Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology

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Abstract
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. Trypanosoma cruzi is a flagellated protozoan parasite whose life cycle includes stages alternating between insect and mammal hosts. The membrane of the infective trypomastigote is equipped to allow survival in blood and to invade cells where it proliferates. Here we provide a novel description of the localization in separate microdomains of the trans-sialidase and mucins, enzyme and substrate, respectively, that play the main role in the crucial scavenging of sialic acids from the host, which allows parasite survival and infection. Even though these proteins, key in the parasite biology, share similar anchoring to the membrane, their puzzling segregation evidences the complexity of the trypanosome virulence mechanisms and protein trafficking. Instead of as soluble proteins, they are shed in microvesicles, a finding relevant to their known role in the pathogenesis of T. cruzi infection. This work provides an integrated view that explores these phenomena at the nanoscale and allows to build a model for the trypomastigote membrane physiology.

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