Bacterial Sensing and Response to Chloride as a Novel Tuberculosis Drug Target

 描述(申请人提供)：结核分枝杆菌(Mtb)是一种极其成功的病原体，仍然是一个关键的公共卫生问题，每年感染三分之一的人口并导致约150万人死亡。巨噬细胞是结核分枝杆菌的主要宿主细胞，细菌将吞噬小体转变为复制利基的能力是 对慢性感染的建立和维持至关重要。这依赖于结核分枝杆菌对吞噬体内信号的感知和适当的反应，这里提出的研究建立在我发现氯(Cl-)作为一种新的环境线索的基础上，结核分枝杆菌在感染期间会做出反应。我进一步鉴定了结核分枝杆菌中的一个氯离子通道，并观察到在小鼠感染模型中，缺乏该通道的结核分枝杆菌突变体的定植能力严重减弱。我的研究提出了一个新的概念，即感知和/或维持丰富的离子(如Cl-)的适当平衡可以深刻地影响病原体建立成功感染的能力，并表明结核分枝杆菌生理学的这一方面代表了开发与当前方法协同的原创和潜在有效的结核病治疗策略的新靶点。在这里，我试图(I)利用我的氯反应报告Mtb菌株的遗传和化学生物学方法，阐明管理Mtb对Cl-的感知和反应的调控网络，并(Ii)利用我最近构建的一种新的复制报告Mtb菌株，使用全基因组突变分析，研究环境线索，如Cl-，如何影响Mtb的复制状态。我长期致力于研究，我的职业目标是在一家学术机构领导我自己的研究小组。我的研究将采取多学科的方法来探测宿主-病原体的相互作用，特别是从病原体的角度，了解它如何定植其特定的微粒子的基本方面，并利用这一知识来研究这些关键节点如何被操纵，以在感染期间改变平衡，有利于宿主。这一奖项的指导阶段将使我能够扩大我的方法论技能，并进一步发展我的研究技能和研究计划。完成专注于阐明Mtb的氯离子调节网络的遗传和化学筛选将进一步为我提供工具(Mtb突变体和调节Mtb的氯离子反应的化学探针)和结果，随着我过渡到独立的位置，将从这些工具和结果中开发新的项目。在独立阶段，我还将开始在一个不同的概念框架中讨论结核分枝杆菌对特定环境线索的感知和反应，特别是询问它可能如何与细菌复制的调控联系在一起，而不依赖于营养的可获得性。该奖项的指导阶段将进行 在康奈尔大学的大卫·罗素博士的实验室里。罗素实验室和康奈尔大学为我的研究和培训提供了理想的环境，拥有独特的资源，使我能够开展拟议的研究，并继续获得对我成为一名成功的独立调查员的目标至关重要的其他方面的技能，如实验室管理、教学和科学沟通技能。此外，我还组建了一个咨询委员会，在氯离子通道和转运蛋白、信号转导系统潜在的化学生物学以及MTB复制和单细胞成像方面拥有不同的专业知识，以在K99阶段和我过渡到R00阶段期间提供进一步的支持。这里提出的研究整合了遗传和化学生物学研究，将阐明宿主细胞环境线索与结核分枝杆菌感染的建立和维持之间的相互作用，对于了解结核分枝杆菌的生命周期以及为开发新的协同治疗方法确定潜在的目标至关重要。与拟议的研究和职业发展计划的其他方面相结合，它将确保我拥有成功实现建立自己的实验室和使用多学科方法进行微生物发病机制研究的目标所需的技能集和基础数据。

 DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb) is an extremely successful pathogen that remains a key public health problem, infecting one-third of the human population and causing ~1.5 million deaths a year. The macrophage serves as a major host cell for Mtb, and the bacterium's ability to turn the phagosome into a replicative niche is critical for the establishment and maintenance of chronic infection. This is dependent on Mtb sensing and responding appropriately to intraphagosomal signals, and the research proposed here builds on my discovery of chloride (Cl-) as a novel environmental cue that Mtb responds to during infection. I have further identified a Cl- channel in Mtb, and observe that a Mtb mutant deficient in this channel is severely attenuated for colonization in a murine infection model. My studies raise the novel concept that the sensing and/or maintenance of an appropriate balance of an abundant ion like Cl- can profoundly impact a pathogen's ability to establish a successful infection, and suggest that this aspect of Mtb physiology represents a new target for the development of original and potentially potent tuberculosis treatment strategies synergistic with current approaches. Here, I seek to (i) elucidate the regulatory network governing Mtb's sensing and response to Cl-, using both genetic and chemical biology approaches that exploit my Cl--responsive reporter Mtb strain, and (ii) examine how environmental cues, like Cl-, can impact on Mtb's replication status, using genome-wide mutational analyses together with a novel replication reporter Mtb strain that I recently constructed. I have a longstanding dedication to research, and my career goal is to direct my own research group at an academic institution. My research will take a multi-disciplinary approach to probe host-pathogen interactions especially from the pathogen's perspective, to understand fundamental aspects of how it can colonize its specific microniche, and exploit this knowledge to examine how these critical nodes may be manipulated to shift the balance during infection in favor of the host. The mentored phase of this award will allow me to expand my methodological repertoire, and further develop my research skills and research program. Completion of genetic and chemical screens focused on elucidating Mtb's Cl- regulatory network will further provide me with tools (Mtb mutants and chemical probes that modulate Mtb's Cl- response) and results from which new projects will be developed as I transition to an independent position. During the independent phase, I will also begin to address Mtb's sensing and response to specific environmental cues in a different conceptual framework, in particular interrogating how it may be linked to regulation of bacterial replication, independent of nutrient availability. The mentored phase of this award will be carried out in the lab of Dr. David Russell at Cornell University. The Russell lab and Cornell University provide an ideal environment for my research and training, with unique resources that will enable me to carry out the proposed research, and continue to acquire skills in other facets crucial to my goal of becoming a successful independent investigator, such as lab management, teaching, and scientific communication skills. In addition, I have assembled an advisory committee with diverse expertise in Cl- channels and transporters, in chemical biology underlying signal transduction systems, and in Mtb replication and single cell imaging, to provide further support during the K99 phase and as I transition to the R00 phase. The research proposed here integrates genetic and chemical biology studies that will shed light on the interplay between host cellular environmental cues and Mtb's establishment and maintenance of infection, insights essential to understanding Mtb's life cycle, and for identifying potential targts for the development of new synergistic treatment methods. Combined with other aspects of the research and career development plan proposed, it will ensure that I have both the skill set and groundwork data needed to successfully accomplish my goals of establishing my own lab and pursuing microbial pathogenesis research using a multi-disciplinary approach.