Oscar Campetella

The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is encoded by multiple polymorphic tandemly organized genes located on different chromosomes

We demonstrate that cruzipain, the major cysteine proteinase of Trypanosoma cruzi epimastigotes, is encoded by a large number of tandemly arranged genes. Restriction enzyme analysis of 20 clones containing complete repeat units of the gene, as well as sequencing of 2 of these clones, and comparison with previously published partial sequences, indicated that the sequence is conserved among the repeat units, although polymorphisms clearly exist. The repeat units contain an intergenic region of 528 bp and coding regions for pre- and pro-enzyme, a central domain and a C-terminal extension. The predicted amino acid sequences of these regions indicated a sequence identity of 30, 60, 70 and 36%, respectively, when the T. cruzi sequence was compared with the sequence of a similar cysteine proteinase from Trypanosoma brucei. Studies by pulsed field gel electrophoresis, complemented with restriction analysis, indicated that the clusters are located on 2-4 different chromosomes in several parasite isolates.

Structural basis of sialyltransferase activity in trypanosomal sialidases

The intracellular parasite Trypanosoma cruzi, the etiological agent of Chagas disease, sheds a developmentally regulated surface trans‐sialidase, which is involved in key aspects of parasite–host cell interactions. Although it shares a common active site architecture with bacterial neuraminidases, the T.cruzi enzyme behaves as a highly efficient sialyltransferase. Here we report the crystal structure of the closely related Trypanosoma rangeli sialidase and its complex with inhibitor. The enzyme folds into two distinct domains: a catalytic β‐propeller fold tightly associated with a lectin‐like domain. Comparison with the modeled structure of T.cruzi trans‐sialidase and mutagenesis experiments allowed the identification of amino acid substitutions within the active site cleft that modulate sialyltransferase activity and suggest the presence of a distinct binding site for the acceptor carbohydrate. The structures of the Trypanosoma enzymes illustrate how a glycosidase scaffold can achieve efficient glycosyltransferase activity and provide a framework for structure‐based drug design.

Trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors RORγt and Ahr that leads to IL-17 production by activated B cells

We identified B cells as a major source for rapid, innate-like interleukin 17 (IL-17) production in vivo in response to Trypanosoma cruzi infection. IL-17+ B cells exhibited a plasmablast phenotype, outnumbered TH17 cells and were required for optimal response to this pathogen. Using both murine and human primary B cells, we demonstrate that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell surface mucin, CD45, leading to Btk-dependent signaling and IL-17A or IL-17F production via an ROR-γt and AHR-independent transcriptional program. Our combined data suggest that generation of IL-17+ B cells may be an unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.Here we identified B cells as a major source of rapid, innate-like production of interleukin 17 (IL-17) in vivo in response to infection with Trypanosoma cruzi. IL-17+ B cells had a plasmablast phenotype, outnumbered cells of the TH17 subset of helper T cells and were required for an optimal response to this pathogen. With both mouse and human primary B cells, we found that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell-surface mucin CD45, which led to signaling dependent on the kinase Btk and production of IL-17A or IL-17F via a transcriptional program independent of the transcription factors RORγt and Ahr. Our combined data suggest that the generation of IL-17+ B cells may be a previously unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.

Differential Expression of a Virulence Factor,the trans-Sialidase, by the Main Trypanosoma cruzi Phylogenetic Lineages

The clinical outcome of Chagas disease is highly variable, mainly because of the heterogeneity of Trypanosoma cruzi, a parasite for which 2 major phylogenetic groups (I and II) were recently defined. Epidemiological and immunological data indicate that the prevalence of T. cruzi II in patients living in the southern cone of South America correlates with the alterations caused by Chagas disease. We report here that infection with T. cruzi II isolates induces 100% mortality in mice, in contrast to infection with T. cruzi I isolates, in which almost all mice enter the chronic phase even when a 1000-fold higher inoculum is administered. Trypomastigotes from T. cruzi II strains express and shed significantly higher amounts of trans-sialidase than do those from the T. cruzi I lineage. Disorganization of the thymus histoarchitecture associated with the circulating enzyme was observed after infection with T. cruzi II strains, in contrast to transient thymus lesions found in mice infected with T. cruzi I strains. Therefore, trans-sialidase becomes the first T. cruzi virulence factor identified that is differentially expressed by the main parasite groups and that contributes to their contrasting behaviors.

