Nuclear trafficking of the anti-apoptotic<i>Coxiella burnetii</i>effector protein AnkG requires binding to p32 and Importin-α1

Schäfer, Walter; Eckart, Rita A.; Schmid, Benedikt; Cagköylü, Hasret; Hof, Kerstin; Muller, Yves A.; Amin, Bushra; Lührmann, Anja

doi:10.1111/cmi.12634

Cited by 46 publications

(67 citation statements)

References 58 publications

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“…AnkX exploits another eukaryotic process, farnesylation, to mediate its association with the Legionella-containing vacuole (81). Finally, Coxiella burnetii AnkG binds both host p32 and importin-␣1 in order to traffic into the nucleus and inhibit apoptosis (82,83).…”

Section: Discussionmentioning

confidence: 99%

Orientia tsutsugamushi Modulates Endoplasmic Reticulum-Associated Degradation To Benefit Its Growth

et al. 2018

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Orientia tsutsugamushi, an obligate intracellular bacterium that is auxotrophic for the aromatic amino acids and histidine, causes scrub typhus, a potentially deadly infection that threatens 1 billion people. O. tsutsugamushi growth is minimal during the first 24 to 48 h of infection but its growth becomes logarithmic thereafter. How the pathogen modulates cellular functions to support its growth is poorly understood. The unfolded protein response (UPR) is a cytoprotective pathway that relieves endoplasmic reticulum (ER) stress by promoting ER-associated degradation (ERAD) of misfolded proteins. Here, we show that O. tsutsugamushi invokes the UPR in the first 48 h and benefits from ER stress in an amino acid-dependent manner. O. tsutsugamushi also impedes ERAD during this time period. By 72 h, ER stress is alleviated and ERAD proceeds unhindered. Sustained inhibition of ERAD using RNA interference results in an O. tsutsugamushi growth defect at 72 h that can be rescued by amino acid supplementation. Thus, O. tsutsugamushi temporally stalls ERAD until ERAD-derived amino acids are needed to support its growth. The O. tsutsugamushi effector Ank4 is linked to this phenomenon. Ank4 interacts with Bat3, a eukaryotic chaperone that is essential for ERAD, and is transiently expressed by O. tsutsugamushi during the infection period when it inhibits ERAD. Ectopically expressed Ank4 blocks ERAD to phenocopy O. tsutsugamushi infection. Our data reveal a novel mechanism by which an obligate intracellular bacterial pathogen modulates ERAD to satisfy its nutritional virulence requirements.

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Section: Discussionmentioning

confidence: 99%

Orientia tsutsugamushi Modulates Endoplasmic Reticulum-Associated Degradation To Benefit Its Growth

et al. 2018

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“…Additionally, the antiapoptotic effectors CaeB (CBU1532) and AnkG (CBU0781) associate with mitochondria (8,19,20). AnkG is believed to sense apoptotic stress at mitochondria, leading to interaction with p32 and subsequent translocation to the nucleus, where it exerts a strong, physiologically relevant, antiapoptotic phenotype (19,21). Thus, mitochondria represent an important effector target for the control of host viability.…”

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confidence: 99%

A Farnesylated Coxiella burnetii Effector Forms a Multimeric Complex at the Mitochondrial Outer Membrane during Infection

et al. 2017

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Coxiella burnetii, the causative agent of Q fever, establishes a unique lysosome-derived intracellular niche termed the Coxiella-containing vacuole (CCV). The Dot/Icm-type IVB secretion system is essential for the biogenesis of the CCV and the intracellular replication of Coxiella. Effector proteins, translocated into the host cell through this apparatus, act to modulate host trafficking and signaling processes to facilitate CCV development. Here we investigated the role of CBU0077, a conserved Coxiella effector that had previously been observed to localize to lysosomal membranes. CBU0077 was dispensable for the intracellular replication of Coxiella in HeLa and THP-1 cells and did not appear to participate in CCV biogenesis. Intriguingly, native and epitope-tagged CBU0077 produced by Coxiella displayed specific punctate localization at host cell mitochondria. As such, we designated CBU0077 MceA (mitochondrial Coxiella effector protein A). Analysis of ectopically expressed MceA truncations revealed that the capacity to traffic to mitochondria is encoded within the first 84 amino acids of this protein. MceA is farnesylated by the host cell; however, this does not impact mitochondrial localization. Examination of mitochondria isolated from infected cells revealed that MceA is specifically integrated into the mitochondrial outer membrane and forms a complex of approximately 120 kDa. Engineering Coxiella to express either MceA tagged with 3ϫFLAG or MceA tagged with 2ϫhemagglutinin allowed us to perform immunoprecipitation experiments that showed that MceA forms a homo-oligomeric species at the mitochondrial outer membrane during infection. This research reveals that mitochondria are a bona fide target of Coxiella effectors and MceA is a complex-forming effector at the mitochondrial outer membrane during Coxiella infection.KEYWORDS Coxiella burnetii, bacterial effector, mitochondria, prenylation, hostpathogen interactions C oxiella burnetii is the causative agent of human Q fever, a zoonotic infection with a worldwide distribution. The extremely low infectious dose, the ease of aerosol dissemination, and the environmental stability of this pathogen all contribute to Coxiella being classified as a category B select agent by the U.S. Centers for Disease Control and Prevention (1, 2).During human infection, inhaled Coxiella bacteria predominantly infect alveolar macrophages, where they develop a unique intracellular replicative niche termed the Coxiella-containing vacuole (CCV). Internalized bacteria are trafficked through the endocytic pathway until they reach the acidic and hydrolytic confines of the lysosome.

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“…Investigations into the function of C. burnetii effector AnkG, demonstrated the protein is capable of trafficking between the mitochondria and the nucleus via a 'piggy-backing' mechanism to interrupt nuclear apoptotic signalling (38,84). Whilst our proteomics analysis did not capture the enrichment of AnkG at the mitochondrion, it is possible that this interorganelle trafficking had already resulted in translocation of the protein to the nucleus.…”

Section: Discussionmentioning

confidence: 83%

Proteomic identification ofCoxiella burnetiieffector proteins targeted to the host cell mitochondria during infection

Fielden

Scott

Palmer

et al. 2020

Preprint

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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 Coxiella burnetii. 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.

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Nuclear trafficking of the anti-apoptoticCoxiella burnetiieffector protein AnkG requires binding to p32 and Importin-α1

Cited by 46 publications

References 58 publications

Orientia tsutsugamushi Modulates Endoplasmic Reticulum-Associated Degradation To Benefit Its Growth

Orientia tsutsugamushi Modulates Endoplasmic Reticulum-Associated Degradation To Benefit Its Growth

A Farnesylated Coxiella burnetii Effector Forms a Multimeric Complex at the Mitochondrial Outer Membrane during Infection

Proteomic identification ofCoxiella burnetiieffector proteins targeted to the host cell mitochondria during infection

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