Structural Basis of the Interaction of a Trypanosoma cruzi Surface Molecule Implicated in Oral Infection with Host Cells and Gastric Mucin

Host cell invasion and dissemination within the host are hallmarks of virulence for many pathogenic microorganisms. As concerns Trypanosoma cruzi, which causes Chagas disease, the insect vector-derived metacyclic trypomastigotes (MT) initiate infection by invading host cells, and later blood trypomastigotes disseminate to diverse organs and tissues. Studies with MT generated in vitro and tissue culture-derived trypomastigotes (TCT), as counterparts of insect-borne and bloodstream parasites, have implicated members of the gp85/trans-sialidase superfamily, MT gp82 and TCT Tc85-11, in cell invasion and interaction with host factors. Here we analyzed the gp82 structure/function characteristics and compared them with those previously reported for Tc85-11. One of the gp82 sequences identified as a cell binding site consisted of an α-helix, which connects the N-terminal β-propeller domain to the C-terminal β-sandwich domain where the second binding site is nested. In the gp82 structure model, both sites were exposed at the surface. Unlike gp82, the Tc85-11 cell adhesion sites are located in the N-terminal β-propeller region. The gp82 sequence corresponding to the epitope for a monoclonal antibody that inhibits MT entry into target cells was exposed on the surface, upstream and contiguous to the α-helix. Located downstream and close to the α-helix was the gp82 gastric mucin binding site, which plays a central role in oral T. cruzi infection. The sequences equivalent to Tc85-11 laminin-binding sites, which have been associated with the parasite ability to overcome extracellular matrices and basal laminae, was poorly conserved in gp82, compatible with its reduced capacity to bind laminin. Our study indicates that gp82 is structurally suited for MT to initiate infection by the oral route, whereas Tc85-11, with its affinity for laminin, would facilitate the parasite dissemination through diverse organs and tissues.     

Modeling the Trypanosoma cruzi Tc85-11 protein and mapping the laminin-binding site

Trypanosoma cruzi expresses a set of glycoproteins encoded by the gp85/trans-sialidase gene superfamily. In this report a structure model is proposed for a cloned member of the superfamily, the Tc85-11 protein. The structure consists of an N-terminus beta-propeller and a C-terminus beta-sandwich interconnected by an alpha-helix. The recombinant protein, corresponding to the N-domain (Tc85-N), binds to laminin in a selective manner. Six synthetic 20-mer peptides from the N-domain adhere onto the surface of LLC-MK(2) cells and two of these peptides specifically inhibit the Tc85-N/laminin interaction, indicating that they are the laminin-binding sites of the molecule. Thus, Tc85-11 and other related members of the family appear to be good candidates to play an important role in T. cruzi infection via a laminin mediated host-parasite interaction.     

A conserved domain of the gp85/trans-sialidase family activates host cell extracellular signal-regulated kinase and facilitates Trypanosoma cruzi infection

Chagas' disease is a chronic, debilitating and incapacitating illness, caused by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells. Although the mechanism of trypomastigotes interaction with mammalian cells has been intensively studied, a final and integrated picture of the signal transduction mechanisms involved still remains to be elucidated. Our group has previously shown that the conserved FLY domain (VTVXNVFLYNR), present in all members of the gp85/trans-sialidase glycoprotein family coating the surface of trypomastigotes, binds to cytokeratin 18 (CK18) on the surface of LLC-MK(2) epithelial cells, and significantly increases parasite entry into mammalian cells. Now it is reported that FLY, present on the surface of trypomastigotes or on latex beads binds to CK18, promotes dephosphorylation and reorganization of CK18 and activation of the ERK1/2 signaling cascade culminating in an increase of approximately 9-fold in the number of parasites/cell. Inhibition of ERK1/2 phosphorylation completely blocks the adhesion of FLY to cells and blocks by 57% the host cell infection by T. cruzi. Taken together our results indicate that the conserved FLY domain is an important tool that trypomastigotes have evolved to specific exploit the host cell machinery and guarantee a successful infection.     

