IAI Accepts, published online ahead of print on 14 April 2014 Infect. Immun. doi:10.1128/IAI.01204-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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The anti-apoptotic activity of the Coxiella burnetii effector protein AnkG is
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controlled by p32-dependent trafficking
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Rita A. Eckart1, Stephanie Bisle1, Jan Schulze-Luehrmann1, Irene Wittmann1, Jonathan
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Jantsch1, Benedikt Schmid2, Christian Berens3 and Anja Lührmann1*
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1
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Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg,
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Wasserturmstraße 3/5, D-91054 Erlangen, Germany
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Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene,
Lehrstuhl für Biotechnik, Department Biologie, Friedrich-Alexander-Universität
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Erlangen-Nürnberg, Henkestrasse 91, D-91052 Erlangen, Germany
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Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany
Lehrstuhl für Mikrobiologie, Department Biologie, Friedrich-Alexander-Universität
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*Corresponding author:
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Anja Lührmann1
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Phone: (+49) 9131 85 22577; Fax: (+49) 9131 85 1001
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Email: [email protected]
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Running title: AnkG trafficking and apoptosis inhibition
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1
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Abstract
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Intracellular bacterial pathogens frequently inhibit host cell apoptosis to ensure
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survival of their host, thereby allowing bacterial propagation. The obligate intracellular
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pathogen Coxiella burnetii displays anti-apoptotic activity which depends on a functional
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type IV secretion system (T4SS). Accordingly, anti-apoptotic T4SS effector proteins, like
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AnkG, have been identified. AnkG inhibits pathogen-induced apoptosis, possibly by
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binding to the host cell mitochondrial protein p32 (gC1qR). However, the molecular
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mechanism of AnkG activity remains unknown.
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Here, we demonstrate that ectopically expressed AnkG associates with
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mitochondria and traffics into the nucleus after apoptosis induction, although AnkG lacks
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a predicted nuclear localization signal. We identified the p32-interaction region in AnkG
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and constructed an AnkG-mutant (AnkGR22/23S) unable to bind to p32. By using this
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mutant we found that intracellular localization and trafficking of AnkG into the nucleus is
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dependent on binding to p32. Furthermore, we demonstrated that nuclear localization of
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AnkG but not binding to p32 is required for apoptosis inhibition. Thus, the anti-apoptotic
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activity of AnkG is controlled by p32-mediated intracellular trafficking, which in turn
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seems to be regulated by host cell processes that sense stress.
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2
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Introduction
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Coxiella burnetii is the obligate intracellular bacterial agent of human Q-fever, a
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worldwide zoonotic disease (1). Infection in humans occurs by inhalation of infectious
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material transmitted from domestic livestock, and as few as ten bacteria can result in
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disease (2). After bacterial uptake into phagocytic cells, C. burnetii establishes a
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phagolysosomal-like vacuole (3, 4, 5). Importantly, establishing this replicative niche
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requires bacterial protein synthesis (6, 7), suggesting direct involvement of bacterial
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proteins. In agreement with this assumption, the type IV secretion system (T4SS) was
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shown to be essential for intracellular replication (8, 9). The presence of the replicative
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C. burnetii-containing vacuole (CCV) within the cell most likely causes tremendous
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stress for the infected cell, as the CCV almost completely fills the host cell lumen (10).
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Eukaryotic cells often respond to intracellular pathogen invasion and stress induction by
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initiating the intrinsic apoptotic pathway as part of the innate immune defense (11).
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Apoptosis is a programmed cell death pathway crucial for immune system
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maintenance and removal of damaged or infected cells (12). Two main pathways lead to
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apoptosis. The extrinsic cell death pathway is launched in response to stimulation of
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death receptor proteins at the cell surface by extracellular stimuli, while the intrinsic cell
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death pathway is initiated in response to intracellular stimuli (13).
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Apoptosis allows pathogen clearance without inflammation and additionally leads
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to activation of the adaptive immune defense (14, 15). As a countermeasure intracellular
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pathogens have developed multiple mechanisms to inhibit host cell apoptosis (16). C.
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burnetii also interferes with host cell apoptosis (17, 18). How this occurs mechanistically
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is incompletely understood, but effector proteins translocated into the host cell by the
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T4SS are required for protection against apoptosis (8). Importantly, C. burnetii 3
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possesses several anti-apoptotic effector proteins like CaeA, CaeB (19) and AnkG (20).
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How exactly AnkG interferes with the host cell apoptotic machinery has been unknown
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to date. However, the anti-apoptotic activity of AnkG correlates with binding to p32,
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because only the N-terminal fragment of AnkG (amino acids (aa) 1-69), which interacts
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with p32 and inhibits apoptosis, while the C-terminal fragment (aa 70-338) neither
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interacts with p32 nor interferes with host cell death. Reducing the level of p32 in
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mammalian cells made them more resistant to apoptosis, suggesting that p32 is a pro-
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apoptotic protein and that AnkG might function by interfering with this p32-mediated pro-
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apoptotic activity (20).
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Several questions regarding AnkG´s function remained open: Does AnkG
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influence p32 expression? Is the AnkG-p32 interaction direct or indirect? Is the binding
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to p32 necessary for AnkG-mediated inhibition of apoptosis?
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To address these questions we have defined the p32-binding pocket within AnkG
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and created an AnkG mutant that does not bind to p32. Using this and several other
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mutants we demonstrated that AnkG activity is controlled by p32-mediated trafficking,
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which in turn seems to be regulated by cellular stress.
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Materials and Methods.
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Reagents, cell lines and bacterial strains. Unless otherwise noted, chemicals
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were purchased from Sigma Aldrich. Complete Protease inhibitor cocktail mixture and
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Xtreme Gene 9 Transfection Reagent were from Roche. Protein A/G Sepharose was
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from Santa Cruz. Staurosporine was from Cell Signaling. Cell lines were cultured at
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37°C in 5% CO2 in media containing 10% heat-inactivated fetal bovine serum
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(Biochrom) and 1% penicillin-streptomycin (Invitrogen). CHO-FcR cells were grown in 4
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minimal essential medium alpha medium (Invitrogen), HeLa and HEK293 cells were
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maintained in Dulbecco´s modified Eagle´s medium (Invitrogen). Bone marrow derived-
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DCs from C57BL/6 mice were prepared as described (21). Escherichia coli strains DH5α
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and BL21-DE3 were cultivated in Luria-Bertani (LB) broth supplemented with
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kanamycin, or ampicillin where appropriate. L. pneumophila serogroup 1 ΔflaA strains
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were grown as described (20).
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Plasmids and primers. Plasmid and primers used are listed in Tables 1 and 2.
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Plasmid construction. For creation of the constructs AnkG1-91-pCMV-HA,
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AnkG50-338-pCMV-HA, AnkG1-157-pCMV-HA and AnkF-pCMV-HA, the genes were
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amplified from C. burnetii Nine Mile phase II clone 4 genomic DNA by PCR using the
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primers listed in table 2, restricted with the enzymes indicated and ligated with likewise-
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restricted pCMV-HA. For creation of the constructs AnkGR23S-pCMV-HA and
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AnkGR22/23S-pCMV-HA the genes were amplified from AnkGFL-pCMV-HA with primers
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listed in table 2 that were 5’ phosphorylated. The PCR constructs were gel-purified and
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ligated. For cloning of the constructs AnkG1-69-pEGFP and AnkG70-338-pEGFP, the genes
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were amplified from AnkG1-69-pJV400 or AnkG70-338-pJV400 using the primers listed in
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table 2, restricted with the enzymes indicated and ligated with likewise-restricted
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pEGFP. For creation of the constructs AnkGR22/23S-pJV400, NES-AnkG-pJV400 and
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NLS-AnkGR22/23S-pJV400, the genes were amplified from AnkGR22/23S-pEGFP or NES-
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AnkG-pEGFP using the primers listed in table 2, restricted as indicated and ligated with
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likewise-restricted pJV400. For cloning the construct NES-AnkG-pGEFP, the gene was
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amplified from AnkGFL-pEGFP using the primers listed in table 2, restricted with the
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indicated enzymes and ligated with likewise restricted pEGFP. For cloning the construct
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NLS-AnkGR22/23S-pEGFP, the gene was amplified from AnkGR22/23S-pCVM-HA using the 5
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primers listed in table 2, restricted with the indicated enzymes and ligated with likewise
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restricted pEGFP.
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Confocal microscopy. CHO cells were plated on coverslips and were
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transfected with the indicated plasmids. The cells were fixed with 4% paraformaldehyde
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(Alfa Aeser) in PBS (Biochrom), permeabilized with ice-cold methanol, quenched with
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50mM NH4Cl (Roth) in PBS. The cells were mounted using ProLong Gold with DAPI
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(Invitrogen) to visualize the nucleus. For mitochondrial staining, the cells were incubated
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using Mitotracker (Molecular Probes) before fixation. Confocal fluorescence microscopy
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was performed using a Zeiss LSM 700 confocal microscope.
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Nuclear fragmentation assays. Was performed as described (19).
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Co-immunoprecipitation. HEK293 cells were transiently transfected with the
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plasmids indicated. On the following day, the cells were washed with PBS and incubated
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with lysis buffer (20mM HEPES (pH7.5), 200mM NaCl, 1mM EDTA, 0.1% (vol/vol)
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Nonidet P-40, 10% (vol/vol) glycerol, 1x protease inhibitor, 1mM DTT) for 30min on ice.
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After centrifugation the supernatants were incubated with anti-GFP rabbit serum from
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Invitrogen for 2h at 4°C. Complexes were precipitated by adding protein A/G PLUS-
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Agarose and incubated for 45min at 4°C. The beads were washed three times with
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washing buffer (20mM HEPES (pH7.5), 100mM NaCl, 1mM EDTA, 0.1% (vol/vol)
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Nonidet P-40) and samples were analyzed.
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Protein purification. E. coli BL21 (DE3) cells transformed with plasmids
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producing GST, GST-AnkG or His-p32 were grown in LB broth containing ampicillin.
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IPTG was added to the media and samples were incubated for 4h at 30°C. The cells
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were resuspended in PBS containing Protease inhibitor. After disruption by French
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Press, the lysate was incubated in 1% Trition X-100 for 1h at 4°C. Lysates were clarified 6
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by centrifugation at 15000x g for 30min. Proteins were purified using glutathione-
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sepharose or Ni-NTA agarose colums respectively.
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GST-tag pull-down. Purified GST or GST-AnkG were loaded onto glutathione–
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sepharose columns (GE Healthcare), and purified His-p32 was added to the columns.
