toxoplasma gondiiinfection confers resistance against bims ......3512 of cytochrome c in infected...

3511Research Article

IntroductionApoptosis is one of the main defence mechanisms of highereukaryotes against intracellular pathogens. In mammalian hosts, itcan counteract intracellular infection after activation of deathreceptors of the tumour necrosis factor (TNF) superfamily (e.g.Fas/CD95, TNF-R1) or by the targeted release of perforin andgranzymes from granules of cytotoxic T lymphocytes and naturalkiller (NK) cells (Liebermann, 2003; Schütze et al., 2008). Microbialinfection might also provide a stress signal that triggers the intrinsicapoptotic pathway (Williams, 1994; Everett and McFadden, 1999;Labbé and Saleh, 2008). If the infected cell is induced to undergoapoptosis via extrinsic signals or commits ‘suicide’ as an innateresponse, the further development, multiplication and disseminationof the invader might be disturbed. Furthermore, the subsequentuptake and degradation of the apoptotic cell, including its microbialcargo by phagocytes, might initiate T-cell responses against thepathogen (Savill and Fadok, 2000). In order to circumvent theseobstacles, intracellular pathogens have evolved elaboratemechanisms to counteract host-cell apoptosis (Schaumburg et al.,2006).

Toxoplasma gondii is a widespread apicomplexan parasite ofwarm-blooded vertebrates, infecting up to one third of the world’s

population (Tenter et al., 2000). Infection of immunocompetentpatients is normally asymptomatic or causes mild disease, but canbe life-threatening in immunocompromised patients or afterinfection in utero. T. gondii actively invades a wide range of hostcells and subverts major cellular functions in its host (Plattner andSoldati-Favre, 2008), including apoptotic signalling (Lüder et al.,2009). Infection with T. gondii renders host cells resistant to a varietyof pro-apoptotic signals (Nash et al., 1998; Goebel et al., 1999;Goebel et al., 2001; Molestina et al., 2003; Carmen et al., 2006;Vutova et al., 2007; Hippe et al., 2008) and might also counteractthe innate ‘suicide’ program of parasite-infected cells (Lüder andGross, 2005).

We and others have shown previously that T. gondii inhibits therelease of cytochrome c from the intermembrane space of host-cellmitochondria into the cytosol in response to a pro-apoptotic trigger(Goebel et al., 2001; Carmen et al., 2006). Cytosolic cytochrome cinduces activation of a caspase cascade, which leads to the executionof the apoptotic death program (Srinivasula et al., 1998). Toxoplasma-mediated blockade of cytochrome c release therefore has beenconsistently assumed to be crucial for the avoidance of host-cell death.By contrast, opposing models have been proposed for the underlyingmechanisms that could mediate the block in mitochondrial release

In order to accomplish their life style, intracellular pathogens,including the apicomplexan Toxoplasma gondii, subvert theinnate apoptotic response of infected host cells. However, theprecise mechanisms of parasite interference with themitochondrial apoptotic pathway remain unknown. Here, weused the conditional expression of the BH3-only protein BimSto pinpoint the interaction of T. gondii with the intrinsicpathway of apoptosis. Infection of epithelial cells with T. gondiidose-dependently abrogated BimS-triggered release ofcytochrome c from host-cell mitochondria into the cytosol,induction of activity of caspases 3, 7 and 9, and chromatincondensation. Furthermore, inhibition of apoptosis in parasite-infected lymphocytes counteracted death of Toxoplasma-infectedhost cells. Although total cellular levels and mitochondrialtargeting of BimS was not altered by the infection, the activationof pro-apoptotic effector proteins Bax and Bak was stronglyimpaired. Inhibition of Bax and Bak activation by T. gondii was

seen with regard to their conformational changes, the cytosol-to-mitochondria targeting and the oligomerization of Bax butnot their cellular protein levels. Blockade of Bax and Bakactivation was not mediated by the upregulation of anti-apoptotic Bcl-2-like proteins following infection. Further, theBH3-mimetic ABT-737 failed to overcome the Toxoplasma-imposed inhibition of BimS-triggered apoptosis. These resultsindicate that T. gondii targets activation of pro-apoptotic Baxand Bak to inhibit the apoptogenic function of mitochondriaand to increase host-cell viability.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/122/19/3511/DC1

Key words: Apoptosis, Bax, BimS, Mitochondria, Parasite-hostinteraction, Toxoplasma

Summary

Toxoplasma gondii infection confers resistanceagainst BimS-induced apoptosis by preventing theactivation and mitochondrial targeting of pro-apoptotic BaxDiana Hippe1, Arnim Weber2, Liying Zhou3, Donald C. Chang3, Georg Häcker2 and Carsten G. K. Lüder1,*1Institute for Medical Microbiology, Georg-August-University, Kreuzbergring 57, D-37075 Göttingen, Germany2Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München, Trogerstrasse 30, D-81675 Munich, Germany3Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong*Author for correspondence ([email protected])

Accepted 27 July 2009Journal of Cell Science 122, 3511-3521 Published by The Company of Biologists 2009doi:10.1242/jcs.050963

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of cytochrome c in infected cells. These include activation of thephosphoinositide 3 kinase (PI3K) (Kim and Denkers, 2006) and thenuclear factor κB (NF-κB) pathways (Molestina et al., 2003; Payneet al., 2003), the upregulation of anti-apoptotic proteins of the Bcl-2 family (Mcl-1 and A1/Bfl-1) (Goebel et al., 2001; Molestina et al.,2003) and the degradation of proapoptotic Bcl-2 family members,Bax and Bad (Carmen et al., 2006).

