The Type IV Secretion System Effector Protein CirA Stimulates the GTPase Activity of RhoA and Is Required for Virulence in a Mouse Model of Coxiella burnetii Infection

Coxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial forC. burnetiiintracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment ofCoxiella burnetiiinfection and virulence in mammalian cell culture and mouse models of infection.

Download Full-text

Identification of ElpA, a Coxiella burnetii Pathotype-Specific Dot/Icm Type IV Secretion System Substrate

Infection and Immunity ◽

10.1128/iai.02855-14 ◽

2015 ◽

Vol 83 (3) ◽

pp. 1190-1198 ◽

Cited By ~ 11

Author(s):

Joseph G. Graham ◽

Caylin G. Winchell ◽

Uma M. Sharma ◽

Daniel E. Voth

Keyword(s):

Coxiella Burnetii ◽

Q Fever ◽

Parasitophorous Vacuole ◽

Secretion System ◽

Terminal Region ◽

Effector Proteins ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Content Type ◽

Type Iv

Coxiella burnetiicauses human Q fever, a zoonotic disease that presents with acute flu-like symptoms and can result in chronic life-threatening endocarditis. In human alveolar macrophages,C. burnetiiuses a Dot/Icm type IV secretion system (T4SS) to generate a phagolysosome-like parasitophorous vacuole (PV) in which to replicate. The T4SS translocates effector proteins, or substrates, into the host cytosol, where they mediate critical cellular events, including interaction with autophagosomes, PV formation, and prevention of apoptosis. Over 100C. burnetiiDot/Icm substrates have been identified, but the function of most remains undefined. Here, we identified a novel Dot/Icm substrate-encoding open reading frame (CbuD1884) present in allC. burnetiiisolates except the Nine Mile reference isolate, where the gene is disrupted by a frameshift mutation, resulting in a pseudogene. The CbuD1884 protein contains two transmembrane helices (TMHs) and a coiled-coil domain predicted to mediate protein-protein interactions. The C-terminal region of the protein contains a predicted Dot/Icm translocation signal and was secreted by the T4SS, while the N-terminal portion of the protein was not secreted. When ectopically expressed in eukaryotic cells, the TMH-containing N-terminal region of the CbuD1884 protein trafficked to the endoplasmic reticulum (ER), with the C terminus dispersed nonspecifically in the host cytoplasm. This new Dot/Icm substrate is now termed ElpA (ER-localizingproteinA). Full-length ElpA triggered substantial disruption of ER structure and host cell secretory transport. These results suggest that ElpA is a pathotype-specific T4SS effector that influences ER function duringC. burnetiiinfection.

Download Full-text

The Coxiella burnetii Dot/Icm System Creates a Comfortable Home through Lysosomal Renovation

mBio ◽

10.1128/mbio.00226-11 ◽

2011 ◽

Vol 2 (5) ◽

Cited By ~ 20

Author(s):

Hayley J. Newton ◽

Craig R. Roy

Keyword(s):

Coxiella Burnetii ◽

Mutational Analysis ◽

Q Fever ◽

Parasitophorous Vacuole ◽

Culture Conditions ◽

Effector Proteins ◽

Collective Actions ◽

Content Type ◽

Genetic Techniques ◽

Obligate Intracellular Bacteria

ABSTRACTUnderstanding the molecular pathogenesis ofCoxiella burnetii, the causative agent of human Q fever, has historically been hindered by the technical difficulties of genetically manipulating obligate intracellular bacteria. The recent development of culture conditions suitable for axenic propagation ofC. burnetiihas paved the way for the application of a range of genetic techniques to address key questions within the field. Recent studies using mutational analysis have revealed that theC. burnetiiDot/Icm type 4 secretion system (T4SS) is an important virulence determinant that is essential for renovation of a lysosome into a matureCoxiella-containing vacuole (CCV) permissive of intracellular replication. Interestingly, a mutant ofC. burnetiideficient in Dot/Icm function was found to be capable of replicating within the parasitophorous vacuole created byLeishmania amazonensis, which indicates thatC. burnetiireplication is not dependent on the cohort of Dot/Icm effector proteinsper sebut rather that the collective actions of effectors are required to create the specialized niche supportive of replication. Thus, a role for the Dot/Icm T4SS during the intracellular life cycle ofC. burnetiihas been more clearly defined by these studies, which demonstrate that advances in genetic analysis should allow future studies to focus on the intricacies of Dot/Icm effector functions that facilitate development of the unique CCV.

