DDD compounds are trypanocidal against purified intracellular amastigotes.

<p>Total number of parasites in each well was counted to evaluate the cytotoxicity of the compounds <b>1</b>, <b>5</b>, and <b>8</b> against purified ICA (<b>A</b>) and Epi (<b>B</b>). C, controls: Untreated, DMSO, and H₂O₂.</p

Alignment of <i>T</i>. <i>cruzi</i> NMT with NMTs from <i>T</i>. <i>brucei</i>, <i>L</i>. <i>major</i> and human.

<p>The deduced open reading frame of <i>Tc</i>NMT (AI069625) was aligned with <i>Tb</i>NMT (TRYP10.0.001826–6), <i>Lm</i>NMT (AF3059561), and human NMT (<i>Hs</i>NMT) (<i>HUMAN</i>, P30419) using the ClustalW2 multiple sequence alignment program (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>). Strictly conserved residues are shown in red. The insertions in protozoan NMTs (<i>Tc</i>NMT, <i>Tb</i>NMT, and <i>Lm</i>NMT) are underlined. Red boxes indicate key residues involved in myristoyl-CoA binding; black boxes indicate residues involved in peptide binding identified in yeast species. Arrows identify the pocket floor residues in <i>C</i>. <i>albicans</i>.</p

Anti-TcNMT shows no cross-reactivity with human cells.

<p>Immunofluorescence microscopy of non-infected and infected cells 72- and 96-h post-infection stained with anti-TcNMT (red), co-stained with DAPI (blue) to visualize host cell and parasite DNA. Scale bar, 10 μm.</p

DDD compounds inhibit intracellular proliferation of <i>T</i>. <i>cruzi</i>.

<p>(<b>A</b>) Representative images of cells treated with the vehicle control (DMSO), untreated, treated with 800 μM benznidazole (BZ) or 10 μM compound <b>8</b>, stained with Draq5 (left panel), and analyzed by HCI. Artificial images created after segmentation on the fluorescence bioimager (right panel). Host cells are shown in blue, extracellular parasites in red and intracellular parasites in pink. (<b>B</b>) The multiparametric data obtained on a cell-by-cell basis by HCI was analyzed to determine several parameters associated to infection of host cells by <i>T</i>. <i>cruzi</i> including the percentage of cells infected with at least five parasites (percentage of cells in which the parasite proliferated), treated or not with DDD compounds <b>1–8</b>. C, controls: 40, 400, and 800 μM BZ, DMSO, and Untreated.</p

EC₅₀ values and selectivity index (S.I.) of DDD compounds in noninfected and <i>T</i>. <i>cruzi</i>-infected U2OS cells, and purified intracellular amastigotes.

<p>(<b>a</b> and <b>b</b>) Non-infected and infected U2OS cells, respectively, were incubated for 48 h at 37°C with DDD compounds 1–8. (<b>c</b>) Purified ICAs were incubated for 24 h at 37°C with DDD compounds 1–8. (<b>d</b>) Not determined.</p

TcNMT is overexpressed in epimastigotes treated with DDD compounds.

<p>Epi forms were treated for 12 h with or without 10 μM of compound <b>1</b>, <b>5</b>, or <b>8</b>. Levels of NMT expression were confirmed by western blotting using anti-TcNMT. BiP (binding protein) was used as a loading control.</p

DDD compounds specifically inhibit TcNMT.

<p>In-gel western blots of lysates of (<b>A</b>) Epi, (<b>B</b>) ICA, and (<b>C</b>) TCT treated or not with 10 μM of the DDD inhibitor <b>1</b>, <b>5</b>, or <b>8</b>, labeled with myristic acid, azide followed by “click” reaction with biotin alkyne. IRDye 800CW streptavidin was used to detect myristoylated proteins. Arrows indicate protein bands whose labeling was more affected by any of the DDD compounds tested.</p

TcNMT is constitutively expressed in all <i>T</i>. <i>cruzi</i> stages and it is associated to the endoplasmic reticulum.

<p>Immunofluorescence microscopy of Epi, ICA, and TCT. Cells are shown as viewed under phase contrast, visualized for fluorescence with anti-TcNMT (red) and anti-BiP (green), co-stained with DAPI to reveal positions of the nucleus and kinetoplast (blue). Scale bar, 2 μm.</p

α-Gal-NGPs as biomarkers of active CL in patients with <i>L</i>. <i>major</i> infection.

<p>Assessment by chemiluminescent ELISA of α-Gal-containing NGPs and controls (Cysteine-BSA and Galβ-BSA) were immobilized on a microplate and reacted with pools of sera (at 1:100 dilution) from patients with active or cured CL, or heterologous skin (non-CL) infections (n = 5 per group, randomly selected) from an endemic region (Saudi Arabia), as described [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006039#pntd.0006039.ref026" target="_blank">26</a>]. RLU, Relative luminescence units. Error bars indicate S.E.M. of triplicate determinations. The fold difference in reactivity between active CL vs. cured CL, and active CL vs. heterologous infection are indicated. Two-way ANOVA with Tukey’s multiple comparisons: ns, non-significant; (*), <i>P</i><0.05; (**), <i>P</i><0.01; (***), <i>P</i><0.001; (****), <i>P</i><0.0001.</p

Anti-NGP5B antibody response is specific against terminal α-Gal residues.

<p>(A-B) Chemiluminescent ELISA reactivity of mouse serum pools obtained at Boost 3 (n = 6) and endpoint (n = 3) from α1,3GalT-KO mice vaccinated with NGP5B, NGP5B+CpG, CpG, or PBS. Immunized groups are indicated in the legend and antigens on the microplate are shown in the Y-axis. (C) Chemiluminescent ELISA reactivity of mouse serum obtained at Boost 2. NGP5B (125 ng/well) was treated or not with green-coffee bean α-galactosidase. One-way ANOVA (compared with untreated sample): (**), <i>P<</i>0.01; (***), <i>P</i><0.001; (****), <i>P</i><0.0001. (A-C) Error bars indicate S.E.M. of triplicate determinations.</p