Chlamydial Protease-Like Activity Factor and Type III Secreted Effectors Cooperate in Inhibition of p65 Nuclear Translocation

Michael John Patton; Stuart McCorrister; Chris Grant; Garrett Westmacott; Robert Fariss; Pingzhao Hu; Kaiqiong Zhao; Mary Blake; Bill Whitmire; Chunfu Yang; Harlan D Caldwell; Grant McClarty

doi:10.1128/mBio.01427-16

Chlamydial Protease-Like Activity Factor and Type III Secreted Effectors Cooperate in Inhibition of p65 Nuclear Translocation

mBio. 2016 Sep 27;7(5):e01427-16. doi: 10.1128/mBio.01427-16.

Authors

Affiliations

¹ Laboratory of Clinical Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
² National Microbiology Laboratories, Winnipeg, Manitoba, Canada.
³ National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA.
⁴ Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Canada.
⁵ National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA.
⁶ Laboratory of Clinical Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA hcaldwell@niaid.nih.gov Grant.McClarty@umanitoba.ca.
⁷ National Microbiology Laboratories, Winnipeg, Manitoba, Canada hcaldwell@niaid.nih.gov Grant.McClarty@umanitoba.ca.

Abstract

The chlamydial protease-like activity factor (CPAF) is hypothesized to be an important secreted virulence factor; however, challenges in denaturing its proteolytic activity have hampered attempts to identify its legitimate targets. Here, we use a genetic and proteomic approach to identify authentic CPAF targets. Human epithelial cells infected with CPAF-sufficient and CPAF-deficient chlamydiae were lysed using known CPAF-denaturing conditions. Their protein profiles were analyzed using isobaric mass tags and liquid chromatography-tandem mass spectrometry. Comparative analysis of CPAF-sufficient and CPAF-deficient infections identified a limited number of CPAF host and chlamydial protein targets. Host targets were primarily interferon-stimulated gene products, whereas chlamydial targets were type III secreted proteins. We provide evidence supporting a cooperative role for CPAF and type III secreted effectors in blocking NF-κB p65 nuclear translocation, resulting in decreased beta interferon and proinflammatory cytokine synthesis. Genetic complementation of null organisms with CPAF restored p65 nuclear translocation inhibition and proteolysis of chlamydial type III secreted effector proteins (T3SEs). We propose that CPAF and T3SEs cooperate in the inhibition of host innate immunity.

Importance: Chlamydia trachomatis is an important human pathogen responsible for over 100 million infections each year worldwide. Its success as an intracellular pathogen revolves around its ability to evade host immunity. The chlamydial protease-like activity factor (CPAF) is a conserved serine protease secreted into the host cytosol of infected cells that is thought to play an important role in immune evasion. Currently, CPAF's authentic in situ target(s) and mechanism of action in immune evasion are poorly characterized. Using a CPAF-deficient strain and high-throughput proteomics, we report novel CPAF host and chlamydial targets. Host targets were primarily interferon-stimulated genes, whereas chlamydial targets were exclusively type III secreted proteins. We propose a novel mechanism for CPAF and type III secreted proteins in the evasion of host innate immune responses. These findings provide new insights into CPAF's function as a virulence factor and a better understanding of how chlamydiae evade host immunity.