Mechanism of Natural and Unnatural Antimicrobial Peptides

Antimicrobial peptides (AMPs) are innate host defense system that exists throughout the evolutionary spectrum. AMPs permeabilize both Gram negative and Gram positive bacterial cells, while causing relatively little damage to eukaryotic cells. This general mechanism of attack on the cell envelope is relatively insusceptible to bacterial resistance by mutation. AMPs and their analogues are thus appealing drug candidates. The standard bulk assays for AMP efficacy lack the temporal and spatial resolution necessary to dissect the underlying biophysical mechanisms of AMP activity. Better understanding of bacteriostatic (growth-inhibiting) and bactericidal (killing) activity may in turn enhance development of new therapeutic agents.

We are developing single-cell, time-resolved assays that place bacterial “symptoms” during AMP attack on a common time line, with high temporal resolution. Quantitative fluorescence microscopy enables direct observation of: permeabilization of the outer membrane, evidenced by periplasm content leakage; permeabilization of the cytoplasm, evidenced by cytoplasmic content leakage and entry of DNA-sensitive dyes; changes in the morphology of the nucleoid; changes in protein mobility in both periplasm and cytoplasm; and the halting of measurable growth and more! We focus on the detailed biophysical mechanisms by which natural and synthetic antimicrobial peptides degrade bacterial cell membranes, halt growth, and kill bacteria.