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Address correspondence to Georg Häcker, Institute for Medical Microbiology, Technische Universität München, Trogerstr. 9, D-81675 Munich, Germany. Phone: 49-89-4140-4121; Fax: 49-89-4140-4868; email: hacker{at}lrz.tum.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: bacteria • pathogens • cell death • proteasome • mitochondria
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Introduction |
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Cell death by apoptosis is the result of the activation of an intracellular signal transduction pathway. It is becoming increasingly clear that apoptosis plays an important role in the defense against pathogens (3, 4) on the level of both the reaction of an individual host cell to an invading microorganism and the reacting immune system. For instance, in viral infections apoptosis is likely to act as a cellular defense mechanism. This is suggested by the finding that many viruses carry genes whose products inhibit apoptosis (5, 6). A number of bacteria have been found to induce apoptosis in the host's cells (for instance Legionella and Shigella; references 7 and 8). By contrast, a number of earlier studies have found that Chlamydia can decrease sensitivity of an infected cell against apoptotic stimuli (9–12).
The molecular function of the apoptotic pathway has been worked out in some detail. The final events of apoptosis are executed by the caspase proteases (13). In most circumstances, caspase activation requires the release of cytochrome c from the mitochondria into the cytosol (14). This release is governed by the Bcl-2 family of proteins. Bcl-2 proteins can be structurally and functionally divided into the following three groups: inhibitors of apoptosis (Bcl-2, Bcl-x, and others), effectors of cytochrome c release (Bax, Bak, and Bok), and triggers of apoptosis (BH3-only proteins). According to a plausible model, a stimulus to apoptosis activates one or several BH3-only proteins (nine are known at present; reference 15) that in turn activates Bax/Bak by an unknown mechanism. Active Bax/Bak then effect the release of cytochrome c. Bcl-2 blocks apoptosis by sequestering active BH3-only proteins (15).
Earlier studies have begun to map the apoptosis-inhibitory activity from Chlamydia within the apoptotic apparatus. Chlamydia efficiently block the release of cytochrome c from mitochondria upon the induction of apoptosis by external stimuli (9), whereas a death receptor signal that induces apoptosis independently of mitochondria is not inhibited (16). Here, we describe a molecular characterization of the apoptosis-inhibitory activity of Chlamydia in human host cells. We first focused on the BH3-only protein Bim, in part because Bim is bound to the microtubuli cytoskeleton, which is substantially reorganized during chlamydial infection (17). We found evidence that Bim is targeted for proteasomal destruction during chlamydial infection and extend this observation to the BH3-only proteins Puma and Bad. Because infected cells were not protected against active Bim or Puma, the disappearance of these proteins likely is the reason for the resistance of infected cells against apoptosis.
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Materials and Methods |
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Induction of Apoptosis.
Host cells (3 x 105/well in 6-well plates seeded the day before) were infected or not and subjected to UV irradiation. Cells were washed with PBS and then exposed to UV light (1,000 J/m2) in a transilluminator box (cells were 
80% confluent at this point; Stratagene). Medium was added and cells were analyzed at the time points given.
Assay for Nuclear Apoptosis and Caspase Activity.
Apoptosis was detected as described previously (11, 16). In brief, for detection of nuclear apoptosis, cells were stained with 20 µM Hoechst 33258 (Sigma-Aldrich) for 30 min and nuclear morphology was assessed under a fluorescence microscope. 300 nuclei per sample were scored. For detection of caspase 3–like activity, cells were lysed in NP-40 lysis buffer (11, 16), triplicates of aliquots were added to a peptide containing 10 µM of a caspase 3 recognition sequence (DEVD-AMC) in assay buffer containing BSA, Hepes, and fluorimetric substrate. Free AMC was measured after 1 h of incubation at 37°C and values are presented as arbitrary relative fluorescence units (mean ± the SD of triplicate reactions).
Microscopy.
