cAMP signaling in Mycobacterium tuberculosis

描述(由申请人提供)：该项目的长期目标是更好地了解结核分枝杆菌(Mtb)是如何建立感染的，以便能够制定有效的策略来预防它。基因调控是结核分枝杆菌在感染过程中对宿主环境做出成功反应的关键方面。最近的研究发现Mtb编码多达15个腺苷酸环化酶(AC)，这表明cAMP信号在Mtb中构成了一种重要的、但以前未被认识的调节机制。一种多学科的方法将在分子、遗传和细胞水平上解决cAMP信号在结核分枝杆菌中的作用，重点放在与潜伏期和结核分枝杆菌与巨噬细胞相互作用相关的条件上。具体目标包括： 目的1：利用DNA芯片和等基因敲除突变体，筛选低氧时受cAMP依赖转录因子Rv1675c调控的mtb基因； 目的：确定并鉴定结核分枝杆菌转录因子Rv3676调控的第二个可能的cAMP依赖调节子。通过定义Rv3676与其启动子区域中确定的DMA基序结合的作用，将对该调控子的候选成员进行功能评估； 目的3：评价cAMP和哺乳类AC Rv1625c在Mtb与巨噬细胞相互作用中的作用，特别是在细菌复制和运输方面； 目的4：评估一个特定的巨噬细胞诱导的cAMP调节基因在巨噬细胞内和潜伏期相关条件下的调节和功能。 鉴定和鉴定结核分枝杆菌中cAMP介导的基因调控将有助于我们理解建立结核病感染和疾病所需的因素。这项工作将探索结核分枝杆菌新的基因调控网络，并为未来cAMP信号在结核分枝杆菌毒力基因调控中的作用提供重要的基础，从而识别用于结核病疫苗、治疗和/或诊断目的的潜在靶点。

DESCRIPTION (provided by applicant): The long term objective of this project is to better understand how Mycobacterium tuberculosis (Mtb) establishes infection so that effective strategies can be developed to prevent it. Gene regulation is a critical aspect of Mtb's successful response to the host environment during infection. The recent finding that Mtb encodes as many as 15 adenylate cyclases (AC) suggests that cAMP signaling constitutes an important, but previously unrecognized, regulatory mechanism in Mtb. A multidisciplinary approach will address the role of cAMP signaling in Mtb, at the molecular, genetic and cellular levels, with a focus on conditions associated with latency and Mtb's interaction with macrophages. Specific aims include: Aim 1: identify, using DMA microarrays and isogenic knockout mutants, Mtb genes that are regulated by the putative cAMP-dependent transcription factor, Rv1675c, at various levels of cAMP during hypoxia; Aim 2: define and characterize a second likely cAMP-dependent regulon controlled by putative transcription factor Rv3676 in Mtb. Candidate members of this regulon will be functionally assessed by defining the role of Rv3676 binding to a DMA motif identified in their promoter regions; Aim 3: evaluate the roles of cAMP and a mammalian-type AC, Rv1625c, in Mtb's interaction with macrophages, particularly with respect to bacterial replication and trafficking; Aim 4: assess the regulation and function of a specific macrophage-induced, cAMP regulated gene within macrophages and during latency-associated conditions. Identification and characterization of cAMP-mediated gene regulation in Mtb will contribute to our understanding of the factors needed for the establishment of tuberculosis infection and disease. This work will explore a new gene regulatory network in Mtb and provide an important foundation for future work on the role of cAMP signaling in Mtb virulence gene regulation, leading to identification of potential targets for TB vaccines, therapeutics, and/ or diagnostic purposes.