Autoprocessing and self-activation of the secreted protease CPAF in Chlamydia-infected cells

Abstract

The Chlamydia-secreted protease/proteasome-like activity factor (CPAF) is synthesized as a proenzyme (proCPAF) and requires processing for proteolytic activity. Recent structural studies have further demonstrated that CPAF is a serine protease that can undergo autoprocessing and self-activation in a concentration-dependent manner in vitro. However, it is not known how CPAF is processed and activated during chlamydial infection. In the current study, we used a mutant CPAF designated as CPAF(E558A) that is deficient in processing by itself as a substrate to search for putative CPAF activation factor(s) in Chlamydia-infected cells. CPAF(E558A) was processed by the lysates made from Chlamydia-infected cells and the processing activity correlated with the presence of endogenous active CPAF in the fractionated lysate samples. CPAF produced in the Chlamydia-infected cells is required for processing the mutant CPAF(E558A) since the processing activity was removed by depletion with anti-CPAF but not control antibodies. Furthermore, a purified and activated wild type CPAF alone was sufficient for processing CPAF(E558A) and no other chlamydial proteases are required. Finally, fusion tag-induced oligomerization can lead to autoprocessing and self-activation of the wild type CPAF in mammalian cells. These observations together have demonstrated that CPAF undergoes autoprocessing and self-activation during chlamydial infection.

Fig. 1. Correlating the endogenous CPAF activity in Chlamydia-infected cells with the processing of the mutant CPAF(E558A)

(A) Different amounts of lysates made from HeLa cells with (Ct-HeLa) or without (HeLa) chlamydial infection were used to cleave the fusion protein CPAF(E558A)-His in a cell-free degradation assay. The degradation mixtures were detected with antibodies against His tag (anti-His tag, panel a) and CPAF C-terminus (mAb 100a, panel b) and N-terminus (mAb 54b, panel c) in a Western blot. Note that Ct-HeLa lysate contained endogenous CPAFc fragments (indicative of activated CPAF) and processed the CPAF(E558A)-His fusion protein into its C-terminal fragment that contains His tag, designated as CPAF(E558A)c-His. The N-terminal fragment derived from both the endogenous CPAF and mutant CPAF(E558A)-His co-migrate in a single band as CPAFn (panel c). (B) Both the supernatant and pellet fractions prepared from the Chlamydia-infected HeLa cells were used to cleave the CPAF(E558A)-His fusion protein and the cleavage products were detected with the anti-His tag (panel a) and CPAFc (panel b) antibodies. Note that only the supernatant fraction contained the activated endogenous CPAF and cleaved the mutant CPAF(E558A)-His. (C) The supernatant fraction was subjected to further fractionation via a mono-Q column and the eluted fractions (labeled from 13 to 23 on top of the figure) were both detected for the presence of CPAFn (panel a)/CPAFc (panel b) fragments and measured for their ability to cleave Keratin 8 [from a cytosolic extract (CE)], a known CPAF substrate (panel c) and to process the mutant CPAF(E558A) (panel d). Note that fractions# 18,19 and 20 (marked with white arrows in panel d) contained the endogenous active CPAF (panels a & b) with relatively high CPAF activity (panel c) and processed the mutant CPAF fusion protein, correlating the endogenous CPAF activity with the processing of CPAF(E558A)-His. Both 100a and 54b mAbs also detected the endogenous CPAF zymogen and 100a further detected the C-terminal processing intermediate (marked with a white star, panel b). The shortest keratin 8 degradation fragment detected in fraction 16 & 17 likely came from the Q-column-fractionated Chlamydia-infected HeLa lysate samples (marked with white # in panel c).

