The Role of Atomic Force Microscopy in Microbiology: Sensing the Cell Surface

A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Microbial Biotechnology".

Deadline for manuscript submissions: closed (31 December 2022) | Viewed by 14123

Special Issue Editors


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Guest Editor
LAAS-CNRS, CNRS, Univeristé de Toulouse, Toulouse, France
Interests: biotechnology; microscopy; bacteria; surface characterization; cell biology; microbiology; nanobiotechnology; antibiotics; pathogens

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Guest Editor
TBI, Université de Toulouse, INSA, INRAE, CNRS, Toulouse, France
Interests: atomic force microscopy; biological interfaces; microalgae; flotation; molecular interactions; biophysics

Special Issue Information

Dear Colleagues,

Atomic force microscopy is now established as a key technology in microbiology. Imaging living cells at high resolution, probing the nanomechanical properties of cells, sensing their interactions with surfaces or with others microbes, characterizing adhesins at the surface of microbes, and analyzing biofilms structures and architectures are, among many others, the topics that benefit from the contribution of atomic force microscopy (AFM). In this Special Issue, we welcome review and original research papers dedicated to or including AFM data recorded on microbes. The subjects covered include but are not restricted to microbial adhesion, microbial division, biofilm formation, extracellular appendices characterization, and antimicrobial effects. Studies on bacteria, unicellular fungi, and microalgae are welcome.

Dr. Etienne Dague
Dr. Cécile Formosa
Guest Editors

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Keywords

  • Atomic force microscopy
  • Single molecule force spectroscopy
  • Single cell force spectroscopy
  • Microorganisms cell wall
  • Microorganisms mechanobiology
  • Microorganisms adhesion
  • Biofilm formation
  • Imaging

Published Papers (5 papers)

