Thymol Mediates Bactericidal Activity against Staphylococcus aureus by Targeting an Aldo–Keto Reductase and Consequent Depletion of NADPH

Staphylococcus aureus is a common pathogen that can cause life-threatening infections. Treatment of antibiotic-resistant S. aureus infection needs effective antibacterial agents. Thymol, a generally recognized safe natural compound, has potential as an alternative to treat S. aureus infections. However, the targets and mechanisms of action of thymol were not fully understood. Bioinformatics analysis showed that IolS, a predicted aldo–keto reductase (AKR) in S. aureus, could be a potential target of thymol. Isothermal titration calorimetry (ITC) analysis demonstrated that thymol directly binds IolS and amino acid residues (Y30 and L33) are essential for such binding. Deletion of IolS or mutation of Y30A and L33A reduced the bactericidal activity of thymol at the concentration of 200 μg/mL, suggesting that thymol mediates bactericidal activity via binding with IolS. Biochemical analysis showed that addition of thymol significantly increased AKR activity of IolS from 1.6 ± 0.1 to 2.4 ± 0.2 U (p < 0.05). The content of NADPH within S. aureus cells decreased significantly from 105 ± 5 to 72 ± 3 pmol/108 cells (p < 0.05) following thymol treatment at the concentration of 200 μg/mL. Importantly, addition of NADPH could alleviate the bactericidal effect of thymol on S. aureus, indicating that the depletion of NADPH is responsible for thymol-mediated bactericidal activity. Overall, these results demonstrated that thymol could directly bind IolS and increase its AKR activity, leading to the depletion of NADPH and bactericidal effect. AKR activity of IolS could be a promising target for the development of new antimicrobials.