A single tyrosine differentiates active and inactive Trypanosoma cruzi trans-sialidases

Several genes encode members of the Trypanosoma cruzi (Tc) trans-sialidase (TS) family. These proteins contain an enzymatic domain on the N terminus, the only one required for TS activity, and an antigenic domain (SAPA (shed acute phase antigen) amino acid (aa) repeats) on the C terminus. Only some members of this glycoprotein family are enzymatically active. The complete sequence of two clones encoding the enzymatic domain of active and inactive protein from each of two Tc strains has now been obtained. Comparison of these sequences showed a limited divergence among them: 20 out of the 642 deduced aa in the enzymatic domain were found to differ. From these 20 aa, only one was found to be essential for enzymatic activity. A Tyr342 residue is deduced in both active proteins while a His342 is present in both inactive ones. This naturally occurring Tyr342-->His substitution completely abolished the TS activity. In addition to Tyr342, a second deduced aa, Pro231, was found to be necessary for full enzymatic TS activity; a Pro231-->Ala change rendered the TS protein partially active. Fourteen aa residues, including Tyr342, out of the 16 aa in the active site of a sialidase from Salmonella typhimurium are present at the same or very similar positions in the Tc TS.

A major cysteine proteinase is developmentally regulated in Trypanosoma cruzi

Epimastigotes of different stocks of Trypanosoma cruzi contain similar levels of proteinase activity on azocasein; amastigotes and trypomastigotes contain 10-fold lower levels of this proteolytic activity, which seems, therefore, to be developmentally regulated. The proteinase could be detected as a broad band, centered at about 60 kDa, which in some cases resolved into two close bands, in (a) SDS-polyacrylamide gels containing fibrinogen, and (b) Western blots probed with a polyclonal rabbit antiserum prepared against purified cysteine proteinase. No proteinase activity was observed at molecular weights lower than 55 kDa. The results show that the enzyme previously purified is the major cysteine proteinase present in epimastigotes of all stocks of T. cruzi tested.

The trans-sialidase from Trypanosoma cruzi triggers apoptosis by target cell sialylation

The trans‐sialidase, a modified sialidase that transfers sialyl residues among macromolecules, is a unique enzymatic activity expressed by some parasitic trypanosomes being essential for their survival in the mammalian host and/or in the insect vector. The enzyme from Trypanosoma cruzi, the agent of Chagas disease, is found in blood and able to act far from the infection site by inducing apoptosis in cells from the immune system. A central and still unsolved question is whether trans‐sialidase‐mediated addition or removal of sialic acid to/from host acceptor molecules is the event associated with the apoptosis induced by the enzyme. Here we show that lactitol, a competitive inhibitor that precluded the transference of the sialyl residue to endogenous acceptors but not the hydrolase activity of the enzyme, prevented ex vivo and in vivo the apoptosis caused by the trans‐sialidase. By lectin histochemistry, the transference of sialyl residue to the cell surface was demonstrated in vivo and found associated with the apoptosis induction. The sialylation of the CD43 mucin, a key molecule involved in trans‐sialidase‐apoptotic process, was readily detected and also prevented by lactitol on thymocytes. Therefore, lesions induced by trans‐sialidase on the immune system are due to the sialylation of endogenous acceptor molecules.

