Effects of human defensin-α(1)on Trypanosoma cruzi trypomastigotes in vitro.

Human defensin-α(1)is a biologically active peptide exhibiting a dose-dependent trypanocidal effect in vitro against trypomastigotes and amastigotes of Trypanosoma cruzi line Tulahuen. This effect is determined by fragmentation of parasite DNA reducing the capacity of passaged T. cruzi to invade HeLa cells.

A ligand that Trypanosoma cruzi uses to bind to mammalian cells to initiate infection.

We purified a soluble gp83 trans-sialidase (gp83-TSA), from phospholipase C-treated Trypanosoma cruzi trypomastigote membranes, which binds to myoblasts, fibroblasts and macrophages to mediate trypanosome entry. Myoblasts display a single class of receptors for the gp83-TSA present at 4x10(4) per myoblast with a K(d) of 8 nM. Monovalent Fab fragments of the monoclonal antibody 4A4 specific for gp83-TSA inhibit gp83-TSA binding to myoblasts, fibroblasts and macrophages, block the trypanosomes from attaching to and entering these cells and neutralize T. cruzi infection in BALB/c mice. This is the first demonstration that gp83-TSA is a ligand that T. cruzi uses to attach to cells.

Novel mechanism that Trypanosoma cruzi uses to adhere to the extracellular matrix mediated by human galectin-3.

Binding of Trypanosoma cruzi trypomastigotes to laminin is enhanced by galectin-3, a beta-galactoside binding lectin. The galectin-3 enhanced binding of trypanosomes to laminin is inhibited by lactose. Co-immunoprecipitations indicate that galectin-3 binds to the 45, 32 and 30 kDa trypanosome surface proteins. Binding of galectin-3 to the 45, 32 and 30 kDa surface proteins is inhibited by lactose. Polyclonal and a monoclonal antibodies to galectin-3 immunoprecipitated a major 64 kDa trypanosome surface protein. T. cruzi monoclonal antibody to mucin recognized the 45 kDa surface protein. The 45, 32 and 30 kDa surface proteins interact with galectin-3 in order to enhance trypanosome adhesion to laminin.

Signal transduction in human macrophages by gp83 ligand of Trypanosoma cruzi: trypomastigote gp83 ligand up-regulates trypanosome entry through protein kinase C activation.

We found that Trypanosoma cruzi trypomastigote cloned surface ligand (gp83 trans-sialidase) signals macrophages to up-regulate parasite entry by activating protein kinase C (PKC). Incubation of r-gp83 ligand with macrophages activates PKC and this activation is abolished when r-gp83 is depleted by immunoprecipitation with anti-r-gp83 antibodies, which recognize the secreted gp83 of trypomastigotes by immunoblotting. This activation is seen as early as 15 min with maximal activity at 60 min and correlates with the concentration of macrophage cell cytosol. Bisindolylmaleimide I, a PKC inhibitor, abolished the activation of PKC induced by r-gp83 ligand. Incubation of macrophages with r-gp83 ligand significantly enhanced the number of trypanosomes per cell. Bisindolylmaleimide I also inhibited the enhancement of trypomastigote uptake by macrophages induced by the r-ligand. These results demonstrate that T. cruzi uses a novel mechanism to signal cells in the process of trypanosome entry, via a secreted trypanosome ligand which signals macrophages through activation of PKC.

The cysteine-cysteine family of chemokines RANTES, MIP-1alpha, and MIP-1beta induce trypanocidal activity in human macrophages via nitric oxide.