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Research

10 pages, 2755 KiB  
Article
Immobilization of Polyethyleneimine (PEI) on Flat Surfaces and Nanoparticles Affects Its Ability to Disrupt Bacterial Membranes
by Nesha May Octavio Andoy, Meera Patel, Ching Lam Jane Lui and Ruby May Arana Sullan
Microorganisms 2021, 9(10), 2176; https://doi.org/10.3390/microorganisms9102176 - 19 Oct 2021
Cited by 4 | Viewed by 2990
Abstract
Interactions between a widely used polycationic polymer, polyethyleneimine (PEI), and a Gram-negative bacteria, E. coli, are investigated using atomic force microscopy (AFM) quantitative imaging. The effect of PEI, a known membrane permeabilizer, is characterized by probing both the structure and elasticity of [...] Read more.
Interactions between a widely used polycationic polymer, polyethyleneimine (PEI), and a Gram-negative bacteria, E. coli, are investigated using atomic force microscopy (AFM) quantitative imaging. The effect of PEI, a known membrane permeabilizer, is characterized by probing both the structure and elasticity of the bacterial cell envelope. At low concentrations, PEI induced nanoscale membrane perturbations all over the bacterial surface. Despite these structural changes, no change in cellular mechanics (Young’s modulus) was detected and the growth of E. coli is barely affected. However, at high PEI concentrations, dramatic changes in both structure and cell mechanics are observed. When immobilized on a flat surface, the ability of PEI to alter the membrane structure and reduce bacterial elasticity is diminished. We further probe this immobilization-induced effect by covalently attaching the polymer to the surface of polydopamine nanoparticles (PDNP). The nanoparticle-immobilized PEI (PDNP-PEI), though not able to induce major structural changes on the outer membrane of E. coli (in contrast to the flat surface), was able to bind to and reduce the Young’s modulus of the bacteria. Taken together, our data demonstrate that the state of polycationic polymers, whether bound or free—which greatly dictates their overall configuration—plays a major role on how they interact with and disrupt bacterial membranes. Full article
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16 pages, 6698 KiB  
Article
Atomic Force Microscopy to Characterize Antimicrobial Peptide-Induced Defects in Model Supported Lipid Bilayers
by Kathleen W. Swana, Ramanathan Nagarajan and Terri A. Camesano
Microorganisms 2021, 9(9), 1975; https://doi.org/10.3390/microorganisms9091975 - 17 Sep 2021
Cited by 3 | Viewed by 2332
Abstract
Antimicrobial peptides (AMPs) interact with bacterial cell membranes through a variety of mechanisms, causing changes extending from nanopore formation to microscale membrane lysis, eventually leading to cell death. Several AMPs also disrupt mammalian cell membranes, despite their significantly different lipid composition and such [...] Read more.
Antimicrobial peptides (AMPs) interact with bacterial cell membranes through a variety of mechanisms, causing changes extending from nanopore formation to microscale membrane lysis, eventually leading to cell death. Several AMPs also disrupt mammalian cell membranes, despite their significantly different lipid composition and such collateral hemolytic damage hinders the potential therapeutic applicability of the AMP as an anti-microbial. Elucidating the mechanisms underlying the AMP–membrane interactions is challenging due to the variations in the chemical and structural features of the AMPs, the complex compositional variations of cell membranes and the inadequacy of any single experimental technique to comprehensively probe them. (1) Background: Atomic Force Microscopy (AFM) imaging can be used in combination with other techniques to help understand how AMPs alter the orientation and structural organization of the molecules within cell membranes exposed to AMPs. The structure, size, net charge, hydrophobicity and amphipathicity of the AMPs affect how they interact with cell membranes of differing lipid compositions. (2) Methods: Our study examined two different types of AMPs, a 20-amino acid, neutral, α-helical (amphipathic) peptide, alamethicin, and a 13-amino acid, non-α-helical cationic peptide, indolicidin (which intramolecularly folds, creating a hydrophobic core), for their interactions with supported lipid bilayers (SLBs). Robust SLB model membranes on quartz supports, incorporating predominantly anionic lipids representative of bacterial cells, are currently not available and remain to be developed. Therefore, the SLBs of zwitterionic egg phosphatidylcholine (PC), which represents the composition of a mammalian cell membrane, was utilized as the model membrane. This also allows for a comparison with the results obtained from the Quartz Crystal Microbalance with Dissipation (QCM-D) experiments conducted for these peptides interacting with the same zwitterionic SLBs. Further, in the case of alamethicin, because of its neutrality, the lipid charge may be less relevant for understanding its membrane interactions. (3) Results: Using AFM imaging and roughness analysis, we found that alamethicin produced large, unstable defects in the membrane at 5 µM concentrations, and completely removed the bilayer at 10 µM. Indolicidin produced smaller holes in the bilayer at 5 and 10 µM, although they were able to fill in over time. The root-mean-square (RMS) roughness values for the images showed that the surface roughness caused by visible defects peaked after peptide injection and gradually decreased over time. (4) Conclusions: AFM is useful for helping to uncover the dynamic interactions between different AMPs and cell membranes, which can facilitate the selection and design of more efficient AMPs for use in therapeutics and antimicrobial applications. Full article
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11 pages, 3102 KiB  
Communication
Nanomotion Spectroscopy as a New Approach to Characterize Bacterial Virulence
by Maria I. Villalba, Leonardo Venturelli, Ronnie Willaert, Maria E. Vela, Osvaldo Yantorno, Giovanni Dietler, Giovanni Longo and Sandor Kasas
