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

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.