Directed Culturing of Pneumocystis Using Metatranscriptomics

描述（由申请人提供）：在免疫功能低下的人，尤其是艾滋病患者中，对真菌乙氏肺囊虫知之甚少，是致死性肺炎（PCP）的重要原因。由于缺乏培养系统，研究方法和临床管理严重阻碍了耶氏疟原虫肺炎。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，亚转录组学，最近成功地用于指导培养基补充以前不可培养的细菌。该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来识别在增长的肺囊虫种群中高度表达的酶和转运体。这些蛋白的底物和靶点将在已建立的短期体外培养系统中以迭代的方式进行测试，直到确定支持肺囊虫持续繁殖的优化培养物。我们的目标是：1)使用RNA-seq获得受感染大鼠肺的转转录组，其中包括卡氏假体、大鼠肺和大肠杆菌

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