sMode of action of the antimicrobial peptide Mel4 is independent of Staphylococcus aureus cell membrane permeability

Abstract

AbstractMel4 is a novel cationic peptide with potent activity against Gram-positive bacteria. The current study examined the anti-staphylococcal mechanism of action of Mel4 and its precursor peptide melimine. The interaction of peptides with lipoteichoic acid (LTA) and with the cytoplasmic membrane using DiSC(3)-5, Sytox green, Syto-9 and PI dyes were studied. Release of ATP and DNA/RNA from cells exposed to the peptides were determined. Bacteriolysis and autolysin-activated cell death were determined by measuring decreases in OD620nm and killing of Micrococcus luteus cells by cell-free media. Both peptides bound to LTA and rapidly dissipated the membrane potential (within 30 seconds) without affecting bacterial viability. Disturbance of the membrane potential was followed by the release of ATP (50% of total cellular ATP) by melimine and by Mel4 (20%) after 2 minutes exposure (p<0.001). Mel4 resulted in staphylococcal cells taking up PI with 3.9% cells predominantly stained after 150 min exposure, whereas melimine showed 34% staining. Unlike melimine, Mel4 did not release DNA/RNA. Cell-free media from Mel4 treated cells hydrolysed peptidoglycan and produced greater zones of inhibition against M. luteus lawn than melimine treated samples. These findings suggest that pore formation is unlikely to be involved in Mel4-mediated membrane destabilization for Staphylococcci, since there was no significant Mel4-induced PI staining and DNA/RNA leakage. It is likely that the S. aureus killing mechanism of Mel4 involves the release of autolysins followed by cell death. Whereas, membrane interaction is the primary bactericidal activity of melimine, which includes membrane depolarisation, pore formation, release of cellular contents leading to cell death.This work is original, has not been published and is not being considered for publication elsewhere. Part of this manuscript has been presented as a poster presentation in Gordon Research Conference Italy in 2019. There are no conflicts of interest for any of the authors that could have influenced the results of this work. Prof. Mark Willcox holds the patent the for the melimine peptide.