Exploration of quorum sensing in tuberculosis

项目总结 分枝杆菌是人类许多重要疾病的罪魁祸首，包括结核病和麻风。 细菌物种使用细菌间信号或群体感应(QS)来协调基因调控 对他们的环境做出反应。尽管这一事实和众多QS分子的存在 所有分枝杆菌基因组中的调节子(LuxR同源物)已测序，没有群体感应分子 在任何分枝杆菌物种中都被发现。目前发现的唯一潜在的信号分子是 复苏促进因子(RPF)是一种溶菌酶样蛋白，可以刺激潜伏期的再激活 肺结核。作为一个探索性的项目，我们开始寻找从 分枝杆菌。最有可能的原因是信号所需的极低浓度的QS分子，几乎 所有的qs分子都是通过使用一种产生光或色素的传感器菌株来鉴定的。 暴露在QS分子中。因为大多数细菌敏感菌株都是革兰氏阴性，所以这并不令人惊讶 由于它们还没有被成功地用于鉴定分枝杆菌QS分子，我们寻找了一种新的 传感器系统。我们推测，基于链霉菌的传感器与放线菌密切相关 分枝杆菌可能会对分枝杆菌QS分子产生反应。在我们的初步研究中，我们 发现灰色链霉菌和天蓝色链霉菌能对分枝杆菌QS分子产生反应。对.的使用 一种新的qs传感器菌株允许我们纯化其中的两个分子，命名为mai-1和mai-2，确定。 MAI-1的部分结构，并展示MAI-1和MAI-2对毒力相关表型的影响。在……里面 在这个应用中，我们建议进一步分析分枝杆菌中的QS通路，以确定它们在 发病机制。这将通过两个具体目标来实现：1)鉴定分枝杆菌QS 分子。我们的工作假设是，分枝杆菌QS分子在结构上与γ- 链霉菌中丁内酯(GBL)QS分子。我们的初步研究表明，MAI-1和 MAI-2可以在天蓝色链霉菌中触发QS通路，这表明它们之间相互关联，并与GBL有关， 但我们还没有完全确定它们的结构。在这个目标中，我们将利用我们的新型传感器来确定 它们的结构，并确认它们作为信号分子的能力。2)剖析了 分枝杆菌中的QS信号。我们的工作假设是MAI-1和MAI-2是由分枝杆菌产生的 以密度依赖的方式控制基因表达。我们的初步研究开发了一种屏幕 分枝杆菌产生MAI分子，并发现MAI分子促进生物膜的形成， 上皮细胞和巨噬细胞的定植和巨噬细胞内的生长。我们将分析这些路径 参与了分枝杆菌中这些QS分子的合成。这些研究的总体目标是 了解这些QS分子是如何形成的，并影响分枝杆菌致病的能力。

PROJECT SUMMARY Mycobacterium are responsible for many important human diseases, including tuberculosis and leprosy. Bacterial species use inter-bacterial signaling or quorum sensing (QS) to coordinate gene regulation in response to their environment. Despite this fact and the presence of numerous QS molecule responsive regulators (LuxR homologues) in all mycobacterial genomes sequenced, no quorum sensing molecules have been identified in any mycobacterial species. The only potential signaling molecule yet identified is resuscitation promoting factor (Rpf) that is a lysozyme-like protein that can stimulate reactivation of latent tuberculosis. As an exploratory project, we set out in search of the best way to identify QS molecules from mycobacteria. Most likely due to the extremely low concentrations of QS molecules required to signal, nearly all QS molecules have been identified through use of a sensor strain that produces light or pigment upon exposure to QS molecules. Since most bacterial sensor strains are Gram negatives, making it unsurprising that they have not been used successfully to identify mycobacterial QS molecules, we searched for a new sensor system. We reasoned that a sensor based on Streptomyces, an Actinomycetales closely related to Mycobacterium, would be likely to respond to mycobacterial QS molecules. In our preliminary studies, we found that Streptomyces griseus and S. coelicolor can respond to mycobacterial QS molecules. The use of a novel QS sensor strain allowed us to purify two of these molecules, designated MAI-1 and MAI-2, determine partial structure for MAI-1 and demonstrate impacts of MAI-1 and MAI-2 on virulence-related phenotypes. In this application we propose to further analyze the QS pathways in mycobacteria to determine their role in pathogenesis. This will be accomplished through two specific aims: 1) Identification of mycobacterial QS molecules. Our working hypothesis is that mycobacterial QS molecules are structurally related to γ- butyrolactone (GBL) QS molecules in Streptomyces. Our preliminary studies demonstrate that MAI-1 and MAI-2 can trigger QS pathways in S. coelicolor, suggesting that they are related to each other and to GBLs, but we have not yet fully determined their structures. In this aim, we will utilize our novel sensor to determine their structures and confirm their ability to function as signaling molecules. 2) Dissect the mechanisms of QS signaling in mycobacteria. Our working hypothesis is that MAI-1 and MAI-2 are made by mycobacteria to control gene expression in density-dependent fashion. Our preliminary studies developed a screen for production of MAI molecules by mycobacteria and found that MAI molecules enhance biofilm formation, colonization of epithelial cells and macrophages and growth in macrophages. We will analyze the pathways involved in synthesis of these QS molecules in mycobacteria. The overall goal of these studies is to understand how these QS molecules are made and affect the ability of mycobacteria to cause disease.