A conclusive model for counteracting the mitochondrialapoptotic pathway by T. gondii has been elusive due to thecomplexity of upstream pathways that fuel the activation of pro-and anti-apoptotic proteins of the Bcl-2 family. This protein familycomprises three groups with different structural and functionalcharacteristics (Youle and Strasser, 2008). BH3-only proteins (e.g.Bim, Bid, Bad, PUMA) sense pro-apoptotic upstream signals andtheir upregulation or post-transcriptional activation precedespermeabilization of the outer mitochondrial membrane (MOMP)(Letai et al., 2002; Willis et al., 2007; Häcker and Weber, 2007).MOMP is a consequence of the oligomerization of the multidomainBcl-2 executioner proteins Bax and Bak, possibly via poreformation, thereby allowing the release of apoptogenic proteinsincluding cytochrome c into the cytosol (Jürgensmeier et al., 1998;Annis et al., 2005) (reviewed by Lalier et al., 2007). A third groupof multidomain anti-apoptotic family members (e.g. Bcl-2, Mcl-1, Bcl-xL) restrains the activity of Bax and Bak, and overexpressionof Bcl-2 blocks the mitochondrial apoptotic pathway (Yang et al.,1997; Fletcher et al., 2008).

Here, we provide a detailed analysis of events that regulatecytochrome c release and caspase activation in cells infected withT. gondii. Using mutant host cells that express the BH3-only proteinBimS under control of a tetracycline repressor-regulated promoter,we could pinpoint the step of parasite interference with themitochondrial apoptotic pathway to the activation of Bax and itstranslocation to the outer mitochondrial membrane. We also provide

evidence that this Toxoplasma–host-cell interaction counteracts thecell death of parasite-infected cells after activation of the cell-intrinsic pathway of apoptosis.

ResultsInhibition of BimS- and staurosporine-induced apoptosis byT. gondiiWe employed a tetracycline-inducible expression system of BimS(Weber et al., 2007) to determine the level of interference of T.gondii with the mitochondrial apoptotic pathway. Treatment withtetracycline for 3 hours led to chromatin condensation in 78.6±5.6%(mean ± s.e.m., n=3) of non-infected T-REx-HeLa/BimS cells (Fig.1B,C). The condensation of chromatin is characteristic for apoptoticcells and correlated with the induction of BimS by tetracycline (Fig.1A). Infection with T. gondii for 21 hours prior to conditionalexpression of BimS prevented chromatin condensation in ~55% ofthe total cell population (Fig. 1B,C) (P=0.001). The level ofreduction in BimS-induced apoptosis corresponded with infectionrates of 51±2% (mean ± s.e.m., n=2; data not shown) after additionof T. gondii at a parasite to host cell ratio of 20:1. Furthermore,immunofluorescence staining of T. gondii parasites in combinationwith Hoechst staining indicated that parasite-positive (Fig. 1B,arrows) but not parasite-negative cells (Fig. 1B, arrowheads) werelargely protected from BimS-induced apoptosis. Thus, infection withT. gondii efficiently inhibited host-cell apoptosis downstream ofthe BH3-only protein BimS. Chromatin condensation, as inducedin HeLa229 wild-type (HeLa-WT) cells by the kinase inhibitorstaurosporine, was inhibited by prior infection with T. gondii to asimilar extent as observed in tetracycline-treated T-REx-HeLa/BimScells (supplementary material Fig. S1).

Triggering of the death receptor pathway and the perforin-granzyme pathway of apoptosis can lead to rapid egress of T.gondii and, thereby, necrotic death of the host cell (Persson et al.,

Journal of Cell Science 122 (19)

Fig. 1. Infection with T. gondii inhibitsapoptosis triggered by inducible expression ofthe BH3-only protein BimS. T-REx-HeLa/BimS cells were infected with T. gondiiat a parasite-to-host cell ratio of 20:1 for 24hours or were left non-infected. During thefinal 3 hours, cells were treated (Tet) or not(wo Tet) with tetracycline to induce BimSexpression. (A) Expression of BimS aftertreatment of non-infected T-Rex-HeLa/BimScells with 1 μg/ml tetracycline was confirmedby western blotting using anti-Bim (BimEL,large isoform; BimS, small isoform) and anti-actin antibodies. (B,C) Cells were fixed andchromatin condensation was assessed afterHoechst 33258 staining. (B) In order tocompare BimS-induced chromatincondensation in parasite-positive (arrows) andparasite-negative host cells (arrowheads),T. gondii was simultaneously immunolabelled(red fluorescence). n.i., non-infected cells.(C) Bars represent the mean percentages ±s.e.m. of apoptotic cells from threeindependent experiments. Significantdifferences between infected and non-infectedcells are indicated (**P

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2007). In order to determine whether the activation ofmitochondrial apoptosis would also induce necrotic death ofToxoplasma-infected cells, we quantified cell death using stainingwith Annexin V and 7-AAD (7-aminoactinomycin D). Becausesurface labelling of phosphatidylserine by Annexin V on adherentcells is hampered owing to the isolation procedure, we used Jurkatcells for these experiments. After treatment with staurosporine,the percentage of Jurkat cells that stained positive for Annexin Vand negative for AAD (Annexin V+/7-AAD–) (early apoptotic)and to a lesser extent that were Annexin V+/7-AAD+ (lateapoptotic and/or necrotic) increased considerably (Fig. 2A,B).Prior infection with T. gondii dose-dependently decreased thenumber of Annexin V+/7- AAD– cells to background levels at thehighest infection ratio. Importantly, this was not accompanied bya concomitant increase in 7-AAD+ cells. We instead observednecrotic death of Jurkat cells as a consequence of T. gondiiinfection; however, the parasite-mediated increase of AnnexinV+/7-AAD+ cells was lower after pro-apoptotic stimulation thanin untreated controls. Consequently, the number of viable (i.e.Annexin V-/7-AAD– cells) increased in staurosporine-treatedJurkat cells by prior infection (Fig. 2A,B).

Using mutant T. gondii parasites expressing GFP we also showedthat triggering of the mitochondrial apoptotic pathway did not affectthe number of parasite-infected Jurkat cells (Fig. 2C,D).Furthermore, parasite-positive (GFP+) Jurkat cells showed a lowerproportion of Annexin V+ cells after staurosporine treatment thanparasite-negative cells from the same culture (Fig. 2C). Together,these results established that activation of the mitochondrialapoptotic pathway was counteracted by T. gondii, thus increasingthe viability of parasite-infected host cells.

