Effect of Gossypol on Trypomastigotes and Amastigotes of Trypanosoma cruzi

Bloodstream Trypanosoma cruzi trypomastigotes isolated from infected mice undergo reduction of motility and structural damages after 5 to 45 min exposure to gossypol at concentrations ranging from 5 to 50 μM. When 1% serum albumin is added to the incubation medium, no alterations of parasites are observed, even with 100 μM gossypol. Intracellular T. cruzi amastigotes in infected Vero cell cultures exposed to 5 μM gossypol for 2 h do not show changes. Incubation with 5 μM gossypol for 48 h produces complete disruption of host cells; however, the amastigotes they contain show only mineor alterations. The observations indicate that, in protein-rich media, gossypol is complexed into associations which have no activity on the different forms of the T. cruzi biological cycle.     

Purification of tissue forms (Amastigotes) of Trypanosoma cruzi by immunoaffinity chromatography

A rapid and simple method for the purification of amastigotes of Trypanosoma cruzi from spleens of infected mice is described. A protein A-Sepharose 4B immunoadsorbent column bound with antisera to epimastigotes of T. cruzi was used to purify the tissue forms of this parasite. Host cells and debris are not retained, and parasites can be eluted in high yields and purity. Studies of surface glycoproteins and glycolipids of the purified amastigotes with 18 lectins of various specificities revealed the presence on the parasites of receptors for N-acetylglucosamine, N-acetylgalactosamine, D-galactose, and D-mannose binding lectins.     

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.     

Antigenic Differences Among Epimastigotes,Amastigotes and Trypomastigotes of Trypanosoma cruzi*

Antigenic differences were demonstrated among trypomastigotes, amastigotes, and epimastigotes of Trypanosoma cruzi by the indirect fluorescent antibody method. Tests using cross-absorbed sera were included in the study.     

Extracellular Trypanosoma cruzi calreticulin in the host-parasite interplay

Calreticulin (CRT) from vertebrates is a calcium-binding protein present mainly in the endoplasmic reticulum (ER). There, it directs the conformation of proteins and controls calcium levels. This review will focus on several extracellular roles of Trypanosoma cruzi CRT (TcCRT) in relation to its capacity to inhibit the complement system, mediate parasite infectivity, interfere with angiogenesis and, as a possible consequence, with tumor growth. The TcCRT antiangiogenic effect parallels with the capacity of T. cruzi infection to inhibit tumor development in vivo. Thus, the TcCRT, complement, and endothelial cell interactions seem to be an evolutionary adaptation to promote prolonged parasite-host relationships.     

Acylation-dependent Export of Trypanosoma cruzi Phosphoinositide-specific Phospholipase C to the Outer Surface of Amastigotes

Phosphoinositide phospholipase C (PI-PLC) plays an essential role in cell signaling. A unique Trypanosoma cruzi PI-PLC (TcPI-PLC) is lipid-modified in its N terminus and localizes to the plasma membrane of amastigotes. Here, we show that TcPI-PLC is located onto the extracellular phase of the plasma membrane of amastigotes and that its N-terminal 20 amino acids are necessary and sufficient to target the fused GFP to the outer surface of the parasite. Mutagenesis of the predicted acylated residues confirmed that myristoylation of a glycine residue in the 2nd position and acyl modification of a cysteine in the 4th but not in the 8th or 15th position of the coding sequence are required for correct plasma membrane localization in T. cruzi epimastigotes or amastigotes. Interestingly, mutagenesis of the cysteine at the 8th position increased its flagellar localization. When expressed as fusion constructs with GFP, the N-terminal 6 and 10 amino acids fused to GFP are predominantly located in the cytosol and concentrated in a compartment that co-localizes with a Golgi complex marker. The N-terminal 20 amino acids of TcPI-PLC associate with lipid rafts when dually acylated. Taken together, these results indicate that N-terminal acyl modifications serve as a molecular addressing system for sending TcPI-PLC to the outer surface of the cell.     

