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

金黄色葡萄球菌是一种主要的人类病原体，在感染、细胞代谢或抗生素治疗过程中，它必须应对活性氧和电泳性物种(ROS，RES)和低卤酸(Hox)。作为防御机制，金黄色葡萄球菌利用低分子的硫醇杆菌硫醇(BSH)和几种解毒和抗氧化酶，这些酶由基于硫醇的氧化还原传感器控制，通常对多种氧化还原信号做出反应。此前，我们已经探索了氧化还原敏感调节因子HypR、MhqR、QsrR和GbaA的机制和功能，它们是金黄色葡萄球菌感染后的重要防御机制。对苯二酚敏感的MhqR和QsrR阻遏物调节多种双加氧酶和苯醌/偶氮还原酶，这些酶对苯醌、抗生素和氧化剂具有耐药性。然而，这些混杂解毒酶的生理作用和底物特异性在很大程度上是未知的。在这个项目中，我们旨在阐明混杂的乙二醛/双加氧酶和苯醌/偶氮还原酶的生理功能，以及它们在金黄色葡萄球菌在氧化和电泳性胁迫下的生存、抗性和持久性中的作用。我们推测，金黄色葡萄球菌使用具有广泛特异性的混杂酶来主要防御特定的应激源，例如亲电的甲基乙二醛或苯二酚，这些应激源扰乱了硫醇-氧化还原动态平衡，导致继发性ROS的形成。因此，电泳性解毒途径配备了还原酶，如醌/偶氮还原酶，以调节对多种氧化还原信号的抗性，包括ROS、Hox和产生ROS的抗菌剂。我们进一步假设，ROS、HOX或RES导致乙醛酸酶-III HchA的硫醇氧化，HchA转化为氧化还原敏感的伴侣蛋白，以保护细胞蛋白免受氧化蛋白聚集的影响。WP(1)将阐明BSH依赖和BSH非依赖的乙醛酶途径来解毒有毒的电泳体甲基乙醛，从而导致金黄色葡萄球菌的细胞质酸化。WP(2)将研究金黄色葡萄球菌中QsrR和MhqR调节子对苯二酚和氧化剂的保护作用的时间动力学、相互作用和功能。QsrR和MhqR调节基因在耐药SCV、宿存细胞和L型细胞存活中的作用是进一步研究的问题。WP(3)将重点研究对硫醇应激反应最强的SACOL2588-2589操纵子的功能特征，该操纵子编码一个富含半胱氨酸的蛋白和一个8.8 kDa的DUF896(UPF0291)蛋白。总之，该项目将揭示新的生存机制，使其能够适应金黄色葡萄球菌在感染和抗生素治疗过程中遇到的多种氧化还原信号，为设计抗击MRSA感染的新疗法提供指导。

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.