Zinniel Denise K, Fenton Robert J, Halouska Steven, Powers Robert, Barletta Raul G
School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, USA.
J Vis Exp. 2012 Sep 3(67):e3673. doi: 10.3791/3673.
Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment, harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 °C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) H NMR and two-dimensional (2D) H-(13)C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.
结核分枝杆菌是全球范围内人类死亡的主要原因。多重耐药（MDR）和广泛耐药（XDR）菌株的出现，有可能使当前的疾病控制工作脱轨。因此，迫切需要开发比现有药物更有效的药物和疫苗。结核分枝杆菌的基因组已被知晓十多年，但我们对基因功能和必要性的了解仍存在重要空白。此后，许多研究在转录组和蛋白质组水平上进行基因表达分析，以确定药物、氧化剂和生长条件对基因表达全局模式的影响。最终，这些变化的最终反应反映在细菌的代谢组成中，包括几千种小分子化学物质。比较野生型和突变株（未处理或用特定药物处理）的代谢谱，可有效实现靶点鉴定，并可能导致开发具有抗结核活性的新型抑制剂。同样，也可以评估两种或多种条件对代谢组的影响。核磁共振（NMR）是一种强大的技术，用于识别和定量代谢中间体。在本方案中描述了用于NMR代谢组学分析的结核分枝杆菌细胞提取物的制备程序。细胞培养物在适当条件下生长，并需要生物安全3级防护，收获后进行机械裂解，同时保持低温以最大程度地保存代谢物。回收细胞裂解物，过滤除菌，并储存在超低温下。将这些细胞提取物的等分试样接种在Middlebrook 7H9琼脂上，用于形成菌落单位，以验证无活细胞。在37°C孵育两个月后，如果未观察到活菌落，则将样品从防护设施中取出进行下游处理。提取物冻干，重悬于氘代缓冲液中，并注入NMR仪器，获取光谱数据，然后进行统计分析。所描述的程序可应用于一维（1D）H NMR和二维（2D）H-（13）C NMR分析。与色谱方法相比，该方法能提供更可靠的小分子代谢物鉴定以及更可靠、灵敏的细胞提取物代谢组成定量分析。细胞裂解步骤之后所描述程序的变体也可适用于平行蛋白质组分析。