Hep2 cells were grown on glass coverslips, infected with C. trachomatis or left uninfected, and some samples were treated with UV light for apoptosis induction as described above. Cells were fixed with 2% formalin for 30 min and consecutively stained with mouse anti–cytochrome c mAb (Becton Dickinson), FITC- or Cy3-labeled anti–mouse antiserum, and Alexa Fluor 546–labeled mouse antichlamydial LPS antibody (Progen) in PBS containing 1% FCS and 1% saponin. For detection of active Bax, cells were stained with anti-active Bax mAb (6A7; Upstate Biotechnology; reference 19) and Cy3-labeled anti–rabbit antiserum (Dianova) followed by staining with MitoTracker Green FM (Molecular Probes) or antichlamydial LPS antibody. Pictures were obtained with a laser scanning microscope (Carl Zeiss MicroImaging, Inc.). For detection of apoptosis in infected and transfected cells, HeLa cells were infected and transfected as described above. Cells were fixed and stained as described above (antichlamydia and Hoechst). Pictures were taken under a Zeiss microscope and analyzed with Zeiss AxioVision software. Brightness of whole pictures was adjusted electronically.
Determination of Bim mRNA by Microarrays.
HeLa cells were infected with C. trachomatis or C. pneumoniae. mRNA was harvested at various time points as indicated (see Results) and analyzed by a human micro array (U95A; Affymetrix, Inc.) as described recently in detail (20). Bim is represented on the chip by the sequence code 31611_s_at and the GenBank accession no. AF032457.
Detection of Bax and Bak by Flow Cytometry.
HeLa cells were infected with C. trachomatis or not and apoptosis was induced as described above. For some experiments, apoptosis was induced by transfection with expression vectors for enhanced GFP (EGFP) and BimS 10 h after infection. Cells were harvested by trypsinization and stained for active Bax or Bak. Anti-active Bak (amino acids 1–24) was purchased from Oncogene Research Products. Flow cytometry was performed with a FACSCalibur (Becton Dickinson). At least 105 cells per sample were recorded.
Transfection of HeLa Cells.
HeLa cells were plated in 12-well plates (2 x 105 per well) and infected with C. trachomatis as indicated. At the time points shown in the figure legends, cells were transfected in triplicate with 3 µg of control vector pEF, pEF-BimS expression vector, or Puma expression vector (provided by D. Huang, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) together with 0.5 µg CMV-LacZ using ethylene imine polymer solution (Fluka; reference 21). After 3 h, medium was changed and 12–20 h later cells were stained for ß-galactosidase activity. Blue cells were viewed under a microscope and scored alive or dead using morphological criteria (22). For inducible expression, BimS and Puma were cloned by PCR into the vector pCDNA4/TO. When transfected into cells that stably express the tetracycline repressor, the expression can be induced by removal of the suppressor upon the addition of tetracycline. The cell line HeLa Trex was transfected with these constructs or the empty vector, infected, and after 24 h the expression of BimS or Puma was induced by the addition of tetracycline. 20 h later, cells were scored as described above.
Western Blot Analysis.
For Western blot analysis, various cell types were either infected with C. trachomatis, C. pneumoniae, or L. pneumophila 2064, or left uninfected and harvested at the indicated time points. In some experiments, cells were treated with 10 µg/ml rifampin or 500 nM proteasome inhibitor MG132 (Calbiochem). For SDS extracts, 4 x 105 cells were directly lysed in SDS-containing loading buffer. For detergent extracts, 5 x 105 cells were lysed by incubation in 50 µl Triton buffer (1% Triton X-100, 0.05 M Pipes-NaOH, 0.05 M Hepes, pH 7.0, 2 mM MgCl2, 1 mM EDTA, 10 mM DTT, and protease inhibitors; Roche) for 30 min on ice. After centrifugation at 2,000 g at 4°C for 10 min, loading buffer was added and lysates were run on a 12% polyacrylamide gel. Proteins were transferred to nitrocellulose membranes, and membranes were probed with antibodies specific for Bax, Bak (Upstate Biotechnology), Bim (polyclonal from rabbit; Sigma-Aldrich), chlamydial Hsp60 (Affinity BioReagents, Inc.), GST (monoclonal from mouse; provided by H. Flaswinkel, GSF, Neuherberg, Germany), Puma NH2 terminus (rabbit; Sigma-Aldrich), tubulin (Sigma-Aldrich), Bad (Cell Signaling Technology), or GFP (CLONTECH Laboratories, Inc.). Proteins were visualized using peroxidase-conjugated secondary antibodies and a chemiluminescence detection system (PerkinElmer).
Measurement of Proteasomal Activity and In Vitro Bim Degradation.
