Localization of TcTS during differentiation to cell-derived and metacyclic trypomastigotes.

<p>(A) Co-localization of αTcTS and antibodies against GFP in intermediate stages (epimastigote-like) obtained from tissue culture supernatants. (B) TcTS localizes to patches of the plasma membrane in fully differentiated trypomastigotes while GFP-TcRab11 remains in the CVC. (C) Co-localization of αTcTS with GFP-TcRab11 in epimastigotes during transformation into metacyclic stages. Scale bars (C–E) = 10 µm.</p

Cryo-immunoelectron microscopy localization of GFP-TcRab11 and TcTS in amastigotes.

<p>Amastigotes were isolated from HFF at different times p.i., as described under <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004224#s4" target="_blank">Materials and Methods</a>. GFP-TcRab11 and TcTS were detected with goat anti-GFP, and rabbit anti-TcTS antibodies, and donkey anti-goat 18 nm colloidal gold and donkey anti-rabbit 12 nm colloidal gold, respectively. (A–D) Amastigotes obtained after 96 h p.i. Co-localization of antibodies against GFP (<i>arrows</i>) and TcTS (<i>small dots</i>) is evident in the CV bladder (CV) and spongiome (Sp), while TcTS also localizes to the flagellar pocket (FP) and in patches of the plasma membrane. Note in (B) a collapsed bladder and intense labeling of the spongiome. (E–F) Amastigotes obtained 106 h p.i. GFP-TcRab11 localizes to the CV bladder while TcTS localizes to vesicles (V, <i>small arrows</i>) close to the plasma membrane and in patches in the plasma membrane. Scale bars = 500 nm. Note that the patchy appearance of the cytoplasm is due to the absence of glutaraldehyde in the fixative because it abolished labeling of TcTS.</p

Localization of anti-Gal antibodies.

<p>(A) GFP-TcRab11 co-localizes with the anti-Gal antibodies in the CVC of the intermediate forms as detected by polyclonal antibody against GFP (<i>green arrow</i>) and anti-α-galactosyl antibodies from patients with chronic Chagas disease (<i>red arrow</i>), respectively. (B) Anti-Gal antibodies strongly label the surface of fully differentiated tissue culture derived trypomastigotes while GFP-TcRab11 labels the CVC. (C) GFP-TcRab11DN mutants show a punctated cytosolic localization (<i>green</i>) while anti-Gal antibodies (<i>red</i>) localize to the plasma membrane in intermediate stages. Scale bars (A–C) = 10 µm.</p

Localization of surface proteins in GFP-TcRab11OE and GFP-TcRab11DN-expressing parasites.

<p>Antibodies against TcTSSA II (<i>red</i>) co-localize with antibodies against GFP (<i>green</i>) in intermediate forms (A) and amastigotes (B) but not in trypomastigotes expressing GFP-TcRab11, where they localize to the plasma membrane (C). Antibodies against TcTSSA II (D) still localize to the plasma membrane in GFP-TcRab11DN-expressing cells, while antibodies against the H⁺-ATPase (E) maintain their intracellular and plasma membrane localization in GFP-Rab11DN-expressing cells. In (D) and (E) GFP staining localizes to the cytosol. Scale bars = 10 µm.</p

GFP-TcRab11DN localizes to the cytoplasm of different life cycle stages.

<p>(A–C) GFP-TcRab11DN, mimicking the GDP–bound state of the protein has a cytosolic punctate localization in epimastigotes, (A), trypomastigotes (B), and intracelular amastigotes (C), as detected using antibodies against GFP. DNA was stained with DAPI. Bars = 10 µm.</p

Overexpression of GFP-TcRab11DN reduces the surface expression of TcTS.

<p>Tissue culture-derived wild type, and GFP-TcRab11DN-expressing trypomastigotes and intermediate forms were fixed, permeabilized and stained with antibodies against TcTS (A), or both TcTS and GFP (B and C). Labeling of TcTS (<i>red</i>) in fully differentiated trypomastigotes was predominantly in surface patches (A). Labeling of GFP-TcRab11DN (<i>green</i>) was predominantly cytosolic while labeling of TcTS was punctated but did not reach the cell surface in intermediate forms (B) or fully differentiated trypomastigotes (C). (D) The fluorescence intensity of TcTS in the cell surface of tissue culture-derived GFP-TcRab11DN-expressing trypomastigotes was measured in 200 cells in each experiment and expressed as percentage of control (wild-type trypomastigotes). Values are means ± SEM of 3 independent experiments. **<i>p</i><0.05. (E) FACS analysis of fixed GFP-TcRab11DN trypomastigotes reveals a decrease in the surface expression of TcTS as depicted by their lesser fluorescence intensity (DN) in comparison to that of wild type cells (WT). The negative control were unstained wild type trypomastigotes (US) showing background fluorescence. Wild type cells have two peaks of TcTS, suggesting the presence of intermediate stages in these asynchronously growing cultures. Data is representative of the profile analysis of 20,000 cells from 3 independent experiments.</p

Association of CVC proteins with lipid rafts and reduced infectivity of TcRab11DN trypomastigote.

