title: Protein profiles of Escherichia coli and Staphylococcus warneri are altered by photosensitization with cationic porphyrins.
abstract: Oxidative stress induced by photodynamic treatment of microbial cells causes irreversible damages to vital cellular components such as proteins. Photodynamic inactivation (PDI) of bacteria, a promising therapeutic approach for the treatment of superficial and localized skin and oral infections, can be achieved by exciting a photosensitizing agent with visible light in an oxygenated environment. Although some studies have addressed the oxidative alterations of PDI in bacterial proteins, the present study is the first to compare the electrophoretic profiles of proteins of Gram-positive and Gram-negative bacteria, having two structurally different porphyrins, with different kinetics of photoinactivation. The cationic porphyrins 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide (Tri-Py(+)-Me-PF) and 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py(+)-Me) were used to photosensitize Escherichia coli and Staphylococcus warneri upon white light irradiation at an irradiance of 4.0 mW cm(-2). After different photosensitization periods, proteins were extracted from bacteria and analyzed using one-dimensional SDS-PAGE. Apparent molecular weights and band intensities were determined after an irradiation period corresponding to a reduction of 4 log10 in cell viability. After photodynamic treatment, there was a general loss of bacterial proteins, assigned to large-scale protein degradation. Protein loss was more pronounced after PDI with Tri-Py(+)-Me-PF in both bacteria. There was also an increase in the concentration of some proteins as well as an increase in the molecular weight of other proteins. We show that proteins of E. coli and S. warneri are important targets of PDI. Although there is an attempt of cellular response to the PDI-induced damage by overexpression of a limited number of proteins, the damage is lethal. Our results show that changes occurring in the protein pattern during photodynamic treatment are different with the two photosensitizers, which helps to explain the different inactivation kinetics of the two bacteria. SDS-PAGE is a rational approach to assign the type of cellular response to stress that is being induced in the cells.
authors.affiliation: Department of Biology & Centre for Environmental and Marine Studies (CESAM), University of Aveiro, 3810-193 Aveiro, Portugal.
authors.forename: Eliana
authors.initials: E
authors.lastname: Alves
authors.forename: Ana Cristina
authors.initials: AC
authors.lastname: Esteves
authors.forename: António
authors.initials: A
authors.lastname: Correia
authors.forename: Ângela
authors.initials: Â
authors.lastname: Cunha
authors.forename: Maria A F
authors.initials: MA
authors.lastname: Faustino
authors.forename: Maria G P M S
authors.initials: MG
authors.lastname: Neves
authors.forename: Adelaide
authors.initials: A
authors.lastname: Almeida
mesh_terms: D002470:Cell Survival / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D008954:Models, Biological; D015394:Molecular Structure; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology; D011166:Porphyrins / Q000494:pharmacology*; D013210:Staphylococcus / Q000187:drug effects*
chemical_list: D017319:Photosensitizing Agents; D011166:Porphyrins