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异性过程仍然是识别疾病干预新靶点的有前途的策略。在本研究中，我们重点研究了人类恰加斯病的病原体——克氏锥虫。目前可用于治疗恰加斯病的药物是有毒的，并且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）技术获得克氏锥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，允许平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IlluminaZ平台上使用RNA-seq方法对克氏T. cruzi CL Brener（参考菌株）和受感染的人细胞（Aim2）的转录组进行高分辨率测序。广泛的计算工具和基因组分析经验将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T. cruzi和人类基因组。我们将定义反式剪接和聚腺苷化位点，并鉴定在原始注释过程中可能遗漏的新的克氏T. cruzi基因，从而大大增强当前基因模型结构和注释。使用一套算法，将确定四个生命周期阶段中每个克氏锥虫基因的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。对于更复杂的从感染细胞中破译寄生虫和宿主转录组的场景，将优化克氏锥虫感染人类血管平滑肌细胞（VSMC）的条件，以确保寄生虫和宿主转录物的最大覆盖。一旦确定，我们将在VSMC中进行有限时间的感染过程，以初步分析感染背景下寄生虫和宿主细胞基因表达程序的动态性质。这些数据将首次揭示在细胞内感染和人类宿主细胞转录反应中唯一激活的克氏锥虫基因表达程序。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TritrypDB （www.tritrypdb.org）进行传播，这些数据库维护和管理当前的基因组数据。这些研究将为今后在我们自己的实验室以及在更广泛的恰加斯病和细胞内寄生领域的其他实验室进行功能和基因组研究提供坚实的框架。

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.