Signaling Molecules in Mycobacterium tuberculosis

结核分枝杆菌中的信号分子

• 批准号: 8622228
• 负责人: Jeffrey D. Cirillo
• 金额: $ 7.28万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-10 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8622228
• 关键词: Actinomycetales; Affect; Animals; Antibiotics; Bacteria; Bioinformatics; Carbon; Cell Wall; Cells; Characteristics; Communicable Diseases; Communication; Data; Disease; Drug Formulations; Epithelial Cells; Family; Gene Expression; Genes; Genome; Genus Mycobacterium; Goals; Growth; Homologous Gene; Human; Infection; Lead; Length; Lung; Mediating; Metabolism; Microbial Biofilms; Molecular; Muramidase; Mycobacterium tuberculosis; Nutrient; Oxygen; Pathogenesis; Pathway interactions; Phenotype; Physiological; Pigments; Play; Population; Production; Proteins; Publications; Regulatory Pathway; Regulon; Resuscitation; Role; Side; Signal Transduction; Signaling Molecule; Skin; Solid; Streptomyces; Streptomyces griseus; Stress; Structure; System; Testing; Toxin; Tuberculosis; Virulence; Work; base; butyrolactone; gene synthesis; human disease; improved; insight; latent infection; macrophage; member; mycobacterial; novel; pathogen; polyketide synthase; public health relevance; quorum sensing; reactivation from latency; research study; sensor; small molecule

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.

描述（由申请人提供）：结核分枝杆菌（Mtb）是结核病的病原体，结核病是全世界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 由次级代谢过程中合成的小分子介导。在结核分枝杆菌中，唯一确定的信号分子是复苏促进因子 (Rpf)，这是一种可以刺激复制的溶菌酶样蛋白质。我们使用 QS 传感器检查了与 Mtb 密切相关的细菌物种的上清液，重点关注放线菌，特别是链霉菌。几乎所有 QS 分子都已使用传感器进行了鉴定，但所有传感器菌株均为革兰氏阴性菌株，因此之前的研究尚未从 Mtb 中鉴定出 QS 分子也就不足为奇了。我们推断使用链霉菌传感器更有可能识别 Mtb QS 分子。在我们的初步研究中，我们鉴定了两种 QS 分子，MAI-1 和 MAI-2，它们能够在灰色链霉菌和天蓝色链霉菌中诱导 QS 途径，分别诱导孢子形成和有色抗生素产生。 MAI-1 和 MAI-2 的纯化和表型效应分析表明，它们影响 Mtb 的毒力相关表型，包括生物膜形成和宿主细胞感染。此外，我们通过 qRT-PCR 表明 MAI-1 和 MAI-2 调节链霉菌中的 QS 基因表达，并通过微阵列控制参与 Mtb 毒力的几个重要的全局调节子。在拟议的小项目中，我们计划完成初步研究，以便出版和制定一个可靠的更全面的项目来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 是具有修饰脂肪族侧链的环状分子，类似于链霉菌中的 g-丁内酯 (GBL) QS 分子。我们获得了大量有关 MAI-1 和 MAI-2 的结构信息，包括完整的 H-H 和 H-C NMR 以及 MS 和 IR 光谱数据。缺少的两条数据是脂肪族侧链与酚环的连接和侧链长度的确认。为此，我们将完成 MAI-1 和 MAI-2 的大规模纯化，以改进碳-碳 NMR 和 HR-MS 研究。 2) 验证 Mtb 中的 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 均诱导 MprA、DosR 和 ClgR 调节子，这些调节子已被证明在毒力中发挥作用。我们将通过使用 qRT-PCR 确定 MAI 的调节子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 是否参与潜伏期重新激活，正如我们的微阵列研究所表明的那样。