Over-expression of TSSA variants improves trypomastigote-to-amastigote transformation kinetics in <i>T</i>. <i>cruzi</i>.

<p>Purified, CL Brener (panel A) or Sylvio X-10 (panel B) trypomastigotes (5 x 10⁶) of the indicated line were incubated in MEM at pH7, without serum. Samples were taken at different time-points, fixed, and total number of trypomastigotes and amastigotes were counted directly under the light microscope. For each sample, at least 300 parasites were counted and trypomastigotes were expressed as % of total parasites. The results are the average of 3 independent experiments. Asterisks denote significant differences (<i>P</i> < 0.05) to wild type parasites using <i>t</i>-Student test.</p

TSSA is not a sialic acid acceptor and does not behave as a typical <i>T</i>. <i>cruzi</i> mucin.

<p><b>A)</b> Representative images of CL Brener trypomastigotes incubated with Neu5Az<i>α</i>2-3Lac<i>β</i>OMe and processed for immunofluorescence using both mouse mAb anti-FLAG (green) and rabbit TSSA-CL antiserum (red). White arrowheads point to few co-localization spots. Cyan arrowheads indicate TSSA-CL-reactive particles probably secreted to the medium. Bars = 10 μm. <b>B</b> and <b>C)</b> Intact CL Brener trypomastigotes were labeled with Neu5Az<i>α</i>2-3Lac<i>β</i>OMe in the absence (panel B) or presence (panel C) of recombinant <i>trans</i>-Sialidase. The labeled material was fractionated on to anti-FLAG–Sepharose and flow-through (FT) and bound (B) fractions were probed by Western blot. Arrowheads point to a ~25 kDa band, which likely represents the light chain of the anti-FLAG mAb that leaked out of the anti-FLAG-Sepharose. A faint ~10 kDa band of unknown identity is denoted with an asterisk in panel C. <b>D)</b> Butan-1-ol extraction analysis of CL Brener trypomastigotes. Fractions were obtained according to Materials and Methods, and processed for Western blot. <b>E)</b> Conditioned medium from CL Brener trypomastigotes was fractionated using Exoquick kit. Fractions corresponding to parasite pellet (P), soluble molecules (S) and micro-vesicles (MVs) were analyzed by Western blot. Relative molecular mass markers (in kDa) are indicated.</p

TSSA-CL, but not TSSA-Sy, is involved in trypomastigote internalization.

<p><b>A</b> and <b>D)</b> Infection (panel A) or adhesion (panel D) rates of transgenic or wild type trypomastigote lines towards Vero cell monolayers were measured in presence of 100 μg/mL of the indicated peptide or PBS as control. Vero cells grown on 24-well culture plates were added with 1 or 2 x 10⁵ Sylvio X-10 or CL Brener trypomastigote forms (with up to 5% of contaminant amastigote forms), respectively. After 3 h of incubation at 4°C (panel D) or at 37°C (panel A), cells were washed with PBS to remove non-attached parasites, fixed with PBS-PFA immediately (panel D) or after additional 36 h incubation at 37°C (panel A) and processed for Indirect Immunofluorescence assay. In all experiments, the number of infected cells was determined in a total of at least 1,000 DAPI-stained cells. Data are expressed as mean values ± SD of 3 independent experiments, each one performed in duplicate. B) Schematic representation of TSSA-Sy (above) and TSSA-CL (below) sequences expressed as GST-fusion molecules or synthetic peptides, and the residues spanned by each construct (numbers indicate amino acid positions in each sequence relative to the initial Met). Variations between TSSAs and sequences derived thereof are indicated in red (for TSSA-Sy) and green (for TSSA-CL). <b>C)</b> GST-fusion proteins spanning TSSA-CL deletion variants were added to HeLa cells and binding was assessed by means of a monoclonal anti-GST antibody followed by a colorimetric method. In every assay, recombinant GST and GST-TSSA-Sy^24-61 proteins were used as negative control whereas GST-TSSA-CL^24-62 was used as positive control. Reactivity for each protein was normalized to GST-TSSA-CL^24-62 and mean ± SD was calculated from 3 independent assays. Significant differences between the indicated population and GST means (<i>P</i> < 0.05 ANOVA followed by Dunnet’s correction) were denoted with an asterisk. <b>E)</b> Infection rates of wild type trypomastigote lines (indicated below) towards Vero cell monolayers were measured in presence of different transgenic or wild type trypomastigote lines in the upper chamber of the trans-well (indicated above). <b>F)</b> Infection rates of wild type CL Brener trypomastigotes in the presence of conditioned medium (CM) of the indicated trypomastigote line were measured as described above. When indicated, CM was FLAG- or Hemmaglutinin-depleted by affinity chromatography before the flow-through fraction being incubated with wild type CL Brener trypomastigotes. In panels A, C, D and E asterisks denote significant differences (<i>P</i> < 0.05) to the corresponding control values using <i>t</i>-Student test.</p

Western blots of <i>TcSMUG L</i> products from <i>T. cruzi</i>.

<p>A) Extracts of epimastigotes from different parasite stocks (Ad, Adriana; CL, CL Brener; Dm, Dm28c) were probed with either anti-TcSMUG L antibodies or anti-glutamate dehydrogenase (GDH) antiserum. B) ConA-fractionated extracts of Dm28c epimastigotes were probed with anti-TcSMUG L antiserum. ft, flow-through. C) Butan-1-ol extraction analysis of Dm28c delipidated epimastigotes. Fractions, named according to <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002552#pntd.0002552-Almeida1" target="_blank">[19]</a>, were probed with affinity-purified anti-TcSMUG L antibodies. Molecular mass markers (in kDa) are indicated at right. *Denotes aggregates.</p

Effect of surface mucins on <i>ex vivo T. cruzi</i> attachment to the midgut epithelium of <i>Rhodnius prolixus</i>.

<p>Midguts obtained from male fifth-instar nymphs 10 days after the bloodmeal were previously incubated for 30 min in PBS supplemented with the indicated mucin peptides and added with BHI interaction medium containing flagellates (2.5×10⁷/ml). Pre-incubation with mucin peptides was omitted in control (non-treated) group. Adhered epimastigotes were counted per 100 epithelial cells in 10 different fields of each midgut preparation. (A) Pre-incubation in 1 µg/ml of TcSMUG S, TSSA or TcSMUG L. (B) Pre-incubation in 0.01, 0.1 or 1.0 µg/ml of TcSMUG L. (C) Pre-incubation in 0.01, 0.1 or 1.0 µg/ml of TSSA. Each group represents mean ± S.D. of parasites attached in 10 midguts. Asterisk represents experimental groups with statistical significance compared to the control. <i>Trypanosoma cruzi</i> small mucin S (TcSMUG S), <i>Trypanosoma cruzi</i> small mucin L (TcSMUG L) and trypomastigote small surface antigen (TSSA).</p

Effect of surface mucins on <i>T. cruzi in vivo</i> development in the digestive tract of <i>Rhodnius prolixus</i>.

<p>Insects were fed on citrated, complement-inactivated human blood containing 2×10⁵ flagellates/ml. Each mucin peptide was added to the bloodmeal at a concentration of 30 µg/ml and insects dissected as days 7, 14 or 21 post feeding. Each point represents mean±S.D of flagellates/ml in the whole gut of 10 insects. Asterisk represents experimental groups with statistical significance compared to the control.</p

Photomicrographs of posterior midgut epithelial cells of fifth-instar <i>R. prolixus</i> incubated with biotin-labeled peptides.

<p>(A) Light microscopy showing single-globe columnar epithelial cells(white star) and PMM (white arrow). (B) Fluorescence microscopy showing that no demarcation was observed after incubation with avidin-FITC-labeled conjugate alone. Light and fluorescence microscopy, respectively, of samples incubated with biotin-labeled TcSMUG L (C and D), biotin-labeled TcSMUG S (E and F), and biotin-labeled TSSA (G and H). Fluorescence of the surface and nucleolus of the midgut cells is indicated by white and black arrows (respectively). 400×.</p