Hep2 cells were infected with C. trachomatis or left uninfected. After 24 h of infection, cytosolic extracts were prepared as described previously (11). In brief, cells were resuspended in a cytoplasmic extraction buffer (1 mM Na-EGTA, 1 mM Na-EDTA, 1.5 mM MgCl2, 10 mM KCl, 20 mM Hepes-KOH, pH 7.5) containing a proteinase inhibitor mix and 1 mM DTT. After incubation for 1 h on ice, cells were disrupted by passages through a 22-gauge needle. For measurement of proteasomal activity, reactions were set up to contain 400 µg of cytosolic extract in a total of 200 µl of assay buffer (20 mM Tris/HCl, pH 7.5, and 50 mM NaCl) containing a final concentration of 10 µM Suc-LLVY-AMC fluorimetric substrate (proteasome substrate; Bachem). Free AMC was measured at 390 nm (excitation) and 440 nm (emission) every 5 min over 35 min.
GST-BimEL (provided by D. Huang) was expressed in Escherichia coli and purified using standard protocols. GST-CED-4 protein was provided by B. Seiffert (Technical University Munich, Munich, Germany; reference 23). For assessment of proteolytic activity, 400 µg of cytosolic extract was incubated with 0.8 µg GST-BimEL, 0.4 µg GST-CED-4, and 500 µM of proteasome inhibitor MG132 for 1.5 h at 37°C. Western blot analysis was performed as described above.
Assay for Degradation of GFP-BimL Mutants.
BimL was fused to the COOH terminus of EGFP in the vector pEGFP-C1. A number of Bim fragments were generated by PCR and cloned into pEGFP-C2 as COOH-terminal fusions to EGFP (BD Biosciences; details about the cloning procedure are available from the authors upon request). Integrity of PCR products was confirmed by sequencing. Hep2 cells were transfected with the constructs by electroporation (5 x 106 cells with 20 µg DNA at 280 V, 960 µF). Some aliquots were infected with C. trachomatis after 4 h. In some experiments, cells were treated with 500 nM of proteasome inhibitor MG132 (Calbiochem) or 10 µM lactacystin (BIOMOL Research Laboratories, Inc.). 20 h after infection, cells were harvested and GFP fluorescence was detected by flow cytometry with a FACSCalibur (Becton Dickinson). At least 105 cells per sample were recorded.
Online Supplemental Material
Fig. S1 shows micrographs of Hoechst staining of normal or infected cells upon UV irradiation. Fig. S2 demonstrates the inhibition of taxol-induced apoptosis by chlamydial infection. The photos provided in Fig. S3 show the morphology of cells (normal or infected) transfected with an expression vector for Puma. Fig. S4 gives data that show that chlamydial infection also fails to protect cells against short-term (6-h) induction of BimS through tetracyclin. Fig. S5 demonstrates that transfection of BimS causes the mitochondrial release of cytochrome c also in infected cells. Figs. S1–S5 are available at http://www.jem.org/cgi/content/full/jem.20040402/DC1.
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Results |
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80% of transfected normal HeLa cells were dead. In infected cells, the percentage of dead cells upon BimS expression was only marginally reduced, indicating that chlamydial infection did not protect against the direct Bim activity. Similar results were seen when the BH3-only protein Puma was transfected (Fig. 3 B, top, and Fig. S3). To exclude the possibility that transfection killed the cells before Chlamydia had established the protective mechanism, we further used an inducible expression system. HeLa cells expressing a tet repressor were first transfected with the ß-galactosidase reporter and a vector in which either Bim or Puma was placed under a tetracycline-inducible promoter. Cells were then infected with C. trachomatis, 24 h later expression of Bim or Puma was induced, and cells were stained after an additional 20 h. As shown in Fig. 3 B, bottom, the addition of tetracycline caused cell death in a large proportion of reporter-positive cells. Infection failed to protect the cells against cell death caused by the induction of Bim or Puma. Similar results were observed in experiments of short-term induction of Bim (6 h of tetracycline; see Fig. S4). Simultaneous detection of transfection, infection, and nuclear morphology further confirmed that BimS induced apoptosis in cells harboring chlamydial inclusions (Fig. 3, C and D).
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These data show that active Bim (or Puma) can still cause apoptosis in infected cells. The inhibition of apoptosis must therefore involve a block in the activation of Bim/Puma.
Chlamydial Infection Causes the Disappearance of Bim, Puma, and Bad Proteins.
