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

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

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

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

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

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

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

Bootstrapped phylogram of <i>Rhodnius prolixus</i> midgut lectins aligned with their best matches to the NR database.

<p>Bootstrap values above 50% are shown on the branches. The bottom line indicates 10% amino acid sequence divergence between the proteins. <i>R. prolixus</i> sequences are shown by the notation RP followed by a unique number. The remaining sequences were obtained from GenBank and are annotated with the first three letters of the genus name, followed by the first three letters of the species name, followed by their GenBank GI number. One thousand replicates were done for the bootstrap test using the neighbor joining test.</p

Bootstrapped phylogram of <i>Rhodnius prolixus</i> and other cysteinyl proteinases.

<p>Bootstrap values above 50% are shown on the branches. The bottom line indicates 10% amino acid sequence divergence between the proteins. <i>R. prolixus</i> sequences are shown by the notation RP followed by a unique number and have a red circle preceding their names. The remaining sequences, obtained from GenBank, are annotated with the first three letters of the genus name, followed by the first three letters of the species name, followed by their GenBank GI number. One thousand replicates were done for the bootstrap test using the neighbor joining test.</p