Serum Stable and Low Hemolytic Temporin-SHa Peptide Analogs Disrupt Cell Membrane of Methicillin-Resistant Staphylococcus aureus (MRSA)

Abstract

Anti-microbial peptides (AMPs) have attracted major attention due to their potential bio-activities against some multidrug resistant pathogens. The present study evaluated the mechanism of actions of highly potent AMP temporin-SHa analogs, i.e., [G4a]-SHa, [G7a]-SHa, and [G10a]-SHa, against methicillin-resistant Staphylococcus aureus (MRSA) NCTC (13277) with minimum inhibitory concentrations (MICs) of 14.35, 7.16, and 3.58 µM, respectively. These analogs exhibited significant anti-MRSA activity at physiological salt concentration, 30% fetal bovine serum, and 30% human serum. [G4a]-SHa and [G7a]-SHa were non-hemolytic and non-cytotoxic to normal mouse fibroblast 3T3 cell and human Caco-2 cell line. Atomic force microscopy revealed that these analogs have profound effect on the morphological changes in MRSA surface with significant leakage of cell cytoplasmic content. Propidium iodide uptake kinetic assay and (bis-(1,3-dibutylbarbituric acid) trimethine oxonol) DiBAC₄(3) membrane depolarization assay demonstrated that these analogs display a membrane disrupting property, characterized by elevation of plasma membrane permeability and rapid transmembrane potential depolarization. [G10a]-SHa showed a significant anti-biofilm activity against biofilm forming S. aureus (ATCC 6538). Acute in vivo toxicity studies revealed that [G10a]-SHa possesses some toxic effect at 100-mg/kg dose. While [G4a]-SHa at 100 mg/kg, i.p. has no toxic effect even after 48 h, [G7a]-SHa also did not show any toxic effect at the dose of 100 mg/kg, i.p. during 24-h observation of animals. In conclusion, [G4a]-SHa, [G7a]-SHa, and [G10a]-SHa show improved activity against MRSA and stability compared to SHa peptide. Although highly potent, [G10a]-SHa, due to its hemolytic activity, might be more suitable for topical application, whereas [G4a]-SHa and [G7a]-SHa have potential to be used for systemic application.

Keywords: AMPs; Bacterial cell membrane; D-alanine; MDR Staphylococcus aureus; MRSA; Temporin-SHa.