Next, we tested whether chlamydial infection modified the expression of Bim. mRNA expression profiling of HeLa cells was performed at 2, 4, 12, 24, 36, and 48 h after infection with either C. trachomatis or C. pneumoniae (20). Bim mRNA was also analyzed by quantitative PCR and neither method showed a significant reduction during infection. When measured by PCR, the relative expression was 1.18 at 24 h and 0.9 at 48 h (expression during infection with C. trachomatis/expression during mock infection, mean of two experiments). However, Western blot analysis revealed a near-complete disappearance of Bim protein at 24 h after infection with C. trachomatis. This effect was most pronounced in the epitheloid cell lines HeLa and Hep2 (derivatives of the natural host cells of Chlamydia) and the breast carcinoma line MCF-7, but could also be observed easily in the T cell line Jurkat and the pro-myeloid cell line HL60 (infection blocks apoptosis in all of them; Fig. 4 A; reference 16 and not depicted). To determine whether chlamydial protein synthesis was required for this effect, Hep2 cells were infected in the presence of the antibiotic rifampin. Under this protocol, the Chlamydia-induced disappearance of Bim was strongly reduced (the efficiency of inhibition of the bacterial protein synthesis can be estimated by monitoring the production of bacterial Hsp60; Fig. 4, A and B). Bim levels were also investigated in cells infected with C. pneumoniae. As shown in Fig. 4 B, infection with this species also caused the disappearance of Bim, which was prevented by rifampin. To investigate whether this disappearance was specific for chlamydial infection or also occurred in other bacterial infections, Hep2 cells and Jurkat cells were infected with L. pneumophila. Bim protein expression remained unchanged in Legionella-infected cells. Infection was controlled by induction of apoptosis by Legionella as measured as the activation of effector caspases by enzyme assay (Fig. 4 C; reference 32).
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24–26 h of infection (Fig. 4 D and not depicted). Bim is one important member of the BH3-only class of proapoptotic proteins, and the loss of Bim could explain some but not all cases where Chlamydia have been found to protect against apoptosis. A second BH3-only protein whose in vivo importance has been demonstrated recently by gene targeting is Puma (33). Puma is known to be regulated transcriptionally, but can also be detected in normal cells in culture (34). We analyzed expression of Puma and found that Puma protein also disappeared during chlamydial infection (Fig. 4 E). The BH3-only protein Bad also disappeared upon infection (Fig. 4 E), suggesting that a common characteristic of BH3-only proteins was targeted.
Chlamydial Infection Targets Bim for Proteasomal Destruction.
These data suggested that Bim was proteolytically destroyed. The finding that no smaller fragments of Bim could be detected by Western blotting (not depicted) further suggested that Bim was degraded by a proteasomal activity. The proteasome is a large multisubunit protein complex that acts to degrade aged and misfolded cellular proteins. To investigate whether Bim was degraded in the proteasome, the proteasome inhibitor MG132 was used. Hep2 cells were infected with C. trachomatis or left uninfected and treated with MG132. As shown in Fig. 5 A, MG132 was able to inhibit Chlamydia-induced degradation of Bim. Although this finding suggested that Bim was degraded by the cellular proteasome, proteasome activity was not enhanced in cytosolic extracts from infected cells (Fig. 5 B). It is further worth noting that although most cellular proteins (for instance tubulin, caspases, Bcl-2, and others) were not degraded in intact cells (11 and not depicted), proteins other than Bim were digested in cytosolic preparations. During incubation in vitro, not only exogenously added, recombinant Bim, but also endogenous tubulin and a control protein (GST-CED-4) were noticeably degraded. This degradation was blocked by MG132, but not by a mix of conventional protease inhibitors, indicating a proteasome-like activity also in these preparations, albeit less specific (Fig. 5 C). Zhong et al. (35) could identify a protein that they named chlamydial proteasome-like activity factor (CPAF), which is secreted from the inclusion into the host cell cytosol and causes the degradation of host transcription factors like RFX5. Under the conditions used here, RFX5 degradation started at 14 h after infection and was complete at 16 h of infection. The disappearance of Bim started 2 h later (not depicted).
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Discussion |
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Chlamydial infection can protect a host cell against a large number of stimuli. Chlamydia did not afford protection against the one stimulus tested that does not involve mitochondrial cytochrome c release, which is death receptor– (CD95) dependent apoptosis induction in so-called type I cells (16), supporting the concept of a block confined to mitochondrial apoptosis. Apoptosis induced by all the stimuli against which protection by Chlamydia has been reported is also inhibited by Bcl-2. The molecular function of Bcl-2 is still somewhat under debate, but the most plausible role appears to be connected to its ability to bind and sequester active BH3-only proteins (15).
