Fig 1.
Selection of peptides common to all members of the gp85/trans-sialidase family.
(A) 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) 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. (C) 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.
Table 1.
The gp85/TS derived peptides grafted into phage.
Fig 2.
Screening of peptides by phage display.
(A) Flowchart summarizing the pipeline for in silico selection, bacteriophage display and the in vitro functional assay (BRASIL method) for the identification of conserved peptides derived from the gp85/TS family with cell binding properties. (B) Quantification of phage binding to LLC-MK2 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). (C) Peptide competition assay. Effect of synthetic peptide TS9 and the control scramble peptide in phage TS9 binding to LLC-MK2. Phage binding results were normalized relative to the control phage Fd-tet. Mean ± SEM of a representative experiment performed in triplicate are shown.
Fig 3.
Cytokeratin is the receptor to peptide TS9.
(A and B). Affinity chromatography of LLC-MK2 cell extract using immobilized peptide TS9, eluted sequentially with 1% SDS and 8M urea, were analyzed by SDS-PAGE and Coomassie staining (A) or Western-blot using anti-KRT18 antibody (B). In (A), 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) Immunoreactivity of anti-KRT18 antibody with the 1% SDS and 8M urea eluates; recombinant KRT18 was used as positive control. (C) 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).
Fig 4.
Structural analysis of peptides FLY and TS9.
(A) 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 T. rangeli 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). (C) Ribbon diagrams representing the protein structures of the LamG domain from the T. rangeli sialidase (1WCS) and from the 3D modeling of the T. cruzi Tc85-11 protein. The positions of peptides TS9 (blue) and FLY (red) are highlighted in the structure.
Fig 5.
Tc85-11LamG protein binds to cytokeratin.
(A) Purified Tc85-11LamG protein analyzed by SDS-PAGE stained with Coomassie blue and (B) immunoreactivity with an anti-gp85/TS monoclonal antibody. (C and D) Binding of Tc85-11LamG protein to intermediate filament proteins. (D) Peptide competition assay. Effect of synthetic peptides scramble, TS9 and FLY on Tc85-11LamG 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 [C] or two way ANOVA [D]).
Fig 6.
Peptide TS9 inhibits parasite cell adhesion and invasion.
(A and B) Effect of peptide TS9 or the control scramble peptide (200 μM) on LLC-MK2 host cell infection by T. cruzi. In (A) 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) 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). (C) 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. (D) Dose dependence effect of peptide TS9 on LLC-MK2 host cell infection by the parasite. (E) 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). (F) Effect of Tc85-11LamG protein on parasite host cell infection.
Fig 7.
gp85/TS LamG domain putative binding sites.
Ribbon diagrams representing protein structures. In (A), the 3D structure of concanavalin-A (PDB ID 1CVN chain A) with a bound trimannoside molecule is shown. In (B), the tertiary structure of the LamG domain from the T. rangeli 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.