Directed Culturing of Pneumocystis Using Metatranscriptomics

利用宏转录组学定向培养肺孢子虫

• 批准号: 8554433
• 负责人: Melanie T Cushion
• 金额: $ 40.45万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-22 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8554433
• 关键词: Acquired Immunodeficiency Syndrome; Address; Adrenal Cortex Hormones; Amphotericin B; Anti-Tumor Necrosis Factor Therapy; Antifungal Agents; Area; Azoles; Bacteria; Basic Science; Bioinformatics; Biological Assay; Categories; Chronic; Chronic Obstructive Airway Disease; Clinical Management; Clinical Research; Communities; Comorbidity; Culture Media; Cyst; DNA; Data; Development; Diagnosis; Disease; Drug Targeting; Drug resistance; Environment; Enzymes; Folic Acid Antagonists; Gene Expression Profiling; Genes; Genomics; Growth; HIV; Health; Healthcare; Human; Immune response; Immunocompromised Host; In Vitro; Individual; Industrial fungicide; Investigation; Left; Life; Ligands; Lung; Malignant neoplasm of lung; Messenger RNA; Metabolic; Methods; Microbe; Molecular Genetics; Molecular Profiling; Mus; Mutation; Nutrient; Nutritional Requirements; Organism; Patients; Pharmaceutical Preparations; Pharmacotherapy; Pneumocystis; Pneumocystis carinii; Pneumocystis carinii Pneumonia; Pneumonia; Population; Proteins; Rattus; Recurrence; Relapse; Research; Resources; Rodent; Site; Supplementation; System; Systems Biology; Testing; Time; Treatment Failure; Trimethoprim-Sulfamethoxazole; Virulence; antiretroviral therapy; base; clinical Diagnosis; cost; design; drug candidate; drug discovery; fungus; global health; in vitro testing; innovation; microbial community; microorganism; mortality; next generation sequencing; novel; novel strategies; novel therapeutics; pathogen; prophylactic; respiratory; screening; success; tool; transcriptome sequencing; urban area

DESCRIPTION (provided by applicant): The poorly understood fungus Pneumocystis jirovecii is an important cause of lethal pneumonia (PCP) in immunocompromised humans, especially those with AIDS. Research approaches and clinical management of P. jirovecii pneumonia have been significantly hindered by the lack of a culture system. Attempts to develop a continuous in vitro system using standard methods have failed. We will employ a new approach, metatranscriptomics, recently used to successfully direct the supplementation of medium for a previously uncultivable bacterium. This approach will be used for the first time on an uncultivable eukaryotic pathogen. The reduced cost for next generation sequencing (NGS) of mRNA and genomic DNA has revolutionized many aspects of research. We will use NGS of mRNA to identify enzymes and transporters that are highly expressed by growing Pneumocystis populations. The substrates and targets of these proteins will be tested in an established short term in vitro culture system in an iterative fashion until an optimized culture is identified that supports the continuous propagation of Pneumocystis spp. Our objectives are to: 1) Use RNA-seq to obtain the metatranscriptome of infected rat lungs, which includes P. carinii, rat lung, and accompanying microbiota, to identify metabolic gene signatures associated with P. carinii growth and decline; 2) Use approaches of systems biology and structural bioinformatics to determine genes, key players in Pneumocystis proliferation, their possible ligands, substrates and products, to select candidate supplements for testing in vitro; 3) Evaluate modified culture media in a short term in vitro culture assay using a high throughput iterative strategy to identify essential nutrients and their concentrations that support the continuous growth of P. carinii; 4) Validate the in vitro culture using P. murina (mouse derived) and P. jirovecii (human derived) to develop a universal supplementary regime for Pneumocystis growth. Success of this project will fundamentally change the clinical diagnosis of PCP by providing a test for viable Pneumocystis and will also propel clinical and basic research forward by allowing application of powerful molecular genetic tools such as transformation and site directed mutation; facilitate drug discovery; and allow testing, tracking and investigation of drug resistance

描述（由申请人提供）：人们对耶氏肺孢子菌知之甚少，它是导致免疫功能低下的人类（尤其是艾滋病患者）致死性肺炎（PCP）的重要原因。由于缺乏培养系统，耶氏疟原虫肺炎的研究方法和临床管理受到严重阻碍。使用标准方法开发连续体外系统的尝试失败了。我们将采用一种新方法，即宏转录组学，最近用于成功指导为以前无法​​培养的细菌补充培养基。这种方法将首次用于无法培养的真核病原体。 mRNA 和基因组 DNA 下一代测序 (NGS) 成本的降低彻底改变了研究的许多方面。我们将使用 mRNA 的 NGS 来识别不断生长的肺孢子菌种群中高度表达的酶和转运蛋白。这些蛋白质的底物和靶标将以迭代方式在已建立的短期体外培养系统中进行测试，直到确定支持肺孢子虫属持续繁殖的优化培养物。我们的目标是：1) 使用 RNA-seq 获得受感染大鼠肺部的宏转录组，其中包括 P. carinii、大鼠肺和 伴随微生物群，以确定与 P. carinii 生长和衰退相关的代谢基因特征； 2）利用系统生物学和结构生物信息学的方法确定肺孢子虫增殖的基因、关键因子、其可能的配体、底物和产物，以选择候选补充剂进行体外测试； 3) 使用高通量迭代策略在短期体外培养测定中评估改良培养基，以识别 支持卡氏疟原虫持续生长的必需营养素及其浓度； 4) 使用 P. murina（小鼠来源）和 P. jirovecii（人类来源）验证体外培养，以开发肺孢子虫生长的通用补充方案。该项目的成功将通过提供活肺孢子虫检测从根本上改变 PCP 的临床诊断，并将通过允许应用强大的分子遗传学工具（如转化和定点突变）推动临床和基础研究向前发展；促进药物发现；并允许测试、跟踪和调查耐药性