Inhibition of BimS-triggered caspase activation by T. gondiiThe mitochondrial apoptotic pathway crucially depends on caspase9, which in turn activates the executioner caspases 3, 6 and 7.Tetracycline-induced expression of BimS in T-REx-HeLa/BimS cellsstrongly activated caspase 9 (Fig. 3A) and caspases 3 and 7 (Fig.3B). Prior infection of the cells with increasing numbers of T. gondiidose-dependently inhibited BimS-induced caspase 9 (P

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tetracycline-treated cells that had been previously infected with theparasite. Control staining with an antibody recognizing cytochromec oxidase (COX) excluded a contamination of cytosolic fractionswith mitochondria (Fig. 4A). In addition, immunolabelling of actinindicated a similar protein content of the cytosolic and organellarfractions, respectively (Fig. 4A). The redistribution of cytochromec from mitochondria to the cytosol was also strongly decreased afterparasitic infection of HeLa-WT cells or Jurkat cells that had beentreated with staurosporine (supplementary material Fig. S2; and datanot shown). This suggested that the step of parasite interferencewith the mitochondrial pathway was the same after conditional BimSexpression as after staurosporine treatment. We also confirmed thatinfection of T-REx-HeLa/BimS cells with T. gondii did not affect

the conditional expression of BimS (Fig. 4D). Together, these dataindicate that T. gondii blocks the mitochondrial apoptotic pathwayat a step between BimS expression and cytochrome c release.

Because the activity of effector caspases might amplify upstreamregulatory events of the mitochondrial apoptotic pathway (Lakhaniet al., 2006), the decrease of BimS-induced cytosolic cytochromec following parasite infection might also be mediated by differentialactivities of downstream caspases in infected and non-infected cells.To unambiguously exclude this possibility, the subcellularredistribution of cytochrome c was evaluated in the presence ofpan-caspase inhibition. The results showed that infection of T-REx-HeLa/BimS cells with T. gondii also inhibited the redistribution ofcytochrome c into the cytosol in the presence of 20 μM of the


Fig. 3. BimS-triggered proteolyticprocessing and activities of initiator andeffector caspases are dose-dependentlyreduced by T. gondii infection. T-REx-HeLa/BimS cells infected with T. gondii atparasite to host cell ratios of 10:1 (+) or20:1 (++) and non-infected controls (–)were induced to express BimS usingtetracycline. Protein extracts were tested intriplicates for the activities of caspase 9(A) and caspases 3 and 7 (B) byfluorimetrically measuring the cleavage ofLEHD-AMC or DEVD-AMC,respectively. Data represent the meancleavage activities ± s.e.m. from fourindependent experiments. Significantdifferences between T. gondii-infected andnon-infected cells are marked (*P


caspase inhibitor z-VAD-fmk (Fig. 4C). z-VAD-fmk at thatconcentration completely abrogated the DEVD-AMC cleavageactivity of effector caspases 3 and 7 in response to staurosporine(Fig. 4B). We noticed in these experiments that there was somemitochondrial cytochrome c release in non-infected cells afterz-VAD-fmk treatment. Such redistribution, as well as that observedafter tetracycline-induced BimS expression, was neverthelessabrogated by T. gondii. This confirmed that T. gondii interferes withthe mitochondrial apoptotic pathway upstream of MOMP, even ifdownstream caspases are inhibited.

Parasite interference with activation of Bax and BakIntrinsic proapoptotic signals (including the expression of BimS)converge on the activation of the effectors of the Bcl-2 family, i.e.Bax and/or Bak. Therefore, we evaluated the activation of Bax andBak in infected cells and non-infected controls using antibodies thatspecifically recognize an epitope within the N-terminus of theproteins (Bax-NT and Bak-NT, respectively). This epitope has beenshown to be exposed during activation of Bax and Bak (Hsu andYoule, 1997; Lalier et al., 2007). Flow cytometry revealed activationof Bax in ~50% of non-infected T-REx-HeLa/BimS cells after

expression of BimS whereas this was significantly diminished inparasite-infected cells (Fig. 5A,B) (P

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Conditional expression of BimS led to a strong labelling of themajority of non-infected T-REx-HeLa/BimS cells using a Bax-NT-specific antibody (Fig. 5E). The punctate staining pattern wasconsistent with a mitochondrial localization of active Bax (Wolteret al., 1997; Gross et al., 1998) (see also below). By contrast, priorinfection with T. gondii largely abolished the tetracycline-inducedBax-NT reactivity. Parasite-positive T-REx-HeLa/BimS cells wereparticularly protected from Bax activation (Fig. 5E, arrows)whereas parasite-negative cells of infected cell populations werestrongly reactive (Fig. 5E, arrowhead). This suggested thatintracellular parasites are able to block BimS-induced Baxactivation effectively.

Inhibition of mitochondrial targeting and oligomerization of BaxInactive Bax is mainly located in the cytosol and – duringactivation – translocates to mitochondria where it oligomerizes andleads to MOMP (Wolter et al., 1997; Zhou and Chang, 2008). Wefollowed the cellular redistribution of a yellow fluorescent protein(YFP)-Bax fusion construct in T-REx-HeLa/BimS cells. In theabsence of tetracycline, YFP-Bax was diffusely distributed in thecytosol and the nuclei of non-infected cells and this did not changeafter T. gondii-infection (Fig. 6A). After conditional expression ofBimS, clusters of YFP-Bax became apparent in non-infected T-REx-HeLa/BimS cells and correlated with condensed chromatin,as visualized by propidium iodide. The punctate distribution ofYFP-Bax strongly indicated translocation to the mitochondria asshown previously (Zhou and Chang, 2008). Importantly, priorinfection with T. gondii prevented BimS-induced redistribution ofYFP-Bax in parasite-positive cells, whereas YFP-Bax redistributed

in parasite-negative cells of an infected culture in the same wayas in non-infected control cultures (Fig. 6A). Essentially the sameresults were obtained after treatment of T. gondii-infected and non-infected HeLa-WT cells with staurosporine (supplementarymaterial Fig. S3A). In order to exclude any impact of the YFPreporter, we also determined the effect of parasite infection onendogenous Bax in HeLa-WT cells treated or not withstaurosporine. Endogenous Bax was evenly located in the cytosolof untreated control cells (supplementary material Fig. S3B).Remarkably, it appeared more punctate than YFP-Bax, indicatingthat in healthy cells Bax might weakly associate with intracellularmembrane-bound structures including mitochondria as shownpreviously (Antonsson et al., 2001). However, a pro-apoptoticstimulus induced the redistribution of endogenous Bax into largeclusters that were unevenly distributed within non-infected cells,whereas this was prominently inhibited in Toxoplasma-infectedcells (supplementary material Fig. S3B). Quantification of cellswith a punctate Bax staining pattern confirmed that parasiticinfection significantly inhibited the subcellular redistribution ofendogenous Bax following treatment with staurosporine(supplementary material Fig. S3C) (P=0.009).

