Phosphorylation of Calcineurin at a Novel Serine-Proline Rich Region Orchestrates Hyphal Growth and Virulence in Aspergillus fumigatus

PLoS Pathogens

Yohannes G. Asfaw¹  Frédéric Lamoth²  Michael A. Hast³  William J. Steinbach³  Jarrod R. Fortwendel⁴  Praveen R. Juvvadi⁴  Erik J. Soderblom⁴  Christopher Gehrke⁴  M. Arthur Moseley⁵  Erik C. Cook⁶  Trevor P. Creamer⁶ 

[1] Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, United States of America;Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America;Department of Molecular and Cellular Biochemistry and Center for Structural Biology, University of Kentucky, Lexington, Kentucky, United States of America;Department of Pediatrics, Division of Pediatric Infectious Diseases, Duke University Medical Center, Durham, North Carolina, United States of America;Division of Laboratory Animal Resources, Duke University Medical Center, Durham, North Carolina, United States of America;Duke Proteomics Facility, Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, United States of America

关键词: Phosphorylation;    Aspergillus fumigatus;    Serine;    Fungi;    Rice blast fungus;    Mutant strains;    Proline;    Plant fungal pathogens;   

DOI  :  10.1371/journal.ppat.1003564

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

The fungus Aspergillus fumigatus is a leading infectious killer in immunocompromised patients. Calcineurin, a calmodulin (CaM)-dependent protein phosphatase comprised of calcineurin A (CnaA) and calcineurin B (CnaB) subunits, localizes at the hyphal tips and septa to direct A. fumigatus invasion and virulence. Here we identified a novel serine-proline rich region (SPRR) located between two conserved CnaA domains, the CnaB-binding helix and the CaM-binding domain, that is evolutionarily conserved and unique to filamentous fungi and also completely absent in human calcineurin. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of A. fumigatus CnaA in vivo at four clustered serine residues (S406, S408, S410 and S413) in the SPRR. Mutation of the SPRR serine residues to block phosphorylation led to significant hyphal growth and virulence defects, indicating the requirement of calcineurin phosphorylation at the SPRR for its activity and function. Complementation analyses of the A. fumigatus ΔcnaA strain with cnaA homologs from the pathogenic basidiomycete Cryptococcus neoformans, the pathogenic zygomycete Mucor circinelloides, the closely related filamentous fungi Neurospora crassa, and the plant pathogen Magnaporthe grisea, revealed filamentous fungal-specific phosphorylation of CnaA in the SPRR and SPRR homology-dependent restoration of hyphal growth. Surprisingly, circular dichroism studies revealed that, despite proximity to the CaM-binding domain of CnaA, phosphorylation of the SPRR does not alter protein folding following CaM binding. Furthermore, mutational analyses in the catalytic domain, CnaB-binding helix, and the CaM-binding domains revealed that while the conserved PxIxIT substrate binding motif in CnaA is indispensable for septal localization, CaM is required for its function at the hyphal septum but not for septal localization. We defined an evolutionarily conserved novel mode of calcineurin regulation by phosphorylation in filamentous fungi in a region absent in humans. These findings suggest the possibility of harnessing this unique SPRR for innovative antifungal drug design to combat invasive aspergillosis.

【 授权许可】

CC BY