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

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.

Components of the Calcium-Calcineurin Signaling Pathway in Fungal Cells and Their Potential as Antifungal Targets

“…Thus, research on the differences between the calcineurins of fungal and mammalian cells is urgently needed to develop antifungal-specific drugs. Fortunately, Juvvadi et al (119) have found a novel serineproline-rich region (SPRR) that is evolutionarily conserved and unique to filamentous fungi but completely absent in human calcineurin. The SPRR appears to be required for the phosphorylation of calcineurin that enables it to be active and function well.…”

Calcineurin-Crz1 Signaling in Lower Eukaryotes

“…But as Crz1 and its orthologues are restricted to lower eukaryotes and cannot be found in higher eukaryotes (see below), it might be worth investigating this class of transcription factors in more detail to find structures that are common in those proteins and that could serve as potential drug targets. Such a strategy was successfully followed in investigating calcineurin of A. fumigatus (77). Calcineurin of A. fumigatus harbors a novel serine-proline-rich region that is conserved, unique to filamentous fungi, and completely absent in human calcineurin.…”

“…Calcineurin of A. fumigatus harbors a novel serine-proline-rich region that is conserved, unique to filamentous fungi, and completely absent in human calcineurin. This offers the potential to design a specific drug targeting this region (77).…”

Calcineurin in fungal virulence and drug resistance: Prospects for harnessing targeted inhibition of calcineurin for an antifungal therapeutic approach

“…85,92 Although mutations in functional domains of calcineurin have revealed important residues required for localization and function of calcineurin at the septum, the exact mechanism of how calcineurin regulates septation is still unclear. 93,94 A filamentous fungalspecific serine proline rich (SPRR) linker domain between the CnBBH and CaMBD was also identified in the A. fumigatus calcineurin catalytic subunit. 93 This SPRR was phosphorylated at all 4 clustered serine residues and mutations in this region caused hyphal growth and virulence defects, implicating the importance of calcineurin phosphorylation for its function.…”

“…93,94 A filamentous fungalspecific serine proline rich (SPRR) linker domain between the CnBBH and CaMBD was also identified in the A. fumigatus calcineurin catalytic subunit. 93 This SPRR was phosphorylated at all 4 clustered serine residues and mutations in this region caused hyphal growth and virulence defects, implicating the importance of calcineurin phosphorylation for its function. 93 Identification of key regions for calcineurin function that are unique to fungi and absent in humans is a promising step toward the development of novel antifungal therapies.…”