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The columns were washed three times with PBS and bound proteins were eluted with
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10mM glutathione in PBS (pH 9.0). The input, the eluate and the bead fractions were
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analyzedas indicated
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His-tag pull-down. Purified His-p32 was loaded onto Ni-NTA agarose columns
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(GE Healthcare), and GST or GST-AnkG added to the columns. The columns were
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washed with increasing concentrations of imidazol in lysis buffer (50mM Tris-HCl pH 7.5,
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150mM NaCl, 1mM DTT) and bound proteins were eluted with 500mM imidazol in lysis
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buffer. Eluate and input fractions were analyzed as indicated.
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Statistical analysis. The unpaired Student’s t-test was used.
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Legionella pneumophila ΔflaA infection. Dendritic cells derived from C57Bl/6
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mice were infected with the L. pneumophila ΔflaA containing the indicated plasmid as
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described (20). 2h and 10h after infection, cells were lysed and plated on charcoal yeast
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extract plates. The plates were incubated for three days at 37°C and colony forming
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units were counted. Colony number after 2h infection represents the infection efficiency,
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after 10h the survival of the intracellular bacteria.
154 155
Results
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AnkG binds p32 directly. To analyze the interaction of AnkG with p32, we first
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determined whether the binding is direct or indirect. Typically, GST pull-down
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experiments are used to verify direct interactions between two proteins. Thus, we 7
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expressed and purified GST, GST-tagged AnkG and His-tagged p32 from Escherichia
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coli. Purified GST or GST-AnkG was incubated with His-p32 and the putative protein
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complex pulled-down with glutathione-coated sepharose beads. The eluate and bead
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fractions were subjected to SDS-PAGE and stained with Coomassie blue (Fig. 1A).
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Additionally, we analyzed eluate and bead fractions by immunoblot analysis (Fig. 1B).
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As shown in Figs. 1A and 1B, His-p32 is pulled-down by GST-AnkG, but not by GST
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alone. To confirm the direct interaction, we also performed the reverse experiment.
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Thus, purified GST or GST-AnkG and His-p32 were incubated and His-coupled proteins
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were pulled-down with nickel-NTA-coated agarose beads. Immunoblot analysis revealed
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that His-p32 pulled-down GST-AnkG (Fig. 1C), but not GST (data not shown).
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Therefore, binding of AnkG to p32 is direct, because no additional proteins were needed
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for this interaction.
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AnkG does not alter the p32 steady-state protein leveI. AnkG was suggested
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to mediate its anti-apoptotic activity by blocking p32 function (20). Therefore, we first
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analyzed whether the expression of AnkG results in a reduced p32 protein level. Thus,
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the respective p32 protein level of cells ectopically expressing GFP or GFP-AnkG was
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analyzed by immunoblot using an anti-p32 antibody. As shown in Fig. 1D, the p32
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protein level was not altered by GFP-AnkG expression, suggesting that AnkG does not
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act by changing the steady-state protein level of p32. Furthermore, AnkG expression did
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not cause any changes in the intracellular distribution of p32 (Figure 1E).
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AnkG associates with mitochondria and traffics into the nucleus after stress
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induction. The host cell protein p32 is mainly found in the mitochondria (22, 23) and a
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small fraction in the nucleus (24). In order to address the question where the interaction
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between AnkG and p32 occurs within the cell, we analyzed the intracellular localization 8
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of ectopically expressed GFP-AnkG. As demonstrated in Fig. 2A GFP-AnkG showed
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vesicular staining with close association to host cell mitochondria.
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The distribution of p32 is altered by perturbation of the physiological state of the
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cell (23-25). As AnkG interacts with p32, we asked whether AnkG also alters its
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intracellular localization after cellular stress induction. Thus, we treated GFP-AnkG
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expressing cells for different time periods with staurosporine to cause cellular stress and
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analyzed subsequently the intracellular localization of AnkG by immunofluorescence.
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Before treatment the majority of GFP-AnkG was localized in close association with the
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mitochondria and to a lesser degree in the nucleus, although AnkG does not contain a
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predicted nuclear localization signal. After treatment with staurosporine, the intracellular
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localization of GFP-AnkG changed. After 4h GFP-AnkG was mainly present within the
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nucleus and only a minority remained in close association with the mitochondria (Fig.
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2B). These results demonstrate that AnkG traffics into the nucleus after apoptosis-
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induction. Furthermore, it suggests that AnkG requires binding to p32 or another host
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cell protein to get transported into the nucleus, as AnkG does not contain a predicted
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nuclear localization.
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The amino-terminal fragment AnkG1–69 contains one or more regions necessary
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for inhibition of apoptosis and for binding to p32, whereas AnkG70-338 neither binds to p32
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nor inhibits apoptosis (20). If the change in intracellular localization depends on binding
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to p32, the intracellular localization of AnkG70-338 should not change after staurosporine
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treatment. Thus, we analyzed the intracellular localization of AnkG1-69 and AnkG70-338
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after cellular perturbation. As shown in Fig. 2C AnkG1–69 was mainly localized in the
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nucleus under healthy and apoptotic conditions. The nuclear localization of GFP-AnkG1-
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69
under healthy conditions might be due to its small size of 34kDa. GFP-AnkG1-69 can 9
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freely migrate into the nucleus and is most likely actively retained within the nucleus. In
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contrast, AnkG70-338 was mainly localized in the cytoplasm and this localization did not
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change after treatment with staurosporine. These results led to the hypothesis that
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trafficking of AnkG might depend on binding to p32 and that nuclear localization might
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be important for AnkG-mediated apoptosis-inhibition. However, to prove the first
212
hypothesis it was necessary to generate an AnkG mutant unable to bind to p32.
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An arginine-rich region within AnkG is required for binding to p32. To
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narrow down the region within AnkG required for binding to p32 we generated different
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AnkG truncations. We expressed HA-tagged AnkG truncations and GFP-p32 in HEK293
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cells, precipitated proteins from the cell lysates with an anti-GFP antibody and evaluated
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the co-immunoprecipitation of the different AnkG truncations by immunoblot analysis. As
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shown in Fig. 3A HA-AnkG, HA-AnkGΔAnk, HA-AnkG1-157, HA-AnkG1-91 and HA-AnkG1-69 ,
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but not HA-AnkF, HA-AnkG70-338, HA-AnkG50-338 and HA-AnkG29-338 co-precipitated with
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GFP-p32. Thus, the first 28 aa of AnkG are most likely required for binding to p32. This
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N-terminal part contains seven arginine residues. Because p32 was shown to bind to
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arginine-rich regions (26, 27), we generated point mutations within this arginine-rich N-
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terminal part, replacing arginine with serine. To analyze binding of the AnkG mutants to
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p32 co-immunoprecipitation was performed. While HA-AnkG and HA-AnkGR23S co-
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precipitated with GFP-p32, HA-AnkGR22/23S did not (Fig. 3B). Hence, we identified the
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p32-binding region within AnkG and generated an AnkG mutant unable to bind to p32.
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AnkG intracellular localization and trafficking depends on p32 binding. Next,
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we analyzed the intracellular localization of the AnkG mutant AnkGR22/23S by
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immunofluorescence. As shown in Fig. 3C, ectopically expressed GFP-AnkGR22/23S co-
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localized with αtubulin, suggesting that intracellular localization of AnkG is dependent on 10
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p32 binding. To analyze whether intracellular trafficking of AnkG also depends on p32
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binding, we treated GFP-AnkGR22/23S expressing cells for different time periods with
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staurosporine
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immunofluorescence. The majority of GFP-AnkGR22/23S co-localized with αtubulin and to
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a lesser degree to the nucleus. Importantly, the intracellular localization of GFP-
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AnkGR22/23S was not changed by treatment with staurosporine (Fig. 3D). Taken together,
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nuclear localization and trafficking of AnkG depend on its binding to p32.
and
analyzed
the
intracellular
localization
of
AnkGR22/23S
by
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AnkG has to migrate into the nucleus to inhibit apoptosis. Having
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demonstrated that AnkG traffics into the nucleus after apoptosis induction, our goal was
240
to determine whether this nuclear localization is essential for the anti-apoptotic activity of
241
AnkG. Consequently, we expressed a chimera comprising GFP-AnkG fused to the
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nuclear export signal of the HIV-1 Rev protein (GFP-NES-AnkG). This nuclear export
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signal has been used successfully to prevent nuclear import of the Golgi vesicle
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tethering protein p115 (28). GFP-NES-AnkG was excluded from the nucleus (Fig. 4A),
245
but still binds to p32 (Fig. 4B). As shown in Fig. 4C, GFP-NES-AnkG was present
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exclusively in the cytoplasm and did not migrate into the nucleus after apoptosis-
247
induction (Fig. 4C). Thus, GFP-NES-AnkG can be used to analyze whether AnkG
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nuclear localization is required for apoptosis inhibition. Therefore, we ectopically
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produced GFP, GFP-AnkG and GFP-NES-AnkG transiently in CHO cells and treated the
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cells with staurosporine to induce cell death. Nuclear fragmentation was visualized by
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DAPI staining and counted to measure apoptosis. Whereas 35% of cells expressing
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GFP had fragmented nuclei, this number was reduced to 20% in cells expressing GFP-
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AnkG (Fig. 4D). Importantly, 40% of cells expressing GFP-NES-AnkG had fragmented
11
254
nuclei, demonstrating that NES-AnkG does not inhibit apoptosis. Thus, the anti-apoptotic
255
activity of AnkG strictly requires its translocation into the nucleus.
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Neither AnkGR22/23S nor NES-AnkG prevents pathogen-induced apoptosis.
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Next, we asked whether AnkG delivered into the host cell by the T4SS also depends on
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nuclear localization to exert its anti-apoptotic activity. Because C. burnetii harbors
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several anti-apoptotic effector proteins, the construction of an ankG deletion-mutant
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complemented or not with nes-ankG might not provide an answer to this question.
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Instead we employed a gain-of-function analysis using Legionella pneumophila ΔflaA to
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determine whether translocation of different AnkG mutants could prevent apoptosis. L.
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pneumonia ΔflaA caused rapid apoptosis in mouse bone marrow-derived dendritic cells
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(DCs) and, thus, could not replicate in these cells (21). These pathogen-induced
265
incidents were blocked by adding AnkG to the repertoire of L. pneumophila effector
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proteins (20). Therefore, this model can be used to analyze whether AnkG has to bind to
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p32 or whether AnkG has to migrate into the nucleus to inhibit pathogen-induced
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apoptosis. We infected DCs with L. pneumophila ΔflaA containing either the empty
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vector (pJV400), AnkG (pJV400-AnkG), NES-AnkG (pJV400-NES-AnkG) or AnkGR22/23S
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(pJV400-AnkGR22/23S). At 2h and 10h post-infection, the cells were lysed and bacterial
271
colony forming units were counted. As shown in Fig. 5A, bacterial uptake was not
272
affected by the addition of AnkG or any of the AnkG mutants. At 10h post-infection, only
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12% of the initial inoculum of L. pneumophila ΔflaA containing vector alone were
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recovered, suggesting that these bacteria induce apoptosis in their host cells and, thus,
275
are not able to survive and replicate (Fig. 5B). In contrast, nearly 50% of the L.
