Infection of T lymphocytes by Trypanosoma cruzi. Possible role of CD3 and HLA-DR

The mechanisms by which the causative agent of Chagas' disease impair its host's immune response are of paramount importance but poorly understood. Results presented in this paper show for the first time that Trypanosoma cruzi trypomastigotes infect T lymphocytes in vitro and more interestingly in vivo, and that trypomastigotes released from infected cells are infectious. In addition treatment of purified human T lymphocytes with McAb against CD3 and HLA-DR antigens significantly inhibited parasite infection. T. cruzi antigens were detected on the membrane of infected T cells and could therefore represents targets for cytotoxic mechanisms. These results might have important consequences for the understanding of the dramatic disruption of immune response observed during Chagas' disease and more generally provide additional information on T lymphocyte infection by pathogens.     

Specific human antibodies do not inhibit Trypanosoma cruzi oligopeptidase B and cathepsin B, and immunoglobulin G enhances the activity of trypomastigote-secreted oligopeptidase B

Trypanosoma cruzi expresses oligopeptidase B and cathepsin B that have important functions in the interaction with mammalian host cells. In this study, we demonstrated that sera from both chagasic rabbits and humans have specific antibodies to highly purified native oligopeptidase B and cathepsin B. Levels of antibodies to cathepsin B were higher than those observed to oligopeptidase B by absorbance values recorded upon ELISA. We next showed that 90% and 30% of sera from individuals with mucocutaneous leishmaniasis have antibodies that recognize oligopeptidase B and cathepsin B as antigens, respectively. In addition, 55% and 40% of sera from kala-azar patients have antibodies to oligopeptidase B and cathepsin B, respectively. Sera from malaria patients did not recognize the proteases as antigens. Despite high levels of specific antibodies, sera from T. cruzi-infected patients did not inhibit the activities of either oligopeptidase B or cathepsin B. Furthermore, sera or IgG purified from either infected or non-infected individuals enhanced the enzymatic activity of the secreted oligopeptidase B. Oligopeptidase B secreted by trypomastigotes and cathepsin B released upon parasite lysis retain their enzymatic activities and may be associated with Chagas' disease pathogenesis by hydrolyzing host proteins and inducing host immune responses.     

Trypanosoma cruzi-cardiomyocyte interaction: role of fibronectin in the recognition process and extracellular matrix expression in vitro and in vivo

We investigated the involvement of fibronectin (FN) in Trypanosoma cruzi-cardiomyocyte invasion and the extracellular matrix (ECM) components expression during T. cruzi infection in vivo and in vitro. Treatment of trypomastigotes with FN or a synthetic peptide (MRGDS) prior to cardiomyocyte interaction reduced T. cruzi infection, indicating that FN mediates the parasite invasion through its RGD sequence. In murine experimental Chagas' disease, an enhancement of the ECM components was detected in the myocardium during the late acute infection, coinciding with inflammatory infiltrates accumulation. In contrast, highly infected cardiomyocytes displayed a reduction in FN expression in vitro, while laminin spatial distribution was altered. Although it has been demonstrated that cardiomyocytes are able to synthesize cytokines upon T. cruzi infection, our data suggest that matrix remodeling is dependent on cytokines secreted by inflammatory cells recruited in immune response.     

Antibody to Trypanosoma cruzi neuraminidase enhances infection in vitro and identifies a subpopulation of trypomastigotes

A rabbit antibody to the neuraminidase of the infective form of Trypanosoma cruzi identifies a subpopulation of trypomastigotes that expresses neuraminidase. Complement-mediated lysis by the antibody selectively destroys 30 to 40% of the trypomastigotes, supporting the conclusion that the immune antibody binds to a subset of parasites. The trypomastigotes that react with the immune antibody are the only ones expressing neuraminidase because the trypomastigotes that survive complement-mediated lysis are depleted of neuraminidase activity. The enzyme seems to negatively modulate infection in vitro, since infection of host cells by trypomastigotes is enhanced when neuraminidase activity is blocked by antineuraminidase antibody; infection is also enhanced when the infecting trypomastigotes have been depleted of parasites that express neuraminidase. Addition of exogenous neuraminidase (from Vibrio cholerae) to trypomastigotes treated with immune antibody, reverts the enhancement observed when infection takes place in the presence of antibody to T. cruzi neuraminidase only. Addition of V. cholerae neuraminidase in the absence of immune antibodies has no effect on infection. These results show that T. cruzi neuraminidase depresses infection and also suggest that sialic acid is involved in the parasite-host cell interaction. The antibody to T. cruzi neuraminidase recognizes on the surface of live trypomastigotes a set of proteins with high m.w. (165,000 to 200,000) and also two antigens of 79,000 to 82,000. The high m.w. proteins appear to be associated with neuraminidase activity as shown by renaturation experiments of released enzyme fractionated on a sodium dodecyl sulfate-polyacrylamide gel.     

