Dataset Information

Photoinduced membrane damage of E. coli and S. aureus by the photosensitizer-antimicrobial peptide conjugate eosin-(KLAKLAK)2.

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.

SUBMITTER: Johnson GA

PROVIDER: S-EPMC3946741 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Publications

Photoinduced membrane damage of E. coli and S. aureus by the photosensitizer-antimicrobial peptide conjugate eosin-(KLAKLAK)2.

Johnson Gregory A GA Ellis E Ann EA Kim Hansoo H Muthukrishnan Nandhini N Snavely Thomas T Pellois Jean-Philippe JP

PloS one 20140307 3

<h4>Background/objectives</h4>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. Herei ...[more]

PMID: 24608860

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Project description:Parasitic diseases are a neglected and serious problem, especially in underdeveloped countries. Among the major parasitic diseases, Leishmaniasis figures as an urgent challenge due to its high incidence and severity. At the same time, the indiscriminate use of antibiotics by the population is increasing together with resistance to medicines. To address this problem, new antibiotic-like molecules that directly kill or inhibit the growth of microorganisms are necessary, where antimicrobial peptides (AMPs) can be of great help. In this work, the ferrocene molecule, one active compound with low levels of in vivo toxicity, was coupled to the N-terminus of the RP1 peptide (derived from the human chemokine CXCL4), aiming to evaluate how this change modifies the structure, biological activity, and toxicity of the peptide. The peptide and the conjugate were synthesized using the solid phase peptide synthesis (SPPS). Circular dichroism assays in PBS showed that the RP1 peptide and its conjugate had a typical spectrum for disordered structures. The Fc-RP1 presented anti-amastigote activity against Leishmania amazonensis (IC50 = 0.25 μmol L-1). In comparison with amphotericin B, a second-line drug approved for leishmaniasis treatment, (IC50 = 0.63 μmol L-1), Fc-RP1 was more active and showed a 2.5-fold higher selectivity index. The RP1 peptide presented a MIC of 4.3 μmol L-1 against S. agalactiae, whilst Fc-RP1 was four times more active (MIC = 0.96 μmol L-1), indicating that ferrocene improved the antimicrobial activity against Gram-positive bacteria. The Fc-RP1 peptide also decreased the minimum inhibitory concentration (MIC) in the assays against E. faecalis (MIC = 7.9 μmol L-1), E. coli (MIC = 3.9 μmol L-1) and S. aureus (MIC = 3.9 μmol L-1). The cytotoxicity of the compounds was tested against HaCaT cells, and no significant activity at the highest concentration tested (500 μg. mL-1) was observed, showing the high potential of this new compound as a possible new drug. The coupling of ferrocene also increased the vesicle permeabilization of the peptide, showing a direct relation between high peptide concentration and high carboxyfluorescein release, which indicates the action mechanism by pore formation on the vesicles. Several studies have shown that ferrocene destabilizes cell membranes through lipid peroxidation, leading to cell lysis. It is noteworthy that the Fc-RP1 peptide synthesized here is a prototype of a bioconjugation strategy, but it still is a compound with great biological activity against neglected and fish diseases.

Dataset Information

Photoinduced membrane damage of E. coli and S. aureus by the photosensitizer-antimicrobial peptide conjugate eosin-(KLAKLAK)2.

Background/objectives

Methodology/principal findings

Conclusions/significance

Publications

Photoinduced membrane damage of E. coli and S. aureus by the photosensitizer-antimicrobial peptide conjugate eosin-(KLAKLAK)2.

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