Unraveling the role of an essential two-component system in Mycobacterium tuberculosis ionic signal response and host colonization

项目摘要/摘要 结核分枝杆菌(Mtb)在建立宿主慢性感染方面非常成功， 其转录能力对其本地环境作出反应是宿主适应的主要因素。这个建筑群， 宿主内不均匀的离子环境是结核分枝杆菌环境线索的关键来源。特别的 注意，酸性pH(高[H+])和其他丰富的离子信号的相遇，如氯(Cl-)，这是 感受到与pH的协同作用，会在Mtb内触发广泛的转录变化，这对宿主来说是关键的 适应。这些表达变化是由转录调控因子控制的，转录调控因子对结核分枝杆菌 采取协调一致的应对措施，以求持续生存。MTB包含&gt；200已知或带注释的转录因子 然而，其中许多因子的生物学作用尚不清楚。 我们最近发现PrrA是一种转铁蛋白，它调节Mtb对酸性pH的转录反应 氯离子浓度较高。PrrA是一种基本的Tf，包括Mtb中12个双组分系统中的1个，以及 不仅被其同源感受器组氨酸激酶PrrB磷酸化，还被多个丝氨酸/苏氨酸磷酸化 蛋白激酶。这表明PrrA可能调节对多种环境信号的反应，实际上， 我们的数据表明PrrA也影响线粒体对一氧化氮和低氧的反应。尽管PrrA是必不可少的，但其 结核分枝杆菌-宿主相互作用中的功能作用尚不清楚。拟议的研究将涉及以下问题： (I)PrrA的调控是什么，它对全球mtb转录反应的影响是什么 在吞噬体内吗？(2)PrrA在感染方面的作用是什么？两者都是诱导性的 过表达系统和双重诱导蛋白降解/基因沉默系统将被用来揭示 PrrA在结核分枝杆菌感染生物学中的作用此外，C3HeB/FEJ小鼠感染模型 在人类感染中观察到的病变，将与Mtb复制荧光报告程序结合使用 (SSB-GFP)，以确定PrrA在体内对结核分枝杆菌生长的可能差异影响 干酪性坏死性病变。这些研究将不仅提供关于PrrA如何使MTB对 在宿主周围离子环境的背景下，也为治疗靶向开辟了新的途径。 除了对我们对结核分枝杆菌生理学的理解有批判性的帮助外，这项研究还将为我提供 广泛的训练。我的赞助人谭淑敏博士致力于教我所需的技术技能 执行我的项目，以及其他重要技能，例如如何有效地将我的发现与 科学界。正如我的培训计划中所描述的，与我的共同赞助人拉尔夫·伊斯伯格博士一起，我们有 制定了一个全面的计划，为我提供了机会来提高我的写作、演讲和教学能力。我 我还将有机会与其他教授合作，进一步丰富我的实验技能。 这个项目将使我磨练所有的基本技能，从研究概念化到沟通 发现和教学，这是我实现成为一名学术机构教授的目标所必需的。

Project Summary/Abstract Mycobacterium tuberculosis (Mtb) is remarkably successful in establishing chronic infection in a host, with its ability to transcriptionally respond to its local environment a major factor in host adaptation. The complex, non-uniform ionic milieu within the host serves as a critical source of environmental cues for Mtb. Of particular note, the encounter of acidic pH (high [H+]) and other abundant ionic signals such as chloride (Cl-), which is sensed in synergy with pH, triggers extensive transcriptional changes within Mtb that are critical for host adaptation. These expression changes are controlled by transcriptional regulators that are critical for Mtb to mount a coordinated response for continued survival. Mtb contains >200 known or annotated transcription factors (TFs), yet the biological role of many of these TFs remain unknown. We recently identified PrrA as a TF that modulates the transcriptional response of Mtb to both acidic pH and high Cl- concentration. PrrA is an essential TF that comprises 1 of 12 two-component systems in Mtb, and is phosphorylated not only by its cognate sensor histidine kinase PrrB, but also by multiple serine/threonine protein kinases. This suggests that PrrA may regulate response to multiple environmental signals, and indeed, our data indicate that PrrA also impacts Mtb response to nitric oxide and hypoxia. Although PrrA is essential, its functional role in Mtb-host interactions is unknown. The proposed studies will address the following questions: (i) What is the regulon of PrrA and what is its impact on global Mtb transcriptional response to conditions reflective of that within a phagosome? (ii) What is the role of PrrA in the context of infection? Both an inducible overexpression system and a dual inducible protein degradation/gene silencing system will be utilized to uncover the function of PrrA in Mtb infection biology. Further, the C3HeB/FeJ murine infection model, which recapitulates lesions observed in human infections, will be used in combination with the fluorescent Mtb replication reporter (SSB-GFP) to define the possible differential impact of PrrA on Mtb growth in vivo within sublocations of a caseous necrotic lesion. These studies will provide not just insight into how PrrA enables Mtb to respond to the surrounding ionic milieu in the context of the host, but also open up new avenues for therapeutic targeting. In addition to shedding critical light on our understanding of Mtb physiology, this study will provide me extensive training. My sponsor Dr. Shumin Tan is dedicated to teaching me the required technical skills to perform my project, as well as other vital skills such as how to effectively communicate my findings with the scientific community. As described in my training plan, together with my co-sponsor Dr. Ralph Isberg, we have assembled a comprehensive plan with opportunities for me to improve my ability to write, present, and teach. I will also have the opportunity to collaborate with other professors to further diversify my experimental skill set. This project will allow me to hone all the essential skills, from research conceptualization to communication of findings and teaching, necessary for me to reach my goal of becoming a professor at an academic institute.