All d-Lysine Analogues of the Antimicrobial Peptide HPA3NT3-A2 Increased Serum Stability and without Drug Resistance

Jong-Kook Lee,Yoonkyung Park

doi:10.3390/ijms21165632

Abstract

Novel antibiotic drugs are urgently needed because of the increase in drug-resistant bacteria. The use of antimicrobial peptides has been suggested to replace antibiotics as they have strong antimicrobial activity and can be extracted from living organisms such as insects, marine organisms, and mammals. HPA3NT3-A2 ([Ala1,8] HPA3NT3) is an antimicrobial peptide that is an analogue of the HP (2–20) peptide derived from Helicobacter pylori ribosomal protein L1. Although this peptide was shown to have strong antimicrobial activity against drug-resistant bacteria, it also showed lower toxicity against sheep red blood cells (RBCs) and HaCaT cells compared to HPA3NT3. The l-Lys residues of HPA3NT3-A2 was substituted with d-Lys residues (HPA3NT3-A2D; [d-Lys2,5,6,9,10,15] HPA3NT3-A2) to prevent the cleavage of peptide bonds by proteolytic enzymes under physiological conditions. This peptide showed an increased half-life and maintained its antimicrobial activity in the serum against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) (pathogen). Furthermore, the antimicrobial activity of HPA3NT3-A2D was not significantly affected in the presence of mono- or divalent ions (Na+, Mg2+, Ca2+). Finally, l- or d-HPA3NT3-A2 peptides exhibited the strongest antimicrobial activity against antibiotic-resistant bacteria and failed to induce resistance in Staphylococcus aureus after 12 passages.

Highlights

The rate of infectious diseases caused by drug-resistant bacteria is increasing worldwide
We previously studied the HP (2–20) peptide [10], which consists of amino acid residues 2–20 of the parental HP derived from the N-terminus of Helicobacter pylori ribosomal protein L1 and its analogue peptide, HPA3NT3
All l-form lysine residues in HPA3NT3-A2 were replaced with d-enantiomer lysine residues to prevent peptide bond cleavage by proteolytic enzymes, and the resulting peptide was named as HPA3NT3-A2D

Summary

Introduction

The rate of infectious diseases caused by drug-resistant bacteria is increasing worldwide. Studies have shown that the mechanism of most antimicrobial peptides occurs via pore formation in the cell walls of Gram-negative and Gram-positive bacteria such as that shown in the “Toroidal”, “Carpet”, and “Barrel stave” models [11,12] These agents have been shown to be toxic toward mammalian cells at lower concentrations. The tryptophan residue was not substituted because it showed increased antimicrobial activity when the indole side chain interacted strongly with the phospholipid membrane of bacteria [10,13,14] The antimicrobial mechanism of the HPA3NT3-A2 peptide involves the inhibition of protein synthesis by binding to nucleic acids following penetration of the bacterial cell membrane This peptide showed lower cytotoxicity at higher concentrations because of its reduced hydrophobicity compared to the parent peptide [15]

Methods

Results

Conclusion