Download Full-text

Coxiella burnetii Alters Cyclic AMP-Dependent Protein Kinase Signaling during Growth in Macrophages

Infection and Immunity ◽

10.1128/iai.00101-12 ◽

2012 ◽

Vol 80 (6) ◽

pp. 1980-1986 ◽

Cited By ~ 11

Author(s):

Laura J. MacDonald ◽

Richard C. Kurten ◽

Daniel E. Voth

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Coxiella Burnetii ◽

Alveolar Macrophages ◽

Q Fever ◽

Parasitophorous Vacuole ◽

Dependent Protein Kinase ◽

Content Type ◽

Bacterial Agent ◽

Dependent Protein

ABSTRACTCoxiella burnetiiis the bacterial agent of human Q fever, an acute, flu-like illness that can present as chronic endocarditis in immunocompromised individuals. Following aerosol-mediated transmission,C. burnetiireplicates in alveolar macrophages in a unique phagolysosome-like parasitophorous vacuole (PV) required for survival. The mechanisms ofC. burnetiiintracellular survival are poorly defined and a recent Q fever outbreak in the Netherlands emphasizes the need for better understanding this unique host-pathogen interaction. We recently demonstrated that inhibition of host cyclic AMP-dependent protein kinase (PKA) activity negatively impacts PV formation. In the current study, we confirmed PKA involvement in PV biogenesis and probed the role of PKA signaling duringC. burnetiiinfection of macrophages. Using PKA-specific inhibitors, we found the kinase was needed for biogenesis of prototypical PV andC. burnetiireplication. PKA and downstream targets were differentially phosphorylated throughout infection, suggesting prolonged regulation of the pathway. Importantly, the pathogen actively triggered PKA activation, which was also required for PV formation by virulentC. burnetiiisolates during infection of primary human alveolar macrophages. A subset of PKA-specific substrates were differentially phosphorylated duringC. burnetiiinfection, suggesting the pathogen uses PKA signaling to control distinct host cell responses. Collectively, the current results suggest a versatile role for PKA inC. burnetiiinfection and indicate virulent organisms usurp host kinase cascades for efficient intracellular growth.

Download Full-text

Coxiella burnetii Effector Proteins That Localize to the Parasitophorous Vacuole Membrane Promote Intracellular Replication

Infection and Immunity ◽

10.1128/iai.02763-14 ◽

2014 ◽

Vol 83 (2) ◽

pp. 661-670 ◽

Cited By ~ 46

Author(s):

Charles L. Larson ◽

Paul A. Beare ◽

Daniel E. Voth ◽

Dale Howe ◽

Diane C. Cockrell ◽

...

Keyword(s):

