The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo
The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo
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DOI：
10.1111/j.1365-2958.2004.04085.x
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发表时间：
2004-06-01
影响因子：
3.6
通讯作者：
Av-Gay, Y
中科院分区：
文献类型：
--
作者：
Cowley, S;Ko, M;Av-Gay, Y
结核分枝杆菌真核样蛋白丝氨酸/苏氨酸激酶PknG的功能研究基因敲除和表达和生化分析。pknG基因（Rv 0410 c）在大肠杆菌中克隆和表达时，编码功能性激酶。重组蛋白的体外激酶测定表明，PknG可以自磷酸化其激酶结构域以及其30 kDa的C-末端部分，其中包含一个tetratricopeptide（TPR）结构信号基序。Western分析表明，PknG定位于胞浆以及在分枝杆菌膜。pknG基因在M.结核所得突变株导致SCID小鼠死亡延迟，并在体外和感染BALB/c小鼠后显示活力降低。在分枝杆菌生长周期的稳定期和在营养耗尽的培养基中生长时，突变体的生长减少更为明显。PknG缺陷突变体积累谷氨酸和谷氨酰胺。这两种氨基酸的细胞水平达到其亲本菌株水平的约三倍。在DeltapknG突变体中检测到更高细胞水平的含胺糖分子，GlcN-Ins和mycothiol，它们来自谷氨酸。从头谷氨酰胺合成显示减少50%。这与目前的知识一致，表明谷氨酰胺合成受谷氨酸和谷氨酰胺水平的调节。这些数据支持了我们的假设，即PknG介导了M.并将其转化为代谢适应。
The function of the Mycobacterium tuberculosis eukaryotic-like protein serine/threonine kinase PknG was investigated by gene knock-out and by expression and biochemical analysis. The pknG gene (Rv0410c), when cloned and expressed in Escherichia coli, encodes a functional kinase. An in vitro kinase assay of the recombinant protein demonstrated that PknG can autophosphorylate its kinase domain as well as its 30 kDa C-terminal portion, which contains a tetratricopeptide (TPR) structural signalling motif. Western analysis revealed that PknG is located in the cytosol as well as in mycobacterial membrane. The pknG gene was inactivated by allelic exchange in M. tuberculosis. The resulting mutant strain causes delayed mortality in SCID mice and displays decreased viability both in vitro and upon infection of BALB/c mice. The reduced growth of the mutant was more pronounced in the stationary phase of the mycobacterial growth cycle and when grown in nutrient-depleted media. The PknG-deficient mutant accumulates glutamate and glutamine. The cellular levels of these two amino acids reached approximately threefold of their parental strain levels. Higher cellular levels of the amine sugar-containing molecules, GlcN-Ins and mycothiol, which are derived from glutamate, were detected in the DeltapknG mutant. De novo glutamine synthesis was shown to be reduced by 50%. This is consistent with current knowledge suggesting that glutamine synthesis is regulated by glutamate and glutamine levels. These data support our hypothesis that PknG mediates the transfer of signals sensing nutritional stress in M. tuberculosis and translates them into metabolic adaptation.