Cargando…
The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets
Protein kinase A (PKA) signaling plays a critical role in the growth and development of all eukaryotic microbes. However, few direct targets have been characterized in any organism. The fungus Aspergillus fumigatus is a leading infectious cause of death in immunocompromised patients, but the specifi...
Autores principales: | , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Microbiology
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773993/ https://www.ncbi.nlm.nih.gov/pubmed/33323509 http://dx.doi.org/10.1128/mBio.02880-20 |
_version_ | 1783630170405470208 |
---|---|
author | Shwab, E. Keats Juvvadi, Praveen R. Waitt, Greg Shaheen, Shareef Allen, John Soderblom, Erik J. Bobay, Benjamin G. Asfaw, Yohannes G. Moseley, M. Arthur Steinbach, William J. |
author_facet | Shwab, E. Keats Juvvadi, Praveen R. Waitt, Greg Shaheen, Shareef Allen, John Soderblom, Erik J. Bobay, Benjamin G. Asfaw, Yohannes G. Moseley, M. Arthur Steinbach, William J. |
author_sort | Shwab, E. Keats |
collection | PubMed |
description | Protein kinase A (PKA) signaling plays a critical role in the growth and development of all eukaryotic microbes. However, few direct targets have been characterized in any organism. The fungus Aspergillus fumigatus is a leading infectious cause of death in immunocompromised patients, but the specific molecular mechanisms responsible for its pathogenesis are poorly understood. We used this important pathogen as a platform for a comprehensive and multifaceted interrogation of both the PKA-dependent whole proteome and phosphoproteome in order to elucidate the mechanisms through which PKA signaling regulates invasive microbial disease. Employing advanced quantitative whole-proteomic and phosphoproteomic approaches with two complementary phosphopeptide enrichment strategies, coupled to an independent PKA interactome analysis, we defined distinct PKA-regulated pathways and identified novel direct PKA targets contributing to pathogenesis. We discovered three previously uncharacterized virulence-associated PKA effectors, including an autophagy-related protein, Atg24; a CCAAT-binding transcriptional regulator, HapB; and a CCR4-NOT complex-associated ubiquitin ligase, Not4. Targeted mutagenesis, combined with in vitro kinase assays, multiple murine infection models, structural modeling, and molecular dynamics simulations, was employed to characterize the roles of these new PKA targets in growth, environmental and antimicrobial stress responses, and pathogenesis in a mammalian system. We also elucidated the molecular mechanisms of PKA regulation for these effectors by defining the functionality of phosphorylation at specific PKA target sites. We have comprehensively characterized the PKA-dependent phosphoproteome and validated PKA targets as direct regulators of infectious disease for the first time in any pathogen, providing new insights into PKA signaling and control over microbial pathogenesis. |
format | Online Article Text |
id | pubmed-7773993 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | American Society for Microbiology |
record_format | MEDLINE/PubMed |
spelling | pubmed-77739932021-01-05 The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets Shwab, E. Keats Juvvadi, Praveen R. Waitt, Greg Shaheen, Shareef Allen, John Soderblom, Erik J. Bobay, Benjamin G. Asfaw, Yohannes G. Moseley, M. Arthur Steinbach, William J. mBio Research Article Protein kinase A (PKA) signaling plays a critical role in the growth and development of all eukaryotic microbes. However, few direct targets have been characterized in any organism. The fungus Aspergillus fumigatus is a leading infectious cause of death in immunocompromised patients, but the specific molecular mechanisms responsible for its pathogenesis are poorly understood. We used this important pathogen as a platform for a comprehensive and multifaceted interrogation of both the PKA-dependent whole proteome and phosphoproteome in order to elucidate the mechanisms through which PKA signaling regulates invasive microbial disease. Employing advanced quantitative whole-proteomic and phosphoproteomic approaches with two complementary phosphopeptide enrichment strategies, coupled to an independent PKA interactome analysis, we defined distinct PKA-regulated pathways and identified novel direct PKA targets contributing to pathogenesis. We discovered three previously uncharacterized virulence-associated PKA effectors, including an autophagy-related protein, Atg24; a CCAAT-binding transcriptional regulator, HapB; and a CCR4-NOT complex-associated ubiquitin ligase, Not4. Targeted mutagenesis, combined with in vitro kinase assays, multiple murine infection models, structural modeling, and molecular dynamics simulations, was employed to characterize the roles of these new PKA targets in growth, environmental and antimicrobial stress responses, and pathogenesis in a mammalian system. We also elucidated the molecular mechanisms of PKA regulation for these effectors by defining the functionality of phosphorylation at specific PKA target sites. We have comprehensively characterized the PKA-dependent phosphoproteome and validated PKA targets as direct regulators of infectious disease for the first time in any pathogen, providing new insights into PKA signaling and control over microbial pathogenesis. American Society for Microbiology 2020-12-15 /pmc/articles/PMC7773993/ /pubmed/33323509 http://dx.doi.org/10.1128/mBio.02880-20 Text en Copyright © 2020 Shwab et al. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Research Article Shwab, E. Keats Juvvadi, Praveen R. Waitt, Greg Shaheen, Shareef Allen, John Soderblom, Erik J. Bobay, Benjamin G. Asfaw, Yohannes G. Moseley, M. Arthur Steinbach, William J. The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets |
title | The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets |
title_full | The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets |
title_fullStr | The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets |
title_full_unstemmed | The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets |
title_short | The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets |
title_sort | protein kinase a-dependent phosphoproteome of the human pathogen aspergillus fumigatus reveals diverse virulence-associated kinase targets |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773993/ https://www.ncbi.nlm.nih.gov/pubmed/33323509 http://dx.doi.org/10.1128/mBio.02880-20 |
work_keys_str_mv | AT shwabekeats theproteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT juvvadipraveenr theproteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT waittgreg theproteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT shaheenshareef theproteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT allenjohn theproteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT soderblomerikj theproteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT bobaybenjaming theproteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT asfawyohannesg theproteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT moseleymarthur theproteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT steinbachwilliamj theproteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT shwabekeats proteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT juvvadipraveenr proteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT waittgreg proteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT shaheenshareef proteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT allenjohn proteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT soderblomerikj proteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT bobaybenjaming proteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT asfawyohannesg proteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT moseleymarthur proteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets AT steinbachwilliamj proteinkinaseadependentphosphoproteomeofthehumanpathogenaspergillusfumigatusrevealsdiversevirulenceassociatedkinasetargets |