Samuels David J, Wang Zhe, Rhee Kyu Y, Brinsmade Shaun R
Department of Biology, Georgetown University.
Division of Infectious Diseases, Weill Cornell Medical College.
J Vis Exp. 2017 Mar 28(121):55558. doi: 10.3791/55558.
In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.
为了对抗细菌病原体，宿主常常会限制感染部位的营养物质供应。这种限制会改变关键代谢物的丰度，而调节因子会对这些代谢物做出反应，从而调整细胞代谢。近年来，一些蛋白质和RNA已成为毒力基因表达的重要调节因子。例如，CodY蛋白会对支链氨基酸和GTP的水平做出反应，并且在低G+C革兰氏阳性细菌中广泛存在。作为金黄色葡萄球菌中的一种全局调节因子，CodY控制着数十个毒力和代谢基因的表达。我们推测，金黄色葡萄球菌部分地利用CodY来改变其代谢状态，以努力适应宿主环境中可能遇到的营养限制条件。本手稿描述了一种使用液相色谱-质谱联用从金黄色葡萄球菌中提取和分析代谢物的方法，该方案是为了验证这一假设而开发的。该方法还强调了确保严谨性和可重复性的最佳实践，例如在不使用连续恒化器培养的情况下维持生物稳态和恒定通气。相对于USA200甲氧西林敏感的金黄色葡萄球菌分离株UAMS-1亲本菌株，同基因的codY突变体中源自天冬氨酸的氨基酸（如苏氨酸和异亮氨酸）显著增加，而其前体（如天冬氨酸和O-乙酰高丝氨酸）减少。这些发现与通过RNA测序分析获得的转录数据高度相关：在codY缺失突变体中，这些途径中的基因上调了10至800倍。将转录组和代谢组的全局分析相结合，可以揭示细菌在面对环境或营养压力时如何改变其代谢，从而为了解感染期间与营养消耗相关的生理变化提供潜在的见解。此类发现可能为新型抗感染药物和治疗方法的开发铺平道路。
