Effect from dinoponeratoxin M-PONTXDq3a arginine and lysine substituted analogues against Staphylococcus aureus strains

Abstract

The growing incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections is associated with increased mortality rates, which has generated interest in the development of antimicrobial peptides (AMP), such as those found in the giant ant Dinoponera quadríceps. In order to improve the net positive charge and the antibacterial activity of the AMP, amino acids with positive side chain single substituted analogues have been proposed, mainly arginine or lysine. The present work aims to study the antimicrobial activity of the analogues of M-PONTX-Dq3a, a 23 amino acid AMP identified in the D. quadriceps venom. M-PONTX-Dq3a[1-15], a fragment containing the 15 central amino acids, and eight derivatives of single arginine or lysine substituted analogues were proposed. The antimicrobial activity of peptides was evaluated against Staphylococcus aureus ATCC 6538 P (MSSA) and ATCC 33591 (MRSA) strains, followed by minimum inhibitory concentration (MIC), minimum lethal concentration (MLC), and minimum biofilm inhibitory concentration (MBIC) measurement. The membrane permeability was then assessed via crystal violet assay and flow cytometry analysis. The effect of exposure time on microbial viability (Time-Kill) was evaluated. Finally, ultrastructural alterations were evaluated through scanning electron microscopy (SEM). Both arginine-substituted peptides [Arg]³M-PONTX-Dq3a[1-15] and [Arg]⁴M-PONTX-Dq3a[1-15], showed lowest MIC and MLC values (each 0.78 μM). In the biofilm formation assays, the peptide [Arg]³M-PONTX-Dq3a [1-15] showed MBIC of 3.12 μM against the two tested strains. Both peptides were able to alter the membrane permeability approximately by 80%. The treatment with MIC was able to eliminate bacteria after 2 h of contact on the other hand, treatment with half of the MIC, the population of both bacterial strains remained constant for up to 12 h, indicating a possible bacteriostatic effect. The SEM results showed that the treatment with the lowest concentration (0.78 μM) of both peptides caused disruption of the cell membrane, destabilization of the intercellular interaction and the CLM of [Arg]⁴M-PONTX-Dq3a [1-15] resulted in the complete eradication of the bacteria. Thus, this study describes two AMPs active against MSSA and MRSA and also inhibits the biofilm formation of these stains. This study finds [Arg]³M-PONTX-Dq3a[1-15] and [Arg]⁴M-PONTX-Dq3a[1-15] as alternative substances to treat resistant and/or biofilm-forming strains.

Keywords: Antimicrobial peptides; Biofilm; Dinoponera quadriceps; Methicillin-resistant.