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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

基本信息

批准号：
10353429
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
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-16 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10353429
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

项目摘要

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.摘要德尔玛根霉是毛霉病的主要临时病原体，毛霉病是一种威胁生命的真菌感染。免疫功能受损的病人。尽管它在临床上很重要，但由于它的临床重要性，R.delemar仍未得到充分的研究遗传适应性有限，缺乏可用的研究资源。具体地说，R.delemar生产共核菌丝和多核孢子，导致获得稳定的基因特异性的难度很高突变菌株。我们以前使用过连续的菌丝顶端显微分离，并使用CRISPR-Cas9 技术，分离出单核突变株。然而，这种分离方法费时费力，效率低。为了绕过这个障碍，我们提出了菌丝碎裂和原生质体生成，再加上荧光激活细胞分选(FACS)，允许分离单核繁殖体以获得突变体基因转化后的无性系。我们将首先优化菌丝碎片和原生质体生产富集单核细胞(Aim 1a)并用荧光核标记德尔玛乳杆菌菌株通过DNA转化标记(目标1b)。然后，我们将对单核菌丝进行分类和分离通过流式细胞仪分析片段/原生质体(目标2a)。此外，我们还将改进DNA质粒以用于进一步的德尔马氏菌的遗传操作(目标2b)。我们的研究计划的成功实施将提供了一个非常有价值的工具，以促进德尔玛毛霉菌引起的毛霉病机制的遗传学研究。