The allocation of apoptotic stimuli to BH3-only proteins is incomplete. Among the stimuli inhibited by chlamydial infection, UV irradiation is mediated by Bim and possibly Bmf (36), etoposide at least in part by Puma (33), staurosporine by Bim and Puma (25, 33), and TNF and CD95 signal via Bid cleavage (where a mitochondrial contribution is required; references 37 and 38). Our data suggest a model that accounts for the far-reaching antiapoptotic effect of Chlamydia. All BH3-only proteins share the BH3 domain, whose presence is required for the induction of apoptosis. Because the chlamydial activity targets this domain in the Bim protein, all BH3-only proteins might be subjected to proteasomal destruction. This hypothesis was tested on the BH3-only proteins Puma and Bad, which were also found to disappear during chlamydial infection. All of the following proteins of the apoptosis pathway we have investigated do not change in expression: Bax, Bak (above), Bcl-2, Bcl-x, Mcl-1, Bid, caspase 3, caspase 8, caspase 9, Apaf-1, Hsp70, and cytochrome c (11, 16, and not depicted). Up-regulation of the antiapoptotic protein C-IAP2 has been reported during chlamydial infection (39). However, IAP act at the level of caspases and, accordingly, our results do not support a role for IAP in inhibition against apoptosis by Chlamydia.
Genetically modified mice lacking either Bim (25) or Puma (33) show a clear protection against some, but not all, apoptotic stimuli. No double mutant mice have been tested, but the combined deficiency of Bim and Puma might well explain the greater share of apoptosis protection of Chlamydia-infected cells. During CD95 signaling, caspase 8 activation and Bid cleavage were unaffected by chlamydial infection (16). Bid is present in an inactive state and is activated by cleavage through caspase 8. Accordingly, the BH3-domain in Bid is normally inactive and only exposed by this cleavage event (40). Therefore, it might be speculated that the active Bid (tBid), but not the inactive form, is also recognized via its BH3 domain and targeted for destruction, which would explain the protection against CD95-induced apoptosis (the antibody used to detect Bid in the above study did not recognize tBid). Such a general BH3-targeting approach of Chlamydia would explain the almost general protection of infected cells against apoptotic stimuli.
We still do not know what the chlamydial effector is and how degradation is achieved. The inhibition of bacterial protein synthesis with rifampin prevented the disappearance (above) and precluded the development of protection against apoptosis (11). Most likely, this means that rifampin inhibits the production of the chlamydial BH3-only, protein-degrading factor. The sensitivity to MG132 and lactacystin indicates the involvement of a proteasomal activity and immediately suggests targeting to the cellular proteasome. However, a chlamydial factor has been purified and partially characterized, named CPAF. The activity of CPAF has been shown to be both necessary and sufficient to cause degradation of cellular transcription factors such as RFX5, even when purified recombinant proteins were used (35). RFX5 degradation slightly preceded Bim degradation during chlamydial infection. It is conceivable that CPAF also mediates degradation of BH3-only proteins. How CPAF works on a molecular level is still unclear, but it is also inhibited by inhibitors of the proteasome. Recognition by the proteasome requires modification of a protein by ligation with the abundant small cellular protein ubiquitin. Ubiquination involves a series of molecular interactions with ubiquitin-transferring enzymes and factors that convey specificity (41). Bim is targeted at an internal structure and its degradation is probably complete because no fragments could be detected by Western blotting and because the fluorescence of the attached EGFP also disappeared in a lactacystin/MG132-sensitive manner. Therefore, it is possible that the chlamydial activity causes a modification of Bim, which then leads to the destruction by the cellular proteasome. Interference with the cellular ubiquitination/SUMOylation system has been described by an effector protein from Yersinia (42). Although mechanism and effect here appear to be opposite (disruption of proteasomal targeting), it provides an example of where bacteria use the strategy of interfering with this host cell pathway.
Inhibition of apoptosis in infected cells is a mechanism that is often used by viruses. Many viruses carry genes whose products interfere with the host cell's apoptosis machinery (43, 44). Chlamydia share a virus' need for integrity of the host cell. They differ in that unlike Chlamydia, viruses pick up their genetic material from host cells. It is in accordance with this model that Chlamydia use a mechanism to inhibit apoptosis that is not found as a regulatory means in host cells.
