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

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 a...

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Autores principales: 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
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Publicado: American Society for Microbiology 2016
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spelling oai:doaj.org-article:35d9cc8f8cee4a74bea69bb767d997452021-11-15T15:50:15ZChlamydial Protease-Like Activity Factor and Type III Secreted Effectors Cooperate in Inhibition of p65 Nuclear Translocation10.1128/mBio.01427-162150-7511https://doaj.org/article/35d9cc8f8cee4a74bea69bb767d997452016-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01427-16https://doaj.org/toc/2150-7511ABSTRACT 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.Michael John PattonStuart McCorristerChris GrantGarrett WestmacottRobert FarissPingzhao HuKaiqiong ZhaoMary BlakeBill WhitmireChunfu YangHarlan D. CaldwellGrant McClartyAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 7, Iss 5 (2016)
institution DOAJ
collection DOAJ
language EN
topic Microbiology
QR1-502
spellingShingle Microbiology
QR1-502
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
Chlamydial Protease-Like Activity Factor and Type III Secreted Effectors Cooperate in Inhibition of p65 Nuclear Translocation
description 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.
format article
author 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
author_facet 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
author_sort Michael John Patton
title Chlamydial Protease-Like Activity Factor and Type III Secreted Effectors Cooperate in Inhibition of p65 Nuclear Translocation
title_short Chlamydial Protease-Like Activity Factor and Type III Secreted Effectors Cooperate in Inhibition of p65 Nuclear Translocation
title_full Chlamydial Protease-Like Activity Factor and Type III Secreted Effectors Cooperate in Inhibition of p65 Nuclear Translocation
title_fullStr Chlamydial Protease-Like Activity Factor and Type III Secreted Effectors Cooperate in Inhibition of p65 Nuclear Translocation
title_full_unstemmed Chlamydial Protease-Like Activity Factor and Type III Secreted Effectors Cooperate in Inhibition of p65 Nuclear Translocation
title_sort chlamydial protease-like activity factor and type iii secreted effectors cooperate in inhibition of p65 nuclear translocation
publisher American Society for Microbiology
publishDate 2016
url https://doaj.org/article/35d9cc8f8cee4a74bea69bb767d99745
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