Photoinduced Membrane Damage of E. coli and S. aureus by the Photosensitizer-Antimicrobial Peptide Conjugate Eosin-(KLAKLAK)2

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Abstract

Background/Objectives

Upon irradiation with visible light, the photosensitizer-peptide conjugate eosin-(KLAKLAK) ₂ kills a broad spectrum of bacteria without damaging human cells. Eosin-(KLAKLAK) ₂ therefore represents an interesting lead compound for the treatment of local infection by photodynamic bacterial inactivation. The mechanisms of cellular killing by eosin-(KLAKLAK) ₂, however, remain unclear and this lack of knowledge hampers the development of optimized therapeutic agents. Herein, we investigate the localization of eosin-(KLAKLAK) ₂ in bacteria prior to light treatment and examine the molecular basis for the photodynamic activity of this conjugate.

Methodology/Principal Findings

By employing photooxidation of 3,3-diaminobenzidine (DAB), (scanning) transmission electron microscopy ((S)TEM), and energy dispersive X-ray spectroscopy (EDS) methodologies, eosin-(KLAKLAK) ₂ is visualized at the surface of E. coli and S. aureus prior to photodynamic irradiation. Subsequent irradiation leads to severe membrane damage. Consistent with these observations, eosin-(KLAKLAK) ₂ binds to liposomes of bacterial lipid composition and causes liposomal leakage upon irradiation. The eosin moiety of the conjugate mediates bacterial killing and lipid bilayer leakage by generating the reactive oxygen species singlet oxygen and superoxide. In contrast, the (KLAKLAK) ₂ moiety targets the photosensitizer to bacterial lipid bilayers. In addition, while (KLAKLAK) ₂ does not disrupt intact liposomes, the peptide accelerates the leakage of photo-oxidized liposomes.

Conclusions/Significance

Together, our results suggest that (KLAKLAK) ₂ promotes the binding of eosin Y to bacteria cell walls and lipid bilayers. Subsequent light irradiation results in membrane damage from the production of both Type I & II photodynamic products. Membrane damage by oxidation is then further aggravated by the (KLAKLAK) ₂ moiety and membrane lysis is accelerated by the peptide. These results therefore establish how photosensitizer and peptide act in synergy to achieve bacterial photo-inactivation. Learning how to exploit and optimize this synergy should lead to the development of future bacterial photoinactivation agents that are effective at low concentrations and at low light doses.

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Author and article information

Contributors

Michael Hamblin: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2014

Publication date (Electronic): 7 March 2014

Volume: 9

Issue: 3

Electronic Location Identifier: e91220

Affiliations

[1 ]Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas, United States of America

[2 ]Microscopy & Imaging Center, Texas A&M University, College Station, Texas, United States of America

MGH, MMS, United States of America

Author notes

* E-mail: pellois@ 123456tamu.edu

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: GAJ JPP. Performed the experiments: GAJ EAE HK NM TS. Analyzed the data: GAJ EAE HK JPP. Contributed reagents/materials/analysis tools: GAJ EAE HK. Wrote the paper: GAJ JPP.

Article

Publisher ID: PONE-D-13-52288

DOI: 10.1371/journal.pone.0091220

PMC ID: 3946741

PubMed ID: 24608860

SO-VID: 9cd7a757-5b6e-4060-9aad-fd635821a62f

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 11 December 2013

Date accepted : 7 February 2014

Page count

Pages: 14

Funding

This work was supported by the Robert A. Welch Foundation (Grant A-1769) ( http://www.welch1.org/) and the Norman Hackerman Advanced Research Program ( http://www.thecb.state.tx.us/index.cfm?objectid=E55F9EE7-E488-6873-7D535561D9B426B8). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.