This paper describes a new role for the cysteine-cysteine (CC) chemokines RANTES, MIP-1alpha, and MIP-1beta on human macrophage function, which is the induction of nitric oxide (NO)-mediated trypanocidal activity. In a previous report, we showed that RANTES, MIP-1alpha and MIP-1beta enhance Trypanosoma cruzi uptake and promote parasite killing by human macrophages (M. F. Lima, Y. Zhang, and F. Villalta, Cell. Mol. Biol. 43:1067-1076, 1997). Here we study the mechanism by which RANTES, MIP-1alpha, and MIP-1beta activate human macrophages obtained from healthy individuals to kill T. cruzi. Treatment of human macrophages with different concentrations of RANTES, MIP-1alpha, and MIP-1beta enhances T. cruzi trypomastigote phagocytosis in a dose peak response. The optimal response induced by the three CC chemokines is attained at 500 ng/ml. The macrophage trypanocidal activity induced by CC chemokines can be completely inhibited by L-N-monomethyl arginine (L-NMMA), a specific inhibitor of the L-arginine:NO pathway, but not by its D-enantiomer. Culture supernatants of chemokine-treated human macrophages contain increased NO2- levels, and NO2- production is also specifically inhibited by L-NMMA. The amount of NO2- induced by these chemokines in human macrophages is comparable to the amount of NO2- induced by gamma interferon. The killing of trypomastigotes by NO in cell-free medium is blocked by an NO antagonist or a NO scavenger. This data supports the hypothesis that the CC chemokines RANTES, MIP-1alpha, and MIP-1beta activate human macrophages to kill T. cruzi via NO, which is an effective trypanocidal mechanism.

Signal transduction in human macrophages by gp83 ligand of Trypanosoma cruzi: trypomastigote gp83 ligand up-regulates trypanosome entry through the MAP kinase pathway.

We found that Trypanosoma cruzi trypomastigote cloned surface ligand (gp83 trans-sialidase) signals human macrophages to up-regulate parasite entry by inducing tyrosine phosphorylation of MAP kinase. Preincubation of human macrophages with r-gp83 transsialidase significantly enhanced both the percentage of phagocytosed trypanosomes and the number of trypanosomes per cell in a concentration dependent fashion. Incubation of r.gp83 with macrophages induced tyrosine phosphorylation of several macrophage proteins. This enhancement was inhibited by genistein, a tyrosine kinase inhibitor. The r-trypanosome ligand enhanced tyrosine phosphorylation of ERK1 and this enhancement was specifically inhibited by the inhibitor of MAP kinase phosphorylation, PD 98059, or by genistein. PD 98050 or genistein also inhibited the enhancement of trypomastigote uptake by macrophages induced by the r-ligand. These results indicate that T. cruzi uses a novel mechanism to signal cells in the process of trypanosome entry, via a secreted trypanosome ligand which signals macrophages through the MAP kinase pathway.

Beta-chemokines that inhibit HIV-1 infection of human macrophages stimulate uptake and promote destruction of Trypanosoma cruzi by human macrophages.

Recently beta-chemokines have been shown to inhibit HIV-1 infection of human macrophages. Here, we show that the beta-chemokines RANTES, MIP-1alpha and MIP-1beta enhance the uptake and cause intracellular destruction of Trypanosoma cruzi trypomastigotes by human macrophages obtained from healthy individuals. The trypanosome enhancing uptake and the trypanocidal effect induced by these beta-chemokines in human macrophages are abrogated by neutralizing antibodies to RANTES, MIP-1alpha and MIP-beta, whereas irrelevant antibodies of the same class do not affect these parameters. These results indicate that the effects seen are beta-chemokine specific. Pretreatment of human macrophages with RANTES, MIP-1alpha and MIP-1beta induced strong tyrosine phosphorylation of several proteins, suggesting that signal transduction events are involved in enhanced trypanosome uptake and parasite killing. Taken together these results suggest that the beta-chemokines RANTES, MIP-1alpha and MIP-1beta, might play a beneficial role in parasite clearance and destruction in individuals infected with T. cruzi. Alternatively, these three beta-chemokines may play a beneficial role in individuals concurrently infected with T. cruzi and HIV-1.

Purification of a 74-kilodalton surface glycoprotein from heart myoblasts that inhibits binding and entry of Trypanosoma cruzi into heart cells.

We have identified and purified a 74 kDa surface glycoprotein from heart myoblasts that specifically binds to Trypanosoma cruzi trypomastigotes, and inhibits the attachment and internalization of trypomastigotes into these cells. The native form of the 74 kDa glycoprotein was purified to apparent homogeneity by preparative scale isoelectrofocusing and anion exchange chromatography. Pre-incubation of trypomastigotes with soluble 74 kDa glycoprotein strongly inhibited the binding and internalization of trypomastigotes into heart myoblast monolayers in a concentration dependent-manner. Pre-incubation of heart myoblast monolayers with antibodies specific to the purified 74 kDa glycoprotein also strongly inhibited trypomastigote binding and internalization into heart cells in a concentration dependent manner. These results support the notion that the surface 74 kDa glycoprotein is a target molecule on heart myoblast cells to which T. cruzi binds in order to invade them.

