Proteomic Identification of Coxiella burnetii Effector Proteins Targeted to the Host Cell Mitochondria During Infection.
Proteomic Identification of Coxiella burnetii Effector Proteins Targeted to the Host Cell Mitochondria During Infection.
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感染过程中靶向宿主细胞线粒体的伯氏杆菌效应蛋白的蛋白质组学鉴定。
DOI：
10.1074/mcp.ra120.002370
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Stojanovski D
中科院分区：
文献类型：
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作者：
Fielden LF;Scott NE;Palmer CS;Khoo CA;Newton HJ;Stojanovski D
宿主细胞的调节对于微生物病原体的存活和复制是不可或缺的。几种细胞内细菌病原体在感染期间通过复杂的蛋白质易位系统将称为“效应蛋白”的细菌蛋白质递送到宿主细胞中，所述蛋白质易位系统操纵细胞过程和功能。单个效应器的功能贡献的特征很差，特别是在具有大量效应蛋白谱系的细胞内细菌病原体中。技术警告限制了在天然感染期间研究这些蛋白质的能力，许多效应蛋白仅被证明在标记版本的过表达期间易位。在这里，我们开发了一种新的策略来检查感染背景下的效应蛋白。我们耦合一个广泛的，无偏见的蛋白质组学为基础的屏幕与细胞器纯化研究宿主-病原体之间发生的相互作用的宿主细胞cavion和革兰氏阴性，Q热病原体贝氏柯克斯体。我们鉴定了四种新的靶向性贝氏柯克斯体效应蛋白，将线粒体柯克斯体效应蛋白（Mce）B更名为E。异位表达的蛋白质的亚细胞定位的检查证实了它们的线粒体定位，证明了我们的方法的鲁棒性。随后的生化分析和亲和富集蛋白质组学的这些效应蛋白，MceC，揭示了蛋白质定位于内膜，并可以与线粒体质量控制机制的组件。我们的研究采用高灵敏度蛋白质组学研究细胞内宿主-病原体相互作用，提供了一个强大的策略，以检查在天然感染的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，以提供整个感染过程中效应动力学的丰富图谱。线粒体纯化和蛋白质组学研究宿主-病原体相互作用。定量蛋白质组学揭示柯克斯体效应蛋白在线粒体。对天然感染期间效应蛋白靶向的见解。进一步表征的MceC揭示了本地化和相互作用。一个广泛的，无偏见的蛋白质组学为基础的屏幕与细胞器纯化，以研究宿主-病原体之间发生的相互作用的宿主细胞cancelon和Q热病原体贝氏柯克斯体。这揭示了在感染期间在线粒体处的柯克斯体效应蛋白的子集。我们的研究采用高灵敏度蛋白质组学，提供了一个强大的策略来检查在原生感染的效应蛋白的亚细胞定位。
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. Mitochondrial purification and proteomics to study host–pathogen interactions. Quantitative proteomics reveals Coxiella effector proteins at mitochondria. Insights into effector protein targeting during native infection. Further characterization of MceC reveals localization and interaction. A broad, unbiased proteomics-based screen with organelle purification to study the host–pathogen interactions occurring between the host cell mitochondrion and the Q fever pathogen Coxiella burnetii. This reveals a subset of Coxiella effector proteins at mitochondria during infection. Our study adapts high-sensitivity proteomics, providing a robust strategy to examine the subcellular localization of effector proteins during native infection.