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）是结核病的病因，是全球人类中最重要的传染病之一。尽管法定人数（QS）通常在毒力中起着重要作用，但关于MTB如何交流的了解很少。 QS是由在继发代谢过程中合成的小分子介导的。在MTB中，发现的唯一信号分子是促进因子（RPF），这是一种可以刺激复制的溶菌酶样蛋白。我们在与MTB密切相关的细菌物种中使用QS传感器检查了上清液，重点是放线菌，尤其是链霉菌。几乎所有QS分子都使用传感器鉴定出来，但所有传感器菌株都是革兰氏骨负的，这毫不奇怪，以前的研究尚未鉴定出MTB的QS分子。我们认为，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，我们确定了两个QS分子MAI-1和MAI-2，它们具有分别诱导Griseus链霉菌和卵形链球链球链球链球链球菌诱导孢子形成和色素化抗生素产生的能力。 MAI-1和MAI-2的表型作用的纯化和分析表明，它们会影响MTB的毒力相关表型，包括生物膜形成和宿主细胞感染。此外，我们已经表明，MAI-1和MAI-2通过QRT-PCR调节链霉菌中的QS基因表达，并控制微阵列中与MTB毒力有关的几种重要的全球调节。在拟议的小型项目中，我们计划完成初步研究，以允许出版和制定一个稳定的更全面的项目，以研究MTB和其他分枝杆菌中的QS。这些实验将通过两个特定的目的来完成：1）MAI-1和MAI-2的完整结构。我们的工作假设是，分枝杆菌MAI是环形分子，具有改良的脂肪族侧链，类似于链霉菌中的G叔丁基酮（GBL）QS分子。我们已经获得了有关MAI-1和MAI-2的大量结构信息，包括完整的H-H和H-C NMR以及MS和IR光谱数据。缺少的两个数据是确认脂肪族侧链与酚环和侧链长度的联系。在此目标中，我们将完成对MAI-1和MAI-2的大规模纯化，以允许改进碳NMR和HR-MS研究。 2）在MTB中验证MAI-1和MAI-2毒力网络。我们的工作假设是，MAI-1和MAI-2诱导了已知与毒力有关的关键调节网络。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，这些调节已证明在毒力中起作用。我们将使用QRT-PCR确定MAI控制的程度来验证这些数据，并在诱导这些调节的生长条件下确认MAI的产生。正如我们的微阵列研究所暗示的那样，我们的长期目标是深入了解MAI是否参与潜伏期的重新激活。