Nucleic acid changes during photodynamic inactivation of bacteria by cationic porphyrins Eliana Alves a , Maria A. F. Faustino b , João P. C. Tomé b , Maria G. P. M. S. Neves b,⇑ , Augusto C. Tomé b , José A. S. Cavaleiro b , Ângela Cunha a , Newton C. M. Gomes a , Adelaide Almeida a,⇑ a Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal b Department of Chemistry & QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal article info Article history: Received 27 February 2013 Revised 22 April 2013 Accepted 24 April 2013 Available online 11 May 2013 Keywords: Escherichia coli Staphylococcus warneri Cationic porphyrins Photodynamic inactivation Reactive oxygen species Genomic DNA RNA abstract Light activation of photosensitizing dyes in presence of molecular oxygen generates highly cytotoxic reactive oxygen species leading to cell inactivation. Nucleic acids are molecular targets of this photody- namic action but not considered the main cause of cell death. The in vivo effect of the photodynamic pro- cess on the intracellular nucleic acid content of Escherichia coli and Staphylococcus warneri was evaluated herein. Two cationic porphyrins (Tetra-Py + -Me and Tri-Py + -Me-PF) were used to photoinactivate E. coli (5.0 lM; 10 8 cells mL 1 ) and S. warneri (0.5 lM; 10 8 cells mL 1 ) upon white light irradiation at 4.0 mW cm 2 for 270 min and 40 min, respectively. Total nucleic acids were extracted from photosensi- tized bacteria after different times of irradiation and analyzed by agarose gel electrophoresis. The double- stranded DNA was quantified by fluorimetry and the porphyrin binding to bacteria was determined by spectrofluorimetry. E. coli was completely photoinactivated with both porphyrins (5.0 lM), whereas S. warneri was only completely inactivated by Tri-Py + -Me-PF (0.5 lM). The hierarchy of nucleic acid changes in E. coli was in the order: 23S rRNA > 16S rRNA > genomic DNA. The nucleic acids of S. warneri were extensively reduced after 5 min with Tri-Py + -Me-PF but almost unchanged with Tetra-Py + -Me after 40 min of irradi- ation. The amount of Tri-Py + -Me-PF bound to E. coli after washing the cells is higher than Tetra-Py + -Me and the opposite was observed for S. warneri. The binding capacity of the photosensitizers is not directly related to the PDI efficiency or nucleic acid reduction and this reduction occurs in parallel with the decrease of surviving cells. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The photodynamic inactivation (PDI) of bacteria refers to the destruction of these microorganisms by the concerted action of light, molecular oxygen and a photosensitizer (PS). The interaction among these agents can occur via two competitive mechanisms: interaction between the light-excited PS with a substrate (type I mechanism) or with molecular oxygen (type II mechanism). Type I mechanism produces hydrogen peroxide and free radicals and type II mechanism originates singlet oxygen. 1–3 The reactive oxy- gen species originated (free radicals and singlet oxygen) are highly cytotoxic and cause structural and molecular damage to cells. 4 A direct and efficient inactivation of Gram-negative bacteria is accomplished with the use of cationic PSs. 5–9 These can interact electrostatically with the negatively charged constituents of the outer wall (lipoproteins, lipopolysaccharides) which facilitates their binding and entry. 10 On the other side, inactivation of Gram-positive bacteria is achieved with neutral, anionic and cationic PSs which readily cross the relatively porous layers of peptidoglycan that constitute these cells. 10 However, the cascade of damages involved in the bacterial PDI is not yet fully elucidated. It is generally accepted that damages occur mainly at the level of cytoplasmic membrane and cell wall constituents (multi-tar- geted photodamage) and that the nucleic acids are not the main or determining target of PDI 11–16 because DNA photocleavage is observed when cells are already largely photoinactivated or no longer viable. 14,17 Such statement is based on studies with easily photoinactivated bacterial strains that are defective for DNA repair mechanisms 16 or have very efficient DNA repair systems. 13,15,18 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.04.065 Abbreviations: PDI, photodynamic inactivation; PS, photosensitizer. ⇑ Corresponding authors. Tel.: +351 234 370 710 (M.G.P.M.S.N.), tel.: +351 234 370 784 (A.A.). E-mail addresses: gneves@ua.pt (M.G.P.M.S. Neves), aalmeida@ua.pt (A. Almeida). Bioorganic & Medicinal Chemistry 21 (2013) 4311–4318 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc