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J. Biol. Chem., Vol. 281, Issue 42, 31495-31501, October 20, 2006

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The Secreted Protease Factor CPAF Is Responsible for Degrading Pro-apoptotic BH3-only Proteins in Chlamydia trachomatis-infected Cells^*

Mustak Pirbhai, Feng Dong, Youmin Zhong, Kelvin Z. Pan, and Guangming Zhong¹

From the Department of Microbiology and Immunology, University of Texas Health Science Center, San Antonio, Texas 78229 and the Department of Microbiology and Immunology, Medical College of Ohio, Toledo, Ohio 43614

Received for publication, March 24, 2006 , and in revised form, August 21, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chlamydia trachomatis has evolved a profound anti-apoptotic activity that may aid in chlamydial evasion of host defense. The C. trachomatis anti-apoptotic activity has been correlated with blockade of mitochondrial cytochrome c release, inhibition of Bax and Bak activation, and degradation of BH3-only proteins. This study presents evidence that a chlamydia-secreted protease factor designated CPAF is both necessary and sufficient for degrading the BH3-only proteins. When the C. trachomatis-infected cell cytosolic extracts were fractionated by column chromatography, both the CPAF protein and activity elution peaks overlapped with the BH3-only protein degradation activity peak. Depletion of CPAF with a CPAF-specific antibody removed the BH3-only protein degradation activity from the infected cell cytosolic extracts, whereas depletion with control antibodies failed to do so. Notably, recombinant CPAF expressed in bacteria was able to degrade the BH3-only proteins, whereas CPAF mutants similarly prepared from bacteria failed to do so. Finally, bacterium-expressed CPAF also degraded the human BH3-only protein Puma purified from bacteria. These results demonstrate that CPAF contributes to the chlamydial anti-apoptotic activity by degrading the pro-apoptotic BH3-only Bcl-2 subfamily members.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chlamydia trachomatis infection is a leading cause of both preventable blindness in developing countries and sexually transmitted diseases worldwide. Urogenital infection with C. trachomatis can lead to complications such as pelvic inflammatory diseases, ectopic pregnancy, and infertility in women and reactive arthritis in men. The precise pathogenic mechanisms of C. trachomatis-induced diseases are still unclear despite decades of extensive research efforts. It is thought that the inflammatory responses triggered by C. trachomatis-infected cells can significantly contribute to the chlamydia-induced pathologies in humans (1–3), suggesting that the root cause of C. trachomatis pathogenicity may lie in the chlamydial ability to invade host cells and to maintain long-term survival in the infected hosts.

C. trachomatis has evolved a unique intracellular biphasic life cycle (4) and is able to both productively replicate and persistently survive within a cytoplasmic vacuole of eukaryotic cells (5–7). A typical chlamydial infection starts with entry of an infectious elementary body into host cells via endocytosis. An elementary body differentiates into a noninfectious but metabolically active reticulate body, which multiplies and differentiates back to elementary bodies. The mature elementary bodies are then released for spreading to the adjacent host cells. The entire growth cycle takes 48–72 h to complete in vitro during productive infection, whereas a persistent infection can last for a long period of time in vivo. To ensure its long-term survival in the infected hosts, chlamydia has also acquired the ability to evade host defense mechanisms (8–12). For example, a chlamydia-secreted protein with proteolytic activity designated CPAF (chlamydial protease/proteasome-like activity factor) is responsible for degrading host transcription factors such as RFX5 required for major histocompatibility complex antigen expression (11), which may allow chlamydia to escape efficient immune detection. CPAF can also cleave cytokeratin 8 (13), which may facilitate chlamydial vacuole expansion and intravacuolar growth. Interestingly, CPAF is synthesized as a proenzyme by chlamydia and has to be processed into intramolecular dimers to acquire proteolytic activity (14, 15). However, it is still unknown whether CPAF targets other cellular proteins and what the CPAF substrate specificity is. To evade host immune effector mechanisms, chlamydia has evolved a profound antiapoptotic activity to suppress host cell apoptosis in both productively (8) and persistently (16) infected cells. However, the precise mechanisms of the chlamydial anti-apoptotic activity are still unknown. An obvious question is whether CPAF is also involved in the chlamydial anti-apoptotic activity.

Host cell apoptosis is a highly regulated form of cell death that plays vital roles in many biological processes, including remodeling tissues during development and eliminating the infected cells during host defense responses. Two major apoptosis pathways have been identified, and they are the intrinsic (17) and extrinsic (18) pathways. In the intrinsic pathway, the intracellular death signals induce mitochondrial release of apoptogenic factors such as cytochrome c that participate in the formation of the apoptosome, leading to activation of downstream effector caspases, including caspase-3, -6, and -7. The activated effector caspases can cleave a wide range of cellular molecules, including the DNA-repairing enzyme poly(ADP-ribose) polymerase, and also activate endonucleases, finally leading to irreversible nuclear apoptosis. On the other hand, the extrinsic pathway is triggered by the ligation of death receptors with ligands from extracellular sources, resulting in the formation of a death-inducing signaling complex that causes activation of upstream caspases such as caspase-8. The activated caspase-8 can either directly activate the downstream effector caspases to cause terminal apoptosis in the so-called type I cells such as the B lymphoblastoid cell line SKW6.4 or simultaneously enter the intrinsic mitochondrial pathway via the generation of cleaved Bid (19) to amplify the receptor-transmitted death signals to achieve terminal apoptosis in type II cells such as the T cell line Jurkat (20, 21). A recent study has demonstrated that the effector caspases can also amplify cell death potency via a positive feedback pathway by promoting mitochondrial release of cytochrome c (22). It is obvious that mitochondria play a critical role in controlling eukaryotic cell apoptosis. The Bcl-2 family members regulate mitochondrial release of apoptogenic factors, including cytochrome c (23–25). There are several dozens of members in the Bcl-2 family, and they share at least one common Bcl-2 homology (BH)² domain. A prototype of Bcl-2 protein has four BH domains, including BH4, BH3, BH1, and BH2 from the N to C termini, and Bcl-2 family members can be categorized into three subfamilies based on their structural and functional characteristics (23, 26, 27). The anti-apoptotic Bcl-2 subfamily members (including Mcl-1, Bcl-2, and Bcl-x_L) share three to four BH domains. The proapoptotic multidomain Bcl-2 subfamily members (including Bax and Bak) share more than one BH domain. Activation of Bax or Bak can cause mitochondrial cytochrome c release and apoptosis (28–31). The pro-apoptotic BH3-only Bcl-2 subfamily members (including Bid, Bad, Bik, Puma, Bim, Bmf, Noxa, and Hrk) share only the BH3 domain (32). How these Bcl-2 family members interact with each other to regulate mitochondrial cytochrome c release is not entirely clear. It is thought that the BH3-only proteins that are normally associated with intracellular organelles can sense cell death signals from either extrinsic (e.g. Bid) or intrinsic (e.g. Bim, Bmf, Puma, and Bik) sources by undergoing transcriptional and/or post-translational changes and translocating to mitochondria (23, 27, 32). These BH3-only proteins can transmit death signals to mitochondria by inhibiting the anti-apoptotic Bcl-2 subfamily members and/or activating the pro-apoptotic multidomain Bcl-2 family members such as Bax and Bak (23, 32).

To understand the mechanisms of the chlamydial anti-apoptotic activity, recent studies have correlated the chlamydial anti-apoptotic activity with blockade of mitochondrial cytochrome c release (8, 33), inhibition of pro-apoptotic Bax and Bak activation (34), and degradation of BH3-only proteins (35–37). However, it is still not known how the host pro-apoptotic BH3-only proteins are degraded in C. trachomatis-infected cells. In this study, we demonstrate that CPAF, a chlamydia-secreted protease factor that has been shown previously to degrade host transcription factors (9–11) and cytokeratin 8 (13), is also responsible for degrading the BH3-only proteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Culture and Chlamydial Infection—HeLa cells (American Type Culture Collection, Manassas, VA) were grown in a growth medium consisting of Dulbecco's modified Eagle's medium (Invitrogen) and 10% fetal calf serum (Novo-Tech, Grand Island, NY) in a humidified incubator supplied with 5% CO₂. C. trachomatis LGV2 (L2) was grown and purified as described previously (38) and used to infect HeLa cells at a multiplicity of infection of 5 or as indicated in individual experiments. The infection was carried out by directly diluting the stock organisms in the growth medium. The infected cultures were harvested 40 h after infection or as indicated in individual experiments. For the inhibition experiments, the inhibitors lactacystin (an irreversible proteasomal inhibitor known to inhibit CPAF; Chemicon, Temecula, CA) and Ala-Ala-Phe-chloromethyl ketone (a cell-permeable serine protease inhibitor; BIOMOL, Plymouth Meeting, PA) were added to the cultures during chlamydial infection.

