Phosphorylation status of α-tubulin upon adhesion of trypomastigotes to laminin, fibronectin or BSA.

<p>(<b>A</b>) Representative immunoblotting of immunoprecipitated α-tubulin phosphorylated (p-α-tubulin) during the times indicated. (<b>B</b>) Quantitation of 3 independent experiments as described in (<b>A</b>); asterisk represents a comparison between the 5 min and 120 min points by the Student's t-test with p<0.001, indicating a progressive α-tubulin dephosphorylation. (<b>C</b>) Representative immunoblotting of phosphorylated (pS, pT, pY, top) and total (bottom) soluble (S) and insoluble (INS) immunoprecipitated α-tubulin from trypomastigotes incubated for 2h with BSA, laminin (L) or fibronectin (F). (<b>D</b>) Calculation of the phosphorylation ratio relative to BSA from the experiments in (<b>C</b>); ratio: relative intensity of phosphorylated-tubulin treatment/control (BSA); on the left, 49.1 corresponds to the molecular mass standard in kDa.</p

Phosphorylation status of PAR proteins upon incubation of trypomastigotes to laminin, fibronectin or BSA.

<p>(<b>A</b>) Representative immunoblotting of phosphorylated (pS, pT, pY) and PAR proteins immunoprecipitated during the incubation time. (<b>B</b>) Quantitation of 3 independent experiments as described in (<b>A</b>); asterisk represents a comparison between the 5 min and 120 min points by the Student's t-test with p<0.05, indicating a progressive PAR dephosphorylation. (<b>C</b>) Representative immunoblotting of phosphorylated (pS, pT, pY, top) and total (bottom) soluble (S) and insoluble (INS) immunoprecipitated PAR proteins from trypomastigotes incubated for 2 h with BSA, laminin (L) or fibronectin (F). (<b>D</b>) Calculation of the phosphorylation ratio relative to BSA from the experiments in (<b>C</b>); ratio: relative intensity of phosphorylated-PAR treatment/control (BSA); on the left, 80 corresponds to the molecular mass standard in kDa.</p

Trypomastigote proteins modified by phosphorylation/dephosphorylation upon adhesion to laminin or fibronectin.

<p>Incubation was for 2 h: (<b>A</b>) Phosphorylated proteins were stained with Pro-Q Diamond. (<b>B</b>) total protein profile developed with colloidal Coomassie blue staining; molecular mass markers (kDa) are shown on the ordinates; spots with variation in phosphorylation status are circled in red. (<b>C</b>) profile of phosphorylated and dephosphorylated spots upon 2 h incubation. (<b>D</b>) functional distribution of proteins from C.</p

Phosphorylation status of ERK1/2 in trypomastigotes incubated with laminin, fibronectin or BSA.

<p>(<b>A</b>) Representative immunoblotting of phosphorylated ERK 1/2 (p-ERK 1/2) or GAPDH protein during the times indicated. (<b>B</b>) Calculation of the ERK 1/2 phosphorylation ratio for each experimental point relative to BSA from 2 experiments as in (<b>A</b>); on the left, 49.1 and 34.8 correspond to molecular mass standards in kDa. Asterisks represent a comparison between the 5 min and 120 min points by the Student's t-test with p<0.05 for fibronectin and p<0.2 for laminin.</p

Phenotype of <i>T. cruzi</i> trypomastigotes incubated with fibronectin, laminin or BSA.

<p>Incubation was for 2 h: (<b>A</b>) Localization of phosphoproteins is shown by the reactivity with anti-pS, -pT, -pY antibodies (red); nucleus and kinetoplast stained with DAPI (blue) and DIC images were also shown; white bars represent 3.2 µm. (<b>B</b>) Quantitation of parasite modifications (arrows) due to treatment with fibronectin and laminin, respectively, as compared to BSA treatment is shown; 6 fields with approximately 40 parasites each have been examined; asterisks represent a p<0.001 when experimental points were compared with the control by the Student's t-test.</p

<i>Trypanosoma cruzi</i> Binds to Cytokeratin through Conserved Peptide Motifs Found in the Laminin-G-Like Domain of the gp85/Trans-sialidase Proteins

