Involvement of host cell heparan sulfate proteoglycan in Trypanosoma cruzi amastigote attachment and invasion.

Cell surface glycosaminoglycans (GAGs) play an important role in the attachment and invasion process of a variety of intracellular pathogens. We have previously demonstrated that heparan sulfate proteoglycans (HSPG) mediate the invasion of trypomastigote forms of Trypanosoma cruzi in cardiomyocytes. Herein, we analysed whether GAGs are also implicated in amastigote invasion. Competition assays with soluble GAGs revealed that treatment of T. cruzi amastigotes with heparin and heparan sulfate leads to a reduction in the infection ratio, achieving 82% and 65% inhibition of invasion, respectively. Other sulfated GAGs, such as chondroitin sulfate, dermatan sulfate and keratan sulfate, had no effect on the invasion process. In addition, a significant decrease in infection occurred after interaction of amastigotes with GAG-deficient Chinese Hamster Ovary (CHO) cells, decreasing from 20% and 28% in wild-type CHO cells to 5% and 9% in the mutant cells after 2 h and 4 h of infection, respectively. These findings suggest that amastigote invasion also involves host cell surface heparan sulfate proteoglycans. The knowledge of the mechanism triggered by heparan sulfate-binding T. cruzi proteins may provide new potential candidates for Chagas disease therapy.

Multicellular spheroids of bone marrow stromal cells: a three-dimensional in vitro culture system for the study of hematopoietic cell migration.

Cell fate decisions are governed by a complex interplay between cell-autonomous signals and stimuli from the surrounding tissue. In vivo cells are connected to their neighbors and to the extracellular matrix forming a complex three-dimensional (3-D) microenvironment that is not reproduced in conventional in vitro systems. A large body of evidence indicates that mechanical tension applied to the cytoskeleton controls cell proliferation, differentiation and migration, suggesting that 3-D in vitro culture systems that mimic the in vivo situation would reveal biological subtleties. In hematopoietic tissues, the microenvironment plays a crucial role in stem and progenitor cell survival, differentiation, proliferation, and migration. In adults, hematopoiesis takes place inside the bone marrow cavity where hematopoietic cells are intimately associated with a specialized three 3-D scaffold of stromal cell surfaces and extracellular matrix that comprise specific niches. The relationship between hematopoietic cells and their niches is highly dynamic. Under steady-state conditions, hematopoietic cells migrate within the marrow cavity and circulate in the bloodstream. The mechanisms underlying hematopoietic stem/progenitor cell homing and mobilization have been studied in animal models, since conventional two-dimensional (2-D) bone marrow cell cultures do not reproduce the complex 3-D environment. In this review, we will highlight some of the mechanisms controlling hematopoietic cell migration and 3-D culture systems.

Interaction of Trypanosoma cruzi with heart muscle cells: ultrastructural and cytochemical analysis of endocytic vacuole formation and effect upon myogenesis in vitro.

The process of interaction of bloodstream trypomastigotes of three different strains of Trypanosoma cruzi with heart mouse muscle cells in primary cultures, was analyzed. Differences were found in the ability of the parasites to infect the cells. Those from the Colombiana strain were more infective than those from the Y and CL strains. Infection of the cells with parasites of the Colombiana strain, but not with those of the Y strain, interfered with the normal myogenic process. Transmission electron microscopy of thin sections of heart muscle cells kept in contact with parasites for 18 h showed that many parasites are found within membrane-bounded endocytic vacuoles. Cytochemical localization of Ca2+-Mg2+-ATPase, adenylate cyclase and anionic sites (labelled with cationized ferritin) indicate that these components of the plasma membrane are not found in the membrane which lines the endocytic vacuole.

Characterization and localization of an ATP-diphosphohydrolase on the external surface of the tegument of Schistosoma mansoni.