Coxiella Burnetii ◽

Parasitophorous Vacuole ◽

Coiled Coil ◽

Bacterial Pathogen ◽

Effector Proteins ◽

Intracellular Replication ◽

Intracellular Growth ◽

Content Type ◽

Cell Cytosol ◽

Pv Generation

The intracellular bacterial pathogenCoxiella burnetiidirects biogenesis of a parasitophorous vacuole (PV) that acquires host endolysosomal components. Formation of a PV that supportsC. burnetiireplication requires a Dot/Icm type 4B secretion system (T4BSS) that delivers bacterial effector proteins into the host cell cytosol. Thus, a subset of T4BSS effectors are presumed to direct PV biogenesis. Recently, the PV-localized effector protein CvpA was found to promoteC. burnetiiintracellular growth and PV expansion. We predict additionalC. burnetiieffectors localize to the PV membrane and regulate eukaryotic vesicle trafficking events that promote pathogen growth. To identify these vacuolar effector proteins, a list of predictedC. burnetiiT4BSS substrates was compiled using bioinformatic criteria, such as the presence of eukaryote-like coiled-coil domains. Adenylate cyclase translocation assays revealed 13 proteins were secreted in a Dot/Icm-dependent fashion byC. burnetiiduring infection of human THP-1 macrophages. Four of the Dot/Icm substrates, termedCoxiellavacuolarprotein B (CvpB), CvpC, CvpD, and CvpE, labeled the PV membrane and LAMP1-positive vesicles when ectopically expressed as fluorescently tagged fusion proteins.C. burnetiiΔcvpB, ΔcvpC, ΔcvpD, and ΔcvpEmutants exhibited significant defects in intracellular replication and PV formation. Genetic complementation of the ΔcvpDand ΔcvpEmutants rescued intracellular growth and PV generation, whereas the growth ofC. burnetiiΔcvpBand ΔcvpCwas rescued upon cohabitation with wild-type bacteria in a common PV. Collectively, these data indicateC. burnetiiencodes multiple effector proteins that target the PV membrane and benefit pathogen replication in human macrophages.

Download Full-text

Host Pathways Important for Coxiella burnetii Infection Revealed by Genome-Wide RNA Interference Screening

mBio ◽

10.1128/mbio.00606-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 77

Author(s):

Justin A. McDonough ◽

Hayley J. Newton ◽

Scott Klum ◽

Rachel Swiss ◽

Hervé Agaisse ◽

...

Keyword(s):

Coxiella Burnetii ◽

Secretion System ◽

Effector Proteins ◽

Host Factors ◽

Content Type ◽

Host Determinants ◽

Genome Wide ◽

Type Ivb Secretion ◽

Type Ivb Secretion System ◽

Bacterial Effector Proteins

ABSTRACTCoxiella burnetiiis an intracellular pathogen that replicates within a lysosome-like vacuole. A Dot/Icm type IVB secretion system is used byC. burnetiito translocate effector proteins into the host cytosol that likely modulate host factor function. To identify host determinants required forC. burnetiiintracellular growth, a genome-wide screen was performed using gene silencing by small interfering RNA (siRNA). Replication ofC. burnetiiwas measured by immunofluorescence microscopy in siRNA-transfected HeLa cells. Newly identified host factors included components of the retromer complex, which mediates cargo cycling between the endocytic pathway and the Golgi apparatus. Reducing the levels of the retromer cargo-adapter VPS26-VPS29-VPS35 complex or retromer-associated sorting nexins abrogatedC. burnetiireplication. Several genes, when silenced, resulted in enlarged vacuoles or an increased number of vacuoles withinC. burnetii-infected cells. Silencing of theSTX17gene encoding syntaxin-17 resulted in a striking defect in homotypic fusion of vacuoles containingC. burnetii, suggesting a role for syntaxin-17 in regulating this process. Lastly, silencing host genes needed forC. burnetiireplication correlated with defects in the translocation of Dot/Icm effectors, whereas, silencing of genes that affected vacuole morphology, but did not impact replication, did not affect Dot/Icm translocation. These data demonstrate thatC. burnetiivacuole maturation is important for creating a niche that permits Dot/Icm function. Thus, genome-wide screening has revealed host determinants involved in sequential events that occur duringC. burnetiiinfection as defined by bacterial uptake, vacuole transport and acidification, activation of the Dot/Icm system, homotypic fusion of vacuoles, and intracellular replication.IMPORTANCEQ fever in humans is caused by the bacteriumCoxiella burnetii. Infection withC. burnetiiis marked by its unique ability to replicate within a large vacuolar compartment inside cells that resembles the harsh, acidic environment of a lysosome. Central to its pathogenesis is the delivery of bacterial effector proteins into the host cell cytosol by a Dot/Icm type IVB secretion system. These proteins can interact with and manipulate host factors, thereby leading to creation and maintenance of the vacuole that the bacteria grow within. Using high-throughput genome-wide screening in human cells, we identified host factors important for several facets ofC. burnetiiinfection, including vacuole transport and membrane fusion events that promote vacuole expansion. In addition, we show that maturation of theC. burnetiivacuole is necessary for creating an environment permissive for the Dot/Icm delivery of bacterial effector proteins into the host cytosol.

