Abstract
Background/ObjectivesUpon 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 FindingsBy 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/SignificanceTogether, 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.
Highlights
The rising incidence of drug resistant pathogens emphasizes the urgent need for new approaches to antimicrobial killing [1,2,3,4]
We expected that eosin(KLAKLAK)2 could lead to DAB polymerization and that a dark contrast visualized by electron microscopy (EM) would indicate where eosin(KLAKLAK)2 localizes in bacteria
We aimed to identify how the conjugate eosin-(KLAKLAK)2, a compound containing both an ROSgenerator and an antimicrobial peptide, kills bacteria
Summary
The rising incidence of drug resistant pathogens emphasizes the urgent need for new approaches to antimicrobial killing [1,2,3,4]. One alternative to traditional antibiotics for topical microbial killing is photodynamic inactivation (PDI), a therapeutic strategy that combines photosensitizers (PS) and light. In this approach, PS are compounds that produce reactive oxygen species (ROS) upon irradiation [5]. PDI is not adequate for the treatment of systemic infections. PDI represents a possible long-term approach for the treatment of local infections. Applications of PDI to infections of the skin/soft tissues and surgical sites may prove to be valuable when considering that these infections account for ,7–10% of hospitalized patient infections [23] and 20–31% [24,25] of healthcare-associated infections, respectively. PDI could play an important role in these contexts to prevent, or reduce the likelihood of, subsequent systemic infections after passage of organisms from the initial infection sites into the bloodstream [26]
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