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对于结核病发病机理至关重要。一次MTB 被内在化，吞噬体是巨噬细胞和细菌之间界面的点，但是我们 几乎了解吞噬体在MTB感染过程中如何演变以及这些变化可能如何发展 将宿主/病原体平衡转移到或远离控制。全面的蛋白质组学分析将提供 在感染过程中进入吞噬体成分的窗口以及在组成之间的差异 能够控制感染和允许MTB生长的巨噬细胞中的吞噬体。迄今为止， 对含MTB的吞噬体膜上单个蛋白的有针对性研究已经确定了一些 吞噬状态的相关性。但是，含MTB的系统，全面的蛋白质组学研究 吞噬体受到限制。在某种程度上，这种限制来自可用的技术方法。最多 含MTB的吞噬体的研究依赖于该亚细胞分数的纯化。实现纯度 这一部分充其量是具有挑战性的，并且在可用的技术的限制下更加困难 BSL3设置。还使用了含有纯化的MTB产品的珠子来隔离吞噬体 分数，但是单个细菌产物对完整的MTB表面的表面尚不清楚。填补空白 我们对巨噬细胞MTB感染中吞噬体组成和进化的理解，我们建议 建立在蛋白质工程和比较蛋白质组学的最新进展的基础上 成功地应用于其他生物学问题：使用混杂的，分泌的生物素的接近标记 联络涡轮增压。我们将首先优化该系统以用于MTB；然后，我们将其应用于构图 在促进或无法促进感染控制的条件下，含MTB的吞噬体的吞噬体。在AIM 1中， 我们将系统地测试涡轮增压的分泌和表面定位策略，以设计MTB菌株 最接近连接实验的最佳。在AIM 2中，我们将优化蛋白质组的分析方法 用我们的工程压力进行分析。优化后，我们将介绍和比较吞噬体 用刺激治疗的巨噬细胞中的组成，这些刺激促进了MTB的不同程度的控制。在AIM 3中，我们 将使用该系统来介绍和比较感染野生型的巨噬细胞的吞噬体成分 MTB或两个缺乏与吞噬膜相互作用的毒力因子的突变体。实现 这项工作，我们预计已经开发并验证了一个用于分析MTB-组成的系统 含有高时空和空间分辨率的吞噬体。我们希望已经确定关键差异 在控制或无法控制感染的吞噬体之间。我们预计我们的结果将推动未来 各个吞噬因素对控制感染的贡献的机理研究。此外，我们 预期这些结果最终将为未来的宿主指导的抗TB疗法提供信息，从而增强宿主控制 感染。