Regulation of Intrinsic Caspofungin Resistance in C. neoformans

摘要： 隐球菌病每年造成数十万人死亡，大多数发生在资源贫乏的环境中。C. 新变形杆菌对棘白菌素类抗真菌药物表现出内在耐药性，消除了这类安全有效的 抗隐球菌的抗真菌药耐药机制尚不清楚，但它是 与药物靶点抑制无关，因为靶蛋白β-1，3-葡聚糖合酶Fks 1对药物靶点抑制敏感。 药我们已经确定了卡泊芬净敏感性的调节因子。控制FKS 1的新型人Puf 4 mRNA水平的稳定性，并且puf 4突变体是卡泊芬净耐药的。推定翻译后缺失 Puf 4的调节剂，蛋白质精氨酸甲基转移酶（PRMT）Rmt 5，赋予对 这表明精氨酸甲基化在调节Puf 4功能和卡泊芬净中的作用。 灵敏度我们的科学前提是Puf 4通过转录后调节卡泊芬净的敏感性。 Puf 4调控FKS 1 mRNA的能力依赖于其甲基化 status.我们将研究Puf 4的调控机制，包括其与FKS 1 mRNA 5' UTR的结合， 以及这种相互作用对翻译和mRNA稳定性的影响（目的1）。然后我们将研究 Puf 4甲基化对其调节FKS 1 mRNA的能力的影响，包括rmt 5缺失对FKS 1甲基化后的影响 转录调控，Puf 4上甲基化位点的鉴定和诱变的使用， 确定甲基化对效应子功能的影响（目的2）。PRMT是已知的药物靶标。 确定Rmt 5及其假定靶点Puf 4控制卡泊芬净敏感性的机制可能 导致增强棘白菌素抗隐球菌活性的连续疗法。

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.