Cationic Amphipathic Antimicrobial Peptides Perturb the Inner Membrane of Germinated Spores Thus Inhibiting Their Outgrowth

The mode of action of four cationic amphipathic antimicrobial peptides (AMPs) was evaluated against the non-pathogenic, Gram-positive, spore-forming bacterium, Bacillus subtilis. The AMPs were TC19, TC84, BP2, and the lantibiotic Nisin A. TC19 and TC84 were derived from the human thrombocidin-1. Bactericidal peptide 2 (BP2) was derived from the human bactericidal permeability increasing protein (BPI). We employed structured illumination microscopy (SIM), fluorescence microscopy, Alexa 488-labeled TC84, B. subtilis mutants producing proteins fused to the green fluorescent protein (GFP) and single-cell live imaging to determine the effects of the peptides against spores. TC19, TC84, BP2, and Nisin A showed to be bactericidal against germinated spores by perturbing the inner membrane, thus preventing outgrowth to vegetative cells. Single cell live imaging showed that the AMPs do not affect the germination process, but the burst time and subsequent generation time of vegetative cells. Alexa 488-labeled TC84 suggested that the TC84 might be binding to the dormant spore-coat. Therefore, dormant spores were also pre-coated with the AMPs and cultured on AMP-free culture medium during single-cell live imaging. Pre-coating of the spores with TC19, TC84, and BP2 had no effect on the germination process, and variably affected the burst time and generation time. However, the percentage of spores that burst and grew out into vegetative cells was drastically lower when pre-coated with Nisin A, suggesting a novel application potential of this lantibiotic peptide against spores. Our findings contribute to the understanding of AMPs and show the potential of AMPs as eventual therapeutic agents against spore-forming bacteria.