Fig. 1. Correlating the endogenous CPAF activity in Chlamydia-infected cells with the processing of the mutant CPAF(E558A)

(A) Different amounts of lysates made from HeLa cells with (Ct-HeLa) or without (HeLa) chlamydial infection were used to cleave the fusion protein CPAF(E558A)-His in a cell-free degradation assay. The degradation mixtures were detected with antibodies against His tag (anti-His tag, panel a) and CPAF C-terminus (mAb 100a, panel b) and N-terminus (mAb 54b, panel c) in a Western blot. Note that Ct-HeLa lysate contained endogenous CPAFc fragments (indicative of activated CPAF) and processed the CPAF(E558A)-His fusion protein into its C-terminal fragment that contains His tag, designated as CPAF(E558A)c-His. The N-terminal fragment derived from both the endogenous CPAF and mutant CPAF(E558A)-His co-migrate in a single band as CPAFn (panel c). (B) Both the supernatant and pellet fractions prepared from the Chlamydia-infected HeLa cells were used to cleave the CPAF(E558A)-His fusion protein and the cleavage products were detected with the anti-His tag (panel a) and CPAFc (panel b) antibodies. Note that only the supernatant fraction contained the activated endogenous CPAF and cleaved the mutant CPAF(E558A)-His. (C) The supernatant fraction was subjected to further fractionation via a mono-Q column and the eluted fractions (labeled from 13 to 23 on top of the figure) were both detected for the presence of CPAFn (panel a)/CPAFc (panel b) fragments and measured for their ability to cleave Keratin 8 [from a cytosolic extract (CE)], a known CPAF substrate (panel c) and to process the mutant CPAF(E558A) (panel d). Note that fractions# 18,19 and 20 (marked with white arrows in panel d) contained the endogenous active CPAF (panels a & b) with relatively high CPAF activity (panel c) and processed the mutant CPAF fusion protein, correlating the endogenous CPAF activity with the processing of CPAF(E558A)-His. Both 100a and 54b mAbs also detected the endogenous CPAF zymogen and 100a further detected the C-terminal processing intermediate (marked with a white star, panel b). The shortest keratin 8 degradation fragment detected in fraction 16 & 17 likely came from the Q-column-fractionated Chlamydia-infected HeLa lysate samples (marked with white # in panel c).

Fig. 1. Correlating the endogenous CPAF activity in Chlamydia-infected cells with the processing of the mutant CPAF(E558A)

(A) Different amounts of lysates made from HeLa cells with (Ct-HeLa) or without (HeLa) chlamydial infection were used to cleave the fusion protein CPAF(E558A)-His in a cell-free degradation assay. The degradation mixtures were detected with antibodies against His tag (anti-His tag, panel a) and CPAF C-terminus (mAb 100a, panel b) and N-terminus (mAb 54b, panel c) in a Western blot. Note that Ct-HeLa lysate contained endogenous CPAFc fragments (indicative of activated CPAF) and processed the CPAF(E558A)-His fusion protein into its C-terminal fragment that contains His tag, designated as CPAF(E558A)c-His. The N-terminal fragment derived from both the endogenous CPAF and mutant CPAF(E558A)-His co-migrate in a single band as CPAFn (panel c). (B) Both the supernatant and pellet fractions prepared from the Chlamydia-infected HeLa cells were used to cleave the CPAF(E558A)-His fusion protein and the cleavage products were detected with the anti-His tag (panel a) and CPAFc (panel b) antibodies. Note that only the supernatant fraction contained the activated endogenous CPAF and cleaved the mutant CPAF(E558A)-His. (C) The supernatant fraction was subjected to further fractionation via a mono-Q column and the eluted fractions (labeled from 13 to 23 on top of the figure) were both detected for the presence of CPAFn (panel a)/CPAFc (panel b) fragments and measured for their ability to cleave Keratin 8 [from a cytosolic extract (CE)], a known CPAF substrate (panel c) and to process the mutant CPAF(E558A) (panel d). Note that fractions# 18,19 and 20 (marked with white arrows in panel d) contained the endogenous active CPAF (panels a & b) with relatively high CPAF activity (panel c) and processed the mutant CPAF fusion protein, correlating the endogenous CPAF activity with the processing of CPAF(E558A)-His. Both 100a and 54b mAbs also detected the endogenous CPAF zymogen and 100a further detected the C-terminal processing intermediate (marked with a white star, panel b). The shortest keratin 8 degradation fragment detected in fraction 16 & 17 likely came from the Q-column-fractionated Chlamydia-infected HeLa lysate samples (marked with white # in panel c).