276
pneumophila ΔflaA encoding AnkG were recovered, suggesting that AnkG delivered into
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the host cell by the L. pneumophila T4SS is able to disrupt pathogen-induced apoptosis 12
278
in DC, in agreement with a previous report (20). Neither L. pneumophila ΔflaA encoding
279
NES-AnkG nor L. pneumophila ΔflaA encoding AnkGR22/23S seemed to inhibit pathogen-
280
induced apoptosis in DCs, as demonstrated by recovery rates of less than 10%. These
281
results support our previous findings and suggest that AnkG depends on binding to p32
282
for proper localization and trafficking into the nucleus and that the nuclear localization is
283
essential for inhibition of host cell apoptosis.
284
The intracellular trafficking, but not the anti-apoptotic activity of AnkG,
285
depends on binding to p32. The previous experiments did not clarify whether the
286
binding to p32 is also necessary for AnkG-mediated anti-apoptotic activity. To address
287
this question we constructed a chimera by fusing the SV40 large T antigen nuclear
288
localization signal (29) to the amino-terminus of AnkGR22/23S (GFP-NLS-AnkGR22/23S).
289
Ectopic expression of this construct displays nuclear localization (Fig. 5C). Next, we
290
ectopically produced GFP, GFP-AnkG and GFP-NLS-AnkGR22/23S transiently in CHO
291
cells and treated the cells with staurosporine to induce cell death. Nuclear fragmentation
292
was visualized by DAPI staining and counted to measure apoptosis. Whereas 35% of
293
cells expressing GFP had fragmented nuclei, this number was reduced to 23% in cells
294
expressing GFP-AnkG (Fig. 5D). Importantly, 22% of cells expressing GFP-NLS-
295
AnkGR22/23S had fragmented nuclei. Thus, AnkG depends on binding to p32 for proper
296
localization and trafficking, but not for anti-apoptotic activity.
297 298
Discussion
299
The elimination of infected cells via apoptosis is an evolutionarily conserved
300
defense mechanism (30). So it is not surprising that many intracellular pathogens have
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developed mechanisms to counter apoptosis-induction by their host cells (16). Several 13
302
intracellular pathogens inject effector proteins into the host cell to prevent premature
303
host cell death. However, their molecular mechanisms of action are distinct (31). Here
304
we analyzed the anti-apoptotic activity of AnkG. We showed that the effector protein
305
AnkG localizes in association with the host cell mitochondria in unstressed cells (Fig.
306
2A). This is in contrast to a report showing that mCherry-AnkG co-localized with
307
microtubules (32). The difference in localization of AnkG cannot be explained by the cell
308
line used, because both studies used HeLa cells. The only other difference is the tag
309
used. However, we have not detected any tag-dependent differences in the intracellular
310
localization of AnkG so far. GFP-, HA- and myc-tagged AnkG all displayed the same
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intracellular localization in HeLa and CHO-FcR cells (data not shown). Interestingly,
312
ectopically expressed GFP-AnkGR22/23S co-localized with tubulin (Fig. 3C), and thus
313
displays the same intracellular localization as reported for mCherry-AnkG (32). This
314
localization is surprising, as one would predict that AnkG unable to bind p32 would
315
display cytoplasmic localization. Furthermore, after staurosporine treatment GFP-NES-
316
AnkG displays partial co-localization with tubulin (data not shown). Therefore, it can be
317
speculated that microtubule-association might play a role in AnkG activity under certain
318
cellular conditions.
319
There are several anti-apoptotic type III or type IV secretion system effector
320
proteins that target the host cell mitochondria, the central organelle of the intrinsic
321
apoptotic pathway. Such targeting of the mitochondria by bacterial proteins seems to be
322
evolutionarily conserved, as plant pathogens also target the mitochondria to suppress
323
the hypersensitive response, a form of programmed cell death (33). As shown in Fig. 2A
324
AnkG only partially co-localized with mitochondria, suggesting that this effector protein is
325
not transported into the mitochondria, as it has been shown for Ats1 and PorB. Ats1 14
326
from Anaplasma phagocytophilum uses the mitochondrial import machinery to get
327
transported into the mitochondria (34), while the meningococcal PorB associate with a
328
porin located in the outer mitochondrial membrane (35). Importantly, the anti-apoptotic
329
activity of Ats1 correlates with mitochondrial import (34). AnkG, in clear contrast, has to
330
get transported into the nucleus to act anti-apoptotically (Fig. 4D). Interestingly, this
331
transport into the nucleus, which depends on the ability of AnkG to bind to p32, only
332
happens under apoptotic or stress conditions (Fig. 2B and 3D). This leads to the
333
hypothesis that AnkG primarily targets the mitochondria to sense host cell apoptotic
334
stress and then hitchhikes to the nucleus, the organelle of activity. As a consequence it
335
can be concluded that the activity of AnkG is adjusted by a host cell stress sensor which
336
regulates the transport process.
337
For intracellular trafficking of AnkG from the mitochondria to the nucleus and,
338
thus, for activity control, binding to p32 is essential. This is in agreement with a report
339
that proposed that p32 is involved in bridging a signaling pathway that extends from the
340
mitochondria to the cell nucleus (23). However, once AnkG is within the nucleus, binding
341
to p32 is not needed for anti-apoptotic activity (Fig. 5D). This result suggests that AnkG
342
must instead interfere with a nuclear function to prevent host cell death. There are
343
several effector proteins known to target the host cell nucleus. The Chlamydia
344
trachomatis effector protein NUE is a histone methyltransferase targeting histones (36).
345
AnkA from A. phagocytophilum mediates epigenetic changes at the CYBB promotor
346
(37), leading to a global down-regulation of host defense genes (38). How AnkG
347
modulates nuclear function has to be determined, but the activity of AnkG is clearly
348
regulated by host cell stress signaling and p32-dependent trafficking. This is, to our
15
349
knowledge the first example that an anti-apoptotic effector protein is regulated by host
350
cell protein-mediated trafficking.
351
The question how effector proteins are regulated has only rarely been
352
investigated. There are several avenues of regulation thinkable: 1) regulation by time
353
point and dosage of translocation; 2) regulation by modulation through other effector
354
proteins; 3) regulation by host cell dependent modification (phosphorylation, lipidation,
355
sumoylation etc.). Examples already exist for the latter scenario. It was shown that
356
intracellular localization, and thereby the function of Legionella pneumophila effector
357
proteins containing a CAAX motif are affected by lipidation through the host cell
358
farnesyltransferase and class I geranylgeranyltransferase (39). The Helicobacter pylori
359
T4SS effector protein CagA is phosphorylated by the host cell tyrosine kinases Src and
360
Abl. Phosphorylated CagA can then modulate various signaling cascades associated
361
with cell polarity, cell proliferation, actin-cytoskeletal rearrangements, cell elongation,
362
disruption of tight and adherence junctions, pro-inflammatory responses and apoptosis
363
inhibition (40). Here, we have identified a fourth possibility to regulate the activity of
364
effector proteins: regulation by stress sensing and intracellular trafficking. In our opinion,
365
more knowledge about host cell requirements for regulation of effector proteins is
366
needed. This knowledge will not only help to understand microbial pathogenesis better,
367
but will also allow us to develop new strategies for therapy. The first steps down this
368
avenue have already been made. An exemplified study showed that identifying host cell
369
signaling pathways required for bacterial survival might help to control infection (41).
370 371
Acknowledgements
16
372
This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the
373
Collaborative Research Initiative 796 (SFB796; to A.L. and C.B.) and through the Priority
374
Programme SPP1580 (to A.L.) as well as by the ERA-NET PathoGenoMics 3rd call (to
375
A.L.). We thank Dr. Christian Bogdan for his valuable comments on the manuscript.
376 377
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499 500
Figure legends
501
Figure 1: AnkG binds directly to the host cell protein p32 and does not alter its steady
502
state protein level. (A) Glutathione–sepharose columns with GST-AnkG or GST alone
503
were incubated with His-p32. Eluate (E1-E4) and bead (beads) fractions were resolved
504
by SDS-PAGE and stained with Coomassie blue. (B) Glutathione–sepharose columns
505
with GST-AnkG or GST alone were incubated with His-p32. Input, eluate (E1-E4) and
506
bead (beads) fractions were subjected to immunoblot analysis using anti-GST and anti-
507
p32 antibodies. (C) Ni-NTA agarose columns with His-p32 were incubated with GST or
508
with GST-AnkG. Eluate (E1-E4) and input were subjected to immunoblot analysis using 22
509
anti-GST and anti-His antibodies. (D) HEK293 cells were transfected with plasmids
510
encoding GFP or GFP-tagged AnkG. Protein extracts were separated by SDS-PAGE,
511
transferred to a PVDF membrane and probed with antibodies directed against GFP, p32
512
and actin. One representative immunoblot out of at least three independent experiments
513
is shown. (E) HeLa cells were transiently transfected with plasmids encoding GFP or
514
GFP-tagged AnkG. The cells were treated with Mitotracker (red) followed by fixation and
515
permeabilization. P32 was stained with a specific primary antibody and a secondary
516
dye405 labeled antibody (blue). Figure 2: Intracellular localization of AnkG. (A)
517
Representative
518
transfected with a plasmid encoding GFP-tagged AnkG (green). The cells were treated
519
with Mitotracker (red) followed by fixation, permeabilization and staining of the nuclei
520
with DAPI (blue). (B) CHO-FcR cells transiently transfected with GFP-tagged AnkG were
521
incubated with 2µM staurosporine. After the indicated time-points cells were fixed and
522
the intracellular localization of AnkG was analyzed in at least 100 transfected cells per
523
sample using confocal microscopy from eight independent experiments. * p< 0.001, n.s.
524
not significant (p=0.055) (C) Representative immunofluorescence micrographs show
525
CHO-FcR cells expressing GFP-tagged AnkG, -AnkG1-69 or -AnkG70-338 (green). The
526
cells were incubated with staurosporine followed by fixation, permeabilization and
527
staining of the nuclei with DAPI (blue).
