Transcriptome profiling of Trypanosoma cruzi and its host cell

克氏锥虫及其宿主细胞的转录组分析

• 批准号: 8265242
• 负责人: BARBARA A BURLEIGH
• 金额: $ 19.57万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8265242
• 关键词: Adverse effects; Algorithms; Biological; Biology; Blood Vessels; Cells; Chagas Disease; Chronic; Complementary DNA; Complex; Country; DNA Sequence; Data; Data Set; Databases; Development; Disease; Ensure; Exhibits; Future; Genbank; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Genome; Goals; Growth; Guidelines; Health; Heart failure; Human; Human Genome; Immigrant; Individual; Infection; Intervention; Laboratories; Latin America; Length; Libraries; Life Cycle Stages; Maintenance; Mammalian Cell; Maps; Mediating; Messenger RNA; Modeling; Molecular; Molecular Profiling; Nature; Oligonucleotide Microarrays; Parasites; Parasitic Diseases; Pathogenesis; Pattern; Pharmaceutical Preparations; Phenotype; Polyadenylation; Prevention; Process; Public Health; RNA; Research Personnel; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Site; Smooth Muscle Myocytes; Solid; Staging; Structure; Technology; Time; Trans-Splicing; Transcript; Trypanosoma cruzi; United States; Vaccines; Virulence; Work; combat; experience; functional genomics; genome-wide; insight; intracellular parasitism; invertebrate host; monolayer; next generation; novel; pathogen; programs; public health relevance; reconstruction; response; tissue culture; tool; transcriptomics

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.

描述（由申请人提供）：利用基因组规模的方法来鉴定病原体特异性过程仍然是鉴定疾病干预新靶点的有前途的策略。在本研究中，我们专注于动质体原生动物寄生虫克氏锥虫，人类恰加斯病的病原体。目前可用的治疗南美锥虫病的药物是有毒的，在感染的慢性阶段缺乏疗效。这项合作研究的目标是利用下一代测序（NGS）技术来获得T的四个主要发育阶段的第一个全面的基因表达谱数据。cruzi（目标1），并建立允许在细胞内寄生虫及其宿主细胞中平行分析基因表达程序的条件（目标2）。为了实现这些目标，我们将对T. cruzi CL Brener（参考菌株）和感染的人细胞（Aim 2）的细胞系中，通过在IlluminaZ平台上使用RNA-seq方法。一系列广泛的计算工具和基因组分析的经验将被应用于从测序数据重建全长转录本，并将其映射到T。cruzi和人类基因组。我们将确定反式剪接和多聚腺苷酸化位点，并确定新的T。cruzi基因，这些基因在原始注释中可能被遗漏，从而极大地增强了当前的基因模型结构和注释。使用一套算法，将确定每个T的mRNA丰度。cruzi基因在四个生命周期阶段的每一个阶段，使我们能够确定与寄生虫的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组的更复杂的情况，将优化T。cruzi感染人血管平滑肌细胞（VSMC）以确保寄生虫和宿主转录物的最大覆盖。一旦建立，我们将在VSMC中进行有限时间的感染过程，以获得感染背景下寄生虫和宿主细胞基因表达程序的动态性质的初步分析。这些数据将提供T. Cruzi基因表达程序，其在细胞内感染的情况下与人宿主细胞的转录应答一起沿着被独特激活。这项研究的结果，包括增强的注释和表达谱数据，将以主要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，其中维护和管理当前的基因组数据。这些研究将为我们自己的实验室以及更广泛的恰加斯病和细胞内寄生虫领域的未来功能和基因组研究提供坚实的框架。 公共卫生相关性：查加斯病是一种热带寄生虫病，在慢性感染原生动物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，恰加斯病在流行国家是一个特别重要的公共卫生问题，在美国也是一个新出现的移民健康问题。本研究的目的是利用新的DNA测序技术来确定在大肠杆菌感染的T。cruzi阶段在全基因组范围内。这项工作的结果将提供新的见解T。cruzi发病机制，并指导有效预防或控制恰加斯病的努力。

项目成果

Transcriptome Remodeling in Trypanosoma cruzi and Human Cells during Intracellular Infection.

• DOI: 10.1371/journal.ppat.1005511
• 发表时间: 2016-04
• 期刊: PLoS pathogens
• 影响因子: 6.7
• 作者: Li Y;Shah-Simpson S;Okrah K;Belew AT;Choi J;Caradonna KL;Padmanabhan P;Ndegwa DM;Temanni MR;Corrada Bravo H;El-Sayed NM;Burleigh BA
• 通讯作者: Burleigh BA