Novobiocin-Induced Ultrastructural Changes and Antagonism of DNA Synthesis in Trypanosoma cruzi Amastigotes Growing in Cell-Free Medium

The antimicrobial agent novobiocin, an inhibitor of the bacterial enzyme topoisomerase II (DNA gyrase), is known to antagonize Trypanosoma cruzi amastigotes growing in cell-free medium. To determine sites of antagonism of novobiocin, the effects of drug on parasite ultrastructure and incorporation of radiolabeled precursors of DNA, RNA and protein into macromolecules were determined. The predominant ultrastructural abnormality seen after exposure to 0.40 mM novobiocin for 24 h was the presence of electron-dense clumps in the mitochondrion-kinetoplast organelle in 95 of 257 (37%) of cells, in comparison to no clumps seen in 110 drug-free cells. In addition, in the nucleus, the karyosome was less distinct than in control cells and appeared to merge with the chromatin. In the radiolabcling studies, incorporation of thymidine was inhibited in a dose-dependent fashion by novobiocin (0.16–0.80 mM) in a range of drug concentrations that also inhibited parasite growth. For 0.16 and 0.24 mM novobiocin, incorporation of thymidine was inhibited up to 65% relative to drug-free control cells while uptake of uridine and leucine was unaltered. We interpret these ultrastructure and precursor-incorporation studies as suggesting that (i) the mitochondrion-kinetoplast and possibly the nucleus are sites of novobiocin antagonism of T. cruzi amastigotes and (ii) that novobiocin appears to antagonize DNA synthesis within these organisms. Whether the drug target is topoisomerase II, however, is as yet unknown.     

Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes

Epimastigote forms of Trypanosoma cruzi (the etiologic agent of Chagas disease) internalize and store extracellular macromolecules in lysosome-related organelles (LROs) called reservosomes, which are positive for the cysteine protease cruzipain. Despite the importance of endocytosis for cell proliferation, macromolecule internalization remains poorly understood in the most clinically relevant proliferative form, the intracellular amastigotes found in mammalian hosts. The main obstacle was the lack of a simple method to isolate viable intracellular amastigotes from host cells. In this work we describe the fast and efficient isolation of viable intracellular amastigotes by nitrogen decompression (cavitation), which allowed the analysis of amastigote endocytosis, with direct visualization of internalized cargo inside the cells. The method routinely yielded 5x10⁷ amastigotes—with typical shape and positive for the amastigote marker Ssp4—from 5x10⁶ infected Vero cells (48h post-infection). We could visualize the endocytosis of fluorescently-labeled transferrin and albumin by isolated intracellular amastigotes using immunofluorescence microscopy; however, only transferrin endocytosis was detected by flow cytometry (and was also analyzed by western blotting), suggesting that amastigotes internalized relatively low levels of albumin. Transferrin binding to the surface of amastigotes (at 4°C) and its uptake (at 37°C) were confirmed by binding dissociation assays using acetic acid. Importantly, both transferrin and albumin co-localized with cruzipain in amastigote LROs. Our data show that isolated T. cruzi intracellular amastigotes actively ingest macromolecules from the environment and store them in cruzipain-positive LROs functionally related to epimastigote reservosomes.     

Protein kinase CK1 from Trypanosoma cruzi

A protein kinase activity, which uses casein as a substrate, has been purified to homogeneity from the epimastigote stage of Trypanosoma cruzi, by sequential chromatography on Q sepharose, heparin sepharose, phenyl sepharose, and alpha-casein agarose. An apparent molecular weight of 36,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography and sedimentation analyses demonstrated that the purified native enzyme is a monomer with a sedimentation coefficient of 2.9 S. The hydrodynamic parameters indicated that the shape of the protein is globular with a frictional ratio f/f(o) = 1.36 and a Stokes radius of 27.7 A. When two selective peptide substrates for protein kinases CK1 and CK2 were used (RRKDLHDDEEDEAM. SITA and RRRADDSDDDDD, respectively), the purified kinase was shown to predominantly phosphorylate the CK1-specific peptide. Additionally, the enzyme was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide, a specific inactivator of CK1s from mammals. Based on these results, we concluded that the purified kinase corresponds to a parasite CK1.     

