权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular dissection of vesiculogenesis in Mycobacterium tuberculosis

结核分枝杆菌囊泡发生的分子解剖

基本信息

批准号：
9090007
负责人：
Rafael Prados-Rosales
金额：
$ 20.88万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-15 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9090007
关键词：
Affect Anabolism Antigens Attenuated Bacteria Binding Biochemical Biological Assay Bone Marrow Cell Wall Cell membrane Cell physiology Cell surface Cells Classification Co-Immunoprecipitations Complement Complex Cytoplasmic Protein Defect Dissection Dot Immunoblotting Electron Microscopy Event Face Family Generations Genes Genus Mycobacterium Goals Gram-Positive Bacteria Growth Human Hydrophobic Surfaces Immune Sera Immune system Impairment In Vitro Infection Inflammatory Response Knock-out Knowledge Label Lead Libraries Link Lipids Lipoproteins Lung Mass Spectrum Analysis Measures Membrane Metabolic Methods Microarray Analysis Microbe Microscopic Molecular Morbidity - disease rate Morphology Motivation Mus Mycobacterium Infections Mycobacterium tuberculosis Pathogenesis Pathway interactions Peptidoglycan Phagocytosis Phagosomes Phenotype Physiology Primary Infection Process Production Proteins Public Health Radioactivity Reporting Role Specificity Spleen System TLR2 gene Techniques Testing Tuberculosis Uncertainty Vesicle Virulence Virulence Factors aerosolized base cell envelope controlled release cytokine exosome extracellular fighting fungus genetic variant improved in vivo insight macrophage mortality mutant mycobacterial novel therapeutics novel vaccines pathogen physical separation public health relevance response screening stress tolerance vesicular release

项目摘要

DESCRIPTION (provided by applicant): Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide. Mycobacterium tuberculosis (M.tb), the causative agent of TB, is capable of establishing primary infections that often become latent and can persist despite an intact immune system. It is well known that M.tb possesses multiple highly evolved mechanisms to manipulate host cellular machinery and evade the host immune system. We have contributed to expanding the knowledge of M.tb physiology through the discovery of an alternative antigen release mechanism based on membrane vesicles (MV). Compositional analysis of MV revealed enrichment in polar lipids and lipoproteins. MV release events were visualized during in vitro and in vivo infections. MV stimulation of macrophages and mouse elicited inflammatory response dependent on Toll-like receptor 2 (TLR-2). The combination of MV stimulation with an aerosolized infection in mice produced a `Koch phenomenon' promoting the growth of mycobacteria in lungs and spleen, suggesting a link between MV and M.tb pathogenesis. Nevertheless a stronger association between vesiculation and virulence in M.tb remains to be established. We recently contributed to characterizing the phenotype of the Rv0431 M.tb mutant, which manifested a hyper- inflammatory response in macrophages due to overproduction of MV. This finding suggests that MV production is a regulated process and given the intrinsic complexity of the process, additional genes are likely to be involved. This application proposes a molecular dissection of vesiculogenesis in M.tb. Initially, we will identify genes responsible for vesicle production by screening a library of M.tb transposon insertion mutants using a sensitive, high-throughput dot blot system to detect MV in low-volume cultures. The screening assay uses antiserum capable of recognizing vesicles whose specificity is imparted by the physical separation techniques used to prepare the immunogen. Mutants with increased MV production, low MV production and non-producing mutants will be characterized by microscopic methods to gain insight into ultrastructural changes, and biochemical and mass spectrometry based-techniques will be employed to identify potentially altered MV composition. Further, we will investigate the role of Rv0431 in vesiculogenesis. The approach proposed here will allow identification of additional genetic variants affected in vesicle production, which willbe essential for studying their role in the pathogenesis of mycobacterial infection.

描述（由申请人提供）：结核病 (TB) 仍然是全世界发病和死亡的主要原因。结核分枝杆菌 (M.tb) 是结核病的病原体，能够引起原发感染，这种感染通常呈潜伏状态，尽管免疫系统完好无损，但仍能持续存在。众所周知，结核分枝杆菌拥有多种高度进化的机制来操纵宿主细胞机器并逃避宿主免疫系统。通过发现基于膜囊泡 (MV) 的替代抗原释放机制，我们为扩大结核分枝杆菌生理学知识做出了贡献。 MV 的成分分析揭示了极性脂质和脂蛋白的富集。在体外和体内感染期间，MV 释放事件被可视化。巨噬细胞和小鼠的 MV 刺激引起依赖于 Toll 样受体 2 (TLR-2) 的炎症反应。 MV 刺激与小鼠雾化感染相结合产生了“科赫现象”，促进了肺和脾中分枝杆菌的生长，表明 MV 和 M.tb 发病机制之间存在联系。然而，结核分枝杆菌中的水疱和毒力之间的更强关联仍有待确定。我们最近致力于表征 Rv0431 M.tb 突变体的表型，该突变体由于 MV 过度产生而在巨噬细胞中表现出过度炎症反应。这一发现表明，MV 的产生是一个受调控的过程，并且考虑到该过程的内在复杂性，可能涉及其他基因。本申请提出了结核分枝杆菌囊泡发生的分子解剖。最初，我们将确定通过使用敏感的高通量斑点印迹系统筛选 M.tb 转座子插入突变体文库来检测小体积培养物中的 MV，从而确定负责囊泡产生的基因。筛选测定使用能够识别囊泡的抗血清，其特异性是由用于制备免疫原的物理分离技术赋予的。 MV 产生增加、MV 产生低和不产生 MV 的突变体将通过显微镜方法进行表征，以深入了解超微结构的变化，并且将采用基于生化和质谱的技术来识别潜在改变的 MV 组成。此外，我们将研究 Rv0431 在囊泡发生中的作用。这里提出的方法将允许识别影响囊泡产生的其他遗传变异，这对于研究它们在分枝杆菌感染发病机制中的作用至关重要。