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: 10117183
• 负责人: Amy K Barczak
• 金额: $ 21万
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10117183
• 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机制的核心。一旦结核病 吞噬体是巨噬细胞和细菌之间的界面点，但我们 我们对结核杆菌感染过程中吞噬体如何演变以及这些变化如何可能 使宿主/病原体平衡向控制方向或远离控制方向移动。全面的蛋白质组分析将提供一个 在感染过程中吞噬体组成的窗口和 在一些实施方案中，Mtb是能够控制感染的巨噬细胞中的吞噬体和允许Mtb生长的吞噬体。到目前为止， 对含结核分枝杆菌的吞噬体膜上的单个蛋白质的靶向研究已经确定了一些 吞噬体状态的相关物。然而，系统的，全面的蛋白质组学研究， 吞噬体是有限的。这一限制部分是由于现有的技术办法造成的。最 含MTB的吞噬体的研究依赖于这种亚细胞级分的纯化。达到纯度 这一部分充其量是具有挑战性的，并且在现有技术的限制下更加困难。 BSL 3设置。含有纯化的Mtb产物的珠粒已另外用于分离吞噬体 部分，但如何以及个别细菌产品代表完整的结核分枝杆菌表面是不清楚的。填补…的空白 根据我们对巨噬细胞Mtb感染中吞噬体组成和演变的了解，我们建议 利用蛋白质工程和比较蛋白质组学的最新进展， 成功地应用于其他生物学问题：使用混杂的，分泌的生物素的邻近标记 连接酶TurboID。我们将首先优化该系统以用于Mtb;然后将其应用于分析组合物 在促进或不能促进感染控制的条件下，含结核分枝杆菌的吞噬体的。在目标1中， 我们将系统地测试TurboID的分泌和表面定位策略，以工程化Mtb菌株 对于邻近连接实验是最佳的。在目标2中，我们将优化蛋白质组的分析方法 我们的工程菌株进行分析优化后，我们将分析和比较吞噬体 在用促进不同程度的Mtb控制的刺激物处理的巨噬细胞中，在目标3中，我们 将使用该系统来分析和比较来自感染野生型的巨噬细胞的吞噬体组成 结核分枝杆菌或两种缺乏与吞噬体膜相互作用的毒力因子的突变体。一旦实现 这项工作，我们预计已经开发和验证了一个系统，用于分析结核分枝杆菌的组成， 具有高时间和空间分辨率的吞噬体。我们希望能够确定关键的差异 控制或无法控制感染的吞噬体之间的联系。我们预计，我们的成果将推动未来 单个吞噬体因子对控制感染的作用的机制研究。我们还 预计这些结果将最终为未来增强宿主控制的宿主导向抗结核治疗提供信息 感染