Singh Amit, Crossman David K, Mai Deborah, Guidry Loni, Voskuil Martin I, Renfrow Matthew B, Steyn Adrie J C
Department of Microbiology, Centers for Free Radical Biology, AIDS Research, and Emerging Infections and Emergency Preparedness, University of Alabama at Birmingham, Birmingham, Alabama, USA.
PLoS Pathog. 2009 Aug;5(8):e1000545. doi: 10.1371/journal.ppat.1000545. Epub 2009 Aug 14.
The metabolic events associated with maintaining redox homeostasis in Mycobacterium tuberculosis (Mtb) during infection are poorly understood. Here, we discovered a novel redox switching mechanism by which Mtb WhiB3 under defined oxidizing and reducing conditions differentially modulates the assimilation of propionate into the complex virulence polyketides polyacyltrehaloses (PAT), sulfolipids (SL-1), phthiocerol dimycocerosates (PDIM), and the storage lipid triacylglycerol (TAG) that is under control of the DosR/S/T dormancy system. We developed an in vivo radio-labeling technique and demonstrated for the first time the lipid profile changes of Mtb residing in macrophages, and identified WhiB3 as a physiological regulator of virulence lipid anabolism. Importantly, MtbDeltawhiB3 shows enhanced growth on medium containing toxic levels of propionate, thereby implicating WhiB3 in detoxifying excess propionate. Strikingly, the accumulation of reducing equivalents in MtbDeltawhiB3 isolated from macrophages suggests that WhiB3 maintains intracellular redox homeostasis upon infection, and that intrabacterial lipid anabolism functions as a reductant sink. MtbDeltawhiB3 infected macrophages produce higher levels of pro- and anti-inflammatory cytokines, indicating that WhiB3-mediated regulation of lipids is required for controlling the innate immune response. Lastly, WhiB3 binds to pks2 and pks3 promoter DNA independent of the presence or redox state of its [4Fe-4S] cluster. Interestingly, reduction of the apo-WhiB3 Cys thiols abolished DNA binding, whereas oxidation stimulated DNA binding. These results confirmed that WhiB3 DNA binding is reversibly regulated by a thiol-disulfide redox switch. These results introduce a new paradigmatic mechanism that describes how WhiB3 facilitates metabolic switching to fatty acids by regulating Mtb lipid anabolism in response to oxido-reductive stress associated with infection, for maintaining redox balance. The link between the WhiB3 virulence pathway and DosR/S/T signaling pathway conceptually advances our understanding of the metabolic adaptation and redox-based signaling events exploited by Mtb to maintain long-term persistence.
在感染期间，与结核分枝杆菌（Mtb）维持氧化还原稳态相关的代谢事件仍知之甚少。在此，我们发现了一种新的氧化还原切换机制，通过该机制，Mtb WhiB3在特定的氧化和还原条件下，差异调节丙酸盐进入复杂毒力多酮化合物聚酰基海藻糖（PAT）、硫脂（SL-1）、分枝菌酸二霉菌酸酯（PDIM）以及受DosR/S/T休眠系统控制的储存脂质三酰甘油（TAG）的同化作用。我们开发了一种体内放射性标记技术，并首次证明了驻留在巨噬细胞中的Mtb的脂质谱变化，同时确定WhiB3是毒力脂质合成代谢的生理调节因子。重要的是，MtbDeltawhiB3在含有毒性水平丙酸盐的培养基上显示出增强的生长，从而表明WhiB3参与了对过量丙酸盐的解毒。引人注目的是，从巨噬细胞中分离出的MtbDeltawhiB3中还原当量的积累表明，WhiB3在感染时维持细胞内氧化还原稳态，并且细菌内脂质合成代谢起着还原剂汇的作用。感染MtbDeltawhiB3的巨噬细胞产生更高水平的促炎和抗炎细胞因子，这表明WhiB3介导的脂质调节对于控制先天免疫反应是必需的。最后，WhiB3独立于其[4Fe-4S]簇的存在或氧化还原状态与pks2和pks3启动子DNA结合。有趣的是，脱辅基WhiB3半胱氨酸硫醇的还原消除了DNA结合，而氧化则刺激了DNA结合。这些结果证实，WhiB3的DNA结合受硫醇-二硫键氧化还原开关的可逆调节。这些结果引入了一种新的范例机制，描述了WhiB3如何通过调节Mtb脂质合成代谢以响应与感染相关的氧化还原应激，促进向脂肪酸的代谢切换，从而维持氧化还原平衡。WhiB3毒力途径与DosR/S/T信号通路之间的联系在概念上推进了我们对Mtb利用代谢适应和基于氧化还原的信号事件来维持长期存活的理解。