Column Chromatography—To correlate CPAF proteolytic activity with BH3-only protein degradation, a cytosolic protein preparation from chlamydia-infected HeLa cells (L2S100) (11) was subjected to fractionation on a Mono Q ion exchange column (Amersham Biosciences). An ÅKTA Model 10 purifier (GE Healthcare) was used to run the column. A salt gradient was used to elute the Mono Q column. The eluted fractions were monitored for the presence of both CPAF and host proteasomes by Western blotting (see below), and CPAF proteolytic activity and BH3-only protein degradation were measured using a cell-free cleavage assay (see below).

Cell-free Cleavage Assay—The cell-free assays were carried out as described previously (11, 13). The enzyme preparations or purified enzymes were mixed with the desired substrate in the presence or absence of the inhibitor lactacystin or the solvent Me₂SO alone (Sigma). All reactions were carried out in phosphate-buffered saline at 37 °C for 1 h or as indicated in individual experiments. The enzyme sources included the cytosolic S100 samples made from HeLa cells with (L2S100) or without (HeLaS100) chlamydial infection, the column-fractionated samples, the remaining supernatants after antibody depletion, the antibody-precipitated pellets, and the various versions of recombinant glutathione S-transferase (GST)-CPAF created as described previously (14, 15). The substrate preparations included crude nuclear extracts containing RFX5 and cytosolic extracts containing keratin 8 and various BH3-only proteins as well as a purified recombinant GST-human Puma fusion protein (see below). The nuclear extract was made as described previously (10). Briefly, normal HeLa cells were homogenized in a Dounce homogenizer to break cytoplasmic membranes, and pellets were repeatedly washed with a buffer consisting of 1% Nonidet P-40 and 150 mM NaCl in 50 mM Tris (pH 8.0) plus a protease inhibitor mixture (phenylmethylsulfonyl fluoride at a final concentration of 1 mM, leupeptin at 20 µM, pepstatin A at 1.6 µM, and aprotinin at 1.7 µg/ml; all from Sigma) to remove cytosolic/membrane proteins as much as possible. The final washed nuclear pellets were extracted with a buffer consisting of 0.5 M NaCl and 1% Triton X-100 in 20 mM Tris (pH 8.0). The cytosolic extract was made as described previously (13) by extracting 1–2 x 10⁷ HeLa cells with 1 ml of a buffer consisting of 1% Nonidet P-40, 0.5% Triton X-100, and 150 mM NaCl in 50 mM Tris (pH 8.0) plus the protease inhibitor mixture. The GST-human Puma fusion was generated by cloning the human Puma cDNA (accession number AF354654 [GenBank] ; ncbi.nlm.nih.gov/entrez) into a pGEX-4T1 vector (kindly provided by Dr. Chun Wu, Abgent, San Diego, CA), and the GST fusion protein expressed in bacteria was purified with glutathione-agarose beads (Amersham Biosciences) following the manufacturer's instructions.

View larger version (69K): [in this window] [in a new window]   FIGURE 1. Correlation of BH3-only protein degradation with CPAF activity by column chromatography. L2S100 made from HeLa cells infected with C. trachomatis serovar L2 (multiplicity of infection of 5) was loaded onto a Mono Q column, and the Mono Q column eluates were collected into different tubes as indicated (Q column fraction numbers). Both the L2S100 and fractionated samples were analyzed by Western blotting for the presence of CPAF and proteasomal proteins (upper two panels) and were also used as the source of enzyme in a cell-free degradation assay to measure the degradation activities for RFX5 (nuclear extract used as the substrate) and the BH3-only proteins Puma, Puma, Bik, and BimL (cytosolic extract used as the substrate) as indicated on the right. The residual (R) target molecules were detected using the corresponding antibodies as indicated on the left. Note that the degradation activities for both RFX5 and BH3-only proteins correlated with the presence of CPAF, but not host proteasomes.

Immunoprecipitation Assay—The immunoprecipitation assay was carried out as described previously (40). For this study, the assay was used to deplete antigens from extracts with specific antibodies. L2S100 samples were mixed with protein A/G-agarose-immobilized antibody complexes. After a 1-h incubation at room temperature, the agarose pellets were spun down, and the remaining supernatants were re-precipitated with a fresh set of the corresponding antibody complexes to completely remove the corresponding antigens in the supernatants. The final remaining supernatants and the precipitates were used as the source of enzymes in the cell-free degradation assay. mAb 54b, mAb MC22 (IgG3) (11), and mAb MCP21 were used to precipitate CPAF, the chlamydial major outer membrane protein, and host proteasome -subunit HC3, respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Correlation of BH3-only Protein Degradation with CPAF Activity—To test whether a previously identified chlamydial protease factor, CPAF, is involved in the degradation of the pro-apoptotic BH3-only proteins in chlamydia-infected cells, we used a column chromatography approach to analyze the relationship between these two (Fig. 1). The C. trachomatis-infected cell cytosolic extracts (L2S100) were subjected to fractionation via Mono Q column chromatography because it is known that CPAF can both bind to and be eluted from a Mono Q column (10, 11). The elution peaks of both CPAF protein and CPAF activity (measured as RFX5 degradation) overlapped with those of BH3-only protein degradation, including the degradation of Puma, Puma, Bik, and BimL, whereas the elution peak of host proteasomes was delayed, demonstrating a correlation between BH3-only protein degradation and CPAF, but not host proteasomes. However, this experiment could not determine whether CPAF indeed participates in the degradation of the host BH3-only proteins.

CPAF Is Required for BH3-only Protein Degradation in C. trachomatis-infected Cells—To test whether CPAF is necessary for degrading the BH3-only proteins, we first tested whether lactacystin, an irreversible proteasomal inhibitor that is known to inhibit CPAF activity, is able to block BH3-only protein degradation activity in the C. trachomatis-infected cell cytosol (Fig. 2A). When a small amount of the C. trachomatis-infected cell cytosol sample (L2S100) was used as the source of enzyme, a partial degradation of Puma and Puma (lane 3) was observed, whereas a large amount caused complete disappearance of Puma and Puma (lane 4). However, addition of lactacystin (but not the solvent Me₂SO alone) to the reaction mixture even containing the large amount of L2S100 restored most of Puma and Puma (lanes 5 and 6), demonstrating that lactacystin-sensitive proteolytic activity is mainly responsible for the degradation of the BH3-only proteins. We next used a depletion experimental approach to further evaluate the necessity of CPAF to degrade BH3-only proteins (Fig. 2B). Intact L2S100 degraded both Puma and Puma (lanes 2 and 3). However, the residual supernatant after precipitation with mAb 54b-conjugated beads lost the degradation activity (lane 4), whereas the antibody pellet retained the degradation activity (lane 5). Because of the co-migration of the immunoglobulin light chain and Puma, only the Puma degradation was clearly measurable by Western blotting. Precipitation with antibody recognizing either host proteasomes (mAb MCP21) or the chlamydial major outer membrane protein (mAb MC22) failed to remove the degradation activity from the L2S100 supernatants. The low level of degradation activity associated with the control antibody pellets may be due to the nonspecific binding of CPAF to the beads.

CPAF Is Sufficient for BH3-only Protein Degradation in C. trachomatis-infected Cells—The next question we asked is whether CPAF alone is able to degrade the BH3-only proteins. We first used the CPAF fusion protein purified from bacteria as the source of enzyme to carry out the cell-free degradation assay (Fig. 3A). The GST-CPAF fusion protein completely degraded both Puma and Puma, but left a degradation fragment (lane 4). The residual degradation fragment was not uniquely generated by the GST-CPAF fusion protein, but represented an intermediate fragment of Puma digested by both GST-CPAF and endogenous CPAF. This is because the degradation fragment left in the GST-CPAF digestion mixture was equivalent to the longer degradation fragment generated by endogenous CPAF when used at a low concentration (Fig. 2A, lane 3; and Fig. 3B, lane 4). More important, the GST-CPAF-mediated degradation of Puma was completely inhibited by lactacystin, but not by Me₂SO (Fig. 3A, lanes 5 and 6), suggesting that a CPAF-like activity in the GST-CPAF fusion protein was responsible for degrading Puma.

To exclude the possible involvement of any contaminating proteolytic activity from the bacteria in the degradation of the host BH3-only proteins, we compared the degradation activity of wild-type GST-CPAF with that of two mutant GST-CPAF fusion proteins (GST-CPAF(YVAD) and GST-CPAF(L281G)). Both GST-CPAF(YVAD) and GST-CPAF(L281G)) have been shown previously to lack CPAF activity (14, 15). The three GST fusion proteins were immobilized on beads and monitored for total protein amount on a Coomassie Blue-stained gel. All three fusion proteins were loaded onto the gel in three different bead volumes, and protein staining revealed that the three GST fusion proteins were present in equivalent amounts (Fig. 4A). A duplicate set of bead aliquots was used as the source of enzyme to degrade the BH3-only proteins in a cell-free degradation assay (Fig. 4B). Because cytokeratin 8 (keratin 8) is a known substrate of CPAF (13), it was used as a positive control. Wild-type GST-CPAF displayed some degradation activity for both Puma and Bik when 8 µl of beads was used and strong degradation activity when 32 µl of beads was used. However, neither of the mutant GST-CPAF proteins showed any degradation activity regardless of the bead volumes used. Because all three GST-CPAF fusion proteins were similarly purified from bacterial cultures, this experiment demonstrated that it is unlikely that bacterial contaminants contributed to BH3-only protein degradation in any significant way.

