Directed Culturing of Pneumocystis Using Metatranscriptomics

利用宏转录组学定向培养肺孢子虫

基本信息

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
项目类别：
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

项目摘要

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）：较知的肺炎肺炎藻（Jirovecii）是免疫功能低下的人，尤其是患有艾滋病的人，是致命性肺炎（PCP）的重要原因。缺乏培养系统的研究方法和肺炎假单胞菌肺炎的临床管理受到了极大的阻碍。尝试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，即元文字组学，最近用于成功地将培养基补充以前无法培养的细菌。这种方法将首次在无法养设的真核病原体上使用。 MRNA和基因组DNA的下一代测序（NGS）的成本降低彻底改变了研究的许多方面。我们将使用NG的mRNA来鉴定通过肺炎囊体种群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的方式在体外培养系统中进行测试，直到确定优化的培养物，以支持肺炎孢子菌的连续传播。我们的目标是：1）使用RNA-Seq获得感染大鼠肺的元转录组，其中包括Carinii，大鼠肺和伴随微生物群，以鉴定与Carinii生长和下降相关的代谢基因特征； 2）使用系统生物学和结构生物信息学的方法来确定基因，肺炎胸膜增殖的主要参与者，可能的配体，底物和产品，以选择用于体外测试的候选补充剂； 3）在短期内使用高通量迭代策略来评估改良的培养基，以识别基本的营养素及其浓度，以支持carinii的持续生长； 4）使用P. murina（小鼠衍生）和Jirovecii（人类衍生）验证体外培养，以开发一种用于肺炎囊肿生长的通用补充方案。该项目的成功将通过提供可行的肺炎胸膜的测试从根本上改变PCP的临床诊断，并通过允许应用强大的分子遗传工具（例如转化和位置的突变）来推动临床和基础研究。促进毒品发现；并允许对耐药性进行测试，跟踪和调查