Staphylococcus aureus, a major human pathogen causes a wide range of disease syndromes, including life-threatening endocarditis, meningitidis and pneumonia. According to the Centers for Disease Control and Prevention this bacterium has been reported to be the most significant cause of serious infections in the United States . S. aureus is able to cause and develop an infection very efficiently due to its ability to produce a few dozen of virulence factors, on one hand, and an ease of antibiotic resistance development, on the other. The most dangerous are methicillin-resistant S. aureus (MRSA) strains, constituting 50% of hospital-aquired isolates as well as emerging vancomycin-resistant variants, isolated from some hospital settings .
Among several virulence factors, S. aureus produces enzymes responsible for resistance against oxidative stress, like catalase and superoxide dismutase (Sod). Sod converts superoxide anion (O2·-) into hydrogen peroxide (H2O2), a less potent biological oxidant, which is further decomposed by catalase to water and ground state oxygen. Sod enzyme is produced in response to the presence of reactive oxygen species (ROS) generated endogenously as an effect of oxygen metabolism or, exogenously produced by neutrophils and macrophages. Superoxide anion, which is the product of oxygen reduction, reacts with hydrogen peroxide within the bacterial cell and produces free hydroxyl radical (.OH), the most dangerous oxygen species able to interact with virtually any organic substance in the cell. Superoxide anion can reduce hypochlorus acid (HOCl) arose as a result of H2O2 interaction with phagocyte-derived peroxidases, and further form .OH .
The classification of Sod enzymes is based on the type of transition metal present in their active center, including manganese (Mn), iron (Fe), copper (Cu) and a few years ago a nickel (Ni)-containing Sod was described, originally isolated from the cytoplasm of Streptomyces seoulensis [4, 5]. In the Escherichia coli bacterium model, the presence of three Sods were described: Fe- and Mn- Sods localized in the cytoplasm, whereas in the periplasm copper-zinc (Cu-Zn) SOD was detected . S. aureus produces three Sod enzymes, encoded by two genes, sodA and sodM [7, 8]. The particular subunits form two kinds of Sod homodimers, i.e. SodA-SodA and SodM-SodM as well as SodA-SodM heterodimers, easily distinguishable on native gels stained for Sod activity . Both, SodA and SodM subunits are believed to possess Mn ions as a cofactor in the active site. Manganese is now believed to play a crucial role in a variety of cellular processes including stress responses . In a range of bacterial pathogens, Mn is recognized as having a major effect on virulence [10, 11]. Apart from participating in several enzyme functions, Mn complexes with phosphate and lactate were demonstrated to scavenge ROS .
The role of Sod in the pathogenesis of many bacteria was proved. In S. aureus however, the results are not unambiguous. The very first analyses of antioxidant enzymes and staphylococcal virulence showed no correlation . Similarly, in a mouse abscess model resulting from S. aureus infection, inactivation of sodA gene, recognized as the main Sod activity in S. aureus, had no impact on staphylococcal virulence . Moreover, mouse kidney infection was not attenuated after sodM gene inactivation . On the other hand, examination of a range of virulent versus non-virulent S. aureus clinical isolates, showed statistically significant higher Sod activity in the first group studied . Karavolos et al. tested the role of Sod in a mouse subcutaneous model of infection and claimed that mutants deprived of either SodA, SodM or both activities had significantly reduced virulence compared to S. aureus wild-type SH1000 strain .
As bacteria replicate very quickly, the possibility of mutant selection which effectively deals with antibiotic treatment rises. An alarming increase in antibiotic resistance spreading among pathogenic bacteria inclines to search for alternative therapeutic options, for which resistance cannot be developed easily. One such option is photodynamic inactivation of bacteria (PDI). This method involves the use of non toxic dyes, so called photosensitizers (PS), which become excited upon visible light of an appropriate wavelength and eventually a number of ROS are formed . As a consequence of ROS action, which are known to cause severe damage to DNA, RNA, proteins, and lipids, bacterial cells die. Two oxidative mechanisms can occur after light activation of a photosensitizer. When the photosensitizer interacts with a biomolecule, free radicals (type I mechanism), and/or singlet molecular oxygen (1O2) (type II mechanism) are produced, which are responsible for cell inactivation . In the case of porphyrin-based photosensitizers, 1O2 seems to be the main ROS generated upon photoexcitation, although O2
-, .OH are also implicated . In a very elegant study by Hoebeke et al., the photochemical action of bacteriochlorin a, a structural analog of protoporphyrin IX, was also demonstrated to be based on both, type I and type II mechanism of action in a 1:1 proportion . Several lines of evidence indicate the effectiveness of PDI in vitro against both Gram-positive and -negative species [21, 22]. It was also demonstrated that photodynamic inactivation may be applied to inactivate bacterial virulence factors, which represents an advantage over topical antibiotic treatments .
In our previous reports we observed that the S. aureus response to PDI is strain-dependent. Among clinical isolates some were killed in 99,999%, whereas others in only about 20% in protoporphyrin-based PDI . To understand if the antioxidant enzyme status may be involved in the S. aureus response to PDI, we checked the survival rate of the isogenic sod mutants of S. aureus and compared the activities of Sods in response to PDI on the protein as well as gene expression level.