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: 10663318
• 负责人: Samantha Lynn Bell
• 金额: $ 46.61万
• 依托单位: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
• 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/10663318
• 关键词: Affect; Antibacterial Response; Antibiotic Resistance; Automobile Driving; Autophagocytosis; Bacillus; Biochemical; Biochemistry; Biological; Biological Process; Biophysical Process; Biophysics; Cell Nucleus; Cell membrane; Cells; Cellular biology; Communicable Diseases; Complex; Cytoplasm; Data; Defense Mechanisms; 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; Macrophage; Membrane; Microbiology; Mission; Modification; Molecular; Molecular Biology; Mycobacterium tuberculosis; Natural Immunity; Organelles; Pathogenesis; Patient-Focused Outcomes; Patients; Persons; Phase; Phenotype; Phosphorylation; Physical condensation; 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; combat; design; experimental study; fighting; fluorescence imaging; genetic approach; human pathogen; in vivo; innate immune pathways; innovation; interdisciplinary approach; live cell imaging; novel; novel therapeutics; optogenetics; pathogen; 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.

项目摘要 在我们对控制人类与自然之间相互作用的复杂过程的理解上， 结核分枝杆菌和巨噬细胞。本申请的总体目标是研究新的生物物理学方法如何在细胞内产生生物效应。 相分离现象影响生物过程，特别是在Mtb感染的情况下。一 需要对识别和应答病原体的分子有详细的了解， 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）是一种令人难以置信的成功和致命的人类 感染了全球四分之一人口的病原体结核分枝杆菌与巨噬细胞的相互作用 激活了许多先天免疫途径，我们对这些复杂的网络是如何激活的了解有限。 的宿主感应分子被调节以协同工作。此外，只有一小部分的许多 分泌的效应子。结核病具有明确的功能。最近的研究表明， 液-液相分离在生物和细胞中的重要性， 分离成离散的液滴来改变它们在细胞中的定位和功能。参与宿主的几种蛋白质 回应M。结核感染，如cGAS，TBK 1，p62和LC 3，已被发现在不同的阶段分离。 体外，但体内相分离如何影响宿主对感染的反应是未知的。初步研究已经 发现这些和其他先天免疫蛋白在M.结核病感染细胞， 类似于相分离的液滴。该建议的核心假设是，在感染后，病原体- 传感和翻译后修饰诱导宿主蛋白质的相分离， 这些缩合事件与其自身的相分离PE/PPE蛋白质。在这里，结合了小说 光遗传学工具，活细胞荧光成像，宿主和细菌遗传学将被用来探测 宿主蛋白（Aim 1）和Mtb蛋白（Aim 2）相分离的生物学后果。此外，本发明还提供了一种方法， 定向和无偏见的遗传学方法将用于探测如何翻译后修饰， 特别是泛素化，有助于Mtb感染期间的相分离（Aim 3）。这种方法 其创新之处在于，它使用新颖的工具来专门且精确地调制相位分离，以便连接 这种生物物理过程与有意义的细胞表型。这项研究意义重大，因为它 这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。 通过增强宿主反应感染。