It has been suggested that at later stages of chlamydial infection, apoptosis is induced through the activation of Bax (30) and the clearance of Chlamydia during vaginal infection of mice was accelerated in mice lacking Bax (45). Experimentally, Bax could directly (in the absence of BH3-only proteins) be activated through changes in the intracellular milieu such as pH changes or oxidative stress (46, 47). If this occurred at late stages of the infection and led to the uptake of the infected dead cell by phagocytes or surrounding cells, such a mechanism might aid the spreading of the infection.
Why do Chlamydia possess the ability to block cell death? Obviously, there is no need for Chlamydia to protect the cell against stimuli like staurosporine. An antiapoptotic capacity only makes sense if there is apoptosis that occurs during infection and has to be inhibited. It could be argued that Chlamydia need to prolong the lifespan of the host cell; however, the natural host cells of Chlamydia, epithelial cells, probably live longer than a normal replication cycle would take. To us, it appears more likely that Chlamydia also have the potential to induce apoptosis. The cell is probably able to pick up on the presence of Chlamydia and undergo apoptosis. It has been shown that the development of the chlamydial inclusion involves restructuring of the cytoskeleton, including microtubular components (see for instance reference 17). Because Bim is sequestered to the cytoskeleton and initiates apoptosis upon its release from this location, Bim release might be the defense mechanism that a cell uses against chlamydial infection and that has to be overcome by the degradation of Bim.
It is a plausible speculation that the inhibition of apoptosis is important for the growth of the bacteria. Most chlamydial infections clear spontaneously, but there is evidence of chronic infections that may even occur in and contribute to the development of atherosclerotic lesions (48). In vitro studies further suggest that chronic infections might be insensitive to antibiotics (49). Therapies directed at an interference with chlamydial antiapoptotic activities could therefore turn out helpful to prevent sequelae of protracted infections with these bacteria.
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Acknowledgments |
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This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to G. Häcker).
The authors have no conflicting financial interests.
Submitted: 2 March 2004
Accepted: 16 August 2004
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References |
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1 Grayston, J.T., and S. Wang. 1975. New knowledge of chlamydiae and the diseases they cause. J. Infect. Dis. 132:87–105.[Medline]
2 Grayston, J.T. 1992. Chlamydia pneumoniae, strain TWAR pneumonia. Annu. Rev. Med. 43:317–323.[CrossRef][Medline]
3 Vaux, D.L., G. Haecker, and A. Strasser. 1994. An evolutionary perspective on apoptosis. Cell. 76:777–779.[CrossRef][Medline]
4 Vaux, D.L., and A. Strasser. 1996. The molecular biology of apoptosis. Proc. Natl. Acad. Sci. USA. 93:2239–2244.
5 Roulston, A., R.C. Marcellus, and P.E. Branton. 1999. Viruses and apoptosis. Annu. Rev. Microbiol. 53:577–628.[CrossRef][Medline]
6 Everett, H., and G. McFadden. 2002. Poxviruses and apoptosis: a time to die. Curr. Opin. Microbiol. 5:395–402.[CrossRef][Medline]
7 Muller, A., J. Hacker, and B.C. Brand. 1996. Evidence for apoptosis of human macrophage-like HL-60 cells by Legionella pneumophila infection. Infect. Immun. 64:4900–4906.[Abstract]
8 Zychlinsky, A., M.C. Prevost, and P.J. Sansonetti. 1992. Shigella flexneri induces apoptosis in infected macrophages. Nature. 358:167–169.[CrossRef][Medline]
9 Fan, T., H. Lu, H. Hu, L. Shi, G.A. McClarty, D.M. Nance, A.H. Greenberg, and G. Zhong. 1998. Inhibition of apoptosis in chlamydia-infected cells: blockade of mitochondrial cytochrome c release and caspase activation. J. Exp. Med. 187:487–496.
10 Rajalingam, K., H. Al-Younes, A. Muller, T.F. Meyer, A.J. Szczepek, and T. Rudel. 2001. Epithelial cells infected with Chlamydophila pneumoniae (Chlamydia pneumoniae) are resistant to apoptosis. Infect. Immun. 69:7880–7888.
11 Fischer, S.F., C. Schwarz, J. Vier, and G. Hacker. 2001. Characterization of antiapoptotic activities of Chlamydia pneumoniae in human cells. Infect. Immun. 69:7121–7129.
