Biosynthesis and recycling of nicotinamide cofactors in Mycobacterium tuberculosis -: An essential role for NAD in nonreplicating bacilli
Biosynthesis and recycling of nicotinamide cofactors in Mycobacterium tuberculosis -: An essential role for NAD in nonreplicating bacilli
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DOI：
10.1074/jbc.m800694200
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发表时间：
2008-07-11
影响因子：
4.8
通讯作者：
Barry, Clifton E., III
中科院分区：
文献类型：
--
作者：
Boshoff, Helena I. M.;Xu, Xia;Barry, Clifton E., III
尽管在其基因组中存在明显编码NAD挽救特异性酶的基因，但以前一直认为结核分枝杆菌只能从头合成NAD。对新生合成和可能的挽救途径基因的转录分析表明，在体内和体外低氧条件下，挽救途径基因表达上调。[C-14]直接从受感染小鼠的肺部或受感染的巨噬细胞分离的结核分枝杆菌的烟酰胺掺入试验表明，与体外需氧培养的细胞相比，在体内适应的细胞中掺入外源烟酰胺是非常有效的。采用体外酶活性测定和等位基因交换研究相结合的方法，对两种可能的烟酸磷酸核糖转移酶PncB1(Rv1330c)和PncB2(Rv0573c)进行了研究。这些研究表明，两者都在辅因子挽救中发挥作用。在体外活跃复制过程中，去掉外源烟酰胺后，新生途径中的突变体死亡。细胞死亡是由辅因子饥饿和细胞氧化还原稳态的破坏引起的，因为限制NAD会损害电子传递。NAD合成酶的抑制剂是循环和从头合成途径的共同关键酶，在活跃生长和非复制的结核分枝杆菌中，其杀菌效果与从头合成途径突变体的突然NAD饥饿相同。这些研究证明了生物体在维持NAD水平方面的可塑性，并确立了通用途径的两种酶对于活动性和潜伏性结核病都是有吸引力的化疗靶点。
Despite the presence of genes that apparently encode NAD salvage-specific enzymes in its genome, it has been previously thought that Mycobacterium tuberculosis can only synthesize NAD de novo. Transcriptional analysis of the de novo synthesis and putative salvage pathway genes revealed an up-regulation of the salvage pathway genes in vivo and in vitro under conditions of hypoxia. [C-14] Nicotinamide incorporation assays in M. tuberculosis isolated directly from the lungs of infected mice or from infected macrophages revealed that incorporation of exogenous nicotinamide was very efficient in in vivo-adapted cells, in contrast to cells grown aerobically in vitro. Two putative nicotinic acid phosphoribosyltransferases, PncB1 (Rv1330c) and PncB2 (Rv0573c), were examined by a combination of in vitro enzymatic activity assays and allelic exchange studies. These studies revealed that both play a role in cofactor salvage. Mutants in the de novo pathway died upon removal of exogenous nicotinamide during active replication in vitro. Cell death is induced by both cofactor starvation and disruption of cellular redox homeostasis as electron transport is impaired by limiting NAD. Inhibitors of NAD synthetase, an essential enzyme common to both recycling and de novo synthesis pathways, displayed the same bactericidal effect as sudden NAD starvation of the de novo pathway mutant in both actively growing and nonreplicating M. tuberculosis. These studies demonstrate the plasticity of the organism in maintaining NAD levels and establish that the two enzymes of the universal pathway are attractive chemotherapeutic targets for active as well as latent tuberculosis.