The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets.

Abstract

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.IMPORTANCE PKA is essential for the virulence of eukaryotic human pathogens. Understanding PKA signaling mechanisms is therefore fundamental to deciphering pathogenesis and developing novel therapies. Despite its ubiquitous necessity, specific PKA effectors underlying microbial disease remain unknown. To address this fundamental knowledge gap, we examined the whole-proteomic and phosphoproteomic impacts of PKA on the deadly fungal pathogen Aspergillus fumigatus to uncover novel PKA targets controlling growth and virulence. We also defined the functional consequences of specific posttranslational modifications of these target proteins to characterize the molecular mechanisms of pathogenic effector regulation by PKA. This study constitutes the most comprehensive analysis of the PKA-dependent phosphoproteome of any human pathogen and proposes new and complex roles played by PKA signaling networks in governing infectious disease.

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Published Version (Please cite this version)

10.1128/mbio.02880-20

Publication Info

Shwab, E Keats, Praveen R Juvvadi, Greg Waitt, Shareef Shaheen, John Allen, Erik J Soderblom, Benjamin G Bobay, Yohannes G Asfaw, et al. (2020). The Protein Kinase A-Dependent Phosphoproteome of the Human Pathogen Aspergillus fumigatus Reveals Diverse Virulence-Associated Kinase Targets. mBio, 11(6). pp. e02880–e02820. 10.1128/mbio.02880-20 Retrieved from https://hdl.handle.net/10161/28885.

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Scholars@Duke

Soderblom

Erik James Soderblom

Associate Research Professor of Cell Biology

Director, Proteomics and Metabolomics Core Facility

Bobay

Benjamin Bobay

Assistant Professor in Radiology

I am the Assistant Director of the Duke University NMR Center and an Assistant Professor in the Duke Radiology Department. I was originally trained as a structural biochemist with an emphasis on utilizing NMR and continue to use this technique daily helping collaborators characterize protein structures and small molecules through a diverse set of NMR experiments. Through the structural characterization of various proteins, from both planta and eukaryotes, I have developed a robust protocol of utilizing computational biology for describing binding events, mutations, post-translations modifications (PTMs), and/or general behavior within in silico solution scenarios. I have utilized these techniques in collaborations ranging from plant pathologists at the Swammerdam Institute for Life Sciences department at the University of Amsterdam to biomedical engineers at North Carolina State University to professors in the Pediatrics department at Duke University. These studies have centered around the structural and functional consequences of PTMs (such as phosphorylation), mutation events, truncation of multi-domain proteins, dimer pulling experiments, to screening of large databases of ligands for potential binding events. Through this combination of NMR and computational biology I have amassed 50 peer-reviewed published articles and countless roles on scientific projects, as well as the development of several tutorials concerning the creation of ligand databases and high-throughput screening of large databases utilizing several different molecular dynamic and computational docking programs.


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