Abstract
AbstractModulation 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.
Highlights
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
The rapidly expanding field of high-sensitivity mass spectrometry coupled to subcellular organelle isolation presents a solution to the study of endogenous host–pathogen interactions occurring during infection, for bacterial pathogens harboring large effector repertoires, where functional redundancy exists within the effector cohort
label-free quantitation (LFQ) mass spectrometry mitochondria of C. burnetiimCherry-infected THP-1 cells were analyzed by liquid chromatography mass spectrometry (LC-MS) on an Orbitrap Lumos mass spectrometer coupled to a Dionex Ultimate 3000 Ultra-Performance Liquid Chromatography using a two-column chromatography set up composed of a PepMap100 C18 20 mm × 75 μm trap and a PepMap C18 500 mm × 75 μm analytical column (Thermo Fisher Scientific)
Summary
A broad, unbiased proteomicsbased 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 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. In consideration of the large cohort of bacterial proteins delivered to the host cell by C. burnetii and the essentiality of multiple mitochondrial functions to cellular homeostasis, we hypothesized that additional effector proteins would localize to the organelle during infection. Development and use of this unbiased proteomics-based technique has allowed us to uncover host–pathogen interactions between C. burnetii and the mitochondria in the context of native infection and provides an exciting platform for future exploration of effector interactions with host cell organelles
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