Objective

This study investigated the effects of allylpyrocatechol (APC), the major component in ethanolic extract of Piper betle, on key oxidative stress resistance enzymes important for the survival of Staphylococcus aureus, a major pathogen in the human host.

Methods

Effects of APC on expressions of genes encoding catalase (katA), superoxide dismutases (SODs), including sodA and sodM, and alkyl hydroperoxide reductase (ahpC) in S. aureus were quantitated by RT-qPCR in reference to gyrA and 16S rRNA. Corresponding activities of the enzymes were also investigated. The Livak analysis was performed for verification of gene-fold expression data. Effects of APC on intracellular and extracellular reactive oxygen species (ROS) levels were determined using the nitroblue tetrazolium (NBT) reduction assay.

Results

APC-treated S. aureus cells had higher sodA and sodM transcripts at 1.5-fold and 0.7-fold expressions respectively with corresponding increase in total SOD activity of 12.24 U/mL compared to untreated cells, 10.85 U/mL (P<0.05). Expression of ahpC was highest in APC-treated cells with 5.5-fold increased expression compared to untreated cells (P<0.05). Correspondingly, ahpC activity was higher in APC-treated cells at 0.672 (A_310nm) compared to untreated cells which was 0.394 (A_310nm). In contrast, katA expression was 1.48-fold and 0.33-fold lower respectively relative to gyrA and 16S rRNA. Further, APC-treated cells showed decreased catalase activity of 1.8 × 10^-4 (U/L or μmol/(min·L)) compared to untreated cells, which was 4.8 × 10^-4 U/L (P<0.05). Absorbance readings (A_575nm) for the NBT reduction assay were 0.709 and 0.695 respectively for untreated and treated cells, which indicated the presence of ROS. APC-treated S. aureus cells had lower ROS levels both extracellularly and intracellularly, but larger amounts remained intracellularly compared to extracellular levels with absorbances of 0.457 and 0.137 respectively (P<0.05).

Conclusion

APC induced expressions of both sodA and sodM, resulting in increased total SOD activity in S. aureus. Higher sodA expression indicated stress induced intracellularly involving O₂^-, presumably leading to higher intracellular pools of H₂O₂. A concommittant decrease in katA expression and catalase activity possibly induced ahpC expression, which was increased the highest in APC-treated cells. Our findings suggest that in the absence of catalase, cells are propelled to seek an alternate pathway involving ahpC to reduce stress invoked by O₂^- and H₂O₂. Although APC reduced levels of ROS, significant amounts eluded its antioxidative action and remained intracellularly, which adds to oxidative stress in treated cells.

Key words: Allylpyrocatechol, Catalase, Superoxide dismutase, Alkyl hydroperoxide reductase, Reactive oxygen species, Staphylococcus aureus, Piper betle