Human platelets express and are activated by galectin-8

Gals (galectins) are proteins with glycan affinity that are emerging as mediators of atherosclerosis. Despite the similarities in structure and sequence, different Gals exert distinct effects on their target cells. We have shown that Gal-1 triggers platelet activation, suggesting a role for Gals in thrombus formation. Since Gal-8 is expressed upon endothelial activation and also contributes to inflammation, to understand further the role of these lectins in haemostasis, we evaluated the effect of Gal-8 on human platelets. Gal-8 bound specific glycans in the platelet membrane and triggered spreading, calcium mobilization and fibrinogen binding. It also promoted aggregation, thromboxane generation, P-selectin expression and granule secretion. GP (glycoprotein) αIIb and Ib-V were identified as putative Gal-8 counter-receptors by MS. Studies performed using platelets from Glanzmanns thromboasthenia and Bernard-Soulier syndrome patients confirmed that GPIb is essential for transducing Gal-8 signalling. Accordingly, Src, PLC2γ (phospholipase C2γ), ERK (extracellular-signal-regulated kinase) and PI3K (phosphoinositide 3-kinase)/Akt downstream molecules were involved in the Gal-8 signalling pathway. Gal-8 fragments containing either the N- or C-terminal carbohydrate-recognition domains showed that activation is exerted through the N-terminus. Western blotting and cytometry showed that platelets not only contain Gal-8, but also expose Gal-8 after thrombin activation. These findings reveal Gal-8 as a potent platelet activator, supporting a role for this lectin in thrombosis and inflammation.

Galectin-8 provides costimulatory and proliferative signals to T lymphocytes.

Galectin (Gal) constitute a family of carbohydrate‐recognizing molecules ubiquitously expressed in mammals. In the immune system, they regulate many processes such as inflammation, adhesion, and apoptosis. Here, we report the expression in the spleen of the two same Gal‐8 splice variants described previously in the thymus. Gal‐8 was found to induce two separate biological activities on T lymphocytes: a robust naive CD4+ T cell proliferation in the absence of antigen and notably, a costimulatory signal that synergized the cognate OVA peptide in DO11.10 mice transgenic for TCROVA. The antigen‐independent proliferation induced by Gal‐8 displayed increased expression of pro‐ and anti‐inflammatory cytokines, thus suggesting the polyclonal expansion of Th1 and Th2 clones. The costimulatory effect on antigen‐specific T cell activation was evidenced when the Gal and the peptide were assayed at doses suboptimal to induce T cell proliferation. By mass spectra analysis, several integrins and leukocyte surface markers, including CD45 isoforms, as well as other molecules specific to macrophages, neutrophils, and platelets, were identified as putative Gal‐8 counter‐receptors. Gal‐8 triggered pZAP70 and pERK1/2. Moreover, pretreatment with specific inhibitors of CD45 phosphatase or ERK1/2 prevented its antigen‐dependent and ‐independent T cell‐proliferative activities. This seems to be associated with the agonistic binding to CD45, which lowers the activation threshold of the TCR signaling pathway. Taken together, our findings support a distinctive role for locally produced Gal‐8 as an enhancer of otherwise borderline immune responses and also suggest that Gal‐8 might fuel the reactivity at inflammatory foci.

Identification of glycoproteins targeted by Trypanosoma cruzi trans-sialidase, a virulence factor that disturbs lymphocyte glycosylation

Trypanosoma cruzi, the agent of the American trypanosomiasis or Chagas disease, bypasses its lack of de novo synthesis of sialic acids by expressing a surface-anchored trans-sialidase. This enzyme transfers sialic acid residues from the hosts sialylglycoconjugates to the parasites galactosylglycoconjugates. In addition to carrying out a pivotal role in parasite persistence/replication within the infected mammal, the trans-sialidase is shed into the bloodstream and induces alterations in the host immune system by modifying the sialylation of the immune cells. A major obstacle to understand these events is the difficulty to identify the transferred sialic acid among all those naturally occurring on the cell surface. Here, we report the use of azido-modified unnatural sialic acid to identify those molecules that act as cell surface acceptors of the sialyl residue in the trans-sialidase-catalyzed reaction, which might then be involved in the immune alterations induced. In living parasites, we readily observed the transfer of azido-sialic acid to surface mucins. When evaluating mouse thymocytes and splenocytes as acceptors of the azido-sugar, a complex pattern of efficiently tagged glycoproteins was revealed. In both leukocyte populations, the main proteins labeled were identified as different CD45 isoforms. Disruption of the cell architecture increased the number and the molecular weight distribution of azido-sialic acid tagged proteins. Nevertheless, CD45 remained to be the main acceptor. Mass spectrometry assays allowed us to identify other acceptors, mainly integrins. The findings reported here provide a molecular basis to understand the abnormalities induced in the immune system by the trans-sialidase during T. cruzi infection.