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

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

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

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

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

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

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

Monitoring of the Parasite Load in the Digestive Tract of <i>Rhodnius prolixus</i> by Combined qPCR Analysis and Imaging Techniques Provides New Insights into the Trypanosome Life Cycle

<div><p>Background</p><p>Here we report the monitoring of the digestive tract colonization of <i>Rhodnius prolixus</i> by <i>Trypanosoma cruzi</i> using an accurate determination of the parasite load by qPCR coupled with fluorescence and bioluminescence imaging (BLI). These complementary methods revealed critical steps necessary for the parasite population to colonize the insect gut and establish vector infection.</p><p>Methodology/Principal Findings</p><p>qPCR analysis of the parasite load in the insect gut showed several limitations due mainly to the presence of digestive-derived products that are thought to degrade DNA and inhibit further the PCR reaction. We developed a real-time PCR strategy targeting the <i>T</i>. <i>cruzi</i> repetitive satellite DNA sequence using as internal standard for normalization, an exogenous heterologous DNA spiked into insect samples extract, to precisely quantify the parasite load in each segment of the insect gut (anterior midgut, AM, posterior midgut, PM, and hindgut, H). Using combined fluorescence microscopy and BLI imaging as well as qPCR analysis, we showed that during their journey through the insect digestive tract, most of the parasites are lysed in the AM during the first 24 hours independently of the gut microbiota. During this short period, live parasites move through the PM to establish the onset of infection. At days 3–4 post-infection (p.i.), the parasite population begins to colonize the H to reach a climax at day 7 p.i., which is maintained during the next two weeks. Remarkably, the fluctuation of the parasite number in H remains relatively stable over the two weeks after refeeding, while the populations residing in the AM and PM increases slightly and probably constitutes the reservoirs of dividing epimastigotes.</p><p>Conclusions/Significance</p><p>These data show that a tuned dynamic control of the population operates in the insect gut to maintain an equilibrium between non-dividing infective trypomastigote forms and dividing epimastigote forms of the parasite, which is crucial for vector competence.</p></div

Comparison of time-course development of epimastigotes and trypomastigotes expressing luciferase in the digestive tract of <i>R</i>. <i>prolixus</i> during the first 24 h p.i., in the presence or absence of <i>R</i>. <i>rhodnii</i>.

<p>(A) Representative values of the luminescence emission for epimastigotes and trypomastigotes at 6 h and 24 h. Epi 6 h vs. Epi 24 h, P<0.01; Trypo 6 h vs. Trypo 24 h, P<0.0001. ANOVA followed by Tukey's multiple comparisons test. (B) Quantification of the BLI signal emitted by adult insects infected with various amounts of epimastigotes (Epi) or trypomastigotes (Trypo). (C) Quantification of the BLI signal emitted by gut microbiota-free first instar nymphs fed with epimastigote (Epi) or trypomastigote (Trypo) forms (10⁷ cells/ml), in the presence or absence of <i>R</i>. <i>rhodnii</i>. In each stage ± bacteria, 6 h vs. 24 h, P<0.001. ANOVA followed by Tukey's multiple comparisons test.</p