Regulation of Intrinsic Caspofungin Resistance in C. neoformans

摘要： 隐球菌病每年造成数十万人死亡，其中大部分发生在资源匮乏的环境中。C。 新生杆菌对棘球菌素类抗真菌药物表现出内在的耐药性，消除了这一安全有效的类别 来自抗隐球菌库的抗真菌药物。抗药性的机制尚不清楚，但它是 作为靶蛋白的β-1，3-葡聚糖合成酶FKS1与药物靶标抑制无关，对药物靶标抑制很敏感。 毒品。我们已经在新生芽孢杆菌中发现了一个控制卡泊芬净敏感性的调节子，Puf4，它控制着FKS1 PUF4突变株对卡泊芬净有抗性。删除推定的翻译后 Puf4的调节蛋白精氨酸甲基转移酶(PRMT)Rmt5使人对 卡泊芬净，提示精氨酸甲基化在Puf4功能和卡泊芬净调节中的作用 敏感度。我们的科学前提是Puf4通过转录后转录控制卡泊芬净的敏感性 PUF4调控FKS1的能力依赖于其甲基化 状态。我们将研究Puf4的调控机制，包括它与FKS1基因5‘端非编码区的结合 以及这种相互作用对翻译和信使核糖核酸稳定性的影响(目标1)。然后我们将调查 PUF4甲基化对FKS1mRNA调控能力的影响包括rmt5缺失对FKS1后基因表达的影响 转录调控、Puf4上甲基化位点的鉴定和突变的应用 确定甲基化对效应器功能的影响(目标2)。PRMT是已知的药物靶标。 确定Rmt5及其假定靶点Puf4控制卡泊芬净敏感性的机制可能 导致辅助治疗，以增强棘球菌素的抗隐球菌活性。

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.