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：
10332017
负责人：
Jason Malcolm Rauceo
金额：
$ 11.91万
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/10332017
关键词：
Address 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 Biology Molecular Genetics Morphogenesis Nematoda Organelles 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 Savings Signal Transduction Source Stress Students Study models System Testing Time Training Vertebrates Virulence Yeasts base candidemia college cost effective drug efficacy flotillin fungus gene discovery genome editing human pathogen in vivo inhibitor/antagonist 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.

线粒体是细胞主要产生能量的细胞器，协调关键活动，例如如信号传导、细胞凋亡和磷脂合成。这些过程对于生存和发展至关重要白色念珠菌（人类最常见的真菌病原体）的发病机制。白色念珠菌呼吸与脊椎动物相比，它通过三种不同的途径发生，并且与毒力特性相关，例如如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控制白色念珠菌响应环境应激和抗真菌生存的分子机制药物。我们之前发现涉及线粒体功能的基因类别是高度转录的响应渗透压和细胞壁应激，基因 SLP3（stomatin 样蛋白 3）显着上调。 SLP3 是保守的 SPFH（Stomatin、Prohibitin、Flotillin、HflK/HflC）蛋白的成员超家族。在真核生物中，SPFH 蛋白是线粒体重要过程所必需的，例如呼吸、线粒体自噬和细胞凋亡并介导多种寄生虫的致病性。我们的目标是该提案旨在确定白色念珠菌 SPFH 蛋白家族在线粒体功能中的作用。我们是第一个证明 Slp3p 过量产生会破坏线粒体膜电位，特别是在长期暴露于氧化应激后，会引发细胞凋亡样死亡。然而， Slp3p 的分子功能仍然未知。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协调线粒体功能。这个假设是基于对哺乳动物 SPFH 蛋白的观察以及我们已发表的初步发现。人类SPFH 当使用荧光显微镜观察时，复合物显示为点状焦点。我们发现Slp3p形式质膜上的斑点。此外，我们在 Slp2p、Phb1p 和 Phb2p 和我们的 Slp2p-GFP 融合蛋白形成线粒体斑点。我们将利用高通量分子遗传学和细胞方法可经济高效且节省时间地测定 SPFH 蛋白定位、蛋白质复合物组成、细胞功能以及在白色念珠菌感染中的作用。我们将创造 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 表位标记菌株并进行 Co-IP 和 LC-MS/MS 分析以鉴定假定的菌株 SPFH 蛋白结合伴侣。我们将通过 CRISPR-Cas9 基因组编辑创建 SPFH 突变体在生长和细胞测定中表征突变菌株的表型特征。我们将检查 SPFH 的功能使用新型无脊椎动物感染系统的白色念珠菌发病机制。我们的研究结果将解决一个问题理解白色念珠菌生物学领域，并为研究病原真菌中的 SPFH 蛋白提供模型。该提案具有创新性，因为我们将在一个关键但效果不佳的背景下描述 SPFH 家族的特征。白色念珠菌生物学的理解领域：线粒体功能的分子框架。