Western Blot.

The Western blot assay was carried out as we described previously 7. Mouse antibodies were used to detect the NH₂-terminal portion of CPAF (CPAFn) fragment (CPAFn54b; mIgG1; data not shown), chlamydial major outer membrane protein (MOMP) (MC22; mIgG3; data not shown), eukaryotic HSP70 (IgG2a; Santa Cruz Biotechnology, Inc.), and 20S proteasome subunits (AFFINITI Research Products Limited). Rabbit antibodies were used to detect RFX5 (Rockland Immunochemicals), USF-1, and USF-2 (Santa Cruz Biotechnology, Inc.).

Cell-free Degradation Assay.

The cell-free degradation assay was performed as described previously 4. To generate fusion proteins for the cell-free assay, chlamydial DNA sequences coding for CPAF or CPAF fragments were cloned into a pGEX vector (Amersham Pharmacia Biotech) and expressed as fusion proteins with the glutathione S-transferase (GST) as fusion partner. The fusion proteins were purified with glutathione-conjugated agarose beads as described in the manufacturer's manual (Amersham Pharmacia Biotech). The following procedure was used to prepare nuclear extracts (NEs) as substrate (containing RFX5 and USF-1 and USF-2) for the cell-free assay. Normal HeLa cells were dounced to break cytoplasmic membranes and the pellets were repeatedly washed with a buffer consisting of 1% NP-40 and 150 mM NaCl in 50 mM Tris, pH 8.0, plus a protease inhibitor cocktail to remove cytosol/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 NEs thus prepared are essentially free of 20S proteasome components as detected on Western blot (data not shown). The human RFX5 gene from pREP-4/RFX5 plasmid (provided by Dr. P.J. van den Elsen, Leiden University Medical Center, Leiden, The Netherlands) was cloned into the pGEX vector and RFX5 was expressed in the JM109 Escherichia coli strain as a GST fusion protein. The GST-RFX5 fusion protein was purified to homogeneity using glutathione-conjugated agarose beads as described above. The purified GST-RFX5 was used as substrate in the cell-free degradation assays.

Immunofluorescence Staining Assay.

Immunofluorescence detection of CPAF in chlamydia-infected cells was carried out as described previously 6 7. In brief, HeLa cell monolayer was infected with Chlamydia trachomatis L2 for 30 h. The monolayer, after it was fixed with paraformaldehyde (Sigma-Aldrich) and permeabilized with Saponin (Sigma-Aldrich), was costained with Hoechst 32258 (blue), anti-MOMP antibody MC22 (probed with an FITC-conjugated, mouse IgG3-specific secondary antibody), and anti-CPAFn antibody 54b (probed with a Cy3-conjugated, mouse IgG1-specific secondary antibody). Images were acquired individually for each stain in gray using a Cooker digital camera connected to an AX70 Olympus microscope, and the single-color images were merged in frame into the triple-color image using the software Image Pro.

CPAF Is Both Necessary and Sufficient for Degradation of Host Transcription Factors.

We have previously demonstrated that the chlamydial proteasome–like activity does not reflect the function of host proteasome components 4. When fractions obtained from a size exclusion chromatography column were probed with an antibody recognizing CPAFn, the chlamydial proteasome–like activity measured with RFX5 degradation was found to correlate very well with the presence of the CPAFn fragment but not host proteasome components (Fig. 2 A). To test whether CPAF is necessary for the chlamydial proteasome–like activity, we used the anti-CPAFn antibody to precipitate CPAFn from chlamydia-infected HeLa cell lysate and measured the RFX5 degradation activity of the precipitated pellets as well as the activity of the supernatants after the antibody depletion (Fig. 2 B). The total lysate of chlamydia-infected HeLa cells efficiently degraded RFX5. However, the supernatants remaining after antibody-mediated depletion of CPAFn completely lost the ability to degrade RFX5, whereas RFX5 degradation activity was readily detected in the precipitated material. Depletion of RFX5 degradation activity by anti-CPAFn was specific, because a control antibody recognizing the MOMP failed to precipitate this activity from the lysates. To monitor the efficiency of the antibody precipitation, radiolabeled lysate from chlamydia-infected HeLa cells was similarly precipitated with anti-CPAFn (I° precipitation) and the supernatant after the first precipitation was reprecipitated (II° precipitation) with the same reagent (Fig. 2 C). Autoradiography after SDS-PAGE of the material in both the I° and II° immunoprecipitates revealed that the anti-CPAFn antibody effectively removed both the CPAFn and CPAFc fragments from the lysates during the I° precipitation, because the II° immunoprecipitate showed minimal amounts of CPAF fragments. The control anti-MOMP antibody efficiently precipitated MOMP but not CPAF molecules from the lysates (Fig. 2 C). Together, these experiments demonstrate that CPAF or CPAF-associated material is necessary for RFX5 degradation activity in chlamydia-infected cell lysates.