To verify that T. gondii interferes with activation of Bax, we alsodetermined its dimerization-oligomerization, which is a prerequisitefor MOMP (Annis et al., 2005; Antonsson et al., 2001). Conditionalexpression of BimS led to the formation of Bax dimers and trimersin non-infected control cells, as revealed after cross-linking withEGS (Fig. 6B). Minor amounts of Bax dimers in cells not treatedto express BimS suggested spontaneous oligomerization of Bax toa limited extent or some leakiness of the tetracycline-regulated


Fig. 6. BimS-induced intracellular redistribution of YFP-Bax chimera and oligomerization of WT-Bax are inhibited after infection with T. gondii. T-REx-HeLa/BimS cells transfected with an YFP-Bax reporter construct (A) or those expressing WT-Bax (B) were infected with T. gondii at a parasite to host cell ratio of20:1 or were left non-infected (n.i.). During the last 3 hours of the infection period (24 hours), cells were induced to express BimS using tetracycline (Tet). (A) Cellswere fixed and labelled with propidium iodide and T. gondii-specific antiserum and appropriate secondary antibody. Representative images (two independentexperiments) of each labelling were obtained by confocal laser scanning microscopy and superimposed (overlay). (B) Alternatively, cells were lysed and proteinextracts treated with EGS in order to preserve protein-protein interactions. EGS-treated cell extracts and untreated controls (wo EGS) were separated by SDS-PAGE and analysed by immunoblotting using antibodies recognizing Bax or actin. Scale bars: 12 μm.

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promoter. Importantly, infection with T. gondii almost completelyabolished the formation of dimers and trimers (Fig. 6B). Controlwestern blots of proteins not cross-linked with EGS confirmed thatT. gondii does not alter the total cellular content of Bax, as alreadyindicated (Fig. 6B; Fig. 5D).

Inhibition of cytochrome c release is independent of de novosynthesis and activity of anti-apoptotic Bcl-2 proteinsUpregulation of anti-apoptotic Bcl-2-like proteins followinginfection with T. gondii could keep Bax or Bak in an inactivestate. To test this hypothesis the release of cytochrome c into thecytosol was evaluated in the presence of actinomycin D. Becausethe transcription inhibitor would also prevent the upregulation ofBimS by tetracycline, we used in these experiments HeLa-WT cellsthat were treated with staurosporine. The results showed that T.gondii inhibited the translocation of cytochrome c in the presenceof actinomycin D as efficiently as in its absence (Fig. 7A). RT-PCR analyses of interleukin 2 (IL-2) transcripts from Jurkat cellsthat had been activated by phytohemagglutinin (PHA) and phorbol12-myristate 13-acetate (PMA) confirmed a complete blockadeof transcription by actinomycin D (Fig. 7B). As describedpreviously (Goebel et al., 2001), treatment with actinomycin Dalone also led to the accumulation of cytochrome c in the cytosolof non-infected cells, whereas this was prevented in HeLa cellspreviously infected with T. gondii (Fig. 7A). The results indicatedthat blockade of mitochondrial cytochrome c release after parasiteinfection generally does not depend on the transcription machinery

of the host cell. This is further supported by the finding thatinfection of T-REx-HeLa/BimS cells with T. gondii at increasingmultiplicities-of-infection (MOIs) did not upregulate the totalcellular levels of Bcl-2, Mcl-1, Bcl-x and A1/Bfl-1 as comparedto non-infected controls (Fig. 7C).

In order to further exclude an impact of anti-apoptotic Bcl-2family proteins during T. gondii-mediated inhibition of apoptosis,we employed the BH3 mimetic ABT-737. ABT-737 is a cell-permeable small compound that binds to the hydrophobic Bcl-2homology 3 (BH3)-binding groove of Bcl-2, Bcl-XL and Bcl-w,thereby abrogating their anti-apoptotic activity and priming cellsfor death (Oltersdorf et al., 2005). Remarkably, treatment of T-REx-HeLa/BimS cells with 0.1 or 1 μM ABT-737 dose-dependently activated effector caspases 3 and 7 in non-infectedcells whereas infection with T. gondii completely abrogated suchactivity (Fig. 7D). The enantiomer (i.e. the stereoisomer)exhibited little activity even at the highest concentration used(Fig. 7D, open bars). ABT-737 at 1 μM also enhanced caspaseactivity that had been induced in non-infected cells bytetracycline-induced BimS expression. By contrast, it did notsensitize T. gondii-infected cells for increased BimS-inducedactivation of caspases 3 and 7 (Fig. 7D), indicating that theparasite inhibited the mitochondrial apoptotic pathwayindependently of anti-apoptotic Bcl-2, Bcl-XL or Bcl-w.Treatment of Toxoplasma-infected and non-infected HeLa-WTcells or Jurkat cells with ABT-737 alone or together withstaurosporine yielded similar results (data not shown).