12 Hacker, G., and S.F. Fischer. 2002. Bacterial anti-apoptotic activities. FEMS Microbiol. Lett. 211:1–6.[Medline]
13 Nicholson, D.W., and N.A. Thornberry. 1997. Caspases: killer proteases. Trends Biochem. Sci. 22:299–306.[CrossRef][Medline]
14 Green, D.R., and J.C. Reed. 1998. Mitochondria and apoptosis. Science. 281:1309–1312.
15 Bouillet, P., and A. Strasser. 2002. BH3-only proteins-evolutionarily conserved proapoptotic Bcl-2 family members essential for initiating programmed cell death. J. Cell Sci. 115:1567–1574.
16 Fischer, S.F., T. Harlander, J. Vier, and G. Hacker. 2004. Protection against CD95-induced apoptosis by chlamydial infection at a mitochondrial step. Infect. Immun. 72:1107–1115.
17 Clausen, J.D., G. Christiansen, H.U. Holst, and S. Birkelund. 1997. Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection. Mol. Microbiol. 25:441–449.[CrossRef][Medline]
18 Garduno, R.A., E. Garduno, and P.S. Hoffman. 1998. Surface-associated hsp60 chaperonin of Legionella pneumophila mediates invasion in a HeLa cell model. Infect. Immun. 66:4602–4610.
19 Hsu, Y.T., and R.J. Youle. 1997. Nonionic detergents induce dimerization among members of the Bcl-2 family. J. Biol. Chem. 272:13829–13834.
20 Hess, S., C. Rheinheimer, F. Tidow, G. Bartling, C. Kaps, J. Lauber, J. Buer, and A. Klos. 2001. The reprogrammed host: Chlamydia trachomatis-induced up-regulation of glycoprotein 130 cytokines, transcription factors, and antiapoptotic genes. Arthritis Rheum. 44:2392–2401.[CrossRef][Medline]
21 Verhagen, A.M., P.G. Ekert, M. Pakusch, J. Silke, L.M. Connolly, G.E. Reid, R.L. Moritz, R.J. Simpson, and D.L. Vaux. 2000. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell. 102:43–53.[CrossRef][Medline]
22 Miura, M., H. Zhu, R. Rotello, E.A. Hartwieg, and J. Yuan. 1993. Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell. 75:653–660.[CrossRef][Medline]
23 Seiffert, B.M., J. Vier, and G. Hacker. 2002. Subcellular localization, oligomerization, and ATP-binding of Caenorhabditis elegans CED-4. Biochem. Biophys. Res. Commun. 290:359–365.[CrossRef][Medline]
24 Puthalakath, H., D.C. Huang, L.A. O'Reilly, S.M. King, and A. Strasser. 1999. The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex. Mol. Cell. 3:287–296.[CrossRef][Medline]
25 Bouillet, P., D. Metcalf, D.C. Huang, D.M. Tarlinton, T.W. Kay, F. Kontgen, J.M. Adams, and A. Strasser. 1999. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science. 286:1735–1738.
26 Wei, M.C., W.X. Zong, E.H. Cheng, T. Lindsten, V. Panoutsakopoulou, A.J. Ross, K.A. Roth, G.R. MacGregor, C.B. Thompson, and S.J. Korsmeyer. 2001. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science. 292:727–730.
27 Antonsson, B., S. Montessuit, B. Sanchez, and J.C. Martinou. 2001. Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J. Biol. Chem. 276:11615–11623.
28 Nechushtan, A., C.L. Smith, I. Lamensdorf, S.H. Yoon, and R.J. Youle. 2001. Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J. Cell Biol. 153:1265–1276.
29 Griffiths, G.J., L. Dubrez, C.P. Morgan, N.A. Jones, J. Whitehouse, B.M. Corfe, C. Dive, and J.A. Hickman. 1999. Cell damage–induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J. Cell Biol. 144:903–914.
30 Perfettini, J.L., J.C. Reed, N. Israel, J.C. Martinou, A. Dautry-Varsat, and D.M. Ojcius. 2002. Role of Bcl-2 family members in caspase-independent apoptosis during Chlamydia infection. Infect. Immun. 70:55–61.
31 O'Connor, L., A. Strasser, L.A. O'Reilly, G. Hausmann, J.M. Adams, S. Cory, and D.C. Huang. 1998. Bim: a novel member of the Bcl-2 family that promotes apoptosis. EMBO J. 17:384–395.[CrossRef][Medline]
32 Gao, L.Y., and Y. Abu Kwaik. 1999. Activation of caspase 3 during Legionella pneumophila-induced apoptosis. Infect. Immun. 67:4886–4894.