Download Full-text

Dependency of Coxiella burnetii Type 4B Secretion on the Chaperone IcmS

Journal of Bacteriology ◽

10.1128/jb.00431-19 ◽

2019 ◽

Vol 201 (23) ◽

Cited By ~ 2

Author(s):

Charles L. Larson ◽

Paul A. Beare ◽

Robert A. Heinzen

Keyword(s):

Host Cell ◽

Coxiella Burnetii ◽

Legionella Pneumophila ◽

Q Fever ◽

Secretion System ◽

Host Cells ◽

Growth Defect ◽

Wild Type ◽

Signal Sequences ◽

Content Type

ABSTRACT Macrophage parasitism by Coxiella burnetii, the cause of human Q fever, requires the translocation of proteins with effector functions directly into the host cell cytosol via a Dot/Icm type 4B secretion system (T4BSS). Secretion by the analogous Legionella pneumophila T4BSS involves signal sequences within the C-terminal and internal domains of effector proteins. The cytoplasmic chaperone pair IcmSW promotes secretion and binds internal sites distinct from signal sequences. In the present study, we investigated requirements of C. burnetii IcmS for host cell parasitism and effector translocation. A C. burnetii icmS deletion mutant (ΔicmS) exhibited impaired replication in Vero epithelial cells, deficient formation of the Coxiella-containing vacuole, and aberrant T4BSS secretion. Three secretion phenotypes were identified from a screen of 50 Dot/Icm substrates: IcmS dependent (secreted by only wild-type bacteria), IcmS independent (secreted by both wild-type and ΔicmS bacteria), or IcmS inhibited (secreted by only ΔicmS bacteria). Secretion was assessed for N-terminal or C-terminal truncated forms of CBU0794 and CBU1525. IcmS-inhibited secretion of CBU1525 required a C-terminal secretion signal whereas IcmS-dependent secretion of CBU0794 was directed by C-terminal and internal signals. Interchange of the C-terminal 50 amino acids of CBU0794 and CBU1525 revealed that sites within the C terminus regulate IcmS dependency. Glutathione S-transferase-tagged IcmSW bound internal sequences of IcmS-dependent and -inhibited substrates. Thus, the growth defect of the C. burnetii ΔicmS strain is associated with a loss of T4BSS chaperone activity that both positively and negatively regulates effector translocation. IMPORTANCE The intracellular pathogen Coxiella burnetii employs a type 4B secretion system (T4BSS) that promotes growth by translocating effectors of eukaryotic pathways into host cells. T4BSS regulation modeled in Legionella pneumophila indicates IcmS facilitates effector translocation. Here, we characterized type 4B secretion by a Coxiella ΔicmS mutant that exhibits intracellular growth defects. T4BSS substrates demonstrated increased, equivalent, or decreased secretion by the ΔicmS mutant relative to wild-type Coxiella. Similar to the Legionella T4BSS, IcmS dependency in Coxiella was determined by C-terminal and/or internal secretion signals. However, IcmS inhibited secretion of some effectors by Coxiella that were previously shown to be translocated by Legionella. Thus, Coxiella has a unique IcmS regulatory mechanism that both positively and negatively regulates T4BSS export.

Download Full-text

Noncanonical Inhibition of mTORC1 byCoxiella burnetiiPromotes Replication within a Phagolysosome-Like Vacuole

mBio ◽

10.1128/mbio.02816-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 9

Author(s):

Charles L. Larson ◽

Kelsi M. Sandoz ◽

Diane C. Cockrell ◽

Robert A. Heinzen

Keyword(s):

