title: Real-time attack of LL-37 on single Bacillus subtilis cells.
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.
authors: Department of Chemistry, University of Wisconsin Madison, Madison WI 53706, USA. barns@wisc.edu
authors: Kenneth J
authors: KJ
authors: Barns
authors: James C
authors: JC
authors: Weisshaar
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D008564:Membrane Potentials / Q000187:drug effects; D050296:Microbial Viability / Q000187:drug effects; D008856:Microscopy, Fluorescence; D013997:Time Factors; D059008:Time-Lapse Imaging; D014741:Video Recording; D054804:Cathelicidins
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D054804:Cathelicidins