Extracellular matrix derived from Trypanosoma cruzi infected endothelial cells directs phenotypic expression.

Infection of confluent human umbilical vein endothelial cells by the parasite Trypanosoma cruzi results in the appearance of an altered heparan sulfate proteoglycan (HSPG) isolated from the extracellular matrix of infected endothelial cells (ECMi). HSPG from ECMi differed from HSPG obtained from the extracellular matrix of uninfected endothelial cells (ECMu) by virtue of an 8-10-fold increase in sulfation and a different elution pattern using DEAE Sepharose chromatography. Analysis of the HSPG that binds to acidic fibroblast growth factor (aFGF) revealed that infection increased the proportion of HSPG that binds to aFGF by 35%. Heparitinase and alkaline borohydride treatment of aFGF-binding HSPG and chromatographic resolution on Sepharose CL4B column revealed an infection-associated 10-fold increase in sulfation of the GAG side chain with no significant change in the migration of the core protein. In addition, the aFGF binding HSPG isolated from ECMi demonstrated a markedly attenuated synergistic mitogenic activity with aFGF in a cell proliferation assay. All of the infection associated changes in HSPG could be demonstrated in HSPG obtained from uninfected endothelial cell cultures grown on ECMi. Hence, the ECMi is associated with signals capable of modulating the ECM associated metabolism of uninfected endothelial cells. This facility of ECMi was also shown to extend to patterns of Gs protein synthesis as revealed by Western blot analysis. The observation that the ECM produced by infected endothelial cells can direct the synthetic patterns of uninfected endothelial cells in a manner uniquely observed in infected endothelial cells suggests a plausible pathway by which infection of only a few cells can ultimately result in the coordinate responses of neighboring uninfected cells.     

Human humoral immunity to hsp70 during Trypanosoma cruzi infection

Immunologic screening of cDNA expression libraries has been widely used for the identification of DNA sequences encoding the immunologically relevant proteins of many pathogenic microorganisms. For reasons that are not entirely clear, sequences encoding 70-kDa heat shock and related proteins (hsp70), which are among the most highly conserved proteins known, have routinely been identified by this approach. Consequently, hsp70 proteins have been proposed to be involved in the autoimmune processes thought responsible for the pathogenesis of the diseases caused by some of these organisms, e.g., chronic Trypanosoma cruzi infection (Chagas' disease). Therefore, we investigated whether hsp70 might be a specific target of the human humoral immune response to T. cruzi infection, and, if so, whether humoral autoimmunity to hsp70 might play a role in pathogenesis. We found that hsp70 is indeed a major polypeptide Ag in Chagas' disease, but that the antibodies to T. cruzi hsp70 do not react with human hsp70--even though the proteins display 73% amino acid sequence identify. These results indicate that self-tolerance to hsp70 is maintained during chronic T. cruzi infection and strongly argue against a role for humoral autoimmunity to hsp70 in the pathogenesis of Chagas' disease.     

Regulation of host cell cyclin D1 by Trypanosoma cruzi in myoblasts

Infection with the parasite Trypanosoma cruzi causes Chagas disease. In this study we demonstrated that there was an increase in cyclin D1 expression in T. cruzi (Tulahuen strain)-infected myoblasts. To examine a possible mechanism for the increased cyclin D1 expression we transfected L6E9 myoblasts with cyclin D1 luciferase reporter constructs and infected with T. cruzi. There was no evidence of an increase in promoter activity. Additionally, quantitative PCR did not demonstrate any change in cyclin D1 message during infection. Moreover, we demonstrated that the cyclin D1 protein was significantly stabilized after infection. Collectively, these data indicate that infection with T. cruzi increases cyclin D1 protein abundance post-translationally.     

Beta-interferon inhibits cell infection by Trypanosoma cruzi

Preparations containing alpha/beta-interferon produced by L-929 cells were found to inhibit the capacity of bloodstream forms of Trypanosoma cruzi to associate with and infect mouse peritoneal macrophages or rat heart myoblasts. Marked reductions in the number of parasites per cell as well as in the percentage of cells associated with the trypanosomes were systematically observed in cultures of these cells that contained interferon. The inhibitory effect was abrogated in the presence of specific antibodies against alpha/beta-interferon, and purified beta-interferon induced a similar inhibitory effect, indicating that the active principle in the preparation was indeed interferon. Pretreatment of the parasites with alpha/beta-interferon reduced their infectivity for untreated host cells, whereas pretreatment of either type of host cell had no consequence on the interaction. The effect of interferon on the trypanosomes was reversible; the extent of the inhibitory effect was significantly reduced after 20 min, and was undetectable after 60 min when macrophages were used as host cells. Longer periods of time were required for the inhibitory effect to begin to subside (60 min) and to become undetectable or insignificant (120 min) when rat heart myoblasts were used. The results of additional studies performed with purified preparations of alpha- or beta-interferon revealed that only the latter was inhibitory of cell-parasite association. Because interferon is known to be produced shortly after T. cruzi infection and its administration has been shown to have a marked protective effect against this infection, our results suggest that the latter may involve inhibition of cell infection by interferon.     

Developmental Life Cycle of Leishmania—Cultivation and Characterization of Cultured Extracellular Amastigotes1

ABSTRACT. The biochemistry and immunology of Leishmania promastigotes has been extensively studied; this is due primarily to the facility with which this stage, in contrast to the amastigotes stage, can be maintained in axenic culture. Several attempts to axenically culture lines of Leishmania amastigotes have been reported in the literature. This paper summarizes methods of adaptation (low pH, elevated temperature and culture medium) and characterization of several axenic lines of Leishmania amastigotes. Based on morphological, biological, immunological and biochemical evidence, these organisms appear to resemble amastigotes from infected macrophages or tissue. The axenically cultured amastigotes appear to be distinct from shocked (heat, serum deprivation, stressed) Leishmania promastigotes in the plethora of proteins synthesized, growth (multiplication) in culture, and developmental regulation observed. These data suggest that Leishmania organisms have a significant developmental response to certain signals (pH, temperature) mimicking their in vivo macrophage milieu. The response to other environmental parameters characteristic of the host-macrophage remain to be determined. These axenically cultured amastigotes should be of interest for further immunological, biochemical and developmental investigations of the disease-maintaining stage of this parasite.     

Unambiguous identification of histone H1 in Trypanosoma cruzi

The existence of histone H₁ has been questioned in Trypanosomatids. We report here the presence of a histone H₁ in the chromatin of Trypanosoma cruzi. This protein was purified by narrow-bore reversed phase HPLC and its amino acid composition analyzed and compared with histones H₁ from other species. Furthermore, the purified chromosomal protein was digested with proteases and the amino acid sequences of the resulting peptides were analyzed by the automated Edman degradation. The sequences obtained were found to present a high degree of homology when compared to the carboxy terminal domain of other known histones H₁.     

Delimitation of Trypanosoma cruzi zymodemes by numerical taxonomy

Summary Based on the absence or presence of 24 isoenzyme characters, Brazilian Trypanosoma cruzi stocks can be separated by methods of numerical taxonomy into three principal, distinct groups; these groups correspond to the three T. cruzi zymodemes previously reported from north Brazil. A distinction is drawn between taxonomy, which may be of zoological interest only, and identification of medically important groups according to one or a few distinguishing characters.This paper is reprinted from Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, 238–242.This paper is reprinted from Transactions of the Royal Society of Tropical Medicine and Hygiene, 74, 238–242.