Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to Isoniazid In Vivo
Stringent Response Factors PPX1 and PPK2 Play an Important Role in Mycobacterium tuberculosis Metabolism, Biofilm Formation, and Sensitivity to Isoniazid In Vivo
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DOI：
10.1128/aac.01139-16
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发表时间：
2016-11-01
影响因子：
4.9
通讯作者：
Karakousis, Petros C.
中科院分区：
文献类型：
--
作者：
Chuang, Yu-Min;Dutta, Noton K.;Karakousis, Petros C.
结核分枝杆菌仍然是全球健康威胁，主要是由于抗生素治疗持续时间过长，导致医疗不依从和耐药性的出现。这种延长的治疗可能是由于结核分枝杆菌持续存在，其表现出抗生素耐受性。无机多磷酸盐[poly(P)]是结核分枝杆菌严格反应介导抗生素耐受性的关键调节分子。多磷酸激酶 PPK1 负责结核分枝杆菌中的聚（P）合成，而外切多磷酸酶 PPX1 和 PPX2 以及 GTP 合酶 PPK2 负责聚（P）水解。在本研究中，我们通过液相色谱-串联质谱法表明，缺乏ppx1或ppk2的聚（P）积累的结核分枝杆菌突变株的细胞内甘油-3-磷酸（G3P）和1-脱氧-木酮糖-5-磷酸水平显着降低。实时 PCR 显示每个突变体中 G3P 合成途径中的基因表达降低。 ppx1 缺陷突变体还表现出三羧酸循环中代谢物的显着积累，以及精氨酸和 NADH 代谢的改变。每个聚(P)积累菌株均显示出生物膜形成缺陷，而ppk2的缺乏与白花丹素和美罗培南的敏感性增加有关，ppx1的缺乏导致对氯法齐明的敏感性增强。表达ppx1和ppk2的DNA疫苗，以及结核分枝杆菌严格反应的另外两个成员，结核分枝杆菌rel和sigE，没有表现出针对结核分枝杆菌气溶胶攻击的保护活性，但疫苗诱导的免疫增强了慢性结核病小鼠模型中异烟肼的杀灭活性。总之，结核分枝杆菌严格反应的聚(P)调节因子在结核分枝杆菌代谢、生物膜形成和体内抗生素敏感性中发挥重要作用。
Mycobacterium tuberculosis remains a global health threat largely due to the lengthy duration of curative antibiotic treatment, contributing to medical nonadherence and the emergence of drug resistance. This prolonged therapy is likely due to the presence of M. tuberculosis persisters, which exhibit antibiotic tolerance. Inorganic polyphosphate [poly(P)] is a key regulatory molecule in the M. tuberculosis stringent response mediating antibiotic tolerance. The polyphosphate kinase PPK1 is responsible for poly( P) synthesis in M. tuberculosis, while the exopolyphosphatases PPX1 and PPX2 and the GTP synthase PPK2 are responsible for poly(P) hydrolysis. In the present study, we show by liquid chromatography-tandem mass spectrometry that poly(P)-accumulating M. tuberculosis mutant strains deficient in ppx1 or ppk2 had significantly lower intracellular levels of glycerol-3-phosphate (G3P) and 1-deoxy-xylulose-5-phosphate. Real-time PCR revealed decreased expression of genes in the G3P synthesis pathway in each mutant. The ppx1-deficient mutant also showed a significant accumulation of metabolites in the tricarboxylic acid cycle, as well as altered arginine and NADH metabolism. Each poly(P)-accumulating strain showed defective biofilm formation, while deficiency of ppk2 was associated with increased sensitivity to plumbagin and meropenem and deficiency of ppx1 led to enhanced susceptibility to clofazimine. A DNA vaccine expressing ppx1 and ppk2, together with two other members of the M. tuberculosis stringent response, M. tuberculosis rel and sigE, did not show protective activity against aerosol challenge with M. tuberculosis, but vaccine-induced immunity enhanced the killing activity of isoniazid in a murine model of chronic tuberculosis. In summary, poly(P)-regulating factors of the M. tuberculosis stringent response play an important role in M. tuberculosis metabolism, biofilm formation, and antibiotic sensitivity in vivo.