Fig. 7. Inhibiton of Bax and Bak activation by T. gondii occurs independently of de novo host-cell transcription of anti-apoptotic Bcl-2 family members and is notabrogated by the BH3 mimetic ABT-737. Jurkat (A,B) or T-REx-HeLa/BimS cells (C,D) were infected with T. gondii at parasite to host cell ratios of 30:1 (A, ++) or10:1 (C, +) and 20:1 (C, ++; D, cross-hatched bars), respectively, and were treated with staurosporine (Stau) or tetracycline (Tet). Non-infected controls were run inparallel (A,C, –; D, black bars). (A,B) Host-cell transcription was inhibited by 20μg/ml actinomycin D starting at 1 hour prior to parasitic infection (A) or at 1 hourprior to activation of Jurkat cells with PMA along with PHA (B). (D) Alternatively, cells were incubated with increasing amounts of ABT-737 (0, 0.1, or 1μM) or itsenantiomer (E, 1μM) starting at 2 hours prior to tetracycline treatment. (A) Digitonin-soluble and digitonin-insoluble fractions containing cytosolic andmitochondrial proteins, respectively, were separated by SDS-PAGE and analysed by immunoblotting using primary antibodies directed against cytochrome c (Cyt c),COX or actin. (B) The abrogation of host-cell transcription by treatment with actinomycin D was confirmed by RT-PCR analyses of IL-2 and β-actin mRNA.(C) Complete cellular extracts were analysed for levels of anti-apoptotic Bcl-2 family members by SDS-PAGE and western blotting. (D) Protein extracts were testedin triplicates for the activities of caspases 3 and 7 by fluorimetrically measuring the cleavage of DEVD-AMC. Data represent the mean cleavage activities ± s.e.m.

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Redistribution of Bax is inhibited despite mitochondriallocalization of BimSWe have shown recently that triggering of apoptosis by BimSrequires its mitochondrial localization (Weber et al., 2007). Tofurther elucidate the interference of T. gondii with BimS-triggeredapoptosis we determined the subcellular distribution of BimS, Baxand cytochrome c. Following treatment of T-REx-HeLa/BimS cellswith tetracycline, BimS levels increased within 2 hours and werefurther upregulated until 4 hours of treatment (Fig. 8). Importantly,it exclusively localized to the mitochondria-containing fraction andthis was not altered in Toxoplasma-infected cells as compared touninfected controls. By contrast, Bax redistributed from the cytosolto the mitochondria in non-infected but not T. gondii-infected cells(Fig. 8), thus confirming the analyses done by immunofluorescencemicroscopy (see above). This clearly showed that T. gondii infectionlargely prevented Bax translocation to the mitochondria, whereasmitochondrial BimS targeting was not affected by the parasite.Colocalization of BimS and Bax in the heavy-organelle fraction ofnon-infected cells coincided with an increasing release ofcytochrome c into the cytosol (Fig. 8). By contrast, MOMP andcytochrome c release was largely prevented in T. gondii-infectedcells.

DiscussionWe demonstrate here that Toxoplasma inhibits the mitochondrialapoptotic pathway via interference with mitochondrial translocationand/or activation of the effector molecules Bax and Bak. This isachieved without altering the levels of pro- or anti-apoptoticregulators of the host, thus suggesting direct parasite interferencewith Bax and Bak. Remarkably, the mitochondrial localization ofthe BH3-only protein BimS did not suffice to recruit Bax to theouter mitochondrial membrane nor to induce its activation inparasitized host cells. Finally, triggering of the intrinsic pathway ofapoptosis does not induce cell death of parasite-infected cells, thusincreasing the viability of host cells.

We pinpointed the level of manipulation of the mitochondrialapoptotic pathway by T. gondii by using the conditional expressionof BimS. This BH3-only protein is the shortest out of three main

Bim splice variants and is normally expressed at only low levels(O’Connor et al., 1998). The latter might be due to the fact thatBimS – in contrast to its longer counterparts – is considered to beconstitutively active and not subject to any post-translationalregulation (Marani et al., 2002; Weber et al., 2007). By employingthe enforced expression of BimS, we could thus largely exclude anyimpact of T. gondii on the various mechanisms of post-translationalregulation of BH3-only proteins, i.e. phosphorylation, proteolyticcleavage or sequestration (see Youle and Strasser, 2008).Furthermore, we also excluded a parasite impact on the variousupstream pathways that sense cellular stress signals and integratethem into a core signal at the level of the BH3-only proteins.Remarkably, T. gondii was nevertheless capable of efficientlyinhibiting BimS-induced apoptosis, indicating parasite interferencedownstream of BimS expression. Because we extended the previousfindings of a parasite-mediated blockade of cytochrome c releasefollowing pro-apoptotic signalling (Goebel et al., 2001; Carmen etal., 2006) to BimS-induced apoptosis we could narrow down thelevel of parasite interference to a step between BimS expressionand MOMP. This notion was further substantiated using pan-caspaseinhibition. It excluded the possibility that differential activities ofeffector caspases in the presence or absence of T. gondii (Keller etal., 2006) were responsible for the parasite-mediated blockade ofpro-apoptotic signalling upstream of MOMP via a feed-backregulatory pathway.

It has to be stressed that the mechanisms we describe do notseem to be restricted to BimS-induced apoptosis because similarresults were observed after infection of wild-type HeLa and/or Jurkatcells that had been treated with staurosporine. We therefore proposethat it is a general mechanism of T. gondii to inhibit both themitochondrial apoptotic pathway induced by various cell-intrinsicstress signals (Nash et al., 1998; Goebel et al., 1999; Goebel et al.,2001; Kim and Denkers, 2006) and also the mitochondrialamplification loop following death receptor ligation (Hippe et al.,2008). Importantly, quantification of cell-death phenotypes ofparasite-infected Jurkat cells established that activation of theintrinsic pathway of apoptosis did not induce necrotic death ofparasite-infected host cells. Following ligation of death receptorsor targeted release of the content of cytotoxic granules from NKor T lymphocytes, including perforin and granzymes, Persson et al.recently described a Ca2+-dependent Toxoplasma egress andsubsequent lytic death of the host cell (Persson et al., 2007). It wastherefore proposed that absence of apoptotic markers after infectionwith Toxoplasma could also be due to a necrotic cell death ofparasite-infected host cells (Persson et al., 2007). However, severalindependent lines of evidence argue against this possibility occurringafter triggering of the mitochondrial apoptotic pathway. Neither didthe number of parasite-infected cells decrease after pro-apoptoticstimulation, nor did the percentage of cells that died by necrosisspecifically increase after treatment of parasite-infected cells withstaurosporine. Conversely, the number of viable parasite-infectedcells was augmented due to an anti-apoptotic activity, whichsuggests that this parasite-host interaction might indeed facilitatethe intracellular development of the parasite. Furthermore, cytosoliclevels of various proteins investigated in this study were unaffectedin Toxoplasma-infected cells treated to undergo apoptosis. Finally,the morphology of parasite-infected host cells treated with pro-apoptotic stimuli was inconsistent with necrotic cell death. Thus,induction of necrosis due to T. gondii egress from host cells can bediscounted as contributing considerably to the blockade of apoptosisthat was induced via the mitochondrial signalling cascade.


