Mapping antigenic motifs in the trypomastigote small surface antigen from Trypanosoma cruzi

Abstract

The trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease.

FIG 1

Mapping of antigenic sequences in TSSA-CL. (A) Peptide chips composed of 15mer peptides overlapping by 14 residues spanning the complete sequence of TSSA-CL were probed, in duplicate, with 2 different IgG samples purified from chronic Chagasic sera. The mean reactivity toward every TSSA-CL-derived peptide (in arbitrary units [A.U.] of fluorescence) and the sequence of the peptide showing the highest reactivity for each IgG sample are indicated. (B) Schematic illustration of TSSA-CL showing the predicted signal peptide (SP) and the GPI-anchoring signal (GPI). The sequences derived from TSSA-CL that were expressed as GST-fusion molecules and the residues spanned by each construct (numbers indicate amino acid positions in each sequence relative to the initial Met) are indicated at the bottom. (C) Schematic diagram showing the anti-TSSA-CL reactivity profile obtained for each sample. Individual serum samples from chronic Chagasic patients (n = 51) were tested toward TSSA-CL deletion variants by ELISA. The gray square boxes indicate positive reactivity, and the black square boxes indicate negative reactivity. The white square boxes indicate nontested molecules. The fraction of positive serum samples/total assayed serum samples is indicated to the right. The identity of pooled sera used to purify IgG sample 1 and IgG sample 2 (see panel A) are indicated by black and gray arrows, respectively. (D) Competitive ELISAs. Eight individual serum samples from chronic Chagasic patients not tested in panel C were preincubated with 2 μg of p36-50 (light gray columns), p30-44 (dark gray columns), p30-50 (striped columns), or a scrambled version of p30-50 (black columns) for 30 min and then added to GST-TSSA^24-62-coated ELISA plates. Absorbance of a control sample preincubated with PBS (white columns) was taken as 100% binding. *, significant differences between the population means (P < 0.001 after Bonferroni's correction).

FIG 2

Receiver operating characteristic (ROC) curve analysis of the ELISA results using TSSA-CL-derived reagents. (A) Dot plot analysis of ELISA results using a GST-fusion protein spanning residues 24 to 62 of TSSA-CL (TSSA-CL^24-62) and a synthetic peptide spanning residues 30 to 50 from the same molecule (p30-50). The ELISA plates were coated with the indicated antigen and incubated with 70 serum samples from chronic Chagas-positive individuals (+) or 38 noninfected individuals (−). The mean ± SD for each group is indicated. *, significant differences between the population means (P < 0.001 after Bonferroni's correction). (B) Reactivity values in panel A were used to generate ROC curves; the area under the ROC curve (AUC) is indicated for each antigen. The values in parentheses indicate the 95% confidence intervals.