title: Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.
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.
authors.affiliation: Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina and Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
authors.forename: Andrés B
authors.initials: AB
authors.lastname: Lantos
authors.affiliation: Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina and Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
authors.forename: Giannina
authors.initials: G
authors.lastname: Carlevaro
authors.affiliation: Laboratorio de Nanoscopías Fotónicas, INQUIMAE, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Araoz
authors.affiliation: Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina and Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
authors.forename: Pablo
authors.initials: P
authors.lastname: Ruiz Diaz
authors.affiliation: Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina and Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
authors.forename: María de Los Milagros
authors.initials: Mde L
authors.lastname: Camara
authors.affiliation: Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina and Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Buscaglia
authors.affiliation: Laboratorio de Nanoscopías Fotónicas, INQUIMAE, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: Mariano
authors.initials: M
authors.lastname: Bossi
authors.affiliation: Department of Chemistry, University of California, Davis, Davis, California, United States of America.
authors.forename: Hai
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Chemistry, University of California, Davis, Davis, California, United States of America.
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Stanford University and Howard Hughes Medical Institute, Stanford, California, United States of America.
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Bertozzi
authors.affiliation: Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina and Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
authors.forename: Juan
authors.initials: J
authors.lastname: Mucci
authors.affiliation: Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín, San Martín, Buenos Aires, Argentina and Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.
authors.forename: Oscar
authors.initials: O
authors.lastname: Campetella
mesh_terms: D000818:Animals; D055252:Cell-Derived Microparticles / Q000378:metabolism; D014355:Chagas Disease / Q000378:metabolism / Q000469:parasitology*; D004195:Disease Models, Animal; D006023:Glycoproteins / Q000378:metabolism; D006790:Host-Parasite Interactions / Q000502:physiology*; D007091:Image Processing, Computer-Assisted; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D008853:Microscopy / Q000379:methods; D008856:Microscopy, Fluorescence; D009077:Mucins / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D009439:Neuraminidase / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000378:metabolism* / Q000472:pathogenicity*; D014774:Virulence
chemical_list: D006023:Glycoproteins; D009077:Mucins; C069715:trans-sialidase; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid