Stimulatory effects of leukotriene B4 on macrophage association with and intracellular destruction of Trypanosoma cruzi

The effects of leukotriene B4 (LTB4) on mouse peritoneal macrophage (MPH) association with and destruction of Trypanosoma cruzi were studied. The presence of 10(-8) to 10(-6) M LTB4 in co-cultures of MPH and T. cruzi enhanced their association (a term meaning surface binding and internalization), as evidenced by increases in the percentage of MPH associating with trypanosomes and the number of parasites per 100 MPH. Pretreatment of either parasites or MPH with LTB4 increased their association with the untreated counterpart, suggesting that the enhancement was a composite of effects on both cells. The effect of LTB4 on MPH was reversible, because normal levels of MPH-parasite association were recorded 60 min after the LTB4 pretreatment. However, the enhancement was demonstrable after the MPH were incubated with LTB4 for up to 24 hr, indicating that the effect lasted if LTB4 was present. Pretreatment with LTB4 also increased the capacity of MPH to take up glutaraldehyde-killed T. cruzi or latex beads, suggesting that LTB4 stimulated phagocytosis. Pretreatment of rat heart myoblasts--which are not phagocytic--with LTB4 also increased parasite association, suggesting that phagocytic ability was not an absolute requirement for production of the enhancement and that LTB4-induced alterations of the cell membrane facilitating parasite invasion may also be involved. An effect of LTB4 on MPH cytotoxicity was denoted by an increased rate of intracellular parasite killing. Two inhibitors of guanylate cyclase abrogated the enhancing effect of LTB4, suggesting that increased MPH levels of cyclic GMP--known to be increased by LTB4--mediated the effect. Because inflammatory cells, such as are found in acute chagasic lesions, are known to produce increased amounts of leukotrienes, the stimulatory effects of LTB4 could contribute to host defense against T. cruzi infection.     

Effects of treatment of trypomastigote forms of Trypanosoma cruzi or host cells with ethidium bromide on cell infection and intracellular fate of the parasite

The effects of treatment of virulent blood forms of Trypanosoma cruzi with ethidium bromide (EtBr)-an intercalating drug that inhibits DNA synthesis-on parasite association with (a term to mean surface binding plus internalization) and multiplication within different types of host cells were investigated. EtBr markedly reduced the extent of T. cruzi association with Vero cells or rat heart myoblasts (RHM) as evidenced by significant decreases in both the number of flagellates per cell and the percentage of infected cells with respect to control values obtained with organisms treated with medium alone. In contrast, treatment of Vero cells with EtBr had no significant consequence on the extent of cell-T. cruzi association and did not affect the capacity of the parasites to transform into amastigotes and multiply intracellularly. Very few organisms were able to gain access to the cytoplasms of the host cells after treated with 1 X 10(-5) M EtBr but these were virtually unable to multiply intracellularly. Parasites treated with 1 X 10(-6) M EtBr multiplied at a slower rate than medium-treated organisms. Unlike untreated trypomastigotes, parasites treated with 1 X 10(-5) M EtBr were unable to transform into amastigotes in a cell-free medium that supported the growth of untreated organisms. A marked reduction in the rate of amastigote multiplication was seen in cells with an established infection when they were treated with EtBr. These results suggest that ongoing DNA synthesis by T. cruzi is required for it to effectively bind and infect host cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Trypanosoma cruzi: parasite-induced release of lysosomal enzymes by human polymorphonuclear leukocytes

The release of beta-glucuronidase and lysozyme from human polymorphonuclear leukocytes (PMN) engaged in phagocytosis and lysis of Trypanosoma cruzi epimastigotes was studied in the presence or absence of chagasic serum. Lysosomal enzyme release was enhanced when parasites were sensitized with serum from a chronic Chagas' patient, increased up to 3 hr of incubation at 28 C, and depended on the PMN:parasite ratio. The release of lysosomal enzymes was determined by the presence of 2 mM cyanide, 2 microM azide, 3 mM amobarbital, and 1 mM phenylbutazone. These drugs inhibited the killing of sensitized T. cruzi by interfering with the oxidative microbicidal mechanisms of PMN without affecting the uptake of the parasites. Lysosomal enzyme release occurred in the presence of cyanide and azide, indicating that in these cases the enzymatic release was unrelated to the killing of the parasites. Amobarbital and phenylbutazone, which stabilize PMN membranes, inhibited the release of beta-glucuronidase and lysozyme by PMN. The addition of 10 micrograms/ml of cytochalasin B inhibited the phagocytosis and killing of sensitized T. cruzi by PMN but increased the enzymatic release by effector cells. Since cytochalasin B did not affect the close contact between PMN and parasites, it appears that the enzymes released to the extracellular milieu were not toxic to noningested parasites. Furthermore, the lysosomal enzymes did not lyse bystander unsensitized parasites. Therefore, the release of lysosomal enzymes during the interaction of T. cruzi epimastigotes and PMN seems to be related to the triggering event of the phagocytic process and does not bear a cause-effect relationship with parasite death.     