Fig. 2. The endogenous CPAF in Chlamydia-infected HeLa cells is required for processing the CPAF(E558A)-His fusion protein

(A) The lysate supernatant (sup) made from Chlamydia-infected HeLa cells (Ct-HeLa) with or without treatment with lactacystin at the indicated concentrations shown on top of the figure were used to digest the CPAF(E558A)-His fusion protein and the digestion products were detected with the anti-His tag antibody as described in Fig.1A legend. Note that lactacystin at the final concentration of 100uM completely blocked the processing CPAF(E558A)-His (lane 8). (B) Aliquots of the Chlamydia-infected HeLa (Ct-HeLa) lysate supernatants (sup) were depleted with or without antibodies as indicated on top of the figure before mixing with the substrate CPAF(E558A)-His fusion protein. The antibodies used for depletion include mAb 100a (recognizing CPAFc), mAb 54b (recognizing CPAFn) and rabbit antiserum raised with C. trachomatis serovar D MOMP (RαMOMP). The digestion products were monitored for both the residual His-tagged fusion protein/fragment (panel a) and the endogenous CPAFc (panel b) and MOMP (panel c) fragments in Western blot. Note that these antibodies efficiently depleted the corresponding specific antigens without affecting the irrelevant antigens (panel b & c) but only the anti-CPAF antibody depletion removed the processing activity from Chlamydia-infected cell samples.

Fig. 2. The endogenous CPAF in Chlamydia-infected HeLa cells is required for processing the CPAF(E558A)-His fusion protein

(A) The lysate supernatant (sup) made from Chlamydia-infected HeLa cells (Ct-HeLa) with or without treatment with lactacystin at the indicated concentrations shown on top of the figure were used to digest the CPAF(E558A)-His fusion protein and the digestion products were detected with the anti-His tag antibody as described in Fig.1A legend. Note that lactacystin at the final concentration of 100uM completely blocked the processing CPAF(E558A)-His (lane 8). (B) Aliquots of the Chlamydia-infected HeLa (Ct-HeLa) lysate supernatants (sup) were depleted with or without antibodies as indicated on top of the figure before mixing with the substrate CPAF(E558A)-His fusion protein. The antibodies used for depletion include mAb 100a (recognizing CPAFc), mAb 54b (recognizing CPAFn) and rabbit antiserum raised with C. trachomatis serovar D MOMP (RαMOMP). The digestion products were monitored for both the residual His-tagged fusion protein/fragment (panel a) and the endogenous CPAFc (panel b) and MOMP (panel c) fragments in Western blot. Note that these antibodies efficiently depleted the corresponding specific antigens without affecting the irrelevant antigens (panel b & c) but only the anti-CPAF antibody depletion removed the processing activity from Chlamydia-infected cell samples.

Fig. 3. Processing of the mutant CPAF(E558A) by a purified wild type CPAF

The CPAF(E558A)-His fusion protein was digested with a wild type GST-CPAF(Wt), a lack of function mutant GST-CPAF(L281G) and an irrelevant GST fusion protein as indicated on top of the figure. Lysates from HeLa cells with (Ct-HeLa) or without chlamydial infection were used as positive and negative controls. The digestion products were subjected to Western blot detection with an anti-His tag mAb. Note that only the wild type GST-CPAF (known to contain activated CPAF fragments) and the Chlamydia-infected HeLa lysates processed the mutant CPAF(E558A)-His fusion protein into the His-tagged C-terminus of CPAF designated as CPAF(E558A)c-His as indicated on the right side of the figure.