View larger version (53K): [in this window] [in a new window]   FIGURE 2. CPAF is required for BH3-only protein degradation in C. trachomatis-infected cells. A, CPAF-containing L2S100 as the source of enzyme (HeLaS100 lacking CPAF used as a negative control; 2 µg/reaction) was used to digest the BH3-only protein-containing cytosolic extract (CE; 2 µg/reaction) made from normal HeLa cells in a cell-free degradation assay. L2S100 was used at high (H; 5 µg/reaction), middle (M; 1 µg/reaction), and low (L; 0.2 µg/reaction) concentrations. The irreversible proteasomal inhibitor lactacystin was used at a final concentration of 1 µM, and the solvent Me2SO (DMSO) alone was used as a control at an equal volume. The total volume of each reaction was 20 µl, and the whole reaction was loaded into SDS gel to detect the residual (R) BH3-only proteins using specific antibodies on a Western blot. Note that partial digestion of Puma and Puma with a long degradation band was achieved at 0.2 µg of L2S100, whereas complete digestion of Puma and Puma with a short degradation band was achieved at 5µg of L2S100; digestion was completely inhibited by lactacystin, but not by Me2SO alone. B, either intact L2S100 at both high (5 µg/reaction) and low (0.2 µg/reaction) concentrations or the remaining L2S100 supernatants after precipitation with antibody-conjugated beads or the precipitated beads as the source of enzyme were used to digest the Puma-containing cytosolic extract in the cell-free degradation assay, and the residual Puma and Puma substrates were detected by Western blotting. Note that intact L2S100 completely digested Puma and Puma at both high and low concentrations, but left a longer degradation fragment at the low concentration. The remaining L2S100 supernatants after precipitation with CPAF-specific mAb 54b, but not with mAbs recognizing host proteasome -subunit HC3 (mAb MCP21) and the chlamydial major outer membrane protein (mAb MC22), were no longer able to digest Puma. Because of the co-migration of Puma and the immunoglobulin light chain (IgL), only Puma was detectable in the reaction containing the antibody-conjugated bead pellets. The mAb 54b-conjugated beads completely degraded full-length Puma with a short degradation fragment. The partial degradation of Puma with a long degradation band by the mAb MCP21- and mAb MC22-conjugated beads may be due to contamination of the beads by a small amount of CPAF. ns, nonspecific band.

View larger version (43K): [in this window] [in a new window]   FIGURE 3. Recombinant GST-CPAF is able to degrade the BH3-only proteins from normal HeLa cells. A, the cell-free degradation assay was carried out as described in the legend to Fig. 2A, except that a recombinant GST-CPAF fusion protein was also used as the source of enzyme to digest the Puma-containing cytosolic extract (CE). Note that GST-CPAF completely digested Puma with a long degradation fragment, which is equivalent to the digestion pattern generated by L2S100 at 0.2 µg/reaction (see Fig. 2B, lane 3). DMSO, Me2SO. B, the cell-free degradation assay was carried as described in the legend to Fig. 2A, except that varying amounts of both L2S100 and GST-CPAF fusion protein were used as the sources of enzymes. L (low), 0.05 µg/reaction; M (middle), 0.5 µg/reaction; H (high), 4 µg/reaction. ns, nonspecific band.

View larger version (47K): [in this window] [in a new window]   FIGURE 4. Recombinant GST-CPAF fusion proteins lacking CPAF proteolytic activity are no longer able to digest the BH3-only proteins. A, recombinant wild-type (wt) GST-CPAF or mutants with an insertion of sequence YVAD or with an L281G substitution were purified on glutathione-conjugated beads from bacterial cultures, and the beads were loaded onto an SDS gel in different amounts. Note that all three GST-CPAF fusion proteins were in equivalent amounts. B, a duplicate set of the three GST-CPAF fusion proteins was used as the source of enzyme to digest the BH3-only protein-containing cytosolic extract in the cell-free degradation assay, and the final reaction mixtures were analyzed for residual BH3-only proteins in the cytosolic extract by Western blotting. Note that the wild-type GST-CPAF fusion protein degraded the BH3-only proteins Puma and Bik as well as the positive control substrate cytokeratin 8, whereas the mutant fusion proteins failed to do so. ns, nonspecific band.

