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

Praveen R. Juvvadi, Christopher Gehrke, Jarrod Fortwendel, Frédéric Lamoth, Erik J. Soderblom, Erik C. Cook, Michael A. Hast, Yohannes G. Asfaw, M. Arthur Moseley, Trevor P. Creamer, William J. Steinbach

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Article numbere1003564
JournalPLoS Pathogens
Volume9
Issue number8
DOIs
StatePublished - Aug 1 2013

Fingerprint

Aspergillus fumigatus
Calcineurin
Proline
Serine
Virulence
Phosphorylation
Calmodulin
Growth
Fungi
Magnaporthe
Mucor
Phosphopeptides
Basidiomycota
Neurospora crassa
Cryptococcus neoformans
Aspergillosis
Drug Design
Phosphoprotein Phosphatases
Protein Folding
Immunocompromised Host

All Science Journal Classification (ASJC) codes

  • Parasitology
  • Microbiology
  • Immunology
  • Molecular Biology
  • Genetics
  • Virology

Cite this

Phosphorylation of Calcineurin at a Novel Serine-Proline Rich Region Orchestrates Hyphal Growth and Virulence in Aspergillus fumigatus. / Juvvadi, Praveen R.; Gehrke, Christopher; Fortwendel, Jarrod; Lamoth, Frédéric; Soderblom, Erik J.; Cook, Erik C.; Hast, Michael A.; Asfaw, Yohannes G.; Moseley, M. Arthur; Creamer, Trevor P.; Steinbach, William J.

In: PLoS Pathogens, Vol. 9, No. 8, e1003564, 01.08.2013.

Research output: Contribution to journalArticle

Juvvadi, PR, Gehrke, C, Fortwendel, J, Lamoth, F, Soderblom, EJ, Cook, EC, Hast, MA, Asfaw, YG, Moseley, MA, Creamer, TP & Steinbach, WJ 2013, 'Phosphorylation of Calcineurin at a Novel Serine-Proline Rich Region Orchestrates Hyphal Growth and Virulence in Aspergillus fumigatus', PLoS Pathogens, vol. 9, no. 8, e1003564. https://doi.org/10.1371/journal.ppat.1003564
Juvvadi, Praveen R. ; Gehrke, Christopher ; Fortwendel, Jarrod ; Lamoth, Frédéric ; Soderblom, Erik J. ; Cook, Erik C. ; Hast, Michael A. ; Asfaw, Yohannes G. ; Moseley, M. Arthur ; Creamer, Trevor P. ; Steinbach, William J. / Phosphorylation of Calcineurin at a Novel Serine-Proline Rich Region Orchestrates Hyphal Growth and Virulence in Aspergillus fumigatus. In: PLoS Pathogens. 2013 ; Vol. 9, No. 8.
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