M. Tuberculosis Influences Host Matrix Metalloproteinases and Granuloma Formation

描述（由申请人提供）：结核分枝杆菌是一种古老的病原体，在现代人群中继续蓬勃发展，每年在全球造成190万人死亡。此外，结核分枝杆菌是一种主要的与艾滋病相关的机会致病菌。结核病的标志性特征是肉芽肿，是肺组织内明确的细胞聚集物，围绕并包含最初的感染灶。肉芽肿的形成一直被认为是宿主对结核杆菌的主要防御；然而，这种结构也可以作为长期维持受感染个体内活分枝杆菌的储存库，最近的研究表明，结核分枝杆菌启动肉芽肿发展的能力是这种生物体的重要毒力决定因素。因此，促进肉芽肿形成的分子因子代表了可能破坏结核分枝杆菌在人群中有效感染和持续存在的能力的潜在靶标。最近，我们的实验室发现了一种结核分枝杆菌编码的腺苷酸环化酶（Rv0386），该酶负责直接注射到宿主细胞中产生3',5'-环腺苷单磷酸（cAMP），初步数据表明，这种分枝杆菌衍生的cAMP可能影响肉芽肿发生的信号事件。我们假设结核分枝杆菌cAMP的主动外排影响宿主反应，促进肉芽肿结构内有利于分枝杆菌生存的局部环境，我们设计了三个具体的研究目的来解决这一假设。首先，我们将研究结核分枝杆菌如何通过细菌膜分泌cAMP进入宿主细胞。cAMP分子的亲水性阻止了它在生物膜上的扩散，我们将利用广泛的突变体收集来鉴定结核分枝杆菌在肉汤培养过程中对cAMP外排重要的基因。其次，我们将评估分枝杆菌衍生的cAMP如何影响宿主细胞信号事件，这些事件涉及基质金属蛋白酶（MMPs）的产生和分泌。这些酶负责肺细胞外基质的分解，这一过程对肉芽肿的形成至关重要。我们建议使用结核分枝杆菌腺苷酸环化酶突变株（JHU-0386）和一个补充菌株，分析cAMP在培养的人单核细胞结核分枝杆菌感染后对人MMPs基因和蛋白表达的作用，以及导致这种表达的信号事件。第三，我们将利用JHU-0386突变体来表征分枝杆菌来源的cAMP在新型兔结核模型体内肉芽肿形成和发展中的作用。总的来说，为了进一步了解结核分枝杆菌的发病机制和寻找新的治疗靶点，本项目结合体外和体内实验，研究结核分枝杆菌感染后肺肉芽肿发展中涉及的细菌和宿主细胞因素。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis is an ancient pathogen that continues to flourish within the modern human population, causing 1.9 million deaths annually across the globe. Additionally, M. tuberculosis is a major AIDS- related opportunistic pathogen. The hallmark feature of tuberculosis, the granuloma, is a defined cellular aggregate within the lung tissue, surrounding and containing the initial focus of infection. Granuloma formation has long been understood as the primary host defense against M. tuberculosis; however, this structure also serves as a reservoir for the long-term maintenance of viable mycobacteria within an infected individual, and recent studies indicate that the ability of M. tuberculosis to initiate granuloma development is an important virulence determinant for this organism. Therefore, molecular factors that contribute to formation of the granuloma represent potential targets that could disrupt the ability of M. tuberculosis to efficiently infect and persist within the human population. Recently, our laboratory has characterized an M. tuberculosis-encoded adenylate cyclase (Rv0386) responsible for the production of 3', 5'-cyclic adenosine monophosphate (cAMP) that is directly injected into the host cell, and preliminary data indicate that this mycobacterial-derived cAMP may affect signaling events involved in development of the granuloma. We hypothesize that the active efflux of cAMP from M. tuberculosis influences the host response to promote a local environment favorable for mycobacterial survival within the granuloma structure, and we have designed three specific research aims to address this hypothesis. First, we will examine how M. tuberculosis secretes cAMP across the bacterial membrane into the host cell. The hydrophilic nature of the cAMP molecule precludes its diffusion across biological membranes, and we will utilize an extensive mutant collection to identify M. tuberculosis genes important for cAMP efflux during growth in broth culture. Second, we will evaluate how the mycobacterial-derived cAMP affects host cell signaling events involved in the production and secretion of matrix metalloproteinases (MMPs). These enzymes are responsible for breakdown of the lung extracellular matrix, a process that is critical for granuloma formation. We propose to use the M. tuberculosis adenylate cyclase mutant (JHU-0386), as well as a complemented strain, to analyze the role of cAMP on the gene and protein expression of human MMPs, as well as on the signaling events leading to this expression, following M. tuberculosis infection in cultured human monocytic cells. Third, we will utilize the JHU-0386 mutant to characterize the role of the mycobacterial-derived cAMP during in vivo granuloma formation and development within a novel rabbit model of tuberculosis. Overall, in the endeavor to further understand M. tuberculosis pathogenesis and identify new therapeutic targets against this organism, this project combines in vitro and in vivo experiments to study both bacterial and host cell factors involved in development of the lung granuloma following an M. tuberculosis infection.