Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.  Methodology/Principal Findings: The T. rangeli haploid genome is ,24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heatshock proteins.  Conclusions/Significance: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets

Transferrin-Associated Lipoplexes as Gene Delivery Systems: Relevance of Mode of Preparation and Biophysical Properties

Abstract The successful application of gene therapy depends highly on understanding the properties of gene carriers and their correlation with the ability to mediate transfection. An important parameter that has been described to improve transfection mediated by cationic liposomes involves association of ligands to cationic liposome–DNA complexes (lipoplexes). In this study, ternary complexes composed of 1,2-dioleoyl-3-(trimethylammonium) propane:cholesterol, plasmid DNA and transferrin (Tf, selected as a paradigm of a ligand) were prepared under various conditions, namely, in medium with different ionic strengths (HEPES-buffered saline [HBS] or dextrose), at different lipid/DNA (+/–) charge ratios and using different modes for component addition. We investigated the effect of these formulation parameters on transfection (in the absence and presence of serum), size of the complexes, degree of DNA protection and extent of their association with cells (in terms of both lipid and DNA). Our results show that all the tested parameters influenced to some extent the size of the complexes and their capacity to protect the carried genetic material, as well as the levels of cell association and transfection. The best transfection profile was observed for ternary complexes (Tf-complexes) prepared in high ionic strength solution (HBS), at charge ratios close to neutrality and according to the following order of component addition: cationic liposomes–Tf–DNA. Interestingly, in contrast to what was found for dextrose–Tf-complexes, transfection mediated by HBS-Tf-complexes in the presence of serum was highly enhanced

General characteristics of the <i>T. rangeli</i> genome.

<p>* Excluding proteins of unknown function.</p><p>General characteristics of the <i>T. rangeli</i> genome.</p

The RNAi machinery is not active in <i>Trypanosoma rangeli</i>.

<p>Western blot analysis of eGFP silencing via siRNA in <i>T. rangeli</i> and Vero cells expressing eGFP. For the Western blot assays, anti-GFP and anti-alpha tubulin antibodies were used. In each blot, wild-type cells (1), eGFP cells (2), eGFP cells transfected with Mock siRNA (3), eGFP cells transfected with EGFP-S1 DS Positive Control (IDT)(4) and eGFP cells transfected with eGFP antisense siRNA (5) are shown sequentially. The experiments were performed in biological triplicates.</p

Synteny analysis between <i>Trypanosoma rangeli</i> scaffolds and organized contig ends of <i>T. cruzi</i>.

<p>The blue lines represent regions of homology between the contigs. Annotated genes and other sequence characteristics are indicated by colored boxes. Arrows indicate sense transcription. <b>A</b>. Comparison between Scaffold Tr 61 (4,000–53,457 nt) and TcChr27-P (794,000–850,241 nt). <b>B</b>. Comparison between Scaffold Tr 115 (136,482–164,482 nt) and TcChr33-S (975,000–1,041,172 nt). Contig ends were oriented in the 5′ to 3′ direction according to the TriTrypDB assemblies of <i>T. cruzi</i> scaffolds. The accession numbers of the annotated sequences in the <i>T. cruzi</i> scaffolds (TriTrypDB) are displayed below the sequences.</p

Evolutionary history of the Trypanosomatidae family obtained through a phylogenomic approach using (<b>A</b>) the neighbor joining (NJ) or (<b>B</b>) the maximum likelihood (ML) methods.

<p>In the NJ results, the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) is shown next to the branches. In the ML results, each internal branch indicates, as a percentage, how often the corresponding cluster was found among the 1,000 intermediate trees. The scale bar represents the number of amino acid substitutions per site.</p

<i>In vitro</i> tolerance to hydrogen peroxide is significantly lower in <i>Trypanosoma rangeli</i> than <i>T. cruzi</i>.

<p>Epimatigote forms were cultured for 3 days in the presence of different concentrations of hydrogen peroxide, and the percentages of live parasites were determined using a model Z1 Coulter Counter. Mean values ± standard deviations from three independent experiments conducted in triplicate are indicated.</p

Comparative number of genes per multicopy gene family in <i>T. rangeli</i> and <i>T. cruzi</i>.

<p>Comparative number of genes per multicopy gene family in <i>T. rangeli</i> and <i>T. cruzi</i>.</p

Number of gene clusters shared by the <i>T. rangeli</i>, <i>T. cruzi</i>, <i>T. brucei</i> and <i>L. major</i> genomes.

<p>Analyzes were performed using the following genome versions and gene numbers retrieved from the TriTrypDB: <i>Leishmania major</i> Friedlin (V. 7.0/8,400 genes), <i>Trypanosoma brucei</i> TREU927 (V. 5.0/10,574 genes), <i>Trypanosoma cruzi</i> CL Brener Esmeraldo (V. 7.0/10,342 genes) and Non-Esmeraldo (V. 7.0/10,834 genes). A total of 7,613 <i>T. rangeli</i> genes were used. BBH analysis used a cut-off value of 1e-05, positive similarity type and similarity value of 40% following manual trimming for comparison with COG analysis in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003176#pntd.0003176-ElSayed1" target="_blank">[55]</a> generating the numbers in the rectangles.</p