of membranes and cell walls in buffer, force spectroscopy probes the interactions of the individual surface molecules and appendages. Published: January 12, 2017 19
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In this issue of ACS Nano, Kumar et al.4 report a novel AFM method to image the organization of single proteins in native bacterial membrane vesicles named chromatophores. While classical experiments require curved membranes to be ruptured and immobilized to a substrate, this methodology enables researchers to probe curved membranes directly in their native state.
supramolecular architecture of photosynthetic membranes is modulated in response to light.6 AFM imaging is generally conducted on flat samples that are immobilized on supporting substrates. For curved membranes like chromatophores, detergents may be used to disrupt and to flatten the vesicles, but such treatments may alter the membrane organization. Also, it is unclear whether the underlying substrate may influence the organization and dynamics of membrane components. Therefore, it is challenging to image membrane vesicles directly in their native, curved states. Kumar et al. lowered the forces applied between the tip and sample in both lateral and normal directions by using the small cantilever tapping mode AFM with optimized scanning parameters and buffer conditions (Figure 2). Small cantilevers yield faster feedback responses and lower the thermal noise. Long-range electrostatic forces were screened by adjusting the salt concentration. This tour de force enabled them to image chromatophores from Rhodobacter sphaeroides with sufficient resolution to distinguish individual proteins. The fine organization of photosystem complexes could be resolved, and proteins in the chromatophores were found to be translationally and rotationally static over several minutes.
In this issue of ACS Nano, Kumar et al. report a novel AFM method to image the organization of single proteins in native bacterial membrane vesicles named chromatophores. IMAGING MEMBRANES AND CELLS AFM imaging has provided three-dimensional views of microbial structures with nanoscale resolution.3 Unlike in other microscopies, the specimen is imaged in buffer without the need for staining, labeling, or fixation. When imaging in contact mode, sample damage may occur due to the force applied by the scanning tip. In tapping mode, the tip is oscillated while scanning over the surface, thereby reducing lateral forces during imaging. These modes have enabled researchers to visualize the organization and assembly of major cell-wall constituents directly in live cells, such as surface-layer proteins and peptidoglycan (Figure 1, left). Resolutions in the 5−25 nm range are routinely obtained on cells that are relatively rigid and well-structured. On soft cells, like Gram-negative bacteria, the resolution is poorer because the cell is damaged due to the local pressure applied by the tip, or loosely bound molecules, such as polysaccharides, contaminate the tip. The best resolution so far has been achieved on purified membranes. The relative rigidity and ordering of membranes make them well-suited for high-resolution studies (down to 1 nm resolution). Since the pioneering study of the Engel group on native Escherichia coli OmpF porin surfaces,5 the quality of highresolution imaging on bacterial membranes has been continuously improving. Widely investigated prototypes are the intracytoplasmic membranes of purple photosynthetic bacteria. In an elegant study, Scheuring and colleagues revealed how the
This new methodology will be applicable to a wide range of curved samples, including membrane vesicles and bacterial cells. What are the challenges ahead? The slow imaging speed of AFM makes it difficult to study biomolecular and cellular dynamics. During the past decade, there have been tremendous advances in improving the time resolution, now giving access to dynamic processes.7 By using small cantilevers and improved electronics, high-speed (HS-) AFMs can now image biological structures such as bacterial membranes with millisecond resolution, as was recently demonstrated by the Ando and Scheuring teams. For instance, HS-AFM captured dynamic structural changes of the light-driven proton pump bacteriorhodopsin in response to light8 and enabled researchers to study the diffusion of OmpF porin,9 a channel-forming protein found in the E. coli outer membrane. Individual OmpF subunits were identified, and the motion of multiple trimers was monitored. Today, it is not yet known whether HS-AFM will be truly useful
Figure 2. AFM imaging of native chromatophores. (a) High-resolution topographic image and (b) corresponding high-pass-filtered image of a chromatophore vesicle in the native state. Scale bar: 20 nm. Reproduced with permission from ref 4. Copyright 2016 American Chemical Society. 20
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for live-cell imaging because of the highly complex and corrugated nature of cell surfaces. The future will tell us whether the technology should be restricted to well-defined purified structures, or whether it has a true potential for the dynamic imaging of live cells. Another possibility to increase time resolution is to use another scanning probe, near-field scanning optical microscopy. Here, a metal-coated tapered optical fiber with a nanoscale aperture is used to observe dynamic processes on labeled membranes with a spatial resolution of 30 nm and submillisecond time resolution. Although this method has been used for probing membrane heterogeneities in animal cells,10 further developments are needed to apply it to microbiology. It will also be challenging to establish correlated platforms combining force and optical nanoscopy methods,3 in order to probe the localization and interactions of cellular components simultaneously. Finally, AFM is essentially a surface imaging technique, meaning that, in principle, it cannot probe intracellular structures. However, new modalities are emerging, in which subsurface information can be obtained by making use of the nonlinear nanomechanical coupling between the probe and the sample,11 suggesting that one day, AFM might be able to “see” inside the cell.
