M. Tuberculosis Influences Host Matrix Metalloproteinases and Granuloma Formation

描述(申请人提供)：结核分枝杆菌是一种古老的病原体，在现代人中继续蓬勃发展，每年导致全球190万人死亡。此外，结核分枝杆菌是一种与艾滋病相关的主要机会性病原体。肺结核的标志特征是肉芽肿，它是肺组织内的一种明确的细胞集合体，包围并包含最初的感染灶。长期以来，肉芽肿的形成一直被认为是对抗结核分枝杆菌的主要宿主防御；然而，这种结构也是在感染者体内长期维持活性分枝杆菌的储存库，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的一个重要的毒力决定因素。因此，导致肉芽肿形成的分子因素是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点。最近，我们实验室鉴定了一种结核分枝杆菌编码的腺苷环化酶(Rv0386)，它负责产生3‘，5’-环磷酸腺苷(CAMP)，并直接注射到宿主细胞中，初步数据表明，这种分枝杆菌衍生的cAMP可能影响参与肉芽肿发展的信号事件。我们假设结核分枝杆菌cAMP的主动外流影响宿主的反应，以促进肉芽肿结构中有利于分枝杆菌生存的局部环境，我们设计了三项具体的研究旨在解决这一假设。首先，我们将研究结核分枝杆菌如何通过细菌膜将cAMP分泌到宿主细胞中。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集合来鉴定在肉汤培养中生长过程中对cAMP外流至关重要的结核分枝杆菌基因。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，这些信号事件涉及基质金属蛋白酶(MMPs)的产生和分泌。这些酶负责分解肺细胞外基质，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环化酶突变体(JHU-0386)和一个互补株来分析cAMP对人MMPs基因和蛋白表达的作用，以及在培养的人单核细胞感染结核分枝杆菌后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衍生的cAMP在一种新的兔结核病模型中在体内肉芽肿形成和发展中的作用。总之，为了进一步了解结核分枝杆菌的发病机制，并确定针对这种微生物的新的治疗靶点，本项目结合了体外和体内实验，研究了细菌和宿主细胞因素与结核分枝杆菌感染后肺肉芽肿的发展有关。

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.