Mechanisms Promoting Cellular Tolerance to Fungistats

总结 真菌感染引起显著的发病率和死亡率，特别是在免疫功能低下的个体中。 大多数感染最初都是用氟康唑或相关的唑类抗真菌药物治疗的，这些药物都是针对固醇的 在内质网中的生物合成酶，并阻止病原体的生长，而不直接 这些抑真菌剂的一个严重限制是耐药性的出现，除了潜在的 戒断后复发。值得注意的是，唑类可以被其他药物转化为杀真菌剂， 抑制蛋白磷酸酶钙调磷酸酶。钙调神经磷酸酶抑制剂对耐药性影响不大 机制或改变真菌抑制剂的效力。相反，它们改变了耐受机制， 病原体在长期的抗真菌攻击中存活。以前的研究集中在抑真菌剂如何触发 钙调磷酸酶的激活。该项目旨在揭示钙调磷酸酶的下游效应物， 调节对抑真菌剂的耐受性。两种无偏倚的筛选方法将用于帮助定义新的 钙调神经磷酸酶依赖性耐受机制的组成部分。首先，我们将利用质谱仪 一种鉴定模型酵母中改变磷酸化状态的磷酸化蛋白的方法， 钙调磷酸酶抑制剂在暴露于模型抑真菌剂（ER应激物）。第二，我们将开发一种新的 遗传学方法，并在人类机会致病菌中进行首次全基因组遗传筛查 光滑念珠菌鉴定特异性调节氟康唑耐受性的基因。基因调控 对氟康唑的耐药性也将被鉴定和分类。这种方法，称为爱马仕插入 热等静压（HIP），包括C. glabrata基因组使用转座子， 插入位点的Illumina测序。这些无偏方法在不同酵母中的组合 暴露于不同抑真菌剂类别的物种提供了基础耐受性的补充观点 机制总之，一组共同的基因/蛋白质被揭示出来，它们的活性对钙调神经磷酸酶有反应， 抗真菌剂应激细胞和调节耐受性。我们提出了一系列的遗传、生化和细胞生物学 在这两种酵母物种中进行实验，以测试关于它们彼此相互作用的几种假设，以及它们之间的相互作用。 钙调神经磷酸酶依赖性耐受机制中的作用顺序。这些实验预计将 揭示了级联反应中至少5种新的组分：合成肌醇的激酶 焦磷酸，蛋白激酶CK 2，ER酶神经酰胺合酶及其产物，以及一种推定的 神经酰胺活化蛋白磷酸酶。因此，该项目提供了对新疗法的直接见解 它可以杀死真菌病原体，同时建立一个耐受机制的范例， 广泛地在自然界。

Summary Fungal infections cause significant morbidity and mortality, particularly in immunocompromised individuals. Most infections are initially treated with Fluconazole or a related azole-class antifungal, which all target sterol biosynthesis enzymes in the endoplasmic reticulum and arrests growth of the pathogen without directly killing it. A serious limitation of these fungistats is the emergence of resistance, in addition to potential for relapse upon withdrawal. Remarkably, azoles can be converted to fungicides by other drugs that specifically inhibit the protein phosphatase calcineurin. The calcineurin inhibitors do not strongly affect resistance mechanisms or change the potency of fungistats. Instead they alter tolerance mechanisms that help the pathogens survive long-term antifungal assaults. Previous studies have focused on how fungistats trigger the activation of calcineurin. This project aims to reveal the downstream effectors of calcineurin that specifically regulate tolerance to the fungistats. Two unbiased screening approaches will be utilized to help define new components of the calcineurin-dependent tolerance mechanism. First, we will utilize a mass spectrometry approach to identify phospho-proteins in a model yeast that change phosphorylation state in response to calcineurin inhibitors during exposure to model fungistats (ER stressors). Second, we will develop a novel genetic approach and conduct the first genome-wide genetic screens in the human opportunistic pathogen Candida glabrata to identify genes that specifically regulate tolerance to Fluconazole. Genes that regulate resistance to Fluconazole also will be identified and categorized. This approach, termed Hermes insertion profiling (HIP), involves in vivo random mutagenesis of the C. glabrata genome using a transposon and Illumina sequencing of the insertion sites. The combination of these unbiased approaches in different yeast species exposed to different fungistat classes provides complementary views of the underlaying tolerance mechanism. Together, a common set of genes/proteins is unveiled whose activities respond to calcineurin in fungistat-stressed cells and regulate tolerance. We propose a series of genetic, biochemical, and cell biological experiments in both yeast species to test several hypotheses about their interactions with one another and their order of action within the calcineurin-dependent tolerance mechanism. These experiments are expected to reveal at least 5 new components in the cascade that act sequentially: the kinases that synthesize inositol pyrophosphates, the protein kinase CK2, the ER enzyme ceramide synthase and its product, and a putative ceramide-activated protein phosphatase. The project therefore provides immediate insights into new therapies that can kill fungal pathogens while establishing a paradigm for tolerance mechanisms that may operate broadly in nature.