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.
We have previously correlated Chlamydia trachomatis antiapoptotic activity with the blockade of mitochondrial cytochrome c release and the inhibition of Bax and Bak activation. We now report that C. trachomatis infection leads to degradation of Bik, Puma, and Bim, three upstream proapoptotic BH3-only proteins of the Bcl-2 family that can transmit death signals to mitochondria by inhibiting the Bcl-2 antiapoptotic proteins and/or activating the Bcl-2 proapoptotic members, such as Bax and Bak. This observation has provided new information on the chlamydial antiapoptosis mechanisms.
SummaryA chlamydia-secreted protein designated CPAF (chlamydial proteasome-like activity factor) was shown previously to degrade host transcriptional factors (e.g. RFX5) required for major histocompatibility (MHC) gene activation. Although CPAF is encoded by a single open reading frame (ORF) in the chlamydial genome, two fragments designated CPAFn and CPAFc were the main products purified. The current study was designed to test whether cleavage of CPAF into CPAFn and CPAFc is a physiological process required for CPAF proteolytic activity. Pulse-chase experiments revealed that CPAF was initially synthesized in chlamydia-infected cells as a 70 kDa fulllength protein and rapidly cleaved into CPAFn and c fragments. Full-length CPAF expressed via a transgene in mammalian cells remained uncleaved and had no proteolytic activity, whereas CPAF expressed in Escherichia coli cells was processed and possessed RFX5 degradation activity. CPAF mutants deficient in processing even when expressed by E. coli failed to degrade RFX5. More importantly, the RFX5 degradation activity was partially restored when the mutant CPAF was artificially induced to undergo cleavage. These observations together have demonstrated that cleavage of CPAF is both necessary and sufficient for CPAF activity.
We have previously shown that individuals infected with Chlamydia trachomatis can develop a robust antibody response to a chlamydia-secreted protein (designated chlamydial proteasome/protease-like activity factor [CPAF]). We now report that sera collected from these infected individuals neutralized the proteolytic activity of CPAF. Depletion of the serum sample with CPAF proteins to remove the CPAF-specific antibodies effectively blocked the neutralization, whereas similar depletion with the HSP60 proteins failed to do so. We further demonstrated that the CPAF central region covering residues 200 to 338 was predominantly recognized by the human neutralization antibodies. The significance of the CPAF neutralization antibodies generated in chlamydia-infected individuals is discussed.
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