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 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 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 Identification of a Family of Vibrio Type III Secretion System Effectors That Contain a Conserved Serine/Threonine Kinase Domain

mSphere ◽  
2021 ◽  
Author(s):  
N. Plaza ◽  
I. M. Urrutia ◽  
K. Garcia ◽  
M. K. Waldor ◽  
C. J. Blondel
Keyword(s):  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Kinase Domain ◽  
Secretion System ◽  
Effector Proteins ◽  
Serine Threonine Kinase ◽  
Content Type ◽  
Threonine Kinase ◽  
Type Iii ◽  
Infected Cells

Vibrio parahaemolyticus is the leading bacterial cause of seafood-borne gastroenteritis worldwide. The pathogen relies on a type III secretion system to deliver a variety of effector proteins into the cytosol of infected cells to subvert cellular function.

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 IntracellularStaphylococcus aureusModulates Host Central Carbon Metabolism To Activate Autophagy

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
pp. e00374-18 ◽  
Author(s):  
Natalia Bravo-Santano ◽  
James K. Ellis ◽  
Luis M. Mateos ◽  
Yolanda Calle ◽  
Hector C. Keun ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Host Cell ◽  
Carbon Metabolism ◽  
Central Carbon Metabolism ◽  
Metabolic Changes ◽  
Autophagic Flux ◽  
Content Type ◽  
Central Carbon ◽  
Infected Cells ◽  
Intracellular Proliferation

ABSTRACTStaphylococcus aureusis a facultative intracellular pathogen that invades and replicates within many types of phagocytic and nonphagocytic cells. During intracellular infection,S. aureusis capable of subverting xenophagy and escaping to the cytosol of the host cell. Furthermore, drug-induced autophagy facilitates the intracellular replication ofS. aureus, but the reasons behind this are unclear. Here, we have studied the host central carbon metabolism duringS. aureusintracellular infection. We found extensive metabolic rerouting and detected several distinct metabolic changes that suggested starvation-induced autophagic flux in infected cells. These changes included increased uptake but lower intracellular levels of glucose and low abundance of several essential amino acids, as well as markedly upregulated glutaminolysis. Furthermore, we show that AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) phosphorylation levels are significantly increased in infected cells. Interestingly, while autophagy was activated in response toS. aureusinvasion, most of the autophagosomes detected in infected cells did not contain bacteria, suggesting thatS. aureusinduces the autophagic flux during cell invasion for energy generation and nutrient scavenging. Accordingly, AMPK inhibition haltedS. aureusintracellular proliferation.IMPORTANCEStaphylococcus aureusescapes from immune recognition by invading a wide range of human cells. Once the pathogen becomes intracellular, the most important last resort antibiotics are not effective. Therefore, novel anti-infective therapies against intracellularS. aureusare urgently needed. Here, we have studied the physiological changes induced in the host cells byS. aureusduring its intracellular proliferation. This is important, because the pathogen exploits the host cell’s metabolism for its own proliferation. We find thatS. aureusseverely depletes glucose and amino acid pools, which leads to increased breakdown of glutamine by the host cell in an attempt to meet its own metabolic needs. All of these metabolic changes activate autophagy in the host cell for nutrient scavenging and energy generation. The metabolic activation of autophagy could be used by the pathogen to sustain its own intracellular survival, making it an attractive target for novel anti-infectives.

scholarly journals LtpD Is a Novel Legionella pneumophila Effector That Binds Phosphatidylinositol 3-Phosphate and Inositol Monophosphatase IMPA1

2013 ◽  
Vol 81 (11) ◽  
pp. 4261-4270 ◽  
Author(s):  
Clare R. Harding ◽  
Corinna Mattheis ◽  
Aurélie Mousnier ◽  
Clare V. Oates ◽  
Elizabeth L. Hartland ◽  
...  
Keyword(s):  
Host Cell ◽  
Legionella Pneumophila ◽  
Galleria Mellonella ◽  
Effector Proteins ◽  
Lung Epithelial Cells ◽  
Systematic Analysis ◽  
Content Type ◽  
Bacterial Replication ◽  
Phosphatidylinositol 3

ABSTRACTThe Dot/Icm type IV secretion system (T4SS) ofLegionella pneumophilais crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of theLegionella-containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD471-626, which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD471-626bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P]in vitroand colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae ofGalleria mellonella, and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide-bindingL. pneumophilaeffector that has a role in intracellular bacterial replication.

scholarly journals The anti-apoptotic Coxiella burnetii effector protein AnkG is a strain specific virulence factor

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Walter Schäfer ◽  
Teresa Schmidt ◽  
Arne Cordsmeier ◽  
Vítor Borges ◽  
Paul A. Beare ◽  
...  
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Cell Apoptosis ◽  
Effector Proteins ◽  
Host Cells ◽  
Infection Model ◽  
Effector Protein ◽  
Host Cell Apoptosis ◽  
Bacterial Replication ◽  
Apoptotic Activity

Abstract The ability to inhibit host cell apoptosis is important for the intracellular replication of the obligate intracellular pathogen Coxiella burnetii, as it allows the completion of the lengthy bacterial replication cycle. Effector proteins injected into the host cell by the C. burnetii type IVB secretion system (T4BSS) are required for the inhibition of host cell apoptosis. AnkG is one of these anti-apoptotic effector proteins. The inhibitory effect of AnkG requires its nuclear localization, which depends on p32-dependent intracellular trafficking and importin-α1-mediated nuclear entry of AnkG. Here, we compared the sequences of ankG from 37 C. burnetii isolates and classified them in three groups based on the predicted protein size. The comparison of the three different groups allowed us to identify the first 28 amino acids as essential and sufficient for the anti-apoptotic activity of AnkG. Importantly, only the full-length protein from the first group is a bona fide effector protein injected into host cells during infection and has anti-apoptotic activity. Finally, using the Galleria mellonella infection model, we observed that AnkG from the first group has the ability to attenuate pathology during in vivo infection, as it allows survival of the larvae despite bacterial replication.

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 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 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.

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