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

Bill Whitmire,Chunfu Yang,Alan G Barbour,Pingzhao Hu,Stuart Mccorrister,Michael John Patton,Robert Fariss,Grant Mcclarty,Kaiqiong Zhao,Mary Blake,Chris Grant,Harlan D Caldwell,Garrett Westmacott

doi:10.1128/mbio.01427-16

Abstract

ABSTRACTThe 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.

Highlights

The chlamydial protease-like activity factor (CPAF) is hypothesized to be an important secreted virulence factor; challenges in denaturing its proteolytic activity have hampered attempts to identify its legitimate targets
We show that CPAF and T3SEs function cooperatively in suppressing innate immune signaling by inhibiting p65 nuclear translocation
Over the past 2 decades, dozens of protein targets were put forth as potential candidates; recent reports have shown that insufficient denaturation of CPAF results in nonspecific proteolysis of proteins during sample preparation [15]

Summary

Introduction

The chlamydial protease-like activity factor (CPAF) is hypothesized to be an important secreted virulence factor; challenges in denaturing its proteolytic activity have hampered attempts to identify its legitimate targets. Genetic complementation of null organisms with CPAF restored p65 nuclear translocation inhibition and proteolysis of chlamydial type III secreted effector proteins (T3SEs). The chlamydial proteaselike 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. 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. Its primary function is to secrete type III secreted effector proteins (T3SEs) into the host cytosol, which modulate cell functions to ensure pathogen survival [4]. Activation of the RB midcycle injectisome and the function of midcycle T3SEs are poorly characterized [7]

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Results

Conclusion