Integrating Global Responses to Nutrient Limitation in Gram-positive Bacteria

7.项目概要/摘要。细菌毒力基因的表达通常与细菌的致病性相关。 营养素的作用，但如何营养素的可用性和由此产生的生理反应的信号是共同的， 排序不清楚。在这一知识空白被填补之前，代谢多样性细菌，如葡萄球菌， 金黄色葡萄球菌将继续引起危险的医院获得性感染。申请人的长期目标是领导一个 一个独立的学术研究小组，研究细菌如何整合和响应 细胞内代谢物（代谢组）重新配置代谢以适应环境变化， 导致疾病。该项目的目标是增强现有的遗传和生物化学专业知识， 通过全球技术来分析基因表达、细胞内代谢物和通量，并且，在这样做时， 滴定全局调节子CodY的活性并推断其在S.金黄色。的核心 这个项目是假设，在细胞内池的支链氨基酸和GTP的波动 导致CodY活动的一系列，对营养限制产生分级反应，最终导致 代谢适应和毒力的发展。这一假设基于初步研究， 确定了真正的细胞内代谢物，控制CodY活性在活细胞中，并揭示了层次或- 三个基因的组织。该项目的基本原理是， 如果我们要了解细菌致病的生理起源，代谢和毒力是必不可少的。 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- ing，将掌握基于质谱的代谢组学和恒化器培养， 代谢和毒力基因的表达模式。aureus，同时获得关键的学术培训需要 在由以下人员组成的指导委员会的指导下，开展成功的独立学术生涯： 细菌生理学、生物化学和系统生物学专家。掌握了修炼和基因， 致病性S.金黄色葡萄球菌沿着高通量方法将使R 00期间的努力 阶段，以量化S.金黄色葡萄球菌CodY调节子诱导生理应激反应系统 tems.这种方法是创新的，因为连续的细菌培养模拟了营养限制性细菌生态位， 这些实验将把毒力基因的表达放在正常的环境中， S的行为。金黄色葡萄球菌在宿主的营养限制条件下。此外， 全局代谢物池和CodY活性将提供先前无法实现的转录组与 代谢组。该项目意义重大，因为它将增加我们对遗传亲- 克的代谢适应和毒力基因的表达是相互关联和相互依赖的。一个更 对这些联系的透彻理解也可能提供潜在的新的治疗策略。的 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.