AFM shed new light into the binding mechanisms (strength, specificity, elasticity) of a variety of adhesion proteins.19−21 In the context of skin, a novel assay was developed to localize and to quantify the nanoscale interaction forces between single S. aureus bacteria and human skin.22 The topography and bacterialbinding properties of skin cells could be mapped at high spatiotemporal resolution, revealing strong attractive forces between bacterial adhesion proteins and target ligands on skin corneocytes. This approach offers promising prospects for understanding the functions of the skin microbiome and for studying the molecular basis of skin diseases such as atopic dermatitis.
STUDYING FORCES IN SINGLE MOLECULES AND CELLS In force spectroscopy, the forces acting on the tip are measured in order to study the localization, elasticity, and interactions of single molecules, either on purified samples or on live cells (Figure 1, right). Force−distance (FD) curves are recorded by measuring the deflection of the cantilever, while the tip is moved up and down. AFM tips functionalized with ligands can be employed to probe cell surface receptors, providing new insights into their biophysical properties.12 In addition, by recording spatially resolved FD curves, researchers can map the distribution of receptors, a method known as affinity imaging first described by the Gaub group.13 Lectin-modified tips could localize single glycolipids in the membrane of red blood cells. More recently, affinity imaging demonstrated that microbial adhesion proteins form nanoscale domains on cell surfaces that are used to strength cell adhesion.14 Notably, force spectroscopy may also be applied to whole cells, offering a means to study heterogeneity and interactions in cellular communities. In 2000, the Gaub group15 attached live Dictyostelium discoideum cells to AFM cantilevers to study the roles of glycoproteins in cell−cell interactions. Since then, singlecell force spectroscopy has been instrumental in studying the forces driving microbial adhesion and biofilm formation. Many microbial pathogens are known to grow as multicellular communities on tissues and implanted devices, leading to biofilm infections that are difficult to eradicate. Understanding and controlling the mechanisms of biofilm formation may contribute to new antimicrobial strategies. Beaussart et al. developed a reliable method for preparing bacterial cell probes for force spectroscopy, using colloidal cantilevers and a bioinspired polydopamine wet adhesive.16 Using these probes, the forces between the oral bacterium Streptococcus mutans and salivary agglutinin were quantified, revealing a cooperative binding mechanism between the adhesion protein P1 and selfassociated agglutinins.17 Single-cell AFM was also applied to the fungal pathogen Candida glabrata, showing the key role of the cell-adhesion protein Epa6 in mediating attachment to hydrophobic substrates.18 Several studies recently focused on Staphylococcus epidermidis and Staphylococcus aureus, two biofilmforming pathogens involved in hospital-acquired infections.
While force spectroscopy offers exciting new experiments in biology, there are still a number of problems to solve in the coming years. FD-based imaging such as affinity imaging is limited by poor temporal and lateral resolution, i.e., images at 30 nm resolution are typically obtained in 20 min. Recently, novel multiparametric imaging methods have been developed,23 in which spatially resolved FD curves are obtained at much higher frequency by oscillating the AFM tip. As a result, the structure, properties, and interactions of the sample are imaged at the speed of topographic imaging. Recent examples of multiparametric imaging experiments include mapping single bacteriophages at the surface of living bacteria,24 imaging the adhesion and elasticity of bacterial pathogens,21,22 and the nanomechanical mapping of the binding events between viruses and animal cells.25 We anticipate that multiparametric imaging will be increasingly used in microbiology to understand how cellular components assemble at the cell surface and how they respond to force. In single-cell force spectroscopy, the low throughput of current assays severely limits their use in microbiology, particularly for screening applications where fast detection is needed. Preparation of microbial cell probes is time-consuming, meaning collecting statistically significant data sets is difficult. Thus, there is much interest in implementing reliable highthroughput single-cell methods for microbial adhesion and biofilm studies. The recently developed FluidFM technology is a powerful solution to this problem.26 Use of microchanneled cantilevers with nanosized apertures enables the quick and reversible manipulation of single cells under physiological conditions. The hollow cantilever is connected to a pressure controller, enabling its operation in liquid as a force-controlled nanopipette under optical control. As a result, many cells can be probed in a short time frame, which is a key asset when it comes to quantifying complex, multiple interactions. Lastly, an interesting research direction for the coming years will be to establish AFM-based force spectroscopy as a platform for screening antiadhesion compounds for therapy. Here, AFM could help identify new antiadhesion agents like peptides and antibodies that are capable of blocking microbial infections, including those caused by multidrug-resistant organisms. In a
In a nutshell, AFM-based force spectroscopy is a powerful technology to study cellular interactions at the cellular and molecular levels, complementing traditional molecular biology and genetic methods and optical and electron microscopy techniques.
