Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors

Abstract

Microbial pathogens have been selected for the capacity to evade or manipulate host responses in order to survive after infection. Chlamydia, an obligate intracellular pathogen and the causative agent for many human diseases, can escape T lymphocyte immune recognition by degrading host transcription factors required for major histocompatibility complex (MHC) antigen expression. We have now identified a chlamydial protease- or proteasome-like activity factor (CPAF) that is secreted into the host cell cytosol and that is both necessary and sufficient for the degradation of host transcription factors RFX5 and upstream stimulation factor 1 (USF-1). The CPAF gene is highly conserved among chlamydial strains, but has no significant overall homology with other known genes. Thus, CPAF represents a unique secreted protein produced by an obligate intracellular bacterial pathogen to interfere with effective host adaptive immunity.

Figure 1

Purification and sequence identification of CPAF. (A) A cytosolic protein preparation from chlamydia-infected HeLa cells (HeLa L2 S100) was subjected to three consecutive column separations as listed in the figure. Fractions collected from each column were monitored for both degradation activity in a cell-free assay and total protein profiles on SDS-polyacrylamide gels. Fractions with degradation activity were pooled and loaded to the next column for further fractionation. Two dominant protein bands that correlated with the degradation activity from the final Mono Q column were excised for protein sequence identification. “+” indicates partial digestion of the substrate and “++” indicates complete loss of substrate. “−” indicates that the substrate band is intact compared with the substrate alone sample. (B) List of tryptic peptides that match the chlamydial ORF CT858-encoded protein (sequence data are available from http://www. ncbi.nlm.nih.gov/Entrez/protein.html under accession no. A71461). Amino acid residues are represented using the single letter codes and numbered according to their positions in CPAF sequence. Tryptic fragments derived from the top band match the COOH-terminal portion whereas those from the bottom band match the NH₂-terminal portion of CPAF. The top band was designated as CPAFc and the bottom band as CPAFn.

Figure 1

Purification and sequence identification of CPAF. (A) A cytosolic protein preparation from chlamydia-infected HeLa cells (HeLa L2 S100) was subjected to three consecutive column separations as listed in the figure. Fractions collected from each column were monitored for both degradation activity in a cell-free assay and total protein profiles on SDS-polyacrylamide gels. Fractions with degradation activity were pooled and loaded to the next column for further fractionation. Two dominant protein bands that correlated with the degradation activity from the final Mono Q column were excised for protein sequence identification. “+” indicates partial digestion of the substrate and “++” indicates complete loss of substrate. “−” indicates that the substrate band is intact compared with the substrate alone sample. (B) List of tryptic peptides that match the chlamydial ORF CT858-encoded protein (sequence data are available from http://www. ncbi.nlm.nih.gov/Entrez/protein.html under accession no. A71461). Amino acid residues are represented using the single letter codes and numbered according to their positions in CPAF sequence. Tryptic fragments derived from the top band match the COOH-terminal portion whereas those from the bottom band match the NH₂-terminal portion of CPAF. The top band was designated as CPAFc and the bottom band as CPAFn.

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.

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.

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.

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.

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.

Figure 3

Intracellular distribution of CPAF during chlamydial infection. (A) Infected or uninfected HeLa cells were fractionated into cytosolic (S100) and nuclear (NE) fractions as described in Materials and Methods. The fractionated cellular samples and purified chlamydial organisms were evaluated for RFX5 degradation activity in a cell-free assay (top row) or analyzed for the presence of CPAFn (second row), MOMP (third row), or host heat shock protein 70 (HSP70; last row) using the corresponding antibodies. HeLa L2 S100 contained the highest RFX5 degradation activity, with a small amount of activity detected in the NE prepared from the same infected cells (lane HeLa L2 NE). The purified chlamydial organisms in the form of either the infectious elementary body (lane L2 EB) or the metabolically active but noninfectious reticulate body (lane L2 RB) contained no CPAF or CPAF activity. (B) Immunofluorescence detection of CPAF in chlamydia-infected cells. HeLa cell monolayer was infected with chlamydia organisms for 30 h. The monolayer was costained with Hoechst 32258 for DNA (blue), anti-MOMP antibody MC22 (probed with an FITC-conjugated, mouse IgG3-specific secondary antibody; green), and anti-CPAFn antibody 54b (probed with a Cy3-conjugated, mouse IgG1-specific secondary antibody; red). Images were acquired individually for each stain in gray (top row) 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 (bottom) using the software Image Pro. Note that the anti-CPAFn antibody only stained the cytosol (red) of the cells with chlamydial inclusions (green).

Figure 3

Intracellular distribution of CPAF during chlamydial infection. (A) Infected or uninfected HeLa cells were fractionated into cytosolic (S100) and nuclear (NE) fractions as described in Materials and Methods. The fractionated cellular samples and purified chlamydial organisms were evaluated for RFX5 degradation activity in a cell-free assay (top row) or analyzed for the presence of CPAFn (second row), MOMP (third row), or host heat shock protein 70 (HSP70; last row) using the corresponding antibodies. HeLa L2 S100 contained the highest RFX5 degradation activity, with a small amount of activity detected in the NE prepared from the same infected cells (lane HeLa L2 NE). The purified chlamydial organisms in the form of either the infectious elementary body (lane L2 EB) or the metabolically active but noninfectious reticulate body (lane L2 RB) contained no CPAF or CPAF activity. (B) Immunofluorescence detection of CPAF in chlamydia-infected cells. HeLa cell monolayer was infected with chlamydia organisms for 30 h. The monolayer was costained with Hoechst 32258 for DNA (blue), anti-MOMP antibody MC22 (probed with an FITC-conjugated, mouse IgG3-specific secondary antibody; green), and anti-CPAFn antibody 54b (probed with a Cy3-conjugated, mouse IgG1-specific secondary antibody; red). Images were acquired individually for each stain in gray (top row) 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 (bottom) using the software Image Pro. Note that the anti-CPAFn antibody only stained the cytosol (red) of the cells with chlamydial inclusions (green).