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).