Inhibition of uptake of Tf by TL in epimastigote forms of <i>T</i>. <i>cruzi</i>.

<p>Trypanosomes preincubated with biotinylated TL in the presence of 20 μM FMK-024 (25 μg/ml) and in the absence (A, left panel) or presence of competing chitin hydrolysate (A, right panel), were then incubated with Tf Alexa-594 for 5 or 30 min at 27°C. Cells were then fixed and treated for fluorescence microscopy. Similar incubations wherein TL was substituted by GSLII (B) were performed to assess the specificity of the TL labeling. Furthermore, live parasites preincubated with DyLight 488-TL and 20 μM protease inhibitor (FMK-024) for 5 min and then incubated for 60 min in the presence of Alexa Fluor 594 conjugated Tf showed a lectin labeling in the cytostome/cytopharynx (arrowhead), while no Tf labeling (red signal) was observed in these conditions (C, upper panel). In presence of a molar excess of chitin hydrolysate an intense labeling of Tf exclusively concentrate into reservosomes (arrow) while no green signal corresponding to TL was observed anymore (C, lower panel). Inhibition of trypanosomes Tf uptake with TL was furthermore quantified by flow cytometry (D). The TL signal was dropping from 913 to 273 of mfi in the absence or presence of chitin hydrolysate, respectively (D, left histogram). Conversely, Tf signal was increasing from 597 to 3793 of mfi in the absence or presence of chitin hydrolysate, respectively (D, right histogram).</p

Localization of TL and GSLII binding sites in <i>T</i>. <i>cruzi</i>.

<p>Endocytosis kinetics of fluorescent Alexa Fluor 594 conjugated Tf was performed in order to follow <i>T</i>. <i>cruzi</i> endocytic pathway from the flagellar pocket/cytostome to the reservosomes. Parasites were fixed at different time points and probed with biotinylated TL (A), biotinylated ricin (B) or Alexa 488 conjugated GSLII (C). The addition of chitin hydrolysate clearly shows inhibition of TL and GSLII staining. (A) Co-localization of biotinylated-TL (green) and Tf (red). (B) Co-localization of biotinylated-ricin (green) and Tf (red). Addition of 200 mM galactose abolished the ricin staining. (C) Co-localization of Alexa 488 conjugated GSLII (green) and Tf (red). (D) Co-localization of Alexa 488 conjugated GSLII (green) and TcJ6 (red). (E) Co-localization of Alexa 488 conjugated GSLII (green) and anti-BiP (red). (F) GSLII blotting of cell extracts enriched by GSLII chromatography. GSLII blots of <i>T</i>. <i>cruzi</i> CHAPS- and Triton-soluble (CHAPS+Triton X-114) cell lysate fractions were enriched by GSLII chromatography and then treated (+) or not (-) with PNGase F. Blots were probed with biotinylated-GSLII. The GSLII blot indicates the presence of <i>N-</i>acetylglucosamine modification in both soluble and membrane fractions. Treatment of the fractions with PNGase F decreased the reactivity of GSLII confirming <i>N</i>-glycoprotein type modification.</p

TL blotting on Tf and glycophorin.

<p>Different amounts of proteins (up to 5 μg) were loaded. The lectin blot analysis indicates that TL does not recognize Tf but reacts with the sialoglycoprotein glycophorin.</p

Subcellular localization of TL-binding sites in <i>T cruzi</i> by transmission electron microscopy (TEM).

<p>Parasites were incubated for 5 min in PSG medium in presence (F) or absence (A-E) of BSA-gold as endocytic tracer (10 nm). Cells were fixed and processed for ultrathin frozen sectioning (Tokayasu method, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163302#pone.0163302.ref042" target="_blank">42</a>]). Cryosections were sequentially probed with biotinylated TL, rabbit anti-biotin antibodies, protein A-gold (5 nm) and finally mounted in methyl cellulose-uranyl acetate films. Representative images are shown. K: kinetoplast, M: mitochondrion, R: reservosome, N: nucleus, FP: flagellar pocket, F: flagellum, G: golgi, Cy: cytostome. Arrows and arrowhead, point to gold particles that mark the presence of TL binding sites and BSA-gold particles, respectively. Asterisk show TL-binding matrix near the opening of the cytostome. Bars = 200 nm.</p

Tomato lectin blotting and fluorescence microscopy analyses.

<p>(A) TL blotting on total protein extracts of three developmental forms of <i>T</i>. <i>cruzi</i>. Similar amounts of proteins (around 50 μg) from three <i>T</i>. <i>cruzi</i> stages were loaded (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163302#sec002" target="_blank">Material and Methods</a>). The same membrane blot was revealed with ponceau red as loading control. The lectin blot analyses indicate that TL-binding glycoproteins are significantly present in epimastigote forms. E: epimastigote, T: trypomastigote, A: amastigote. (B) Fluorescence microscopy of three developmental forms of <i>T</i>. <i>cruzi</i> probed with biotinylated tomato lectin. Arrows indicate the position of nucleus (N) and kinetoplast (K) stained in blue by DAPI. E: epimastigote; M: metacyclic, T: trypomastigote, A: amastigote. Bars scales represent 2μm. (C) TL blotting on total extract of <i>T</i>. <i>brucei</i> bloodstream forms (10⁶ cells) vs <i>T</i>. <i>cruzi</i> epimastigote forms (5 x10⁶ cells). (D) TL blots of <i>T</i>. <i>cruzi</i> CHAPS- and Triton-soluble (CHAPS+Triton X-114) cell lysate fractions. Fractions were enriched by TL chromatography and then treated (+) or not (-) with PNGase F and T represents the total cell lysate. Blots were either probed with TL (upper panel) or anti-TcrCATL (lower panel). The TL blot indicates the presence of <i>N</i>-glycan modification in both soluble and membrane fractions. Treatment of the fractions with PNGase F abolished the reactivity of TL confirming <i>N</i>-glycoprotein type modification. The lower panel shows the presence of TcrCATL, a poly-LacNAc-modified glycoprotein, in both fractions. PNGase F treatment results in the appearance of a lower band corresponding to the loss of the N-glycosylation. Apparent molecular weights are indicated in kDa on the left.</p

