title: Fine-tuning recA expression in Staphylococcus aureus for antimicrobial photoinactivation: importance of photo-induced DNA damage in the photoinactivation mechanism.
abstract: Bacterial cell envelope is generally accepted as the primary target for a photo-induced oxidative stress. It is plausible that DNA damage occurs during the antimicrobial photoinactivation. Here we investigate the correlation between DNA damage and photoinactivation by evaluating the level of RecA-based DNA repair system in Staphylococcus aureus. By using exogenous photosensitizers (new methylene blue (NMB), toluidine blue O (TBO), 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), zinc phthalocyanine (ZnPc), Rose Bengal (RB)) and ALA-induced endogenous porphyrin-dependent blue light (405 nm), several outcomes were observed: (i) an increase of DNA damage (from gel electrophoresis in DNA damage assay), (ii) an increase of recA expression (luminescence assay in recA-lux strain), and (iii) an increase of RecA protein level (Western blotting). When recA expression was repressed by novobiocin, or abolished by deleting the gene, S. aureus susceptibility towards photoinactivation was increased at approximately a hundred-fold. The absence of RecA increases DNA damage to yield bactericidal effect. In novobiocin-resistant mutant (gyrB), as opposed to wild type, neither RecA protein level nor cell's susceptibility was affected by photoinactivation (when novobiocin is present). This is to suggest that GyrB-dependent inhibition mediated recA repression. Therefore, we have established the role of RecA in DNA damage during photoinactivation. With the use of rifampicin mutation frequency and Ames tests, we demonstrated that photoinactivation did not increase S. aureus mutagenesis and potentially is not mutagenic toward eukaryotic cells. The results suggest that the treatment is considered safe. In conclusion, we provide an evidence that recA inhibitor may serve as therapeutic adjuvant for antimicrobial photoinactivation. Clinical relevance of our findings warrants further investigations.
authors.affiliation: Laboratory of Molecular Diagnostics, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland. mariusz.grinholc@biotech.ug.edu.pl.
authors.forename: Mariusz
authors.initials: M
authors.lastname: Grinholc
authors.affiliation: Laboratory of Molecular Diagnostics, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Rodziewicz
authors.affiliation: Laboratory of Molecular Diagnostics, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Forys
authors.affiliation: Laboratory of Molecular Diagnostics, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Rapacka-Zdonczyk
authors.affiliation: Department of Biotechnology, Division of Plant Protection and Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland.|Laboratory of Human Physiology, Medical University of Gdansk, Tuwima 15, 80-210, Gdansk, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Kawiak
authors.affiliation: Department of Biotechnology, Division of Plant Protection and Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Domachowska
authors.affiliation: Laboratory of Biophysics, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Golunski
authors.affiliation: Interfaculty Institute of Microbiology and Infection Medicine, University of Tubingen, Wlfriede-Aulhorn-Strasse 6, 72076, Tubingen, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Wolz
authors.affiliation: Outreach, Research Training and Minority Science Program, Ayala School of Biological Sciences, University of California, 333 Steinhaus Hall, Irvine, CA, 92697-2525, USA.
authors.forename: Lili
authors.initials: L
authors.lastname: Mesak
authors.affiliation: Institute of Medical Microbiology, University Hospital Münster, Domagkstr. 10, 48149, Münster, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Becker
authors.affiliation: Laboratory of Molecular Diagnostics, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland.
authors.forename: Krzysztof P
authors.initials: KP
authors.lastname: Bielawski
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism*; D004249:DNA Damage / Q000528:radiation effects*; D027081:DNA Gyrase / Q000235:genetics; D017353:Gene Deletion; D009675:Novobiocin / Q000378:metabolism; D017319:Photosensitizing Agents / Q000378:metabolism*; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000528:radiation effects*
chemical_list: D000900:Anti-Bacterial Agents; D017319:Photosensitizing Agents; D009675:Novobiocin; D011938:Rec A Recombinases; D027081:DNA Gyrase
keywords: DNA damage; Endogenous porphyrins; Mutagenesis; Photoinactivation; RecA; Staphylococcus aureus