Investigating the role of liquid-liquid phase separation in the interaction between Mycobacterium tuberculosis and macrophages

研究液-液相分离在结核分枝杆菌与巨噬细胞相互作用中的作用

• 批准号: 10675829
• 负责人: Samantha Lynn Bell
• 金额: $ 22万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-10 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10675829
• 关键词: Affect; Antibacterial Response; Antibiotic Resistance; Automobile Driving; Autophagocytosis; Bacillus; Biochemical; Biochemistry; Biological; Biological Process; Biophysical Process; Biophysics; Cell Nucleus; Cells; Cellular biology; Communicable Diseases; Complex; Cytoplasm; Data; Development; Disease; Disease Outcome; Event; Galactose Binding Lectin; Health; Human; Immune; Immune response; Immune system; In Vitro; Individual; Infection; Infection Control; Innate Immune Response; Knowledge; Link; Liquid substance; Membrane; Microbiology; Mission; Modification; Molecular; Molecular Biology; Mycobacterium tuberculosis; Natural Immunity; Organelles; Pathogenesis; Patient-Focused Outcomes; Patients; Persons; Phase; Phenotype; Phosphorylation; Physical condensation; Plasma; Polyubiquitination; Population; Post-Translational Protein Processing; Process; Proteins; Proteomics; Reaction; Recording of previous events; Regulation; Reporting; Research; Role; Signal Transduction; Stimulator of Interferon Genes; Structure; TBK1 gene; Testing; Therapeutic; Training; Tuberculosis; Ubiquitination; United States National Institutes of Health; Work; bacterial genetics; base; combat; design; experimental study; fighting; fluorescence imaging; genetic approach; human pathogen; in vivo; innate immune pathways; innovation; interdisciplinary approach; live cell imaging; macrophage; novel; novel therapeutics; optogenetics; pathogen; phase 2 testing; prevent; response; tool

PROJECT SUMMARY There is a fundamental gap in our understanding of the complex processes that govern the interactions between Mtb and macrophages. The overall objective of this application is to investigate how the novel biophysical phenomenon of phase separation impacts biological processes, specifically in the context of Mtb infection. A detailed knowledge of the molecules that recognize and respond to pathogens is required to reveal how cells fight infection; therefore, there is a critical need to understand how phase separation may influence or control innate immune responses. Mycobacterium tuberculosis (Mtb) is an incredibly successful and deadly human pathogen that infects one-quarter of the world's population. While interaction of Mtb bacilli and macrophages activates numerous innate immune pathways, we have a limited understanding of how these complex networks of host sensing molecules are regulated to work cooperatively. Furthermore, only a small subset of the many secreted effectors used by M. tuberculosis have well-characterized functions. Recent studies have illuminated the biological and cellular importance of liquid-liquid phase separation, a process by which proteins condense into discrete droplets to alter their localization and function in a cell. Several proteins involved in the host response to M. tuberculosis infection, like cGAS, TBK1, p62, and LC3, have been found to phase separate in vitro, but how in vivo phase separation impacts host responses to infection is unknown. Preliminary studies have found that these and other innate immune proteins form circular puncta in M. tuberculosis-infected cells that resemble phase separated droplets. The central hypothesis of this proposal is that upon infection, pathogen- sensing and post-translational modifications induce phase separation of host proteins and that Mtb modulates these condensation events with its own phase-separating PE/PPE proteins. Here, a combination of novel optogenetics tools, live cell fluorescent imaging, and host and bacterial genetics will be employed to probe the biological consequences of phase separation of host proteins (Aim 1) and Mtb proteins (Aim 2). In addition, directed and unbiased genetics approaches will be used to probe how post-translational modifications, and especially ubiquitination in particular, contributes to phase separation during Mtb infection (Aim 3). This approach is innovative in that it uses novel tools to specifically and precisely modulate phase separation in order to link this biophysical process with meaningful cellular phenotypes. The proposed research is significant because it will greatly expand our understanding of how macrophages destroy Mtb and advance efforts to combat Mtb infection via enhancing host responses.

项目概要 我们对控制之间相互作用的复杂过程的理解存在根本差距 Mtb 和巨噬细胞。该应用程序的总体目标是研究新型生物物理学如何 相分离现象会影响生物过程，特别是在结核分枝杆菌感染的情况下。一个 需要详细了解识别病原体并对其做出反应的分子，以揭示细胞如何 对抗感染；因此，迫切需要了解相分离如何影响或控制 先天免疫反应。结核分枝杆菌 (Mtb) 是一种极其成功且致命的人类细菌 感染世界四分之一人口的病原体。结核杆菌与巨噬细胞的相互作用 激活许多先天免疫途径，我们对这些复杂网络如何激活的了解有限 宿主传感分子被调节以协同工作。此外，只有一小部分 结核分枝杆菌使用的分泌效应器具有明确的功能。最近的研究表明 液-液相分离（蛋白质凝结的过程）的生物学和细胞重要性 形成离散的液滴以改变它们在细胞中的定位和功能。与宿主相关的几种蛋白质 对结核分枝杆菌感染的反应，如 cGAS、TBK1、p62 和 LC3，被发现在 体外相分离，但体内相分离如何影响宿主对感染的反应尚不清楚。初步研究有 发现这些和其他先天免疫蛋白在结核分枝杆菌感染的细胞中形成圆形点， 类似于相分离的液滴。该提议的中心假设是，一旦感染，病原体- 传感和翻译后修饰诱导宿主蛋白的相分离，并且 Mtb 调节 这些缩合事件与其自身的相分离 PE/PPE 蛋白有关。这里结合了新颖 将采用光遗传学工具、活细胞荧光成像以及宿主和细菌遗传学来探测 宿主蛋白（目标 1）和 Mtb 蛋白（目标 2）相分离的生物学后果。此外， 将使用定向和公正的遗传学方法来探讨翻译后修饰如何进行，以及 尤其是泛素化，有助于 Mtb 感染期间的相分离（目标 3）。这种做法 其创新之处在于它使用新颖的工具来专门且精确地调节相分离，以便链接 这种生物物理过程具有有意义的细胞表型。拟议的研究意义重大，因为它 将极大地扩展我们对巨噬细胞如何破坏 Mtb 的理解并推进对抗 Mtb 的努力 通过增强宿主反应来感染。