Integrating Global Responses to Nutrient Limitation in Gram-positive Bacteria

7.项目摘要/摘要。细菌毒力基因的表达通常与呼气相关联- 营养物质的释放，但营养物质可利用性的信号和由此产生的生理反应是如何共同作用的 坐标尚不清楚。在这一知识差距弥合之前，新陈代谢多样性的细菌，如葡萄球菌 金黄色葡萄球菌将继续导致危险的医院获得性感染。申请者的长期目标是领导一个 独立学术研究小组，研究细菌如何整合和响应由 细胞内代谢产物(代谢体)重新调整新陈代谢以适应环境变化和 引发疾病。该项目的目标是增加现有的遗传和生化专业知识。 通过全球技术来分析基因表达、细胞内代谢物和通量，并且在这样做的过程中， 滴定全球调节因子Cody的活性，并推断其在金黄色葡萄球菌中的调节等级。在…的核心 这个项目是假设细胞内支链氨基酸和GTP池的波动 导致一系列Cody活动，对营养限制产生分级反应，最终导致 代谢适应与毒力的发展。这一假设建立在初步研究的基础上 确定了控制活细胞中Cody活性的真正的细胞内代谢物，并揭示了分级或- 三个基因的组织。该项目基本原理是全面了解共同管制 如果我们要了解细菌致病的生理起源，新陈代谢和毒力的研究是必不可少的。 创世纪。在塔夫茨大学医学院的指导(K99)阶段，大规模并行序列- 将掌握ING、基于质谱仪的代谢组学和恒化器培养，以绘制交叉图 金黄色葡萄球菌的代谢和毒力基因表达模式，同时获得所需的关键学术培训 在由以下成员组成的指导委员会的指导下开始成功的独立学术生涯 细菌生理学、生物化学和系统生物学方面的专家。掌握栽培和遗传育种技术 致病性金黄色葡萄球菌的剔除以及高通量方法将使在R00期间的努力成为可能 在诱导生理应激反应系统时，量化金黄色葡萄球菌Cody调节子的变化。 Temes.这种方法是创新的，因为连续的细菌培养模仿了限制营养的细菌生态位-- ES在人体和实验中将毒力基因表达放在正常的背景下 金黄色葡萄球菌在寄主营养限制条件下的行为。此外，两者之间的相关性 全球代谢物库和Cody活性将提供以前无法实现的转录组与 代谢物。这个项目意义重大，因为它将增加我们对基因亲本是如何 克的代谢适应和毒力基因表达是相互关联和相互依赖的。A更多 彻底了解这些联系也可能提供潜在的新的治疗策略。这个 独立之路奖将提供实现这些目标所需的时间和资源。

7. Project Summary/Abstract. Expression of bacterial virulence genes often correlates with the exhaus- tion of nutrients, but how the signaling of nutrient availability and the resulting physiological responses are co- ordinated is unclear. Until this gap in knowledge is closed, metabolically diverse bacteria like Staphylococcus aureus will continue to cause perilous hospital-acquired infections. The applicant's long-term goal is to lead an independent academic research group studying how bacteria integrate and respond to information provided by intracellular metabolites (the metabolome) to reconfigure metabolism to adapt to environmental changes and cause disease. The objective of this project is to augment existing genetic and biochemical expertise with high- throughput global techniques to analyze gene expression, intracellular metabolites and flux, and, in doing so, titrate the activity of the global regulator CodY and deduce its regulatory hierarchy in S. aureus. At the heart of this project is the hypothesis that fluctuations in the intracellular pools of branched-chain amino acids and GTP result in a spectrum of CodY activities that produce a graded response to nutrient limitation, culminating in metabolic adaptation and the development of virulence. This hypothesis is based on preliminary studies that identified the true intracellular metabolites that control CodY activity in living cells and revealed hierarchical or- ganization for three genes. The rationale for this project is that comprehensive knowledge of the co-regulation of metabolism and virulence is essential if we are to understand the physiological origins of bacterial patho- genesis. During the mentored (K99) phase at Tufts University School of Medicine, massively parallel sequenc- ing, mass spectrometry-based metabolomics and chemostat cultivation will be mastered to map intersecting metabolic and virulence gene expression patterns in S. aureus, while gaining critical scholarly training needed to launch a successful independent academic career with guidance from a mentoring committee composed of experts in bacterial physiology, biochemistry and systems biology. Mastering the cultivation and genetic ma- nipulation of pathogenic S. aureus along with high-throughput methods will enable efforts during the R00 phase to quantify changes in the S. aureus CodY regulon upon induction of physiological stress response sys- tems. The approach is innovative, because continuous bacterial cultures mimic nutrient-limiting bacterial nich- es in the human body and the experiments will place virulence gene expression in the context of the normal behavior of S. aureus under the nutrient-limiting conditions of the host. Furthermore, correlations between global metabolite pools and CodY activity will provide a previously unattainable linkage of the transcriptome to the metabolome. The project is significant because it will increase our understanding of how the genetic pro- grams of metabolic adaptation and virulence gene expression are interrelated and interdependent. A more thorough understanding of these connections may also offer potentially novel therapeutic strategies. The Pathway to Independence Award will provide the time and resources needed to achieve these goals.