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.

Antimicrobial blue light photoinactivation of Pseudomonas aeruginosa: Quorum sensing signaling molecules, biofilm formation and pathogenicity

“…The mechanism underlying the bactericidal activity of aBL is unclear. A common hypothesis is that visible aBL excites endogenous photosensitizing compounds that occur naturally within microbial cells, inducing the production of cytotoxic ROS . Within the present work, we made an effort to detect the photosensitizing molecule within P. aeruginosa cells and indicated that PPIX or PPIX‐like derivatives are mainly responsible for the bactericidal effect of aBL.…”

Antimicrobial blue light inactivation of pathogenic microbes: State of the art

“…The hypothesis regarding the mechanism of action of aBL was further endorsed by DNA manipulation to knockout the porphyrin synthesis pathway of a microbe sensitive to aBL and then the comparison of the aBL susceptibilities between the knockout mutant and its parental strain (Galbis-Martinez et al, 2012; Grinholc et al, 2015). It was also reported that the endogenous porphyrins patterns in vitro (e.g., porphyrin quantity and porphyrin species) are highly affected by the culturing conditions (e.g., time of culturing, passaging, culture media) (Fyrestam et al, 2016).…”

“…Grinholc et al (Grinholc et al, 2015) compared the aBL efficacy towards a wild-type S. aureus reference strain, which is capable of producing endogenous porphyrins, and its isogenic knockout mutant, which lacks the ability to produce endogenous porphyrins due to the knockout of porphyrin biosynthetic pathway. Exposure of the wildtype S. aureus strain to 10 J/cm 2 aBL at 405 nm resulted in >2.5-log 10 CFU reduction.…”

Blue light treatment ofPseudomonas aeruginosa: Strong bactericidal activity, synergism with antibiotics and inactivation of virulence factors

“… 24 In our previous study examining the S. aureus wild-type reference strain NCTC 8325–4, which is capable of endogenous porphyrin production, and an isogenic knockout mutant (8325–4 Δ hemB ) in which endogenous porphyrin production was blocked, we demonstrated the requirement for endogenous porphyrins for the bactericidal activity of a 405-nm light treatment. 29 …”