Microorganisms 2021, 9(8), 1545; https://doi.org/10.3390/microorganisms9081545 - 21 Jul 2021
Cited by 5 | Viewed by 2125
Abstract
Atomic force microscopy (AFM)-based nanomotion detection is a label-free technique that has been used to monitor the response of microorganisms to antibiotics in a time frame of minutes. The method consists of attaching living organisms onto an AFM cantilever and in monitoring its [...] Read more.
Atomic force microscopy (AFM)-based nanomotion detection is a label-free technique that has been used to monitor the response of microorganisms to antibiotics in a time frame of minutes. The method consists of attaching living organisms onto an AFM cantilever and in monitoring its nanometric scale oscillations as a function of different physical-chemical stimuli. Up to now, we only used the cantilever oscillations variance signal to assess the viability of the attached organisms. In this contribution, we demonstrate that a more precise analysis of the motion pattern of the cantilever can unveil relevant medical information about bacterial phenotype. We used B. pertussis as the model organism, it is a slowly growing Gram-negative bacteria which is the agent of whooping cough. It was previously demonstrated that B. pertussis can expresses different phenotypes as a function of the physical-chemical properties of the environment. In this contribution, we highlight that B. pertussis generates a cantilever movement pattern that depends on its phenotype. More precisely, we noticed that nanometric scale oscillations of B. pertussis can be correlated with the virulence state of the bacteria. The results indicate a correlation between metabolic/virulent bacterial states and bacterial nanomotion pattern and paves the way to novel rapid and label-free pathogenic microorganism detection assays. Full article
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17 pages, 2473 KiB  
Article
Extracellular Polymeric Substance Protects Some Cells in an Escherichia coli Biofilm from the Biomechanical Consequences of Treatment with Magainin 2
by Helen M. Greer, Kanesha Overton, Megan A. Ferguson, Eileen M. Spain, Louise E. O. Darling, Megan E. Núñez and Catherine B. Volle
Microorganisms 2021, 9(5), 976; https://doi.org/10.3390/microorganisms9050976 - 30 Apr 2021
Cited by 3 | Viewed by 2267
Abstract
Bacterial biofilms have long been recognized as a source of persistent infections and industrial contamination with their intransigence generally attributed to their protective layer of extracellular polymeric substances (EPS). EPS, consisting of secreted nucleic acids, proteins, and polysaccharides, make it difficult to fully [...] Read more.
Bacterial biofilms have long been recognized as a source of persistent infections and industrial contamination with their intransigence generally attributed to their protective layer of extracellular polymeric substances (EPS). EPS, consisting of secreted nucleic acids, proteins, and polysaccharides, make it difficult to fully eliminate biofilms by conventional chemical or physical means. Since most bacteria are capable of forming biofilms, understanding how biofilms respond to new antibiotic compounds and components of the immune system has important ramifications. Antimicrobial peptides (AMPs) are both potential novel antibiotic compounds and part of the immune response in many different organisms. Here, we use atomic force microscopy to investigate the biomechanical changes that occur in individual cells when a biofilm is exposed to the AMP magainin 2 (MAG2), which acts by permeabilizing bacterial membranes. While MAG2 is able to prevent biofilm initiation, cells in an established biofilm can withstand exposure to high concentrations of MAG2. Treated cells in the biofilm are classified into two distinct populations after treatment: one population of cells is indistinguishable from untreated cells, maintaining cellular turgor pressure and a smooth outer surface, and the second population of cells are softer than untreated cells and have a rough outer surface after treatment. Notably, the latter population is similar to planktonic cells treated with MAG2. The EPS likely reduces the local MAG2 concentration around the stiffer cells since once the EPS was enzymatically removed, all cells became softer and had rough outer surfaces. Thus, while MAG2 appears to have the same mechanism of action in biofilm cells as in planktonic ones, MAG2 cannot eradicate a biofilm unless coupled with the removal of the EPS. Full article
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11 pages, 4623 KiB  
Article
A Non-Destructive, Tuneable Method to Isolate Live Cells for High-Speed AFM Analysis
by Christopher T. Evans, Sara J. Baldock, John G. Hardy, Oliver Payton, Loren Picco and Michael J. Allen
Microorganisms 2021, 9(4), 680; https://doi.org/10.3390/microorganisms9040680 - 25 Mar 2021
Cited by 6 | Viewed by 3165
Abstract
Suitable immobilisation of microorganisms and single cells is key for high-resolution topographical imaging and study of mechanical properties with atomic force microscopy (AFM) under physiologically relevant conditions. Sample preparation techniques must be able to withstand the forces exerted by the Z range-limited cantilever [...] Read more.
Suitable immobilisation of microorganisms and single cells is key for high-resolution topographical imaging and study of mechanical properties with atomic force microscopy (AFM) under physiologically relevant conditions. Sample preparation techniques must be able to withstand the forces exerted by the Z range-limited cantilever tip, and not negatively affect the sample surface for data acquisition. Here, we describe an inherently flexible methodology, utilising the high-resolution three-dimensional based printing technique of multiphoton polymerisation to rapidly generate bespoke arrays for cellular AFM analysis. As an example, we present data collected from live Emiliania huxleyi cells, unicellular microalgae, imaged by contact mode High-Speed Atomic Force Microscopy (HS-AFM), including one cell that was imaged continuously for over 90 min. Full article
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