ArtinM triggers TLR2-mediated cell activation.

<p>HEK293A cells were transfected with the ectodomain of human TLR2 (A) or human TLR4 (B), and the necessary co-receptors (CD14, CD36, and MD-2), as well as an NF-κB reporter construct and a <i>Renilla</i> luciferase control reporter plasmid. The cells were then stimulated with different concentrations of ArtinM (15.6, 156, and 780 nM) at 37°C for 18 h. In (A), MALP-2 (50 ng/mL) was used as the positive control. In (B), LPS (1 µg/mL) was used as the positive control; the addition of polymyxin B (100 µg/mL) to LPS served as another control. In both A and B, medium and an empty vector were used as the negative controls. The luciferase activity was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">materials and methods</a>. Statistical comparisons between unstimulated and stimulated cells were performed using a one-way analysis of variance followed by Bonferroni's multiple comparison test. * p<0.05.</p

TLR2 mediates the cytokine production induced by ArtinM.

<p>IL-12p40 and IL-10 levels in the cell culture supernatants of adherent spleen cells (A and B) or peritoneal macrophages (C and D) from WT (white bars) and TLR2-KO (black bars) mice were measured by ELISA. Adherent spleen cells were stimulated with ArtinM (156 nM) or P3C4 (1 µg/mL) for 24 h, while the peritoneal macrophages were incubated with ArtinM (39 nM) or P3C4 (1 µg/mL) for 48 h. Statistical comparisons between unstimulated and stimulated cells were performed using a one-way analysis of variance followed by Bonferroni's multiple comparison test. *p<0.05.</p

ArtinM induces the activation of TLR2/1- and TLR2/6-expressing cells.

<p>HEK293A cells were transfected with TLR2/1 (A and C) or TLR2/6 (B and D), co-receptors, an NF-κB reporter construct, and a <i>Renilla</i> luciferase reporter plasmid as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#pone-0098512-g002" target="_blank">Figure 2</a>. The transfected cells were stimulated with ArtinM (15.6, 156, and 780 nM) at 37°C for 4 h. Medium and cells transfected with an empty vector were used as the negative controls. The positive controls were P3C4 (1 nM) for TLR2/1 activation (A and C) and FSL1 (0.1 nM) for TLR2/6 activation (B and D). The luciferase activity (A and B) was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">materials and methods</a>. IL-8 levels in the culture supernatants (C and D) were measured by ELISA. Statistical comparisons between the cells incubated with medium and the cells stimulated with ArtinM were performed with a one-way analysis of variance followed by Bonferroni's multiple comparison test. * p<0.05.</p

Enhanced TLR2 relative expression by ArtinM-stimulated macrophages.

<p>Peritoneal macrophages from C57BL/6 mice were incubated with ArtinM (39 nM) for 5 h. Medium was used as a negative control and P3C4 (1 µg/mL) was used as a positive control. RNA from macrophages were isolated and used for qRT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">Materials and Methods</a>. The results are expressed as the relative expression of TLR2 after quantification using the ΔΔCt method and normalized to β-actin expression. Statistical comparisons between stimulated cells and unstimulated were performed with one-way analysis of variance followed by Bonferroni's multiple comparison test. ** p<0.01.</p

ArtinM binding to TLR2 depends on sugar recognition.

<p>(A and B) Peritoneal macrophages from C57BL/6 mice were incubated with biotinylated ArtinM after pre-incubation with anti-TLR2 antibody or non-specific IgG. ArtinM binding was detected with streptavidin-FITC and analyzed by flow cytometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">materials and methods</a>. Results are expressed as the percentage of cells positive for ArtinM binding (A) and MFI (median fluorescence intensity) (B). (C) The dependence of ArtinM-TLR2 binding on carbohydrate recognition used anti-TLR2 antibody coated onto 96-well microplates (5 µg/mL) to capture TLR2 from a cellular extract of peritoneal macrophages. Biotinylated ArtinM (40 µg/mL), previously incubated with the indicated concentrations of Manα1-3 [Manα1-6] Man or Galα(1,6)Galα(1,6)Gluα(1,2)Fru, was added to the wells. After washing, ArtinM binding was detected by neutravidin-AP, and signal was developed with <i>p</i>-nitrophenyl phosphate. Results are expressed in O.D as the mean ± SEM. Statistical comparisons between cells incubated or not with carbohydrates were performed with one-way analysis of variance followed by Bonferroni's multiple comparison test. *p<0.05.</p

Cell signaling molecules that possibly mediate the macrophage activation induced by ArtinM.

<p>Peritoneal macrophages were pretreated with the inhibitors PD98059 (p42/44 MAPK or ERK 1/2), SB202190 (p38 MAPK), SP600125 (c-Jun N-terminal kinase), and LY-294002 (PI3K), or with medium alone (absence of inhibitor) and then stimulated with ArtinM (39 nM). After 48 h, IL-12p40 and IL-10 levels in the culture supernatants were assessed by ELISA. (A) Cytokine production reported as pg/mL (mean ± SEM) and statistical comparison were performed using a one-way analysis of variance followed by Bonferroni's multiple comparison test. *p<0.05. (B) Inhibition of ArtinM induced cytokine production after pre-incubation with the indicated inhibitors. The results are presented as the percent inhibition, which represents the ratio between uninhibited cells and those pretreated with inhibitors.</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

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

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

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