Get access

Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches

Authors

  • Emily A. Snavely,

    1. Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University Medical Center, Durham, NC, USA
    Search for more papers by this author
  • Marcela Kokes,

    1. Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University Medical Center, Durham, NC, USA
    Search for more papers by this author
  • Joe Dan Dunn,

    1. Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University Medical Center, Durham, NC, USA
    Search for more papers by this author
  • Hector A. Saka,

    1. Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University Medical Center, Durham, NC, USA
    Current affiliation:
    1. Department of Clinical Biochemistry, School of Chemistry, Cordoba National University, Córdoba, Argentina
    Search for more papers by this author
  • Bidong D. Nguyen,

    1. Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University Medical Center, Durham, NC, USA
    Search for more papers by this author
  • Robert J. Bastidas,

    1. Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University Medical Center, Durham, NC, USA
    Search for more papers by this author
  • Dewey G. McCafferty,

    1. Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University Medical Center, Durham, NC, USA
    2. Department of Chemistry, Duke University, Durham, NC, USA
    Search for more papers by this author
  • Raphael H. Valdivia

    Corresponding author
    1. Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University Medical Center, Durham, NC, USA
    • Correspondence

      Raphael H. Valdivia, Department of Molecular Genetics, Microbiology and Center for Microbial Pathogenesis, Duke University Medical Center, DUMC 3580, Durham, NC 27710, USA

      Tel.: +1 919 668 3881

      fax: +1 919 684 2790

      e-mail: Raphael.valdivia@duke.edu

    Search for more papers by this author

  • This exciting study is a sorely needed advance that will be of significant interest to those who follow the field of Chlamydia pathogenesis. Although the data primarily refute prior reported roles of CPAF, the rationale is clear in the context of prior work. There are also new activities attributed to CPAF, and the reduced infectivity of the CPAF mutants is a new finding. The genetic approach used here will become the gold standard for the field.

Abstract

The secreted Chlamydia protease CPAF cleaves a defined set of mammalian and Chlamydia proteins in vitro. As a result, this protease has been proposed to modulate a range of bacterial and host cellular functions. However, it has recently come into question the extent to which many of its identified substrates constitute bona fide targets of proteolysis in infected host cell rather than artifacts of postlysis degradation. Here, we clarify the role played by CPAF in cellular models of infection by analyzing Chlamydia trachomatis mutants deficient for CPAF activity. Using reverse genetic approaches, we identified two C. trachomatis strains possessing nonsense, loss-of-function mutations in cpa (CT858) and a third strain containing a mutation in type II secretion (T2S) machinery that inhibited CPAF activity by blocking zymogen secretion and subsequent proteolytic maturation into the active hydrolase. HeLa cells infected with T2S or CPAF C. trachomatis mutants lacked detectable in vitro CPAF proteolytic activity and were not defective for cellular traits that have been previously attributed to CPAF activity, including resistance to staurosporine-induced apoptosis, Golgi fragmentation, altered NFκB-dependent gene expression, and resistance to reinfection. However, CPAF-deficient mutants did display impaired generation of infectious elementary bodies (EBs), indicating an important role for this protease in the full replicative potential of C. trachomatis. In addition, we provide compelling evidence in live cells that CPAF-mediated protein processing of at least two host protein targets, vimentin filaments and the nuclear envelope protein lamin-associated protein-1 (LAP1), occurs rapidly after the loss of the inclusion membrane integrity, but before loss of plasma membrane permeability and cell lysis. CPAF-dependent processing of host proteins correlates with a loss of inclusion membrane integrity, and so we propose that CPAF plays a role late in infection, possibly during the stages leading to the dismantling of the infected cell prior to the release of EBs during cell lysis.

Get access to the full text of this article

Ancillary