528
Figure 3: Identification of the p32 binding site. (A and B) HEK293 cells were co-
529
transfected with plasmids encoding GFP-tagged p32 and the indicated HA-tagged AnkG
530
mutants (HA-tagged AnkF was used as negative control). The proteins were precipitated
531
from the cell lysates with an anti-GFP antibody. Immunoblot analysis was used to detect
532
p32 (anti-GFP) and Ank-proteins (anti-HA) in the lysates (pre-IP) and precipitates (IP).
immunofluorescence
micrographs
show
HeLa
cells
transiently
23
533
(C) Representative immunofluorescence micrographs show HeLa cells expressing GFP-
534
tagged AnkGR22/23S (green). The cells were fixed, permeabilized and stained with anti-
535
tubulin antibody (red) and DAPI (blue). (D) CHO-FcR cells expressing GFP-tagged
536
AnkGR22/23S were incubated with 2µM staurosporine. After the indicated time points cells
537
were fixed and the localization of AnkG was analyzed in at least 100 transfected cells
538
per sample from four independent experiments using confocal microscopy. n.s. not
539
significant
540
Figure 4: AnkG has to migrate into the nucleus to inhibit staurosporine-induced
541
apoptosis. (A) Representative immunofluorescence micrograph show CHO-FcR cells
542
expressing GFP-tagged NES-AnkG (green). The cells were treated with Mitotracker
543
(red) followed by fixation, permeabilization and staining of the nuclei with DAPI (blue).
544
(B) HEK293 cells were co-transfected with plasmids encoding the indicated GFP-tagged
545
AnkG mutants or GFP as negative control. The proteins were precipitated from the cell
546
lysates with an anti-GFP antibody. Immunoblot analysis was used to detect endogenous
547
p32 (anti-p32) or AnkG (anti-AnkG) in the lysates (pre-IP) and precipitates (IP). (C)
548
CHO-FcR cells expressing GFP-tagged NES-AnkG were incubated with 2µM
549
staurosporine. After the indicated time-points cells were fixed and the localization of
550
AnkG was analyzed in at least 100 transfected cells per sample from three independent
551
experiments using confocal microscopy. n.s. not significant (D) CHO-FcR cells
552
expressing GFP, GFP- AnkG or -NES-AnkG were treated with staurosporine for 4h. The
553
cells were fixed, permeabilized and the nuclei were stained with DAPI. The nuclear
554
morphology of at least 100 GFP expressing cells was scored in four independent
555
experiments. n.s. not significant * p< 0.02.
24
556
Figure 5: Neither AnkGR22/23S nor NES-AnkG can prevent pathogen-induced apoptosis
557
(A/B) Dendritic cells were infected with Legionella pneumophila ΔflaA containing the
558
indicated plasmids. The data shown are from one representative experiment of three
559
experiments with similar results. Shown is the bacterial uptake after 2 hours infection (A)
560
and the relative number of intracellular bacteria 10 hours after infection compared to the
561
2 hour value. **p= 0.001 (C) Representative immunofluorescence micrograph shows
562
CHO-FcR cells expressing GFP-NLS-AnkGR22/23S (green) incubated with mitotracker
563
(red) followed by fixation, permeabilization and staining of the nuclei with DAPI (blue).
564
(D) CHO-FcR cells expressing GFP, GFP-AnkG or GFP-NLS-AnkGR22/23S were treated
565
with 2µM staurosporine for 4h. After treatment the cells were fixed, permeabilized and
566
the nuclei were stained with DAPI. The nuclear morphology was scored of at least 100
567
GFP expressing cells in three independent experiments. * p< 0.01.
568 569
Tables
570 Plasmid AnkGFL-pCMV-HA AnkG∆Ank-pCMV-HA AnkG1-69-pCMV-HA AnkG70-338-pCMV-HA AnkG1-91-pCMV-HA AnkG50-338-pCMV-HA AnkG1-157-pCMV-HA AnkGR23S-pCMV-HA AnkGR22/23S-pCMV-HA AnkF-pCMV-HA pGEX-5X AnkG-pGEX-5X p32-pET16b pEGFP AnkG-pEGFP p32-pEGFP AnkG1-69-pEGFP AnkG70-338-pEGFP
Primer
329/339 331/340 307/310 590/591 590/592 79/229 184/185
329/338 332/340
Reference 20 20 20 20 This study This study This study This study This study This study Amersham 20 This study Clontech 20 20 This study This study
25
AnkGR22/23S-pEGFP NES-AnkG-pEGFP NLS-AnkGR22/23S-pEGFP pJV400 pJV400-AnkG pJV400-AnkGR22/23S pJV400-NES-AnkG
571
665/34 746/400 373/26 696/26
This study This study This study 20 20 This study This study
Table 1: Primer numbers are as in Table 2.
572 Number 26 34 79 184 185 229 307 310 329 330 331 332 338 339 340 373 400 590 591 592 665 696 746
573
Sequence 5' AAGGCGCGCCTCACCGAGGACTAGACAG 5’ AAGGATCCTCACCGAGGACTAGACAGA 5’ CCGGTACCCTACCGCTGGAAGCCGC 5’ CCCATATGCTGCACACCGACGGAGAC 5’ CCGGATCCCTACTGGCTCTTGACAAAACT 5’ CCGAATTCATGTGCAATACCAACATGTCT 5’ CCGGTACCATGAGTAGACGTGAGACTCC 5’ CCGGTACCTTATTTATATTTGATTTTCACATCAGC 5' CCAAGATCTCTATGAGTAGACGTGAGACTCC 5’ CCAAGATCTCTATGGGACATCCTGTAAGAAGAAG 5’ CCAAGATCTCTATGTCGTTTGAAATACTCATAAATGC 5’ CCAAGATCTCTATGCTTCGCGGGGATTCTTTTCA 5' CCGGTACCTCAGTAGTTTTTTATTATGCTCAAGCT 5’ CCGGTACCTCAGAAATCCGTCTTTGGCGGTA 5' CCGGTACCTCACCGAGGACTAGACAGA 5' CCGGCCGGCCATGAGTAGACGTGAGACTCC 5' CCGGTACCTCACCGAGGACTAGACAGA P-5’ CGTTGAGGATATTGTGCTAGTGGGAGTCTACGTCTAC TCAT P-5’ CGACAGGAACTCGAACGCCGAGAAGTAGATTGAGCC GAAAA P-5’CGACAGGAACTCGAACGCCGAGTAGTAGATTGAGC CGAAAA 5’ CCGGTACCGCCTCCAGCAGCCTCCCCTGGAGGACTGACCCT GAGTAGACGTGAGACTCCCACTAGC 5’ CCGGCCGGCCATGCTCCAGCTGCCTCCCC 5’ CCACTCAGATCTCTCCTAAGAAGAAAAGGAAGGTTAGT AGACGTGAGACTCCCACTAGCACAA
Site AscI BamHI KpnI NdeI BamHI EcoRI KpnI KpnI BglII BglII BglII BglII KpnI KpnI KpnI FseI KpnI KpnI FseI BglII
Table 2. Underlined denotes the location of the restriction site.
26
GST-AnkG
E1
E2
E3
E4
anti-GST anti-GFP
anti-HIS
HIS-p32 GST
D
nk G
GST-AnkG + HIS-p32
G FP -A
C
GST + His-p32
G FP
GST-AnkG + His-p32
E1 E2 E3 E4 Beads E1 E2 E3 E4 Beads
G ST -A n H IS kG -p 32
A
HIS-tag pull-down
anti-p32
input
anti-actin GST-tag pull-down
E G ST H is -p 3
2
B
p32
GFP
Mitotracker
Merge
p32
GFP-AnkG
Mitotracker
Merge
E1 E2 E3 E4 Beads anti-GST anti-p32 GST-tag pull-down 10µm
G ST H Ank is -p G 32
input
E1 E2 E3 E4 Beads anti-GST anti-p32
input
GST-tag pull-down
10µm
A GFP-AnkG
Mitotracker
Merge
Zoom
10µm
B % phenotype
125
*** ***
100
n.s.
75 50
Mitochondrial associa!on
25
nuclear
0 Mock
30
120
240
min staurosporine
GFP-AnkG
GFP-AnkG1-69
GFP-AnkG 70-338
Mock
C
10µm
10µm
Staurosporine
10µm
10µm
10µm
10µm
22
S
A
-A
nk G
R
23 R
W T
-A H
H
A
A H
H
nk G
nk G
nk F
-A
-A A
-A A
/2 3
S
S /2 3 22
S
nk G
R
22 R
W T
nk G
nk G
-A A
-A
nk F A
-A H
A H
H
50
nk G -A
A H
H
38 -3
38
B
70
-3
-3 29
-A A H
H
A
-A
nk G
nk G -A
A H
nk G
1-
91 1-
1-
H
A
-A
nk G
nk G -A
A H
69
7 15
nk ∆A
W T
nk G
nk G H
A
-A
-A A H
38
A
pre-IP
anti-HA anti-HA anti-GFP
pre-IP amino acid AnkG AnkG R23S AnkG R22/23S
18 19 20 21 22 23 24 25 26 27 28 T R T P R R R L S R K T R T P R S R L S R K T R T P S S R L S R K
IP
anti-HA
IP
D
n.s.
anti-GFP
GFP-AnkGR22/23S
αTubulin
100
% phenotype
C Merge
n.s. n.s.
75 50
Co-localiza!on with Tubulin
25
nuclear
0 Mock 10µm
30
120
240
min staurosporine
A GFP-NES-AnkG
Mitotracker
Merge
G FP -
G FP
G FP -
A nk G G FP -A nk G G FP R 22 -N / ES 23S -A nk G G FP
B
A nk G G FP -A nk G G FP R 22 -N / ES 23S -A nk G
10µm 10µm
anti-AnkG anti-p32
pre-IP
IP
C
n.s. n.s. n.s.
% phenotype
100 75 50 cytosolic 25
nuclear
0 Mock
30
120
min staurosporine
D
n.s.
% fragmented nuclei
50
*
40 30 20 10
240
-A ES -N FP G G nk
-A FP G
FP G
G nk
5
10 0
0
* * 20
10
0
Merge Mitotracker GFP-NLS-AnkG
G nk -A ES /23S -N 22 00 GR V4 nk pJ 0-A 0 G V4 nk pJ 0-A 0 V4 pJ 0 40
C
30
20
% survival
40
bacterial *103/well
10
V pJ
G nk -A ES /23S -N 22 00 GR V4 nk pJ 0-A 0 G V4 nk pJ 0-A 0 V4 pJ 0 40 V pJ
40
D
30
% fragmented nuclei
15
* 50 20
60
B n.s. 25
A
-A LS -N FP kG G An FP G FP G
G nk
R
22
3 /2
S
1
The anti-apoptotic activity of the Coxiella burnetii effector protein AnkG is
2
controlled by p32-dependent trafficking
3
Rita A. Eckart1, Stephanie Bisle1, Jan Schulze-Luehrmann1, Irene Wittmann1, Jonathan
4
Jantsch1, Benedikt Schmid2, Christian Berens3 and Anja Lührmann1*
5 6
1
7
Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg,
8
Wasserturmstraße 3/5, D-91054 Erlangen, Germany
9
2
Mikrobiologisches Institut – Klinische Mikrobiologie, Immunologie und Hygiene,
Lehrstuhl für Biotechnik, Department Biologie, Friedrich-Alexander-Universität
10
Erlangen-Nürnberg, Henkestrasse 91, D-91052 Erlangen, Germany
11
3
12
Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany
Lehrstuhl für Mikrobiologie, Department Biologie, Friedrich-Alexander-Universität
13 14
*Corresponding author:
15
Anja Lührmann1
16
Phone: (+49) 9131 85 22577; Fax: (+49) 9131 85 1001
17
Email: [email protected]
18 19
Running title: AnkG trafficking and apoptosis inhibition
20
1
21
Abstract
22
Intracellular bacterial pathogens frequently inhibit host cell apoptosis to ensure
23
survival of their host, thereby allowing bacterial propagation. The obligate intracellular
24
pathogen Coxiella burnetii displays anti-apoptotic activity which depends on a functional
25
type IV secretion system (T4SS). Accordingly, anti-apoptotic T4SS effector proteins, like
26
AnkG, have been identified. AnkG inhibits pathogen-induced apoptosis, possibly by
27
binding to the host cell mitochondrial protein p32 (gC1qR). However, the molecular
28
mechanism of AnkG activity remains unknown.
