9 research outputs found
<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
gp85/TS LamG domain putative binding sites.
<p>Ribbon diagrams representing protein structures. In (<b>A</b>), the 3D structure of concanavalin-A (PDB ID 1CVN chain A) with a bound trimannoside molecule is shown. In (<b>B</b>), the tertiary structure of the LamG domain from the <i>T</i>. <i>rangeli</i> sialidase (1WCS, amino acids 426–624) is shown. Arrows indicate the conserved peptides TS9 and FLY constituting the possible cytokeratin binding (red); and a putative carbohydrate binding site identified by structural analogy to the concanavalin-A molecule.</p
Peptide TS9 inhibits parasite cell adhesion and invasion.
<p><b>(A and B)</b> Effect of peptide TS9 or the control scramble peptide (200 μM) on LLC-MK<sub>2</sub> host cell infection by <i>T</i>. <i>cruzi</i>. In <b>(A)</b> the number of infected cells is indicated. Values are expressed as relative the number of infected cells (vehicle-only treatment values were set to 1). Mean ± SEM of a representative experiment performed in triplicate are shown. In <b>(B)</b> the number of parasites per infected host cells is shown. Data are presented in box plots in which the boxes define the 25th and 75th percentiles, with a line at a median and whiskers defining the maximum and minimum values of experiments performed in triplicate (*** p<0.001, one-way ANOVA). <b>(C)</b> Representative pictures of microscope fields used for the quantification of parasite invasion. Cells were stained with DAPI. The arrows indicate infected cells with amastigotes in the cytoplasm. (<b>D</b>) Dose dependence effect of peptide TS9 on LLC-MK<sub>2</sub> host cell infection by the parasite. (<b>E</b>) Infection assay performed at 4°C to prevent parasite from entering host cells in the presence or absence of peptides TS9 or scramble (200 μM). (<b>F</b>) Effect of Tc85-11<sup>LamG</sup> protein on parasite host cell infection.</p
Cytokeratin is the receptor to peptide TS9.
<p><b>(A and B)</b>. Affinity chromatography of LLC-MK<sub>2</sub> cell extract using immobilized peptide TS9, eluted sequentially with 1% SDS and 8M urea, were analyzed by SDS-PAGE and Coomassie staining <b>(A)</b> or Western-blot using anti-KRT18 antibody <b>(B)</b>. In <b>(A)</b>, proteins with calculated masses of 49, 53, 59 and 66 kDa identified by Coomassie staining (arrows a-d, respectively) were excised from the gel and analyzed by mass spectrometry. <b>(B)</b> Immunoreactivity of anti-KRT18 antibody with the 1% SDS and 8M urea eluates; recombinant KRT18 was used as positive control. <b>(C)</b> Binding of phage TS9, FLY and Fd-tet (insertless phage) to BSA, recombinant human cytokeratins 8, 14, 18, 19 and 20, vimentin or gelatin. Mean ± SEM of a representative experiment performed in triplicate are shown (* indicates p<0.05, two-way ANOVA).</p
Screening of peptides by phage display.
<p><b>(A)</b> Flowchart summarizing the pipeline for <i>in silico</i> selection, bacteriophage display and the <i>in vitro</i> functional assay (BRASIL method) for the identification of conserved peptides derived from the gp85/TS family with cell binding properties. <b>(B)</b> Quantification of phage binding to LLC-MK<sub>2</sub> cells using the BRASIL methodology and quantitative PCR (qPCR). Phage binding results were normalized relative to the control phage Fd-tet (insertless phage). Mean ± standard error of the means (SEM) from four samples are shown. Significant cell binding was observed for phage FLY and TS9 (* p<0.05 and *** p<0.001; one way ANOVA, N = 4). <b>(C)</b> Peptide competition assay. Effect of synthetic peptide TS9 and the control scramble peptide in phage TS9 binding to LLC-MK<sub>2</sub>. Phage binding results were normalized relative to the control phage Fd-tet. Mean ± SEM of a representative experiment performed in triplicate are shown.</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
The gp85/TS derived peptides grafted into phage.
<p>The gp85/TS derived peptides grafted into phage.</p
Tc85-11<sup>LamG</sup> protein binds to cytokeratin.
<p><b>(A)</b> Purified Tc85-11<sup>LamG</sup> protein analyzed by SDS-PAGE stained with Coomassie blue and <b>(B)</b> immunoreactivity with an anti-gp85/TS monoclonal antibody. <b>(C and D)</b> Binding of Tc85-11<sup>LamG</sup> protein to intermediate filament proteins. <b>(D)</b> Peptide competition assay. Effect of synthetic peptides scramble, TS9 and FLY on Tc85-11<sup>LamG</sup> binding to KRT18 protein. The results were normalized by the negative control gelatin. Mean ± SEM of a representative experiment performed in triplicate are shown (* p<0.05, ** p<0.01***, p<0.001; one way ANOVA [<b>C</b>] or two way ANOVA [<b>D</b>]).</p