An ATP-diphosphohydrolase (EC 3.6.1.5) was identified in the tegumental fraction isolated from Schistosoma mansoni worms. Both ATP and ADP were hydrolyzed to AMP at similar rates by the enzyme. Other nucleotides were also degraded by the tegument enzyme, revealing a broad substrate specificity. Electrophoretic separation of tegumental proteins under non-denaturing conditions followed by addition of ATP or ADP as substrate revealed a single band of activity with similar mobility. In addition, similar heat-inactivation profiles were obtained for ATPase or ADPase activities, indicating that a single enzyme is responsible for degrading both nucleotides. The enzyme was not inhibited by vanadate, levamisole, tetramisole, ouabain or sodium azide. The ADPase activity was not affected by adenosine (5')-pentaphospho-(5')-adenosine (Ap5A) or by an excess of glucose and hexokinase used as an ATP-trapping system, thus excluding the presence of any significant adenylate kinase activity. The ATP-diphosphohydrolase displayed micromolar affinities for both Mg2+ and Ca2+, and the calcium-activated enzyme was inhibited by millimolar Mg2+. In intact live worms a calcium phosphate precipitate was formed on the outer tegumental surface upon incubation of the worms with either ATP or ADP, indicating the ectolocalization of this enzyme. In addition, ultrastructural histochemical localization of the enzyme was obtained. A distinct deposition of lead phosphate granules on the outer surface of the tegument was observed by electron microscopy, in the presence of either ATP or ADP as substrate. It is suggested that the ATP-diphosphohydrolase could regulate the concentration of purine nucleotides around the parasites and hence enable them to escape the host hemostasis by preventing ADP-induced platelet activation.

Inhibitors of the major cysteinyl proteinase (GP57/51) impair host cell invasion and arrest the intracellular development of Trypanosoma cruzi in vitro.

Peptidyl diazomethane (PDAM) derivatives, a class of irreversible inhibitors for cysteine proteinase, were screened for the ability to impair Trypanosoma cruzi invasion and intracellular development in primary cultures of heart muscle cells (HMC). T. cruzi GP57/51, a purified cysteinyl proteinase, and the substrate Z-Phe-Arg-NHMec were used to determine inhibition rate constants (k'+2) by continuous kinetic assays. The k'+2 values ranged from 25,400 to 2,800. The best inhibitors of GP57/51 had bulky hydrophobic residues in the P1 position (in addition to P2), the S1 sub-site specificity of the enzyme being thus similar to mammalian cathepsin L. The effects of these PDAM on parasite infectivity were then investigated. The ability to invade HMC was markedly impaired when trypomastigotes were briefly exposed to 10 microM of Z-(S-Bzl)Cys-Phe-CHN2. Striking effects were observed when PDAM were added to HMC cultures that had been previously infected with trypomastigotes: Z-(S-Bzl)Cys-Phe-CHN2 with an IC50 of 0.4 microM, and less markedly Z-Phe-Phe-CHN2 and Z-Tyr-Phe-CHN2 (or Z-Phe-Tyr-CHN2) blocked amastigote replication as well as their transformation into trypomastigotes, thereby arresting intracellular development. Bz-Phe-Gly-CHN2, in contrast, failed to display antiparasite activity. Direct characterization of the target cysteinyl proteinase was sought, by incubating viable amastigotes or infected HMC with Z-[125I]Tyr-Phe-CHN2. Affinity labeling implicated GP57/51 as the major cysteinyl proteinase target for this probe. We propose that T. cruzi intracellular development is critically dependent on GP57/51 (cruzipain). Selective inhibitors for this cysteinyl proteinase may have therapeutic potential.

Interaction of bloodstream, tissue culture-derived and axenic culture-derived trypomastigotes of Trypanosoma cruzi with macrophages.

Mouse macrophages were infected with bloodstream, tissue culture-derived and axenic culture-derived trypomastigotes of the Y and the CL strains of Trypanosoma cruzi. The percentage of infected cells, the mean number of parasites per cell and the incorporation index were determined after 2 h of interaction. Longer periods of interaction were used to evaluate the fate of the different trypomastigotes inside the macrophage. It was observed that the incorporation of T. cruzi by the macrophages was high for tissue culture-derived trypomastigotes, intermediate for axenic culture-derived trypomastigotes and low for bloodstream trypomastigotes. For the three types of trypomastigotes, a larger number of macrophages were infected with parasites from the Y than the CL strain. These results suggest that the ability to infect macrophages is a basic characteristic of each strain which is maintained when the parasites are transferred from the vertebrate of the invertebrate host to in vitro systems.

