Effects of D-Lysine Substitutions on the Activity and Selectivity of Antimicrobial Peptide CM15.

Heather M Kaminski,Jimmy B Feix

doi:10.3390/polym3042088

Abstract

Despite their potent antimicrobial activity, the usefulness of antimicrobial peptides (AMPs) as antibiotics has been limited by their toxicity to eukaryotic cells and a lack of stability in vivo. In the present study we examined the effects of introducing D-lysine residues into a 15-residue hybrid AMP containing residues 1-7 of cecropin A and residues 2-9 of melittin (designated CM15). Diastereomeric analogs of CM15 containing between two and five D-lysine substitutions were evaluated for their antimicrobial activity, lysis of human erythrocytes, toxicity to murine macrophages, ability to disrupt cell membranes, and protease stability. All of the analogs caused rapid permeabilization of the Staphylococcus aureus cell envelope, as indicated by uptake of SYTOX green. CM15 also permeabilized the plasma membrane of RAW264.7 macrophages, but this was substantially diminished for the D-lysine containing analogs. The introduction of D-lysine caused moderate decreases in antimicrobial activity for all analogs studied. However, D-Lys substitution produced a much more pronounced reduction in toxicity to eukaryotic cells, leading to marked improvements in antimicrobial efficacy for some analogs. Circular dichroism studies indicated a progressive loss of helical secondary structure upon introduction of D-lysine residues, and there was a good correspondence between helical content and eukaryotic cell cytotoxicity. Overall, these studies show that the biological activity of CM15 analogs containing D-lysine depends on both the number and position of D-Lys substitutions, and that such substitutions can dramatically lower toxicity to eukaryotic cells with only minimal decreases in antimicrobial activity.

Highlights

The extensive use of antibiotics over the past sixty years has led to an increased prevalence of antibiotic resistance in both hospital- and community-acquired infections [1,2,3,4,5,6], giving rise to a critical need for the development of new approaches for treatment of bacterial infections
We find that changes in the biological activity of CM15 depend on both the number and position of D-lysine substitutions, and that such D-amino acid substitutions can dramatically lower toxicity to eukaryotic cells with only minimal decreases in antimicrobial activity
To examine the effects of disrupting peptide secondary structure on the antimicrobial activity and cytotoxicity of CM15, we designed a set of peptides containing D-amino acid substitutions at lysine residues (Table 1)

Summary

Introduction

The extensive use of antibiotics over the past sixty years has led to an increased prevalence of antibiotic resistance in both hospital- and community-acquired infections [1,2,3,4,5,6], giving rise to a critical need for the development of new approaches for treatment of bacterial infections. AMPs are typically 12–50 amino acids in length, contain from 2–9 positively charged residues, and adopt amphipathic secondary structures upon binding to membranes [7,8,9,10,11,12,13]. Their positive charge allows AMPs to interact more strongly with the highly negatively-charged membranes of bacteria as opposed to the nearly neutral plasma membranes of eukaryotic cells [12,13], and their amphipathic secondary structure facilitates partitioning into the membrane bilayer [14]. Some AMPs do interact with intracellular targets [11], even these must cross the membrane in order to exert their biological effect

Methods

Results

Conclusion