A superfamily of Trypanosoma cruzi surface antigens

Several genes o f Trypanosoma cruzi encode surface antigens that include an amino acid motif that is conserved among bacterial neurominidases. Oscar Campetella, Daniel Sdnchez, Juan Jose Cazzulo and Alberto Carlos Frasch here suggest grouping these gene families in a superfamily.     

Signaling pathways that regulate Trypanosoma cruzi infection and immune response

Current understanding of key cellular pathways, which are activated by the interaction between T. cruzi and host immunity, is crucial for controlling T. cruzi infection and also for limiting the development of the immunopathological symptoms of Chagas´ disease. Here, we focus on recent advances in the knowledge of modulation of innate receptors such as TLRs and NLRs, especially NLRP3, by T. cruzi in different cells of the immune system. On the other hand, the modulation of macrophage activation may be instrumental in allowing parasite persistence and long-term host survival. In this sense, we discuss the importance of the metabolism of two amino acids: L-arginine and tryptophan, and evaluate the role of iNOS, arginase and IDO enzymes in the regulation of innate and adaptive immune response during this infection; and, finally, we also discuss how T. cruzi exploits the AhR, mTOR and Wnt signaling pathways to promote their intracellular replication in macrophages, thus evading the host's immune response.     

Trypanosoma cruzi invade a mammalian epithelial cell in a polarized manner

We have determined that parasite entry into host cells can be influenced by cell polarity using a DNA probe to quantitate the infection of cultured Madin-Darby canine kidney (MDCK) epithelial cells by Trypanosoma cruzi, the agent of Chagas' disease. Confluent MDCK cells are polarized, with their plasma membrane separated by tight junctions into two domains, apical and basolateral. We show that T. cruzi forms corresponding to the insect infective stages (metacyclics) and the vertebrate blood stages (trypomastigotes) enter confluent MDCK cells preferentially through their basolateral domains. Sparsely plated MDCK cells are less polarized and are better infected than confluent cells. Scanning electron microscopy showed that 92% +/- 4% of the parasites entered at the edges of cells.     

The cAMP receptor protein of Trypanosoma cruzi

The widest used method to determine cAMP-binding activity in cell-free extracts (Gilman, A. G. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 305-312) underestimated by about 10-fold the total amount of [3H]cAMP bound by crude extracts of cultured forms of Trypanosoma cruzi (epimastigotes), when compared with the results obtained by the modified Millipore filter technique described by Doskeland and Ueland (Doskeland, S. O., and Ueland, P.M. (1977) Biochem. J. 165, 561-573). After column chromatography on DEAE-Sephacel, the cAMP-binding activity eluted as a single symmetrical peak at about 210 mM NaCl, totally separated from two peaks of cAMP-independent phosphotransferase activities which eluted, respectively, at 90 and 270 mM NaCl. These two protein kinases showed similar specificities for exogenous substrates, preferring in this order: protamine greater than casein greater than histone H2b. Photoaffinity labeling of cell-free extracts with 8-azido[32P]adenosine 3':5'-monophosphate and autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis specifically labeled only one protein of Mr = 62,000. From these results it is concluded that T. cruzi epimastigotes possess one single cAMP-binding protein of monomeric Mr = 62,000, not associated with a phosphotransferase activity and probably very different in nature to known regulatory subunits of protein kinases, given the results obtained with the Millipore filtration technique.     

TcZFP1: a CCCH zinc finger protein of Trypanosoma cruzi that binds poly-C oligoribonucleotides in vitro

We have identified two zinc finger proteins of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease in humans. These proteins, named tcZFP1 and tcZFP2, share the unusual zinc finger motif (CCCH) found in a diverse range of RNA-binding proteins involved in various aspects of the control of cell homeostasis and differentiation. We report here the functional expression of a recombinant tcZFP1, and the relative affinity and stability of the specific complexes formed between the protein and synthetic oligoribonucleotides containing C-rich sequences.     

TcDJ1, a putative mitochondrial DnaJ protein in Trypanosoma cruzi

A full length cDNA encoding a novel Trypanosoma cruzi DnaJ protein was cloned and characterized. The 324 amino acid protein encoded by the cDNA (TcDJ1) displays a characteristic J-domain, but lacks the Gly-Phe and zinc finger regions present in some other DnaJ proteins. Relative to four other T. cruzi DnaJ proteins, TcDJ1 has an amino terminal extension containing basic and hydroxylated residues characteristic of mitochondrial import peptides. A T. cruzi transfectant expressing epitope-tagged TcDJ1 was generated and subcellular fractions were produced. Western blot analysis revealed that the protein has a molecular mass of 29 kDa and is found in the mitochondrial fraction. The expression of TcDJ1 is developmentally regulated since the levels of both mRNA and protein are much higher in epimastigotes (replicative form) than in metacyclic trypomastigotes (infective form). Thus it may participate in mitochondrial biosynthetic processes in this organism.     

The innate immune response to Trypanosoma cruzi infection

Trypanosoma cruzi infection is a major public health problem in Latin America. The host innate immune system plays a pivotal role in the recognition of T. cruzi infection and the subsequent development of adaptive immunity. In this review, we focus on the TLR-dependent and -independent innate immune responses to T. cruzi.     

