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

2017 ◽  
Vol 85 (5) ◽  
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.

scholarly journals Dot/Icm Type IVB Secretion System Requirements for Coxiella burnetii Growth in Human Macrophages

mBio ◽  
2011 ◽  
Vol 2 (4) ◽  
Author(s):  
Paul A. Beare ◽  
Stacey D. Gilk ◽  
Charles L. Larson ◽  
Joshua Hill ◽  
Christopher M. Stead ◽  
...  
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Secretion System ◽  
Mononuclear Phagocytes ◽  
Productive Infection ◽  
Content Type ◽  
Human Macrophages ◽  
Type Ivb Secretion ◽  
Type Ivb Secretion System

ABSTRACTCentral to Q fever pathogenesis is replication of the causative agent,Coxiella burnetii, within a phagolysosome-like parasitophorous vacuole (PV) in mononuclear phagocytes.C. burnetiimodulates PV biogenesis and other host cell functions, such as apoptotic signaling, presumably via the activity of proteins delivered to the host cytosol by a Dot/Icm type IVB secretion system (T4BSS). In this study, we utilized aC. burnetiistrain carrying IcmD inactivated by theHimar1transposon to investigate the requirements for Dot/Icm function inC. burnetiiparasitism of human THP-1 macrophage-like cells. TheicmD::Tn mutant failed to secrete characterized T4BSS substrates, a defect that correlated with deficient replication, PV development, and apoptosis protection. Restoration of type IVB secretion and intracellular growth of theicmD::Tn mutant required complementation withicmD,-J, and-B, indicating a polar effect of the transposon insertion on downstreamdot/icmgenes. Induction oficmDJBexpression at 1 day postinfection resulted inC. burnetiireplication and PV generation. Collectively, these data prove that T4BSS function is required for productive infection of human macrophages byC. burnetii. However, illustrating the metabolic flexibility ofC.burnetti, theicmD::Tn mutant could replicate intracellularly when sequestered in a PV generated by wild-type bacteria, where Dot/Icm function is provided intrans, and within a phenotypically similar PV generated by the protozoan parasiteLeishmania amazonensis, where host cells are devoid of Dot/Icm T4BSS effector proteins.IMPORTANCECoxiella burnetii, the cause of human Q fever, is the only bacterial pathogen known to replicate in a vacuole resembling a phagolysosome. The organism manipulates host macrophages to promote the biogenesis of a vacuolar compartment permissive for growth. By analogy to the well-established cellular microbiology ofLegionella pneumophila, the Dot/Icm type IVB secretion system ofC. burnetiiis implicated as a critical virulence factor in host cell modification that delivers proteins with effector functions directly into the host cell cytosol. Using new genetic tools, we verify that Dot/Icm function is essential for productive infection of human macrophages byC. burnetii. Interestingly, despite the production of homologous secretion systems,L. pneumophilaandC. burnetiihave strikingly different temporal requirements for Dot/Icm function during their respective infectious cycles.

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

2015 ◽  
Vol 83 (3) ◽  
pp. 1190-1198 ◽  
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.

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

2014 ◽  
Vol 83 (2) ◽  
pp. 661-670 ◽  
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.

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

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
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.

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

2019 ◽  
Vol 201 (23) ◽  
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.

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

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
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.

scholarly journals Coxiella burnetiiBlocks Intracellular Interleukin-17 Signaling in Macrophages

2018 ◽  
Vol 86 (10) ◽  
Author(s):  
Tatiana M. Clemente ◽  
Minal Mulye ◽  
Anna V. Justis ◽  
Srinivas Nallandhighal ◽  
Tuan M. Tran ◽  
...  
Keyword(s):  
Host Cell ◽  
Q Fever ◽  
Protective Role ◽  
Effector Proteins ◽  
Antimicrobial Protein ◽  
Interleukin 17 ◽  
Dependent Manner ◽  
Cell Infection ◽  
Content Type ◽  
Infected Cells

ABSTRACTCoxiella burnetiiis an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires theCoxiellatype IVB secretion system (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets ofCoxiellaT4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with aCoxiellaT4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild-type (WT) bacteria, suggesting thatCoxiellaT4BSS effector proteins downregulate the expression of these genes. In addition, the interleukin-17 (IL-17) signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 duringCoxiellainfection is unknown. We found that IL-17 kills intracellularCoxiellain a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed the increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT- or mock-infected cells, including the proinflammatory cytokine genesIl1a,Il1b, andTnfa, the chemokine genesCxcl2andCcl5, and the antimicrobial protein geneLcn2. We further confirmed that theCoxiellaT4BSS downregulates macrophage CXCL2/macrophage inflammatory protein 2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest thatCoxielladownregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response.

