Atomic force microscopy to elucidate how peptides disrupt membranes

Atomic force microscopy is an increasingly attractive tool to study how peptides disrupt membranes. Often performed on reconstituted lipid bilayers, it provides access to time and length scales that allow dynamic investigations with nanometre resolution. Over the last decade, AFM studies have enabled visualisation of membrane disruption mechanisms by antimicrobial or host defence peptides, including peptides that target malignant cells and biofilms. Moreover, the emergence of high-speed modalities of the technique broadens the scope of investigations to antimicrobial kinetics as well as the imaging of peptide action on live cells in real time. This review describes how methodological advances in AFM facilitate new insights into membrane disruption mechanisms

Imaging live bacteria at the nanoscale: comparison of immobilisation strategies

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

Imaging Protein Fibers at the Nanoscale and In Situ.

Protein self-assembly offers a rich repertoire of tools and technologies. However, despite significant progress in this area, a deterministic measure of the phenomenon, which might lead to predictable relationships between protein components, assembly mechanisms, and ultimately function, is lacking. Often the challenge relates to the choice of the most informative and precise measurements that can link the chemistry of the building blocks with the resulting assembly, ideally in situ and in real time. Using the example of protein fibrillogenesis-a self-assembly process fundamental to nearly every aspect of biological organization, from viral assembly to tissue restoration-this chapter demonstrates how protein self-assembly can be visually and precisely measured while providing measurement protocols applicable to other self-assembly systems

CREIM: Coffee Ring Effect Imaging Model for Monitoring Protein Self-Assembly in Situ

Protein self-assembly is fundamental to nanotechnology. Self-assembling structures are produced under static in vitro conditions typically forming over hours. In contrast, hydrodynamic intracellular environments employ far shorter time scales to compartmentalize highly concentrated protein solutions. Herein, we exploit the radial capillary flow within a drying sessile droplet (the coffee ring effect) to emulate dynamic native environments and monitor an archetypal protein assembly in situ using high-speed super-resolution imaging. We demonstrate that the assembly can be empirically driven to completion within minutes to seconds without apparent changes in supramolecular morphology. The model offers a reliable tool for the diagnosis and engineering of self-assembling systems under nonequilibrium conditions

Single Cell Killing Kinetics Differentiate Phenotypic Bacterial Responses to Different Antibacterial Classes

This is the final version. Available on open access from the American Society for Microbiology via the DOI in this recordData availability. We have made available a step-by-step experimental protocol for the fabrication and handling of microfluidic devices for investigating the interactions between the antimicrobial agents and individual cells (74). Data supporting the conclusions of this article will be made available by the authors to any qualified researcher upon request.With the spread of multidrug-resistant bacteria, there has been an increasing focus on molecular classes that have not yet yielded an antibiotic. A key capability for assessing and prescribing new antibacterial treatments is to compare the effects antibacterial agents have on bacterial growth at a phenotypic, single-cell level. Here, we combined time-lapse microscopy with microfluidics to investigate the concentration-dependent killing kinetics of stationary-phase Escherichia coli cells. We used antibacterial agents from three different molecular classes, β-lactams and fluoroquinolones, with the known antibiotics ampicillin and ciprofloxacin, respectively, and a new experimental class, protein Ψ-capsids. We found that bacterial cells elongated when treated with ampicillin and ciprofloxacin used at their minimum inhibitory concentration (MIC). This was in contrast to Ψ-capsids, which arrested bacterial elongation within the first two hours of treatment. At concentrations exceeding the MIC, all the antibacterial agents tested arrested bacterial growth within the first 2 h of treatment. Further, our single-cell experiments revealed differences in the modes of action of three different agents. At the MIC, ampicillin and ciprofloxacin caused the lysis of bacterial cells, whereas at higher concentrations, the mode of action shifted toward membrane disruption. The Ψ-capsids killed cells by disrupting their membranes at all concentrations tested. Finally, at increasing concentrations, ampicillin and Ψ-capsids reduced the fraction of the population that survived treatment in a viable but nonculturable state, whereas ciprofloxacin increased this fraction. This study introduces an effective capability to differentiate the killing kinetics of antibacterial agents from different molecular classes and offers a high content analysis of antibacterial mechanisms at the single-cell level. IMPORTANCE Antibiotics act against bacterial pathogens by inhibiting their growth or killing them directly. Different modes of action determine different antibacterial responses, whereas phenotypic differences in bacteria can challenge the efficacy of antibiotics. Therefore, it is important to be able to differentiate the concentration-dependent killing kinetics of antibacterial agents at a single-cell level, in particular for molecular classes which have not yielded an antibiotic before. Here, we measured single-cell responses using microfluidics-enabled imaging, revealing that a novel class of antibacterial agents, protein Ψ-capsids, arrests bacterial elongation at the onset of treatment, whereas elongation continues for cells treated with β-lactam and fluoroquinolone antibiotics. The study advances our current understanding of antibacterial function and offers an effective strategy for the comparative design of new antibacterial therapies, as well as clinical antibiotic susceptibility testing.Biotechnology and Biological Sciences Research Council (BBSRC)Medical Research Council (MRC)UK Department for Business, Energy and Industrial Strategy (BEIS