Enzymatically inactive trans-sialidase from Trypanosoma cruzi binds sialyl and beta-galactopyranosyl residues in a sequential ordered mechanism

Host/parasite interaction mediated by carbohydrate/lectin recognition results in the attachment to and invasion of host cells and immunoregulation, enabling parasite replication and establishment of infection. Trypanosoma cruzi, the protozoan responsible for Chagas disease, expresses on its surface a family of enzymatically active and inactive trans-sialidases. The parasite uses the active trans-sialidase for glycoprotein sialylation in an unusual trans-glycosylation reaction. Inactive trans-sialidase is a sialic acid-binding lectin that costimulates host T cells through leucosialin (CD43) engagement. The co-mitogenic effect of trans-sialidase can be selectively abrogated by N-acetyllactosamine, suggesting the presence of an additional carbohydrate binding domain for galactosides, in addition to that for sialic acid. Here we investigated the interaction of inactive trans-sialidase in the presence of beta-galactosides. By using NMR spectroscopy, we demonstrate that inactive trans-sialidase has a beta-galactoside recognition site formed following a conformational switch induced by sialoside binding. Thus prior positioning of a sialyl residue is required for the beta-galactoside interaction. When an appropriate sialic acid-containing molecule is available, both sialoside and beta-galactoside are simultaneously accommodated in the inactive trans-sialidase binding pocket. This is the first report of a lectin recognizing two distinct ligands by a sequential ordered mechanism. This uncommon binding behavior may play an important role in several biological aspects of T. cruzi/host cell interaction and could shed more light into the catalytic mechanism of the sialic acid transfer reaction of enzymatically active trans-sialidase.     

In vivo infection by Trypanosoma cruzi: the conserved FLY domain of the gp85/trans-sialidase family potentiates host infection

Trypanosoma cruzi is a protozoan parasite that infects vertebrates, causing in humans a pathological condition known as Chagas' disease. The infection of host cells by T. cruzi involves a vast collection of molecules, including a family of 85 kDa GPI-anchored glycoproteins belonging to the gp85/trans-sialidase superfamily, which contains a conserved cell-binding sequence (VTVXNVFLYNR) known as FLY, for short. Herein, it is shown that BALB/c mice administered with a single dose (1 μg/animal, intraperitoneally) of FLY-synthetic peptide are more susceptible to infection by T. cruzi, with increased systemic parasitaemia (2-fold) and mortality. Higher tissue parasitism was observed in bladder (7·6-fold), heart (3-fold) and small intestine (3·6-fold). Moreover, an intense inflammatory response and increment of CD4+ T cells (1·7-fold) were detected in the heart of FLY-primed and infected animals, with a 5-fold relative increase of CD4+CD25+FoxP3+ T (Treg) cells. Mice treated with anti-CD25 antibodies prior to infection, showed a decrease in parasitaemia in the FLY model employed. In conclusion, the results suggest that FLY facilitates in vivo infection by T. cruzi and concurs with other factors to improve parasite survival to such an extent that might influence the progression of pathology in Chagas' disease.     

Trypanosoma cruzi: monoclonal antibodies to the surface glycoprotein superfamily differentiate subsets of the 85-kDa surface glycoproteins and confirm simultaneous expression of variant 85-kDa surface glycoproteins.

Most surface glycoproteins expressed by mammalian-stage forms of Trypanosoma cruzi are homologous to the parasite's trans-sialidase and therefore are members of the parasite's trans-sialidase superfamily. Few members of this superfamily have trans-sialidase activity. The SA85-1 family is a subfamily of the trans-sialidase superfamily whose members lack trans-sialidase activity. The function of these non-trans-sialidase members remains unknown. In this report a series of monoclonal and polyclonal antibodies to the SA85-1 glycoproteins is presented. The mAbs define distinct subgroups of SA85-1 glycoproteins, and these distinct subgroups are simultaneously expressed by individual trypomastigotes, supporting previous studies indicating that multiple SA85-1 glycoproteins and trans-sialidase superfamily glycoproteins are simultaneously expressed by each trypomastigote. In addition, the antibodies define two major subsets of the SA85-1 family (subset 1 and subset 2) based on differences in migration in SDS-PAGE; the subsets do not appear to be created by differences in glycosylation. Subset 1 migrates slower and is spontaneously released or shed preferentially from the parasite surface compared to subset 2. In addition, subset 1 is attached to the trypomastigote surface by a GPI linkage. Since these glycoprotein subsets are differentially expressed, they may have different functions.     

