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

Inactive trans-Sialidase Expression in iTS-null Trypanosoma cruzi Generates Virulent Trypomastigotes

Disclosing virulence factors from pathogens is required to better understand the pathogenic mechanisms involved in their interaction with the host. In the case of Trypanosoma cruzi several molecules are associated with virulence. Among them, the trans-sialidase (TS) has arisen as one of particular relevance due to its effect on the immune system and involvement in the interaction/invasion of the host cells. The presence of conserved genes encoding for an inactive TS (iTS) isoform is puzzlingly restricted to the genome of parasites from the Discrete Typing Units TcII, TcV, and TcVI, which include highly virulent strains. Previous in vitro results using recombinant iTS support that this isoform could play a different or complementary pathogenic role to that of the enzymatically active protein. However, direct evidence involving iTS in in vivo pathogenesis and invasion is still lacking. Here we faced this challenge by transfecting iTS-null parasites with a recombinant gene that allowed us to follow its expression and association with pathological events. We found that iTS expression improves parasite invasion of host cells and increases their in vivo virulence for mice as shown by histopathologic findings in heart and skeletal muscle

The Trypomastigote Small Surface Antigen (TSSA) regulates Trypanosoma cruzi infectivity and differentiation.

BackgroundTSSA (Trypomastigote Small Surface Antigen) is an antigenic, adhesion molecule displayed on the surface of Trypanosoma cruzi trypomastigotes. TSSA displays substantial sequence identity to members of the TcMUC gene family, which code for the trypomastigote mucins (tGPI-mucins). In addition, TSSA bears sequence polymorphisms among parasite strains; and two TSSA variants expressed as recombinant molecules (termed TSSA-CL and TSSA-Sy) were shown to exhibit contrasting features in their host cell binding and signaling properties.Methods/principle findingsHere we used a variety of approaches to get insights into TSSA structure/function. We show that at variance with tGPI-mucins, which rely on their extensive O-glycoslylation to achieve their protective function, TSSA seems to be displayed on the trypomastigote coat as a hypo-glycosylated molecule. This has a functional correlate, as further deletion mapping experiments and cell binding assays indicated that exposition of at least two peptidic motifs is critical for the engagement of the 'adhesive' TSSA variant (TSSA-CL) with host cell surface receptor(s) prior to trypomastigote internalization. These motifs are not conserved in the 'non-adhesive' TSSA-Sy variant. We next developed transgenic lines over-expressing either TSSA variant in different parasite backgrounds. In strict accordance to recombinant protein binding data, trypomastigotes over-expressing TSSA-CL displayed improved adhesion and infectivity towards non-macrophagic cell lines as compared to those over-expressing TSSA-Sy or parental lines. These phenotypes could be specifically counteracted by exogenous addition of peptides spanning the TSSA-CL adhesion motifs. In addition, and irrespective of the TSSA variant, over-expression of this molecule leads to an enhanced trypomastigote-to-amastigote conversion, indicating a possible role of TSSA also in parasite differentiation.Conclusion/significanceIn this study we provided novel evidence indicating that TSSA plays an important role not only on the infectivity and differentiation of T. cruzi trypomastigotes but also on the phenotypic variability displayed by parasite strains

Over-expression of TSSA variants improves trypomastigote-to-amastigote transformation kinetics in <i>T</i>. <i>cruzi</i>.

<p>Purified, CL Brener (panel A) or Sylvio X-10 (panel B) trypomastigotes (5 x 10⁶) of the indicated line were incubated in MEM at pH7, without serum. Samples were taken at different time-points, fixed, and total number of trypomastigotes and amastigotes were counted directly under the light microscope. For each sample, at least 300 parasites were counted and trypomastigotes were expressed as % of total parasites. The results are the average of 3 independent experiments. Asterisks denote significant differences (<i>P</i> < 0.05) to wild type parasites using <i>t</i>-Student test.</p

TSSA is not a sialic acid acceptor and does not behave as a typical <i>T</i>. <i>cruzi</i> mucin.

