Trypanosoma cruzi, the flagellate protozoan agent of
Chagas disease or
American trypanosomiasis, is unable to synthesize
sialic acids de novo.
Mucins and
trans-sialidase (TS) are substrate and
enzyme, respectively, of the glycobiological system that scavenges
sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote
glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural
sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired
sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as
mucins by biochemical procedures and
protein markers. Together with determining their shedding and turnover rates, we also report that several
membrane proteins, including TS and its substrates, both
glycosylphosphatidylinositol-anchored
proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated
mucins, indicating that two species of glycosylated
mucins do exist, which are segregated at the parasite surface. Moreover, sialylated
mucins were included in
lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate
mucins, a role played by the shed TS instead.
Phosphatidylinositol-
phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.