Role of the gp85/trans-sialidases in Trypanosoma cruzi tissue tropism: preferential binding of a conserved peptide motif to the vasculature in vivo

Background

Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas'' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature.

Methods

Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied.

Findings and Conclusions

Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.     

trans-Sialidase from Trypanosoma cruzi binds host T-lymphocytes in a lectin manner

Trypanosoma cruzi, the protozoan parasite responsible for Chagas' disease, expresses on its surface an uncommon membrane-bound sialidase, known as trans-sialidase. trans-Sialidase is the product of a multigene family encoding both active and inactive proteins. We report here that an inactive mutant of trans-sialidase physically interacts with CD4(+) T cells. Using a combination of flow cytometry and immunoprecipitation techniques, we identified the sialomucin CD43 as a counterreceptor for trans-sialidase on CD4(+) T cells. Using biochemical, immunological, and spectroscopic approaches, we demonstrated that the inactive trans-sialidase is a sialic acid-binding protein displaying the same specificity required by active trans-sialidase. Taken together, these results suggest that inactive members of the trans-sialidase family can physically interact with sialic acid-containing molecules on host cells and could play a role in host cell/T. cruzi interaction.     

Only some members of a gene family in Trypanosoma cruzi encode proteins that express both trans-sialidase and neuraminidase activities.

Trypomastigotes, the blood stage form of the human parasite Trypanosoma cruzi, contain an enzyme on their surface, trans-sialidase, which catalyses the transfer of sialic acid from host glycoconjugates to acceptors on its own cell surface. At least a subset of the sialic acid-bearing acceptor molecules are involved in parasite invasion of host cells, an essential step in the life cycle of the parasite. Another trypomastigote surface enzyme that affects host cell invasion is neuraminidase and recent evidence suggests that both trans-sialidase and neuraminidase activities may be expressed by the same proteins on the parasite surface. We describe here the isolation and expression of several members of a trans-sialidase--neuraminidase gene family from T.cruzi. One of the isolated genes does indeed encode a protein with both trans-sialidase and neuraminidase activities, while other members of the gene family encode closely related proteins that express neither enzymatic activity. Chimeric protein constructs combining different portions of active and inactive genes identified a region of the gene necessary for enzymatic activity. Sequence analysis of this portion of the gene revealed a limited number of amino acid differences between the predicted active and inactive gene products.     

Expression of a membrane-bound form of Trypanosoma cruzi trans-sialidase in baculovirus-infected insect cells: a potential tool for sialylation of glycoproteins produced in the baculovirus-insect cells system.

A chimeric protein containing the catalytic domain of Trypanosoma cruzi trans-sialidase, the transmembrane domain of the major envelope glycoprotein of the baculovirus (gp67), and the signal peptide of ecdysteroid glucosyltransferase of the baculovirus was expressed under the control of the very late promoter p10 in baculovirus-infected lepidopteran cells. The recombinant protein was found to be enzymatically active. Three days after infection, equal amounts of activity were found associated to the plasma membrane and in the infection medium, both forms having the same apparent molecular weight and being N-glycosylated. When exogenous galactosylated acceptors (lactose or asialo-alpha1-acid glycoprotein) were added in the culture medium of cells infected with the recombinant baculovirus in the presence of a sialylated donor, a sialylation could be observed. Therefore, we propose the use of trans-sialidase as a potential tool for sialylation of glycoconjugates in the baculovirus-insect cells system.     

Costimulation of host T lymphocytes by a trypanosomal trans-sialidase: involvement of CD43 signaling

Trans-sialidase is a membrane-bound and shed sialidase from Trypanosoma cruzi, the protozoan parasite responsible for Chagas disease. We investigated the role of soluble trans-sialidase on host CD4+ T cell activation. Trans-sialidase activated naive CD4+ T cells in vivo. Both enzymatically active and inactive recombinant trans-sialidases costimulated CD4+ T cell activation in vitro. Costimulation resulted in increased mitogen-activated protein kinase activation, proliferation, and cytokine synthesis. Furthermore, active and inactive trans-sialidases blocked activation-induced cell death in CD4+ T cells from T. cruzi-infected mice. By flow cytometry, inactive trans-sialidase bound the highly sialylated surface Ag CD43 on host CD4+ T cells. Both costimulatory and antiapoptotic effects of trans-sialidases required CD43 signaling. These results suggest that trans-sialidase family proteins are involved in exacerbated host T lymphocyte responses observed in T. cruzi infection.     

Relevance of the diversity among members of the Trypanosoma cruzi trans-sialidase family analyzed with camelids single-domain antibodies

The sialic acid present in the protective surface mucin coat of Trypanosoma cruzi is added by a membrane anchored trans-sialidase (TcTS), a modified sialidase that is expressed from a large gene family. In this work, we analyzed single domain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitory single-domain antibody fragments were obtained by phage display selection, taking advantage of a screening strategy using an inhibition test instead of the classic binding assay. Four single domain antibodies displaying strong trans-sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity-determining region 3 length (17 residues) and have very similar sequences. This result indicates that they likely derived from a unique clone. Probably there is only one structural solution for tight binding inhibitory antibodies against the TcTS used for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes. These results suggest that the presence of a large and diverse trans-sialidase family might be required to prevent the inhibitory response against this essential enzyme and might thus constitute a novel strategy of T. cruzi to evade the host immune system.     

A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage-specific epitope required for invasion of mammalian cells.

When trypomastigotes of T. cruzi emerge from cells of the mammalian host, they contain little or no sialic acids on their surfaces. However, rapidly upon entering the circulation, they express a unique cell surface trans-sialidase activity. This enzyme specifically transfers alpha (2-3)-linked sialic acid from extrinsic host-derived macromolecules to parasite surface molecules, leading to the assembly of Ssp-3, a trypomastigote-specific epitope. The T. cruzi trans-sialidase does not utilize cytidine 5' monophospho-N-acetylneuraminic acid as a donor substrate, but readily transfers sialic acid from exogenously supplied alpha (2-3)-sialyllactose. Monoclonal antibodies that recognize sialic acid residues of Ssp-3 inhibit attachment of trypomastigotes to host cells, suggesting that the unusual trans-sialidase provides Ssp-3 with structural features required for target cell recognition.     

Genomic analyses, gene expression and antigenic profile of the trans-sialidase superfamily of Trypanosoma cruzi reveal an undetected level of complexity

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a highly debilitating human pathology that affects millions of people in the Americas. The sequencing of this parasite's genome reveals that trans-sialidase/trans-sialidase-like (TcS), a polymorphic protein family known to be involved in several aspects of T. cruzi biology, is the largest T. cruzi gene family, encoding more than 1,400 genes. Despite the fact that four TcS groups are well characterized and only one of the groups contains active trans-sialidases, all members of the family are annotated in the T. cruzi genome database as trans-sialidase. After performing sequence clustering analysis with all TcS complete genes, we identified four additional groups, demonstrating that the TcS family is even more heterogeneous than previously thought. Interestingly, members of distinct TcS groups show distinctive patterns of chromosome localization. Members of the TcSgroupII, which harbor proteins involved in host cell attachment/invasion, are preferentially located in subtelomeric regions, whereas members of the largest and new TcSgroupV have internal chromosomal locations. Real-time RT-PCR confirms the expression of genes derived from new groups and shows that the pattern of expression is not similar within and between groups. We also performed B-cell epitope prediction on the family and constructed a TcS specific peptide array, which was screened with sera from T. cruzi-infected mice. We demonstrated that all seven groups represented in the array are antigenic. A highly reactive peptide occurs in sixty TcS proteins including members of two new groups and may contribute to the known cross-reactivity of T. cruzi epitopes during infection. Taken together, our results contribute to a better understanding of the real complexity of the TcS family and open new avenues for investigating novel roles of this family during T. cruzi infection.     

Heterologous expression of a Trypanosoma cruzi surface glycoprotein (gp82) in mammalian cells indicates the existence of different signal sequence requirements and processing

Metacyclic trypomastigotes of Trypanosoma cruzi express a developmentally regulated 82 kDa surface glycoprotein (gp82) that has been implicated in the mammalian cell invasion. When the non-infective epimastigote stage of the parasite was transfected with a vector containing the gp82 gene, an 82 kDa surface glycoprotein, which was indistinguishable from the metacyclic stage protein, was expressed. In contrast, when the same gene was expressed in transfected mammalian cells, although a large amount of protein was produced, it was not imported into the endoplasmic reticulum and glycosylated. This blockage in targeting and processing could be partially compensated for by the addition of a virus haemagglutinin signal peptide to the amino terminus of gp82. Thus, the requirements for membrane protein processing are distinct in mammals and T. cruzi, and an intrinsic feature of the gp82 prevents subsequent sorting to the mammalian cell surface. These results could be useful in the development of new DNA vaccines against T. cruzi employing parasite genes encoding immunodominant surface glycoproteins.     

Synthetic pentapeptide from the B1 chain of laminin promotes B16F10 melanoma cell migration

Laminin is a basement membrane-specific glycoprotein that promotes cell adhesion, proliferation, differentiation, and tumor cell migration. Synthetic peptides from the amino acid sequence deduced from a cDNA clone of the B1 chain of laminin were tested for their ability to promote the migration of B16F10 melanoma cells. A peptide, CDPGYIGSR, that is able to mediate epithelial cell attachment to laminin was found to promote migration, and the constituent pentapeptide YIGSR was also active but to a lesser degree. This nine-amino acid peptide blocked migration of melanoma cells to laminin but had no effect on migration to fibronectin. These data suggest that the cell-binding site and migration site on laminin share a common sequence that is unique to laminin.     

Trypanosoma cruzi prolyl oligopeptidase Tc80 is involved in nonphagocytic mammalian cell invasion by trypomastigotes

Trypanosoma cruzi is an intracellular protozoan parasite able to invade a wide variety of mammalian cells. To have access to the target organs/cells, the parasite must cross the basal laminae and the extracellular matrix (ECM). We previously characterized an 80-kDa proteinase (Tc80) secreted by the infective trypomastigotes that hydrolyzes native collagens and might be involved in infection by degrading ECM components. Here, we present evidence indicating a role for Tc80 in the invasion of nonphagocytic cells. Tc80 was classified as a member of the prolyl oligopeptidase (POP) family of serine proteases and was also found to hydrolyze fibronectin. Selective inhibitors for POP Tc80 were synthesized that blocked parasite entry into cells. Blockage occurred when trypomastigotes were preincubated with irreversible inhibitors but not after host cell preincubation, and the blockage correlated with inhibition of POP Tc80 activity in treated parasites. These data and the enzyme location inside a vesicular compartment close to the flagellar pocket, a specialized domain in endocytosis/exocytosis, strongly suggest a role for POP Tc80 in the maturation of parasite protein(s) and/or, after secretion, in a local action on parasite or host cell/ECM components required for invasion.     

Adhesion of Trypanosoma cruzi Trypomastigotes to Fibronectin or Laminin Modifies Tubulin and Paraflagellar Rod Protein Phosphorylation

Background

The unicellular parasite Trypanosoma cruzi is the causative agent of Chagaś disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored.

Methodology/Principal Findings

Viable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin.

Conclusions/Significance

Herein it is shown, for the first time, that paraflagellar rod proteins and α-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.     

Trypanosoma cruzi trans-sialidase and cell invasion

Trypanosoma cruzi does not synthesize sialic acid but does contain a trans-sialidase, an enzyme capable of transferring sialic acid between host glycoconjugates and the parasite. Sialic acids are negatively charged carbohydrates attached to the terminal non-reducing end of glycoproteins and glycolipids, and their presence can dramatically influence many cell-surface recognition processes. Since sialic acids have been implicated in several ligand-receptor interactions, including the interaction of pathogenic viruses, bacteria and protozoans with their hosts, the expression of trans-sialidase and the acquisition of sialic acid by T. cruzi may be relevant to the interaction of the parasite with the host, and consequently may influence the pathobiology of Chagas disease. In this review, Sergio Schenkman and Daniel Eichinger discuss recent data about the structure and function of T. cruzi trans-sialidase.