Atomic force microscopy to elucidate how peptides disrupt membranes

Hammond, Kenneth R.; Ryadnov, Maxim G.; Hoogenboom, Bart W.

doi:10.1016/j.bbamem.2020.183447

Cited by 42 publications

(33 citation statements)

References 182 publications

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“…In contrast, CL unrimmed pores present no features above the upper leaflet of the bilayer. Instead, these dynamic structures appear topographically similar to those induced by the antimicrobial peptide melittin and its derivatives (10,28,29). The rimmed CL pores are quite distinct from the unrimmed pores.…”

Section: Discussionmentioning

confidence: 91%

The C. albicans virulence factor Candidalysin polymerizes in solution to form membrane pores and damage epithelial cells

Russell

Schaefer

Dixson

et al. 2021

Preprint

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The pathogenic fungus Candida albicans causes severe invasive candidiasis. C. albicans infection requires the action of the virulence factor Candidalysin (CL), which damages the plasma membrane of the target human cells. However, the molecular mechanism that CL uses to permeabilize membranes is poorly understood. We employed complementary biophysical, modeling, microscopy, and cell biology methods to reveal that CL forms membrane pores using a unique molecular mechanism. Unexpectedly, it was observed that CL readily assembles into linear polymers in solution. The basic structural unit in polymer formation is a CL 8-mer, which is sequentially added into a string configuration. Finally, the linear polymers can close into a loop. Our data indicate that CL loops spontaneously insert into the membrane to become membrane pores. We identified a CL mutation (G4W) that inhibited the formation of polymers in solution and prevented formation of pores in different synthetic lipid membranes systems. Studies in epithelial cells showed that G4W CL failed to activate the danger response signaling pathway, a hallmark of the pathogenic effect of CL. These results indicate that CL polymerization in solution is a necessary step for the damage of cellular membranes. Analysis of thousands of CL pores by atomic force microscopy revealed the co-existence of simple depressions and complex pores decorated with protrusions. Imaging and modeling indicate that the two types of pores are formed by CL molecules assembled into alternate orientations. We propose that this structural rearrangement represents a maturation mechanism that might stabilize pore formation to achieve more robust cellular damage. Taken together, the data show that CL uses a previously unknown mechanism to damage membranes, whereby pre-assembly of CL loops in solution directly leads to formation of membrane pores. Our investigation not only unravels a new paradigm for the formation of membrane pores, but additionally identifies CL polymerization as a novel therapeutic target to treat candidiasis.

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Section: Discussionmentioning

confidence: 91%

The C. albicans virulence factor Candidalysin polymerizes in solution to form membrane pores and damage epithelial cells

Russell

Schaefer

Dixson

et al. 2021

Preprint

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“…In order to understand the fundamentals of how this important class of molecules function, as well as to effectively deploy AMPs in the clinic, it is critical to address the aforementioned gaps between the in vivo function of AMPs, with detailed studies of AMP mechanism in model lipid systems. This objective is starting to be addressed with a variety of approaches that provided high resolution data on AMPs interacting with whole cells, including atomic force microscopy (45)(46)(47), electron microscopy (48,49), Fourier Transform InfraRed (FTIR) spectroscopy (46), differential scanning calorimetry (DSC) (21), and confocal microscopy with fluorescently labeled peptides (50,51). However, the rest of this mini-review will focus on 2 H solid state NMR studies of AMPs interacting with whole cells.…”

Section: Bridging Biophysical and Functional Studiesmentioning

confidence: 99%

Deuterium Solid State NMR Studies of Intact Bacteria Treated With Antimicrobial Peptides

Booth

2021

Front. Med. Technol.

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Solid state NMR has been tremendously useful in characterizing the structure and dynamics of model membranes composed of simple lipid mixtures. Model lipid studies employing solid state NMR have included important work revealing how membrane bilayer structure and dynamics are affected by molecules such as antimicrobial peptides (AMPs). However, solid state NMR need not be applied only to model membranes, but can also be used with living, intact cells. NMR of whole cells holds promise for helping resolve some unsolved mysteries about how bacteria interact with AMPs. This mini-review will focus on recent studies using 2H NMR to study how treatment with AMPs affect membranes in intact bacteria.

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“…The insertion state can be followed by pore expansion causing critical membrane disruption and eventually leading to cell lysis. 12,14…”

Section: Introductionmentioning

confidence: 99%

“…The insertion state can be followed by pore expansion causing critical membrane disruption and eventually leading to cell lysis. 12,14 Leakage studies using reconstituted synthetic unilamellar vesicles provide a straightforward probe of the antimicrobial activity of membrane active AMPs. Typically, a fluorescent probe is encapsulated in the lumen of a phospholipid vesicle, and a stable (non-decaying) fluorescence signal from the vesicle indicates that the lipid membrane remains intact.…”

Section: Introductionmentioning

confidence: 99%

Measuring thousands of single vesicle leakage events reveals the mode of action of antimicrobial peptides

Nahas

Fletcher

Hammond

et al. 2021

Preprint

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Host defense or antimicrobial peptides hold promise for providing new pipelines of effective antimicrobial agents. Their activity quantified against model phospholipid membranes is fundamental to a detailed understanding of their structure-activity relationships. However, existing characterization assays lack the resolution necessary to achieve this insight. Leveraging a highly parallelized microfluidic platform for trapping and studying thousands of giant unilamellar vesicles, we conducted quantitative long-term microscopy studies to monitor the membrane-disruptive activity of archetypal antimicrobial peptides with a high spatiotemporal resolution. We described the modes of action of these peptides via measurements of the disruption of the vesicle population under the conditions of continuous peptide dosing using a range of concentrations, and related the observed modes with the molecular activity mechanisms of these peptides. The study offers an effective approach for characterizing membrane-targeting antimicrobial agents in a standardized manner, and for assigning specific modes of action to the corresponding antimicrobial mechanisms.

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Atomic force microscopy to elucidate how peptides disrupt membranes

Cited by 42 publications

References 182 publications

The C. albicans virulence factor Candidalysin polymerizes in solution to form membrane pores and damage epithelial cells

The C. albicans virulence factor Candidalysin polymerizes in solution to form membrane pores and damage epithelial cells

Deuterium Solid State NMR Studies of Intact Bacteria Treated With Antimicrobial Peptides

Measuring thousands of single vesicle leakage events reveals the mode of action of antimicrobial peptides

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