GST-δ-AmastinH specifically binds to HeLa cells in a dose-dependent saturable manner.

<p><b>A. SDS-PAGE showing the purity of recombinant GST-δ-amastinH:</b> 1; Molecular weigh markes, in kiloDaltons; 2: GST alone; 3: purified recombinant GST-δ-AmastinH; 4 total extract of induced <i>E. coli</i>. <b>B: Recombinant GST-δ-AmastinH binds to HeLa cells</b>. Increasing concentrations of GST-AmastinH or GST (negative control) were added to wells in ELISA plates containing adhered and fixed HeLa cells. After washing, cells were sequentially incubated with anti-GST antibodies and anti-rabbit IgG conjugated to peroxidase. The bound enzyme was revealed by <i>o-</i>phenylenediamine as a substrate. Representative results of two independent experiments are shown. *p<0.05.</p

Delta-amastin is more abundant in less infective <i>T. cruzi</i> extracellular amastigotes. A.

<p><b>mRNAs in EAs of the G strain are less abundant when compared to EAs of the CL-Brener clone </b><b><i>T. cruzi</i></b><b>.</b> Transcript levels were determined by quantitative real-time PCR using SYBR® Green I chemistry. qRT-PCR was performed on RNA samples from EAs of G and CL strains. The comparative mRNA levels were determined after normalization with GAPDH amplicons. Standard deviations are derived from three replicates. *p<0.05 <b>B. mRNA corresponding to amastin is preferentially expressed in amastigotes from CL Brener clone.</b> Northern blot analyses of total RNA (10 µg) from <i>T. cruzi</i> epimastigotes (E), trypomastigotes (T) and amastigotes (A) from CL-Brener clone or the G <i>T. cruzi</i> strain was submitted to electrophoresis and blotted on nylon membranes by standard procedures. Each blot was hybridized with amastin probe previously labeled with [α-^32P]-dCTP. To determine equal loading of RNA, the 1.2% agarose/MOPS/formaldehyde gel was stained with ethidium bromide (bottom panel).</p

Delta-amastin interferes with EA-HeLa cell interaction. A: GST-AmastinH inhibited host cell invasion by <i>T. cruzi</i> EAs.

<p>Prior to invasion, HeLa cells were treated for 1 h with 5 µg/ml of GST (white column) or GST-δ-AmastinH (dark column). The parasites were then added to HeLa cells and the invasion proceeded for 2 h. The number of internalized parasites was counted in a total of at least 300 cells. <b>B: EAs (G strain) overexpressing amastin showed lower infectivity toward HeLa cells.</b> EAs of the G strain expressing high levels of δ-amastin (black column) showed a significant decrease in cell invasion when compared to wild type parasites (clear column) or parasites expressing only GFP (gray column). The invasion proceeded for 2 h. The values are shown as means ± standard deviations of two independent experiments performed in duplicate. *p<0.05.</p

Schematic proposed model for the role of δ-amastin in <i>T. cruzi</i> virulence.

<p>The model indicates two distinct parasites, EAs expressing high levels of amastin was represented by dotted line membrane of amastigotes (left cell side) whereas the low amastin expression was represented by spaced dots in the amastigotes membrane (right cell side), during EA invasion and differentiation processes of <i>T. cruzi</i> virulence in a hypothetical host cell. <b>1.</b> EAs expressing more amastin (left) show a lower infectivity rate when compared with parasites expressing lower levels of the protein. <b>2.</b> High levels of amastin accelerate the transformation of amastigotes into TCTs (left).</p

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

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

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

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

Representation of the telomeric and subtelomeric regions of <i>Trypanosoma rangeli</i>, <i>T. cruzi</i> and <i>T. brucei</i>.

<p>The two types of telomeres identified in <i>T. rangeli</i> and two others representing the heterogeneity of <i>T. cruzi</i> chromosome ends are shown. The size of the subtelomeric region, which extends between the telomeric hexamer repeats and the first internal core genes of the trypanosomes, is indicated below each map. Boxes indicate genes and/or gene arrays. The maps are not to scale. The <i>T. brucei</i> and <i>T. cruzi</i> maps were adapted from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003176#pntd.0003176-ElSayed1" target="_blank">[55]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003176#pntd.0003176-MoraesBarros1" target="_blank">[98]</a>.</p