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

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.

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Mel4 is a novel cationic peptide with potent activity against Gram-positive bacteria. The 36 current study examined the anti-staphylococcal mechanism of action of Mel4 and its precursor 37 peptide melimine. The interaction of peptides with lipoteichoic acid (LTA) and with the 38 cytoplasmic membrane using DiSC(3)-5, Sytox green, Syto-9 and PI dyes were studied. 39 Release of ATP and DNA/RNA from cells exposed to the peptides were determined. INTRODUCTION membranes by forming pores by various mechanisms called "barrel stave" or "toroidal pore" 83 or through disintegration of lipids via the "carpet model" [21]. Cytoplasmic membrane collapse 84 can result in leakage of cellular contents such as potassium ions, ATP and DNA/RNA which 85 in turn may lead to cell death [22,23]. Some AMPs translocate across the cell membrane and 86 inhibit DNA/RNA or protein synthesis [24,25]. AMPs can also kill Gram positive bacteria by 87 activating cell wall bound autolytic enzymes known as autolysins [26]. LTAs bind to autolysins 88 in bacterial cell walls and control their activity. AMPs can disturb the regulatory function 89 ofLTA resulting in unregulated autolysin activity which then acts on the peptidoglycan chains 90 and peptide bridges of murein [27]. 91 Melimine is a cationic hybrid peptide of melittin and protamine [28]. It shows broad spectrum 92 antimicrobial activity against Gram-negative and Gram-positive bacteria (including MRSA), 93 fungi and protozoan such as Acanthamoeba without inducing resistance in bacteria [28,29] Oxoid, Basingstoke, UK) and cells were harvested following washing with phosphate buffer 122 saline (PBS, NaCl 8 g/L, KCl 0.2 g/L, Na2HPO4 1.4 g/L, KH2PO4 0.24 g/L; pH 7.4) then 123 diluted in the PBS containing 1/1000 TSB to OD600nm 0.05-0.06 (1× 10 7 colony forming units 124 (CFU)/ml confirmed upon retrospective plate counts on TS agar (Oxoid)). Cells prepared this 125 way were used in most experiments except for assessing the minimum inhibitory and 126 bactericidal concentrations, measuring the release of DiSC3-5 from cells, and the autolytic 127 activity assay.    Penetration of these two dyes through compromised membrane occurs through pores and 165 differences may indicate different pore structures/sizes created by peptides.

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Cytoplasmic membrane depolarization was performed as described by Rasul et al.,[30] with The bacteria-lytic potential of the two peptides was evaluated using two different bacterial 214 inoculums 1× 10 8 CFU/ml (OD660 0.1) and 3 × 10 10 CFU/ml. The smaller inoculum size was 215 used to see whether OD620nm was measurable at the concentration of cells used in all other 216 assays. However, no measurable optical density change at 660nm was obtained, and so a larger 217 inoculum of 3 × 10 10 CFU/ml was used. The larger inoculum was obtained by adjusting 218 OD620nm to 0.3, and bacterial numbers (CFU/ml) were confirmed upon retrospective plate count.

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Melimine and Mel4 were added at their MICs and MBCs. Bacterial cultures were immediately 220 mixed and then diluted 1:1000 in TSB. The OD620nm was measured and additional readings 221 were taken at 30, 60, 90, 120 minutes, 6.5 and 24 h after incubating at room temperature.    Triton-X 100 resulted emission of higher Sytox green fluorescence compared to either peptides 305 within 10 minutes (p<0.001).

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The membrane damaging effect of these peptides was also assessed with S. aureus 38 only by Syto-9 staining and 37.7% having high levels of both Syto-9 and PI staining. However, when 323 looking at the flow cytometry readout (Fig. 4) there did appear to be a general increase in the 324 percentage of double stained cells, but the gating did not bin cells with sufficiently fine detail.

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There was a difference in the kinetics of cell death with the positive control Triton-X 100 (1%), 326 which after 30 minutes stained 16.34% cells with high PI and increased up to 24.94 % after 327 150 min incubation. With buffer treated (negative control) cells > 86% were stained with high 328 levels of Syto-9 and low levels of PI even after 150 minutes incubation (Fig. 4).  At low bacterial concentrations (1× 10 8 CFU/ml), both the peptides showed no effect on optical 351 density. This was probably due to bacterial concentration was too low to detect any change in 352 optical density. However, when higher inoculum size of 1× 10 10 CFU/ml was used and treated 353 with various concentration of peptides, a significant reduction in optical density was observed 354 at 6.5 hours (Fig. 7). At this time point, melimine reduced optical density by 21±1% at its MIC 355 and 31±4% at its MBC compared to buffer treated control (p<0.001) (Fig. 7). Melimine lysed 356 more than 42% cells after 24 h. A similar trend was observed for Mel4 which reduced optical 357 density by 22±3 % and 25±2 % at its MICs and MBCs respectively after 6.5 h (Fig. 7). After The killing kinetics of AMPs was determined by exposing S. aureus to their MIC and MBC.

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Melimine reduced the number of cells by 1.1 log10 at its MIC and 1.2 log10 CFU/ml at its MBC 364 after 2 h of incubation (Fig. 8). At its MIC, after 2 hours melimine continued to reduce the 365 numbers of live cells, but the rate of reduction diminished after 12 hours. The initial rate of 366 reduction from 0-2 h was 6.6 CFU log10/ml/hour, from 2-12 hours was 5.9 CFU log10/ml/hour 367 and from 12-24 h was 2.9 CFU log10/ml/hour. At its MBC, the initial rate of reduction was 6.7 368 CFU log10/ml/hour, then from 2-24 h the rate remained constant at 5.7 CFU log10/ml/hour.

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Mel4 at its MIC did not significantly reduce bacterial viability within the first 2 h. The rate of 370 reduction from 2-12 h was 5.6 CFU log10/ml/hour and 3.8 CFU log10/ml/hour from 12-24 h. At 371 its MBC the rate of reduction was also negligible initially (0-2 h), however, from 2-24 h the 372 rate of reduction increased to 5.5 CFU log10/ml/hour. Killing rate of melimine from 2-24 was 373 significantly higher than Mel4 (p<0.001). The bacterial viability remained unaffected in control 374 (buffer treated) over 24 h of the experiment. It is likely that the amphipathic characteristics of melimine allowed disruption of the cell 461 membranes and pore formation that resulted in ATP and DNA release and ultimately cell death.

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However, Mel4 showed less interaction with cell membranes and its killing of S. aureus was 463 more likely due to activation autolysins along with minimum membrane disruption.