Department of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, Victoria, Australia.
Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.
Mol Cell Proteomics. 2021;20:100005. doi: 10.1074/mcp.RA120.002370. Epub 2020 Dec 3.
Modulation of the host cell is integral to the survival and replication of microbial pathogens. Several intracellular bacterial pathogens deliver bacterial proteins, termed "effector proteins" into the host cell during infection by sophisticated protein translocation systems, which manipulate cellular processes and functions. The functional contribution of individual effectors is poorly characterized, particularly in intracellular bacterial pathogens with large effector protein repertoires. Technical caveats have limited the capacity to study these proteins during a native infection, with many effector proteins having only been demonstrated to be translocated during over-expression of tagged versions. Here, we developed a novel strategy to examine effector proteins in the context of infection. We coupled a broad, unbiased proteomics-based screen with organelle purification to study the host-pathogen interactions occurring between the host cell mitochondrion and the Gram-negative, Q fever pathogen Coxiella burnetii. We identify four novel mitochondrially-targeted C. burnetii effector proteins, renamed Mitochondrial Coxiella effector protein (Mce) B to E. Examination of the subcellular localization of ectopically expressed proteins confirmed their mitochondrial localization, demonstrating the robustness of our approach. Subsequent biochemical analysis and affinity enrichment proteomics of one of these effector proteins, MceC, revealed the protein localizes to the inner membrane and can interact with components of the mitochondrial quality control machinery. Our study adapts high-sensitivity proteomics to study intracellular host-pathogen interactions, providing a robust strategy to examine the subcellular localization of effector proteins during native infection. This approach could be applied to a range of pathogens and host cell compartments to provide a rich map of effector dynamics throughout infection.
宿主细胞的调控对于微生物病原体的存活和复制至关重要。几种胞内细菌病原体在感染过程中通过复杂的蛋白转运系统将细菌蛋白（称为“效应蛋白”）输送到宿主细胞中，从而操纵细胞过程和功能。个别效应蛋白的功能贡献尚未得到充分描述，特别是在具有大量效应蛋白库的胞内细菌病原体中。技术上的限制限制了在天然感染期间研究这些蛋白质的能力，许多效应蛋白仅在标记版本过表达时才被证明可转运。在这里，我们开发了一种新的策略来研究感染背景下的效应蛋白。我们将广泛的、基于蛋白质组学的无偏筛选与细胞器纯化相结合，研究宿主细胞线粒体与革兰氏阴性、Q 热病原体柯克斯体之间发生的宿主-病原体相互作用。我们鉴定了四种新的靶向线粒体的 C. 伯内特效应蛋白，分别命名为 Mitochondrial Coxiella effector protein (Mce) B 至 E。异位表达蛋白的亚细胞定位研究证实了它们的线粒体定位，证明了我们方法的稳健性。随后对其中一种效应蛋白 MceC 的生化分析和亲和富集蛋白质组学分析表明，该蛋白定位于内膜，并可以与线粒体质量控制机制的成分相互作用。我们的研究将高灵敏度蛋白质组学应用于研究细胞内宿主-病原体相互作用，提供了一种强大的策略来研究天然感染过程中效应蛋白的亚细胞定位。这种方法可以应用于一系列病原体和宿主细胞区室，为整个感染过程中效应蛋白动力学提供丰富的图谱。
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