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

Autor: Jarrod R. Fortwendel, Praveen R. Juvvadi, Yohannes G. Asfaw, Erik C. Cook, William J. Steinbach, M. Arthur Moseley, Frédéric Lamoth, Christopher Gehrke, Trevor P. Creamer, Michael A. Hast, Erik J. Soderblom
Rok vydání: 2013
Předmět:
lcsh:Immunologic diseases. Allergy
Male
Hyphal growth
Antifungal Agents
Calmodulin
Amino Acid Motifs
Calcineurin Inhibitors
Immunology
Hyphae
Antifungal drug
Biology
Models
Biological
Microbiology
Neurospora crassa
Aspergillus fumigatus
Fungal Proteins
Serine
Mice
03 medical and health sciences
Virology
Molecular Cell Biology
Genetics
Animals
Aspergillosis
Humans
Phosphorylation
lcsh:QH301-705.5
Molecular Biology
030304 developmental biology
2. Zero hunger
0303 health sciences
030306 microbiology
Calcineurin
Antifungal Agents/chemistry
Antifungal Agents/therapeutic use
Aspergillosis/drug therapy
Aspergillosis/enzymology
Aspergillosis/genetics
Aspergillus fumigatus/enzymology
Aspergillus fumigatus/genetics
Calcineurin/chemistry
Calcineurin/immunology
Calcineurin/metabolism
Fungal Proteins/antagonists & inhibitors
Fungal Proteins/chemistry
Fungal Proteins/genetics
Fungal Proteins/metabolism
Hyphae/enzymology
Hyphae/genetics
Protein Structure
Tertiary
biology.organism_classification
3. Good health
lcsh:Biology (General)
Biochemistry
biology.protein
Parasitology
lcsh:RC581-607
Research Article
Zdroj: PLoS Pathogens
PLoS pathogens, vol. 9, no. 8, pp. e1003564
PLoS pathogens
PLoS Pathogens, Vol 9, Iss 8, p e1003564 (2013)
ISSN: 1553-7374
DOI: 10.1371/journal.ppat.1003564
Popis: 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.
Author Summary Invasive fungal infections are a leading cause of death in immunocompromised patients. Translating molecular understanding into tangible clinical benefit has been difficult due to the fact that fungal pathogens and their hosts have similar physiology. The calcineurin pathway is an important signaling cascade in all eukaryotes, and calcineurin inhibitors are powerful immunosuppressants that have revolutionized medicine. Through both genetic and pharmacologic inhibition, we have established that calcineurin is vital for invasive fungal disease. Although the currently available calcineurin inhibitors are active in vitro against the major invasive fungal pathogens, they are also immunosuppressive in the host, limiting therapeutic effectiveness. Here we defined an evolutionarily conserved novel mode of calcineurin regulation by phosphorylation in filamentous fungi that is responsible for virulence in the opportunistic human pathogen, Aspergillus fumigatus. This phosphorylation occurs on a cluster of four serine residues located in a unique serine-proline rich domain of calcineurin that is absent in humans. This finding of a new fungal-specific mechanism controlling hyphal growth and virulence represents a new potential target for antifungal drug therapy.
Databáze: OpenAIRE
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