Host Cell ◽

Coxiella Burnetii ◽

Secretion System ◽

Effector Proteins ◽

Nutrient Deprivation ◽

Autophagic Flux ◽

Content Type ◽

Mechanistic Target Of Rapamycin ◽

Infected Cells ◽

Bacterial Replication

ABSTRACTThe Q fever agentCoxiella burnetiiis a Gram-negative bacterium that invades macrophages and replicates inside a specialized lysosomal vacuole. The pathogen employs a type 4B secretion system (T4BSS) to deliver effector proteins into the host cell that modify theCoxiella-containing vacuole (CCV) into a replication-permissive niche. Mature CCVs are massive degradative organelles that acquire lysosomal proteins. Inhibition of mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) kinase by nutrient deprivation promotes autophagy and lysosome fusion, as well as activation of the transcription factors TFE3 and TFEB (TFE3/B), which upregulates expression of lysosomal genes. Here, we report thatC. burnetiiinhibits mTORC1 as evidenced by impaired localization of mTORC1 to endolysosomal membranes and decreased phosphorylation of elF4E-binding protein 1 (4E-BP1) and S6 kinase 1 in infected cells. Infected cells exhibit increased amounts of autophagy-related proteins protein 1A/1B-light chain 3 (LC3) and p62 as well as of activated TFE3. However,C. burnetiidid not accelerate autophagy or block autophagic flux triggered by cell starvation. Activation of autophagy or transcription by TFE3/B increased CCV expansion without enhancing bacterial replication. By contrast, knockdown of tuberous sclerosis complex 1 (TSC1) or TSC2, which hyperactivates mTORC1, impaired CCV expansion and bacterial replication. Together, these data demonstrate that specific inhibition of mTORC1 byC. burnetii, but not amplified cell catabolism via autophagy, is required for optimal pathogen replication. These data reveal a complex interplay between lysosomal function and host cell metabolism that regulatesC. burnetiiintracellular growth.IMPORTANCECoxiella burnetiiis an intracellular pathogenic bacterium that replicates within a lysosomal vacuole. Biogenesis of theCoxiella-containing vacuole (CCV) requires effector proteins delivered into the host cell cytosol by the type 4B secretion system (T4BSS). Modifications to lysosomal physiology required for pathogen replication within the CCV are poorly understood. Mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) is a master kinase that regulates lysosome structure and function. Nutrient deprivation inhibits mTORC1, which promotes cell catabolism in the form of accelerated autophagy and increased lysosome biosynthesis. Here, we report thatC. burnetiigrowth is enhanced by T4BSS-dependent inhibition of mTORC1 that does not activate autophagy. Canonical inhibition of mTORC1 by starvation or inhibitor treatment that induces autophagic flux does not benefitC. burnetiigrowth. Furthermore, hyperactivation of mTORC1 impairs bacterial replication. These findings indicate thatC. burnetiiinhibition of mTORC1 without accelerated autophagy promotes bacterial growth.

Download Full-text

Development of anEx VivoTissue Platform To Study the Human Lung Response to Coxiella burnetii

Infection and Immunity ◽

10.1128/iai.00012-16 ◽

2016 ◽

Vol 84 (5) ◽

pp. 1438-1445 ◽

Cited By ~ 12

Author(s):

Joseph G. Graham ◽

Caylin G. Winchell ◽

Richard C. Kurten ◽

Daniel E. Voth

Keyword(s):

