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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1-IDM-S (03))
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Joy, Deirdre A
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Harvard University
Schools of Public Health
United States
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Li, Yuan; Shah-Simpson, Sheena; Okrah, Kwame et al. (2016) Transcriptome Remodeling in Trypanosoma cruzi and Human Cells during Intracellular Infection. PLoS Pathog 12:e1005511