Mechanisms Promoting Cellular Tolerance to Fungistats

促进细胞对抑菌剂耐受的机制

• 批准号: 10406233
• 负责人: KYLE W CUNNINGHAM
• 金额: $ 39.44万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-15 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10406233
• 关键词: Affect; Antifungal Agents; Azole resistance; Azoles; Binding; Biochemical; Biological; Biological Assay; Biological Models; Bypass; Calcineurin; Calcineurin inhibitor; Candida glabrata; Cells; Cellular Stress; Ceramides; Cessation of life; Clinic Visits; Clinical; Clinics and Hospitals; Cyclosporine; Data; Diphosphates; Drug Tolerance; Endoplasmic Reticulum; Enzymes; Eukaryota; Exposure to; FK506; Failure; Fluconazole; Fluconazole resistance; Gene Proteins; Genes; Genetic; Genetic Screening; Genome; Growth; Human; Immune system; Immunocompromised Host; Immunosuppression; Individual; Industrial fungicide; Infection; Inositol; Knock-out; Learning; Mass Spectrum Analysis; Measurement; Modeling; Morbidity - disease rate; Mutagenesis; Mutation; Mycoses; Nature; Pathogenicity; Pathway interactions; Persons; Pharmaceutical Preparations; Phosphoproteins; Phosphorylation; Phosphotransferases; Protein Kinase; Protein phosphatase; Recovery; Relapse; Resistance; Saccharomyces cerevisiae; Series; Signal Transduction; Site; Sterol Biosynthesis Pathway; Stress; T-Lymphocyte; Testing; Therapeutic; Toxin; Withdrawal; Yeast Model System; Yeasts; assault; base; calcineurin phosphatase; casein kinase II; deep sequencing; dihydroceramide desaturase; emerging pathogen; endoplasmic reticulum stress; enzyme biosynthesis; experimental study; functional genomics; fungus; genetic approach; genetic resistance; genome wide screen; genome-wide; improved; in vivo; inhibitor; insight; mimetics; mortality; new technology; novel; novel therapeutics; opportunistic pathogen; pathogen; pathogenic fungus; phosphoproteomics; resistance mechanism; response; screening; stressor; tool; tumor

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.

摘要 真菌感染会导致严重的发病率和死亡率，特别是在免疫功能低下的人中。 大多数感染最初都使用氟康唑或相关的唑类抗真菌药物治疗，这些药物都针对类固醇。 内质网中的生物合成酶，不直接抑制病原菌的生长 杀了它。这些抑菌剂的一个严重限制是出现抗药性，除了潜在的 戒毒后复发。值得注意的是，氮唑可以通过其他特定的药物转化为杀菌剂 抑制蛋白磷酸酶钙调神经磷酸酶。钙调神经磷酸酶抑制剂对耐药性影响不大 作用机制或改变抑菌剂的效力。相反，他们改变了宽容机制，帮助 病原体在长期的抗真菌攻击中存活下来。此前的研究主要集中在真菌抑制剂如何触发 钙调神经磷酸酶的激活。该项目旨在揭示钙调神经磷酸酶的下游效应，特别是 规范对真菌保护者的容忍度。将使用两种无偏见的筛选方法来帮助定义新的 钙调神经磷酸酶依赖的耐受机制的组成部分。首先，我们将利用质谱仪 一种在模型酵母中鉴定磷酸化状态响应的磷酸化蛋白的方法 钙调神经磷酸酶抑制剂在暴露于模型真菌(内质网应激源)的过程中。第二，我们将开发一部小说 遗传学方法并在人类机会性病原体中进行第一次全基因组遗传筛选 光滑假丝酵母菌鉴定特异性调节对氟康唑耐受性的基因。调控基因 对氟康唑的耐药性也将被鉴定和归类。这种方法被称为Hermes插入 侧写(HIP)，涉及使用转座子和 插入位点的Illumina测序。这些公正的方法在不同的酵母中的组合 暴露于不同真菌类的物种提供了关于基础耐受性的互补观点 机制。共同揭示了一组共同的基因/蛋白质，它们的活动对钙调神经磷酸酶有反应。 真菌抑制细胞的生长并调节耐受性。我们提出了一系列遗传、生化和细胞生物学 在这两种酵母菌中进行实验，以检验关于它们之间相互作用的几个假设以及它们的 钙调神经磷酸酶依赖的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个顺序起作用的新成分：合成肌醇的激酶 焦磷酸，蛋白激酶CK2，ER酶神经酰胺合成酶及其产物，以及一种推定的 神经酰胺激活的蛋白磷酸酶。因此，该项目提供了对新疗法的即时洞察 可以杀死真菌病原体，同时建立一种可能起作用的耐受机制的范例 大体上是自然界的。