Signaling Molecules in Mycobacterium tuberculosis

描述(申请人提供)：结核分枝杆菌(Mtb)是结核病的病原体，结核病是全球人类最重要的传染病之一。尽管群体感应(QS)通常在毒力中发挥重要作用，但人们对结核分枝杆菌如何沟通知之甚少。QS是由次生代谢过程中合成的小分子介导的。在结核分枝杆菌中，唯一发现的信号分子是复苏促进因子(RPF)，这是一种类似溶菌酶的蛋白质，可以刺激复制。我们使用QS传感器检测了与结核分枝杆菌密切相关的细菌物种的上清液，重点是放线菌，特别是链霉菌。几乎所有的QS分子都是用传感器鉴定出来的，但所有的传感器菌株都是革兰氏阴性，所以以前的研究没有从结核分枝杆菌中鉴定出QS分子就不足为奇了。我们推测，使用链霉菌传感器将更有可能识别Mtb QS分子。在我们的初步研究中，我们发现了两个QS分子MAI-1和MAI-2，它们分别能够在灰色链霉菌和天蓝色链霉菌中诱导QS通路，分别诱导产孢子和产色素抗生素。MAI-1和MAI-2的纯化和表型效应分析表明，它们影响结核分枝杆菌毒力相关的表型，包括生物膜的形成和宿主细胞的感染。此外，我们还通过qRT-PCR发现MAI-1和MAI-2调控了链霉菌中qs基因的表达，并通过基因芯片技术控制了与Mtb毒力相关的几个重要的全局调控基因。在拟议的小型项目中，我们计划完成我们的初步研究，以便出版和制定一个坚实的、更全面的项目，研究结核分枝杆菌和其他分枝杆菌物种的QS。这些实验将通过两个具体的目标来完成：1)MAI-1和MAI-2的完整结构。我们的工作假设是，分枝杆菌MAI是带有修饰脂肪侧链的环状分子，类似于链霉菌中的g-丁内酯(GBL)QS分子。我们已经获得了大量关于MAI-1和MAI-2的结构信息，包括完整的H-H和H-C核磁共振以及MS和IR光谱数据。缺少的两条数据是脂族侧链与酚环连接的确认和侧链的长度。为此，我们将完成MAI-1和MAI-2的大规模纯化，以改进碳-碳核磁共振和HR-MS研究。2)验证结核分枝杆菌MAI-1和MAI-2毒力网络。我们的工作假设是MAI-1和MAI-2诱导已知参与毒力的关键调控网络。我们的微阵列分析发现，MAI-1和MAI-2都可以诱导MprA、DosR和ClgR调节子，这些调节子已经被证明在毒力中发挥作用。我们将通过使用qRT-PCR确定MAI对调节子的控制程度来验证这些数据，并确认在诱导这些调节子的生长条件下MAI的产生。我们的长期目标是深入了解MAI是否参与从潜伏期重新激活，正如我们的微阵列研究所表明的那样。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most important infectious diseases in humans worldwide. Very little is understood regarding how Mtb communicates, despite the fact that quorum sensing (QS) normally plays an important role in virulence. QS is mediated by small molecules synthesized during secondary metabolism. In Mtb, the only signaling molecule identified is resuscitation promoting factor (Rpf), a lysozyme-like protein that can stimulate replication. We examined supernatants using QS sensors in bacterial species closely related to Mtb, focusing on Actinomycetales, particularly Streptomyces. Nearly all QS molecules have been identified using sensors, but all sensor strains are Gram negatives, making it unsurprising that previous studies have not identified QS molecules from Mtb. We reasoned that use of a Streptomyces sensor would be more likely to identify Mtb QS molecules. In our preliminary studies we identified two QS molecules, MAI-1 and MAI-2, that have the ability to induce QS pathways in Streptomyces griseus and S. coelicolor inducing sporulation and pigmented antibiotic production, respectively. Purification and analysis of the phenotypic effects of MAI-1 and MAI-2 demonstrated that they impact virulence-related phenotypes of Mtb including biofilm formation and host cell infection. Furthermore, we have shown that MAI-1 and MAI-2 regulate QS gene expression in Streptomyces by qRT-PCR and control several important global regulons involved in Mtb virulence by microarray. In the proposed small project, we plan to complete our preliminary studies to allow publication and formulation of a solid more comprehensive project to study QS in Mtb and other mycobacterial species. These experiments will be accomplished through two specific aims: 1) Complete structures of MAI-1 and MAI-2. Our working hypothesis is that the mycobacterial MAI are ringed molecules with modified aliphatic side chains similar to g-butyrolactone (GBL) QS molecules in Streptomyces. We have obtained a great deal of structural information regarding MAI-1 and MAI-2, including full H-H and H-C NMR as well as MS and IR spectral data. The two pieces of data missing are confirmation of the linkage of the aliphatic side chain to the phenolic ring and length of the side chains. In this aim, we will complete large-scale purification of MAI- 1 and MAI-2 to allow improved carbon-carbon NMR and HR-MS studies. 2) Validate MAI-1 and MAI-2 virulence networks in Mtb. Our working hypothesis is that MAI-1 and MAI-2 induce key regulatory networks that are known to be involved in virulence. Our microarray analyses found that MAI-1 and MAI-2 both induce MprA, DosR and ClgR regulons that have been shown to play a role in virulence. We will validate these data by determining the extent of regulon control by the MAI using qRT-PCR and confirm production of MAI under growth conditions that induce these regulons. Our long-term goal is to gain insight into whether MAI are involved in reactivation from latency, as suggested by our microarray studies.