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

项目总结/摘要 结核分枝杆菌（Mtb）在宿主中建立慢性感染方面非常成功， 其转录响应其局部环境的能力是宿主适应的主要因素。综合体， 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 注意，遇到酸性pH值（高[H+]）和其他丰富的离子信号，如氯化物（Cl-），这是 与pH协同作用，触发Mtb内广泛的转录变化，这些变化对宿主 适应这些表达变化是由转录调节因子控制的，转录调节因子对结核分枝杆菌 为继续生存做出协调一致的反应。MTB包含>200种已知或注释的转录因子 (TFs)然而，许多这些TF的生物学作用仍然未知。 我们最近鉴定出PrrA作为TF，其调节结核分枝杆菌对酸性pH和酸性pH的转录反应。 Cl-浓度高。PrrA是Mtb中12个双组分系统之一的重要TF， 不仅被其同源传感器组氨酸激酶PrrB磷酸化，而且被多个丝氨酸/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，事实上， 我们的数据表明PrrA也影响结核分枝杆菌对一氧化氮和缺氧的反应。尽管PrrA至关重要，但其 在结核杆菌-宿主相互作用中的功能作用是未知的。拟议的研究将处理下列问题： (i)什么是PrrA的调节子，它对全球结核分枝杆菌转录反应的影响是什么？ 在吞噬体中(ii)PrrA在感染中的作用是什么？既有诱导性的 过表达系统和双重诱导蛋白降解/基因沉默系统将用于揭示 PrrA在结核分枝杆菌感染生物学中的作用。此外，C3 HeB/FeJ鼠感染模型，其概括了 将与荧光Mtb复制报告基因组合使用 （SSB-GFP），以确定PrrA对Mtb体内生长的可能差异影响， 干酪样坏死病变。这些研究不仅将提供对PrrA如何使Mtb对结核病的反应的深入了解， 在宿主的背景下，周围的离子环境，而且还开辟了新的治疗靶向的途径。 除了对我们对结核病生理学的理解提出批评外，这项研究还将为我提供 广泛的训练。我的赞助商Shumin Tan博士致力于教我所需的技术技能， 执行我的项目，以及其他重要的技能，如如何有效地沟通我的发现与 科学界。正如我的培训计划中所描述的，与我的共同赞助商Ralph Isberg博士一起，我们 我制定了一个全面的计划，为我提供了提高写作、演讲和教学能力的机会。我 我也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， 研究成果和教学，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.