29
Here, we demonstrate that ectopically expressed AnkG associates with
30
mitochondria and traffics into the nucleus after apoptosis induction, although AnkG lacks
31
a predicted nuclear localization signal. We identified the p32-interaction region in AnkG
32
and constructed an AnkG-mutant (AnkGR22/23S) unable to bind to p32. By using this
33
mutant we found that intracellular localization and trafficking of AnkG into the nucleus is
34
dependent on binding to p32. Furthermore, we demonstrated that nuclear localization of
35
AnkG but not binding to p32 is required for apoptosis inhibition. Thus, the anti-apoptotic
36
activity of AnkG is controlled by p32-mediated intracellular trafficking, which in turn
37
seems to be regulated by host cell processes that sense stress.
38
2
39
Introduction
40
Coxiella burnetii is the obligate intracellular bacterial agent of human Q-fever, a
41
worldwide zoonotic disease (1). Infection in humans occurs by inhalation of infectious
42
material transmitted from domestic livestock, and as few as ten bacteria can result in
43
disease (2). After bacterial uptake into phagocytic cells, C. burnetii establishes a
44
phagolysosomal-like vacuole (3, 4, 5). Importantly, establishing this replicative niche
45
requires bacterial protein synthesis (6, 7), suggesting direct involvement of bacterial
46
proteins. In agreement with this assumption, the type IV secretion system (T4SS) was
47
shown to be essential for intracellular replication (8, 9). The presence of the replicative
48
C. burnetii-containing vacuole (CCV) within the cell most likely causes tremendous
49
stress for the infected cell, as the CCV almost completely fills the host cell lumen (10).
50
Eukaryotic cells often respond to intracellular pathogen invasion and stress induction by
51
initiating the intrinsic apoptotic pathway as part of the innate immune defense (11).
52
Apoptosis is a programmed cell death pathway crucial for immune system
53
maintenance and removal of damaged or infected cells (12). Two main pathways lead to
54
apoptosis. The extrinsic cell death pathway is launched in response to stimulation of
55
death receptor proteins at the cell surface by extracellular stimuli, while the intrinsic cell
56
death pathway is initiated in response to intracellular stimuli (13).
57
Apoptosis allows pathogen clearance without inflammation and additionally leads
58
to activation of the adaptive immune defense (14, 15). As a countermeasure intracellular
59
pathogens have developed multiple mechanisms to inhibit host cell apoptosis (16). C.
60
burnetii also interferes with host cell apoptosis (17, 18). How this occurs mechanistically
61
is incompletely understood, but effector proteins translocated into the host cell by the
62
T4SS are required for protection against apoptosis (8). Importantly, C. burnetii 3
63
possesses several anti-apoptotic effector proteins like CaeA, CaeB (19) and AnkG (20).
64
How exactly AnkG interferes with the host cell apoptotic machinery has been unknown
65
to date. However, the anti-apoptotic activity of AnkG correlates with binding to p32,
66
because only the N-terminal fragment of AnkG (amino acids (aa) 1-69), which interacts
67
with p32 and inhibits apoptosis, while the C-terminal fragment (aa 70-338) neither
68
interacts with p32 nor interferes with host cell death. Reducing the level of p32 in
69
mammalian cells made them more resistant to apoptosis, suggesting that p32 is a pro-
70
apoptotic protein and that AnkG might function by interfering with this p32-mediated pro-
71
apoptotic activity (20).
72
Several questions regarding AnkG´s function remained open: Does AnkG
73
influence p32 expression? Is the AnkG-p32 interaction direct or indirect? Is the binding
74
to p32 necessary for AnkG-mediated inhibition of apoptosis?
75
To address these questions we have defined the p32-binding pocket within AnkG
76
and created an AnkG mutant that does not bind to p32. Using this and several other
77
mutants we demonstrated that AnkG activity is controlled by p32-mediated trafficking,
78
which in turn seems to be regulated by cellular stress.
79 80
Materials and Methods.
81
Reagents, cell lines and bacterial strains. Unless otherwise noted, chemicals
82
were purchased from Sigma Aldrich. Complete Protease inhibitor cocktail mixture and
83
Xtreme Gene 9 Transfection Reagent were from Roche. Protein A/G Sepharose was
84
from Santa Cruz. Staurosporine was from Cell Signaling. Cell lines were cultured at
85
37°C in 5% CO2 in media containing 10% heat-inactivated fetal bovine serum
86
(Biochrom) and 1% penicillin-streptomycin (Invitrogen). CHO-FcR cells were grown in 4
87
minimal essential medium alpha medium (Invitrogen), HeLa and HEK293 cells were
88
maintained in Dulbecco´s modified Eagle´s medium (Invitrogen). Bone marrow derived-
89
DCs from C57BL/6 mice were prepared as described (21). Escherichia coli strains DH5α
90
and BL21-DE3 were cultivated in Luria-Bertani (LB) broth supplemented with
91
kanamycin, or ampicillin where appropriate. L. pneumophila serogroup 1 ΔflaA strains
92
were grown as described (20).
93
Plasmids and primers. Plasmid and primers used are listed in Tables 1 and 2.
94
Plasmid construction. For creation of the constructs AnkG1-91-pCMV-HA,
95
AnkG50-338-pCMV-HA, AnkG1-157-pCMV-HA and AnkF-pCMV-HA, the genes were
96
amplified from C. burnetii Nine Mile phase II clone 4 genomic DNA by PCR using the
97
primers listed in table 2, restricted with the enzymes indicated and ligated with likewise-
98
restricted pCMV-HA. For creation of the constructs AnkGR23S-pCMV-HA and
99
AnkGR22/23S-pCMV-HA the genes were amplified from AnkGFL-pCMV-HA with primers
100
listed in table 2 that were 5’ phosphorylated. The PCR constructs were gel-purified and
101
ligated. For cloning of the constructs AnkG1-69-pEGFP and AnkG70-338-pEGFP, the genes
102
were amplified from AnkG1-69-pJV400 or AnkG70-338-pJV400 using the primers listed in
103
table 2, restricted with the enzymes indicated and ligated with likewise-restricted
104
pEGFP. For creation of the constructs AnkGR22/23S-pJV400, NES-AnkG-pJV400 and
105
NLS-AnkGR22/23S-pJV400, the genes were amplified from AnkGR22/23S-pEGFP or NES-
106
AnkG-pEGFP using the primers listed in table 2, restricted as indicated and ligated with
107
likewise-restricted pJV400. For cloning the construct NES-AnkG-pGEFP, the gene was
108
amplified from AnkGFL-pEGFP using the primers listed in table 2, restricted with the
109
indicated enzymes and ligated with likewise restricted pEGFP. For cloning the construct
110
NLS-AnkGR22/23S-pEGFP, the gene was amplified from AnkGR22/23S-pCVM-HA using the 5
111
primers listed in table 2, restricted with the indicated enzymes and ligated with likewise
112
restricted pEGFP.
113
Confocal microscopy. CHO cells were plated on coverslips and were
114
transfected with the indicated plasmids. The cells were fixed with 4% paraformaldehyde
115
(Alfa Aeser) in PBS (Biochrom), permeabilized with ice-cold methanol, quenched with
116
50mM NH4Cl (Roth) in PBS. The cells were mounted using ProLong Gold with DAPI
117
(Invitrogen) to visualize the nucleus. For mitochondrial staining, the cells were incubated
118
using Mitotracker (Molecular Probes) before fixation. Confocal fluorescence microscopy
119
was performed using a Zeiss LSM 700 confocal microscope.
120
Nuclear fragmentation assays. Was performed as described (19).
121
Co-immunoprecipitation. HEK293 cells were transiently transfected with the
122
plasmids indicated. On the following day, the cells were washed with PBS and incubated
123
with lysis buffer (20mM HEPES (pH7.5), 200mM NaCl, 1mM EDTA, 0.1% (vol/vol)
124
Nonidet P-40, 10% (vol/vol) glycerol, 1x protease inhibitor, 1mM DTT) for 30min on ice.
125
After centrifugation the supernatants were incubated with anti-GFP rabbit serum from
126
Invitrogen for 2h at 4°C. Complexes were precipitated by adding protein A/G PLUS-
127
Agarose and incubated for 45min at 4°C. The beads were washed three times with
128
washing buffer (20mM HEPES (pH7.5), 100mM NaCl, 1mM EDTA, 0.1% (vol/vol)
129
Nonidet P-40) and samples were analyzed.
130
Protein purification. E. coli BL21 (DE3) cells transformed with plasmids
131
producing GST, GST-AnkG or His-p32 were grown in LB broth containing ampicillin.
132
IPTG was added to the media and samples were incubated for 4h at 30°C. The cells
133
were resuspended in PBS containing Protease inhibitor. After disruption by French
134
Press, the lysate was incubated in 1% Trition X-100 for 1h at 4°C. Lysates were clarified 6
135
by centrifugation at 15000x g for 30min. Proteins were purified using glutathione-
136
sepharose or Ni-NTA agarose colums respectively.
137
GST-tag pull-down. Purified GST or GST-AnkG were loaded onto glutathione–
138
sepharose columns (GE Healthcare), and purified His-p32 was added to the columns.
139
The columns were washed three times with PBS and bound proteins were eluted with
140
10mM glutathione in PBS (pH 9.0). The input, the eluate and the bead fractions were
141
analyzedas indicated
142
His-tag pull-down. Purified His-p32 was loaded onto Ni-NTA agarose columns
143
(GE Healthcare), and GST or GST-AnkG added to the columns. The columns were
144
washed with increasing concentrations of imidazol in lysis buffer (50mM Tris-HCl pH 7.5,
145
150mM NaCl, 1mM DTT) and bound proteins were eluted with 500mM imidazol in lysis
146
buffer. Eluate and input fractions were analyzed as indicated.