Purification of a Trypanosoma cruzi trypomastigote 60-kilodalton surface glycoprotein that primes and activates murine lymphocytes.

We have purified a glycoprotein with a relative molecular mass of 60 kDa and present on the surface of Trypanosoma cruzi trypomastigotes and studied its ability to prime and stimulate the proliferation of murine spleen cells. T. cruzi trypomastigote membrane proteins were separated by preparative isoelectrofocusing. A trypomastigote 60-kDa surface protein with an isoelectric point of 4.2 was enriched by chromatofocusing and was readily purified in native form to homogeneity by gel filtration on a Superose column by use of a fast protein liquid chromatography system. Biotinylated wheat germ agglutinin, Ricinus communis agglutinin, and Datura stramonium agglutinin bound to blots containing the purified trypomastigote 60-kDa surface protein, indicating that this protein was glycosylated. The purified trypomastigote 60-kDa glycoprotein was recognized by antibodies produced during human infection, and immunoglobulin G against the purified glycoprotein immunoprecipitated a biotinylated 60-kDa molecule from the surface of trypomastigotes but not epimastigotes. Specific immunoglobulin G against the 60-kDa glycoprotein also increased the uptake of trypomastigotes and promoted parasite killing by macrophages. The purified 60-kDa glycoprotein was able to specifically activate primed lymphocytes, since there was a significant increase in [3H]thymidine incorporation by spleen cells obtained from CBA mice primed with this glycoprotein, with respect to control values. Furthermore, the 60-kDa glycoprotein did not stimulate unprimed spleen cells, indicating that the lymphoproliferation induced by this glycoprotein was specific and was not due to polyclonal activation. Our findings indicate that this T. cruzi trypomastigote 60-kDa surface glycoprotein primes and activates lymphocytes, which could lead to a beneficial immune response in the host.

Attachment of Trypanosoma cruzi to host cells: a monoclonal antibody recognizes a trypomastigote stage-specific epitope on the gp 83 required for parasite attachment.

A set of monoclonal antibodies against the purified surface gp 83 of T. cruzi trypomastigotes was produced and the ability of these monoclonals to inhibit the attachment of trypomastigotes to heart myoblasts was investigated. Western blots of solubilized trypomastigotes, epimastigotes or amastigotes probed with this set of monoclonal antibodies show that the gp 83 is present in invasive trypomastigotes, but not in non-invasive epimastigotes or amastigotes. One monoclonal antibody (Mab 4A4) from this set inhibits the attachment of trypomastigotes to heart myoblasts, whereas the others (MAbs 2H6, 4B9, 2D11) do not. These results show that the Mab 4A4 recognizes an epitope on the gp 83 of invasive trypomastigotes required for parasite binding to host cells.

Changes in polypeptide expression following Trypanosoma cruzi differentiation from trypomastigotes to amastigotes.

Changes in the dynamic of expression of polypeptides following the differentiation from infective trypomastigotes to multiplicative amastigote forms of Trypanosoma cruzi were mapped by two-dimensional gel electrophoresis and quantitatively analyzed by laser densitometry. Following the differentiation from trypomastigotes to amastigotes the expression of the polypeptides 212, 183, 176, 149, 50-55, 43, 39, 34 and 28 kDa is turned off in multiplicative amastigotes, whereas the expression of the polypeptides 80, 66 (p.Is. 6.75-7.50), 42 and 38 kDa is turned on. After complete differentiation from trypomastigotes to amastigotes the expression of the polypeptides 43, 42, 33, 32, 29 and 23 kDa is up-regulated in amastigotes, whereas the expression of the acidic polypeptides 66 (p.Is. 6.27-6.64), 45-48 and 41-43 kDa is down-regulated.

Purification of Trypanosoma cruzi surface proteins involved in adhesion to host cells.

We have identified four surface 83 kDa proteins of pI values 6.3, 6.4, 6.5 and 6.6 in T. cruzi trypomastigotes which specifically bind to rat heart myoblasts. These proteins were purified by isoelectric focusing and anion-exchange chromatography in an FPLC system. These 83 kDa proteins inhibit the attachment of trypomastigotes to myoblasts in a concentration-dependent manner, indicating that these trypomastigote proteins mediate the attachment of trypomastigotes to heart myoblasts.

