Directed Culturing of Pneumocystis Using Metatranscriptomics

描述(由申请人提供)：鲜为人知的真菌--圣伊氏肺孢子虫是免疫功能低下的人类，尤其是艾滋病患者中致死性肺炎(PCP)的重要原因。缺乏培养系统严重阻碍了日本血吸虫肺炎的研究方法和临床管理。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，元转录组学，最近用于成功地指导对以前不可培养的细菌的培养基的补充。这种方法将首次用于一种不可培养的真核病原体。新一代信使核糖核酸和基因组DNA测序成本的降低使许多方面的研究发生了革命性的变化。我们将使用mRNA的NGS来识别在不断增长的肺孢子虫种群中高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的方式进行测试，直到确定支持肺孢子虫持续繁殖的优化培养。我们的目标是：1)利用rna-seq获得感染大鼠肺的转录组，包括卡氏肺吸虫、大鼠肺和 结合微生物群，鉴定与卡氏肺孢子虫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关键基因、可能的配体、底物和产物，以选择候选补充剂进行体外试验；3)使用高通量迭代策略在短期内对改良的培养基进行评估，以确定 支持卡氏肺孢子虫持续生长的必需营养素及其浓度；4)验证使用P.Murina(鼠源性)和P.jirovecii(人源性)的体外培养，以开发一种通用的肺孢子虫生长补充方案。该项目的成功将从根本上改变PCP的临床诊断，为活的肺孢子虫提供测试，并通过允许应用强大的分子遗传工具，如转化和定点突变，推动临床和基础研究；促进药物发现；以及允许测试、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