Fig. 8. T. gondii inhibits cellular redistribution of Bax without affectingmitochondrial levels of BimS. T-REx-HeLa/BimS cells were infected with T.gondii at a parasite to host cell ratio of 20:1 for 24 hours (+) or were left non-infected as indicated. Cells were treated for 2 or 4 hours with tetracycline toinduce expression of BimS or were left untreated (0). Digitonin-soluble anddigitonin-insoluble fractions containing cytosolic and mitochondrial proteins,respectively, were separated by SDS-PAGE and analysed by immunoblottingusing primary antibodies directed against Bim, Bax, cytochrome c (Cyt c),COX or actin.

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Bax and/or Bak are crucial in permeabilizing the outermitochondrial membrane. Following Toxoplasma infection, eventsthat preceded MOMP induced by BimS expression were prominentlyinhibited. This included the conformational change of Bax and Bak,translocation of a YFP-Bax reporter as well as endogenous wild-type Bax from the cytosol to mitochondria, and the oligomerizationof Bax. Importantly, the total protein levels of Bax and Bak werenot altered after parasitic infection. This indicated that Toxoplasmatargets the functionality of Bax and Bak rather than decreasing theirexpression and, thereby, contradicted a previous report (Carmen etal., 2006). Because a quantification of the cellular Bax levels wasnot provided in that study and the functional consequences on Baxactivation were not delineated (Carmen et al., 2006), the impact ofBax on the reduced cytochrome c release following Toxoplasmainfection remained elusive. One might also speculate that the useof different Toxoplasma strains (type I RH in the study by Carmenand coworkers; type II NTE in the current study) explains thisdiscrepancy because both lineages have been shown to differentiallymodulate host-cell signalling (Saeij et al., 2007; Kim et al., 2006).The results presented herein nevertheless provide strong evidencethat Toxoplasma is capable of exclusively inhibiting the activationof Bax and Bak without reducing their total protein levels.

In healthy cells, Bax is largely cytosolic and has to translocateto mitochondria in order to initiate MOMP (Wolter et al., 1997;Zhou and Chang, 2008). In addition, changes of the proteinconformation, including exposure of an N-terminal domain (Hsuand Youle, 1997; Lalier et al., 2007), accompany mitochondrialtranslocation although the exact order of events is unknown. Incontrast to Bax, inactive Bak is constitutively bound to mitochondriaand a conformational change might represent the first step duringactivation. Blockade of BimS-mediated subcellular redistributionof Bax, as well as exposure of a normally hidden N-terminal epitopeof Bax and Bak in T. gondii-infected cells, collectively pointstowards parasite interference with an initial trigger of activation.Of note, a common signal thus appears to initiate conformationalchanges of Bak at the mitochondrial membrane as well astranslocation plus conformational changes of Bax because bothprocesses were efficiently inhibited by Toxoplasma infection.Whereas the nature of this trigger has not yet been conclusivelyidentified, two opposing models are controversially discussed(Youle and Strasser, 2008; Chipuk and Green, 2008).

The ‘displacement model’ proposes that Bax and Bak arerestrained inactive by anti-apoptotic Bcl-2-like proteins and areunleashed when the inhibitors are bound by BH3-only proteins(Willis et al., 2007; Youle and Strasser, 2008). Thus, upregulationof anti-apoptotic Bcl-2-like proteins following infection providesone possible means to counteract the mitochondrial apoptoticpathway. However, several independent lines of evidence argueagainst this possibility. First, cytochrome c release was efficientlyinhibited by T. gondii in the absence of transcriptional activity bythe host cell; second, the levels of Bcl-2, Mcl-1, Bcl-XL and A1were not altered in T. gondii-infected cells as compared to non-infected controls; and third, T. gondii inhibited BimS-mediatedcaspase activity in the presence of the BH3 mimetic ABT-737 asefficiently as in its absence. Together, these data provide clearevidence that upregulation of anti-apoptotic Bcl-2 family membersafter T. gondii infection is not required to prevent host-cell death.

The second model, the ‘direct activation model’, postulates thatbinding by distinct BH3-only proteins, specifically Bim, truncatedBid (tBid) and Puma activates Bax and Bak (Chipuk and Green,2008; Häcker and Weber, 2007). However, after enforced BimS

expression as employed in this study, BimS quantitatively localizedinto the heavy-organelle fraction containing mitochondria, whereasBax stayed in the cytosol and only translocated to mitochondriaduring prolonged BimS expression. It is thus completely unclearhow mitochondrial BimS could directly activate cytosolic Bax totranslocate to the outer mitochondrial membrane.

Of particular interest, the mitochondrial targeting of BimS wascompletely unaffected in T. gondii-infected cells, whereastranslocation and activation of Bax was at the same time efficientlyinhibited (see above). Thus, mitochondrial localization of BimS mightindeed be required to induce MOMP as reported (Weber et al., 2007),but does not suffice to recruit and activate Bax in T. gondii-infectedcells as shown herein. The blockade of Bax activation andredistribution can either result from direct binding of a parasite effectormolecule to Bax (and Bak), or else from parasite interference withthe triggering of Bax and Bak activation. After immunoprecipitation,we were unable to detect parasite proteins associated with Bax orBak when using a polyclonal anti-Toxoplasma serum (data notshown). Furthermore, preservation of protein complexes using EGSfailed to detect an increase in the molecular weight of Bax followinginfection.

