title: Photodynamic oxidation of Staphylococcus warneri membrane phospholipids: new insights based on lipidomics.
abstract: BACKGROUND
  The photodynamic process involves the combined use of light and a photosensitizer, which, in the presence of oxygen, originates cytotoxic species capable of oxidizing biological molecules, such as lipids. However, the effect of the photodynamic process in the bacterial phospholipid profile by a photosensitizer has never been reported. A lipidomic approach was used to study the photodynamic oxidation of membrane phospholipids of Staphylococcus warneri by a tricationic porphyrin [5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin triiodide, Tri-Py(+)-Me-PF].
  
  
  METHODS
  S. warneri (10(8) colony forming units mL(-1)) was irradiated with white light (4 mW cm(-2), 21.6 J cm(-2)) in the presence of Tri-Py(+)-Me-PF (5.0 μM). Non-photosensitized bacteria were used as control (irradiated without porphyrin). After irradiation, total lipids were extracted and separated by thin-layer chromatography (TLC). Isolated fractions of lipid classes were quantified by phosphorus assay and analyzed by mass spectrometry (MS): off-line TLC/ESI-MS, hydrophilic interaction (HILIC)-LC/MS and MS/MS.
  
  
  RESULTS
  The most representative classes of S. warneri phospholipids were identified as phosphatidylglycerols (PGs) and cardiolipins (CLs). Lysyl-phosphatidylglycerols (LPGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs) and phosphatidic acids (PAs) were also identified. After photodynamic treatment, an overall increase in the relative abundance of PGs was observed as well as the appearance of new oxidized species from CLs, including hydroxy and hydroperoxy derivatives. Formation of high amounts of lipid hydroperoxides was confirmed by FOX2 assay. Photodynamic oxidation of phospholipid standards revealed the formation of hydroperoxy and dihydroperoxy derivatives, confirming the observed CL oxidized species in S. warneri.
  
  
  CONCLUSIONS
  Membrane phospholipids of S. warneri are molecular targets of the photoinactivation process induced by Tri-Py(+) -Me-PF. The overall modification in the relative amount of phospholipids and the formation of lipid hydroxides and hydroperoxides indicate the lethal damage caused to photosensitized bacterial cells.
authors.affiliation: Centre for Environmental and Marine Studies, Department of Biology, University of Aveiro, Aveiro, Portugal.
authors.forename: Eliana
authors.initials: E
authors.lastname: Alves
authors.forename: Tânia
authors.initials: T
authors.lastname: Melo
authors.forename: Cláudia
authors.initials: C
authors.lastname: Simões
authors.forename: Maria A F
authors.initials: MA
authors.lastname: Faustino
authors.forename: João P C
authors.initials: JP
authors.lastname: Tomé
authors.forename: Maria G P M S
authors.initials: MG
authors.lastname: Neves
authors.forename: José A S
authors.initials: JA
authors.lastname: Cavaleiro
authors.forename: Angela
authors.initials: A
authors.lastname: Cunha
authors.forename: Newton C M
authors.initials: NC
authors.lastname: Gomes
authors.forename: Pedro
authors.initials: P
authors.lastname: Domingues
authors.forename: M Rosário M
authors.initials: MR
authors.lastname: Domingues
authors.forename: Adelaide
authors.initials: A
authors.lastname: Almeida
mesh_terms: D000704:Analysis of Variance; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000528:radiation effects; D002853:Chromatography, Liquid; D008027:Light; D008054:Lipid Peroxides / Q000032:analysis / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects / Q000528:radiation effects; D010743:Phospholipids / Q000032:analysis / Q000737:chemistry* / Q000528:radiation effects; D055668:Photochemical Processes; D017319:Photosensitizing Agents / Q000494:pharmacology; D011166:Porphyrins / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization; D013210:Staphylococcus / Q000737:chemistry* / Q000187:drug effects / Q000528:radiation effects; D005464:Fluorobenzenes; D005466:Fluorocarbons
chemical_list: D008054:Lipid Peroxides; D010743:Phospholipids; D017319:Photosensitizing Agents; D011166:Porphyrins; C549552:5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin triiodide; D005464:Fluorobenzenes; D005466:Fluorocarbons