Deciphering the roles of promiscuous enzymes in the defense against electrophiles and oxidative stress in Staphylococcus aureus

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Staphylococcus aureus is a major human pathogen, which has to cope with reactive oxygen and electrophile species (ROS, RES) and hypohalous acids (HOX) during infection, cellular metabolism or antibiotics treatment. As defense mechanisms, S. aureus utilizes the low molecular weight thiol bacillithiol (BSH) and several detoxification and antioxidant enzymes, which are controlled by thiol-based redox sensors and often respond to multiple redox signals. Previously, we have explored the mechanisms and functions of the redox-sensing regulators HypR, MhqR, QsrR and GbaA, which are important defense mechanisms under infections in S. aureus. The quinone-sensing MhqR and QsrR repressors regulate multiple dioxygenases and quinone/azo reductases, which confer resistance against quinones, antibiotics and oxidants. However, the physiological roles and substrate specificities of such promiscuous detoxification enzymes are largely unknown. In this project, we aim to elucidate the physiological functions of promiscuous glyoxalases/dioxygenases and quinone/azo reductases and their roles in survival, resistance and persistence under oxidative and electrophile stress in S. aureus. We hypothesize that S. aureus employs promiscuous enzymes with broad specificities, to defend mainly against the specific stressor, e.g. electrophilic methylglyoxal or quinones, which disturb the thiol-redox homeostasis, leading to secondary ROS formation. Thus, electrophile detoxification pathways are equipped with reducing enzymes, such as quinone/azo reductases to mediate resistance against multiple redox signals, including ROS, HOX and ROS-generating antimicrobials. We further hypothesize that ROS, HOX or RES lead to thiol-oxidation of the glyoxalase-III HchA, which is converted to a redox-sensitive chaperone to protect cellular proteins against oxidative protein aggregation. WP (1) will elucidate the BSH-dependent and BSH-independent glyoxalase pathways for detoxification of the toxic electrophile methyglyoxal, causing cytoplasmic acidification in S. aureus. WP (2) will investigate the temporal dynamics, cross-talks and functions of the QsrR and MhqR regulons in protection against quinones and oxidants in S. aureus. The role of the QsrR and MhqR regulons for the survival of antibiotic resistant SCVs, persister cells and L-forms are further research questions. WP (3) will focus on the functional characterization of the most strongly thiol-stress responsive SACOL2588-2589 operon, which encodes a Cys-rich protein and a small 8.8 kDa DUF896 (UPF0291) protein. Altogether, this project will uncover novel survival mechanisms enabling the adaptation towards multiple redox signals, encountered by S. aureus during infections and antibiotic treatments providing leads for the design of novel therapeutics to combat MRSA infections.