Phenotypic analysis of genes whose mRNA accumulation is dependent on calcineurin in Aspergillus fumigatus

Abstract

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 subunit B. A mutant of Aspergillus 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 DeltacalA mutant strains. Our results showed that the mitochondrial copy number is reduced in the DeltacalA mutant strain, and the mutant has increased alternative oxidase (aoxA) mRNA accumulation and activity. Furthermore, we identified four genes that encode transcription factors that have increased mRNA expression in the DeltacalA mutant. Deletion mutants for these transcription factors had reduced susceptibility to itraconazole, caspofungin, and sodium dodecyl sulfate (SDS).

The Aspergillus fumigatus CrzA Transcription Factor Activates Chitin Synthase Gene Expression during the Caspofungin Paradoxical Effect

“…Previously, four TFs encoded by the genes htfA (Afu4g10110), zfpA (Afu8g05010), zfpB (Afu1g10230), and zipD (Afu2g03280) were found to be upregulated in the presence of calcium (27, 28). Deletion of only zipD resulted in a reduced CPE and increased sensitivity to high levels of calcium (Fig.…”

Involvement of an Alternative Oxidase in Oxidative Stress and Mycelium-to-Yeast Differentiation in Paracoccidioides brasiliensis

“…The MTT metabolic assay was performed according to the method of Malavazi et al (36), with some modifications. Cells were washed three times with PBS, and the amount equivalent to 400 micrograms of total protein was added to each well with 10 l of MTT solution (5 mg/ml in PBS) in a final volume of 160 l. The mixture was incubated in the dark for 2 h at 37°C, and after that, the 96-well plate was centrifuged at 3,600 ϫ g for 10 min.…”

The Aspergillus fumigatus cell wall integrity signaling pathway: drug target, compensatory pathways, and virulence

“…In the last years, several transcriptomics studies of A. fumigatus signal transduction mutants were published (Malavazi et al, 2009; Jain et al, 2011; Müller et al, 2012; Macheleidt et al, 2015). The generated data suggest potential interactions of the CWI signaling cascade with other signal transduction pathways.…”

“…For example, in a microarray analysis of the Δ mpkA strain, two calcium/calmodulin dependent kinases were found to be differentially regulated in the mutant compared to the wild type under stress conditions induced by glucanex, which lyses the CW (Jain et al, 2011). Furthermore, a microarray hybridisation approach comparing the transcriptional profile of the calcineurin mutant Δ calA with the wild type, found the two MAP kinase kinase-encoding genes mkk2 , involved in CWI signaling, and pbs2 , involved in the HOG pathway, to be significantly down-regulated in the mutant strain (Malavazi et al, 2009). Data indicate once more a potential co-regulation of central signaling pathways in A. fumigatus .…”