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

In Vitro and In Vivo High-Throughput Assays for the Testing of Anti-Trypanosoma cruzi Compounds

The treatment of Trypanosoma cruzi infection (the cause of human Chagas disease) remains a significant challenge. Only two drugs, both with substantial toxicity, are available and the efficacy of these dugs is often questioned – in many cases due to the limitations of the methods for assessing efficacy rather than to true lack of efficacy. For these reasons relatively few individuals infected with T. cruzi actually have their infections treated. In this study, we report on innovative methods that will facilitate the discovery of new compounds for the treatment of T. cruzi infection and Chagas disease. Utilizing fluorescent and bioluminescent parasite lines, we have developed in vitro tests that are reproducible and facile and can be scaled for high-throughput screening of large compound libraries. We also validate an in vivo screening test that monitors parasite replication at the site of infection and determines the effectiveness of drug treatment in less than two weeks. More importantly, results in this rapid in vivo test show strong correlations with those obtained in long-term (e.g. 40 day or more) treatment assays. The results of this study remove one of the obstacles for identification of effective and safe compounds to treat Chagas disease

High Throughput Selection of Effective Serodiagnostics for Trypanosoma cruzi infection

The diagnosis of Trypanosoma cruzi infection (the cause of human Chagas disease) is difficult because the symptoms of the infection are often absent or non-specific, and because the parasites themselves are usually below the level of detection in the infected subjects. Therefore, diagnosis generally depends on the measurement of T. cruzi–specific antibodies produced in response to the infection. However, current methods to detect anti–T. cruzi antibodies are relatively poor. In this study, we have conducted a broad screen of >400 T. cruzi proteins to identify those proteins which are best able to detect anti–T. cruzi antibodies. Using a set of proteins selected by this screen, we were able to detect 100% of >100 confirmed positive human cases of T. cruzi infection, as well as suspect cases that were negative using existing tests. This protein panel was also able to detect apparent changes in infection status following drug treatment of individuals with chronic T. cruzi infection. The results of this study should allow for significant improvements in the detection of T. cruzi infection and better screening methods to avoid blood transfusion–related transmission of the infection, and offer a crucial tool for determining the success or failure of drug treatment and other intervention strategies to limit the impact of Chagas disease

Luminescent <i>T. cruzi</i> imaged at various times post-treatment.

<p>(A) Mice (10 per group) were infected in the footpad with 1×10⁵<i>T. cruzi</i> bioluminescent trypomastigotes. For all images shown the color scale ranges from blue (with a minimum set at 60 photons/s/cm²/sr) to red (maximum of 3000 photons/s/cm2/sr). (B) Quantification of luminescent signal from mice in panel A.</p

<i>In vitro</i> amastigote growth assays using tdTomato parasites.

<p>(A) Amastigotes growth in Vero cells grown in 96 well plates over time in the presence of benznidazole (n = 8). (B) Comparison of IC50 calculations in response to EXO2-04 in 96 and 384 well plates at 3 days of treatment (n = 4). (C) Amastigote growth assay in 96 or 384 well plates using the Colombiana and TCC strain of <i>T. cruzi</i> expressing tdTomato fluorescent protein at 3 days of treatment (n = 8).</p

Plasmids used in the generation of fluorescent and bioluminescent <i>T. cruzi</i>.

<p>Schematic representation of (A) the pTrex-Neo-tdTomato plasmid, and (B) the pLew90β-GW/T7/PARP SAS/luciferase/Aldolase 3′UTR plasmid used for generation of the <i>T. cruzi</i> reporter lines.</p

<i>In vitro</i> epimastigote growth assays using tdTomato parasites.

<p>(A) Epimastigotes growth over time in the presence of benznidazole at the indicated concentrations and comparison of measurement of drug inhibition of epimastigote growth by fluorescence and visual counting by hemacytometer. (B) Intra-assay analysis (left) showing the low variation among wells with the same drug concentration (n = 4). Inter-assay analysis (right) showing the low variation among IC₅₀ curves from individual assays. (C) IC₅₀ calculation in response to benznidazole and the EXO2 derivatives activity against epimastigotes after 3 days of treatment/culture.</p

Rapid suppression of parasitemia following drug-treatment is a poor indicator of drug efficacy and parasitological cure.

<p>(A) Evolution of parasitemia after infection with 1×10³ CL strain of <i>T. cruzi</i> on day 0 in untreated (▪), BZ-40 (▵), POS (○), NTLA-1 (▴), or BIS767 (□) treated mice. “BIS767, BZ-40, POS and NTLA-1” bars below x axis indicate period of treatments. (B) Parasitemias in untreated or treated mice at 120dpi, after administration of the immunosuppressant cyclophosphamide (cy) (days 105, 108, 111, 113 and 117).</p