147
Statistical analysis. The unpaired Student’s t-test was used.
148
Legionella pneumophila ΔflaA infection. Dendritic cells derived from C57Bl/6
149
mice were infected with the L. pneumophila ΔflaA containing the indicated plasmid as
150
described (20). 2h and 10h after infection, cells were lysed and plated on charcoal yeast
151
extract plates. The plates were incubated for three days at 37°C and colony forming
152
units were counted. Colony number after 2h infection represents the infection efficiency,
153
after 10h the survival of the intracellular bacteria.
154 155
Results
156
AnkG binds p32 directly. To analyze the interaction of AnkG with p32, we first
157
determined whether the binding is direct or indirect. Typically, GST pull-down
158
experiments are used to verify direct interactions between two proteins. Thus, we 7
159
expressed and purified GST, GST-tagged AnkG and His-tagged p32 from Escherichia
160
coli. Purified GST or GST-AnkG was incubated with His-p32 and the putative protein
161
complex pulled-down with glutathione-coated sepharose beads. The eluate and bead
162
fractions were subjected to SDS-PAGE and stained with Coomassie blue (Fig. 1A).
163
Additionally, we analyzed eluate and bead fractions by immunoblot analysis (Fig. 1B).
164
As shown in Figs. 1A and 1B, His-p32 is pulled-down by GST-AnkG, but not by GST
165
alone. To confirm the direct interaction, we also performed the reverse experiment.
166
Thus, purified GST or GST-AnkG and His-p32 were incubated and His-coupled proteins
167
were pulled-down with nickel-NTA-coated agarose beads. Immunoblot analysis revealed
168
that His-p32 pulled-down GST-AnkG (Fig. 1C), but not GST (data not shown).
169
Therefore, binding of AnkG to p32 is direct, because no additional proteins were needed
170
for this interaction.
171
AnkG does not alter the p32 steady-state protein leveI. AnkG was suggested
172
to mediate its anti-apoptotic activity by blocking p32 function (20). Therefore, we first
173
analyzed whether the expression of AnkG results in a reduced p32 protein level. Thus,
174
the respective p32 protein level of cells ectopically expressing GFP or GFP-AnkG was
175
analyzed by immunoblot using an anti-p32 antibody. As shown in Fig. 1D, the p32
176
protein level was not altered by GFP-AnkG expression, suggesting that AnkG does not
177
act by changing the steady-state protein level of p32. Furthermore, AnkG expression did
178
not cause any changes in the intracellular distribution of p32 (Figure 1E).
179
AnkG associates with mitochondria and traffics into the nucleus after stress
180
induction. The host cell protein p32 is mainly found in the mitochondria (22, 23) and a
181
small fraction in the nucleus (24). In order to address the question where the interaction
182
between AnkG and p32 occurs within the cell, we analyzed the intracellular localization 8
183
of ectopically expressed GFP-AnkG. As demonstrated in Fig. 2A GFP-AnkG showed
184
vesicular staining with close association to host cell mitochondria.
185
The distribution of p32 is altered by perturbation of the physiological state of the
186
cell (23-25). As AnkG interacts with p32, we asked whether AnkG also alters its
187
intracellular localization after cellular stress induction. Thus, we treated GFP-AnkG
188
expressing cells for different time periods with staurosporine to cause cellular stress and
189
analyzed subsequently the intracellular localization of AnkG by immunofluorescence.
190
Before treatment the majority of GFP-AnkG was localized in close association with the
191
mitochondria and to a lesser degree in the nucleus, although AnkG does not contain a
192
predicted nuclear localization signal. After treatment with staurosporine, the intracellular
193
localization of GFP-AnkG changed. After 4h GFP-AnkG was mainly present within the
194
nucleus and only a minority remained in close association with the mitochondria (Fig.
195
2B). These results demonstrate that AnkG traffics into the nucleus after apoptosis-
196
induction. Furthermore, it suggests that AnkG requires binding to p32 or another host
197
cell protein to get transported into the nucleus, as AnkG does not contain a predicted
198
nuclear localization.
199
The amino-terminal fragment AnkG1–69 contains one or more regions necessary
200
for inhibition of apoptosis and for binding to p32, whereas AnkG70-338 neither binds to p32
201
nor inhibits apoptosis (20). If the change in intracellular localization depends on binding
202
to p32, the intracellular localization of AnkG70-338 should not change after staurosporine
203
treatment. Thus, we analyzed the intracellular localization of AnkG1-69 and AnkG70-338
204
after cellular perturbation. As shown in Fig. 2C AnkG1–69 was mainly localized in the
205
nucleus under healthy and apoptotic conditions. The nuclear localization of GFP-AnkG1-
206
69
under healthy conditions might be due to its small size of 34kDa. GFP-AnkG1-69 can 9
207
freely migrate into the nucleus and is most likely actively retained within the nucleus. In
208
contrast, AnkG70-338 was mainly localized in the cytoplasm and this localization did not
209
change after treatment with staurosporine. These results led to the hypothesis that
210
trafficking of AnkG might depend on binding to p32 and that nuclear localization might
211
be important for AnkG-mediated apoptosis-inhibition. However, to prove the first
212
hypothesis it was necessary to generate an AnkG mutant unable to bind to p32.
213
An arginine-rich region within AnkG is required for binding to p32. To
214
narrow down the region within AnkG required for binding to p32 we generated different
215
AnkG truncations. We expressed HA-tagged AnkG truncations and GFP-p32 in HEK293
216
cells, precipitated proteins from the cell lysates with an anti-GFP antibody and evaluated
217
the co-immunoprecipitation of the different AnkG truncations by immunoblot analysis. As
218
shown in Fig. 3A HA-AnkG, HA-AnkGΔAnk, HA-AnkG1-157, HA-AnkG1-91 and HA-AnkG1-69 ,
219
but not HA-AnkF, HA-AnkG70-338, HA-AnkG50-338 and HA-AnkG29-338 co-precipitated with
220
GFP-p32. Thus, the first 28 aa of AnkG are most likely required for binding to p32. This
221
N-terminal part contains seven arginine residues. Because p32 was shown to bind to
222
arginine-rich regions (26, 27), we generated point mutations within this arginine-rich N-
223
terminal part, replacing arginine with serine. To analyze binding of the AnkG mutants to
224
p32 co-immunoprecipitation was performed. While HA-AnkG and HA-AnkGR23S co-
225
precipitated with GFP-p32, HA-AnkGR22/23S did not (Fig. 3B). Hence, we identified the
226
p32-binding region within AnkG and generated an AnkG mutant unable to bind to p32.
227
AnkG intracellular localization and trafficking depends on p32 binding. Next,
228
we analyzed the intracellular localization of the AnkG mutant AnkGR22/23S by
229
immunofluorescence. As shown in Fig. 3C, ectopically expressed GFP-AnkGR22/23S co-
230
localized with αtubulin, suggesting that intracellular localization of AnkG is dependent on 10
231
p32 binding. To analyze whether intracellular trafficking of AnkG also depends on p32
232
binding, we treated GFP-AnkGR22/23S expressing cells for different time periods with
233
staurosporine
234
immunofluorescence. The majority of GFP-AnkGR22/23S co-localized with αtubulin and to
235
a lesser degree to the nucleus. Importantly, the intracellular localization of GFP-
236
AnkGR22/23S was not changed by treatment with staurosporine (Fig. 3D). Taken together,
237
nuclear localization and trafficking of AnkG depend on its binding to p32.
and
analyzed
the
intracellular
localization
of
AnkGR22/23S
by
238
AnkG has to migrate into the nucleus to inhibit apoptosis. Having
239
demonstrated that AnkG traffics into the nucleus after apoptosis induction, our goal was
240
to determine whether this nuclear localization is essential for the anti-apoptotic activity of
241
AnkG. Consequently, we expressed a chimera comprising GFP-AnkG fused to the
242
nuclear export signal of the HIV-1 Rev protein (GFP-NES-AnkG). This nuclear export
243
signal has been used successfully to prevent nuclear import of the Golgi vesicle
244
tethering protein p115 (28). GFP-NES-AnkG was excluded from the nucleus (Fig. 4A),
245
but still binds to p32 (Fig. 4B). As shown in Fig. 4C, GFP-NES-AnkG was present
246
exclusively in the cytoplasm and did not migrate into the nucleus after apoptosis-
247
induction (Fig. 4C). Thus, GFP-NES-AnkG can be used to analyze whether AnkG
248
nuclear localization is required for apoptosis inhibition. Therefore, we ectopically
249
produced GFP, GFP-AnkG and GFP-NES-AnkG transiently in CHO cells and treated the
250
cells with staurosporine to induce cell death. Nuclear fragmentation was visualized by
251
DAPI staining and counted to measure apoptosis. Whereas 35% of cells expressing
252
GFP had fragmented nuclei, this number was reduced to 20% in cells expressing GFP-
253
AnkG (Fig. 4D). Importantly, 40% of cells expressing GFP-NES-AnkG had fragmented
11
254
nuclei, demonstrating that NES-AnkG does not inhibit apoptosis. Thus, the anti-apoptotic
255
activity of AnkG strictly requires its translocation into the nucleus.
256
Neither AnkGR22/23S nor NES-AnkG prevents pathogen-induced apoptosis.
257
Next, we asked whether AnkG delivered into the host cell by the T4SS also depends on
258
nuclear localization to exert its anti-apoptotic activity. Because C. burnetii harbors
259
several anti-apoptotic effector proteins, the construction of an ankG deletion-mutant
260
complemented or not with nes-ankG might not provide an answer to this question.
261
Instead we employed a gain-of-function analysis using Legionella pneumophila ΔflaA to
262
determine whether translocation of different AnkG mutants could prevent apoptosis. L.
263
pneumonia ΔflaA caused rapid apoptosis in mouse bone marrow-derived dendritic cells
264
(DCs) and, thus, could not replicate in these cells (21). These pathogen-induced
265
incidents were blocked by adding AnkG to the repertoire of L. pneumophila effector
266
proteins (20). Therefore, this model can be used to analyze whether AnkG has to bind to
267
p32 or whether AnkG has to migrate into the nucleus to inhibit pathogen-induced
268
apoptosis. We infected DCs with L. pneumophila ΔflaA containing either the empty
269
vector (pJV400), AnkG (pJV400-AnkG), NES-AnkG (pJV400-NES-AnkG) or AnkGR22/23S
270
(pJV400-AnkGR22/23S). At 2h and 10h post-infection, the cells were lysed and bacterial
271
colony forming units were counted. As shown in Fig. 5A, bacterial uptake was not
272
affected by the addition of AnkG or any of the AnkG mutants. At 10h post-infection, only
273
12% of the initial inoculum of L. pneumophila ΔflaA containing vector alone were
274
recovered, suggesting that these bacteria induce apoptosis in their host cells and, thus,
275
are not able to survive and replicate (Fig. 5B). In contrast, nearly 50% of the L.
