Investigating the molecular mechanism of caffeine and antifungal resistance in Schizosaccharomyces pombe epimutants

Abstract

Fungal pathogens are a growing threat to human health, food security and ecosystem biodiversity. Emergence of isolates resistant to the very limited antifungals render treatment of fungal infections increasingly challenging. Understanding the mechanisms by which fungi develop resistance to antifungals and how they respond to external insults is of utmost importance for the informed deployment of existing, and development of new antifungal agents. In the fission yeast Schizosaccharomyces pombe genes embedded in histone H3 lysine 9 methylation (H3K9me) heterochromatin, deposited by the sole H3K9- methyltransferase Clr4, are transcriptionally silenced. It has been previously demonstrated that S. pombe epimutants resistant to caffeine (CAF) can be isolated. Epimutants are defined here as unstable resistant (UR) isolates that lose resistance in the absence of insult. In UR isolates, genes conferring caffeine susceptibility are silenced via the formation of heterochromatin islands, without any alteration of the underlying DNA sequence. In this thesis, in order to investigate the mechanism by which silencing of genes under the heterochromatin islands results in resistance, I employed genetic mutants that phenocopy specific epimutants. Two genes whose heterochromatin mediated repression results in resistance phenotype encode mitochondrial proteins: ppr4+, which encodes Ppr4 the translation regulator of [cox1+] and cup1+, which encodes LYR-domain protein Cup1.

Mutants of these genes display mitochondrial dysfunction and increased reactive oxygen species. Elevated oxidative stress triggers transcriptional reprogramming via the fission yeast oxidative stress response pathway, leading to upregulation of efflux pumps and antioxidant genes, resulting in caffeine and multidrug cross-resistance, explaining the phenotype of epimutants.