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Gene expression response to the antifungal compound plakortide F acid in S. cerevisiae

ABSTRACT: Plakortide F acid (PFA), a marine-derived polyketide endoperoxide, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In the present study, transcriptional profiling coupled with mutant and biochemical analyses were conducted using the model organism Saccharomyces cerevisiae, to investigate the mechanism of action of this compound. PFA elicited a transcriptome response indicative of a Ca2+ imbalance, affecting the expression of genes known to be responsive to altered cellular calcium levels. Several additional lines of evidence obtained supported a role for Ca2+ in PFA's activity. First, mutants lacking calcineurin and various Ca2+ transporters including pumps (Pmr1 and Pmc1) and channels (Cch1 and Mid1) showed increased sensitivity to PFA. In addition, the calcineurin inhibitors FK506 and cyclosporine A strongly enhanced PFA activity in wild-type cells. Furthermore, PFA activated the transcription of a lacZ reporter driven by the calcineurin-dependent response element. Finally, elemental analysis indicated a significant increase in intracellular calcium levels in PFA-treated cells. Collectively, our results demonstrate that PFA mediates its antifungal activity by perturbing Ca2+ homeostasis, thus representing a potentially novel mechanism distinct from that of currently used antifungal agents. DNA microarray analysis of gene expression response to the antifungal compound plakortide F acid in yeast

ORGANISM(S): Schizosaccharomyces pombe Saccharomyces cerevisiae S288C Saccharomyces cerevisiae

PROVIDER: GSE26925 | GEO | 2011/01/28

SECONDARY ACCESSION(S): PRJNA136071

REPOSITORIES: GEO

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Project description:Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeast Schizosaccharomyces pombe. Genome-wide studies of the Crz1 and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This paper identified an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2, and tunicamycin treatment, as well as a âpmr1 genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of the Saccharomyces cerevisiae ortholog Crz1. These genes were functionally enriched for Crz1-conserved processes such as cell wall biosynthesis. Overexpression of prz1+ increased resistance to the cell wall degradation enzyme zymolyase, likely from upregulation of the O-mannosyltransferase encoding gene omh1+. Loss of omh1+ abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss of prz1+ resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the âprz1 strain was abrogated by the loss of gsf2+ or cbf12+. This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes between Crz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional-regulatory network. We generated 3 overexpression microarrays and 2 deletion microarrays with dye swaps, and 2 deletion microarrays with drug treatments with dye swaps, each with a biological replicate performed as a dye swap. The effect of the mutant strains were all compared to wild type or empty vector strains, except for one experiment where the prz1 deletion mutant was compared to the pmr1 deletion mutant.

Project description:Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca2+ signaling responses. In Cryptococcus neoformans, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in Saccharomyces cerevisiae and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that Crz1 is dephosphorylated by calcineurin, and crz1Δ mutants display phenotypes intermediate between wild-type and calcineurin mutants. RNA-sequencing revealed 102 genes that are regulated in a calcineurin/Crz1-dependent manner at 37°C. 99 genes were down-regulated in cna1Δ and crz1Δ mutants, indicating these genes are normally activated by the calcineurin/Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. Surprisingly, only five genes have orthologs known to be calcineurin/Crz1-dependent in S. cerevisiae. 393 genes are independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Taken together, these results indicate that the calcineurin/Crz1-dependent pathway controls a transcriptional circuit that has been extensively rewired in C. neoformans compared to S. cerevisiae. Given the intermediate crz1Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output has been extensively rewired to promote fungal virulence.

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Gene expression response to the antifungal compound plakortide F acid in S. cerevisiae

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