High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism.
High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism.
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高分辨率表型分析定义了对分枝杆菌生长和胆固醇分解代谢必不可少的基因。
DOI：
10.1371/journal.ppat.1002251
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发表时间：
2011-09
期刊：
影响因子：
6.7
通讯作者：
Sassetti CM
中科院分区：
文献类型：
--
作者：
Griffin JE;Gawronski JD;Dejesus MA;Ioerger TR;Akerley BJ;Sassetti CM
构成细胞新陈代谢的途径是高度相互联系的，个别酶的变化可能会产生深远的影响。因此，测量基因表达的全球图谱方法在预测个体功能丧失将如何影响细胞方面价值有限。在这项工作中，我们使用了一种新的全球表型图谱方法来直接定义结核分枝杆菌生长所需的基因。采用高密度诱变和深度测序相结合的方法，对暴露于不同条件下的复杂突变体库的组成进行了研究。这使得对结核分枝杆菌在体外生长所必需的基因进行了明确的识别，并被证明是对以前的方法的重大改进。为了进一步探索在宿主中持续存在所需的功能，我们定义了利用胆固醇所必需的途径，胆固醇是感染期间的关键碳源。我们鉴定的基因中，很少有以前通过转录图谱与这种适应有关，并且只有一小部分在已知的编码类固醇分解代谢功能的染色体区域编码。这些基因的比例出人意料地高得出人意料地超过了先前显示的小鼠组织中细菌生长所需的基因的比例。因此，这种单一的营养变化在对宿主的适应中占了很大一部分。这项工作提供了迄今为止最全面的甾醇分解代谢途径的遗传特征，提示了未确定的毒力基因的假定作用，并精确地定位了编码潜在药物靶点的基因。细菌病原体对感染过程中所遇到的环境的适应需要细胞生理学的大规模重塑。感染后遇到的主要变化之一是营养，因为细菌被迫利用从宿主那里清除的化合物。结核分枝杆菌具有将宿主胆固醇作为碳源和能量来源的不同寻常的能力，这种能力是动物模型中持久性所必需的。然而，我们对参与类固醇降解的生化途径的无知限制了我们评估这种碳转移在塑造细菌新陈代谢状态方面的重要性。在这项工作中，我们开发了一种新的方法来同时定量描述数千个突变体的适应度。这使得能够利用胆固醇作为碳源来识别细菌生长所需的每个细菌基因。对这些必需基因产物组成的途径的重建表明，对胆固醇的适应需要广泛的代谢变化。这些基因占细菌功能的很大一部分，对动物组织的生长至关重要，这表明这种细胞内病原体的生理是由这种环境中可用的碳源决定的。
The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis. A combination of high-density mutagenesis and deep-sequencing was used to characterize the composition of complex mutant libraries exposed to different conditions. This allowed the unambiguous identification of the genes that are essential for Mtb to grow in vitro, and proved to be a significant improvement over previous approaches. To further explore functions that are required for persistence in the host, we defined the pathways necessary for the utilization of cholesterol, a critical carbon source during infection. Few of the genes we identified had previously been implicated in this adaptation by transcriptional profiling, and only a fraction were encoded in the chromosomal region known to encode sterol catabolic functions. These genes comprise an unexpectedly large percentage of those previously shown to be required for bacterial growth in mouse tissue. Thus, this single nutritional change accounts for a significant fraction of the adaption to the host. This work provides the most comprehensive genetic characterization of a sterol catabolic pathway to date, suggests putative roles for uncharacterized virulence genes, and precisely maps genes encoding potential drug targets. The adaptation of a bacterial pathogen to the environments encountered during infection requires the wholesale remodeling of cellular physiology. One of the major changes encountered upon infection is nutritional, as the bacterium is forced to utilize compounds scavenged from the host. Mycobacterium tuberculosis has the unusual ability to use host cholesterol as a source of carbon and energy, and this capacity is required for persistence in animal models. However, our ignorance of the biochemical pathways involved in sterol degradation has limited our ability to assess the importance of this carbon shift in shaping the metabolic state of the bacterium. In this work, we developed a new method to quantitatively profile the fitness of thousands of mutants simultaneously. This allowed the identification of each bacterial gene that is required for the bacterium to grow using cholesterol as a carbon source. Reconstruction of the pathways comprised by these essential gene products revealed that adaptation to cholesterol required widespread metabolic changes. These genes account for a significant fraction of the bacterial functions important for growth in animal tissues, suggesting that the physiology of this intracellular pathogen is shaped by carbon sources available in this environment.
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DOI：
10.1084/jem.20041429
发表时间：
2004-12-20
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Matsunaga I;Bhatt A;Young DC;Cheng TY;Eyles SJ;Besra GS;Briken V;Porcelli SA;Costello CE;Jacobs WR Jr;Moody DB
通讯作者：
Moody DB
影响因子：
2.1
作者：
Brzostek, Anna;Dziadek, Bozena;Dziadek, Jaroslaw
通讯作者：
Dziadek, Jaroslaw
影响因子：
7
作者：
Langridge, Gemma C.;Phan, Minh-Duy;Turner, A. Keith
通讯作者：
Turner, A. Keith
影响因子：
82.9
作者：
Muñoz-Elías, EJ;McKinney, JD
通讯作者：
McKinney, JD
影响因子：
46.9
作者：
Badarinarayana, V;Estep, PW;Church, GM
通讯作者：
Church, GM