Modulation of the host cell is integral to the survival and replication of microbial pathogens. Several intracellular bacterial pathogens deliver a cohort of bacterial proteins, termed effector proteins into the host cell during infection by sophisticated protein translocation systems which manipulate cellular processes and functions. Despite the importance of these proteins during infection the functional contribution of individual effectors is poorly characterised, 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 present development of 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 Coxiellaburnetii. We identify 4 novel mitochondrially-targeted C.burnetii effector proteins, renamed Mitochondrial Coxiella effector protein (Mce) B to E. Examination of the subcellular localisation of ectopically expressed proteins in epithelial cells confirmed the mitochondrial localisation, demonstrating the robustness of our approach. Subsequent biochemical analysis and affinity enrichment proteomics of one of these effector proteins, MceC, revealed the protein is imported into mitochondria and can interact with components of the mitochondrial quality control machinery. Our study adapts high-sensitivity proteomics to the study of intracellular host-pathogen interactions occurring during infection, providing a robust strategy to examine the sub-cellular localisation 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.

中文翻译：

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感染过程中靶向宿主细胞线粒体的伯氏柯氏杆菌效应蛋白的蛋白质组学鉴定

宿主细胞的调节对于微生物病原体的存活和复制是必不可少的。几种细胞内细菌病原体在感染过程中会通过操纵细胞过程和功能的复杂蛋白质转运系统，将一批称为效应蛋白的细菌蛋白质传递到宿主细胞中。尽管这些蛋白质在感染期间很重要，但单个效应子的功能贡献却很难被表征，特别是在具有大量效应子蛋白库的细胞内细菌病原体中。技术上的警告限制了在天然感染过程中研究这些蛋白质的能力，许多效应蛋白仅在过表达的标签版本中才被证明是易位的。在这里，我们提出了一种在感染情况下检查效应蛋白的新策略。我们将广泛，无偏见的基于蛋白质组学的筛查与细胞器纯化相结合，以研究宿主细胞线粒体与革兰氏阴性Q热病原体Coxiellaburnetii之间发生的宿主-病原体相互作用。我们确定4新型线粒体靶向的C.burnetii效应蛋白，重命名为线粒体Coxiella效应蛋白（Mce）B到E。上皮细胞中异位表达蛋白的亚细胞定位检查证实了线粒体定位，证明了我们方法的稳健性。这些效应蛋白之一MceC的后续生化分析和亲和力富集蛋白质组学 揭示了该蛋白质被导入线粒体，并且可以与线粒体质量控制机制的组成部分相互作用。我们的研究使高灵敏度蛋白质组学适应于感染过程中发生的细胞内宿主与病原体相互作用的研究，提供了一种强有力的策略来检查天然感染过程中效应蛋白的亚细胞定位。该方法可应用于多种病原体和宿主细胞区室，以在整个感染过程中提供丰富的效应子动态图。