Does concanavalin A treatment of host cells enhance or inhibit their association with Trypanosoma cruzi trypomastigotes?

Concanavalin A (Con A) has frequently been used as a probe of cell surface molecules that mediate cell-cell interactions. There have been conflicting reports that Con A treatment of vertebrate host cells can subsequently increase or reduce the level of association (surface attachment and penetration) of Trypanosoma cruzi trypomastigotes with these cells. In this work, we have established that the type of effect produced by treatment of host cells with Con A depended on whether or not fetal bovine serum was present during the interaction of trypomastigotes and host cells; Con A treatment reduced host cell association with T. cruzi in the presence of the serum, but increased it when the serum was absent. In addition, ovalbumin, a glycoprotein with a high mannose content and the ability to specifically bind to Con A, was found capable of altering the effect of Con A treatment of host cells on parasite association levels in a manner similar to fetal bovine serum. These results suggested that glycoproteins present in the serum can modulate the effect of Con A, possibly by blocking free sites remaining on the Con A molecules which had bound to the surface of host cells. If free binding sites on the Con A molecule remained unblocked, they could conceivably form bridges between host cells and parasites resulting in an artifactual enhancement of their level of association in serum-free medium.     

In vitro multiplication of Toxoplasma gondii and Trypanosoma cruzi in mouse, rat, and hamster astrocytes

The infection and multiplication of Toxoplasma gondii and Trypanosoma cruzi were compared in primary cultures of white rat, mouse and hamster astrocytes. These cells were cultured on cover slides and infected with T. gondii tachyzoites or T. cruzi blood trypomastigotes. Results show that hamster astrocytes are more susceptible to the multiplication of both parasites than rat and mouse cells. There was no statistical difference between the T. gondii infection in rat and mouse astrocytes (p < 0.05), and this suggests an important role of other mechanisms or cells in the white rat natural resistance to this parasite. Because the hamster astrocytes are less resistant to these parasites multiplication and not necessarily to the invasion, any difference observed could be due to an intracellular effect: hamster brain astrocytes favor survival and multiplication of these parasites.     

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.     

Human chagasic serum contains antibodies capable of inhibiting Trypanosoma cruzi egress from tissue culture cells

Previous studies at our laboratory have shown that an antibody (antiegressin) present in the serum of chronically infected mice is capable of inhibiting the egress of Trypanosoma cruzi from infected BALB/c fibroblasts. We have used this in vitro system to evaluate whether human chagasic serum is also capable of inhibiting T. cruzi egress. BALB/c fibroblasts were infected with tissue culture-derived parasites. Five-percent solutions of the individual human serum samples in culture medium were added to the wells, and the number of parasites released was determined at day 5 after infection. The cells cultured with serum from infected individuals released between 37% and 72% fewer parasites than those cultured with control serum. A similar reduction in parasite egress resulted from incubation with the protein-A purified IgG fraction from 3 of these human samples. Immunocytochemical staining employing antineuraminidase antibodies supported the notion that the reduction in parasite levels is due to inhibition at the point of parasite egress. These results indicate that human serum of individuals infected with T. cruzi is capable of inhibiting release of the parasite from infected tissue culture cells and that the phenomenon of egress-inhibition may be relevant during infection of human subjects.     

The role of Ca2+ in the process of cell invasion by intracellular parasites

In order to replicate, many parasites must invade host cells. Changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) of different parasites and tissue culture cells during their interaction have been studied. An increase in cytosolic Ca(2+) in Trypanosoma cruzi trypomastigotes occurs after association of the parasites with host cells. Ca(2+) mobilization in the host cells also takes place upon contact with T. cruzi trypomastigotes, Leishmania donovani amastigotes or Plasmodium falciparum merozoites. When Ca(2+) transients are prevented by intracellular Ca(2+) chelators, a decrease in parasite association to host cells is observed. This reveals the importance of [Ca(2+)](i) in the process of parasite-host cell interaction, as discussed here by Roberto Docampo and Silvia Moreno.     