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.     

Characterization of a Trypanosoma cruzi C3 binding protein with functional and genetic similarities to the human complement regulatory protein, decay-accelerating factor.

Evasion of the complement system by microorganisms is an essential event in the establishment of infection. In the case of Trypanosoma cruzi, the causative agent of Chagas disease, resistance to complement-mediated lysis is a developmentally regulated characteristic. Infectious trypomastigotes are resistant to complement-mediated lysis in the absence of immune antibodies, whereas the insect forms (epimastigotes) are sensitive to lysis via the alternative complement pathway. We have purified a developmentally regulated, trypomastigote glycoprotein, gp160, and shown that it has complement regulatory activity. The T. cruzi gp160 restricts complement activation by binding the complement component C3b and inhibiting C3 convertase formation. The protein is anchored in the parasite membrane via a glycosyl phosphatidylinositol linkage, similar to the human complement regulatory protein, decay-accelerating factor. Using anti-gp160 antibodies we have isolated a bacteriophage lgt11 clone expressing a portion of the gp160 gene that shares significant DNA sequence homology with the human DAF gene. These results provide functional, biochemical, and genetic evidence that the T. cruzi gp160 is a member of the C3/C4 binding family of complement regulatory proteins, and that gp160 may provide the infectious trypomastigotes with a means of evading the destructive effects of complement.     

Cell mediated immunity in Chagas' disease. Trypanosoma cruzi antigens induce suppression of the in vitro proliferative response of mononuclear cells.

The partial suppression of the cell-mediated immune response by Trypanosoma cruzi antigens in patients with Chagas' disease is demonstrated in a costimulation assay with T. cruzi antigens and Mycobacterium tuberculosis purified protein derivative (PPD) or Tetanus toxoid (TT). Mononuclear cells from 13 patients with chagasic infection without evidence of heart disease, 10 patients with chagasic cardiomyopathy and 7 healthy blood bank donors were stimulated with antigen A (autoclaved epimastigotes), PPD, TT, PPD + A, PPD + TT and TT + A. The average percentage of suppression induced by costimulation of mononuclear cells with PPD and antigen A was 47.1% in patients with chagasic infection without heart disease (INF), 38.8% in patients with chagasic cardiomyopathy (CDM) and 23.3% in healthy controls. Similar values were observed when living trypomastigotes were used. A costimulatory study with PPD and TT, PPD and A and TT and A was carried out in 8 patients with chagasic infection, in order to evaluate the possibility that this difference could be due to a nonspecific inhibitory effect. The mean suppression induced by TT + PPD was -8.9, with TT + A was 52.7 and with PPD + A was 50.1. The data reported show that T. cruzi antigens induce a specific suppression of the proliferative response of mononuclear cells, that might be relevant to the persistence of the parasite in the host.     

Complement-mediated lysis of Trypanosoma cruzi trypomastigotes by human anti-alpha-galactosyl antibodies.

