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Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis
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Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

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Cleavage-dependent activation of a chlamydia-secreted protease

Feng Dong1, Mustak Pirbhai, Youmin Zhong

  • 1Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA.

Molecular Microbiology
|May 29, 2004
PubMed

Insights

Chlamydia-secreted protein CPAF (chlamydial proteasome-like activity factor) must be cleaved into fragments to degrade host RFX5, which is essential for MHC gene activation. This cleavage is necessary and sufficient for CPAF

Area of Science:

  • Microbiology
  • Molecular Biology
  • Immunology

Background:

  • Chlamydia-secreted protein CPAF degrades host transcriptional factors like RFX5.
  • RFX5 is crucial for activating major histocompatibility (MHC) gene expression.
  • CPAF is encoded by a single ORF but purified as two fragments: CPAFn and CPAFc.

Purpose of the Study:

  • To determine if CPAF cleavage is a physiological requirement for its proteolytic activity.
  • To investigate the role of CPAF processing in the degradation of host factors.

Main Methods:

  • Pulse-chase experiments in infected cells.
  • Expression of full-length CPAF and mutants in mammalian cells and E. coli.
  • Assessing RFX5 degradation activity of processed and unprocessed CPAF.

Main Results:

  • CPAF is synthesized as a 70 kDa protein and rapidly cleaved into CPAFn and CPAFc in infected cells.
  • Full-length CPAF expressed in mammalian cells remained uncleaved and inactive.
  • Processed CPAF from E. coli degraded RFX5, while unprocessed mutants did not.
  • Partial restoration of RFX5 degradation activity was observed upon artificial cleavage induction in mutants.

Conclusions:

  • CPAF cleavage into CPAFn and CPAFc is a necessary and sufficient step for its proteolytic activity.
  • This processing is critical for CPAF's ability to degrade host RFX5 and impact MHC gene activation.

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