Trypanosoma cruzi: productive infection is not allowed by chorionic villous explant from normal human placenta in vitro

We hypothesize that a sustained infection of Trypanosoma cruzi into placental tissue might be diminished. Human placental chorionic villi and VERO cells as controls were co-cultured with T. cruzi. Parasites occupied 0.0137% at 3h, 0.0224% (24h), and 0.0572% (72 h) of the total chorionic villi area analyzed and some few placental samples were negative to parasite DNA, whereas 52% of VERO cells were infected at 3h and parasites occupied 0.57%, at 24h the parasite area was of 2.78% and at 72 h was of 3.32%. There were no live parasites in placenta-T. cruzi culture media at 72 h of co-culture. There were significantly increased dead parasites when T. cruzi was treated with unheated culture media coming from placental explants and fewer dead parasites when pre-heated culture media were employed. CONCLUSION: Low productive infection by T. cruzi into placental tissue associated with no viable parasites in the culture media partially due to placental thermo labile substances.     

The natural heterogeneity of trypanosoma cruzi: Biological and medical implications

Trypanosoma cruzi is a heterogeneous group of parasites. The imposition of natural or artificial pressures can result in the selection of subsets of the population with concomitant changes in characteristics used to evaluate the group. In order to ascertain the extent of heterogeneity, stocks of single-cell clones were prepared from various sources. Selected cell biological, biochemical, immunochemical, parasitological, and histopathological parameters of these clones have been studied. A ten-fold difference in the rate of growth of the epimastigote stage of T cruzi clones has been observed. The extracellular growth rates of the clones correlate with the rate of growth of the obligate intracellular amastigote stage and consequently, the length of intracellular cycle of the parasite. A 40% difference in the amount of total DNA/parasite has been found between clones. Although the amount of DNA/kinetoplast and nucleus varies between clones, the major contribution to the differences in total DNA/parasite appears to be the nucleus. From 16 to 35 antigens have been demonstrated in the T cruzi clones assayed to date. Five to seven of these antigens are common to all of the stocks assayed. However, both isolate- and clone-specific antigens have also been demonstrated. The susceptibility of inbred strains of mice to T cruzi clones varies with the clone of the parasite. These data imply that the genetics of the parasite as well as the host modulate both the course and outcome of a T cruzi infection. The influence of monosaccharides on the receptor-mediated infection of vertebrate cells by trypomastigotes of T cruzi also varies between clones. The implications of these findings upon our concept and understanding of present and future problems in Chagas disease are discussed.     

Tumour necrosis factor (TNF)-mediated NF-κB activation facilitates cellular invasion of non-professional phagocytic epithelial cell lines by Trypanosoma cruzi

At the site of infection, pro-inflammatory cytokines locally produced by macrophages infected with Trypanosoma cruzi can activate surrounding non-professional phagocytes such as fibroblasts, epithelial and endothelial cells, which can be further invaded by the parasite. The effect of secreted soluble factors on the invasion of these cells remains, however, to be established. We show here that two epithelial cell lines become significantly susceptible to the infection by the Y strain of T. cruzi after tumour necrosis factor (TNF) treatment. The increase in the invasion was correlated with the increasing concentration of recombinant TNF added to cultures of HEK293T or LLC-MK2 cells. Supernatants taken from PMA-differentiated human monocytes infected with T. cruzi also increased the permissiveness of epithelial cells to subsequent infection with the parasite, which was inhibited by a TNF monoclonal antibody. Furthermore, the permissiveness induced by TNF was inhibited by TPCK, and led to significant decrease in the number of intracellular parasites, providing evidence that activation of NF-κB induced by TNF favours the invasion of the epithelial cell lines by T. cruzi through yet an unidentified mechanism. Our data indicate that soluble factors released from macrophages early in the infection favours T. cruzi invasion of non-professional phagocytic cells.     