276
pneumophila ΔflaA encoding AnkG were recovered, suggesting that AnkG delivered into
277
the host cell by the L. pneumophila T4SS is able to disrupt pathogen-induced apoptosis 12
278
in DC, in agreement with a previous report (20). Neither L. pneumophila ΔflaA encoding
279
NES-AnkG nor L. pneumophila ΔflaA encoding AnkGR22/23S seemed to inhibit pathogen-
280
induced apoptosis in DCs, as demonstrated by recovery rates of less than 10%. These
281
results support our previous findings and suggest that AnkG depends on binding to p32
282
for proper localization and trafficking into the nucleus and that the nuclear localization is
283
essential for inhibition of host cell apoptosis.
284
The intracellular trafficking, but not the anti-apoptotic activity of AnkG,
285
depends on binding to p32. The previous experiments did not clarify whether the
286
binding to p32 is also necessary for AnkG-mediated anti-apoptotic activity. To address
287
this question we constructed a chimera by fusing the SV40 large T antigen nuclear
288
localization signal (29) to the amino-terminus of AnkGR22/23S (GFP-NLS-AnkGR22/23S).
289
Ectopic expression of this construct displays nuclear localization (Fig. 5C). Next, we
290
ectopically produced GFP, GFP-AnkG and GFP-NLS-AnkGR22/23S transiently in CHO
291
cells and treated the cells with staurosporine to induce cell death. Nuclear fragmentation
292
was visualized by DAPI staining and counted to measure apoptosis. Whereas 35% of
293
cells expressing GFP had fragmented nuclei, this number was reduced to 23% in cells
294
expressing GFP-AnkG (Fig. 5D). Importantly, 22% of cells expressing GFP-NLS-
295
AnkGR22/23S had fragmented nuclei. Thus, AnkG depends on binding to p32 for proper
296
localization and trafficking, but not for anti-apoptotic activity.
297 298
Discussion
299
The elimination of infected cells via apoptosis is an evolutionarily conserved
300
defense mechanism (30). So it is not surprising that many intracellular pathogens have
301
developed mechanisms to counter apoptosis-induction by their host cells (16). Several 13
302
intracellular pathogens inject effector proteins into the host cell to prevent premature
303
host cell death. However, their molecular mechanisms of action are distinct (31). Here
304
we analyzed the anti-apoptotic activity of AnkG. We showed that the effector protein
305
AnkG localizes in association with the host cell mitochondria in unstressed cells (Fig.
306
2A). This is in contrast to a report showing that mCherry-AnkG co-localized with
307
microtubules (32). The difference in localization of AnkG cannot be explained by the cell
308
line used, because both studies used HeLa cells. The only other difference is the tag
309
used. However, we have not detected any tag-dependent differences in the intracellular
310
localization of AnkG so far. GFP-, HA- and myc-tagged AnkG all displayed the same
311
intracellular localization in HeLa and CHO-FcR cells (data not shown). Interestingly,
312
ectopically expressed GFP-AnkGR22/23S co-localized with tubulin (Fig. 3C), and thus
313
displays the same intracellular localization as reported for mCherry-AnkG (32). This
314
localization is surprising, as one would predict that AnkG unable to bind p32 would
315
display cytoplasmic localization. Furthermore, after staurosporine treatment GFP-NES-
316
AnkG displays partial co-localization with tubulin (data not shown). Therefore, it can be
317
speculated that microtubule-association might play a role in AnkG activity under certain
318
cellular conditions.
319
There are several anti-apoptotic type III or type IV secretion system effector
320
proteins that target the host cell mitochondria, the central organelle of the intrinsic
321
apoptotic pathway. Such targeting of the mitochondria by bacterial proteins seems to be
322
evolutionarily conserved, as plant pathogens also target the mitochondria to suppress
323
the hypersensitive response, a form of programmed cell death (33). As shown in Fig. 2A
324
AnkG only partially co-localized with mitochondria, suggesting that this effector protein is
325
not transported into the mitochondria, as it has been shown for Ats1 and PorB. Ats1 14
326
from Anaplasma phagocytophilum uses the mitochondrial import machinery to get
327
transported into the mitochondria (34), while the meningococcal PorB associate with a
328
porin located in the outer mitochondrial membrane (35). Importantly, the anti-apoptotic
329
activity of Ats1 correlates with mitochondrial import (34). AnkG, in clear contrast, has to
330
get transported into the nucleus to act anti-apoptotically (Fig. 4D). Interestingly, this
331
transport into the nucleus, which depends on the ability of AnkG to bind to p32, only
332
happens under apoptotic or stress conditions (Fig. 2B and 3D). This leads to the
333
hypothesis that AnkG primarily targets the mitochondria to sense host cell apoptotic
334
stress and then hitchhikes to the nucleus, the organelle of activity. As a consequence it
335
can be concluded that the activity of AnkG is adjusted by a host cell stress sensor which
336
regulates the transport process.
337
For intracellular trafficking of AnkG from the mitochondria to the nucleus and,
338
thus, for activity control, binding to p32 is essential. This is in agreement with a report
339
that proposed that p32 is involved in bridging a signaling pathway that extends from the
340
mitochondria to the cell nucleus (23). However, once AnkG is within the nucleus, binding
341
to p32 is not needed for anti-apoptotic activity (Fig. 5D). This result suggests that AnkG
342
must instead interfere with a nuclear function to prevent host cell death. There are
343
several effector proteins known to target the host cell nucleus. The Chlamydia
344
trachomatis effector protein NUE is a histone methyltransferase targeting histones (36).
345
AnkA from A. phagocytophilum mediates epigenetic changes at the CYBB promotor
346
(37), leading to a global down-regulation of host defense genes (38). How AnkG
347
modulates nuclear function has to be determined, but the activity of AnkG is clearly
348
regulated by host cell stress signaling and p32-dependent trafficking. This is, to our
15
349
knowledge the first example that an anti-apoptotic effector protein is regulated by host
350
cell protein-mediated trafficking.
351
The question how effector proteins are regulated has only rarely been
352
investigated. There are several avenues of regulation thinkable: 1) regulation by time
353
point and dosage of translocation; 2) regulation by modulation through other effector
354
proteins; 3) regulation by host cell dependent modification (phosphorylation, lipidation,
355
sumoylation etc.). Examples already exist for the latter scenario. It was shown that
356
intracellular localization, and thereby the function of Legionella pneumophila effector
357
proteins containing a CAAX motif are affected by lipidation through the host cell
358
farnesyltransferase and class I geranylgeranyltransferase (39). The Helicobacter pylori
359
T4SS effector protein CagA is phosphorylated by the host cell tyrosine kinases Src and
360
Abl. Phosphorylated CagA can then modulate various signaling cascades associated
361
with cell polarity, cell proliferation, actin-cytoskeletal rearrangements, cell elongation,
362
disruption of tight and adherence junctions, pro-inflammatory responses and apoptosis
363
inhibition (40). Here, we have identified a fourth possibility to regulate the activity of
364
effector proteins: regulation by stress sensing and intracellular trafficking. In our opinion,
365
more knowledge about host cell requirements for regulation of effector proteins is
366
needed. This knowledge will not only help to understand microbial pathogenesis better,
367
but will also allow us to develop new strategies for therapy. The first steps down this
368
avenue have already been made. An exemplified study showed that identifying host cell
369
signaling pathways required for bacterial survival might help to control infection (41).
370 371
Acknowledgements
16
372
This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the
373
Collaborative Research Initiative 796 (SFB796; to A.L. and C.B.) and through the Priority
374
Programme SPP1580 (to A.L.) as well as by the ERA-NET PathoGenoMics 3rd call (to
375
A.L.). We thank Dr. Christian Bogdan for his valuable comments on the manuscript.
376 377
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378 379
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18. Voth DE, Howe D, Heinzen RA. 2007. Coxiella burnetii inhibits apoptosis in
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mitochondrial level. Cell. Microbiol. 15: 675-687.
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20. Lührmann A, Nogueira CV, Carey KL, Roy CR. 2010. Inhibition of pathogen-
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21. Dedio J, Jahnen-Dechent W, Bachmann M, Müller-Esterl W. 1998. The
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26. Beatch MD, Everitt JC, Law LJ, Hobman TC. 2005. Interactions between
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rickettsial pathogen Anaplasma phagocytophilum. PLoS Pathog. 5: e1000488.
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39. Ivanov SS, Charron G, Hang HC, Roy CR. 2010. Lipidation by the host
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725-730.
499 500
Figure legends
501
Figure 1: AnkG binds directly to the host cell protein p32 and does not alter its steady
502
state protein level. (A) Glutathione–sepharose columns with GST-AnkG or GST alone
503
were incubated with His-p32. Eluate (E1-E4) and bead (beads) fractions were resolved
504
by SDS-PAGE and stained with Coomassie blue. (B) Glutathione–sepharose columns
505
with GST-AnkG or GST alone were incubated with His-p32. Input, eluate (E1-E4) and
506
bead (beads) fractions were subjected to immunoblot analysis using anti-GST and anti-
507
p32 antibodies. (C) Ni-NTA agarose columns with His-p32 were incubated with GST or
508
with GST-AnkG. Eluate (E1-E4) and input were subjected to immunoblot analysis using 22
509
anti-GST and anti-His antibodies. (D) HEK293 cells were transfected with plasmids
510
encoding GFP or GFP-tagged AnkG. Protein extracts were separated by SDS-PAGE,
511
transferred to a PVDF membrane and probed with antibodies directed against GFP, p32
512
and actin. One representative immunoblot out of at least three independent experiments
513
is shown. (E) HeLa cells were transiently transfected with plasmids encoding GFP or
514
GFP-tagged AnkG. The cells were treated with Mitotracker (red) followed by fixation and
515
permeabilization. P32 was stained with a specific primary antibody and a secondary
516
dye405 labeled antibody (blue). Figure 2: Intracellular localization of AnkG. (A)
517
Representative
518
transfected with a plasmid encoding GFP-tagged AnkG (green). The cells were treated
519
with Mitotracker (red) followed by fixation, permeabilization and staining of the nuclei
520
with DAPI (blue). (B) CHO-FcR cells transiently transfected with GFP-tagged AnkG were
521
incubated with 2µM staurosporine. After the indicated time-points cells were fixed and
522
the intracellular localization of AnkG was analyzed in at least 100 transfected cells per
523
sample using confocal microscopy from eight independent experiments. * p< 0.001, n.s.
524
not significant (p=0.055) (C) Representative immunofluorescence micrographs show
525
CHO-FcR cells expressing GFP-tagged AnkG, -AnkG1-69 or -AnkG70-338 (green). The
526
cells were incubated with staurosporine followed by fixation, permeabilization and
527
staining of the nuclei with DAPI (blue).
