Adaptation of Escherichia Coli Spheroplasts to the Characterization of Antimicrobial Peptides

Abstract

With activity against a broad range of microorganisms and low susceptibility to the resistance mechanisms developed by multidrug resistant organisms, naturally-occurring antimicrobial peptides (AMPs) have the potential to serve as effective antimicrobial agents to combat the ever-increasing number of antibiotic-resistant illnesses. In addition to various spectroscopic measurements, antimicrobial activity assays, and molecular modeling, the mechanisms of action of novel AMPs can be further characterized using confocal microscopy. This imaging technique utilizes fluorescently-labeled peptides or fluorophore-containing peptide conjugates to examine the localization of antimicrobial peptides in bacterial cells, providing insight into a peptide's mechanism of action. However, the small size and the rod shape of bacteria make it difficult to acquire clear and conclusive images to determine the localization of the peptide in the bacterial cell. The present study focuses on the adaptation of Escherichia coli spheroplasts, which are spherical and at least four times larger than normal bacteria, as a model for the characterization of the mechanisms of action of AMPs. The larger size of the spheroplasts increases the ease of focusing on the target and allows clearer images with increased resolution and more convincing evidence of peptide localization. The spherical shape of the spheroplasts allows for equivalent images to be taken from any angle of the sample. During the optimization process for this approach, we found that the presence of the stabilizing agent, divalent magnesium cation, affects the translocation of cell-penetrating AMPs. Ongoing work is focused on finding a suitable replacement for magnesium in spheroplast stabilization. The successful adaptation of E. coli spheroplasts protocols with confocal microscopy will allow the system to be used for the further characterization of a wide variety of molecules, including AMPs and other cell-penetrating, non-antimicrobial peptides.