Potent Activity of Hybrid Arthropod Antimicrobial Peptides Linked by Glycine Spacers.

Miray Tonk,Alejandro Cabezas-Cruz,Andreas Vilcinskas,James J Valdés

doi:10.3390/ijms22168919

Abstract

Arthropod antimicrobial peptides (AMPs) offer a promising source of new leads to address the declining number of novel antibiotics and the increasing prevalence of multidrug-resistant bacterial pathogens. AMPs with potent activity against Gram-negative bacteria and distinct modes of action have been identified in insects and scorpions, allowing the discovery of AMP combinations with additive and/or synergistic effects. Here, we tested the synergistic activity of two AMPs, from the dung beetle Copris tripartitus (CopA3) and the scorpion Heterometrus petersii (Hp1090), against two strains of Escherichia coli. We also tested the antibacterial activity of two hybrid peptides generated by joining CopA3 and Hp1090 with linkers comprising two (InSco2) or six (InSco6) glycine residues. We found that CopA3 and Hp1090 acted synergistically against both bacterial strains, and the hybrid peptide InSco2 showed more potent bactericidal activity than the parental AMPs or InSco6. Molecular dynamics simulations revealed that the short linker stabilizes an N-terminal 310-helix in the hybrid peptide InSco2. This secondary structure forms from a coil region that interacts with phosphatidylethanolamine in the membrane bilayer model. The highest concentration of the hybrid peptides used in this study was associated with stronger hemolytic activity than equivalent concentrations of the parental AMPs. As observed for CopA3, the increasing concentration of InSco2 was also cytotoxic to BHK-21 cells. We conclude that AMP hybrids linked by glycine spacers display potent antibacterial activity and that the cytotoxic activity can be modulated by adjusting the nature of the linker peptide, thus offering a strategy to produce hybrid peptides as safe replacements or adjuncts for conventional antibiotic therapy.

Highlights

The increasing prevalence of multidrug-resistant (MDR) bacteria and the lack of novel antibiotics in the development pipeline threaten healthcare systems worldwide and have prompted the search for antimicrobial candidates, those with new mechanisms of action against Gram-negative bacteria [1,2]
We investigated the role of glycine spacers on the antibacterial activity of the hybrid peptide by constructing variants separated by two (InSco2) or six (InSco6) glycine residues
We found that the greater hydrophobicity of the hybrid peptides, especially InSco2, increased their hemolytic and cytotoxic activity compared to the parental Antimicrobial peptides (AMPs), equivalent to a loss of selectivity

Summary

Introduction

The increasing prevalence of multidrug-resistant (MDR) bacteria and the lack of novel antibiotics in the development pipeline threaten healthcare systems worldwide and have prompted the search for antimicrobial candidates, those with new mechanisms of action against Gram-negative bacteria [1,2]. Antimicrobial peptides (AMPs) are especially promising due to their potent antimicrobial activity and their ability to neutralize toxins [3]. These membrane-active peptides, 10–50 amino acids in length, display activity against bacteria, fungi, viruses and parasites, and are key components of the vertebrate and invertebrate innate immune system [4]. The non-ribosomal antibiotic peptides produced by bacteria and fungi differ from the ribosomal peptides found in higher eukaryotes, and the term AMP usually refers to these latter molecules. AMPs can be assigned to four categories according to their structure and function [5]: linear α-helical peptides, β-sheet peptides, peptides that contain unusually high numbers of specific amino acid residues such as proline or glycine, and mixed α-helix/β-sheet peptides [5]. Where secondary structures and disulfide bridges are present, these are often necessary for AMP activity [6,7]

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Discussion

Conclusion