Genetic bypass of Aspergillus nidulans crzA function in calcium homeostasis.

After dephosphorylation by the phosphatase calcineurin, the fungal transcription factor CrzA enters the nucleus and activates the transcription of genes responsible for calcium homeostasis and many other calcium-regulated activities. A lack of CrzA confers calcium-sensitivity to the filamentous fungus Aspergillus nidulans. To further understand calcium signaling in filamentous fungi and to identify genes that interact genetically with CrzA, we selected for mutations that were able to suppress crzAΔ calcium intolerance and identified three genes. Through genetic mapping, gene sequencing, and mutant rescue, we were able to identify these as cnaB (encoding the calcineurin regulatory subunit), folA (encoding an enzyme involved in folic acid biosynthesis, dihydroneopterin aldolase), and scrC (suppression of crzA(-), encoding a hypothetical protein). By using a calcium indicator, Fluo-3, we were able to determine that the wild-type and the suppressor strains were either able to regulate intracellular calcium levels or were able to take up and or store calcium correctly. The increased expression of calcium transporters, pmcA and/or pmcB, in suppressor mutants possibly enabled tolerance to high levels of calcium. Our results suggest that a cnaB suppressor mutation confers calcium tolerance to crzAΔ strains through restoration of calcium homeostasis. These results stress that in A. nidulans there are calcineurin-dependent and CrzA-independent pathways. In addition, it is possible that CrzA is able to contribute to the modulation of folic acid biosynthesis.

Functional characterization of the Aspergillus fumigatusPHO80 homologue.

Phosphate is an ion that is essential for fungal growth. The systems for inorganic phosphate (P(i)) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of P(i) at normal or high external P(i) concentrations) and a high-affinity (activated in response to P(i) starvation). Here, as an initial step to understand the PHO pathway in Aspergillus fumigatus, we characterized the PHO80 homologue, PhoB(PHO80). We show that the DeltaphoB(PHO80) mutant has a polar growth defect (i.e., a delayed germ tube emergence) and, by phenotypic and phosphate uptake analyses, establish a link between PhoB(PHO80), calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild-type and DeltaphoB(PHO80) mutant cells identify Afu4g03610 (phoD(PHO84)), Afu7g06350 (phoE(PHO89)), Afu4g06020 (phoC(PHO81)), and Afu2g09040 (vacuolar transporter Vtc4) as more expressed both in the DeltaphoB(PHO80) mutant background and under phosphate-limiting conditions of 0.1mM P(i). Epifluorescence microscopy revealed accumulation of poly-phosphate in DeltaphoB(PHO80) vacuoles, which was independent of extracellular phosphate concentration. Surprisingly, a phoD(PHO84) deletion mutant is indistinguishable phenotypically from the corresponding wild-type strain. mRNA analyses suggest that protein kinase A absence supports the expression of PHO genes in A. fumigatus. Furthermore, DeltaphoB(PHO80) and DeltaphoD(PHO84) mutant are fully virulent in a murine low dose model for invasive aspergillosis.

Functional characterization of the Aspergillus fumigatus CRZ1 homologue, CrzA.

The protein phosphatase calcineurin is an important mediator connecting calcium-dependent signalling to various cellular responses in multiple organisms. In fungi calcineurin acts largely through regulating Crz1p-like transcription factors. Here we characterize an Aspergillus fumigatus CRZ1 homologue, CrzA and demonstrate its mediation of cellular tolerance to increased concentrations of calcium and manganese. In addition to acute sensitivity to these ions, and decreased conidiation, the crzA null mutant suffers altered expression of calcium transporter mRNAs under high concentrations of calcium, and loss of virulence when compared with the corresponding complemented and wild-type strains. We use multiple expression analyses to probe the transcriptional basis of A. fumigatus calcium tolerance identifying several genes having calA and/or crzA dependent mRNA accumulation patterns. We also demonstrate that contrary to previous findings, the gene encoding the Aspergillus nidulans calcineurin subunit homologue, cnaA, is not essential and that the cnaA deletion mutant shares the morphological phenotypes observed in the corresponding A. fumigatus mutant, DeltacalA. Exploiting the A. nidulans model system, we have linked calcineurin activity with asexual developmental induction, finding that CrzA supports appropriate developmental induction in a calcineurin and brlA-dependent manner in both species.