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

Time-lapse fluorescence microscopy of single, growing Bacillus subtilis cells with 2-12 s 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 2 mu M LL-37, the mean single-cell growth rate decreases, but membrane permeabilization does not occur. At 4 mu M 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 0.5 frame/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 2 mu M LL-37 is a recoverable symptom, while the abrupt cell shrinkage observed at 4 mu M LL-37 is not. (C) 2013 Elsevier B.V. All rights reserved.