Comparative mode of action of antimicrobial peptide melimine and its derivative Mel4 against Pseudomonas aeruginosa

Abstract

Society for Microbiology annual meetings in 2017. There are no conflicts of interest for any 31 of the authors that could have influenced the results of this work. Prof. Mark Willcox holds 32 the patent for the melimine peptide. 33 2 ABSTRACT 34Melimine and Mel4 are chimeric cationic peptides with broad spectrum antimicrobial 35 activity, and recent investigations have shown that they are highly biocompatible with animal 36 model and human clinical trials. The current study examined the mechanism of action of 37 these two antimicrobial peptides against P. aeruginosa with a series of investigations. 38 Antimicrobial activities were determined by MIC and MBC. Endotoxin neutralization was 39 determined using the LAL assay, effect on the cytoplasmic membrane was evaluated using 40 DiSC(3)-5 and Sytox green stains, and Syto-9 and PI dyes using flow cytometry. Release of 41 cytoplasmic materials (ATP and DNA/RNA) were determined using ATP luminescence and 42 increase in OD 260nm . The ability to lyse bacteria was studied by measuring a decrease in 43 OD 620nm . The MIC of the peptides remained low against P. aeruginosa strains, which showed 44 efficient neutralization of LPS, indicating their role in the anti-pseudomonas and LPS binding 45 activities. Both AMPs rapidly (starting at 30 seconds) depolarized P. aeruginosa cytoplasmic 46 membrane leading to reduction in viability. Melimine was responsible for more ATP release 47 (75%) compared to Mel4 (36%) (P<0.001) following two minutes exposure. For both 48 peptides, Sytox green entered cells after five minutes of incubation. Flow cytometry 49 demonstrated that both the AMPs permeabilized the cell membrane at 30 minutes and 50 followed by increasing permeability. Similar results were found with DNA/RNA release 51 experiments. Overall, melimine showed higher ability of membrane disruption, cell lysis 52 compared to Mel4 (P<0.001). Knowledge regarding mechanism of action of these two AMPs 53 would be helpful in making them as anti-pseudomonas drug. 54KEYWORDS P. aeruginosa, Antimicrobial peptides, mechanism of action, membrane 55 disruption 56 57 3 58 59 Pseudomonas aeruginosa is a metabolically versatile ubiquitous Gram-negative opportunistic 60 pathogen that can cause infections in animals and plants (1). P. aeruginosa is responsible for 61 localized to systemic infections in humans, which can be life threatening. Over the years, P. 62 aeruginosa has become one of the most frequent causative agents of nosocomial infections, 63 associated with substantial morbidity and mortality (2). The current standards of care to treat 64 P. aeruginosa infections are not effective (3) as its outer membrane offers low permeability, 65 which limits the penetration of antibiotics into the bacterial cells thereby increase antibiotic 66 resistance (4, 5). Given the severity of P. aeruginosa infections and the limited antimicrobial 67 arsenal with which to treat them, finding new alternative antimicrobials with unique 68 mechanisms of action is urgently required (6). 69 Antimicr...

Mechanism of Action of Surface Immobilized Antimicrobial Peptides Against Pseudomonas aeruginosa

“…The results of the interaction of surface-bound melimine and Mel4 with P. aeruginosa was similar to that found with these peptides in solution (Yasir et al, 2019). However, the time course of interaction and disruption of cell membranes by FIGURE 4 | Inner membrane permeabilization.…”

“…If 11% of the melimine was released into the 200 µl of bacterial suspension, this would be equivalent of approximately 0.17 nmole/ml, and similarly 14% of Mel4 in the bacterial suspension is equivalent to appriximately 0.26 nmole/ml. These concentrations of potentially free peptide are much lower than the minimum inhibitory concentration (MIC) for melimine (66-132 nmole/ml) or Mel4 (26.6-106.5 nmole/ml) (Yasir et al, 2019) and so very unlikely to have had substantial effects on the results, indicating that the results are very likely to be due covalently attached peptide only. Previous results had confirmed that free melimine adopts a more ordered structure in a membrane mimetic environment (Rasul et al, 2010), but the confirmation of Mel4 in membrane mimetic environments had not been previously investigated.…”

“…Cytoplasmic membrane depolarization caused by surface bound AMPs was determined (Hilpert et al, 2009;Yasir et al, 2019). Prior to exposure to surface bound AMPs, bacteria were labeled with 4 µM membrane potential sensitive dye DiSC3-5 (Sigma Aldrich) in the presence of 0.5 mM EDTA.…”

“…The effect of surface bound peptides on the release of nucleic acid was also examined (Yasir et al, 2019). Bacteria suspended in HEPES were incubated with peptide-coated and uncoated surfaces at 37 • C for 10 h. Aliquots (200 µl) were drawn at 2 h intervals, and filtered through 0.22 µm membranes (Merck, Tullagreen, Carrigtwohill, Ireland).…”

“…The mechanism of action of both AMPs in solution involves interaction with lipopolysaccharide, permeabilization of inner membrane and release of cellular contents such as ATP and nucleic acid leading to lysis of pseudomonal cells (Rasul et al, 2010;Yasir et al, 2019). How surface bound melimine and Mel4 interacts and kills bacteria and whether their antimicrobial activity after immobilization is similar to that when they are free in solution is unknown.…”