Surface-targeted lysosomal membrane glycoprotein-1 (Lamp-1) enhances lysosome exocytosis and cell invasion by Trypanosoma cruzi

To gain entry into non-phagocytic cells, Trypanosoma cruzi trypomastigotes recruit lysosomes to the host cell surface. Lysosome fusion at the site of parasite entry leads to the formation of a parasitophorous vacuole with lysosomal properties. Here, we show that increased expression of the lysosomal membrane glycoprotein Lamp-1 at the cell surface renders CHO cells more susceptible to trypomastigote invasion in a microtubule-dependent fashion. Mutation of critical residues in the lysosome-targeting motif of Lamp-1 abolished the enhancement of T. cruzi invasion. This suggests that interactions dependent on Lamp-1 cytoplasmic tail motifs, and not the surface-exposed luminal domain, modulate T. cruzi entry. Measurements of Ca2+-triggered exocytosis of lysosomes in these cell lines revealed an enhancement of beta-hexosaminidase release in cells expressing wild-type Lamp-1 on the plasma membrane; this effect was not observed in cell lines transfected with Lamp-1 cytoplasmic tail mutants. These results also implicate Ca2+-regulated lysosome exocytosis in cell invasion by T. cruzi and indicate a role for the Lamp-1 cytosolic domain in promoting more efficient fusion of lysosomes with the plasma membrane.     

Modulation of macrophage interaction with Trypanosoma cruzi by phospholipase A2-sensitive components of the parasite membrane

The presence of phospholipase A2 (PLA2) significantly increased the association between Trypanosoma cruzi and macrophages. This effect reflected alterations to the parasite membrane since it was reproduced only when the parasite but not the macrophage was pretreated with PLA2. That PLA2 activity was responsible for the noted enhancement was indicated by the ability of the specific substrate phosphatidylcholine to block it. The presence of the PLA2 inhibitors quinacrine, 4-bromophenacyl bromide or phentermine markedly inhibited parasite-macrophage association. Quinacrine also inhibited association of the parasite with a non-phagocytic host cell. These results suggested a role for endogenous PLA2 in the initial stages of cell infection by T. cruzi.     

Stimulatory effects of retinoic acid on macrophage interaction with blood forms of Trypanosoma cruzi: involvement of transglutaminase activity

The effects of retinoic acid (RA; vitamin A acid) on macrophage function were investigated by measuring the capacities of mouse peritoneal macrophages to associate with (i.e., bind and internalize) and kill the unicellular parasite Trypanosoma cruzi. The presence of 10(-8) to 10(-6) M RA in co-cultures of macrophages and blood forms of the parasite markedly increased their interaction as evidenced by significant increases in both the percentage of phagocytes associating with parasites and the average number of parasites per 100 cells. A similar effect was produced when either the macrophages or the trypanosomes were pretreated with RA, suggesting that both cell types could contribute to the noted effect. Although RA might have enhanced parasite-macrophage association by binding to both, its ability to stimulate phagocytosis was independently evidenced by a significant increase in the uptake of latex particles. RA-treated macrophages also took up larger numbers of dead T cruzi, denoting that parasite viability (i.e., infectivity) was not necessary for the production of the RA effect. The minimum pretreatment time for RA to significantly stimulate macrophage association with T. cruzi was 30 min, although a 45-min pretreatment was necessary for a maximal effect to be seen under our experimental conditions. The RA effect was reversible because, once optimally induced, it remained demonstrable for only 30 to 60 min after removal of the reagent; however, the effect persisted for at least 3 hr if RA was not removed. Transglutaminase activity appeared to be involved in the RA effect, because the latter was abrogated when the macrophages were treated with RA in the presence of cystamine, methylamine, or monodansylcadaverine, all of which inhibit transglutaminase activity by different mechanisms. RA also increased the capacity of macrophages to kill parasites internalized before the treatment. This cytotoxic capacity was inhibited by catalase, indicating that H2O2 played a role in the killing mechanism. RA treatment significantly increased the proportion of macrophages capable of reducing nitroblue tetrazolium. The present results indicated that RA was capable of activating macrophages, leading to greater uptake and killing of a protozoan parasite.     

A role for protease activity and host-cell permeability during the process of Trypanosoma cruzi egress from infected cells

The mechanism by which Trypanosoma cruzi egresses from infected cells at the end of the intracellular replication cycle is not understood. This study explored the role of T. cruzi-derived proteases and host-cell membrane permeability during the parasite's egress process. Treatment with a fluoromethyl ketone, known to inhibit the parasite's major protease, significantly reduced parasite egress. In addition, in the late stages of intracellular infection, cells infected with T. cruzi showed increased permeability as evidenced by dye exclusion tests. Furthermore, parasites could be antibody stained inside host cells without chemical permeabilization of the plasma membrane. These results suggest that in advanced stages of the intracellular cycle of T. cruzi, the host cells lose membrane integrity. Previous studies in our laboratory have found that antibodies present in sera of mice chronically infected with T. cruzi (antiegressin) bind the surface of infected cells and reduce parasite egress. In agreement with these reports, western blot analysis showed that several proteins in infected cell membrane extracts reacted with antibodies from infected mouse serum. The findings reported herein might have implications in the process of T. cruzi egress, as well as in the mechanism of action of antiegressin.     

