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Localization of antimicrobial peptides on polymerized liposomes leading to their enhanced efficacy against Pseudomonas aeruginosa.

ABSTRACT: Antimicrobial peptide IG-25 (a truncated version of LL-37 of the cathelicidin family) tethering an azido-capped poly(ethylene glycol) chain at the N-terminus was site-specifically attached to alkynyl-terminated polymerized liposomes using copper catalyzed "click" reaction, leading to an 18 fold enhancement in efficacy against Pseudomonas aeruginosa when compared to LL-37 without any increase in cytotoxicity to human corneal epithelial cells.

SUBMITTER: Kumar A

PROVIDER: S-EPMC3235330 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Localization of antimicrobial peptides on polymerized liposomes leading to their enhanced efficacy against Pseudomonas aeruginosa.

Kumar Amit A Kolar Satya S SS Zao Meriong M McDermott Alison M AM Cai Chengzhi C

Molecular bioSystems 20110113 3

Antimicrobial peptide IG-25 (a truncated version of LL-37 of the cathelicidin family) tethering an azido-capped poly(ethylene glycol) chain at the N-terminus was site-specifically attached to alkynyl-terminated polymerized liposomes using copper catalyzed "click" reaction, leading to an 18 fold enhancement in efficacy against Pseudomonas aeruginosa when compared to LL-37 without any increase in cytotoxicity to human corneal epithelial cells. ...[more]

PMID: 21229178

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Project description:Bacterial colonization and biofilm development on medical devices can lead to infection. Antimicrobial peptide-coated surfaces may prevent such infections. Melimine and Mel4 are chimeric cationic peptides showing broad-spectrum antimicrobial activity once attached to biomaterials and are highly biocompatible in animal models and have been tested in Phase I and II/III human clinical trials. These peptides were covalently attached to glass using an azidobenzoic acid linker. Peptide attachment was confirmed using X-ray photoelectron spectroscopy and amino acid analysis. Mel4 when bound to glass was able to adopt a more ordered structure in the presence of bacterial membrane mimetic lipids. The ability of surface bound peptides to neutralize endotoxin was measured along with their interactions with the bacterial cytoplasmic membrane which were analyzed using DiSC(3)-5 and Sytox green, Syto-9, and PI dyes with fluorescence microscopy. Leakage of ATP and nucleic acids from cells were determined by analyzing the surrounding fluid. Attachment of the peptides resulted in increases in the percentage of nitrogen by 3.0% and 2.4%, and amino acid concentrations to 0.237 nmole and 0.298 nmole per coverslip on melimine and Mel4 coated surfaces, respectively. The immobilized peptides bound lipopolysaccharide and disrupted the cytoplasmic membrane potential of Pseudomonas aeruginosa within 15 min. Membrane depolarization was associated with a reduction in bacterial viability by 82% and 63% for coatings melimine and Mel4, respectively (p < 0.001). Disruption of membrane potential was followed by leakage of ATP from melimine (1.5 ± 0.4 nM) or Mel4 (1.3 ± 0.2 nM) coated surfaces compared to uncoated glass after 2 h (p < 0.001). Sytox green influx started after 3 h incubation with either peptide. Melimine coatings yielded 59% and Mel4 gave 36% PI stained cells after 4 h. Release of the larger molecules (DNA/RNA) commenced after 4 h for melimine (1.8 ± 0.9 times more than control; p = 0.008) and after 6 h with Mel4 (2.1 ± 0.2 times more than control; p < 0.001). The mechanism of action of surface bound melimine and Mel4 was similar to that of the peptides in solution, however, their immobilization resulted in much slower (approximately 30 times) kinetics.

Project description:BackgroundPre-elafin/trappin-2 is a human innate defense molecule initially described as a potent inhibitor of neutrophil elastase. The full-length protein as well as the N-terminal "cementoin" and C-terminal "elafin" domains were also shown to possess broad antimicrobial activity, namely against the opportunistic pathogen P. aeruginosa. The mode of action of these peptides has, however, yet to be fully elucidated. Both domains of pre-elafin/trappin-2 are polycationic, but only the structure of the elafin domain is currently known. The aim of the present study was to determine the secondary structures of the cementoin domain and to characterize the antibacterial properties of these peptides against P. aeruginosa.ResultsWe show here that the cementoin domain adopts an α-helical conformation both by circular dichroism and nuclear magnetic resonance analyses in the presence of membrane mimetics, a characteristic shared with a large number of linear polycationic antimicrobial peptides. However, pre-elafin/trappin-2 and its domains display only weak lytic properties, as assessed by scanning electron micrography, outer and inner membrane depolarization studies with P. aeruginosa and leakage of liposome-entrapped calcein. Confocal microscopy of fluorescein-labeled pre-elafin/trappin-2 suggests that this protein possesses the ability to translocate across membranes. This correlates with the finding that pre-elafin/trappin-2 and elafin bind to DNA in vitro and attenuate the expression of some P. aeruginosa virulence factors, namely the biofilm formation and the secretion of pyoverdine.ConclusionsThe N-terminal cementoin domain adopts α-helical secondary structures in a membrane mimetic environment, which is common in antimicrobial peptides. However, unlike numerous linear polycationic antimicrobial peptides, membrane disruption does not appear to be the main function of either cementoin, elafin or full-length pre-elafin/trappin-2 against P. aeruginosa. Our results rather suggest that pre-elafin/trappin-2 and elafin, but not cementoin, possess the ability to modulate the expression of some P.aeruginosa virulence factors, possibly through acting on intracellular targets.

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Localization of antimicrobial peptides on polymerized liposomes leading to their enhanced efficacy against Pseudomonas aeruginosa.

Publications

Localization of antimicrobial peptides on polymerized liposomes leading to their enhanced efficacy against Pseudomonas aeruginosa.

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