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Integrin Nanoclusters Function as Nucleation Sites for Cell Adhesion. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 18557−18562. (11) Tetard, L.; Passian, A.; Thundat, T. New Modes for Subsurface Atomic Force Microscopy through Nanomechanical Coupling. Nat. Nanotechnol. 2010, 5, 105−109. (12) Alsteens, D.; Dupres, V.; Klotz, S. A.; Gaur, N. K.; Lipke, P. N.; Dufrêne, Y. F. Unfolding Individual Als5p Adhesion Proteins on Live Cells. ACS Nano 2009, 3, 1677−1682. (13) Grandbois, M.; Dettmann, W.; Benoit, M.; Gaub, H. E. Affinity Imaging of Red Blood Cells using an Atomic Force Microscope. J. Histochem. Cytochem. 2000, 48, 719−724. (14) Alsteens, D.; Garcia, M. C.; Lipke, P. N.; Dufrene, Y. F. ForceInduced Formation and Propagation of Adhesion Nanodomains in Living Fungal Cells. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 20744− 20749. (15) Benoit, M.; Gabriel, D.; Gerisch, G.; Gaub, H. E. Discrete Interactions in Cell Adhesion Measured by Single-Molecule Force Spectroscopy. Nat. Cell Biol. 2000, 2, 313−317. (16) Beaussart, A.; El-Kirat-Chatel, S.; Sullan, R. M.; Alsteens, D.; Herman, P.; Derclaye, S.; Dufrene, Y. F. Quantifying the Forces Guiding Microbial Cell Adhesion Using Single-Cell Force Spectroscopy. Nat. Protoc. 2014, 9, 1049−1055. (17) Sullan, R. M.; Li, J. K.; Crowley, P. J.; Brady, L. J.; Dufrêne, Y. F. Binding Forces of Streptococcus mutans P1 Adhesin. ACS Nano 2015, 9, 1448−1460. (18) El-Kirat-Chatel, S.; Beaussart, A.; Derclaye, S.; Alsteens, D.; Kucharíková, S.; Van Dijck, P.; Dufrêne, Y. F. Force Nanoscopy of Hydrophobic Interactions in the Fungal Pathogen Candida glabrata. ACS Nano 2015, 9, 1648−1655. (19) Herman, P.; El-Kirat-Chatel, S.; Beaussart, A.; Geoghegan, J. A.; Foster, T. J.; Dufrêne, Y. F. The Binding Force of the Staphylococcal Adhesin SdrG is Remarkably Strong. Mol. Microbiol. 2014, 93, 356−368. (20) Herman-Bausier, P.; El-Kirat-Chatel, S.; Foster, T. J.; Geoghegan, J. A.; Dufrêne, Y. F. Staphylococcus aureus Fibronectin-Binding Protein A Mediates Cell−Cell Adhesion through Low-Affinity Homophilic Bonds. mBio 2015, 6, e01413-15. (21) Formosa-Dague, C.; Speziale, P.; Foster, T. J.; Geoghegan, J. A.; Dufrêne, Y. F. Zinc-Dependent Mechanical Properties of Staphylococcus aureus Biofilm-Forming Surface Protein SasG. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 410−415. (22) Formosa-Dague, C.; Fu, Z.-H.; Feuillie, C.; Derclaye, S.; Foster, T. J.; Geoghegan, J. A.; Dufrêne, Y. F. Forces between Staphylococcus aureus and Human Skin. Nanoscale Horiz. 2016, 1, 298−303. (23) Dufrêne, Y. F.; Martínez-Martín, D.; Medalsy, I.; Alsteens, D.; Müller, D. J. Multiparametric Imaging of Biological Systems by ForceDistance Curve-Based AFM. Nat. Methods 2013, 10, 847−854. (24) Alsteens, D.; Trabelsi, H.; Soumillion, P.; Dufrêne, Y. F. Multiparametric Atomic Force Microscopy Imaging of Single Bacteriophages Extruding from Living Bacteria. Nat. Commun. 2013, 4, 2926. (25) Alsteens, D.; Newton, R.; Schubert, R.; Martinez-Martin, D.; Delguste, M.; Roska, B.; Müller, D. J. Nanomechanical Mapping of First Binding Steps of a Virus to Animal Cells. Nat. Nanotechnol. 2016, in press, DOI: 10.1038/nnano.2016.228. (26) Potthoff, E.; Guillaume-Gentil, O.; Ossola, D.; Polesel-Maris, J.; LeibundGut-Landmann, S.; Zambelli, T.; Vorholt, J. A. Rapid and Serial Quantification of Adhesion Forces of Yeast and Mammalian Cells. PLoS One 2012, 7, e52712. (27) Beaussart, A.; Abellán-Flos, M.; El-Kirat-Chatel, S.; Vincent, S. P.; Dufrêne, Y. F. Force Nanoscopy as a Versatile Platform for Quantifying the Activity of Antiadhesion Compounds Targeting Bacterial Pathogens. Nano Lett. 2016, 16, 1299−1307. (28) Herman-Bausier, P.; Valotteau, C.; Pietrocola, G.; Rindi, S.; Alsteens, D.; Foster, T. J.; Speziale, P.; Dufrêne, Y. F. Mechanical Strength and Inhibition of the Staphylococcus aureus Collagen-Binding Protein Cna. mBio 2016, 7, e01529-16.