Trypanosoma cruzi receptors for human transferrin and their role.

Trypanosoma cruzi amastigotes present receptors for human transferrin as indicated by the saturable binding of 125I-transferrin to this form of the parasite. Computerized Scatchard analysis revealed one class of receptors present at 8.1 X 10(4) receptors per amastigote with a Kd of 2.82 microM. Immunofluorescence studies indicate that more than 90% of amastigotes bind human transferrin, whereas trypomastigotes do not. Iron is required for amastigote growth in cell-free medium since deferoxamine, an iron chelator, inhibits amastigote growth. Amastigote growth is restored when deferoxamine is removed from the medium. 59Fe-transferrin, which bound to amastigotes at 4 degrees C for 1 h, was readily dissociated from the parasite surface upon treatment with acid. However, this treatment did not disrupt binding that occurred at 37 degrees C for 1 h. Amastigote growth in cell-free medium is inhibited in ferrotransferrin-depleted serum, and addition of ferrotransferrin but not apotransferrin restores parasite growth. Western blots of solubilized amastigote membranes probed with anti-human transferrin receptor antibody recognize a protein of 200 kDa. This protein is present on the amastigote cell surface; therefore, human transferrin seems to interact with a 200-kDa surface amastigote protein receptor. Iron, which is essential for amastigote growth, thus appears to be delivered to T. cruzi amastigotes by transferrin receptor-mediated endocytosis.

Fibronectin increases Trypanosoma cruzi amastigote binding to and uptake by murine macrophages and human monocytes.

Trypanosoma cruzi amastigotes present receptors for human fibronectin as indicated by the saturable binding of [125I]fibronectin to this form of the parasite. Scatchard analysis indicates that the number of fibronectin receptors per amastigote was 1.3 x 10(3) with a Kd of approximately 2.3 nM. Addition of physiological concentrations of fibronectin to amastigote-macrophage cocultures significantly increased the binding of amastigotes to murine macrophages. This increase was evidenced in both the number of amastigotes bound to macrophages and the percentage of macrophages containing bound amastigotes. The uptake of amastigotes by either murine macrophages or human blood monocytes was also increased in the presence of exogenous fibronectin. The increase induced by fibronectin was blocked when amastigotes were pretreated with the RGDS tetrapeptide of the fibronectin cell attachment site. Furthermore, the ability of fibronectin to enhance amastigote binding to and uptake by macrophages was inhibited by the F(ab')2 fragment of anti-fibronectin immunoglobulin G (IgG) but not by an irrelevant anti-human IgG F(ab')2 fragment. Pretreatment of either amastigotes or macrophages with fibronectin also resulted in a significant increase in amastigote binding to and uptake by macrophages. These results suggest that fibronectin may play a role in facilitating the attachment and ingestion of T. cruzi amastigotes by macrophages and monocytes in chagasic tissue lesions.

Trypanosoma cruzi trypomastigote clones differentially express a parasite cell adhesion molecule.

We have cloned blood trypomastigotes from infected mice and found that Trypanosoma cruzi strains are composed of heterogeneous populations that dramatically vary (more than 100 fold) in their abilities to attach to and enter rat heart myoblasts. Trypomastigote clones were distinctively separated into highly and weakly infective groups presenting higher and lower rates of attachment to myoblasts, respectively. Each trypomastigote clone maintained the same profile of attachment and internalization into heart myoblasts when tested at different periods of time. This pattern did not change when the parasites were incubated in fresh medium before being exposed to heart myoblasts. Highly and weakly infective clones show differences at the cell surface level, particularly with regard to a 83 kDa glycoprotein. We have identified this 83 kDa glycoprotein as the parasite membrane ligand that specifically binds to rat heart myoblasts. The binding of the biotinylated 83 kDa to myoblasts is inhibited by cold excess in Western blots, as indicated by laser densitometry. In addition, the specific binding of this molecule to myoblasts is saturable and is greater in highly than in weakly infective trypomastigote clones. Highly invasive trypomastigote clones express this glycoprotein in more abundance on their surface than weakly infective trypomastigote clones. These results indicate that the 83 kDa glycoprotein present on the surface of T. cruzi trypomastigotes mediates the attachment of the parasite to heart myoblasts.

