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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

基本信息

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
项目类别：
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

项目摘要

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.

项目总结我们对支配相互作用的复杂过程的理解存在着根本性的差距结核分枝杆菌和巨噬细胞。这项应用的总体目标是调查新的生物物理相分离现象影响生物过程，特别是在结核分枝杆菌感染的情况下。一个需要识别和响应病原体的分子的详细知识才能揭示细胞如何抗感染；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(结核分枝杆菌)是一种非常成功且致命的人类。感染世界四分之一人口的病原体。而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如何的宿主感应分子被调节为协同工作。此外，只有一小部分人结核分枝杆菌使用的分泌型效应器具有独特的功能。最近的研究表明液-液分离的生物学和细胞重要性，即蛋白质浓缩的过程变成离散的液滴，改变它们在细胞中的位置和功能。与宿主有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，已被发现分阶段分开。在体外，但体内相分离如何影响宿主对感染的反应尚不清楚。初步研究表明发现这些和其他先天性免疫蛋白在结核分枝杆菌感染细胞中形成环状点状，类似于相分离的液滴。这一提议的中心假设是，一旦感染，病原体- 感知和翻译后修饰诱导宿主蛋白的相分离，Mtb调节这些缩合事件与其自身的相分离PE/PPE蛋白相分离。在这里，结合了小说将使用光遗传学工具、活细胞荧光成像以及宿主和细菌遗传学来探索宿主蛋白相分离的生物学后果(目标1)和结核分枝杆菌蛋白(目标2)。此外, 将使用定向和无偏见的遗传学方法来探索翻译后修饰是如何进行的，以及尤其是泛素化，有助于结核分枝杆菌感染期间的相分离(目标3)。这种方法它的创新之处在于它使用了新的工具来专门和精确地调制相分离，以便将这一生物物理过程具有有意义的细胞表型。这项拟议的研究意义重大，因为它将大大加深我们对巨噬细胞如何破坏结核分枝杆菌的了解，并推动对抗结核杆菌的努力通过增强宿主反应来感染。