Antigen-specific Th1 but not Th2 cells provide protection from lethal Trypanosoma cruzi infection in mice

Infection with Trypanosoma cruzi results in the development of both type 1 and type 2 patterns of cytokine responses during acute and chronic stages of infection. To investigate the role of Th1 and Th2 subsets of CD4(+) T cells in determining the outcome of T. cruzi infection in mice, we have developed T. cruzi clones that express OVA and have used OVA-specific TCR-transgenic T cells to generate OVA-specific Th1 and Th2 cells. BALB/c mice receiving 10(7) OVA-specific Th1 cells and then challenged with OVA-expressing T. cruzi G-OVA.GPI showed significantly lower parasitemia and increased survival in comparison to mice that received no cells. In contrast, recipients of OVA-specific Th2 cells developed higher parasitemias, exhibited higher tissue parasitism and inflammation, and had higher mortality than recipients of Th1 cells after infection with T. cruzi G-OVA.GPI. Mice receiving a mixture of both Th1 and Th2 OVA-specific cells also were not protected from lethal challenge. The protective effect of the OVA-specific Th1 cells was OVA dependent as shown by the fact that transfer of OVA-specific Th1 or Th2 cells failed to alter the course of infection or disease in mice challenged with wild-type T. cruzi. Immunohistochemical analysis of OVA-specific Th1 and Th2 cells at 4, 15, and 30 days postinfection revealed the persistence and expansion of these cells in mice challenged with T. cruzi G-OVA.GPI but not in mice infected with wild-type T. cruzi. We conclude that transfer of Ag-specific Th1 cells but not Th2 cells protect mice from a lethal infection with T. cruzi.     

Mucosal immunization with a ricin toxin B subunit-rotavirus NSP4 fusion protein stimulates a Th1 lymphocyte response

The castor-oil plant Ricinus communis A-B dimeric toxin B subunit (RTB) was genetically linked at its N-terminus with a 90 amino acid peptide from simian rotavirus SA-11 non-structural protein NSP4(90) and produced in Escherichia coli BL21 cells. Biologically active recombinant NSP4(90)-RTB fusion protein was shown to bind glycoprotein asialofetuin receptor molecules in an in vitro enzyme-linked immunosorbent assay (ELISA). Oral inoculation of the purified NSP4(90)-RTB ligand-antigen fusion protein delivered the chimeric protein to intestinal epidermal cells for mucosal immunization against rotavirus infection. Mice fed the NSP4(90)-RTB fusion protein generated higher humoral and intestinal antibody titers than mice inoculated with NSP4(90) alone. Titers of serum IgG2a antibodies were significantly higher than IgG1 titers suggesting a dominant Th1 lymphocyte immune response. ELISA measurement of cytokines secreted from splenocyte isolated from immunized mice confirmed NSP4(90)-RTB fusion protein stimulates a strong Th1 cell-mediated immune response. The experimental results demonstrate that the ricin toxin B subunit N-terminus can be used as a site for delivery of virus antigens to the gut associated lymphoid tissues for RTB-mediated immune stimulation of antiviral mucosal immune responses.     

The vaccine potential of cell surface glycoproteins from Trypanosoma cruzi

An experimental Trypanosoma cruzi 90 kDa cell surface glycoprotein (GP90) vaccine, previously shown to be protective in mice is similarly effective in marmosets (Callithrix jacchus jacchus). Protection in the mouse is completely dependent on the adjuvant saponin and immunological studies confirm that GP90 is intrisically poorly immunogenic. Both specific antibody and cell mediated immunity are potentiated strongly by saponin and the resulting protective immunity is long lasting (six months). It is effective against the naturally infective, insect-metacyclic, form and a range of heterologous T. cruzi strains including a low mouse passage human isolate. Sterile immunity (that is, complete elimination of parasites) was not however, achieved. Evidence is presented that the levels of tissue damage associated with acute infection, as measured by production of auto anti-tissue immunoglobulins, are significantly reduced in GP90-immunized mice. These and other results are discussed in terms of the desired characteristics for vaccine use of T. cruzi antigens.     

Trypanosoma cruzi: identification of a cell surface polysaccharide.

An immunogenic polysaccharide prepared by phenol-water extraction of Trypanosoma cruzi was characterized by polyacrylamide gel electrophoresis and molecular sieve chromatography. The polysaccharide was shown to be a cell surface constituent by adsorption of rabbit anti-polysaccharide serum with live culture forms of the protozoa. The cell surface localization of the antigen was visualized using fluorescein- and ferritin-conjugated antibodies.     

The detection of a spectrin-like protein in Trypanosoma cruzi with a polyclonal antibody

A rabbit serum raised against sheep erythrocyte spectrin stained mainly the flagella of epimastigote forms of the parasitic protozoan Trypanosoma cruzi. In Western blots of T. cruzi proteins solubilized with Nonidet P-40 it recognized mainly a polypeptide doublet with an apparent molecular weight of about 240 kDa, while a pre-immune rabbit serum and a tubulin monoclonal antibody did not. The results are consistent with the idea that a spectrin-like protein may be involved in cytoskeleton-membrane interactions in flagella of T. cruzi.     

