The anaplerotic node is essential for the intracellular survival of Mycobacterium tuberculosis.
The anaplerotic node is essential for the intracellular survival of Mycobacterium tuberculosis.
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DOI：
10.1074/jbc.ra118.001839
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发表时间：
2018-04-13
期刊：
影响因子：
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通讯作者：
Beste DJV
中科院分区：
文献类型：
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作者：
Basu P;Sandhu N;Bhatt A;Singh A;Balhana R;Gobe I;Crowhurst NA;Mendum TA;Gao L;Ward JL;Beale MH;McFadden J;Beste DJV
磷酸烯醇丙酮酸 (PEP)-丙酮酸-草酰乙酸或回补 (ANA) 节点的酶控制糖酵解、糖异生和回补的代谢流。在这里，我们使用遗传、生化和 13C 同位素分析来表征 ANA 节点酶在世界上最成功的细菌病原体结核分枝杆菌 (Mtb) 细胞内存活中的作用。我们发现，丙酮酸羧化酶（PCA）、PEP 羧激酶（PCK）、苹果酸酶（MEZ）和丙酮酸磷酸二激酶（PPDK）这四种 ANA 酶中的每一种在 Mtb 的细胞内生存过程中都发挥着独特且重要的代谢功能。我们发现，除了 PCK 之外，细胞内 Mtb 还需要 PPDK 作为糖异生的替代途径。丙酸和胆固醇解毒也被确定为 PPDK 的基本功能，揭示了 ANA 节点在这些生理上重要的细胞内底物代谢中的意想不到的作用，并强调了这种酶作为结核病 (TB) 特异性药物靶点。我们表明，通过 ANA 节点回补固定 CO2 对于 Mtb 的细胞内生存至关重要，并且 Mtb 拥有能够实现此功能的三种酶（PCA、PCK 和 MEZ）。除了在回补中提供后备作用外，我们还发现 MEZ 在脂质生物合成中也发挥着作用。 MEZ 敲除菌株的细胞壁发生了改变，并且在初始进入巨噬细胞方面存在缺陷。这项工作揭示了 ANA 节点是控制 Mtb 细胞内复制的焦点，它超越了经典的糖异生作用，代表了设计新型抗结核药物的一个有希望的目标。
Enzymes at the phosphoenolpyruvate (PEP)–pyruvate–oxaloacetate or anaplerotic (ANA) node control the metabolic flux to glycolysis, gluconeogenesis, and anaplerosis. Here we used genetic, biochemical, and 13C isotopomer analysis to characterize the role of the enzymes at the ANA node in intracellular survival of the world's most successful bacterial pathogen, Mycobacterium tuberculosis (Mtb). We show that each of the four ANA enzymes, pyruvate carboxylase (PCA), PEP carboxykinase (PCK), malic enzyme (MEZ), and pyruvate phosphate dikinase (PPDK), performs a unique and essential metabolic function during the intracellular survival of Mtb. We show that in addition to PCK, intracellular Mtb requires PPDK as an alternative gateway into gluconeogenesis. Propionate and cholesterol detoxification was also identified as an essential function of PPDK revealing an unexpected role for the ANA node in the metabolism of these physiologically important intracellular substrates and highlighting this enzyme as a tuberculosis (TB)-specific drug target. We show that anaplerotic fixation of CO2 through the ANA node is essential for intracellular survival of Mtb and that Mtb possesses three enzymes (PCA, PCK, and MEZ) capable of fulfilling this function. In addition to providing a back-up role in anaplerosis we show that MEZ also has a role in lipid biosynthesis. MEZ knockout strains have an altered cell wall and were deficient in the initial entry into macrophages. This work reveals that the ANA node is a focal point for controlling the intracellular replication of Mtb, which goes beyond canonical gluconeogenesis and represents a promising target for designing novel anti-TB drugs.