proof of concept study, single-cell force spectroscopy was used to demonstrate the ability of multivalent glycofullerenes to block the adhesion of uropathogenic E. coli bacteria to their carbohydrate receptors.27 Also, the antiadhesion activity of monoclonal antibodies against staphylococcal adhesion proteins was assessed directly on living bacteria, without the need for labeling or purification.28 Some antibodies efficiently blocked bacterial adhesion, suggesting they could be used to prevent biofilm formation.
AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. ORCID
Yves F. Dufrêne: 0000-0002-7289-4248 Notes
The author declares no competing financial interest.
ACKNOWLEDGMENTS Work at the Université catholique de Louvain was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 693630), the National Fund for Scientific Research (FNRS), the FNRS-WELBIO under grant no. WELBIO-CR-2015A-05, the Federal Office for Scientific, Technical and Cultural Affairs (Interuniversity Poles of Attraction Programme), and the Research Department of the Communauté Française de Belgique (search Action). Y.F.D. is a Research Director at the FRS-FNRS. REFERENCES (1) Biteen, J. S.; Blainey, P. C.; Cardon, Z. G.; Chun, M.; Church, G. M.; Dorrestein, P. C.; Fraser, S. E.; Gilbert, J. A.; Jansson, J. K.; Knight, R.; Miller, J. F.; Ozcan, A.; Prather, K. A.; Quake, S. R.; Ruby, E. G.; Silver, P. A.; Taha, S.; van den Engh, G.; Weiss, P. S.; Wong, G. C. L.; et al. Tools for the Microbiome: Nano and Beyond. ACS Nano 2016, 10, 6−37. (2) Gitai, Z. New Fluorescence Microscopy Methods for Microbiology: Sharper, Faster, and Quantitative. Curr. Opin. Microbiol. 2009, 12, 341−346. (3) Xiao, J.; Dufrêne, Y. F. Optical and Force Nanoscopy in Microbiology. Nat. Microbiol. 2016, 1, 16186. (4) Kumar, S.; Cartron, M. L.; Mullin, N.; Qian, P.; Leggett, G. J.; Hunter, C. N.; Hobbs, J. K. Direct Imaging of Protein Organization in an Intact Bacterial Organelle Using High-Resolution Atomic Force Microscopy. ACS Nano 2016, DOI: 10.1021/acsnano.6b05647. (5) Schabert, F.; Henn, C.; Engel, A. Native Escherichia coli OmpF Porin Surfaces Probed by Atomic Force Microscopy. Science 1995, 268, 92−94. (6) Scheuring, S.; Sturgis, J. N. Chromatic Adaptation of Photosynthetic Membranes. Science 2005, 309, 484−487. (7) Ando, T.; Uchihashi, T.; Scheuring, S. Filming Biomolecular Processes by High-Speed Atomic Force Microscopy. Chem. Rev. 2014, 114, 3120−3188. (8) Shibata, M.; Yamashita, H.; Uchihashi, T.; Kandori, H.; Ando, T. High-Speed Atomic Force Microscopy Shows Dynamic Molecular Processes in Photoactivated Bacteriorhodopsin. Nat. Nanotechnol. 2010, 5, 208−212. (9) Casuso, I.; Khao, J.; Chami, M.; Paul-Gilloteaux, P.; Husain, M.; Duneau, J.-P.; Stahlberg, H.; Sturgis, J. N.; Scheuring, S. Characterization of the Motion of Membrane Poteins using High-Speed Atomic Force Microscopy. Nat. Nanotechnol. 2012, 7, 525−529. (10) van Zanten, T. S.; Cambi, A.; Koopman, M.; Joosten, B.; Figdor, C. G.; Garcia-Parajo, M. F. Hotspots of GPI-anchored Proteins and 22
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