No abstract

Background: Antimicrobial Peptides (AMPs) are an attractive alternative to traditional small molecule antibiotics as AMPs typically target the bacterial cell membrane. A Trp-rich peptide sequence derived from water buffalo (Bubalus bubalis), buCATHL4B was previously identified as a broad-spectrum antimicrobial peptide. Objective: In this work, native Trp residues were replaced with other naturally occurring aromatic amino acids to begin to elucidate the importance of these residues on peptide activity. Methods: Minimal Inhibitory Concentration (MIC) results demonstrated activity against seven strains of bacteria. Membrane and bilayer permeabilization assays were performed to address the role of bilayer disruption in the activity of the peptides. Lipid vesicle binding and quenching experiments were also performed to gain an understanding of how the peptides interacted with lipid bilayers. Results: MIC results indicate the original, tryptophan-rich sequence, and the phenylalanine substituted sequences exhibit strong inhibition of bacterial growth. In permeabilization assays, peptides with phenylalanine substitutions have higher levels of membrane permeabilization than those substituted with tyrosine. In addition, one of the two-tyrosine substituted sequence, YWY, behaves most differently in the lowest antimicrobial activity, showing no permeabilization of bacterial membranes. Notably the antimicrobial activity is inherently species dependent, with varying levels of activity against different bacteria. Conclusion: There appears to be little correlation between membrane permeabilization and activity, indicating these peptides may have additional mechanisms of action beyond membrane disruption. The results also identify two sequences, denoted FFF and YYW, which retain antibacterial activity but have markedly reduced hemolytic activity.

The plasma membrane insulates a cell from its outside environment, serving as a selectively impermeable barrier across which only small or hydrophobic molecules can pass. Although the membrane is necessary for life, it is also problematic when useful macromolecules such as antibodies, peptides, polysaccharides, and imaging agents are blocked from entry. Most macromolecules can easily be uptaken by the cell through endocytosis, but remain trapped and eventually degraded within endosomes, which mature into lysosomes. To promote the escape of macromolecules from endosomes prior to their maturation into lysosomes, we used a high throughput screen to discover pH triggered, pore-forming peptides[1]. To determine their mechanism of action, we measured the peptides' membrane binding affinity, secondary structure, and induction of pore-formation in POPC membranes. We identified that at least 5 acidic residues are essential for mediating a change from a soluble, predominantly unfolded, and inactive state at pH 7 to a membrane bound, helical, and active state at pH 5. These peptides are highly potent, with significant macromolecular leakage occurring at concentrations as low as 2 peptides per 1000 lipids. We determined that the peptides behave dynamically, associating with and dissociating from membranes, and consequently form pores on multiple vesicles. By atomic force microscopy imaging, we confirmed that the peptides form macromolecular pores, with diameters as large as 50 nm.