Fig. 4. Processing of the mutant CPAF(E558A) by 29 putative proteases encoded in C. trachomatis genome

All 29 putative chlamydial proteases encoded in C. trachomatis serovar D genome were produced as GST fusion proteins and the purified fusion proteins were checked for quantity and quality on a Coomassie blue-staining SDS-Page gel (panel a). Note that full-length GST chlamydial fusion proteins were purified for all 29 proteases including CPAF although certain levels of degradation fragments and free GST molecules were also detected. A parallel set of the same GST fusion proteins were further used to digest the CPAF(E558A)-His fusion protein in a cell-free assay and the digestion products were detected with an anti-His tag antibody on a Western blot as indicated on the left of the figure (panel b). Note that only the positive control Infected-infected HeLa (Ct-HeLa) lysate (lane 2) and the GST-CPAF fusion protein cleaved the CPAF(E558A)-His fusion protein into the CPAF(E558A)c-His fragment (see arrow pointed along the right side of the figure) and none of other chlamydial proteases was able to do so.

Fig. 5. Induction of CPAF autoprocessing and self-activation in mammalian cells

(A) Mammalian expression vectors pEF/Myc, pDsRed Monomer C1 or pDsRed Express C1 with (pEF/Myc-CPAF, pDsRedMono-CPAF, pDsRedExp-CPAF) or without (pEF/Myc, pDsRedMono or pDsRedExp alone) coding for CPAF as listed on top of the figure were used to transfect 293T cells and 24h after transfection, the whole cell samples were subjected to Western blot detection with the mouse anti-CPAFc mAb 100a (panel a) or rabbit anti-PUMA mAb (panel b). Note that a matured CPAFc fragment identical to that from the Chlamydia-infected HeLa (Ct-HeLa) lysate as indicated on the right side of the figure was detected in samples transfected with CPAF gene encoded in pDsRed but not pEF vectors and more obvious CPAFc appeared in the pDsRedExp-CPAF plasmid-transfected sample. The appearance of CPAFc correlated with PUMA cleavage in the cell samples. (B) The mutant CPAF(E558A) was cloned into the pDsRedExp vector and compared for processing in mammalian cells with the wild type CPAF. The transfection and Western blot detection were carried similarly as described above in legend for (A). Note that only the wild type CPAF sample produced the matured CPAFc fragment (panel a) and displayed Puma cleavage activity (panel b).

Fig. 5. Induction of CPAF autoprocessing and self-activation in mammalian cells

(A) Mammalian expression vectors pEF/Myc, pDsRed Monomer C1 or pDsRed Express C1 with (pEF/Myc-CPAF, pDsRedMono-CPAF, pDsRedExp-CPAF) or without (pEF/Myc, pDsRedMono or pDsRedExp alone) coding for CPAF as listed on top of the figure were used to transfect 293T cells and 24h after transfection, the whole cell samples were subjected to Western blot detection with the mouse anti-CPAFc mAb 100a (panel a) or rabbit anti-PUMA mAb (panel b). Note that a matured CPAFc fragment identical to that from the Chlamydia-infected HeLa (Ct-HeLa) lysate as indicated on the right side of the figure was detected in samples transfected with CPAF gene encoded in pDsRed but not pEF vectors and more obvious CPAFc appeared in the pDsRedExp-CPAF plasmid-transfected sample. The appearance of CPAFc correlated with PUMA cleavage in the cell samples. (B) The mutant CPAF(E558A) was cloned into the pDsRedExp vector and compared for processing in mammalian cells with the wild type CPAF. The transfection and Western blot detection were carried similarly as described above in legend for (A). Note that only the wild type CPAF sample produced the matured CPAFc fragment (panel a) and displayed Puma cleavage activity (panel b).

