Chromosome level assembly of the hybrid Trypanosoma cruzi genome

<p>Abstract</p> <p>Background</p> <p>In contrast to the essentially fully assembled genome sequences of the kinetoplastid pathogens <it>Leishmania major </it>and <it>Trypanosoma brucei </it>the assembly of the <it>Trypanosoma cruzi </it>genome has been hindered by its repetitive nature and the fact that the reference strain (CL Brener) is a hybrid of two distinct lineages. In this work, the majority of the contigs and scaffolds were assembled into pairs of homologous chromosomes based on predicted parental haplotype, inference from TriTryp synteny maps and the use of end sequences from <it>T. cruzi </it>BAC libraries.</p> <p>Results</p> <p>Ultimately, 41 pairs of chromosomes were assembled using this approach, a number in agreement with the predicted number of <it>T. cruzi </it>chromosomes based upon pulse field gel analysis, with over 90% (21133 of 23216) of the genes annotated in the genome represented. The approach was substantiated through the use of Southern blot analysis to confirm the mapping of BAC clones using as probes the genes they are predicted to contain, and each chromosome construction was visually validated to ensure sufficient evidence was present to support the organization. While many members of large gene families are incorporated into the chromosome assemblies, the majority of genes excluded from the chromosomes belong to gene families, as these genes are frequently impossible to accurately position.</p> <p>Conclusion</p> <p>Now assembled, these chromosomes bring <it>T. cruzi </it>to the same level of organization as its kinetoplastid relatives and have been used as the basis for the <it>T. cruzi </it>genome in TriTrypDB, a trypanosome database of EuPathDB. In addition, they will provide the foundation for analyses such as reverse genetics, where the location of genes and their alleles and/or paralogues is necessary and comparative genome hybridization analyses (CGH), where a chromosome-level view of the genome is ideal.</p

From Questions to Effective Answers: On the Utility of Knowledge-Driven Querying Systems for Life Sciences Data

We compare two distinct approaches for querying data in the context of the life sciences. The first approach utilizes conventional databases to store the data and intuitive form-based interfaces to facilitate easy querying of the data. These interfaces could be seen as implementing a set of "pre-canned" queries commonly used by the life science researchers that we study. The second approach is based on semantic Web technologies and is knowledge (model) driven. It utilizes a large OWL ontology and same datasets as before but associated as RDF instances of the ontology concepts. An intuitive interface is provided that allows the formulation of RDF triples-based queries. Both these approaches are being used in parallel by a team of cell biologists in their daily research activities, with the objective of gradually replacing the conventional approach with the knowledge-driven one. This provides us with a valuable opportunity to compare and qualitatively evaluate the two approaches. We describe several benefits of the knowledge-driven approach in comparison to the traditional way of accessing data, and highlight a few limitations as well. We believe that our analysis not only explicitly highlights the specific benefits and limitations of semantic Web technologies in our context but also contributes toward effective ways of translating a question in a researcher's mind into precise computational queries with the intent of obtaining effective answers from the data. While researchers often assume the benefits of semantic Web technologies, we explicitly illustrate these in practice

Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi

<p>Abstract</p> <p>Background</p> <p><it>Trypanosoma cruzi</it>, a kinetoplastid protozoan parasite that causes Chagas disease, infects approximately 15 million people in Central and South America. In contrast to the substantial <it>in silico </it>studies of the <it>T. cruzi </it>genome, transcriptome, and proteome, only a few genes have been experimentally characterized and validated, mainly due to the lack of facile methods for gene manipulation needed for reverse genetic studies. Current strategies for gene disruption in <it>T. cruzi </it>are tedious and time consuming. In this study we have compared the conventional multi-step cloning technique with two knockout strategies that have been proven to work in other organisms, one-step-PCR- and Multisite Gateway-based systems.</p> <p>Results</p> <p>While the one-step-PCR strategy was found to be the fastest method for production of knockout constructs, it does not efficiently target genes of interest using gene-specific sequences of less than 80 nucleotides. Alternatively, the Multisite Gateway based approach is less time-consuming than conventional methods and is able to efficiently and reproducibly delete target genes.</p> <p>Conclusion</p> <p>Using the Multisite Gateway strategy, we have rapidly produced constructs that successfully produce specific gene deletions in epimastigotes of <it>T. cruzi</it>. This methodology should greatly facilitate reverse genetic studies in <it>T. cruzi</it>.</p

Chagas Disease and the London Declaration on Neglected Tropical Diseases

Fil: Tarleton, Rick L.. University of Georgia; Estados Unidos. Chagas Disease Foundation; Estados UnidosFil: Gurtler, Ricardo Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Ecología, Genética y Evolución de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Ecología, Genética y Evolución de Buenos Aires; ArgentinaFil: Urbina, Julio A.. Instituto Venezolano de Investigaciones Científicas; VenezuelaFil: Ramsey, Janine. Instituto Nacional de Salud Pública; MéxicoFil: Viotti, Rodolfo Jorge. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; Argentin

Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure

The ability of the Chagas disease agent Trypanosoma cruzi to resist extended in vivo exposure to highly effective trypanocidal compounds prompted us to explore the potential for dormancy and its contribution to failed drug treatments in this infection. We document the development of non-proliferating intracellular amastigotes in vivo and in vitro in the absence of drug treatment. Non-proliferative amastigotes ultimately converted to trypomastigotes and established infections in new host cells. Most significantly, dormant amastigotes were uniquely resistant to extended drug treatment in vivo and in vitro and could re-establish a flourishing infection after as many as 30 days of drug exposure. These results demonstrate a dormancy state in T. cruzi that accounts for the failure of highly cytotoxic compounds to completely resolve the infection. The ability of T. cruzi to establish dormancy throws into question current methods for identifying curative drugs but also suggests alternative therapeutic approaches.Fil: Sánchez Valdéz, Fernando Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina. University of Georgia; Estados UnidosFil: Poveda Padilla, Angélica Gabriela. University of Georgia; Estados UnidosFil: Wang, Wei. University of Georgia; Estados UnidosFil: Orr, Dylan. University of Georgia; Estados UnidosFil: Tarleton, Rick L.. University of Georgia; Estados Unido

Quantitative 3D Imaging of Trypanosoma cruzi-Infected Cells, Dormant Amastigotes, and T Cells in Intact Clarified Organs

Reliable detection of Trypanosoma cruzi (T. cruzi) in vivo infections have long been needed to understand the complex biology of Chagas disease and to accurately evaluate the outcome of treatment regimens. Here, an integrated pipeline for automated quantification of T. cruzi-infected cells in 3D-reconstructed, cleared organs was developed. Light-sheet fluorescent microscopy allows us to accurately visualize and quantify not only actively proliferating but also dormant T. cruzi parasites and immune effector cells in whole-organs or tissues. Also, CUBIC-HistoVision pipeline to obtain uniform labeling of cleared organs with antibodies and nuclear stains was successfully adopted. Tissue clearing coupled to 3D immunostaining provides an unbiased approach to comprehensively evaluate drug treatment protocols, improve the understanding of the cellular organization of T. cruzi-infected tissues and is expected to advance discoveries related to anti-T. cruzi immune responses, tissue damage and repair in Chagas disease.Fil: Sánchez Valdéz, Fernando Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Padilla, Angel Marcelo. University of Georgia; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bustamante, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Georgia; Estados UnidosFil: Hawkins, Caleb W. D.. University of Georgia; Estados UnidosFil: Tarleton, Rick L.. University of Georgia; Estados Unido

A semi-quantitative GeLC-MS analysis of temporal proteome expression in the emerging nosocomial pathogen Ochrobactrum anthropi

A semi-quantitative gel-based analysis identifies distinct proteomic profiles associated with specific growth points for the nosocomial pathogen Ochrobactrum anthropi

CD8(+) T-Cell Responses to Trypanosoma cruzi Are Highly Focused on Strain-Variant trans-Sialidase Epitopes

CD8(+) T cells are crucial for control of a number of medically important protozoan parasites, including Trypanosoma cruzi, the agent of human Chagas disease. Yet, in contrast to the wealth of information from viral and bacterial infections, little is known about the antigen specificity or the general development of effector and memory T-cell responses in hosts infected with protozoans. In this study we report on a wide-scale screen for the dominant parasite peptides recognized by CD8(+) T cells in T. cruzi–infected mice and humans. This analysis demonstrates that in both hosts the CD8(+) T-cell response is highly focused on epitopes encoded by members of the large trans-sialidase family of genes. Responses to a restricted set of immunodominant peptides were especially pronounced in T. cruzi–infected mice, with more than 30% of the CD8(+) T-cell response at the peak of infection specific for two major groups of trans-sialidase peptides. Experimental models also demonstrated that the dominance patterns vary depending on the infective strain of T. cruzi, suggesting that immune evasion may be occurring at a population rather than single-parasite level

Drug Discovery for Kinetoplastid Diseases : Future Directions

International audienceKinetoplastid parasites have caused human disease for millennia. Significant achievements have been made toward developing new treatments for leishmaniasis (particularly on the Indian subcontinent) and for human African trypanosomiasis (HAT). Moreover, the sustained decrease in the incidence of HAT has made the prospect of elimination a tantalizing reality. Despite the gains, no new chemical or biological entities to treat kinetoplastid diseases have been registered in more than three decades, and more work is needed to discover safe and effective therapies for patients with Chagas disease and leishmaniasis. Advances in tools for drug discovery and novel insights into the biology of the host-parasite interaction may provide opportunities for accelerated progress. Here, we summarize the output from a gathering of scientists and physicians who met to discuss the current status and future directions in drug discovery for kinetoplastid diseases

Discovery of an orally active benzoxaborole prodrug effective in the treatment of Chagas disease in non-human primates

Trypanosoma cruzi, the agent of Chagas disease, probably infects tens of millions of people, primarily in Latin America, causing morbidity and mortality. The options for treatment and prevention of Chagas disease are limited and underutilized. Here we describe the discovery of a series of benzoxaborole compounds with nanomolar activity against extra- and intracellular stages of T. cruzi. Leveraging both ongoing drug discovery efforts in related kinetoplastids, and the exceptional models for rapid drug screening and optimization in T. cruzi, we have identified the prodrug AN15368 that is activated by parasite carboxypeptidases to yield a compound that targets the messenger RNA processing pathway in T. cruzi. AN15368 was found to be active in vitro and in vivo against a range of genetically distinct T. cruzi lineages and was uniformly curative in non-human primates (NHPs) with long-term naturally acquired infections. Treatment in NHPs also revealed no detectable acute toxicity or long-term health or reproductive impact. Thus, AN15368 is an extensively validated and apparently safe, clinically ready candidate with promising potential for prevention and treatment of Chagas disease