<div><p>Background</p><p>Chagas' disease, caused by the protozoan parasite <i>Trypanosoma cruzi</i>, is a disease that affects millions of people most of them living in South and Central Americas. There are few treatment options for individuals with Chagas' disease making it important to understand the molecular details of parasite infection, so novel therapeutic alternatives may be developed for these patients. Here, we investigate the interaction between host cell intermediate filament proteins and the <i>T</i>. <i>cruzi</i> gp85 glycoprotein superfamily with hundreds of members that have long been implicated in parasite cell invasion.</p><p>Methodology/Principal Findings</p><p>An <i>in silico</i> analysis was utilized to identify peptide motifs shared by the gp85 <i>T</i>. <i>cruzi</i> proteins and, using phage display, these selected peptide motifs were screened for their ability to bind to cells. One peptide, named TS9, showed significant cell binding capacity and was selected for further studies. Affinity chromatography, phage display and invasion assays revealed that peptide TS9 binds to cytokeratins and vimentin, and prevents <i>T</i>. <i>cruzi</i> cell infection. Interestingly, peptide TS9 and a previously identified binding site for intermediate filament proteins are disposed in an antiparallel β-sheet fold, present in a conserved laminin-G-like domain shared by all members of the family. Moreover, peptide TS9 overlaps with an immunodominant T cell epitope.</p><p>Conclusions/Significance</p><p>Taken together, the present study reinforces previous results from our group implicating the gp85 superfamily of glycoproteins and the intermediate filament proteins cytokeratin and vimentin in the parasite infection process. It also suggests an important role in parasite biology for the conserved laminin-G-like domain, present in all members of this large family of cell surface proteins.</p></div

The effect of nitric oxide availability on <i>Trypanosoma cruzi</i> protein S-nitrosylation.

<p>Trypomastigotes (1x10⁹) were incubated with ECM (1.5 mg) in phenol red free-MEM, supplemented with 2% FBS, for 2 h at 37°C and 5% CO₂, in presence or absence of 100 μM cPTIO or CysNO. (A) Total protein S-NO was quantified in the parasite lysate using the Saville-Griess method. Asterisks represent a p<0.01 according to one-way ANOVA. (B) Parasite cell viability (1x10⁷/well in a 96 well plate) was carried out by following the reduction of WST-1 reagent (Roche) as described by the manufacturer. Differences are not statistically significant. Values are the mean of three independent experiments.</p

Presence of S-nitrosylated proteins in <i>Trypanosoma cruzi</i> trypomastigotes during adhesion to the extracellular matrix.

<p>Trypomastigotes (1x10⁹) were incubated with ECM (1.5 mg) in phenol red free-MEM, supplemented with 2% FBS, for 2 h at 37°C and 5% CO₂. Parasites were submitted to the immunofluorescence protocol. S-nitrosylated proteins are stained in red, alpha-tubulin in green, and nucleus and kinetoplast in blue (DAPI). The white bar represents 3.2 μm.</p

Selection of peptides common to all members of the gp85/trans-sialidase family.

<p><b>(A)</b> General representation of gp85/TS proteins belonging to the group II. The signal peptides for endoplasmic reticulum (ER) localization and glycophosphatidylinositol (GPI) anchor (green boxes) are indicated; the sialidase ASP-box repeats (orange boxes) and the conserved peptides identified in this study (blue boxes), which include the VTVxNVxLYNRLN motif denominated FLY, are shown. <b>(B)</b> Moving average of nine amino acids for each position in the gp85/TS group II protein alignment. The signal peptide for ER localization (arrow) and the 10 most conserved peptides (*) are indicated. <b>(C)</b> Representation in sequence logo format of the gp85/TS derived peptides (shown within brackets). The letter size indicates amino acid conservation in each position and the color, whether amino acids are polar (green), hydrophobic (black), positively (blue) or negatively (red) charged.</p

Structural analysis of peptides FLY and TS9.

<p><b>(A)</b> Schematic representation of the two conserved domains (Sialidase and LamG) present in the gp85/TS family members. The location of peptides TS9 and FLY are indicated. (B) Sequence alignment of the LamG domain from Tc85-11 and from a <i>T</i>. <i>rangeli</i> sialidase (sequence from PDB ID 1WCS, amino acids 426–624). Identical amino acids are highlighted in blue and conservative changes in green. Peptides TS9 and FLY are shown (boxes). <b>(C)</b> Ribbon diagrams representing the protein structures of the LamG domain from the <i>T</i>. <i>rangeli</i> sialidase (1WCS) and from the 3D modeling of the <i>T</i>. <i>cruzi</i> Tc85-11 protein. The positions of peptides TS9 (blue) and FLY (red) are highlighted in the structure.</p