Integrating Global Responses to Nutrient Limitation in Gram-positive Bacteria

描述（由申请人提供）：细菌毒力基因的表达通常与营养物的耗尽相关，但是营养物可用性的信号传导和细菌毒力基因的表达如何与营养物的耗尽相关。 所产生的生理反应是否协调尚不清楚。在这一知识空白被填补之前，代谢多样性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（代谢组）提供的信息，以重新配置代谢，以适应环境变化并导致疾病。该项目的目的是用高通量全球技术来增强现有的遗传和生物化学专业知识，以分析基因表达、细胞内代谢物和通量，并在此过程中滴定全球调节因子CodY的活性并推断其在S.金黄色。该项目的核心假设是，支链氨基酸和GTP的细胞内池的波动导致CodY活性谱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。这一假设是基于初步研究，确定了真正的细胞内代谢产物，控制CodY活性在活细胞中，并揭示了层次组织的三个基因。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，那么代谢和毒力的共同调节的全面知识是必不可少的。在塔夫茨大学医学院的指导（K99）阶段，将掌握大规模平行测序、基于质谱的代谢组学和恒化器培养，以绘制S.金黄色葡萄球菌，同时获得关键的学术培训，需要启动一个成功的独立的学术生涯的指导委员会组成的专家在细菌生理学，生物化学和系统生物学的指导。掌握致病性S.金黄色葡萄球菌沿着高通量方法将使在R 00阶段的努力，以量化在S.金黄色葡萄球菌CodY调节子诱导生理应激反应系统。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性细菌的生态位，并且实验将把毒力基因表达置于S.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活性之间的相关性将提供转录组与代谢组之间以前无法实现的联系。该项目意义重大，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如何相互关联和相互依赖的理解。对这些联系的更透彻的理解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：这项拟议中的研究与公共卫生有关，因为了解细菌如何将代谢和致病过程联系起来，可能会揭示新的方法，以防止从无害的生活方式转变为有害的生活方式，从而导致可能危及生命的感染。在这条独立之路奖申请中提出的研究和职业发展将揭开细菌疾病的细胞机制，同时为独立的学术生涯提供垫脚石-这些目标与国立综合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.