Antibodies that lyse trypomastigotes in a complement-mediated reaction are believed to be the main participants in the protection against virulent Trypanosoma cruzi. Antibodies with a specificity for alpha-galactosyl-containing determinants--generally called antiGal--were studied to determine their role in the lysis of trypomastigote forms. The titers of antiGal markedly increase in Chagas's disease. In the present study we demonstrate binding of this antibody to T. cruzi and the complement-mediated lysis of trypomastigotes by antiGal. Lysis of metacyclic trypomastigotes by whole Chagasic (Ch) serum or isolated antiGal fractions was equally inhibited by alpha- but not by beta-galactosides. Most of the lytic power of the Ch antiGal as well as of the whole Ch serum was removed by absorption on Synsorb-linked Gal alpha 1, 3Gal beta 1, 4GlcNAc followed by rabbit erythrocyte absorption. The Ch antiGal had a lower affinity for melibiose bound to agarose than for the trisaccharide linked to Synsorb, and was several times more effective in the immunolysis of trypomastigotes than the corresponding antiGal from normal human serum. Lytic antibodies were partly absorbed by Serratia marcescens but not by Escherichia coli O111. A human volunteer immunized with an S. marcescens vaccine elicited a specific antiGal response that was lytic to trypomastigotes (70% lysis). We suggest that in vivo high-affinity antiGal antibody clones, as occur in Ch patients, may significantly contribute to the destruction of the parasite, whereas low-affinity antiGal clones are much less effective in the protection against T. cruzi infection.     

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.     

Trypanosoma cruzi suppresses the ability of activated human lymphocytes to enter the cell cycle

Using an in vitro system in which human peripheral blood mononuclear cells stimulated with the T-lymphocyte mitogen phytohemagglutinin were cocultured with Trypanosoma cruzi trypomastigotes, we demonstrated a marked accumulation of cells in G0/G1a during the 72-hr observation period whereas mitogen-stimulated cells in parallel cultures lacking the parasite readily entered the G1b, S, and G2/M phases. These results suggest that the immunological alterations that occur in acute Chagas' disease may result from an early cell cycle blockade induced by the parasite.     

Trypanosoma cruzi proline racemases are involved in parasite differentiation and infectivity

Polyclonal lymphocyte activation is one of the major immunological disturbances observed after microbial infections and among the primary strategies used by the parasite Trypanosoma cruzi to avoid specific immune responses and ensure survival. T. cruzi is the insect-transmitted protozoan responsible for Chagas' disease, the third public health problem in Latin America. During infection of its mammalian host, the parasite secretes a proline racemase that contributes to parasite immune evasion by acting as a B-cell mitogen. This enzyme is the first described eukaryotic amino acid racemase and is encoded by two paralogous genes per parasite haploid genome, TcPRACA and TcPRACB that give rise, respectively, to secreted and intracellular protein isoforms. While TcPRACB encodes an intracellular enzyme, analysis of TcPRACA paralogue revealed putative signals allowing the generation of an additional, non-secreted isoform of proline racemase by an alternative trans-splicing mechanism. Here, we demonstrate that overexpression of TcPRAC leads to an increase in parasite differentiation into infective forms and in its subsequent penetration into host cells. Furthermore, a critical impairment of parasite viability was observed in functional knock-down parasites. These results strongly emphasize that TcPRAC is a potential target for drug design as well as for immunomodulation of parasite-induced B-cell polyclonal activation.     

Acute heart inflammation: ultrastructural and functional aspects of macrophages elicited by Trypanosoma cruzi infection

The heart is the main target organ of the parasite Trypanosoma cruzi , the causal agent of Chagas' disease, a significant public health issue and still a major cause of morbidity and mortality in Latin America. During the acute disease, tissue damage in the heart is related to the intense myocardium parasitism. To control parasite multiplication, cells of the monocytic lineage are highly mobilized. In response to inflammatory and immune stimulation, an intense migration and extravasation of monocytes occurs from the bloodstream into heart. Monocyte differentiation leads to the formation of tissue phagocytosing macrophages, which are strongly activated and direct host defence. Newly elicited monocyte-derived macrophages both undergo profound physiological changes and display morphological heterogeneity that greatly differs from originally non-inflammatory macrophages, and underlie their functional activities as potent inflammatory cells. Thus, activated macrophages play a critical role in the outcome of parasite infection. This review covers functional and ultrastructural aspects of heart inflammatory macrophages triggered by the acute Chagas' disease, including recent discoveries on morphologically distinct, inflammation-related organelles, termed lipid bodies, which are actively formed in vivo within macrophages in response to T. cruzi infection. These findings are defining a broader role for lipid bodies as key markers of macrophage activation during innate immune responses to infectious diseases and attractive targets for novel anti-inflammatory therapies. Modulation of macrophage activation may be central in providing therapeutic benefits for Chagas' disease control.     