Host-cell attachment by Trypanosoma cruzi: identification of an adhesion molecule.

We have identified an 83 kDa surface glycoprotein in T. cruzi trypomastigotes which specifically binds to rat heart myoblasts. The binding of this molecule to myoblasts is inhibited by excess unlabeled material and saturable. Antibodies against the cell surface of insect trypomastigotes, blood trypomastigotes and produced during human infection recognize the 83 kDa glycoprotein adhesion molecule by immunoblotting, indicating that this molecule that mediates this critical step is immunogenic and is a candidate for vaccination against Chagas' disease.

Extracellular killing of Trypanosoma cruzi amastigotes by human eosinophils.

Granules released from human eosinophils upon interaction with Trypanosoma cruzi amastigotes in vitro were seen attached to the surface of non-internalized parasites by electron microscopy. Amastigote damage was preceded by the binding of eosinophil granule material to its membrane, and eosinophil granule major basic protein (MBP) bound to the parasite surface was readily detectable. Additional evidence of eosinophil cytotoxicity for extracellular amastigotes was the observation that amastigotes trapped between two eosinophils, without being ingested by either one, were destroyed at the interface. Amastigotes isolated from the spleens of infected mice or grown in culture were similarly sensitive to the lytic effects of purified MBP. These results demonstrate the ability of human eosinophils to lyse T. cruzi amastigotes extracellularly in the absence of antibody and suggest that MBP may be involved in the effect. Thus, eosinophils, known to be capable of destroying phagocytosed amastigotes, could also contribute to the clearance of these parasites through extracellular killing.

Insect-borne and culture-derived metacyclic Trypanosoma cruzi: differences in infectivity and virulence.

We report in this paper significant differences in the virulence of insect-derived and cultured metacyclic forms of Trypanosoma cruzi which are morphologically indistinguishable. Mice infected intraperitoneally with 10(3) metacyclic T. cruzi isolated from Rhodnius prolixus showed average parasitemia levels greater than 2 X 10(5) organisms/ml around day 10 post-infection (when first measured) and peak levels recorded on day 16 post-infection exceeded 4 X 10(7) organisms/ml. None of these animals survived after 30 days post-infection. In contrast, in mice infected with 10(3) or 10(4) metacyclic forms from axenic cultures the highest average parasitemia was approximately 10(4) organisms/ml and occurred around day 19 post-infection. In these animals, parasitemias declined with time to become undetectable and no mortality was recorded over the 100-day observation period. There was also a marked difference in the 50% lethal dose of insect- and culture-derived metacyclics. The value for the former was 670 parasites whereas none of the mice infected intraperitoneally with up to 10(6) cultured metacyclics died. These results point to a marked difference in the biological properties of insect-borne and cultured T. cruzi metacyclics under our experimental conditions and caution against extending results obtained with the latter to vector-transmissible metacyclics, at least in infectivity and virulence studies.

Role of membrane N-linked oligosaccharides in host cell interaction with invasive forms of Trypanosoma cruzi.

The removal of N-linked oligosaccharides by peptide-N4-[N-acetyl-beta-glucoseaminyl]asparagine amidase (previously known as aspartoglycosylamine amidohydrolase and abbreviated N-glycanase) from the surface of blood or insect-transmissible forms of Trypanosoma cruzi markedly increased the capacity of these organisms to associate with (i.e., bind and penetrate) either mouse peritoneal macrophages or rat heart myoblasts. This effect was evidenced by a significant elevation in both the percentage of infected host cells and the average number of parasites per 100 cells. Conversely, N-glycanase treatment of either host cell markedly reduced both parameters to levels significantly below those obtained with cells mock treated with medium alone. The N-glycanase effect on the parasites was inhibited by heat inactivation of the enzyme or by the presence of fetuin, an N-glycanase substrate. The enhanced capacity of N-glycanase-treated T. cruzi to engage the host cells started to subside 2 h after the treatment, indicating the reversibility of the effect. The decreased reactivity of N-glycanase-treated macrophages or myoblasts with T. cruzi suggests that N-linked oligosaccharides on these host cells are involved in the initial phase of the cell infection process. Instead, because T. cruzi interacted more effectively with host cells after treatment with N-glycanase, parasite surface N-linked oligosaccharides would seem to interfere with the association.