Mechanism of Action of Surface Immobilized Antimicrobial Peptides Against Pseudomonas aeruginosa


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.

Publication DOI:
Divisions: College of Health & Life Sciences > School of Optometry > Optometry
College of Health & Life Sciences > School of Optometry > Optometry & Vision Science Research Group (OVSRG)
College of Health & Life Sciences
Funding Information: MY acknowledges the Higher Education Commission (HEC) of Pakistan and the University of New South Wales, Australia for provision of a Ph.D. scholarship. The authors are grateful to Dr. Michael J. Carnell Biomedical Imaging Facility (BMIF) at the Universit
Additional Information: © 2020 Yasir, Dutta, Hossain, Chen, Ho, Kuppusamy, Clarke, Kumar and Willcox. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Uncontrolled Keywords: antimicrobial peptides,membrane disruption,mode of action,Pseudomonas aeruginosa,surface immobilization,Microbiology,Microbiology (medical)
Last Modified: 03 Jun 2024 07:44
Date Deposited: 17 Feb 2020 10:03
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Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
https://www.fro ... 2019.03053/full (Publisher URL)
PURE Output Type: Article
Published Date: 2020-01-22
Accepted Date: 2019-12-18
Authors: Yasir, Muhammad
Dutta, Debarun (ORCID Profile 0000-0002-2204-5272)
Hossain, Khondker R.
Chen, Renxun
Ho, Kitty K.K.
Kuppusamy, Rajesh
Clarke, Ronald J.
Kumar, Naresh
Willcox, Mark D.P.



Version: Published Version

License: Creative Commons Attribution

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