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

Abstract

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.

Lysosomal degradation products induce Coxiella burnetii virulence

“…1A). Effector translocation was examined using a reporter C. burnetii Nine Mile phase II (NMII) strain constitutively expressing the known effector protein MceA (10,23) with an N-terminal BlaM fusion and the fluorescent BlaM substrate CCF2-AM. Following excitation at 410 nm, cleavage of CCF2-AM by BlaM leads to a shift in fluorescence emission from 520 nm (green) to 450 nm (blue).…”

Mechanisms of action of Coxiella burnetii effectors inferred from host-pathogen protein interactions

“…We also investigated the potential interactions of CBU0077, which accumulates at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic [ 89 ]. Recent research tag CBU0077 as a complex-forming effector at the mitochondrial outer membrane during Coxiella infection [ 93 ]. Six host proteins interacted with CBU0077, three of which directly related to the reported phenotype (i.e., the junction adhesion molecule AMICA1 [ 94 ], the transmembrane-trafficking protein TMED10, and ACADM as part of the mitochondrial fatty acid beta-oxidation pathway.…”

Coxiella burnetii utilizes both glutamate and glucose during infection with glucose uptake mediated by multiple transporters

“…At both day 3 and day 6 post-infection, bacteria were harvested for labeling experiments as described below. Infections for growth curves in HeLa CCL2 and THP-1 cells were performed as previously described [20].…”

“…Infected HeLa CCL2 and THP-1 cells were stained at day 3 post-infection as previously described [20], using anti-LAMP-1 (Developmental Studies Hybridoma Bank) and anti- Coxiella antibodies (Roy Laboratory, Yale University).…”