DM-GRASP, a novel immunoglobulin superfamily axonal surface protein that supports neurite extension

We have identified a 95 kd cell surface protein, DM-GRASP, that is expressed on a restricted population of axons. Its expression begins early in chick embryogenesis, and within the spinal cord it is localized to axons in the dorsal funiculus, midline floorplate cells, and motoneurons. Antibodies to DM-GRASP impair neurite extension on axons, and purified DM-GRASP supports neurite extension from chick sensory neurons. We have cloned and sequenced the cDNA corresponding to this protein and find that it is a new member of the immunoglobulin superfamily of adhesion molecules. Consequently we have named this protein DM-GRASP, since it is an immunoglobulin-like restricted axonal surface protein that is expressed in the dorsal funiculus and ventral midline of the chick spinal cord.     

Trypanosoma cruzi calreticulin is a lectin that binds monoglucosylated oligosaccharides but not protein moieties of glycoproteins

Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse-chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin-cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-beta-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin-cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.     

Binding specificity of mannose-specific carbohydrate-binding protein from the cell surface of Trypanosoma cruzi

The sugar binding specificity of the recently described mannose-specific carbohydrate-binding proteins (CBP) isolated to homogeneity from both the epimastigote and trypomastigote stages of the pathogenic protozoa Trypanosoma cruzi has been studied by quantitative hapten inhibition of the biotinylated CBPs to immobilized thyroglobulin using model oligosaccharides. The results clearly show a differential specificity toward high-mannose glycans between the CBPs from the two developmental stages. Thus, the isolated CBP from epimastigotes exhibited stronger affinity for higher mannose oligomers containing the Manalpha1-2Manalpha1-6Manalpha1-6 structure. Its affinity decreased, as did the number of mannose residues on the oligomer or removal of the terminal Manalpha1-2-linked mannose. By contrast the CBP isolated from the trypomastigote stage showed about 400-fold lower avidity than the epimastigote form, and contrary to it, it was slightly more specific toward Man5GlcNAc than Man9GlcNAc. Analysis of the interaction of epimastigote-Man-CBP with its ligands by UV difference spectroscopy indicates the existence of an extended binding site in that protein with a large enthalpic contribution to the binding. The thermodynamic parameters of binding were obtained by isothermal titration calorimetry and been found that the DeltaH values to be in good agreement with the van't Hoff values. The binding reactions are mainly enthalpically driven and exhibit enthalpy-enthropy compensation. In addition, analysis of the high-mannose glycans from different parts of the digestive tract of the reduviid insect vector of T. cruzi suggest a role of the CBP in the retention of the epimastigote stage in the anterior portion of the gut.     

The cell surface of Trypanosoma cruzi: a fracture-flip, replica-staining label-fracture survey

Fracture-flip and replica-staining label-fracture were used to study the nanoanatomy and topochemistry of the cell surface of Trypanosoma cruzi. Fracture-flip surface images differentiate the three main developmental stages of T. cruzi. Epimastigotes display a smooth surface, except the cytostome which appears as a clearly demarcated, raised, roughly textured platform. Amastigotes and trypomastigotes are covered by numerous surface particles with diameters ranging from 10 to 20 nm and 15 to 30 nm, respectively. Labeling of concanavalin A receptors showed that the surfaces of amastigotes and trypomastigotes were labeled, with amastigotes displaying the highest density of gold particles. In contrast, epimastigotes were sparsely labeled with exception of the cytostome, where a higher density of labeling coincided with the raised platform seen in fracture-flipped specimens, and with the particle-free area exposed on fracture faces. Labeling of epimastigotes by Ricinus communis I and Wistaria floribunda lectins showed that surface receptors for these lectins were absent from the cytostome.     

The targets of the lytic antibody response against Trypanosoma cruzi

Trypanosoma cruzi trypomastigotes, but not epimastigotes, are normally resistant to the lytic effects of complement from vertebrate hosts susceptible to infection. This resistance facilitates parasite survival and infectivity. During the course of chronic infections, however, the vertebrate hosts produce antibodies that render the trypomastigotes sensitive to lysis, primarily via the alternative complement cascade and amplified by the classical pathway. Here, Greice Krautz, Jessica Kissinger and Antoniana Krettli summarize research on lytic antibodies, and on their respective target(s) on the T. cruzi surface. These targets are useful in tests aimed at the diagnosis of chronic Chagas disease for control of cure after specific treatment and for vaccine development.     

A Trypanosoma cruzi monoclonal antibody that recognizes a superficial tubulin-like antigen

A monoclonal antibody (MAB 10), obtained from mice infected with Trypanosoma cruzi, was found to recognize a superficial antigen in living or fixed parasites. It reacted more strongly with T. cruzi than with related parasites such as T. brucei and Leishmania. In immunoblots it recognized a single trypanosoma polypeptide and also brain tubulin, both of which had the same electrophoretic mobility. Further analysis suggested that the alpha-tubulin subunit contained the epitope recognized by MAB 10. These results suggest that a surface tubulin-like protein is present is T. cruzi.