Fig. 6. Processing of CPAF(E558A) in Chlamydia-infected cells

(A) Mammalian expression vector pEF/Myc coding for the mutant CPAF(E558A) from C. trachomatis [pEF/Myc-CPAF(E558A)-Ct] was used to transfect HeLa cells and 8h after transfection, some transfected cells were infected with C. muridarum organisms. 30h after infection, all transfected cells with or without infection were harvested, some for direct Western blot detection (lanes 5 & 6) while others were incubated with lysates made from either HeLa alone (lane 3) or C. muridarum-infected cells (lane 4) prior to the Western blot detection with the mouse mAbs 100a (panel a) and 54b (panel b) as primary antibodies. Lysates made from either C. trachomatis serovar D- or C. muridarum organism-infected cells were directly loaded as controls (lanes 1 & 2). Note that there was no detection of C-terminal fragment in the CPAF(E558A)-transfected and C. muridarum-infected cell sample (lane 6, panel a) although the C. muridarum-infected cell lysates contained CPAF-Cm (lane 6, panel b) and cleaved CPAF(E558A)-Ct in a cell-free system (lane 4), suggesting that the Chlamydia-secreted CPAF-Cm was unable to access to the plasmid-expressed CPAF(E558A)-Ct in the same infected cells. (B) Mammalian expression vectors pDsRed Monomer C1 coding with CPAF(E558A)-Cm [pDsRedMono-CPAF(E558A)-Cm] were used to transfect HeLa cells and 8h after transfection, the transfectants were infected with C. trachomatis serovar D organisms. 40h after infection, the cell samples were processed for immunofluorescence assay. The plasmid-expressed CPAF(E558A)-Cm was visualized via the fusion tag RFP (red, panel b), the C. trachomatis-secreted CPAF-Ct was labeled with mAb100a and visualized with a goat anti-mouse IgG conjugated with Cy2 (green, panel a) while DAPI was used to visualized DNA (blue). “N” stands for nuclei while “I” for chlamydial inclusion. The selected areas from each panel were shown in the bottom row marked with a1, b1 & c1. Note that the Chlamydia-secreted CPAF-Ct (green or green arrowheads) did not overlap with the plasmid-expressed CPAF(E558)-Cm (red or red arrowheads).

Fig. 6. Processing of CPAF(E558A) in Chlamydia-infected cells

(A) Mammalian expression vector pEF/Myc coding for the mutant CPAF(E558A) from C. trachomatis [pEF/Myc-CPAF(E558A)-Ct] was used to transfect HeLa cells and 8h after transfection, some transfected cells were infected with C. muridarum organisms. 30h after infection, all transfected cells with or without infection were harvested, some for direct Western blot detection (lanes 5 & 6) while others were incubated with lysates made from either HeLa alone (lane 3) or C. muridarum-infected cells (lane 4) prior to the Western blot detection with the mouse mAbs 100a (panel a) and 54b (panel b) as primary antibodies. Lysates made from either C. trachomatis serovar D- or C. muridarum organism-infected cells were directly loaded as controls (lanes 1 & 2). Note that there was no detection of C-terminal fragment in the CPAF(E558A)-transfected and C. muridarum-infected cell sample (lane 6, panel a) although the C. muridarum-infected cell lysates contained CPAF-Cm (lane 6, panel b) and cleaved CPAF(E558A)-Ct in a cell-free system (lane 4), suggesting that the Chlamydia-secreted CPAF-Cm was unable to access to the plasmid-expressed CPAF(E558A)-Ct in the same infected cells. (B) Mammalian expression vectors pDsRed Monomer C1 coding with CPAF(E558A)-Cm [pDsRedMono-CPAF(E558A)-Cm] were used to transfect HeLa cells and 8h after transfection, the transfectants were infected with C. trachomatis serovar D organisms. 40h after infection, the cell samples were processed for immunofluorescence assay. The plasmid-expressed CPAF(E558A)-Cm was visualized via the fusion tag RFP (red, panel b), the C. trachomatis-secreted CPAF-Ct was labeled with mAb100a and visualized with a goat anti-mouse IgG conjugated with Cy2 (green, panel a) while DAPI was used to visualized DNA (blue). “N” stands for nuclei while “I” for chlamydial inclusion. The selected areas from each panel were shown in the bottom row marked with a1, b1 & c1. Note that the Chlamydia-secreted CPAF-Ct (green or green arrowheads) did not overlap with the plasmid-expressed CPAF(E558)-Cm (red or red arrowheads).