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Novel motif in calcineurin catalytic subunit is required for septal localization of calcineurin in Aspergillus fumigatus.

ABSTRACT: Calcineurin heterodimer, comprised of the catalytic (CnaA) and regulatory (CnaB) subunits, localizes at the hyphal tips and septa to direct growth, septation, and disease in the human pathogen Aspergillus fumigatus. Here we discovered a novel motif (FMDVF) required for this critical CnaA septal localization, including residues Phe368, Asp370 and Phe372 overlapping the cyclosporine A-cyclophilin A-binding domain, CnaB-binding helix and the FK506-FKBP12-binding pocket. Mutations in adjacent residues Asn367, Trp374, and Ser375 confer FK506 resistance without impacting CnaA septal localization. Modeling A. fumigatus CnaA confirmed that the FMDVF motif forms a bridge between the two known substrate-binding motifs, PxIxIT and LxVP, and concurrent mutations (F368A D370A; F368A F372A) in the FMDVF motif disrupt CnaA-substrate interaction at the septum.

SUBMITTER: Juvvadi PR

PROVIDER: S-EPMC4764407 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Novel motif in calcineurin catalytic subunit is required for septal localization of calcineurin in Aspergillus fumigatus.

Juvvadi Praveen R PR Pemble Charles W CW Ma Yan Y Steinbach William J WJ

FEBS letters 20160215 4

Calcineurin heterodimer, comprised of the catalytic (CnaA) and regulatory (CnaB) subunits, localizes at the hyphal tips and septa to direct growth, septation, and disease in the human pathogen Aspergillus fumigatus. Here we discovered a novel motif (FMDVF) required for this critical CnaA septal localization, including residues Phe368, Asp370 and Phe372 overlapping the cyclosporine A-cyclophilin A-binding domain, CnaB-binding helix and the FK506-FKBP12-binding pocket. Mutations in adjacent residu ...[more]

PMID: 26864964

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Project description:Calcineurin plays an important role in the control of cell morphology and virulence in fungi. Calcineurin is a serine/threonine-specific protein phosphatase heterodimer consisting of a catalytic subunit A and a regulatory Ca2+/Calmodulin binding subunit. A mutant of A. fumigatus lacking the calcineurin A (calA) catalytic subunit exhibited defective hyphal morphology related to apical extension and branching growth, which resulted in drastically decreased filamentation. Here, we investigated which pathways are influenced by A. fumigatus calcineurin during proliferation by comparatively determining the transcriptional profile of A. fumigatus wild type and delta calA mutant strains. Our results showed that although the mitochondrial function is reduced in the delta calA mutant strain, its respiratory chain is functional and the mutant has increased alternative oxidase (aoxA) mRNA accumulation and activity. Furthermore, we identified several genes that encode transcription factors that have increased mRNA expression in the delta calA mutant and that could be involved in the Cal-CrzA pathway. Deletion mutants for these transcription factors had also reduced susceptibility to itraconazole, caspofungin, and sodium dodcyl sulfate. For the time course microarray experiments, 1.0 x 109 conidia of both A. fumigatus wild type [CEA17 delta akuB(KU80)] and mutant strain (delta calA) were used to inoculate 400 ml of pre-warmed liquid cultures in complete medium (YG) in 1 L erlenmeyer flasks. Cultures were allowed to grow for 6, 8, 12, 18 and 24 hours in a reciprocal shaker (250 rpm) at 37°C. At each time point, germilings were harvested by centrifugation or filtration and total RNA was extracted. Hybridization experiments were competitive using Cy3 or Cy5-labeled probes derived from both wild type and delta calA strain grown for 6, 8, 12, 18 and 24 hours. Hybridizations were performed using the wild type RNA as reference in comparison to the mutant strain (test RNA) for the same time point. Normalized signal intensities were used to generate relative hybridization ratios (query/reference). Following normalization, the values for each gene in-slide replicates were condensed (median variance <0.01), and corresponding flip-dye replicates were averaged to compensate for dye-specific effects (see Supplementary files linked below).

Project description: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 ?cnaA 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.

Dataset Information

Novel motif in calcineurin catalytic subunit is required for septal localization of calcineurin in Aspergillus fumigatus.

Publications

Novel motif in calcineurin catalytic subunit is required for septal localization of calcineurin in Aspergillus fumigatus.

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