Although these data do not exclude the direct binding of a parasiteprotein to Bax and Bak, they support the view that BimS-inducedapoptosis might rely on two distinct signals: expression ofmitochondria-targeted BimS, and the translocation and activationof Bax (the trigger remaining elusive). The fact that T. gondii targetsexclusively Bax (and Bak) activation, but not BimS might representa valuable model for identifying those mechanisms that initiate Baxand Bak activity, recently referred to as the ‘holy grail’ of apoptosisresearch (Youle and Strasser, 2008). In addition, futurecharacterization of the parasite effector molecules and how theyblock the activation of Bax and Bak will provide important insightsinto the interaction of an intracellular pathogen and its host cell.This knowledge might also be applicable to the development ofnovel therapies against apicomplexan parasites.

Materials and MethodsCells, transfection and cultureHeLa229 wild-type (HeLa-WT) cells were cultivated in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS),100 U/ml penicillin, 10 μg/ml streptomycin, 1 mM sodium pyruvate and 1% non-essential amino acids (complete DMEM). T-REx-HeLa cells (Invitrogen) that stablyexpress the BH3-only protein BimS under control of the tetracycline repressor fromthe pCDNA6/TR vector have been described previously (Weber et al., 2007) and werecultivated in complete DMEM supplemented with 5 μg/ml blasticidin and 125 μg/mlzeocin. HeLa-WT and T-REx-HeLa/BimS cells were transiently transfected with 2 μgof the pEYFP-C1 vector (Clontech) containing the human bax gene (Zhou and Chang,2008) using FuGene HD transfection reagent (Roche). For some experiments, JurkatE6.1 T lymphoblasts were cultivated in RPMI (Roswell Park Memorial Institute)medium supplemented with 10% FCS and penicillin and streptomycin.

Parasite infection and induction of apoptosisParasites of the mouse-avirulent type II strain NTE [which was used in all experimentsunless otherwise stated (Gross et al., 1991)] were propagated in L929 fibroblasts ashost cells in RPMI supplemented with 1% FCS and penicillin and streptomycin (asabove). For some experiments, a GFP-expressing mutant of the mouse-virulent typeI strain RH [RH-LDM (Barragan and Sibley, 2002); kindly provided by AntonioBarragan, Karolinska Institutet, Stockholm, Sweden] was used. Parasites wereseparated from host cells by differential centrifugation (Vutova et al., 2007). AdherentHeLa-WT and T-REx-HeLa/BimS cells were infected at parasite-to-host cell ratiosof 10:1 or 20:1, routinely yielding infection rates of 42±1 and 51±2%, respectively,at 24 hours after infection. In order to compensate for the lower infectivity ofsuspension cells, Jurkat cells were infected at a parasite to host cell ratio of 30:1,which led to 54.3±2.5% parasite-positive cells. If not indicated otherwise, infectedcells and non-infected controls were induced to express BimS using 1 μg/mltetracycline or were treated with 1 μM staurosporine at 21 hours post-infection foran additional 3 hours. In some experiments, 20 μM of the pan-caspase inhibitor z-VAD-fmk or 20 μg/ml of actinomycin D was added starting at 1 hour prior to infection.

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The BH3 mimetic ABT-737 or its enantiomer (Oltersdorf et al., 2005) (kindly providedby Steven Elmore, Abbott’s Pharmaceutical Research and Development Division,Abbott Park, IL) was added at 0.1 or 1 μM at 2 hours prior to the induction of apoptosis.

Morphological detection of apoptosisTo assess the condensation of chromatin, T. gondii-infected or uninfected cells werefixed in 4% paraformaldehyde, 0.1 M sodium cacodylate, pH 7.4 for 30 minutes andstained with 50 ng/ml Hoechst 33258 in phosphate-buffered serum (PBS) for 1 hour.In some experiments, parasites were simultaneously visualized using a rabbit anti-Toxoplasma hyperimmune serum and Cy3-conjugated F(ab�)2 fragment anti-rabbitIgG as described below. After being washed and mounted with Mowiol (Calbiochem),at least 500 cells per sample were examined by fluorescence microscopy.

Flow cytometryCell death following induction of apoptosis and infection with T. gondii was quantifiedby flow cytometry. To this end, infected or non-infected Jurkat cells were washedtwice in PBS and then stained with R-phycoerythrin-conjugated Annexin V and 7-aminoactinomycin D (AAD) using the Apoptosis Detection Kit I as recommended(BD Biosciences, Heidelberg, Germany). Cells were analysed using a FACSCaliburflow cytometer (BD Biosciences).

In order to quantify Bax and Bak, T. gondii-infected cells or non-infected controlswere washed in PBS and fixed with 0.5% paraformaldehyde in PBS for 30 minuteson ice. They were then incubated in 50 μg/ml digitonin, 1% FCS in PBS containing10 μg/ml of conformation-specific mouse monoclonal anti-Bax-NT (clone 6A7; BDPharmingen) or anti-Bak-NT (clone TC-100; Calbiochem). Staining with irrelevantisotype control antibody was performed in parallel. After 30 minutes on ice, cellswere washed and then incubated with R-phycoerythrin-conjugated goat anti-mouseIgG before analysis.

Fluorometric caspase activity assaysCaspase activities were determined as described (Vutova et al., 2007). Briefly, infectedand non-infected cells (1�106 cells per sample) were extracted with 50 μl of 1%Nonidet P40, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, and complete protease inhibitorcocktail (EDTA-free; Roche). Aliquots (10 μl) of the supernatants were mixed intriplicates with 90 μl of either 10 μM Ac-DEVD–amino-4-methyl-coumarin (AMC;caspases 3 and 7-specific; Bachem) or 50 μM Ac-LEHD-AMC (caspase-9-specific;Bachem) in 0.1 mg/ml BSA, 0.1% CHAPS, 10 mM HEPES, 50 mM NaCl, 40 mMβ-glycerophosphate, 2 mM MgCl2 and 5 mM EGTA, pH 7.0. The kinetics of substratecleavage was recorded over a period of 60 minutes at 37°C using a Victor V multilabelcounter (Perkin Elmer).

