Mechanisms Promoting Cellular Tolerance to Fungistats

促进细胞对抑菌剂耐受的机制

基本信息

批准号：
10192653
负责人：
KYLE W CUNNINGHAM
金额：
$ 39.48万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
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/10192653
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 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/antagonist insight mimetics mortality new technology novel novel therapeutics opportunistic pathogen pathogen pathogenic fungus phosphoproteomics resistance mechanism response screening stressor tool tumor

项目摘要

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.

概括真菌感染会导致显着的发病率和死亡率，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，这些药物都针对甾醇内质网中的生物合成酶并在不直接作用的情况下阻止病原体的生长杀死它。这些真菌抑制剂的一个严重限制是除了潜在的潜在耐药性之外，还会出现耐药性。停药后复发。值得注意的是，唑类可以通过其他专门针对真菌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不会强烈影响抵抗力机制或改变抑菌剂的效力。相反，它们改变了耐受机制，帮助病原体能够在长期的抗真菌攻击下存活下来。先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子，特别是调节对真菌抑制剂的耐受性。将利用两种公正的筛选方法来帮助定义新的钙依赖磷酸酶依赖性耐受机制的组成部分。首先，我们将利用质谱仪识别模型酵母中磷酸化蛋白的方法，这些磷酸化蛋白响应于改变磷酸化状态暴露于模型真菌抑制剂（ER 应激源）期间的钙调神经磷酸酶抑制剂。其次，我们将开发一部小说遗传方法并在人类机会性病原体中进行首次全基因组遗传筛选光滑念珠菌鉴定特异性调节氟康唑耐受性的基因。调节基因对氟康唑的耐药性也将被识别和分类。这种方法称为 Hermes 插入分析（HIP），涉及使用转座子对 C. glabrata 基因组进行体内随机诱变，插入位点的 Illumina 测序。这些无偏见方法在不同酵母中的组合暴露于不同真菌抑制剂类别的物种提供了潜在耐受性的补充视图机制。共同揭示了一组常见的基因/蛋白质，其活性对神经钙蛋白有反应真菌抑制剂应激细胞并调节耐受性。我们提出一系列遗传、生化和细胞生物学在这两种酵母菌中进行了实验，以测试关于它们之间相互作用的几种假设以及它们的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中至少 5 个依次作用的新成分：合成肌醇的激酶焦磷酸盐、蛋白激酶 CK2、ER 酶神经酰胺合酶及其产物，以及假定的神经酰胺激活蛋白磷酸酶。因此，该项目提供了对新疗法的即时见解可以杀死真菌病原体，同时建立可以运作的耐受机制的范例广义上来说。