Uptake of Dextran in the presence of TL in epimastigote forms of <i>T</i>. <i>cruzi</i>.

<p>Flow cytometry profiles of uptake of Dextran Alexa-647 by trypanosomes in the presence or absence of biotinylated TL. Trypanosomes preincubated (A) or not (B) with biotinylated TL in the presence of 20 μM FMK-024 (25 μg/ml) and in absence (A, left histogram) or presence of competing chitin hydrolysate (A, right histogram), were then incubated with Dextran Alexa-647 for 30 min at 27°C.</p

Comparisons of the identified protein families in three independent proteomic studies [53, 54].

<p>The percentages of different protein families identified in three different studies are compared. The stacked bar chart represents the cumulative distribution of the different fractions shown for each protein family. Functional classification of <i>T</i>. <i>cruzi</i> proteins was performed according to Atwood <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163302#pone.0163302.ref053" target="_blank">53</a>]. Proteins grouped under others and hypothetical were discarded from this comparison.</p

Enrichment of glycoproteins from <i>T</i>. <i>cruzi</i> epimastigote using TL and GSLII affinity chromatography.

<p><i>T</i>.<i>cruzi</i> epimastigote proteins were fractionated by detergent extraction into CHAPS and CHAPS + Triton X-114 fractions. These fractions were loaded either onto agarose-coupled TL or GSLII beads columns and left overnight at 4°C on a rotating device. Whole cell extracts, columns flow-through and eluates were then separated on NuPAGE gels (4–12%) and proteins were revealed by SafeStain blue staining.</p

Overview of different antibody fragments and effect of reconstituted Nb_An05-Fc on <i>in vitro</i> mediated trypanolysis.

<p>(A) Schematic of the different antibody fragments. (1) Nbs are derived from the HCAbs after cloning and selection by phage-display. (2) The monoclonal Nb is reconstituted into an HCAb molecule. (3) and (4) Fab′₂ or Nb′₂ and Fab or Nb fragments are obtained by proteolytic digestion of conventional immunoglobulin or camelid HCAb using papain or pepsin, respectively. B) Percent lysis of monomorphic AnTat1.1 parasites over three hours at 37°C and incubated with Nb_An05 (▴), Nb_An05-Fc (□), Nb_An05-Fc and complement (∇) or without any Nb (▪). C) Percent lysis of monomorphic AnTat1.1 parasites, after incubation with recombinant Nb_An05 (▴), Nb_An05-Fc (□) or with Nb′₂ obtained after pepsin digestion of Nb_An05-Fc (•) and Nb obtained after papain-digestion of Nb_An05-Fc (∘). The percent lysis was calculated relative to the initial parasite number. The data given are typical results from three independent experiments performed in triplicate (±SD). For each experiment 2×10⁵ parasites were incubated with 0.067 nmole Nb, Nb-Fc or Nb′₂ constructs (VSG/antibody ratio of 1∶20).</p

Identification of trypanolytic Nbs. 2×10⁵ monomorphic T. brucei parasites were kept in HMI-9 buffer at 37°C and counted hourly.

<p>A) Percent of lysed parasites at different times after adding 1 µg Nb_An05 (▴), Nb_An06 (•), Nb_An46 (▾), Nb_An33 (○) (VSG∶Nb ratio is 1/20) and a control with no Nb (▪). B) Percent lysis of antigenically distinct trypanosomes (AnTat1.1, MiTat1.1, MiTat1.2, MiTat1.5 and MiTat1.6) after two hours incubation with 1 µg Nb_An05 (light grey bars), Nb_An06 (dashed grey bars), Nb_An46 (dark grey bars), Nb_An33 (white bars) (VSG∶Nb molar ratio of 1∶20) and a control with no Nb (black bars). C) Inhibition of Nb-mediated trypanolysis using three-fold molar excess of AnTat1.1 VSG after two hours incubation with 1 µg Nb_An05 (light grey bars), Nb_An06 (dashed grey bars), Nb_An46 (dark grey bars), Nb_An33 (white bars) (VSG/Nb molar ratio of 1/20) and a control with no Nb (black bars). D) Percent lysed parasites after incubation with Nb_An05 at 1 µg (▴), 0.5 µg (▾), 0.1 µg (□), 0.05 µg (•) and control with no Nb (▪). These data are typical results from three independent experiments performed in triplicate (±SD). E) <i>In vivo</i> effect of Nbs on parasitemia development and survival of C57Bl/6 mice infected with virulent monomorphic AnTat1.1A parasites, injected i.v. with Nb_An46 (▾), Nb_An33 or without Nb treatment (○). Antibody injections were given at daily interval, starting from day one until day four post-infection (arrows). (†: indicates all mice died).</p