Transcriptome profiling of Trypanosoma cruzi and its host cell

描述(由申请人提供)：利用基因组规模的方法来识别病原体特定的过程仍然是识别疾病干预的新靶点的一个有前途的策略。在本研究中，我们将重点放在人类恰加斯病的病原体克氏锥虫上。目前治疗恰加氏病的药物是有毒的，在慢性感染阶段缺乏疗效。这项合作研究的目标是利用下一代测序(NGS)技术获得克鲁兹毛滴虫四个主要发育阶段的第一个全面的基因表达谱数据(目标1)，并建立条件，允许并行分析细胞内寄生虫及其宿主细胞的基因表达程序(目标2)。为了实现这些目标，我们将在IllLumaZ平台上使用RNA-seq方法对克氏锥虫CL Brener(参考菌株)和感染的人类细胞(AIM2)的转录本进行高分辨率测序。一系列广泛的计算工具和基因组分析经验将被应用于从测序数据中重建全长转录本，并将这些转录本映射到克氏锥虫和人类基因组。我们将定义反式剪接和多聚腺苷酸化位点，并识别在原始注释过程中可能遗漏的新的T.ruzi基因，从而极大地增强当前的基因模型结构和注释。使用一套算法，将确定每个克氏锥虫基因在四个生命周期阶段的mRNA丰度，使我们能够识别与寄生虫生物学相关的共表达模式。对于从感染细胞中破译寄生虫和宿主转录本的更复杂的场景，将优化克氏锥虫感染人血管平滑肌细胞(VSMC)的条件，以确保最大限度地覆盖寄生虫和宿主转录本。一旦确定，我们将在VSMC中进行有限时间的感染过程，以初步分析寄生虫和宿主细胞基因在感染背景下的动态性质。这些数据将提供对克氏锥虫基因表达程序的第一次窥探，这些程序在细胞内感染和人类宿主细胞的转录反应的背景下被独特地激活。这项研究的结果，包括增强的注释和表达谱数据，将以原始数据集的形式在现有的公共数据库中传播，并通过Genbank和TritypDB(www.tritcrypdb.org)传播，在那里维护和管理当前的基因组数据。这些研究将为我们自己实验室未来的功能和基因组研究以及更广泛的恰加斯病和细胞内寄生领域的其他研究提供坚实的框架。 公共卫生相关性：恰加斯病是一种热带寄生虫病，在长期感染原虫寄生虫克氏锥虫的个体中发展多年。作为拉丁美洲心力衰竭的主要原因，查加斯病在流行国家是一个特别重要的公共卫生问题，在美国也是一个新的移民健康问题。这项拟议的研究旨在使用新的DNA测序技术在全基因组范围内识别哺乳动物感染的克氏锥虫阶段的表达基因。这项工作的结果将为了解克鲁兹毛滴虫的发病机制提供新的见解，并指导有效预防或控制恰加斯病的努力。

DESCRIPTION (provided by applicant): Exploiting genome-scale approaches to identify pathogen-specific processes remains a promising strategy for the identification of novel targets for disease intervention. In the present study, we focus on the kinetoplastid protozoan parasite Trypanosoma cruzi, the causative agent of human Chagas' disease. The currently available drugs to treat Chagas' disease are toxic and lack efficacy in the chronic stage of infection. The goal of this collaborative study is to exploit the use of Next-Generation Sequencing (NGS) technologies to obtain the first comprehensive gene expression profiling data for the four main developmental stages of T. cruzi (Aim 1) and to establish conditions that will permit the parallel analysis of gene expression programs in both intracellular parasites and their host cells (Aim 2). To achieve these goals, we will conduct high-resolution sequencing of the transcriptomes of T. cruzi CL Brener (the reference strain) and infected human cells (Aim2) by using an RNA-seq approach on the IlluminaZ platform. An extensive array of computation tools and experience in genome analysis will be applied to reconstruct full-length transcripts from the sequencing data and to map these to the T. cruzi and human genomes. We will define trans-splicing and polyadenylation sites and identify novel T. cruzi genes that were likely missed during the original annotation, thereby greatly enhancing current gene model structures and annotations. Using a suite of algorithms, mRNA abundance will be determined for every T. cruzi gene in each of four life cycle stages enabling us to identify co-expression patterns that correlate with the biology of the parasite. For the more complex scenario of deciphering parasite and host transcriptomes from infected cells, conditions will be optimized for T. cruzi infection of human vascular smooth muscle cells (VSMC) to ensure maximal coverage of parasite and host transcripts. Once this is established, we will conduct a limited time course of infection in VSMC to obtain a preliminary analysis of the dynamic nature of parasite and host cell gene expression programs in the context of infection. These data will provide the first glimpse of T. cruzi gene expression programs that are uniquely activated in the context of intracellular infection along with the transcriptional response of the human host cell. Results from this study, including enhanced annotations and expression profiling data, will be disseminated throughout existing public databases in the form of primary datasets and through Genbank and TritrypDB (www.tritrypdb.org), where the current genome data is maintained and curated. These studies will provide a solid framework for future functional and genomic studies in our own laboratories, as well as others in the broader Chagas's disease and intracellular parasitism fields. PUBLIC HEALTH RELEVANCE: Chagas' disease is a tropical parasitic disease that develops over a number of years in individuals that are chronically infected with the protozoan parasite, Trypanosoma cruzi. As a leading cause of heart failure in Latin America, Chagas' disease represents a public health problem of exceptional importance in endemic countries as well as an emerging immigrant health issue in the United States. The proposed study aims to use new DNA sequencing technologies to identify expressed genes in mammalian-infective T. cruzi stages on a genome-wide scale. Results from this work will provide novel insights into T. cruzi pathogenesis and guide efforts toward effective prevention or control of Chagas' disease.