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: 10050787
• 负责人: Samantha Lynn Bell
• 金额: $ 46.61万
• 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/10050787
• 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; Phase; Phenotype; Phosphorylation; Physical condensation; Plasma; Polyubiquitination; Population; Post-Translational Protein Processing; Process; Proteins; Proteomics; Reaction; Recording of previous events; Regulation; Reporting; Research; Role; S Phase; 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.

项目摘要 我们对控制复杂过程的基本差距 MTB和巨噬细胞。该应用的总体目的是研究新型生物物理如何 相分离的现象会影响生物学过程，特别是在MTB感染的背景下。一个 需要对识别和反应病原体的分子的详细知识，以揭示细胞的方式 战斗感染；因此，迫切需要了解相位分离如何影响或控制 先天免疫反应。结核分枝杆菌（MTB）是一个令人难以置信的致命人类 感染世界人口四分之一的病原体。而MTB细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的方式有限 宿主传感分子的调节以合作起作用。此外，只有一小部分 结核分枝杆菌使用的分泌效应子具有良好的功能。最近的研究已经照亮了 液态液相分离的生物学和细胞的重要性，蛋白质凝结的过程 进入离散的液滴以改变其在单元格中的定位和功能。宿主参与的几种蛋白质 对结核分枝杆菌感染的反应，例如CGA，TBK1，P62和LC3，已被发现在 体外，但是体内分离如何影响宿主对感染的反应。初步研究 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感染后，病原体 - 传感和翻译后修饰诱导宿主蛋白的相分离，MTB调节 这些凝结事件与其自身分离的PE/PPE蛋白。在这里，小说的结合 光遗传学工具，活细胞荧光成像以及宿主和细菌遗传学将被用来探测 宿主蛋白相分离（AIM 1）和MTB蛋白（AIM 2）的生物学后果。此外， 定向和无偏的遗传学方法将用于探测翻译后修改和 特别是尤其是泛素化，有助于MTB感染期间的相位分离（AIM 3）。这种方法 具有创新性的是，它使用新颖的工具专门，精确地调节相位分离以链接 具有有意义的细胞表型的生物物理过程。拟议的研究很重要，因为它 将极大地扩展我们对巨噬细胞如何破坏MTB的理解和与MTB作战的努力 通过增强宿主反应感染。