Reassessing the role of CPAF in Chlamydia infections through genetic approaches

EMILY A. SNAVELY; MARCELA KOKES; ROBERT J. BASTIDAS; JOE D. DUNN; BIDONG D. NGUYEN; HECTOR A. SAKA; RAPHAEL H. VALDIVIA

San Antonio, Texas

Encuentro; CBRS 2013: 6th Biennial Meeting of the Chlamydia Basic Research Society; 2013

Chlamydia Basic Research Society

The Chlamydia protease CPAF has been proposed to modulate multiple mammalian cellular functions based on the identification of a broad range of host substrates. Recent findings suggest that CPAF activity can persist after cell lysis and thus confound the extent to which CPAF cleavage and degradation of its putative targets occurs in living cells. Furthermore, small molecule and peptide inhibitors of CPAF have yielded divergent results and their off-target effects are difficult to predict and control. Here we sought to clarify the role played by CPAF in the manipulation of host cell functions by analyzing Chlamydia mutants lacking CPAF activity. From a broad screen of small plaque forming, EMS-derived Chlamydia mutants, we identified a strain with a loss-of-function truncation mutation at the amino-terminal end of CPAF.  CPAF cannot be detected in this strain by western blot or immunofluorescence microscopy, and infected cell lysates do not possess any detectable CPAF activity. Cells infected with these CPAF-deficient strains were still resistant to staurosporine-induced apoptosis, exhibited Golgi fragmentation and were still resistant to reinfection, suggesting that these chlamydial activities are not mediated by CPAF. In contrast, we observed that late in infection remodeling of the intermediate filament protein vimentin was dependent on CPAF. This cleavage correlated with loss of inclusion membrane integrity, suggesting that CPAF may play a role during the late stages of infection.