View larger version (47K): [in this window] [in a new window]   FIGURE 5. Recombinant GST-CPAF is sufficient for degrading the BH3-only protein Puma purified from bacteria. A, human Puma expressed as a GST fusion protein was purified from bacteria, and the purified protein was loaded onto SDS-polyacrylamide gels. After electrophoretic separation, one gel was stained with Coomassie Blue to visualize the total protein (lanes 1–6), and the other gel was transferred onto a nitrocellulose membrane for Western blot detection of GST-Puma (lanes 7–12). Free GST alone was used as a control. Note that a dominant full-length GST-Puma protein was detected despite various degradation fragments and that the anti-Puma antibody did not detect any free GST. B, recombinant GST-human Puma (free of other mammalian protein contamination) was used as the substrate at 0.1 µg/reaction in a cell-free degradation assay. Note that L2S100 (but not HeLaS100) degraded GST-Puma. GST-CPAF on beads was also able to degrade GST-Puma in a dose-dependant manner with partial digestion when GST-CPAF was used at a low concentration (L; 2 µl/reaction) and complete digestion when used at middle (m; 8 µl/reaction) and high (h; 32 µl/reaction) concentrations. DMSO, Me2SO.

View larger version (34K): [in this window] [in a new window]   FIGURE 6. Inhibition of Puma degradation by lactacystin during chlamydial infection. HeLa cells were infected with C. trachomatis L2, and 12 h after infection, the protease inhibitors lactacystin and Ala-Ala-Phe-chloromethyl ketone (AAF; cell-permeable serine protease inhibitor) were added to the cultures at final concentrations of 10 µM. After an additional 36 h of culturing, the cell samples were harvested for Western blot detection of Puma. Puma and Puma and their degradation products are indicated on the right. Note that lactacystin (but not Ala-Ala-Phe-chloromethyl ketone) inhibited Puma degradation. ns, stands for nonspecific band.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chlamydia may be selected to target the BH3-only proteins for inactivating host apoptosis. The pro-apoptotic BH3-only proteins are normally sequestrated from their pro-apoptotic activity by associating with various cytoplasmic organelles and functioning as the silent sensors for intracellular stress signals. The chlamydial inclusion has to expand during chlamydial intracellular growth, and the inclusion expansion inevitably alters the intracellular organization and disrupts the normal distribution of intracellular organelles, which may set off the BH3-only proteins to deliver death/stress signals to mitochondria. It is known that host cell apoptosis is an effector mechanism that controls intracellular infection, and apoptosis of the infected cells during the reticulate body stage may lead to the termination of chlamydial infection. Such a strong selection pressure may allow only the chlamydial organisms able to prevent host cell apoptosis to complete intracellular growth. By the time the inclusion expansion starts to disturb the intracellular environment, CPAF is already secreted into and accumulated in the host cell cytosol. Indeed, at 16–24 h after infection, obvious BH3-only protein degradation is observed (35–37). Efforts are under way to further evaluate whether CPAF alone in the absence of chlamydial infection can degrade the BH3-only proteins inside cells and prevent host cells from undergoing apoptosis. The challenge for this experiment is the difficulty in expressing active CPAF in mammalian cells. Because CPAF is synthesized as a proenzyme and has to be processed into an intramolecular dimer to acquire activity (14, 15), it has been difficult to generate constructs coding for active CPAF in mammalian cells.

CPAF is known to degrade the transcription factors RFX5 and USF-1 and cytokeratin 8. This study has added the BH3-only Bcl-2 family members to the list of CPAF substrates. An obvious question is what determines the substrate specificity of CPAF. When these known substrates are aligned, no common primary amino acid sequence motifs can be identified. We are in the process of determining CPAF substrate specificities using various biochemical approaches, which may potentially allow us to predict new cellular targets of CPAF.

	FOOTNOTES

 
^* This work was supported in part by National Institutes of Health Grants R01 AI57450, R01 AI64537, and R01 AI47997 (to G. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229. Tel.: 210-567-1169; Fax: 210-567-0293; E-mail: Zhongg{at}uthscsa.edu.

² The abbreviations used are: BH, Bcl-2 homology; GST, glutathione S-transferase; mAb, monoclonal antibody.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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