A proteomic approach to understanding phagosome composition in TB infection

了解结核病感染中吞噬体组成的蛋白质组学方法

• 批准号: 9979082
• 负责人: Amy K Barczak
• 金额: $ 25.2万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-02 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979082
• 关键词: Alveolar Macrophages; Antitubercular Agents; Bacteria; Biological; Biotin; Cause of Death; Cells; Cessation of life; Codon Nucleotides; Data; Data Set; Development; ELF3 gene; Engineering; Epidemic; Equilibrium; Event; Evolution; Foundations; Future; Genetic; Genus Mycobacterium; Growth; Human; Immune response; Individual; Infection; Infection Control; Knowledge; Label; Ligase; Ligation; Lipids; Literature; Lysosomes; Membrane; Microscopy; Mycobacterium tuberculosis; Pathogenesis; Phagosomes; Play; Protein Engineering; Proteins; Proteome; Proteomics; Resolution; Role; Route; Shapes; Side; Stimulus; Subcellular Fractions; Surface; System; Technology; Testing; Therapeutic; Time; Tuberculosis; Virulence; Virulence Factors; Virulent; Work; analytical tool; base; comparative; experimental study; global health; macrophage; mutant; mycobacterial; novel; pathogen; prevent; proteomic signature; tool; tuberculosis treatment; vacuolar H+-ATPase

The interaction between a host macrophage and infecting bacterium is central to TB pathogenesis. Once Mtb is internalized, the phagosome is the point of interface between macrophage and bacterium, yet we understand little of how the phagosome evolves in the course of Mtb infection and how those changes might shift the host/pathogen balance toward or away from control. Comprehensive proteomic profiling would offer a window into phagosome composition over the course of infection and the differences in composition between phagosomes in macrophages that are able to control infection and those that permit Mtb growth. To date, targeted studies of individual proteins on the Mtb-containing phagosomal membrane have identified a few correlates of phagosome state. However, systematic, comprehensive proteomic studies of the Mtb-containing phagosome have been limited. In part, this limitation has arisen from available technical approaches. Most studies of Mtb-containing phagosomes have relied on purification of this subcellular fraction. Achieving purity of this fraction is challenging at best, and is more difficult within the constraints of technologies available in a BSL3 setting. Beads containing purified Mtb products have additionally been used to isolate the phagosomal fraction, but how well individual bacterial products represent the intact Mtb surface is not clear. To fill gaps in our understanding of phagosome composition and evolution in Mtb infection of macrophages, we propose to build upon recent advances in protein engineering and comparative proteomics using an approach successfully applied to other biological questions: proximity labeling using the promiscuous, secreted biotin ligase TurboID. We will first optimize this system for use in Mtb; we will then apply it to profile the composition of the Mtb-containing phagosome under conditions that promote or fail to promote control of infection. In Aim 1, we will systematically test strategies for secretion and surface localization of TurboID to engineer an Mtb strain optimal for proximity ligation experiments. In Aim 2, we will optimize analytical approaches to proteome profiling with our engineered strain. Following optimization, we will profile and compare phagosomal composition in macrophages treated with stimuli that promote varying degrees of control of Mtb. In Aim 3, we will use this system to profile and compare phagosomal composition from macrophages infected with wild-type Mtb or two mutants that lack virulence factors that interact with the phagosomal membrane. Upon achieving this work, we anticipate having developed and validated a system for profiling the composition of the Mtb- containing phagosome with high temporal and spatial resolution. We expect to have identified key differences between phagosomes that control or fail to control infection. We anticipate that our results will fuel future mechanistic studies of the contribution of individual phagosomal factors to control of infection. Further, we anticipate these results will ultimately inform future host-directed anti-TB therapeutics that enhance host control of infection.

宿主巨噬细胞和感染细菌之间的相互作用是结核病发病的核心。一次Mtb 是内化的，吞噬小体是巨噬细胞和细菌之间的交界点，但我们 对吞噬小体在结核分枝杆菌感染过程中如何演变以及这些变化可能 改变宿主/病原体的平衡，使之接近或远离控制。全面的蛋白质组学分析将提供 了解感染过程中的吞噬小体组成以及两者之间的组成差异 巨噬细胞中能够控制感染和允许结核分枝杆菌生长的吞噬小体。到目前为止， 对含有结核分枝杆菌的吞噬体膜上个别蛋白的靶向性研究已经确定了几个 吞噬小体状态的相关因素。然而，系统的、全面的含结核分枝杆菌的蛋白质组学研究 吞噬小体受到了限制。在一定程度上，这种限制是由现有的技术方法造成的。多数 对含结核分枝杆菌吞噬小体的研究依赖于对该亚细胞组分的纯化。实现纯净的 这一部分充其量是具有挑战性的，而且在可用技术的限制下更难 BSL3设置。含有纯化的结核分枝杆菌产物的珠子还被用来分离吞噬小体。 部分，但单个细菌产品代表完整的结核分枝杆菌表面的情况如何尚不清楚。填补空白 我们对巨噬细胞感染结核分枝杆菌的吞噬小体组成和进化的理解，我们建议 以蛋白质工程和比较蛋白质组学的最新进展为基础 成功地应用于其他生物学问题：使用混杂的、分泌的生物素进行邻近标记 连接酶涡轮。我们将首先优化此系统以在Mtb中使用；然后我们将应用它来分析成分 在促进或未能促进感染控制的条件下，含有结核分枝杆菌的吞噬小体。在目标1中， 我们将系统地测试TurboID的分泌和表面定位策略，以改造Mtb菌株 最适合近距离结扎实验。在目标2中，我们将优化蛋白质组的分析方法 用我们的基因工程菌株进行分析。在优化之后，我们将对吞噬菌体进行剖析和比较 巨噬细胞经刺激处理后，促进对结核分枝杆菌不同程度控制的成分。在目标3中，我们 将使用这个系统来分析和比较感染野生型病毒的巨噬细胞的吞噬体组成 MTB或两个缺乏与吞噬体膜相互作用的毒力因子的突变体。在实现 这项工作，我们预计已经开发和验证了一个系统，用于分析MTB的成分- 含有高时间和空间分辨率的吞噬小体。我们预计已经确定了主要差异 在控制或不能控制感染的吞噬小体之间。我们预计，我们的业绩将为未来提供动力 个体吞噬因子对控制感染作用的机制研究。此外，我们 预期这些结果将最终为加强宿主控制的未来宿主导向的抗结核病疗法提供信息 感染的可能性。