Regulation of Intrinsic Caspofungin Resistance in C. neoformans

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Abstract: Cryptococcosis causes hundreds of thousands of deaths per year, mostly in resource-poor settings. C. neoformans exhibits intrinsic resistance to echinocandin antifungals, eliminating this safe and effective class of antifungal drug from the anti-cryptococcal arsenal. The mechanism of resistance is unknown, but it is independent of drug target inhibition, as the target protein, the β-1,3-glucan synthase Fks1, is sensitive to the drug. We have identified a regulator of caspofungin sensitivity in C. neoformans, Puf4, that controls the FKS1 mRNA at the level of stability, and a puf4 mutant is caspofungin resistant. Deletion putative post-translational regulator of Puf4, the protein arginine methyltransferase (PRMT) Rmt5, confers hypersensitivity to caspofungin, suggesting a role for arginine methylation in the regulation of Puf4 function and caspofungin sensitivity. Our scientific premise is that Puf4 controls caspofungin sensitivity through post-transcriptional regulation of the FKS1 mRNA, and that the ability of Puf4 to regulate FKS1 is dependent on its methylation status. We will investigate the mechanism of Puf4 regulation including its binding to the FKS1 mRNA 5’ UTR and the effect of that interaction on translation and mRNA stability (Aim 1). We will then investigate the role of Puf4 methylation on its ability to regulate the FKS1 mRNA including the effect of rmt5 deletion on FKS1 post- transcriptional regulation, the identification of methylation sites on Puf4 and the use of mutagenesis to determine the consequence of methylation on effector function (Aim 2). PRMTs are known drug targets. Determining the mechanism by which Rmt5 and its putative target Puf4 control caspofungin sensitivity may lead to adjunctive therapies to potentiate the anti-cryptococcal activity of echinocandins.