<p>(A) Parasite extracts were loaded at the bottom (fraction 9) of a discontinuous Optiprep density gradient and subjected to ultracentrifugation. Fractions were collected and analyzed by anti-GFP and anti-FCaBP immunoblotting. Fractions 2 and 3 contain the lipid raft interface. The TcSNARE2.1GFP (SNARE), GFP-TcRab11 (Rab11), and FCaBP floated to the lipid raft interface. Lanes 6–9 represent the heavier fractions of the GFP and FCaBP derivatives and GFP alone was detected in these fractions. A whole cell lysate (WCL) is included in each panel as a control of loading. Total protein in lysates of GFP-TcSNARE2.1-, GFP-TcRab11- and GFP-expressing epimastigotes were 1.41, 1.3 and 1.39 mg/ml, respectively. (B) <i>T. cruzi</i> expressing GFP-TcRab11 or GFP were solubilized in Triton X-100 at 4°C or 37°C and separated into soluble (S) and insoluble (P) fractions and analyzed by western blotting with anti-GFP. Rab11-GFP partitions in the pellet fraction at 4°C, but is solubilized at 37°C, whereas GFP is only detected in the soluble fraction. (C–D) Effect of TcRab11 overexpression (OE) or mutation (DN) on trypomastigote invasion of host cells. In vitro infection assays were carried out as described under <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004224#s4" target="_blank">Materials and Methods</a>. (E–F) Partial rescue of the infectivity of DN trypomastigotes by their incubation in the presence of active TcTS and sialofetuin, whereas inactive <i>trans</i>-sialidase (iTS) does not rescue the infectivity of GFP-TcRab11DN mutants. Fetuin was present in all samples. Other conditions under <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004224#s4" target="_blank">Materials and Methods</a>. Values in C–F are mean ± SD (n = 3). *, ** and *** indicate that differences are statistically significant compared with respective controls, <i>p</i><0.05 (Ordinary one way ANOVA with Bonferroni post-test).</p

Regulatory volume changes of epimastigotes.

<p>(A–B) Cells were pre-incubated in isosmotic buffer for 3 min and then subjected to hyposmotic (final osmolarity = 150 mOsm) (A) or hyperosmotic (final osmolarity = 650 mOsm) (B) stress. Relative change in cell volume was followed by monitoring absorbance at 550 nm by light scattering. As compared to wild-type cells (WT), cells expressing GFP-TcRab11DN (DN) failed to fully recover their volume after hyposmotic stress and shrank less after hyperosmotic stress, while cells overexpressing GFP-TcRab11 (OE) recovered their volume faster after hyposmotic stress and shrank more after hyperosmotic stress. Values are means ± SD of three different experiments. Asterisks indicate statistically significant differences, <i>p</i><0.05, (Bonferroni's multiple comparison “a posteriori” test of one-way ANOVA) at all time points after induction of osmotic stress. (C–D) Epimastigotes were immobilized on glass slides with poly-lysine and diluted with deionized water to a final osmolarity of 150 mOsm (C) or bathed with hyperosmotic (650 mOsm) buffer (D). Video microscopy data were collected and selected frames are shown. Times indicated in each frame represent 1 second apart after induction of stress. Arrowheads show different dilated compartments that transform into larger bladders at a later time. Results are representative of those obtained from at least three independent experiments. Bars = 10 µm.</p

Co-localization of GFP-TcRab11 and TcTS during amastigote differentiation in human foreskin fibroblasts.

<p>(A) Expression of TcTS becomes apparent at 48 h p.i., when antibodies against TcTS (<i>red</i>) co-localize with GFP-TcRab11, as detected with antibodies against GFP (<i>green</i>). Co-localization progresses to close to 80% of cells by 96 h, and after 106 h co-localization starts to decrease and surface labeling of TcTS is more evident. Scale bars = 10 µm. Insets shows co-localization at high magnification (double). (B) Percentage of amastigotes showing co-localization of TcTS and GFP-Rab11 with time. Two hundred amastigotes were counted in each experiment and results are expressed as means ± SEM (n = 3).</p

Fluorescence microscopy analysis of TcRab11 in different stages of <i>T. cruzi</i>.

<p>(A–C) GFP fusion protein of TcRab11 was detected in the contractile vacuole bladder of epimastigotes (Epi, A), trypomastigotes (Trypo, B), and intracellular amastigotes (Ama, C) using antibodies against GFP. Upper panels show differential interference contrast microscopy (DIC) images merged with DAPI staining of DNA (in <i>blue</i>) and GFP-TcRab11 (in <i>green</i>). Lower panels show fluorescence images. (D) GFP-TcRab11 (<i>green</i>) co-localizes with antibodies against <i>T. cruzi</i> aquaporin 1 (α-AQP, <i>red</i>), a marker for the contractile vacuole, under hyposmotic conditions. (E) Antibodies against TbRab11 (α-Rab11, <i>red</i>) co-localize with GFP-TcRab11 (<i>green</i>). (F) Antibodies against TbRab11 (<i>red</i>) localize to a compartment that resembles the contractile vacuole in (E). DAPI staining is in <i>blue</i>. Arrowheads in D–F show co-localization between antibodies against TcAQP1 and GFP (D), TbRab11 antibody and GFP (E) and labeling with antibodies against TbRab11 (F), respectively. Bars in A–F = 10 µm. (G) Western blot analyses with TbRab11 antibody of lysates of epimastigotes overexpressing GFP-TcRab11 (E-OE), or wild-type epimastigotes (E), trypomastigotes (T) and amastigotes (A) showing bands (<i>arrows</i>) corresponding to the endogenous TcRab11 (24 kDa) and to GFP-TcRab11 (50 kDa). The blots were sequentially probed with αTbRab11 and anti-tubulin antibodies, used as loading control.</p