Isolation of mononuclear propagules from coenocytic hyphae of the mucormycosis pathogen Rhizopus delemar

从毛霉菌病病原体德莱马根霉的共生菌丝中分离单核繁殖体

• 批准号: 10221180
• 负责人: PING WANG
• 金额: $ 7.35万
• 依托单位: LSU HEALTH SCIENCES CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-16 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10221180
• 关键词: Antifungal Therapy; CRISPR/Cas technology; Cell Nucleus; Cell Separation; Cells; Clinical; Coupled; Custom; DNA; Digestion; Disease; Disease Management; Enzymes; Flow Cytometry; Fluorescence; Fluorescence-Activated Cell Sorting; Generations; Genes; Genetic; Genetic Markers; Genetic Transformation; Genetic study; Goals; Hyphae; Immunocompromised Host; Individual; Infection; Knock-out; Label; Life; Mechanics; Methods; Microdissection; Mitotic; Molecular; Mononuclear; Mucormycosis; Mycoses; Nuclear; Organism; Pathogenesis; Patients; Plasmids; Population Heterogeneity; Production; Protoplasts; Reproduction spores; Research; Resources; Rhizopus; Techniques; Technology; asexual; base; fungus; genetic analysis; genetic manipulation; improved; interest; mortality; mutant; new therapeutic target; overexpression; pathogen; pathogenic fungus; plasmid DNA; protein biomarkers; success; therapeutically effective; tool

1. Abstract Rhizopus delemar is the leading casual-agent of mucormycosis, a life-threatening fungal infection in immunocompromised patients. Despite its clinical importance, R. delemar remains understudied due to its limited genetic tractability and lack of available research resources. Specifically, R. delemar produces coenocytic hyphae and multinucleated spores, resulting in a high difficulty obtaining stable gene-specific mutant strains. We have previously employed consecutive hyphal tip microdissection, and with CRISPR-Cas9 techniques, to isolate mononuclear mutant strains. However, this isolation method is laborious and inefficient. To circumvent this obstacle, we propose hyphal fragmentation and protoplast generation, coupled with fluorescence-activated cell sorting (FACS), which allows isolation of mononuclear propagules to obtain mutant clones following genetic transformation robustly. We will first optimize hyphal fragmentation and protoplast production to enrich single nucleated cells (Aim 1a) and label R. delemar strains with fluorescent nuclear markers through DNA transformation (Aim 1b). We will then sort and isolate mononuclear hyphal fragments/protoplasts through FACS analysis (Aim 2a). Additionally, we will improve the DNA plasmid for further genetic manipulation of R. delemar (Aim 2b). Our research plan's successful implementation will provide an invaluable tool to facilitate genetic studies of R. delemar-induced mucormycosis mechanisms.

1.摘要 根霉是毛霉病的主要病原体，毛霉病是一种威胁生命的真菌感染， 免疫功能低下的患者。尽管它的临床重要性，R。delemar仍然研究不足，由于其 有限的遗传易处理性和缺乏可用的研究资源。特别是R.德莱马尔生产 多核菌丝和多核孢子，导致很难获得稳定的基因特异性 突变株我们之前采用了连续的菌丝尖端显微切割，并使用CRISPR-Cas9 技术，分离单核突变株。然而，这种分离方法是费力且低效的。 为了克服这一障碍，我们提出了菌丝破碎和原生质体产生，再加上 荧光激活细胞分选（FACS），其允许分离单核繁殖体以获得突变体 克隆后的遗传转化鲁棒。我们将首先优化菌丝破碎和原生质体 生产以富集单核细胞（Aim 1a）并标记R.核荧光delemar菌株 通过DNA转化标记（目的1b）。然后我们将挑选和分离单核菌丝 片段/原生质体通过FACS分析（目的2a）。此外，我们将改进DNA质粒， 进一步的基因操作R. delemar（目标2b）。我们的研究计划的成功实施将 提供了一个宝贵的工具，以促进遗传研究的R。delemar诱导的毛霉菌病机制。