Glycolipid and protein profiles of normal and Trypanosoma cruzi infected heart muscle cells.

Using Triton X-114, glycolipids and proteins were extracted from heart muscle cells (HMC) infected with Trypanosoma cruzi clone Dm28c and from uninfected HMC, and analysed by SDS-PAGE and high-performance thin-layer chromatography (HPTLC). Two major differences were observed: (a) two proteins with a molecular mass of 92 kDa and 69 kDa were present in the uninfected cells but absent from the infected cells and (b) a 70-90 kDa protein band was detected only in parasitized cells. These differences would seem to constitute alterations taking place during the process of cell recognition and/or parasite interiorization. No differences were observed in the respective glycolipid compositions, of control and infected cells analysed by HPTLC. A glycolipid with the same mobility as the neutral glycolipid isolated from epimastigotes of T. cruzi was detected in the uninfected cells. This finding may lend support to the previously described hypothesis that molecular mimicry is implicated in the cardioneuropathology of Chagas' disease.

Effect of the alkyl-lysophospholipids on the proliferation and differentiation of Trypanosoma cruzi.

Alkyl-lysophospholipids (ALPs), designed as potential immunomodulators, have been shown to be cytotoxic for a variety of tumour cells and are under clinical studies for cancer chemotherapy. ET-18-OCH(3), hexadecylphosphocholine and ilmofosine were assayed against the three forms of Trypanosoma cruzi. Incubation with bloodstream trypomastigotes resulted, under different experimental conditions, in higher activity of the compounds in comparison with crystal violet. The ED(50)/24 h values were 13.4+/-2.8 microM and 11. 7+/-0.6 microM for amastigotes and epimastigotes, respectively. ET-18-OCH(3) (0.3 and 0.6 microM) inhibited the differentiation of epimastigotes to trypomastigotes (Dm28C clone) in the range 40-57%. This drug (3.75-15 microM) also caused a time- and dose-dependent inhibition of the intracellular proliferation of amastigotes in heart muscle cells with ED(50) values of 14.3+/-4.2, 8.9+/-1.9 and 6. 8+/-0.4 microM, after 1, 2 and 3 days of treatment. Pre-treatment of the parasite with this drug inhibited its interiorization into the host cell. Interestingly, the intracellular differentiation of amastigotes to trypomastigotes was not hampered by the drug. The present results demonstrate the lytic effect of ALPs on the three forms of T. cruzi, as well as the inhibition of both the differentiation to the infective form and the proliferation of parasites interiorized in heart cells. Ultrastructural analysis of epimastigotes treated with the three ALPs showed extensive blebing of the flagellar membrane. As described in tumour cells, the membrane seems to be a primary target of the drugs.

Differential effect of amphotericin B on the three evolutive stages of Trypanosoma cruzi and on the host cell-parasite interaction.

1. Amphotericin B (Am.B) was shown to have a direct effect on T. cruzi, with the three forms of the parasite presenting different susceptibilities to the drug in the following order: amastigotes > trypomastigotes > epimastigotes. These differences highlight the importance of using the vertebrate forms of the parasite in tests of new drugs. 2. The treated parasites showed alterations of the plasma membrane, suggesting that, as in fungi, the primary effect of Am.B was probably via formation of complexes with membrane components. 3. When exposed to filipin, another polyene antibiotic, the three parasite forms were observed to present a similar order of susceptibility, with comparable ultrastructural modifications. 4. Higher concentrations of Am.B were required to damage the intracellular parasites in vitro, 2.3 micrograms/ml for parasites inside peritoneal macrophages and 7 micrograms/ml for parasites inside heart muscle cells. 5. Am.B is effective against the parasite, but is also toxic to mammalian cells. Testing of Am.B for the control of Chagas' disease by blood transfusion may be useful, since bloodstream forms are lysed by lower concentrations of the drug than those required to affect intracellular parasites.