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Figure 2

CPAF is both necessary and sufficient for degradation of host transcription factors. (A) HeLa L2 S100 was subjected to Superdex 200 size exclusion column analysis as described previously 4. The fractions were assayed for both RFX5 degradation activity and the presence of either host 20S proteasome subunits or CPAFn fragment. The presence of CPAFn but not host 20S proteasomes correlated with RFX5 degradation activity. (B) The effect of antibody depletion of CPAF on RFX5 degradation activity in chlamydia-infected HeLa cell lysates. Both the anti-CPAFn (54b) and anti-MOMP (MC22) antibodies were used to precipitate proteins from chlamydia-infected HeLa cell lysate. The intact (total) lysate, the supernatant after antibody precipitation, and the proteins precipitated by the antibodies (pellet) were all examined for their ability to degrade RFX5 in a cell-free assay. The anti-CPAFn antibody precipitated RFX5 degradation activity from supernatants to pellets. (C) Radioimmunoprecipitation analysis of proteins precipitated by anti-CPAFn or anti-MOMP antibodies. HeLa cells infected with chlamydia were metabolically labeled and the proteins in the labeled cell lysate were precipitated with antibodies. The supernatant after the first precipitation (I°) was subjected to a second immunoprecipitation (II°) with the same antibodies. Both the anti-CPAFn and anti-MOMP antibodies completely removed the corresponding molecules from the lysates during I° precipitation. The ratio of cell lysate vs. antibody was the same as in Fig. 2 B. (D) Degradation of transcription factors RFX5 and USF-1 by chlamydia-synthesized and bacterium-expressed CPAF in a cell-free assay with or without the inhibitor lactacystin. HeLa L2 S100 (containing chlamydia-synthesized CPAF) was used as positive control, and HeLa S100 was used as a negative control. Bacterium-expressed GST-CPAF was used at a final concentration of either 0.2 (low; sufficient for RFX5 degradation) or 0.6 μM (high; sufficient for both RFX5 and USF-1 degradation). The degradation was inhibitable by the proteasome inhibitor lactacystin (100 μM). A fusion protein containing GST and the COOH-terminal portion of CPAF (GST-CPAFc) failed to degrade any of the nuclear factors even at 4 μM. The anti–USF-2 antibody can detect both USF-1 and USF-2 as described previously (reference 4). Ns, nonspecific binding. (E). Purification of the recombinant human RFX5 from a bacterial expression system. The GST-RFX5 fusion protein was purified with glutathione-agarose beads as described in Materials and Methods. Different amounts of the purified protein were loaded to a 12% polyacrylamide SDS gel. After electrophoresis, the gel was stained with Coomassie blue dye. The GST-RFX5 fusion protein has a MW of ~100 kD. (F). Degradation of the purified human recombinant RFX5 by CPAF in a cell-free assay. The cell-free assay was carried out in the exact same way as described in D, except that 0.5 μg of the purified GST-RFX5 instead of the NEs was used as substrate in some reactions as indicated in the figure. The digestion experiment with the purified GST-RFX5 as substrate was repeated four times and similar results were observed. RFX5 in NEs is defined as endogenous and the bacterium-expressed GST-RFX5 fusion protein defined as recombinant.

To evaluate whether CPAF is sufficient for degrading host transcription factors, the protein encoded by the full-length CPAF gene was expressed as a fusion with GST, using a bacterial system. The purified GST-CPAF was evaluated for its ability to degrade RFX5 in a cell-free assay in comparison to material extracted from infected cells (HeLa L2 S100; Fig. 2 D). The HeLa L2 S100 degraded both the RFX5 and USF-1 transcription factors in NEs, whereas the material from uninfected HeLa cells (HeLa S100) failed to do so. Furthermore, even though the chlamydia proteolytic activity could be fractionated away from host cell 20S proteasomes (Fig. 2 A), the degradation activity of the HeLa L2 S100 was completely inhibited by lactacystin, an irreversible inhibitor of proteasomal proteases. These observations suggest that a chlamydial proteasome–like activity was being measured in this assay, as reported previously 4. More significantly, the purified GST-CPAF was able to degrade both RFX5 and USF-1 and the degradation was inhibitable by lactacystin. A GST fusion protein containing only the COOH-terminal portion of CPAF (GST-CPAFc) failed to degrade either of the host nuclear proteins even at a concentration 20 times higher than that of GST-CPAF (Fig. 2 D). These observations demonstrated that the purified CPAF is sufficient to mediate the degradation of host transcription factors in HeLa cell NEs. However, this set of experiments failed to address whether other components in the NEs also contributed to the degradation activity.

To further confirm that CPAF alone is sufficient for the degradation activity, we expressed the human transcription factor RFX5 in a prokaryotic system as a GST fusion protein. The GST-RFX5 was purified to homogeneity (Fig. 2 E) and the purified protein was used as the substrate for measuring the degradation activity of the cloned CPAF in a cell-free assay (Fig. 2 F). The cloned CPAF completely degraded the endogenous RFX5 in HeLa cell NEs and the degradation activity was inhibited by lactacystin, which is consistent with the observations described above in Fig. 2 D. More importantly, the cloned CPAF also effectively degraded the recombinant human RFX5 purified from a bacterial expression system (Fig. 2 F). Degradation of the recombinant RFX5 was inhibited by lactacystin, suggesting that the same enzymatic activity was responsible for the degradation of both endogenous and recombinant RFX5. A similarly purified control GST-CPAFc fusion protein failed to degrade the recombinant RFX5, suggesting that degradation of RFX5 measured in the assay was not contributed by bacterial contaminants (if there was any) or the GST fusion partner. These observations clearly demonstrate that the chlamydial CPAF alone is sufficient for degrading host RFX5.

We thank Drs. Ronald N. Germain, Joel Baseman, and Feng Liu for reading the manuscript, and Peter J. van den Elsen for providing the RFX5 plasmid.

This work was supported in part by grants (to G. Zhong) from the Medical Research Council of Canada and the National Institutes of Health (R01 AI47997-01).