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识别病原体特异性过程仍然是鉴定新型疾病干预目标的有前途的策略。在本研究中，我们专注于人类chagas疾病的病原体Cruzi Cruzi的动力学原生动物寄生虫锥虫。目前可用于治疗Chagas疾病的药物有毒，并且在慢性感染阶段缺乏功效。这项协作研究的目的是利用下一代测序（NGS）技术的使用，以获取Cruzi T. Cruzi的四个主要发育阶段（AIM 1）的第一个全面的基因表达数据数据（AIM 1），并确定可以在细胞内寄生虫及其宿主细胞中对基因表达程序进行平行分析的条件（AIM 2）。为了实现这些目标，我们将通过在Illuminaz平台上使用RNA-Seq方法对Cruzi Cl Brener（参考应变）和感染的人类细胞的转录组进行高分辨率测序。大量的计算工具和基因组分析中的经验将应用于从测序数据中重建全长转录本，并将其映射到Cruzi和人类基因组中。我们将定义变速箱和聚腺苷酸化位点，并确定可能在原始注释期间遗漏的新型Cruzi基因，从而大大增强了当前的基因模型结构和注释。使用算法套件，将在四个生命周期阶段的每个T. cruzi基因中确定mRNA丰度，从而使我们能够识别与寄生虫生物学相关的共表达模式。对于来自感染细胞的解密寄生虫和宿主转录组的更复杂情况，将对人血管平滑肌细胞（VSMC）的T. cruzi感染进行优化，以确保寄生虫和宿主转录物的最大覆盖率。一旦建立了这一点，我们将在VSMC中进行有限的感染时间，以获得对寄生虫和宿主细胞基因表达程序的动态性质的初步分析。这些数据将提供第一次瞥见Cruzi基因表达程序，这些程序在细胞内感染以及人类宿主细胞的转录反应中被唯一激活。这项研究的结果，包括增强的注释和表达分析数据，将以主要数据集的形式以及通过GenBank和TritryPDB（www.tritrypdb.org）在整个现有的公共数据库中传播，并在其中维护并策划了当前的基因组数据。这些研究将为我们自己的实验室以及更广泛的Chagas病和细胞内寄生虫领域中的未来功能和基因组研究提供一个坚实的框架。 公共卫生相关性：Chagas疾病是一种热带寄生虫病，在长期感染原生动物寄生虫Crypanosoma Cruzi的个体中，多年来发展了多年。作为拉丁美洲心力衰竭的主要原因，查加斯疾病代表了在流行国家以及美国新兴的移民健康问题中非常重要的公共卫生问题。拟议的研究旨在使用新的DNA测序技术来鉴定哺乳动物感染性T. cruzi阶段中表达的基因，以全基因组范围的范围。这项工作的结果将为克鲁济氏菌发病机理提供新的见解，并指导有效预防或控制查加斯疾病的努力。

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