Purpose: Calcium signaling plays a vital role in the growth and pathogenesis of Aspergillus fumigatus. The phosphatase calcineurin regulates the cellular response to calcium in this fungus primarily through activation of the transcription factor CrzA. Dephosphorylation of CrzA by calcineurin enables its translocation into the nucleus and subsequent transcriptional upregulation of key virulence-associated genes. While calcineurin is conserved throughout the eukarya, CrzA is a fungal-specific molecule with only minimal homology to NFAT, its closest mammalian counterpart. As such, CrzA represents a potentially excellent target for the treatment of invasive aspergillosis. However, little is known concerning the mechanisms of CrzA activation.
Methods: To better understand the biochemical mechanisms underlying the control of CrzA by calcineurin, we employed liquid chromatography-tandem mass spectroscopy (LC-MS/MS) to identify specific sites of phosphorylation involved in the activation of this transcription factor. Strains expressing GFP-labeled CrzA were generated in both wild-type and calcineurin A deletion backgrounds and labeled CrzA was isolated from each background using a GFP-Trap affinity purification method. Specific clusters of phosphorylated residues were then mutated via site-directed mutagenesis to phosphomimetic amino acids in order to characterize the functional roles of phosphorylation at these sites with regard to nuclear translocation of CrzA, hyphal growth, conidiation and cell wall stress tolerance.
Results: Using this methodology, we characterized twenty such sites, the majority of which were specific to filamentous fungi and located in a serine-rich region N-terminal to the conserved DNA-binding domain of the protein. We found dephosphorylation of particular serine clusters to be essential for efficient nuclear translocation of CrzA. We also characterized the roles of phosphorylation at each cluster in regulating the hyphal growth, conidiation, and response to cell wall stress of the fungus. Our findings indicate a quantitative effect for phosphorylation of CrzA at multiple clusters in determining its activation state.
Conclusion: This work takes a large step forward in defining the biochemistry of CrzA regulation in A. fumigatus by identifying specific sites of phosphorylation under the control of calcineurin and characterizing the functional significance of phosphorylation at these residues. These findings provide important new insight into the mechanisms of CrzA activation that will likely constitute essential knowledge for the successful pursuit of new antifungal agents targeting this unique regulator of Aspergillus virulence.
Full conference title:
- AAA 8th (2018)