<p><b>A)</b> Representative images of CL Brener trypomastigotes incubated with Neu5Az<i>α</i>2-3Lac<i>β</i>OMe and processed for immunofluorescence using both mouse mAb anti-FLAG (green) and rabbit TSSA-CL antiserum (red). White arrowheads point to few co-localization spots. Cyan arrowheads indicate TSSA-CL-reactive particles probably secreted to the medium. Bars = 10 μm. <b>B</b> and <b>C)</b> Intact CL Brener trypomastigotes were labeled with Neu5Az<i>α</i>2-3Lac<i>β</i>OMe in the absence (panel B) or presence (panel C) of recombinant <i>trans</i>-Sialidase. The labeled material was fractionated on to anti-FLAG–Sepharose and flow-through (FT) and bound (B) fractions were probed by Western blot. Arrowheads point to a ~25 kDa band, which likely represents the light chain of the anti-FLAG mAb that leaked out of the anti-FLAG-Sepharose. A faint ~10 kDa band of unknown identity is denoted with an asterisk in panel C. <b>D)</b> Butan-1-ol extraction analysis of CL Brener trypomastigotes. Fractions were obtained according to Materials and Methods, and processed for Western blot. <b>E)</b> Conditioned medium from CL Brener trypomastigotes was fractionated using Exoquick kit. Fractions corresponding to parasite pellet (P), soluble molecules (S) and micro-vesicles (MVs) were analyzed by Western blot. Relative molecular mass markers (in kDa) are indicated.</p

TSSA-CL, but not TSSA-Sy, is involved in trypomastigote internalization.

<p><b>A</b> and <b>D)</b> Infection (panel A) or adhesion (panel D) rates of transgenic or wild type trypomastigote lines towards Vero cell monolayers were measured in presence of 100 μg/mL of the indicated peptide or PBS as control. Vero cells grown on 24-well culture plates were added with 1 or 2 x 10⁵ Sylvio X-10 or CL Brener trypomastigote forms (with up to 5% of contaminant amastigote forms), respectively. After 3 h of incubation at 4°C (panel D) or at 37°C (panel A), cells were washed with PBS to remove non-attached parasites, fixed with PBS-PFA immediately (panel D) or after additional 36 h incubation at 37°C (panel A) and processed for Indirect Immunofluorescence assay. In all experiments, the number of infected cells was determined in a total of at least 1,000 DAPI-stained cells. Data are expressed as mean values ± SD of 3 independent experiments, each one performed in duplicate. B) Schematic representation of TSSA-Sy (above) and TSSA-CL (below) sequences expressed as GST-fusion molecules or synthetic peptides, and the residues spanned by each construct (numbers indicate amino acid positions in each sequence relative to the initial Met). Variations between TSSAs and sequences derived thereof are indicated in red (for TSSA-Sy) and green (for TSSA-CL). <b>C)</b> GST-fusion proteins spanning TSSA-CL deletion variants were added to HeLa cells and binding was assessed by means of a monoclonal anti-GST antibody followed by a colorimetric method. In every assay, recombinant GST and GST-TSSA-Sy^24-61 proteins were used as negative control whereas GST-TSSA-CL^24-62 was used as positive control. Reactivity for each protein was normalized to GST-TSSA-CL^24-62 and mean ± SD was calculated from 3 independent assays. Significant differences between the indicated population and GST means (<i>P</i> < 0.05 ANOVA followed by Dunnet’s correction) were denoted with an asterisk. <b>E)</b> Infection rates of wild type trypomastigote lines (indicated below) towards Vero cell monolayers were measured in presence of different transgenic or wild type trypomastigote lines in the upper chamber of the trans-well (indicated above). <b>F)</b> Infection rates of wild type CL Brener trypomastigotes in the presence of conditioned medium (CM) of the indicated trypomastigote line were measured as described above. When indicated, CM was FLAG- or Hemmaglutinin-depleted by affinity chromatography before the flow-through fraction being incubated with wild type CL Brener trypomastigotes. In panels A, C, D and E asterisks denote significant differences (<i>P</i> < 0.05) to the corresponding control values using <i>t</i>-Student test.</p

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

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