Trypanosoma cruzi: distribution of fluorescently labeled tubulin and actin in epimastigotes.

Cytoskeletal components were visualized in epimastigote forms of Trypanosoma cruzi by double immunofluorescence microscopy using monospecific antibodies against tubulin and against actin. Intense staining of the flagellum and the edges of the cell body was observed when the cells were stained with anti-tubulin, reflecting the presence of the basal bodies, the flagellar axoneme and the subpellicular microtubules. A less intense staining was seen in the cell body of epimastigotes stained with anti-actin. However, an intense staining was observed with this antibody in the flagellum, in a pattern similar to that observed with anti-tubulin. It is suggested that the paraxial structure, which is formed by a complex array of 6-nm-thick microfilaments is composed, at least in part, of actin.

Concomitant endosome-phagosome fusion and lysis of endosomal membranes account for Pseudomonas aeruginosa survival in human endothelial cells.

P. aeruginosa is selectively internalized by human endothelial cells but is not efficiently killed in the intracellular (IC) compartment. To investigate whether IC survival is associated with failure in bacteria-containing endosome-lysosome (E-L) fusion, endothelial cells were exposed to albumin-colloidal gold complex and to bacterial suspension and submitted to transmission electron microscopy (TEM). Gold granules were detected in P. aeruginosa-containing vacuoles, indicating that E-L fusion had occurred. Bacteria were also seen apparently free in the cell cytoplasm, suggesting disruption of endosome membranes. To ascertain whether phospholipase C (PLC) could account for vacuolar lysis, PLC producing PAO1 and PAK strains were compared with a PLC deficient mutant (PLCN) in their IC survival. All three strains were equally uptaken by the endothelial cells, as determined by the gentamicin exclusion assay. After 3 h of infection, the IC concentration of PAK and PAO1 increased significantly while the concentration of the mutant decreased to 56.8 +/- 18.2% of the viable counts at 1 h of infection. After 5 h, the IC concentration of P. aeruginosa corresponded to 83.1 +/- 34.6%, 109 +/- 22.6% and 26.2 +/- 14.7% of the viable counts detected at 1 h, for PAK, PAO1 and the mutant, respectively. By TEM, while most PAO1-containing vacuoles presented partially lysed membranes, in cells infected with the PLCN mutant bacteria were most often observed in vacuoles with intact membranes. These observations suggest that the IC survival of P. aeruginosa results from a competition between the microbicidal activity of endothelial cells following E-L fusion and the capacity of bacteria to escape from endosomes.

The role of RCA-binding sites in the adhesion of trypanosoma cruzi to heart muscle cells, as revealed by electron spectroscopic imaging.

The involvement of host-cell surface membrane components during T. cruzi-heart muscle cell (HMC) interaction was investigated. We used the lectin RCA I (Ricinus communis), which binds to residues of D-galactose, conjugated with ferritin as a tool to reveal the role played by lectin-binding sites during adhesion of T. cruzi to HMC. With electron spectroscopic imaging (ESI) it was possible to observe a concentration of RCA I-ferritin particles on the surface membrane of HMC at the site of parasite attachment. This suggested that migration of galactosyl residues was occurring during the cellular recognition process, particularly since these particles were absent in the immediate vicinity of the attachment site, while being present in other regions of HMC membrane not related to the attachment sites. No region of the parasite's cell body was observed to have preferential status for the purposes of adhesion to HMC.

Differential expression of the plasma membrane Mg2+ ATPase and Ca2+ ATPase activity during adhesion and interiorization of Leishmania amazonensis in fibroblasts in vitro.

With ultrastructural cytochemistry we localized the activity of the plasma membrane enzyme markers Mg2+ ATPase and Ca2+ ATPase during the interaction between Leishmania amazonensis and in vitro primary culture fibroblasts. The expression of the enzymes was followed during the parasite adhesion and its interiorization. After the interiorization step, a striking difference was seen between the two enzymes studied when the parasite was found within the parasitophorous vacuole in the fibroblast cytoplasm. The activity of the Ca2+ ATPase found at the Leishmania amazonensis plasma membrane during the attachment step of the infection remained also present inside the phagosome, whereas the Mg2+ ATPase activity disappeared. So far, all the reports in the literature referred the presence of Ca2+ ATPase in Leishmania parasite only in the crude ghost plasma membrane. The Ca2+ ATPase present at the parasite plasma membrane may be involved in the regulation of calcium levels inside the phagosome. Further characterization of this Ca2+ ATPase at the plasma membrane of the parasite, when still inside the phagosome, should permit a better understanding of its functional role in maintaining the parasite surface membrane structure necessary for its existence.

The nature of anionic sites and the endocytic pathway in heart muscle cells.

The distribution and internalization of anionic sites in heart muscle cells (HMC) were studied by direct measurements of their zeta potential (ZP) and by ultrastructural cytochemistry. Our data showed that HMC are negatively charged and that their anionic sites are distributed over the entire sarcolemma. Treatments with neuraminidase and trypsin altered the ZP value and also reduced binding of cationized ferritin (CF) to the sarcolemma. Sialic acid was shown to be an important component on the surface of HMC, since its removal reduced the cell surface negative charge by 25%. Phospholipase C did not significantly change the surface charge, nor did it alter HMC reactivity to CF particles when compared with control cells. Endocytosis of anionic sites was investigated using two different protocols that allow follow-up of this dynamic process. Incubation of HMC with cationized ferritin particles at 37 degrees C induced a redistribution of ligand-bound anionic sites, followed by their internalization or detachment. The clustering of anionic sites on the surface of HMC indicates that these cells are characterized by a high level of membrane fluidity. CF particles were localized inside early and late endosomes, lysosomes, and also in ferritin-enriched vesicles near the sarcolemma. An endocytic pathway for anionic sites in HMC is discussed.

Myofibrillar breakdown and cytoskeletal alterations in heart muscle cells during invasion by Trypanosoma cruzi: immunological and ultrastructural study.

The cytoskeletal organization of normal and Trypanosoma cruzi-infected mouse embryo heart muscle cells (HMC) in primary culture was investigated using immunofluorescence and transmission electron microscopy. Fluorescent probes revealed that in the early stages of infection, up to 24 h, the HMC cytoskeleton appeared to accommodate the intracellular parasites perinuclearly, with only a few cells displaying a slight disturbance in the distribution of filaments. However, as the infection progressed (48 to 72 h), microtubules and desmin filaments were disrupted. Breakdown of myofibrils occurred in regions where the parasites were present, followed by formation of actin polygons. Using Triton X-100 treated whole cell mount, we obtained a striking preservation of the three-dimensional architecture of the cytoskeleton. Combining electron spectroscopic imaging (ESI) with contrast tuning, we detected a highly interconnected cytoskeletal network in normal cells, and a loose network in infected cells. Bundles of filaments running under and over the parasites were also observed. Our results demonstrate that T. cruzi infection induces myofibrillar breakdown and destruction of several cytoskeleton filaments in heart muscle cells.

Trypanosoma cruzi-heart muscle cell interaction: the presence of two or more trypomastigotes within a single endocytic vacuole.

Ultrastructural studies of the interaction between T. cruzi clone Dm28c and heart muscle cells (HMC) showed that in the initial phase of the cell recognition process several parasites could be found attached to a focal point on the surface of the host cell. Immediately following this phase, two or more parasites could be detected inside the same endocytic vacuole by electron spectroscopic imaging (ESI).

Glycosylation patterns of human alpha2-macroglobulin: analysis of lectin binding by electron microscopy.