Trypanosoma cruzi: ultrastructural studies of adhesion, lysis and biofilm formation by Serratia marcescens

A few days after blood meal the number of bacteria in the anterior midgut (stomach) of Rhodnius prolixus, a vector of Trypanosoma cruzi, the causative agent of Chagas' disease, increases dramatically. Many of the bloodstream trypomastigotes of the pathogenic protozoan as well as ingested erythrocytes are lysed in the stomach. Incubation of T. cruzi with Serratia marcescens variant SM365, lead to parasite lysis. In the present study, this bacterium rapidly adhered to the protozoan surface through d-mannose recognizing fimbriae and rapidly induced its complete lysis. In contrast, the DB11 variant of the same bacterial species did not adhere and did not induce protozoan lysis. Scanning and transmission electron microscopy revealed that following bacteria-protozoan attachment there is an assembly of long filamentous structures, identified as a biofilm, which connect the protozoan to the bacteria forming bacterial clusters. We conclude that parasite lysis and biofilm formation mechanisms are important for understanding parasite-microbiota interactions in the gut of insect vectors of trypanosomatids.     

Development of an Aptamer-Based Concentration Method for the Detection of Trypanosoma cruzi in Blood

Trypanosoma cruzi, a blood-borne parasite, is the etiological agent of Chagas disease. T. cruzi trypomastigotes, the infectious life cycle stage, can be detected in blood of infected individuals using PCR-based methods. However, soon after a natural infection, or during the chronic phase of Chagas disease, the number of parasites in blood may be very low and thus difficult to detect by PCR. To facilitate PCR-based detection methods, a parasite concentration approach was explored. A whole cell SELEX strategy was utilized to develop serum stable RNA aptamers that bind to live T. cruzi trypomastigotes. These aptamers bound to the parasite with high affinities (8-25 nM range). The highest affinity aptamer, Apt68, also demonstrated high specificity as it did not interact with the insect stage epimastigotes of T. cruzi nor with other related trypanosomatid parasites, L. donovani and T. brucei, suggesting that the target of Apt68 was expressed only on T. cruzi trypomastigotes. Biotinylated Apt68, immobilized on a solid phase, was able to capture live parasites. These captured parasites were visible microscopically, as large motile aggregates, formed when the aptamer coated paramagnetic beads bound to the surface of the trypomastigotes. Additionally, Apt68 was also able to capture and aggregate trypomastigotes from several isolates of the two major genotypes of the parasite. Using a magnet, these parasite-bead aggregates could be purified from parasite-spiked whole blood samples, even at concentrations as low as 5 parasites in 15 ml of whole blood, as detected by a real-time PCR assay. Our results show that aptamers can be used as pathogen specific ligands to capture and facilitate PCR-based detection of T. cruzi in blood.     

Exacerbation of experimental Trypanosoma cruzi infection in mice by concomitant malaria.

The effect of malaria on the chronic phase of Chagas' disease was investigated in mice. The animals were given Plasmodium bergheri-infected red blood cells 2 to 12 months after their initial inoculation with trypomastigotes of 3 different strains of Trypanosoma cruzi (Y. CL and Gilmar). in all the experiments carried out with one of the strains (CL), a somewhat variable but always considerable percentage of mice (average 39%) relapsed in to the acute phase of Chagas' disease. This relapse was characterized by a significant increase in the number of circulating trypomastigotes. Recrudescence was observed also with a 2nd strain of T. cruzi (Gilmar), which is similar in many aspects to the CL strain, e.g. the morphology of blood stages, curved of parasitemia and susceptibility to antibodies in vitro. In mice whose chronic phase was induced by trypomastigotes of the Y strain, malaria infections did not induce a typical acute phas with high parasitemia by T. cruzi. Bloodstream forms of Y parasites differ from those of CL and Gilmar strains morphologically as well as immunologically, i.e. only the Y strain is easily agglutinated and partly inactivated by specific immune serum. In light of this and other known characteristics of the strains used in the present work, the author speculates on mechanisms which allow malaria infections selectively to suppress acquired host resistance to certain strains of T. cruzi.     

