CPAF: a Chlamydial protease in search of an authentic substrate

Abstract

Bacteria in the genus Chlamydia are major human pathogens that cause an intracellular infection. A chlamydial protease, CPAF, has been proposed as an important virulence factor that cleaves or degrades at least 16 host proteins, thereby altering multiple cellular processes. We examined 11 published CPAF substrates and found that there was no detectable proteolysis when CPAF activity was inhibited during cell processing. We show that the reported proteolysis of these putative CPAF substrates was due to enzymatic activity in cell lysates rather than in intact cells. Nevertheless, Chlamydia-infected cells displayed Chlamydia-host interactions, such as Golgi reorganization, apoptosis resistance, and host cytoskeletal remodeling, that have been attributed to CPAF-dependent proteolysis of host proteins. Our findings suggest that other mechanisms may be responsible for these Chlamydia-host interactions, and raise concerns about all published CPAF substrates and the proposed roles of CPAF in chlamydial pathogenesis.

Figure 1. Golgin-84 cleavage does not occur in Chlamydia-infected cells when CPAF is inhibited during cell processing.

(A) Uninfected (0 hpi) and infected cells at time points between 12 and 36 hpi were treated with methyl acetate as a solvent control (top panel) or 150 µM of the CPAF inhibitor clasto-lactacystin (bottom panel) for 1 hour prior to cell lysis in RIPA buffer. Total cell lysates were separated by SDS-PAGE and probed with antibodies against golgin-84 or Erk 1/2 (loading control). (B) Cell-free degradation assay testing for CPAF activity in lysates prepared from the Chlamydia-infected HeLa cells described in Figure 1A. Each infected cell lysate was incubated with a lysate of uninfected HeLa cells as the source of golgin-84 substrate and reactions were analyzed by immunoblotting with golgin-84 antibodies. (C) Lysates of uninfected (0 hpi) or infected cells from different times in the infection were prepared in RIPA buffer (left panel) or by direct lysis in 8M urea (right panel), separated by SDS-PAGE and analyzed with antibodies to golgin-84 or α-tubulin (loading control). (D) Confocal images of uninfected or Chlamydia-infected HeLa cells examined at 18 and 24 hpi. Cells were stained with antibodies to the Golgi marker α-mannosidase II (red), the chlamydial major outer membrane protein MOMP (green) and the DNA dye Hoechst 33342 (blue) to detect Golgi membranes, the chlamydial inclusion and DNA, respectively. Scale bar, 10 µm.

Figure 2. Degradation of BH3-only proteins in Chlamydia-infected cells is prevented by inhibiting CPAF during cell processing.

(A) Lysates of uninfected (0 hpi) or infected HeLa cells were prepared in RIPA buffer (left panel) or by direct lysis in 8M urea (right panel) at the indicated times, separated by SDS-PAGE and analyzed by immunoblotting with antibodies to the proapoptotic BH3-only proteins Puma, Bik, or Bim. Equal loading was monitored for each blot with antibodies to Erk 1/2, but only the loading control for the Puma blot is shown as an example. (B) Uninfected or Chlamydia-infected HeLa cells were treated with 1 µM staurosporine to induce apoptosis, which was monitored by the loss of full-length caspase-3. Immunoblots of the lysates were probed with antibodies to caspase-3, MOMP (marker of Chlamydia infection), or Erk 1/2 (loading control).

Figure 3. Cleavage of intermediate filaments in Chlamydia-infected cells is also dependent on cell processing.

(A) Lysates of uninfected (0 hpi) or infected HeLa cells were prepared in RIPA buffer (left panel) or by direct lysis in 8M urea (right panel) at the indicated times, separated by SDS-PAGE and analyzed by immunoblotting with antibodies to keratin-8, keratin-18, or vimentin. Equal loading for each blot was monitored by blotting for Erk 1/2 (loading control), but only the loading control for keratin-8 is shown. (B) Uninfected and infected HeLa cells at 30 hpi were fixed and stained with antibodies to vimentin (green), the chlamydial major outer membrane protein MOMP (red) and the DNA dye Hoechst 33342 (blue). Representative confocal images are shown. Scale bar, 10 µm.

Figure 4. Proteolysis of four additional CPAF substrates is dependent on cell processing.

Lysates of uninfected (0 hpi) or infected HeLa cells were prepared in RIPA buffer (left panel) or by direct lysis in 8M urea (right panel) at the indicated times, separated by SDS-PAGE and probed with antibodies to the p65/RelA subunit of NFκB, cyclin B1, nectin-1, or RFX5 as indicated. Equal loading for each blot was monitored by blotting for Erk 1/2, but only the loading control for nectin-1 is shown.

Figure 5. CPAF is autocatalytically cleaved into its active form in Chlamydia-infected cells.

Lysates of uninfected (0 hpi) or infected HeLa cells were prepared in RIPA buffer (left panel) or by direct lysis in 8M urea (right panel) at the indicated times, separated by SDS-PAGE and probed with antibodies to the C-terminal fragment of CPAF (CPAFc) and α-tubulin (loading control).