Coxiella Burnetii ◽

Lung Tissue ◽

Human Lung ◽

Q Fever ◽

Ex Vivo ◽

Parasitophorous Vacuole ◽

Inflammasome Activation ◽

Human Lung Tissue ◽

Content Type

Coxiella burnetiiis an intracellular bacterial pathogen that causes human Q fever, an acute debilitating flu-like illness that can also present as chronic endocarditis. Disease typically occurs following inhalation of contaminated aerosols, resulting in an initial pulmonary infection. In human cells,C. burnetiigenerates a replication niche termed the parasitophorous vacuole (PV) by directing fusion with autophagosomes and lysosomes.C. burnetiirequires this lysosomal environment for replication and uses a Dot/Icm type IV secretion system to generate the large PV. However, we do not understand howC. burnetiievades the intracellular immune surveillance that triggers an inflammatory response. We recently characterized human alveolar macrophage (hAM) infectionin vitroand found that avirulentC. burnetiitriggers sustained interleukin-1β (IL-1β) production. Here, we evaluated infection ofex vivohuman lung tissue, defining a valuable approach for characterizingC. burnetiiinteractions with a human host. Within whole lung tissue,C. burnetiipreferentially replicated in hAMs. Additionally, IL-1β production correlated with formation of an apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC)-dependent inflammasome in response to infection. We also assessed potential activation of a human-specific noncanonical inflammasome and found that caspase-4 and caspase-5 are processed during infection. Interestingly, although inflammasome activation is closely linked to pyroptosis, lytic cell death did not occur followingC. burnetii-triggered inflammasome activation, indicating an atypical response after intracellular detection. Together, these studies provide a novel platform for studying the human innate immune response toC. burnetii.

Download Full-text

The Coxiella burnetii Ankyrin Repeat Domain-Containing Protein Family Is Heterogeneous, with C-Terminal Truncations That Influence Dot/Icm-Mediated Secretion

Journal of Bacteriology ◽

10.1128/jb.01656-08 ◽

2009 ◽

Vol 191 (13) ◽

pp. 4232-4242 ◽

Cited By ~ 104

Author(s):

Daniel E. Voth ◽

Dale Howe ◽

Paul A. Beare ◽

Joseph P. Vogel ◽

Nathan Unsworth ◽

...

Keyword(s):

Genome Sequencing ◽

Coxiella Burnetii ◽

Legionella Pneumophila ◽

Mammalian Cells ◽

Q Fever ◽

Parasitophorous Vacuole ◽

Protein Family ◽

Ankyrin Repeat ◽

Reference Isolate ◽

Type Iv

ABSTRACT Coxiella burnetii is an obligate intracellular bacterium that directs biogenesis of a parasitophorous vacuole (PV) for replication. Effectors of PV maturation are likely translocated into the host cytosol by a type IV secretion system (T4SS) with homology to the Dot/Icm apparatus of Legionella pneumophila. Since secreted bacterial virulence factors often functionally mimic the activities of host proteins, prokaryotic proteins with eukaryotic features are considered candidate T4SS substrates. Genes encoding proteins with eukaryotic-type ankyrin repeat domains (Anks) were identified upon genome sequencing of the C. burnetii Nine Mile reference isolate, which is associated with a case of human acute Q fever. Interestingly, recent genome sequencing of the G and K isolates, derived from human chronic endocarditis patients, and of the Dugway rodent isolate revealed remarkable heterogeneity in the Ank gene family, with the Dugway isolate harboring the largest number of full-length Ank genes. Using L. pneumophila as a surrogate host, we identified 10 Dugway Anks and 1 Ank specific to the G and K endocarditis isolates translocated into the host cytosol in a Dot/Icm-dependent fashion. A 10-amino-acid C-terminal region appeared to be necessary for translocation, with some Anks also requiring the chaperone IcmS for secretion. Ectopically expressed Anks localized to a variety of subcellular regions in mammalian cells, including microtubules, mitochondria, and the PV membrane. Collectively, these data suggest that C. burnetii isolates translocate distinct subsets of the Ank protein family into the host cytosol, where they modulate diverse functions, some of which may be unique to C. burnetii pathotypes.

Download Full-text

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

Infection and Immunity ◽

10.1128/iai.01046-16 ◽

2017 ◽

Vol 85 (5) ◽

Cited By ~ 9

Author(s):

Laura F. Fielden ◽

Jennifer H. Moffatt ◽

Yilin Kang ◽

Michael J. Baker ◽

Chen Ai Khoo ◽

...

Keyword(s):

Host Cell ◽

Outer Membrane ◽

Coxiella Burnetii ◽

Q Fever ◽

Effector Proteins ◽

Mitochondrial Outer Membrane ◽

Intracellular Replication ◽

Content Type ◽

Multimeric Complex ◽

Type Ivb Secretion

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 C oxiella 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 3×FLAG or MceA tagged with 2×hemagglutinin 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.

Download Full-text