title: Photoinduced membrane damage of E. coli and S. aureus by the photosensitizer-antimicrobial peptide conjugate eosin-(KLAKLAK)2.
abstract: OBJECTIVE
  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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Johnson
authors.affiliation: Microscopy & Imaging Center, Texas A&M University, College Station, Texas, United States of America.
authors.forename: E Ann
authors.initials: EA
authors.lastname: Ellis
authors.affiliation: Microscopy & Imaging Center, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Hansoo
authors.initials: H
authors.lastname: Kim
authors.affiliation: Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Nandhini
authors.initials: N
authors.lastname: Muthukrishnan
authors.affiliation: Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Thomas
authors.initials: T
authors.lastname: Snavely
authors.affiliation: Department of Biochemistry & Biophysics, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Pellois
mesh_terms: D015100:3,3'-Diaminobenzidine / Q000378:metabolism; D000595:Amino Acid Sequence; D016880:Anisotropy; D023181:Antimicrobial Cationic Peptides / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects* / Q000528:radiation effects; D004801:Eosine Yellowish-(YS) / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects* / Q000528:radiation effects / Q000648:ultrastructure; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008027:Light; D008055:Lipids / Q000032:analysis; D008081:Liposomes / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction / Q000187:drug effects / Q000528:radiation effects; D010455:Peptides / Q000494:pharmacology*; D017319:Photosensitizing Agents / Q000494:pharmacology*; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000528:radiation effects / Q000648:ultrastructure
chemical_list: D023181:Antimicrobial Cationic Peptides; D036341:Intercellular Signaling Peptides and Proteins; C535058:KLA peptide; D008055:Lipids; D008081:Liposomes; D010455:Peptides; D017319:Photosensitizing Agents; D015100:3,3'-Diaminobenzidine; D004801:Eosine Yellowish-(YS)