Exploration of quorum sensing in tuberculosis

项目摘要 分枝杆菌是导致许多重要人类疾病的原因，包括结核病和麻风病。 细菌物种使用细菌间信号传导或群体感应（QS）来协调基因调控， 对环境的反应。尽管存在这一事实和许多QS分子响应 调节因子（LuxR同源物）在所有分枝杆菌基因组测序，没有群体感应分子 在任何分枝杆菌物种中被发现。目前唯一确定的潜在信号分子是 复苏促进因子（Rpf）是一种溶菌酶样蛋白，可以刺激潜伏的 结核作为一个探索性的项目，我们开始寻找从细菌中鉴定QS分子的最佳方法。 分枝杆菌最有可能的原因是信号所需的QS分子浓度极低， 所有的QS分子都是通过使用一种传感器菌株来鉴定的， 暴露于QS分子。由于大多数细菌传感器菌株都是革兰氏阴性， 他们还没有被成功地用于鉴定分枝杆菌QS分子，我们寻找了一个新的 传感器系统我们推断，基于链霉菌的传感器，一种与 分枝杆菌，可能会响应分枝杆菌QS分子。在初步研究中，我们 发现灰色链霉菌和S.腔棘鱼能对分枝杆菌的QS分子作出反应。使用 一种新的QS传感器菌株使我们能够纯化其中两种分子，命名为MAI-1和MAI-2， MAI-1的部分结构，并证明MAI-1和MAI-2对毒力相关表型的影响。在 在本申请中，我们建议进一步分析分枝杆菌中的QS途径，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- 链霉菌中的丁内酯（GBL）QS分子。我们的初步研究表明，MAI-1和 MAI-2可以触发S. coelicolor，表明它们彼此相关，并与GBL相关， 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功能。2)剖析 分枝杆菌中的QS信号传导。我们的工作假设是MAI-1和MAI-2是由分枝杆菌 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筛选 通过分枝杆菌产生MAI分子，并发现MAI分子增强生物膜形成， 上皮细胞和巨噬细胞的定殖以及在巨噬细胞中的生长。我们将分析 参与合成这些QS分子。这些研究的总体目标是 了解这些QS分子是如何产生的，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.