Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins

Background: TolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3–5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen. Methods/Principle findings: Genome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis. Conclusion/Significance: This study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications.Fil: Lobo, Mabel Maite. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Balouz, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Melli, Luciano Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Carlevaro, Giannina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Cortina, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Camara, María de los Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Canepa, Gaspar Exequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Carmona, Santiago Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Altcheh, Jaime Marcelo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Campetella, Oscar Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ciocchini, Andres Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Agüero, Fernan Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Mucci, Juan Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.

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

Molecular and antigenic characterization of Trypanosoma cruzi TolT proteins.

BACKGROUND:TolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3-5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen. METHODS/PRINCIPLE FINDINGS:Genome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis. CONCLUSION/SIGNIFICANCE:This study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications

Atomic force microscopy (AFM).

<p>Trypomastigotes were AFM imaged by tapping mode. Upper panels show height traces and bottom panels show 3D transformation. Special focus was done over the flagellum where heterogeneous and irregular domains following parallel structures along the flagellum could be observed. Arrows indicate neighboring domains to highlight size distribution and domain separation. Flag: flagellum.</p

Sialylated mucins and TS are included in segregated membrane microdomains.

<p>A) Live trypomastigotes were sialylated and FLAG-tagged. Sialylated mucins and TS rendered a dotted pattern on the trypomastigotes surface that did not co-localize (confocal microscopy). Line profiles for sialic acid and TS signal in the flagellum (boxed area) showed that mucins and TS do not co-localize but were rather out-of-phase with each other. GSDIM superresolution fluorescence microscopy performed for sialylated mucins (B) and TS (C) independently showed that mucins were included in domains 90 nm wide and separated 120-500nm from each other. Results for TS were equivalent. Size and distribution of trypomastigote membrane domains explain why under a confocal microscope, restricted to classical light diffraction limits, the domains for TS and mucins were not fully resolved.</p

The membrane of trypomastigotes is complex to the nanometer scale.

<p>Membrane model for <i>T</i>. <i>cruzi</i> trypomastigotes. The surface is packed in microdomains of different size, shape, lipid composition and embedded proteins. Some of these domains are detergent resistant, however this does not imply a functional profile. Mucins are included in DRMs whereas TS is not, thus being segregated in the membrane of trypomastigotes. This challenges the membrane bound TS as the sialylating factor for mucins, a role proposed for the shed TS instead. DRMs embed different proteins, many of them localized to the flagellum. Flagellum domains tend to be smaller and closer together than those in the cell body and suggest an association to the flagellar cytoskeleton. Mucins and TS are shed to the extracellular environment included in microvesicles probably resulting from membrane budding and fission events. Furthermore, TS is shed associated to vesicles instead of as a soluble protein. No hydrolysis of the GPI-anchors occurs in the trypomastigote stage.</p

Sialylated mucins are included in lipid rafts whereas TS is not.

<p>(A) Cold Triton X-100 partition. Sialylated trypomastigotes were lysed at 4°C. Mucins were predominantly recovered in the pellet whereas TS and HSP70, a cytosolic protein, were recovered in the supernatant. (B) Triton X-114 extraction for GPI-anchored proteins. Parasites were lysed at 4°C and detergent and aqueous phases separated at 37°C and analyzed by Western blots. Mucins and TS partitioned in the detergent phase due to their GPI-anchoring. Glutamate Dehydrogenase, a cytosolic protein, was recovered in the aqueous phase. (C) Purification of DRMs by step-gradient ultracentrifugation. Trypomastigotes were lysed in Triton X-100 at 4°C or 37°C and centrifuged in an Optiprep gradient. Mucins floated to the 35%-5% interface (lane 6) only when lysis was done at 4°C indicating its DRM nature in contrast to TS. (D) Living parasites were sialylated from a Neu5Az donor, then treated for membrane fluidization with 1% diethyl ether in phosphate-buffered saline (PBS) and fixed with <i>p</i>-formaldehyde (PFA). Doted labeling for TS and mucins was disrupted only after 90sec treatment even showing colocalization.</p

Selected Hits from Mass Spectrometry Assays from Lipid Rafts Proteins.

<p>Selected Hits from Mass Spectrometry Assays from Lipid Rafts Proteins.</p

Distribution of proteins contained in DRMs.

<p>Proteins identified by mass spectrometry were analyzed by immunofluorescence. KMP, TolT and CLCP were located in domains of the flagellum. ADK-1 displayed a dotted pattern in the cell body and in the flagellum. No co-localization with mucins or with TS was found. Arrow points to an amastigote, this stage remains unsialylated. Bar: 5μm.</p

Mucin sialylation and characterization.

<p>A) Mucins (or any glycoconjugate bearing a terminal β-galactose) may be tagged by the TS using a Neu5Az donor such as Neu5Azα(2–3)LacβOMe. Then the Azide group may be coupled via the Staudinger-Bertozzi chemistry or the Cu²⁺-free click chemistry to obtain a FLAG or biotin tag ready for detection. B) Western blot of Neu5Az <i>trans</i>-sialylated trypomastigote lysates revealing the relative molecular mass distribution of acceptor molecules. A line profile of the blot is also plotted. Neg Ctrl: Negative control. C) Neu5Az <i>trans</i>-sialylated parasites were submitted to organic solvents extraction as described in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005559#ppat.1005559.ref020" target="_blank">20</a>] to determine their mucin nature. Extracted material was subjected to Western blot. F1, F2 and F3 refer to the different purification fractions (for details see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005559#sec010" target="_blank">M&M</a>). D) Neu5Az <i>trans</i>-sialylated trypomastigotes (900x10⁶) were lysed and sialylated proteins pulled-down with anti-FLAG antibodies. Western blots of this material were revealed with anti-TcMUC II antibodies. E-F) Confocal images displaying partial colocalization of anti-FLAG and anti-TcMUC II (E) or anti-αGal (F) labeling at the parasite surface.</p