Chopra Tarun, Hamelin Romain, Armand Florence, Chiappe Diego, Moniatte Marc, McKinney John D
From the ‡School of Life Sciences and
¶Proteomics Core Facility, Swiss Federal Institute of Technology in Lausanne, 1015 Lausanne, Switzerland
Mol Cell Proteomics. 2014 Nov;13(11):3014-28. doi: 10.1074/mcp.M113.034082. Epub 2014 Jul 5.
Mycobacterium tuberculosis has a remarkable ability to persist within the human host as a clinically inapparent or chronically active infection. Fatty acids are thought to be an important carbon source used by the bacteria during long term infection. Catabolism of fatty acids requires reprogramming of metabolic networks, and enzymes central to this reprogramming have been targeted for drug discovery. Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, is often used as a model system because of the similarity of basic cellular processes in these two species. Here, we take a quantitative proteomics-based approach to achieve a global view of how the M. smegmatis metabolic network adjusts to utilization of fatty acids as a carbon source. Two-dimensional liquid chromatography and mass spectrometry of isotopically labeled proteins identified a total of 3,067 proteins with high confidence. This number corresponds to 44% of the predicted M. smegmatis proteome and includes most of the predicted metabolic enzymes. Compared with glucose-grown cells, 162 proteins showed differential abundance in acetate- or propionate-grown cells. Among these, acetate-grown cells showed a higher abundance of proteins that could constitute a functional glycerate pathway. Gene inactivation experiments confirmed that both the glyoxylate shunt and the glycerate pathway are operational in M. smegmatis. In addition to proteins with annotated functions, we demonstrate carbon source-dependent differential abundance of proteins that have not been functionally characterized. These proteins might play as-yet-unidentified roles in mycobacterial carbon metabolism. This study reveals several novel features of carbon assimilation in M. smegmatis, which suggests significant functional plasticity of metabolic networks in this organism.
结核分枝杆菌具有非凡的能力，能够在人类宿主内以临床隐匿或慢性活跃感染的形式持续存在。脂肪酸被认为是细菌在长期感染过程中使用的重要碳源。脂肪酸的分解代谢需要对代谢网络进行重新编程，而参与这种重新编程的关键酶已成为药物研发的靶点。耻垢分枝杆菌是结核分枝杆菌的非致病性近亲，由于这两个物种的基本细胞过程相似，它常被用作模型系统。在这里，我们采用基于定量蛋白质组学的方法，以全面了解耻垢分枝杆菌的代谢网络如何调整以利用脂肪酸作为碳源。对同位素标记蛋白质进行二维液相色谱和质谱分析，共鉴定出3067种高可信度蛋白质。这个数字相当于预测的耻垢分枝杆菌蛋白质组的44%，包括大多数预测的代谢酶。与以葡萄糖为生长底物的细胞相比，162种蛋白质在以乙酸盐或丙酸盐为生长底物的细胞中表现出丰度差异。其中，以乙酸盐为生长底物的细胞中，构成功能性甘油酸途径的蛋白质丰度更高。基因失活实验证实，乙醛酸循环和甘油酸途径在耻垢分枝杆菌中均起作用。除了具有注释功能的蛋白质外，我们还证明了尚未进行功能表征的蛋白质在碳源依赖下的丰度差异。这些蛋白质可能在分枝杆菌的碳代谢中发挥尚未确定的作用。这项研究揭示了耻垢分枝杆菌碳同化的几个新特征，这表明该生物体代谢网络具有显著的功能可塑性。