Infection and invasion mechanisms of Trypanosoma cruzi in the congenital transmission of Chagas' disease: a proposal

Chagas' disease is produced by the haemophlagelated protozoan Trypanosoma cruzi and transmitted by haematophages insects such as Triatoma infestans (vinchuca). Due to vector control, congenital transmission gains importance and is responsible for the presence and expansion of this disease in non-endemic areas. The mechanisms of congenital infection are uncertain. It has been suggested that the parasite reaches the fetus through the bloodstream by crossing the placental barrier, and that congenital Chagas' disease is the result of complex interactions between the immune response, placental factors, and the parasite's characteristics. We review the cellular and molecular mechanisms of infection and invasion of the parasite and how immune and placental factors may modulate this process. Finally, we propose a possible model for the vertical transmission of Chagas' disease.     

The classical activation pathway of the human complement system is specifically inhibited by calreticulin from Trypanosoma cruzi

The high resistance of Trypanosoma cruzi trypomastigotes, the causal agent of Chagas' disease, to complement involves several parasite strategies. In these in vitro studies, we show that T. cruzi calreticulin (TcCRT) and two subfragments thereof (TcCRT S and TcCRT R domains) bind specifically to recognition subcomponents of the classical and lectin activation pathways (i.e., to collagenous tails of C1q and to mannan-binding lectin) of the human complement system. As a consequence of this binding, specific functional inhibition of the classical pathway and impaired mannan-binding lectin to mannose were observed. By flow cytometry, TcCRT was detected on the surface of viable trypomastigotes and, by confocal microscopy, colocalization of human C1q with surface TcCRT of infective trypomastigotes was visualized. Taken together, these findings imply that TcCRT may be a critical factor contributing to the ability of trypomastigotes to interfere at the earliest stages of complement activation.     

Novel strategy in Trypanosoma cruzi cell invasion: implication of cholesterol and host cell microdomains

Trypanosoma cruzi, the etiological agent of Chagas' disease, is an obligatory intracellular parasite in the mammalian host. In order to invade a wide variety of mammalian cells, T. cruzi engages parasite components that are differentially expressed among strains and infective forms. Because the identification of putative protein receptors has been particularly challenging, we investigated whether cholesterol and membrane rafts, sterol- and sphingolipid-enriched membrane domains, could be general host surface components involved in invasion of metacyclic trypomastigotes and extracellular amastigotes of two parasite strains with distinct infectivities. HeLa or Vero cells treated with methyl-beta-cyclodextrin (MbetaCD) are less susceptible to invasion by both infective forms, and the effect was dose-dependent for trypomastigote but not amastigote invasion. Moreover, treatment of parasites with MbetaCD only inhibited trypomastigote invasion. Filipin labeling confirmed that host cell cholesterol concentrated at the invasion sites. Binding of a cholera toxin B subunit (CTX-B) to ganglioside GM1, a marker of membrane rafts, inhibited parasite infection. Cell labeling with CTX-B conjugated to fluorescein isothiocyanate revealed that not only cholesterol but also GM1 is implicated in parasite entry. These findings thus indicate that microdomains present in mammalian cell membranes, that are enriched in cholesterol and GM1, are involved in invasion by T. cruzi infective forms.     

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.     

Attachment of Trypanosoma cruzi trypomastigotes to receptors at restricted cell surface domains

We have used glutaraldehyde-fixed target cells to study the attachment phase of cell invasion by live trypomastigotes of Trypanosoma cruzi, and determined that attachment is polarized and receptor-mediated. T. cruzi trypomastigotes bind much less efficiently to confluent epithelial cells, which are polarized, than to sparse epithelial cells. When the tight junctions of confluent epithelial cells are disrupted by removing Ca2+ from the incubation medium before glutaraldehyde fixation, binding of T. cruzi increases. T. cruzi also shows preference for attachment underneath cells or to the edges of cells. The binding occurs within a few minutes, is saturable, and is influenced by the parasite developmental stage. Fab fragment derived from monoclonal antibodies that immunoprecipitate a 160-kDa molecule present only on the surface of trypomastigotes inhibit adhesion to fixed and live cells. Future characterization of the target cell receptors for this molecule and the use of fixed target cells should facilitate studies of the mechanisms involved in the initial interaction of T. cruzi with its host cells.