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1.
Biochim Biophys Acta. 2016 Apr;1858(4):725-32. doi: 10.1016/j.bbamem.2016.01.003. Epub 2016 Jan 8.

Single-cell, time-resolved study of the effects of the antimicrobial peptide alamethicin on Bacillus subtilis.

Author information

1
Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA.
2
Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA; Molecular Biophysics Program, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706, USA. Electronic address: weisshaar@chem.wisc.edu.

Abstract

Alamethicin is a well-studied antimicrobial peptide (AMP) that kills Gram-positive bacteria. It forms narrow, barrel-stave pores in planar lipid bilayers. We present a detailed, time-resolved microscopy study of the sequence of events during the attack of alamethicin on individual, live Bacillus subtilis cells expressing GFP in the cytoplasm. At the minimum inhibitory concentration (MIC), the first observed symptom is the halting of growth, as judged by the plateau in measured cell length vs time. The data strongly suggest that this growth-halting event occurs prior to membrane permeabilization. Gradual degradation of the proton-motive force, inferred from a decrease in pH-dependent GFP fluorescence intensity, evidently begins minutes later and continues over about 5 min. There follows an abrupt permeabilization of the cytoplasmic membrane to the DNA stain Sytox Orange and concomitant loss of small osmolytes, causing observable cell shrinkage, presumably due to decreased turgor pressure. This permeabilization of the cytoplasmic membrane occurs uniformly across the entire membrane, not locally, on a timescale of 5s or less. GFP gradually leaks out of the cell envelope, evidently impeded by the shrunken peptidoglycan layer. Disruption of the cell envelope by alamethicin occurs in stages, with larger and larger species permeating the envelope as time evolves over tens of minutes. Some of the observed symptoms are consistent with the formation of barrel-stave pores, but the data do not rule out "chaotic pore" or "carpet" mechanisms. We contrast the effects of alamethicin and the human cathelicidin LL-37 on B. subtilis.

KEYWORDS:

Alamethicin; Antimicrobial peptide; Bacillus subtilis; Barrel-stave pore; Fluorescence microscopy; Membrane permeabilization; Real-time imaging; Single-cell imaging

PMID:
26777771
PMCID:
PMC4779711
DOI:
10.1016/j.bbamem.2016.01.003
[Indexed for MEDLINE]
Free PMC Article
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2.
Biochim Biophys Acta. 2013 Jun;1828(6):1511-20. doi: 10.1016/j.bbamem.2013.02.011. Epub 2013 Feb 26.

Real-time attack of LL-37 on single Bacillus subtilis cells.

Author information

1
Department of Chemistry, University of Wisconsin Madison, Madison WI 53706, USA. barns@wisc.edu

Abstract

Time-lapse fluorescence microscopy of single, growing Bacillus subtilis cells with 2-12s time resolution reveals the mechanisms of antimicrobial peptide (AMP) action on a Gram-positive species with unprecedented detail. For the human cathelicidin LL-37 attacking B. subtilis, the symptoms of antimicrobial stress differ dramatically depending on the bulk AMP concentration. At 2μM LL-37, the mean single-cell growth rate decreases, but membrane permeabilization does not occur. At 4μM LL-37, cells abruptly shrink in size at the same time that Sytox Green enters the cytoplasm and stains the nucleoids. We interpret the shrinkage event as loss of turgor pressure (and presumably the membrane potential) due to permeabilization of the membrane. Movies of Sytox Green staining at 0.5frame/s show that nucleoid staining is initially local, more consistent with pore formation than with global permeabilization models. In a novel "growth recovery" assay, cells are incubated with LL-37 for a variable period and then rinsed with fresh growth medium lacking LL-37. The growth rate attenuation observed at 2μM LL-37 is a recoverable symptom, while the abrupt cell shrinkage observed at 4μM LL-37 is not.

PMID:
23454084
PMCID:
PMC3625923
DOI:
10.1016/j.bbamem.2013.02.011
[Indexed for MEDLINE]
Free PMC Article
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