33 Villunger, A., E.M. Michalak, L. Coultas, F. Mullauer, G. Bock, M.J. Ausserlechner, J.M. Adams, and A. Strasser. 2003. p53- and drug-induced apoptotic responses mediated by BH3-only proteins puma and noxa. Science. 302:1036–1038.
34 Han, J., C. Flemington, A.B. Houghton, Z. Gu, G.P. Zambetti, R.J. Lutz, L. Zhu, and T. Chittenden. 2001. Expression of bbc3, a pro-apoptotic BH3-only gene, is regulated by diverse cell death and survival signals. Proc. Natl. Acad. Sci. USA. 98:11318–11323.
35 Zhong, G., P. Fan, H. Ji, F. Dong, and Y. Huang. 2001. Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors. J. Exp. Med. 193:935–942.
36 Puthalakath, H., A. Villunger, L.A. O'Reilly, J.G. Beaumont, L. Coultas, R.E. Cheney, D.C. Huang, and A. Strasser. 2001. Bmf: a proapoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis. Science. 293:1829–1832.
37 Li, H., H. Zhu, C.J. Xu, and J. Yuan. 1998. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell. 94:491–501.[CrossRef][Medline]
38 Luo, X., I. Budihardjo, H. Zou, C. Slaughter, and X. Wang. 1998. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell. 94:481–490.[CrossRef][Medline]
39 Wahl, C., S. Maier, R. Marre, and A. Essig. 2003. Chlamydia pneumoniae induces the expression of inhibitor of apoptosis 2 (c-IAP2) in a human monocytic cell line by an NF-kappaB-dependent pathway. Int. J. Med. Microbiol. 293:377–381.[CrossRef][Medline]
40 McDonnell, J.M., D. Fushman, C.L. Milliman, S.J. Korsmeyer, and D. Cowburn. 1999. Solution structure of the proapoptotic molecule BID: a structural basis for apoptotic agonists and antagonists. Cell. 96:625–634.[CrossRef][Medline]
41 Ciechanover, A. 1998. The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J. 17:7151–7160.[CrossRef][Medline]
42 Orth, K., Z. Xu, M.B. Mudgett, Z.Q. Bao, L.E. Palmer, J.B. Bliska, W.F. Mangel, B. Staskawicz, and J.E. Dixon. 2000. Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease. Science. 290:1594–1597.
43 Cuconati, A., and E. White. 2002. Viral homologs of BCL-2: role of apoptosis in the regulation of virus infection. Genes Dev. 16:2465–2478.
44 Bump, N.J., M. Hackett, M. Hugunin, S. Seshagiri, K. Brady, P. Chen, C. Ferenz, S. Franklin, T. Ghayur, P. Li, et al. 1995. Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35. Science. 269:1885–1888.
45 Perfettini, J.L., D.M. Ojcius, C.W. Andrews Jr., S.J. Korsmeyer, R.G. Rank, and T. Darville. 2003. Role of proapoptotic BAX in propagation of Chlamydia muridarum (the mouse pneumonitis strain of Chlamydia trachomatis) and the host inflammatory response. J. Biol. Chem. 278:9496–9502.
46 Khaled, A.R., K. Kim, R. Hofmeister, K. Muegge, and S.K. Durum. 1999. Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH. Proc. Natl. Acad. Sci. USA. 96:14476–14481.
47 Jungas, T., I. Motta, F. Duffieux, P. Fanen, V. Stoven, and D.M. Ojcius. 2002. Glutathione levels and BAX activation during apoptosis due to oxidative stress in cells expressing wild-type and mutant cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 277:27912–27918.
48 Leinonen, M., and P. Saikku. 2002. Evidence for infectious agents in cardiovascular disease and atherosclerosis. Lancet Infect. Dis. 2:11–17.[CrossRef][Medline]
49 Kutlin, A., P.M. Roblin, and M.R. Hammerschlag. 2002. Effect of prolonged treatment with azithromycin, clarithromycin, or levofloxacin on Chlamydia pneumoniae in a continuous-infection model. Antimicrob. Agents Chemother. 46:409–412.
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, uninfected; 
, C. trachomatis–infected. The appearance of free AMC over time is shown. (C) Nonspecific proteasomal degradation of proteins in cytosolic extracts from infected cells. Cytosolic extracts of cells infected with C. trachomatis or not were incubated for 1.5 h at 37°C with GST-BimEL, GST-CED-4, and 500 nM proteasome inhibitor MG132 as indicated. Western blotting was performed using antibodies against Bim, tubulin, or GST.