Adenosine 3',5'-cyclic monophosphate signaling in Mycobacterium tuberculosis

描述（由申请人提供）：结核病（TB）是一种全球性的毁灭性疾病。这种疾病的病原体，结核分枝杆菌（Mtb），潜伏感染了世界上大约三分之一的人口，每年造成近200万人死亡。此外，与艾滋病毒感染的致命协同作用和耐药菌株的出现使治疗和管理这种疾病的努力进一步复杂化。了解允许结核分枝杆菌对不断变化的宿主环境作出反应的信号通路可能有助于确定迫切需要的新治疗策略。环磷酸腺苷（cAMP）是结核分枝杆菌基因调控的关键信号分子。结核分枝杆菌具有15个腺苷酸环化酶（AC），其中许多在体外响应于不同的环境刺激。这种Mtb AC的大库提出了这些AC是否在体内不同的信号传导途径中起作用的问题。当AC都增加cAMP水平时，如何实现这种特异性是一个尚未解决的主要问题。本申请测试了Mtb cAMP信号传导中存在特异性的假设。此外，本申请测试了Mtb AC是否与两种最近鉴定的cAMP受体蛋白样转录因子（TF）中的任一种差异性地相互作用并激活。我建议，以确定是否AC功能在不同的信号转导通路，不完全重叠使用蛋白质组学的方法，AC缺失菌株生长在高营养，中性pH条件下。我还将评估物理接近特定的AC和两个TF，使用分枝杆菌蛋白片段互补（M-PFC），免疫共沉淀，和间接免疫荧光显微镜。如果cAMP信号的特异性是通过物理接近发生的，我预计特定的AC将结合或定位于一个TF，而不是另一个。我还将通过鉴定基因（使用半定量逆转录聚合酶链反应和染色质免疫沉淀）来表征cAMP信号通路，这些基因通常在转录水平上由特定AC和两种TF中的任一种调节。这些研究的结果将提供关于特异性在Mtb cAMP信号传导中的作用的关键新信息。这些知识可以确定新的结核病药物靶点或治疗策略。公共卫生相关性：每年约有200万人死于结核分枝杆菌感染。迫切需要确定新的结核病药物靶标和改进的治疗策略来控制这一新出现的流行病。

DESCRIPTION (provided by applicant): Tuberculosis (TB) is a devastating disease of global proportions. The causative agent of this disease, Mycobacterium tuberculosis (Mtb), latently infects approximately a third of the world's population and causes nearly two million deaths a year. Additionally, deadly synergy with HIV-infection and the emergence of drug resistant strains have further complicated efforts to treat and manage the disease. Understanding the signaling pathways that allow Mtb to respond to changing host environments will likely facilitate the identification of desperately needed new treatment strategies. Adenosine 3',5'-cyclic monophosphate (cAMP) is a key signaling molecule in Mtb gene regulation. Mtb possesses 15 adenylyl cyclases (ACs), many of which respond to different environmental stimuli in vitro. This large repertoire of Mtb ACs raises the issue of whether these ACs function in distinct signaling pathways in vivo. How such specificity could be achieved when ACs all increase cAMP levels is a major unresolved question. This application tests the hypothesis that specificity exists in Mtb cAMP signaling. Additionally, this application tests whether Mtb ACs differentially interact with and activate either of the two recently identified cAMP receptor protein-like transcription factors (TFs). I propose to determine whether ACs function in distinct signaling pathways that do not completely overlap using a proteomics approach on AC deletion strains grown under high nutrient, neutral pH conditions. I will also assess physical proximity between specific ACs and the two TFs, using mycobacterial protein fragment complementation (M-PFC), coimmunoprecipitation, and indirect immunofluorescence microscopy. If specificity in cAMP signaling is occurring via physical proximity, I expect that specific ACs will bind to or localize with one TF, but not the other. I will also characterize the cAMP signaling pathway by identifying genes (using semi-quantitative reverse transcription polymerase chain reaction and chromatin immunoprecipitation) that are commonly regulated at the transcriptional level by a specific AC and either of the two TFs. Results from these studies will provide critical new information on the role of specificity in Mtb cAMP signaling. Such knowledge could identify new Mtb drug targets or treatment strategies. PUBLIC HEALTH RELEVANCE: Approximately two million people die each year from infection with Mtb. The identification of new Mtb drug targets and improved treatment strategies are sorely needed to control this emerging epidemic.