Leveraging genomic approaches to define sterol transport in Cryptococcus neoformans

利用基因组方法定义新型隐球菌中的甾醇转运

• 批准号: 10727128
• 负责人: Tamara L Doering
• 金额: $ 19.48万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-24 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10727128
• 关键词: Amphotericin B; Antifungal Agents; Area; Back; Basic Science; Binding; Biological Assay; Biology; Carrier Proteins; Cell membrane; Cells; Cessation of life; Cholesterol; Columbidae; Cryptococcus; Cryptococcus neoformans; DNA Sequence Alteration; DNA sequencing; Dangerousness; Defect; Developing Countries; Drug Interactions; Drug Targeting; Endoplasmic Reticulum; Engineering; Equilibrium; Ergosterol; Exhibits; Family; Fluorescence-Activated Cell Sorting; Fungal Components; Future; Genes; Genetic Risk; Genetic Screening; Genome; Genomic approach; Genomics; Glycosphingolipids; Goals; Growth; HIV; Hand; Homologous Gene; Human; Individual; Infection; Investigation; Knowledge; Label; Lipids; Lung; Measures; Mediating; Membrane; Membrane Fluidity; Methods; Morbidity - disease rate; Morphology; Movement; Mutation; Organelles; Organism; Pathogenesis; Phospholipids; Population; Positioning Attribute; Process; Productivity; Protein Family; Proteins; Public Health; Research; Resistance; Saccharomyces cerevisiae; Sterols; Stress; Structure; Surface; System; Therapeutic; Toxic effect; Virulence; Work; Yeast Model System; Yeasts; clinical application; experience; experimental study; fungus; gene discovery; genetic approach; genome-wide; improved outcome; insight; interest; lipid transport; member; mutant; novel; pathogen; pathogenic fungus; patient population; polypeptide; prevent; protein function; skills; success; vesicle transport; whole genome

ABSTRACT Cryptococcus neoformans is a devastating opportunistic fungus that causes hundreds of thousands of deaths each year, mainly in developing countries. The most abundant component of its plasma membrane is ergosterol, which is also a compelling topic of study because it is the target of the most powerful antifungal drugs available. How this compound is transported within the cell has never been studied in C. neoformans, constituting a major gap in our knowledge of a critical lipid. Our preliminary results suggest that novel factors are involved in this movement, and that identifying them may be amenable to genetic approaches. The long-term goal of our research is to define the mechanisms of lipid transport in C. neoformans, to advance our fundamental understanding and improve the outcome of this dangerous infection. In this R21 proposal we focus on ergosterol transport. In Aim 1 we will apply a whole genome-scale genetic screen to discover genes whose products influence ergosterol levels at the plasma membrane. In Aim 2 we will isolate and investigate sequences that suppress a defect in retrograde traffic of ergosterol from the plasma membrane to the endoplas- mic reticulum. In Aim 3 we will address the functions of prioritized genes from the first two aims, including studies of growth, virulence, sterol content, and protein and lipid localization. These experiments will be enabled by our expertise in cryptococcal biology and advice from expert colleagues, which we will integrate into thoughtful and rigorous studies. Together, they will increase our understanding of cryptococcal ergosterol transport and position us for further mechanistic investigations of proteins that we discover to be involved in this process. Current antifungal drugs that target ergosterol and its synthesis are hampered by factors that include toxicity, drug interactions, and resistance. It is therefore critical to find other vulnerabilities in this system that can be productively exploited – our proposed studies are a focused step on this path. Completing them will advance genomic methods for fungal discovery, deepen our knowledge of lipid transport, and open the door to future mechanistic studies.

摘要 新型隐球菌是一种毁灭性的机会性真菌，导致数十万人死亡 每年，主要是在发展中国家。其质膜中最丰富的成分是麦角固醇， 这也是一个引人注目的研究课题，因为它是最强大的抗真菌药物的目标。 这种化合物如何在细胞内转运从未在C中研究过。新生儿，构成了一个主要的 我们对一种关键脂质的认识存在空白。我们的初步结果表明，新的因素参与了这一点 运动，并确定他们可能服从遗传方法。 本研究的长期目标是明确C.新型人， 我们的基本认识，并改善这种危险的感染的结果。在本R21提案中， 重点关注麦角固醇的转运。在目标1中，我们将应用全基因组规模的遗传筛选来发现基因 其产物影响质膜上的麦角固醇水平。在目标2中，我们将分离和研究 抑制麦角固醇从质膜到内质网逆行运输缺陷的序列， 微网在目标3中，我们将讨论前两个目标中优先考虑的基因的功能，包括研究 生长、毒力、甾醇含量、蛋白质和脂质定位。这些实验将通过我们的 隐球菌生物学方面的专业知识和专家同事的建议，我们将把这些知识和建议整合到深思熟虑的， 严谨的研究。总之，他们将增加我们对隐球菌麦角固醇运输和位置的理解 我们将对我们发现的参与这一过程的蛋白质进行进一步的机制研究。 目前针对麦角固醇及其合成的抗真菌药物受到包括毒性， 药物相互作用和耐药性。因此，找到该系统中的其他漏洞至关重要， 我们提出的研究是这条道路上的一个重点步骤。完成这些研究将促进 真菌发现的基因组方法，加深我们对脂质转运的认识，并打开未来的大门 机械研究。