Date Published: July 29, 2019
Publisher: Public Library of Science
Author(s): Muhammad Yasir, Debarun Dutta, Mark D. P. Willcox, Anirban Bhunia.
Mel4 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 lysodeikticus 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. lysodeikticus lawn than melimine treated samples. These findings suggest that pore formation is unlikely to be involved in Mel4-mediated membrane destabilization for staphylococci, 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 depolarization, pore formation, release of cellular contents leading to cell death.
S. aureus is a major cause of infections in both health care and community settings which can produce high levels of mortality and have a high economic burden on society [1, 2]. S. aureus can reside on the skin and in the nasal cavity of 20–50% humans [3, 4] and this poses a risk for subsequent infections . S. aureus is also a major cause of infections of medical devices and can cause 30–40% bacteraemia, surgical wound and implant-related infections [4, 6]. Methicillin-resistant S. aureus (MRSA) now causes over 50% of skin and soft tissue infections . The mortality rate of S. aureus bacteraemia can reach up to 40% . S. aureus associated infections are difficult to treat with currently available antibiotics  partly due to the increase in MRSA which are often very resistant to many different classes of antibiotics [6, 10]. To overcome these problems, new antimicrobials are needed which have unique modes of action and limited potential for resistance development.
The current study demonstrated that the mode of action of S. aureus killing by Mel4 and its precursor melimine were different. A previous study had shown that 30 minutes incubation with melimine can distort S. aureus cells, induce bleb formation and accummulation of cell debris , as well as being able to permeabilise the cytoplasmic membrane of S. aureus in a concentration independent manner . The previous study also showed that melimine was only able to produce <1 log10 reduction in numbers of S. aureus cells when incubated for up to five minutes. The current study demonstrated similar effects, but extended this to demonstrate that melimine could interact with LTA of S. aureus and produce beakage in the cytoplasmic membrane of S. aureus that resulted in leakage of intracellular contents (ATP and DNA/RNA) eventually leading to cell lysis and death. Melimine was also able to liberate a small amount of autolysins from the staphylococcal cells. Whilst Mel4 could also interact with LTA and depolarise the cytoplasmic membrane. It had a weaker effect on producing pores which was evidenced by negligible Sytox green intake by cells, minimum disruption to cytoplasmic membrane compared to melimine and minimum propidium iodide staining. Based on the flow cytometry data it is suggested that it can cause transient cell membrane permeability. In addition, less ATP was released from Mel4-treated cells and no DNA/RNA was released over 150 minutes. However, both the peptides eventually resulted in cell lysis and death after 24 hours exposure. Mel4-treated cells released greater amount of autolysins compared to melimine-treated ones. It is probable that the main killing mechanism of Mel4 was release of autolysins from LTA in cell walls along with membrane depolarization. Live bacteria produce a proton gradient outside the cell membrane, and the protons can accumulate in the cell walls and protonate the D-Alanine ester linkages of LTA . The protonation of D-Ala ester linkages lowers the pH of the cell wall which in turn suppress the activity of autolysins bound to LTA . Upon dissipation of the membrane protentional, deprotonation of the D-Ala ester linkages of LTA raises the pH of the cell wall resulting in activation of autolysins [55, 56]. Moreover, the membrane embedded glycolipid anchor of LTA plays an important role in its inhibitory effect on autolysins . Antimicrobial peptides cause the delocalization of the glycolipid anchor and subsequently release autolysins by disrupting the cell membrane . Therefore, the membrane depolarization caused by melimine and Mel4 may leads to LTA deprotonation and release of LTA which in turn liberates autolysins. It is likely that the amphipathic characteristics of melimine allowed disruption of the cell membranes and pore formation that resulted in ATP and DNA release and ultimately cell death. However, Mel4 showed less interaction with cell membranes and its killing of S. aureus was more likely due to activation autolysins along with minimum membrane disruption. A comprehensive timeline of the mechanism of action of melimine and Mel4 against S. aureus is summarized in Fig 11. Source: http://doi.org/10.1371/journal.pone.0215703