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

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
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.

scholarly journals Antiapoptotic Activity of Coxiella burnetii Effector Protein AnkG Is Controlled by p32-Dependent Trafficking

2014 ◽  
Vol 82 (7) ◽  
pp. 2763-2771 ◽  
Author(s):  
Rita A. Eckart ◽  
Stephanie Bisle ◽  
Jan Schulze-Luehrmann ◽  
Irene Wittmann ◽  
Jonathan Jantsch ◽  
...  
Keyword(s):  
Host Cell ◽  
Nuclear Localization ◽  
Coxiella Burnetii ◽  
Intracellular Localization ◽  
Mitochondrial Protein ◽  
Effector Proteins ◽  
Content Type ◽  
Type Iv ◽  
Antiapoptotic Activity ◽  
Induced Apoptosis

ABSTRACTIntracellular bacterial pathogens frequently inhibit host cell apoptosis to ensure survival of their host, thereby allowing bacterial propagation. The obligate intracellular pathogenCoxiella burnetiidisplays antiapoptotic activity which depends on a functional type IV secretion system (T4SS). Accordingly, antiapoptotic T4SS effector proteins, like AnkG, have been identified. AnkG inhibits pathogen-induced apoptosis, possibly by binding to the host cell mitochondrial protein p32 (gC1qR). However, the molecular mechanism of AnkG activity remains unknown. Here, we demonstrate that ectopically expressed AnkG associates with mitochondria and traffics into the nucleus after apoptosis induction, although AnkG lacks a predicted nuclear localization signal. We identified the p32 interaction region in AnkG and constructed an AnkG mutant (AnkGR22/23S) unable to bind to p32. By using this mutant, we found that intracellular localization and trafficking of AnkG into the nucleus are dependent on binding to p32. Furthermore, we demonstrated that nuclear localization of AnkG but not binding to p32 is required for apoptosis inhibition. Thus, the antiapoptotic activity of AnkG is controlled by p32-mediated intracellular trafficking, which, in turn, seems to be regulated by host cell processes that sense stress.

scholarly journals EirA Is a Novel Protein Essential for Intracellular Replication of Coxiella burnetii

2020 ◽  
Vol 88 (6) ◽  
Author(s):  
Miku Kuba ◽  
Nitika Neha ◽  
Patrice Newton ◽  
Yi Wei Lee ◽  
Vicki Bennett-Wood ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Protein Function ◽  
Galleria Mellonella ◽  
Q Fever ◽  
Axenic Culture ◽  
Febrile Illness ◽  
Intracellular Replication ◽  
Content Type ◽  
Novel Protein

ABSTRACT The zoonotic bacterial pathogen Coxiella burnetii is the causative agent of Q fever, a febrile illness which can cause a serious chronic infection. C. burnetii is a unique intracellular bacterium which replicates within host lysosome-derived vacuoles. The ability of C. burnetii to replicate within this normally hostile compartment is dependent on the activity of the Dot/Icm type 4B secretion system. In a previous study, a transposon mutagenesis screen suggested that the disruption of the gene encoding the novel protein CBU2072 rendered C. burnetii incapable of intracellular replication. This protein, subsequently named EirA (essential for intracellular replication A), is indispensable for intracellular replication and virulence, as demonstrated by infection of human cell lines and in vivo infection of Galleria mellonella. The putative N-terminal signal peptide is essential for protein function but is not required for localization of EirA to the bacterial inner membrane compartment and axenic culture supernatant. In the absence of EirA, C. burnetii remains viable but nonreplicative within the host phagolysosome, as coinfection with C. burnetii expressing native EirA rescues the replicative defect in the mutant strain. In addition, while the bacterial ultrastructure appears to be intact, there is an altered metabolic profile shift in the absence of EirA, suggesting that EirA may impact overall metabolism. Most strikingly, in the absence of EirA, Dot/Icm effector translocation was inhibited even when EirA-deficient C. burnetii replicated in the wild type (WT)-supported Coxiella containing vacuoles. EirA may therefore have a novel role in the control of Dot/Icm activity and represent an important new therapeutic target.

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