Human alpha2-macroglobulin (alpha 2M) is a 720 kDa glycoprotein that presents two ultrastructural conformations: slow (S-alpha 2M) and fast (F-alpha 2M). alpha 2M acts mainly as a proteinase scavenger, but an immunomodulatory role was also proposed. This work studies the effect of desialylation and deglycosylation on the structure patterns of alpha 2M by ultrastructural analysis of lectin-induced aggregates, which represents a new approach that had never been previously used. Transmission electron microscopy (TEM) analysis showed the loss of S-alpha 2M conformation after deglycosylation, indicating that glycosidic side-chains contribute to the molecular stability of S-alpha 2M. TEM proved to be an important tool to analyze the effect of biochemical changes on alpha 2M, yielding an objective qualitative control of its morphological state. Certain carbohydrate residues did not vary between the alpha 2M conformations, since both bound similarly ConA and WGA lectins. However, the binding of PNA and BSI-B(4) was slightly lower in F-alpha 2M than in S-alpha 2M. Among the neuraminidases used to desialylate both conformations of alpha 2M that from Arthrobacter ureafaciens was the most effective. Incubation with the lectins ConA or SNA, respectively specific for mannosyl and sialyl residues, led to dose-dependent patterns of aggregation of alpha 2M molecules, mediated by lectin binding and clearly visualized by TEM.

Cellular effects of reversed amidines on Trypanosoma cruzi.

Aromatic diamidines represent a class of DNA minor groove-binding ligands that exhibit high levels of antiparasitic activity. Since the chemotherapy for Chagas' disease is still an unsolved problem and previous reports on diamidines and related analogues show that they have high levels of activity against Trypanosoma cruzi infection both in vitro and in vivo, our present aim was to evaluate the cellular effects in vitro of three reversed amidines (DB889, DB702, and DB786) and one diguanidine (DB711) against both amastigotes and bloodstream trypomastigotes of T. cruzi, the etiological agent of Chagas' disease. Our data show that the reversed amidines have higher levels of activity than the diguanidine, with the order of trypanocidal activities being as follows: DB889 > DB702 > DB786 > DB711. Transmission electron microscopy analysis showed that the reversed amidines induced many alterations in the nuclear morphology, swelling of the endoplasmic reticulum and Golgi structures, and consistent damage in the mitochondria and kinetoplasts of the parasites. Interestingly, in trypomastigotes treated with the reversed amidine DB889, multiple axoneme structures (flagellar microtubules) were noted. Flow cytometry analysis confirmed that the treated parasites presented an important loss of the mitochondrial membrane potential, as revealed by a decrease in rhodamine 123 fluorescence. Our results show that the reversed amidines have promising activities against the relevant mammalian forms of T. cruzi and display high trypanocidal effects at very low doses. This is especially the case for DB889, which merits further in vivo evaluation.

Disarray of sarcomeric alpha-actinin in cardiomyocytes infected by Trypanosoma cruzi.

Infection with Trypanosoma cruzi causes acute myocarditis and chronic cardiomyopathy. Remarkable changes have been demonstrated in the structure and physiology of cardiomyocytes during infection by this parasite that may contribute to the cardiac dysfunction observed in Chagas' disease. We have investigated the expression of alpha-actinin, an actin-binding protein that plays a key role in the formation and maintenance of Z-lines, during the T. cruzi-cardiomyocyte interaction in vitro. Immunolocalization of alpha-actinin in control cardiomyocytes demonstrated a typical periodicity in the Z line of cardiac myofibrils, as well as its distribution at focal adhesion sites and along the cell-cell junctions. No significant changes were observed in the localization of alpha-actinin after 24 h of infection. In contrast, depletion of sarcomeric distribution of alpha-actinin occurred after 72 h in T. cruzi-infected cardiomyocytes, while no change occurred at focal adhesion contacts. Biochemical assays demonstrated a reduction of 46% and 32% in the expression of alpha-actinin after 24 h and 72 h of infection, respectively. Intracellular parasites were also stained with an anti-alpha-actinin antibody that recognized a protein of 78 kDa by Western blot. Taken together, our data demonstrate a degeneration of the myofibrils in cardiomyocytes induced by T. cruzi infection, rather than a disassembly of the I bands within sarcomeres.