The protozoan parasite, Trypanosoma cruzi, is the causative agent of Chagas disease (CD), which is endemic throughout Latin America, where millions of people are affected. While CD can be accurately diagnosed by polymerase chain reaction (PCR) and serological immunoassays, parasite-derived molecular biomarkers are of great interest, particularly for the evaluation of chemotherapy efficacy. The mammal-dwelling trypomastigote form of T. cruzi expresses glycosylphosphatidylinositol (GPI)-anchored mucin-like glycoproteins, which contain O-glycans with terminal, nonreducing a-galactopyranosyl (aGalp) residues. Previously, one of the mucin O-glycans, Galpa1,3Galpb1,4GlcNAca, has been identified as an immunodominant T. cruzi glycotope. However, the mucin O-glycans (depending on the parasite strain) and/or the GPI anchor also contains appending b-galactofuranosyl (bGalf) residues, which are completely absent on human cells and could, therefore, be part of other T. cruzi glycotopes exploitable as antigens in serological assays. Here we report that sera from chronic CD (CCD) patients show a strong IgG response to two synthetic glycans with terminal, nonreducing bGalf moieties. We took a reversed immunoglycomics approach that entailed the chemical synthesis of two bGalf-containing, T. cruzi GPI-anchor-derived glycans, their conjugation to bovine serum albumin (BSA) to produce neoglycoproteins NGP29b and NGP32b, and their use as antigens in a chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) with sera from CCD patients from Caracas, Venezuela, and Salta, Argentina. Receiver-operating characteristic (ROC) analysis revealed that both, NGP29b, and NGP32b are suitable as diagnostic biomarkers for CCD. NGP29b can distinguish CCD sera from normal human sera (NHS) with 80% sensitivity and 93% specificity. NGP32b comprises a larger glycotope with two nonreducing, terminal bGalf units, and is an even better CCD biomarker, with 88% sensitivity and 96% specificity. The two glycotopes discovered here could prove useful for the follow-up of CCD patients following chemotherapy. Furthermore, our preliminary data show that the combination of NGP32b and the previously described aGalp-containing NGP24b has a higher antibody response in the CL-ELISA when compared with either NGP29b or NGP32b alone.

Specific recognition of β-galactofuranosyl-containing glycans by sera from chronic Chagas disease patients

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