Protein extraction, subcellular fractionation and western blotanalysesTo obtain total cellular lysates, Toxoplasma-infected cells or uninfected controls werelysed in 1% Triton X-100, 150 mM NaCl, 50 mM Tris/HCl pH 8.0, 50 mM NaF, 5mM sodium pyrophosphate, 1 mM PMSF, 1 mM EDTA, 1 mM sodium orthovanadate,and 5 μg/ml each of leupeptin, aprotinin and pepstatin. The subcellular distributionof cytochrome c and Bcl-2 family members was determined after separation of cellsinto digitonin-soluble and digitonin-insoluble fractions (Goebel et al., 2001). Briefly,cells (1�106 cells per sample) were resuspended in PBS, and mixed with an equalvolume of 150 μg/ml digitonin in 0.5 M sucrose. After 30 seconds, heavy organelles,including mitochondria, were pelleted at 14,000 g for 1 minute and supernatants savedas the cytosol-containing digitonin-soluble fraction. The digitonin-insoluble pelletswere then extracted as described above. After centrifugation, soluble proteins of totalcellular extracts or subcellular fractions were separated by standard SDS-PAGE andtransferred to nitrocellulose by semidry blotting. Nonspecific binding sites wereblocked using 5% dry skimmed milk, 0.2% Tween-20, 0.02% NaN3 in PBS, pH 7.4.Membranes were probed overnight at 4°C with rabbit polyclonal anti-caspase-9(directed against the p35 fragment), rabbit polyclonal anti-Mcl-1, rabbit polyclonalanti-A1/Bfl-1 (all from Santa Cruz Biotechnology), goat polyclonal anti-caspase-3(R&D Systems), rabbit polyclonal anti-Bim, mouse monoclonal anti-Bax (clone 3),mouse monoclonal anti-Bak (clone G317-2), mouse monoclonal anti-Bcl-2 (clone4D7), mouse monoclonal anti-Bcl-X (clone 2H12), mouse monoclonal anti-cytochrome c (clone 7H8.2C12; all from BD Pharmingen), mouse monoclonal anti-actin (clone C4; kindly provided by James Lessard, Children’s Hospital MedicalCenter, Cincinnati, OH), or mouse monoclonal anti-COX subunit IV (clone 10G8-D12-C12; Molecular Probes) diluted in 5% dry skimmed milk, 0.05 % Tween-20 inPBS, pH 7.4. Immune complexes were labelled with horseradish-peroxidase-conjugated anti-mouse, anti-rabbit or anti-goat IgG (Dianova) and visualized by ECLchemiluminescence.

Cross-linking of protein complexesIn order to preserve oligomeric Bax complexes, cells were extracted using 2% CHAPS,137 mM NaCl, 10 mM HEPES, 2 mM EDTA, complete protease inhibitor cocktailand 1 mM sodium orthovanadate, pH 7.4 for 30 minutes on ice. Soluble proteinswere cross-linked with 1 mM ethylene glycol-bis(succinimidylsuccinate) (EGS) for30 minutes at room temperature. The reaction was stopped by adding Tris-HCl, pH

7.4, to a final concentration of 20 mM. Protein extracts were analysed byimmunoblotting as described above.

RT-PCRBlockade of transcription by actinomycin D was assessed by analysing IL-2transcripts after activation of Jurkat cells for 24 hours with 5 nM PMA and 1 μg/mlPHA. Briefly, total RNA was isolated and mRNA reverse-transcribed using standardprotocols. IL-2 and β-actin cDNAs were then amplified by PCR using specific primerpairs and analysed by agarose gel electrophoresis.

Immunofluorescence staining and confocal microscopyThe intracellular distribution of WT-Bax or YFP-Bax reporter as well as the invasionand intracellular development of T. gondii were determined by double or tripleimmunofluorescence microscopy. Infected cells or non-infected controls were fixedwith 4% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.4, for 1 hour andquenched for 10 minutes in 50 mM NH4Cl in PBS. Following permeabilization ofthe cells with 0.1 mg/ml saponin and 1% BSA in PBS, they were incubated for 1hour with rabbit anti-Toxoplasma hyperimmune serum alone or along with eitheranti-Bax-NT (clone 6A7) or anti-Bax (clone 3; both from BD Pharmingen) dilutedin PBS containing saponin and BSA. After washing, cells were incubated for 1 hourwith Cy3-conjugated F(ab�)2 fragment anti-rabbit IgG alone, or Cy2-conjugatedF(ab�)2 fragment anti-rabbit IgG in combination with Cy3-conjugated F(ab�)2fragment anti-mouse IgG, or else with Cy5-conjugated F(ab�)2 fragment donkey anti-rabbit IgG (Dianova). In some experiments, total cell populations were stained withpropidium iodide. Cells were examined by confocal laser scanning microscopy usingLeica TCS SP2.

Statistical analysesResults are expressed as means ± s.e.m. of at least three independent experimentsunless otherwise indicated. Significant differences between mean values wereidentified by the Student’s t-test. In order to avoid false positive results due to multiplecomparisons, P-values were multiplied by the number of comparisons (Bonferronicorrection). Corrected P-values of less than 0.05 were considered significant.

We thank Antonio Barragan (Karolinska Institutet, Stockholm,Sweden) and James Lessard, (Children’s Hospital Medical Center,Cincinnati, OH) for kindly providing RH-LDM parasites and anti-actinmonoclonal antibody, respectively. We are also grateful to StevenElmore (Abbott’s Pharmaceutical Research and Development Division,Abbott Park, IL) for the kind gift of ABT-737 and its enantiomer. Thisstudy was supported by the Deutsche Forschungsgemeinschaft (LU777/4-1). We also thank the Karl-Enigk-Stiftung, Hannover, Germanyfor a scholarship to D.H.

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