Studying the Mechanisms of Hybrid Peptides Containing Permeabalizing and Cell Penetrating Domains

Abstract

Antibiotic resistant pathogens cause 2 million infections and 23,000 deaths in the U.S. every year. Bacteria's adaptability to the current drugs on the market necessitates alternative therapeutics, such as antimicrobial peptides (AMPs). AMPs exhibit non-specific bactericidal activity against both gram positive and negative bacteria, selectively killing prokaryotes over eukaryotes. These peptides function by two main mechanistic categories: 1) permeabilization, where the peptide compromises the membrane causing intercellular leakage, and 2) translocation, where the integrity of the membrane is maintained while the peptide enters the cell and disrupts intercellular processes. Our current study examines the effect of combining AMPs that utilize different antimicrobial mechanisms together to form a hybrid peptide. Based on previous work in our lab considering the mechanism of hipposin, we predicted that hybrid peptides made from combining permeabilizing and translocating peptides would generally follow a permeabilizing mechanism. In particular, we considered the activity of hybrid peptides made from parasin, a previously characterized AMP known to work via permeabilization, and DesHDAP1, a designed peptide known to translocate through bacterial membranes. We attached DesHDAP1 to both the N- and C-termini of Parasin, both with and without an alanine linker between the peptides. The activity and mechanism of these hybrid peptides was considered using radial diffusion assays, propidium iodide uptake assays, and confocal microscopy. Confocal microscopy integrated the formation of larger bacterial spheroplasts and the utilization of a membrane specific dye to test for peptide-membrane co-localization. These enhancements have improved image quality and aid in reliable interpretations of translocation versus membrane localization. Overall, the hybrid peptides did demonstrate significant permeabilizing activity and were at least as active as the parent peptides.