Integrating Global Responses to Nutrient Limitation in Gram-positive Bacteria

描述(申请人提供)：细菌毒力基因的表达通常与营养物质的耗尽有关，但营养物质可获得性和营养物质的信号如何 由此产生的生理反应是否协调尚不清楚。在这一知识差距被弥合之前，代谢多样化的细菌，如金黄色葡萄球菌，将继续导致危险的医院获得性感染。申请人的长期目标是领导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(代谢体)提供的信息，以重新配置新陈代谢，以适应环境变化和导致疾病。该项目的目的是利用高通量的全球技术来分析基因表达、细胞内代谢物和通量，以增强现有的遗传和生化专业知识，并在此过程中滴定全球调控因子Cody的活性，并推断其在金黄色葡萄球菌中的调控层次。该项目的核心假设是，细胞内支链氨基酸和GTP池的波动会导致Cody活性的谱产生对营养限制的分级反应，最终导致代谢适应和毒力的发展。这一假说建立在初步研究的基础上，这些研究确定了控制活细胞中Cody激活素的真实细胞内代谢物，并揭示了三个基因的分层组织。这个项目的基本原理是，如果我们要了解细菌致病的生理起源，全面了解新陈代谢和毒力的共同调节是必不可少的。在塔夫茨大学医学院的导师(K99)阶段，将掌握大规模并行测序、基于质谱学的代谢组学和恒化器培养，以绘制金黄色葡萄球菌交叉代谢和毒力基因表达模式，同时在由细菌生理学、生物化学和系统生物学专家组成的导师委员会的指导下，获得必要的关键学术培训，以启动成功的独立学术生涯。掌握致病金黄色葡萄球菌的培养和遗传操作以及高通量方法，将使R00阶段的努力能够量化诱导生理应激反应系统时金黄色葡萄球菌Cody调节子的变化。这种方法是创新的，因为连续的细菌培养模拟了人体内营养限制的细菌生态位，实验将把毒力基因的表达置于金黄色葡萄球菌在宿主营养限制的条件下的正常行为的背景下。此外，全球代谢物库和Cody活性之间的相关性将提供以前无法实现的转录组与代谢组之间的联系。这个项目意义重大，因为它将增加我们对代谢适应和毒力基因表达的遗传程序是如何相互关联和相互依赖的理解。更透彻地了解这些联系也可能提供潜在的新的治疗策略。独立之路奖将提供实现这些目标所需的时间和资源。 公共卫生相关性：拟议中的研究与公共健康相关，因为了解细菌如何将新陈代谢和致病过程联系起来，可能会揭示防止这种从无害的生活方式转变为有害的生活方式的新方法，这种生活方式会导致潜在的危及生命的感染。在这个独立之路奖项申请中提出的研究和职业发展将揭开细菌疾病背后的细胞机制，同时提供一个独立的学术生涯的垫脚石-目标与国家普通医学科学研究所的使命一致。

DESCRIPTION (provided by applicant): Expression of bacterial virulence genes often correlates with the exhaustion 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 activit in living cells and revealed hierarchical organization 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 pathogenesis. During the mentored (K99) phase at Tufts University School of Medicine, massively parallel sequencing, 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 manipulation 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 systems. The approach is innovative, because continuous bacterial cultures mimic nutrient-limiting bacterial niches 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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because understanding how bacteria connect metabolism and disease-causing processes may reveal new ways to prevent the switch from harmless to harmful lifestyles that lead to potentially life-threatening infections. The research and career development proposed in this Pathway to Independence award application will unravel cellular mechanisms underlying bacterial disease while providing a stepping stone to an independent academic career - objectives that align with the mission of the National Institute of General Medical Sciences.