Intracellular growth and metacyclogenesis defects in Trypanosoma cruzi carrying a targeted deletion of a Tc52 protein-encoding allele

We have identified previously a Trypanosoma cruzi gene encoding a protein named Tc52 sharing structural and functional properties with the thioredoxin and glutaredoxin protein family involved in thiol-disulphide redox reactions. Furthermore, we have reported that Tc52 also played a role in T. cruzi-associated immunosuppression observed during Chagas' disease. In an effort to understand further the biological role of Tc52, we used a gene-targeted deletion strategy to create T. cruzi mutants. Although T. cruzi tolerates deletion of one wild-type Tc52 allele, deletion of both genes is a lethal event, indicating that at least one active Tc52 gene is required for parasite survival. Monoallelic disruption of Tc52 (Tc52+/-) resulted in the production of T. cruzi lines that express less Tc52 mRNA and produced lower amounts of Tc52 protein compared with wild-type cells. In axenic cultures, growth rates of epimastigote forms bearing an interrupted allele were not different from those of wild-type parasites. Furthermore, monoallelic disruption of the Tc52 gene did not modify the growth rate of epimastigotes or their sensitivity to inhibition by benznidazole and nifurtimox, the two drugs used to treat Chagasic patients. Moreover, the antimonial drug SbIII, which is known, at least in Leishmania parasites, to be conjugated to a thiol and extruded by an ATP-coupled pump, had a similar effect on wild-type and mutant parasites, being equally sensitive. Hence, parasite drug sensitivity was also observed in clones overexpressing the Tc52 protein as well as in those carrying an antisense plasmid construct. Surprisingly, a significant impairment of the ability of epimastigotes carrying a Tc52 single gene replacement or antisense construct to differentiate into metacyclic trypomastigotes and to proliferate in vitro and in vivo was observed, whereas no significant enhancement of these biological properties was seen in the case of parasites that overexpress Tc52 protein. Moreover, functional complementation of Tc52+/- single mutant or selection of antisense revertant clones demonstrated that the phenotype observed is a direct consequence of Tc52 gene manipulation. Taken together, these results may suggest that Tc52 could participate among other factors in the phenotypic expression of T. cruzi virulence.     

Mechanisms of Trypanosoma cruzi persistence in Chagas disease

Trypanosoma cruzi infection leads to development of chronic Chagas disease. In this article, we provide an update on the current knowledge of the mechanisms employed by the parasite to gain entry into the host cells and establish persistent infection despite activation of a potent immune response by the host. Recent studies point to a number of T. cruzi molecules that interact with host cell receptors to promote parasite invasion of the diverse host cells. T. cruzi expresses an antioxidant system and thromboxane A(2) to evade phagosomal oxidative assault and suppress the host's ability to clear parasites. Additional studies suggest that besides cardiac and smooth muscle cells that are the major target of T. cruzi infection, adipocytes and adipose tissue serve as reservoirs from where T. cruzi can recrudesce and cause disease decades later. Further, T. cruzi employs at least four strategies to maintain a symbiotic-like relationship with the host, and ensure consistent supply of nutrients for its own survival and long-term persistence. Ongoing and future research will continue to help refining the models of T. cruzi invasion and persistence in diverse tissues and organs in the host.     

Trypanosoma cruzi immune evasion mediated by host cell-derived microvesicles

The innate immune system is the first mechanism of vertebrate defense against pathogen infection. In this study, we present evidence for a novel immune evasion mechanism of Trypanosoma cruzi, mediated by host cell plasma membrane-derived vesicles. We found that T. cruzi metacyclic trypomastigotes induced microvesicle release from blood cells early in infection. Upon their release, microvesicles formed a complex on the T. cruzi surface with the complement C3 convertase, leading to its stabilization and inhibition, and ultimately resulting in increased parasite survival. Furthermore, we found that TGF-β-bearing microvesicles released from monocytes and lymphocytes promoted rapid cell invasion by T. cruzi, which also contributed to parasites escaping the complement attack. In addition, in vivo infection with T. cruzi showed a rapid increase of microvesicle levels in mouse plasma, and infection with exogenous microvesicles resulted in increased T. cruzi parasitemia. Altogether, these data support a role for microvesicles contributing to T. cruzi evasion of innate immunity.