528
Figure 3: Identification of the p32 binding site. (A and B) HEK293 cells were co-
529
transfected with plasmids encoding GFP-tagged p32 and the indicated HA-tagged AnkG
530
mutants (HA-tagged AnkF was used as negative control). The proteins were precipitated
531
from the cell lysates with an anti-GFP antibody. Immunoblot analysis was used to detect
532
p32 (anti-GFP) and Ank-proteins (anti-HA) in the lysates (pre-IP) and precipitates (IP).
immunofluorescence
micrographs
show
HeLa
cells
transiently
23
533
(C) Representative immunofluorescence micrographs show HeLa cells expressing GFP-
534
tagged AnkGR22/23S (green). The cells were fixed, permeabilized and stained with anti-
535
tubulin antibody (red) and DAPI (blue). (D) CHO-FcR cells expressing GFP-tagged
536
AnkGR22/23S were incubated with 2µM staurosporine. After the indicated time points cells
537
were fixed and the localization of AnkG was analyzed in at least 100 transfected cells
538
per sample from four independent experiments using confocal microscopy. n.s. not
539
significant
540
Figure 4: AnkG has to migrate into the nucleus to inhibit staurosporine-induced
541
apoptosis. (A) Representative immunofluorescence micrograph show CHO-FcR cells
542
expressing GFP-tagged NES-AnkG (green). The cells were treated with Mitotracker
543
(red) followed by fixation, permeabilization and staining of the nuclei with DAPI (blue).
544
(B) HEK293 cells were co-transfected with plasmids encoding the indicated GFP-tagged
545
AnkG mutants or GFP as negative control. The proteins were precipitated from the cell
546
lysates with an anti-GFP antibody. Immunoblot analysis was used to detect endogenous
547
p32 (anti-p32) or AnkG (anti-AnkG) in the lysates (pre-IP) and precipitates (IP). (C)
548
CHO-FcR cells expressing GFP-tagged NES-AnkG were incubated with 2µM
549
staurosporine. After the indicated time-points cells were fixed and the localization of
550
AnkG was analyzed in at least 100 transfected cells per sample from three independent
551
experiments using confocal microscopy. n.s. not significant (D) CHO-FcR cells
552
expressing GFP, GFP- AnkG or -NES-AnkG were treated with staurosporine for 4h. The
553
cells were fixed, permeabilized and the nuclei were stained with DAPI. The nuclear
554
morphology of at least 100 GFP expressing cells was scored in four independent
555
experiments. n.s. not significant * p< 0.02.
24
556
Figure 5: Neither AnkGR22/23S nor NES-AnkG can prevent pathogen-induced apoptosis
557
(A/B) Dendritic cells were infected with Legionella pneumophila ΔflaA containing the
558
indicated plasmids. The data shown are from one representative experiment of three
559
experiments with similar results. Shown is the bacterial uptake after 2 hours infection (A)
560
and the relative number of intracellular bacteria 10 hours after infection compared to the
561
2 hour value. **p= 0.001 (C) Representative immunofluorescence micrograph shows
562
CHO-FcR cells expressing GFP-NLS-AnkGR22/23S (green) incubated with mitotracker
563
(red) followed by fixation, permeabilization and staining of the nuclei with DAPI (blue).
564
(D) CHO-FcR cells expressing GFP, GFP-AnkG or GFP-NLS-AnkGR22/23S were treated
565
with 2µM staurosporine for 4h. After treatment the cells were fixed, permeabilized and
566
the nuclei were stained with DAPI. The nuclear morphology was scored of at least 100
567
GFP expressing cells in three independent experiments. * p< 0.01.
568 569
Tables
570 Plasmid AnkGFL-pCMV-HA AnkG∆Ank-pCMV-HA AnkG1-69-pCMV-HA AnkG70-338-pCMV-HA AnkG1-91-pCMV-HA AnkG50-338-pCMV-HA AnkG1-157-pCMV-HA AnkGR23S-pCMV-HA AnkGR22/23S-pCMV-HA AnkF-pCMV-HA pGEX-5X AnkG-pGEX-5X p32-pET16b pEGFP AnkG-pEGFP p32-pEGFP AnkG1-69-pEGFP AnkG70-338-pEGFP
Primer
329/339 331/340 307/310 590/591 590/592 79/229 184/185
329/338 332/340
Reference 20 20 20 20 This study This study This study This study This study This study Amersham 20 This study Clontech 20 20 This study This study
25
AnkGR22/23S-pEGFP NES-AnkG-pEGFP NLS-AnkGR22/23S-pEGFP pJV400 pJV400-AnkG pJV400-AnkGR22/23S pJV400-NES-AnkG
571
665/34 746/400 373/26 696/26
This study This study This study 20 20 This study This study
Table 1: Primer numbers are as in Table 2.
572 Number 26 34 79 184 185 229 307 310 329 330 331 332 338 339 340 373 400 590 591 592 665 696 746
573
Sequence 5' AAGGCGCGCCTCACCGAGGACTAGACAG 5’ AAGGATCCTCACCGAGGACTAGACAGA 5’ CCGGTACCCTACCGCTGGAAGCCGC 5’ CCCATATGCTGCACACCGACGGAGAC 5’ CCGGATCCCTACTGGCTCTTGACAAAACT 5’ CCGAATTCATGTGCAATACCAACATGTCT 5’ CCGGTACCATGAGTAGACGTGAGACTCC 5’ CCGGTACCTTATTTATATTTGATTTTCACATCAGC 5' CCAAGATCTCTATGAGTAGACGTGAGACTCC 5’ CCAAGATCTCTATGGGACATCCTGTAAGAAGAAG 5’ CCAAGATCTCTATGTCGTTTGAAATACTCATAAATGC 5’ CCAAGATCTCTATGCTTCGCGGGGATTCTTTTCA 5' CCGGTACCTCAGTAGTTTTTTATTATGCTCAAGCT 5’ CCGGTACCTCAGAAATCCGTCTTTGGCGGTA 5' CCGGTACCTCACCGAGGACTAGACAGA 5' CCGGCCGGCCATGAGTAGACGTGAGACTCC 5' CCGGTACCTCACCGAGGACTAGACAGA P-5’ CGTTGAGGATATTGTGCTAGTGGGAGTCTACGTCTAC TCAT P-5’ CGACAGGAACTCGAACGCCGAGAAGTAGATTGAGCC GAAAA P-5’CGACAGGAACTCGAACGCCGAGTAGTAGATTGAGC CGAAAA 5’ CCGGTACCGCCTCCAGCAGCCTCCCCTGGAGGACTGACCCT GAGTAGACGTGAGACTCCCACTAGC 5’ CCGGCCGGCCATGCTCCAGCTGCCTCCCC 5’ CCACTCAGATCTCTCCTAAGAAGAAAAGGAAGGTTAGT AGACGTGAGACTCCCACTAGCACAA
Site AscI BamHI KpnI NdeI BamHI EcoRI KpnI KpnI BglII BglII BglII BglII KpnI KpnI KpnI FseI KpnI KpnI FseI BglII
Table 2. Underlined denotes the location of the restriction site.
26
GST-AnkG
E1
E2
E3
E4
anti-GST anti-GFP
anti-HIS
HIS-p32 GST
D
nk G
GST-AnkG + HIS-p32
G FP -A
C
GST + His-p32
G FP
GST-AnkG + His-p32
E1 E2 E3 E4 Beads E1 E2 E3 E4 Beads
G ST -A n H IS kG -p 32
A
HIS-tag pull-down
anti-p32
input
anti-actin GST-tag pull-down
E G ST H is -p 3
2
B
p32
GFP
Mitotracker
Merge
p32
GFP-AnkG
Mitotracker
Merge
E1 E2 E3 E4 Beads anti-GST anti-p32 GST-tag pull-down 10µm
G ST H Ank is -p G 32
input
E1 E2 E3 E4 Beads anti-GST anti-p32
input
GST-tag pull-down
10µm
A GFP-AnkG
Mitotracker
Merge
Zoom
10µm
B % phenotype
125
*** ***
100
n.s.
75 50
Mitochondrial associa!on
25
nuclear
0 Mock
30
120
240
min staurosporine
GFP-AnkG
GFP-AnkG1-69
GFP-AnkG 70-338
Mock
C
10µm
10µm
Staurosporine
10µm
10µm
10µm
10µm
22
S
A
-A
nk G
R
23 R
W T
-A H
H
A
A H
H
nk G
nk G
nk F
-A
-A A
-A A
/2 3
S
S /2 3 22
S
nk G
R
22 R
W T
nk G
nk G
-A A
-A
nk F A
-A H
A H
H
50
nk G -A
A H
H
38 -3
38
B
70
-3
-3 29
-A A H
H
A
-A
nk G
nk G -A
A H
nk G
1-
91 1-
1-
H
A
-A
nk G
nk G -A
A H
69
7 15
nk ∆A
W T
nk G
nk G H
A
-A
-A A H
38
A
pre-IP
anti-HA anti-HA anti-GFP
pre-IP amino acid AnkG AnkG R23S AnkG R22/23S
18 19 20 21 22 23 24 25 26 27 28 T R T P R R R L S R K T R T P R S R L S R K T R T P S S R L S R K
IP
anti-HA
IP
D
n.s.
anti-GFP
GFP-AnkGR22/23S
αTubulin
100
% phenotype
C Merge
n.s. n.s.
75 50
Co-localiza!on with Tubulin
25
nuclear
0 Mock 10µm
30
120
240
min staurosporine
A GFP-NES-AnkG
Mitotracker
Merge
G FP -
G FP
G FP -
A nk G G FP -A nk G G FP R 22 -N / ES 23S -A nk G G FP
B
A nk G G FP -A nk G G FP R 22 -N / ES 23S -A nk G
10µm 10µm
anti-AnkG anti-p32
pre-IP
IP
C
n.s. n.s. n.s.
% phenotype
100 75 50 cytosolic 25
nuclear
0 Mock
30
120
min staurosporine
D
n.s.
% fragmented nuclei
50
*
40 30 20 10
240
-A ES -N FP G G nk
-A FP G
FP G
G nk
5
10 0
0
* * 20
10
0
Merge Mitotracker GFP-NLS-AnkG
G nk -A ES /23S -N 22 00 GR V4 nk pJ 0-A 0 G V4 nk pJ 0-A 0 V4 pJ 0 40
C
30
20
% survival
40
bacterial *103/well
10
V pJ
G nk -A ES /23S -N 22 00 GR V4 nk pJ 0-A 0 G V4 nk pJ 0-A 0 V4 pJ 0 40 V pJ
40
D
30
% fragmented nuclei
15
* 50 20
60
B n.s. 25
A
-A LS -N FP kG G An FP G FP G
G nk
R
22
3 /2
S
