Directed Culturing of Pneumocystis Using Metatranscriptomics

描述（由申请人提供）：对耶氏肺孢子虫（Pneumocystis jirovecii）真菌知之甚少，它是免疫功能低下人群（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，耶氏肺孢子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。我们将采用一种新的方法，metatranscriptomics，最近用于成功地直接补充培养基以前不可培养的细菌。这种方法将首次用于不可培养的真核病原体。mRNA和基因组DNA的下一代测序（NGS）成本的降低已经彻底改变了研究的许多方面。我们将使用mRNA的NGS来鉴定在不断增长的肺孢子虫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在已建立的短期体外培养系统中以迭代方式进行检测，直至鉴定出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获得感染的大鼠肺的元转录组，其包括卡氏肺孢子虫、大鼠肺，以及 2）利用系统生物学和结构生物信息学的方法来确定肺孢子虫增殖中的关键参与者基因、其可能的配体、底物和产物，以选择用于体外测试的候选补充剂; 3）使用高通量迭代策略在短期体外培养测定中评估修饰的培养基，以鉴定 支持卡氏肺孢子虫持续生长的必需营养素及其浓度; 4）使用鼠肺孢子虫（小鼠来源的）和耶氏肺孢子虫（人来源的）进行体外培养，以开发肺孢子虫生长的通用补充方案。该项目的成功将从根本上改变PCP的临床诊断，提供一种检测活肺孢子虫的方法，还将推动临床和基础研究向前发展，允许应用强大的分子遗传工具，如转化和定点突变;促进药物发现;并允许测试、跟踪和调查耐药性

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