Control of Mitochondrial Function by SPFH Proteins in Pathogenic Yeast

病原酵母中 SPFH 蛋白对线粒体功能的控制

基本信息

批准号：
10674870
负责人：
Jason Malcolm Rauceo
金额：
$ 12.18万
依托单位：
JOHN JAY COLLEGE OF CRIMINAL JUSTICE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-16 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10674870
关键词：
Antifungal Agents Antifungal Therapy Apoptosis Apoptotic Area Binding Proteins Biological Assay Biology Biomedical Research CRISPR/Cas technology Candida albicans Carbon Cell Wall Cell membrane Cell physiology Cells Cellular Assay Cessation of life Chimeric Proteins Clustered Regularly Interspaced Short Palindromic Repeats Co-Immunoprecipitations Complement Complex Disseminated candidiasis Drug usage Epitopes Eukaryota Eukaryotic Cell Exposure to Family Family member Filament Flow Cytometry Fluorescence Microscopy Funding Genes Genetic Genetic Transcription Goals Growth Hispanic-serving Institution Human Hyphae Infection Intervention Invertebrates Investigation Knowledge Leishmania Life Life Cycle Stages Mammals Mediating Membrane Membrane Potentials Membrane Proteins Mitochondria Molecular Molecular Genetics Morphogenesis Nematoda Organelles Osmosis Oxidative Stress Parasite Control Parasites Pathogenesis Pathogenicity Pathway interactions Patients Phenotype Phospholipids Planarians Plasmodium Process Production Productivity Property Protein Family Protein Sequence Analysis Proteins Proteomics Publishing Reactive Oxygen Species Research Respiration Role Signal Transduction Source Stress Study models System Testing Time Training Underrepresented Students Vertebrates Virulence Yeasts candidemia college cost effective drug efficacy flotillin fungus gene discovery genome editing human pathogen in vivo inhibitor innovation light microscopy member microbial mitochondrial membrane mortality mutant novel pathogen pathogenic fungus programs prohibitin protein complex protein function respiratory response

项目摘要

The mitochondrion is the major energy-producing organelle of the cell and coordinates key activities such as signaling, apoptosis, and phospholipid synthesis. These processes are essential for survival and pathogenesis of Candida albicans, the most common fungal pathogen in humans. C. albicans respiration occurs via three distinct pathways compared to vertebrates and is associated with virulence properties such as morphogenesis and cell wall synthesis. Our long-term research goal is to understand the cellular and molecular mechanisms that govern C. albicans survival in response to environmental stress and antifungal drugs. We previously discovered that gene classes involved mitochondrial functions are highly transcribed in response to osmotic and cell wall stress, and the gene SLP3 (stomatin like protein 3) was significantly upregulated. SLP3 is a member of the conserved SPFH (Stomatin, Prohibitin, Flotillin, HflK/HflC) protein superfamily. In eukaryotes, SPFH proteins are required for essential mitochondrial processes such as respiration, mitophagy, and apoptosis and mediate pathogenicity in several parasites. Our objective in this proposal is to determine the role of the C. albicans SPFH protein family in mitochondrial function. We were the first to demonstrate that Slp3p overproduction disrupted mitochondrial membrane potential and triggered apoptotic-like death specifically following prolonged exposure to oxidative stress. However, the molecular function Slp3p remains unknown. Thus, our central hypothesis is that C. albicans SPFH proteins form membrane complexes to coordinate mitochondrial function. This hypothesis is based upon observations with mammalian SPFH proteins and our published and preliminary findings. Human SPFH complexes appear as punctate foci when viewed using fluorescence microscopy. We found that Slp3p form puncta at the plasma membrane. Further, we identified a mitochondrial targeting signal in Slp2p, Phb1p and Phb2p, and our Slp2p-GFP fusion protein formed mitochondrial puncta. We will utilize a high-throughput molecular genetic and cellular approach that is cost-effective and time-saving to determine SPFH protein localization, protein complex composition, cellular function, and role in C. albicans infection. We will create SPFH-GFP fusion proteins and use fluorescence microscopy to determine cellular localization. We will construct SPFH-epitope-tagged strains and perform Co-IP and LC-MS/MS analyses to identify putative SPFH protein binding partners. We will create SPFH mutants via CRISPR-Cas9 genome editing and phenotypically characterize mutant strains in growth and cellular assays. We will examine SPFH function in C. albicans pathogenesis using a novel invertebrate infection system. Our findings will address a poorly understood area in C. albicans biology and provide a model for studying SPFH proteins in pathogenic fungi. This proposal is innovative as we will characterize the SPFH family in the context of a critical, yet poorly understood area in C albicans biology: the molecular framework underlying mitochondrial function.

线粒体是细胞的主要产能细胞器，并协调关键活动作为信号，凋亡和磷脂合成。这些过程对于生存至关重要白色念珠菌的发病机理，白色念珠菌是人类最常见的真菌病原体。白色念珠菌呼吸与脊椎动物相比，通过三种不同的途径发生，并且与毒力特性有关作为形态发生和细胞壁合成。我们的长期研究目标是了解细胞和响应环境应激和抗真菌的分子机制毒品。我们先前发现，涉及线粒体功能的基因类是高度转录的响应渗透和细胞壁应力，基因SLP3（类似烟素蛋白3）显着上调。 SLP3是保守的SPFH（臭虫，禁止素，氟替肽，HFLK/HFLC）蛋白的成员超家族。在真核生物中，SPFH蛋白是必需的线粒体过程（例如呼吸，线粒体和凋亡并介导了几种寄生虫的致病性。我们的目标建议是确定白色念珠菌SPFH蛋白家族在线粒体功能中的作用。我们是第一个证明Slp3P过度生产破坏了线粒体膜电位和长时间暴露于氧化应激之后，触发了凋亡样死亡。但是，分子功能SLP3P仍然未知。因此，我们的中心假设是白色念珠菌SPFH蛋白形成膜复合物以坐标线粒体功能。该假设基于哺乳动物SPFH蛋白以及我们发表的初步发现的观察结果。人类SPFH 当使用荧光显微镜查看时，复合物作为点状灶。我们发现Slp3P表格质膜上的点。此外，我们在SLP2P，PHB1P和 PHB2P，我们的SLP2P-GFP融合蛋白形成线粒体点。我们将使用高通量具有成本效益且节省时间的分子遗传和细胞方法，以确定SPFH蛋白定位，蛋白质复合物组成，细胞功能以及白色念珠菌感染中的作用。我们将创建 SPFH-GFP融合蛋白并使用荧光显微镜确定细胞定位。我们将构建SPFH - ePitope标记的菌株并执行Co-IP和LC-MS/MS分析以识别推定 SPFH蛋白结合伴侣。我们将通过CRISPR-CAS9基因组编辑创建SPFH突变体，然后表型在生长和细胞测定中表征突变株。我们将检查SPFH功能 C.白色念珠菌发病机理使用新型无脊椎动物感染系统。我们的发现将解决一个糟糕的问题了解白色念珠菌生物学的区域，并提供了研究致病真菌中SPFH蛋白的模型。该提议具有创新性在白色疾病生物学中理解的区域：线粒体功能的分子框架。