Imaging the Effects of Peptide Materials on Phospholipid Membranes by Atomic Force Microscopy

Recent advances in biomolecular design require accurate measurements performed in native or near-native environments in real time. Atomic force microscopy (AFM) is a powerful tool to observe the dynamics of biologically relevant processes at aqueous interfaces with high spatial resolution. Here, we describe imaging protocols to characterize the effects of peptide materials on phospholipid membranes in solution by AFM. These protocols can be used to determine the mechanism and kinetics of membrane-associated activities at the nanoscale

Flowering poration – a synergistic multi-mode antibacterial mechanism by a bacteriocin fold

Bacteriocins are a distinct family of antimicrobial proteins postulated to porate bacterial membranes. However, direct experimental evidence of pore formation by these proteins is lacking. Here we report a multi-mode poration mechanism induced by four-helix bacteriocins, epidermicin NI01 and aureocin A53. Using a combination of crystallography, spectroscopy, bioassays and nanoscale imaging, we established that individual two-helix segments of epidermicin retain antibacterial activity but each of these segments adopts a particular poration mode. In the intact protein these segments act synergistically to balance out antibacterial and hemolytic activities. The study sets a precedent of multi-mode membrane disruption advancing the current understanding of structure-activity relationships in pore-forming proteins

Phase separation in the outer membrane of Escherichia coli

Antimicrobial resistance is particularly prevalent in gram-negative bacteria, as antibiotics that act inside the cells must overcome their outer membrane. So far, technical limitations have prevented us from determining how outer-membrane proteins and lipids are organized to form this functional barrier. Here, we use nanoscale imaging of live bacteria to reveal that the most abundant outer-membrane proteins form a network that spans the entire bacterial surface, leaving only small gaps of phase-separated lipopolysaccharide. This tendency to phase separate is further emphasized by the formation of new domains when phospholipids are mislocated at the surface, rendering cells more susceptible to some antibiotics. Overall, the phase-separated nature of the outer membrane defines a perspective on its integrity and barrier function

Nano-mechanical single-cell sensing of cell-matrix contacts

Extracellular protein matrices provide a rigidity interface exhibiting nano-mechanical cues that guide cell growth and proliferation. Cells sense such cues using actin-rich filopodia extensions which encourage favourable cell–matrix contacts to recruit more actin-mediated local forces into forming stable focal adhesions. A challenge remains in identifying and measuring these local cellular forces and in establishing empirical relationships between them, cell adhesion and filopodia formation. Here we investigate such relationships using a micromanipulation system designed to operate at the time scale of focal contact dynamics, with the sample frequency of a force probe being 0.1 ms, and to apply and measure forces at nano-to-micro Newton ranges for individual mammalian cells. We explore correlations between cell biomechanics, cell–matrix attachment forces and the spread areas of adhered cells as well as their relative dependence on filopodia formation using synthetic protein matrices with a proven ability to induce enhanced filopodia numbers in adherent cells. This study offers a basis for engineering exploitable cell–matrix contacts in situ at the nanoscale and single-cell levels

Antimicrobial peptide capsids of de novo design

The spread of bacterial resistance to antibiotics poses the need for antimicrobial discovery. With traditional search paradigms being exhausted, approaches that are altogether different from antibiotics may offer promising and creative solutions. Here, we introduce a de novo peptide topology that-by emulating the virus architecture-assembles into discrete antimicrobial capsids. Using the combination of high-resolution and real-time imaging, we demonstrate that these artificial capsids assemble as 20-nm hollow shells that attack bacterial membranes and upon landing on phospholipid bilayers instantaneously (seconds) convert into rapidly expanding pores causing membrane lysis (minutes). The designed capsids show broad antimicrobial activities, thus executing one primary function-they destroy bacteria on contact