Fluorescence Investigations on the Attack of Cell-Wall-Deficient Bacteria by Antimicrobial Peptides

Antimicrobial resistance remains a serious global issue, with an increasing prevalence of multi-drug resistant bacteria appearing throughout the biosphere. Evidence of resistance towards our last-line-of-defense antibiotics is also developing, painting a grim outlook for future treatment outcomes. Antimicrobial peptides offer a promising therapeutic alternative to conventional antibiotics, which act by directly permeating and disrupting the bacterial cell membrane. Much contention persists surrounding the precise mechanism of action responsible for cell death, compounded by the complexity of the biological systems under study.One largely forgotten characteristic of bacteria is the L-Form transformation. Under stressful environmental conditions, such as antibiotic exposure, bacteria enhance their survival by shedding their exterior membrane structure, leaving the cytoplasmic membrane unharmed. In this cell-wall-deficient state, known as the L-Form, bacteria continue to metabolize and reproduce. Upon removal of the stressful stimuli, the bacteria can revert to their classical morphology, resuming previous infectious behavior. The relatively simple L-Form morphology is of particular interest, as directly exposing the bacterial cytoplasmic membrane may allow for new therapeutic strategies, bypassing many classical bacterial defenses.In our work, we have investigated the interaction of a model antimicrobial peptide with Pseudomonas aeruginosa L-Forms via several fluorescence microscopy techniques. P. aeruginosa is a notorious pathogen known for its significant resistance capacity towards antibiotics, but its L-Form state is potentially vulnerable to attack. Details of the interaction are provided using 3D super-resolution imaging correlated with independent experiments of spatially resolved fluorescence lifetime information. We use our prior studies on membrane systems of varying complexity to help interpret our recent findings.