Immobilizing live Escherichia coli for AFM studies of surface dynamics

Lonergan, Natalie E.; Britt, L. D.; Sullivan, Claretta J.

doi:10.1016/j.ultramic.2013.10.017

Cited by 31 publications

(25 citation statements)

References 31 publications

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“…In order to begin examining the ultrastructure of the C. albicans cell wall, we used atomic force microscopy (AFM), as it gives high-resolution images of the cell wall in a liquid environment without damaging the sample. This allows real-time imaging of metabolically active samples (50)(51)(52)(53). First, we needed to immobilize the cells on a suitable surface.…”

Section: Resultsmentioning

confidence: 99%

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

et al. 2017

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Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in ␤-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for ␤-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of ␤-(1,3)-glucan to the immune system occurs when the mannan layer is altered or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system.

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Section: Resultsmentioning

confidence: 99%

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

et al. 2017

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“…Previous studies have clearly shown that, depending on the intensity of force applied, bacteria indented by atomic force microscopy (AFM) tips can recover and survive . Other researchers provided evidences on the recovery of bacteria damaged by hypoosmotic stress, too . Thus, it is lucid that bacteria are able to recover the damages in their envelopes up to an extent.…”

Section: Resultsmentioning

confidence: 99%

Synergism of Water Shock and a Biocompatible Block Copolymer Potentiates the Antibacterial Activity of Graphene Oxide

Karahan

Wei

Goh

et al. 2015

Small

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Graphene oxide (GO) is promising in the fight against pathogenic bacteria. However, the antibacterial activity of pristine GO is relatively low and concern over human cytotoxicity further limits its potential. This study demonstrates a general approach to address both issues. The developed approach synergistically combines the water shock treatment (i.e., a sudden decrease in environmental salinity) and the use of a biocompatible block copolymer (Pluronic F-127) as a synergist co-agent. Hypoosmotic stress induced by water shock makes gram-negative pathogens more susceptible to GO. Pluronic forms highly stable nanoassemblies with GO (Pluronic-GO) that can populate around bacterial envelopes favoring the interactions between GO and bacteria. The antibacterial activity of GO at a low concentration (50 μg mL(-1) ) increases from <30% to virtually complete killing (>99%) when complemented with water shock and Pluronic (5 mg mL(-1) ) at ≈2-2.5 h of exposure. Results suggest that the enhanced dispersion of GO and the osmotic pressure generated on bacterial envelopes by polymers together potentiate GO. Pluronic also significantly suppresses the toxicity of GO toward human fibroblast cells. Fundamentally, the results highlight the crucial role of physicochemical milieu in the antibacterial activity of GO. The demonstrated strategy has potentials for daily-life bacterial disinfection applications, as hypotonic Pluronic-GO mixture is both safe and effective.

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“…Different immobilisation techniques also yield different levels of adhesion and are affected by buffer composition to different degrees (Figure 4a). Gelatin and PLL are the most common methods used for immobilisation of bacteria 17,31 . These are cationic protein coatings that promote the attachment of anionic bacteria via electrostatic interactions.…”

Section: Resultsmentioning

confidence: 99%

Imaging live bacteria at the nanoscale: comparison of immobilisation strategies

Benn

Pyne

Ryadnov

et al. 2019

Preprint

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Atomic force microscopy (AFM) provides an effective, label-free technique enabling the imaging of live bacteria under physiological conditions with nanometre precision. However, AFM is a surface scanning technique, and the accuracy of its performance requires the effective and reliable immobilisation of bacterial cells onto substrates. Here, we compare the effectiveness of various chemical approaches to facilitate the immobilisation of Escherichia coli onto glass cover slips in terms of bacterial adsorption, viability and compatibility with correlative imaging by fluorescence microscopy. We assess surface functionalisation using gelatin, poly-L-lysine, Cell-Tak™, and Vectabond®. We describe how bacterial immobilisation, viability and suitability for AFM experiments depend on bacterial strain, buffer conditions and surface functionalisation. We demonstrate the use of such immobilisation by AFM images that resolve the porin lattice on the bacterial surface; local degradation of the bacterial cell envelope by an antimicrobial peptide (Cecropin B); and the formation of membrane attack complexes on the bacterial membrane.

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Immobilizing live Escherichia coli for AFM studies of surface dynamics

Cited by 31 publications

References 31 publications

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

β-(1,3)-Glucan Unmasking in Some Candida albicans Mutants Correlates with Increases in Cell Wall Surface Roughness and Decreases in Cell Wall Elasticity

Synergism of Water Shock and a Biocompatible Block Copolymer Potentiates the Antibacterial Activity of Graphene Oxide

Imaging live bacteria at the nanoscale: comparison of immobilisation strategies

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