Cathelicidin Peptides Restrict Bacterial Growth via Membrane Perturbation and Induction of Reactive Oxygen Species

All metazoans produce antimicrobial peptides (AMPs) that have both broad antimicrobial and immunomodulatory activity. Cathelicidins are AMPs that preferentially kill Gram-negative bacteria in vitro, purportedly by assembling into higher-order structures that perforate the membrane. We utilized high-resolution, single-cell fluorescence microscopy to examine their mechanism of action in real time. Engineered cathelicidins rapidly bound to Gram-negative and Gram-positive cells and penetrated the cytoplasmic membrane. Rapid failure of the peptidoglycan superstructure in regions of active turnover caused leakage of cytoplasmic contents and the formation of membrane-bound blebs. A mutation anticipated to destabilize interactions between cathelicidin subunits had no effect on bactericidal activity, suggesting that cathelicidins have activities beyond perforating the membrane. Nanomolar concentrations of cathelicidins, although not bactericidal, reduced the growth rate of Gram-negative and Gram-positive bacteria. The cells exhibited expression changes in multiple essential processes, including protein synthesis, peptidoglycan biosynthesis, respiration, and the detoxification of reactive oxygen species (ROS). Time-lapse imaging revealed that ROS accumulation preceded bleb formation, and treatments that reduced cellular ROS levels overcame these bactericidal effects. We propose that that the primary effect of cathelicidins is to induce the production of ROS that damage bacterial molecules, leading to slowed growth or cell death. Given their low circulating levels in vivo, AMPs may serve to slow bacterial population expansion so that cellular immunity systems can respond to and battle the infection.