Tissue Culture Trypomastigotes Research Articles

Trypanosoma cruzi175-kDa Protein Tyrosine Phosphorylation Is Associated with Host Cell Invasion

Favoreto, S., Jr., Dorta, M. L., and Yoshida, N. 1998.Trypanosoma cruzi175-kDa protein tyrosine phosphorylation is associated with host cell invasion.Experimental Parasitology89,188–194. We examined the requirement ofTrypanosoma cruziprotein tyrosine phosphorylation for parasite entry into mammalian cells and analyzed the profile of phosphorylated proteins in infective trypomastigotes. Treatment of metacyclic or tissue culture trypomastigotes with genistein, an inhibitor of protein tyrosine kinase activity, significantly inhibited invasion of cultured HeLa cells. A soluble factor, contained in HeLa cell extract and absent in the extract otT. cruzi-resistant K562 cells, greatly enhanced phosphorylation levels of a 175-kDa protein (p175) in trypomastigotes. Genistein inhibited p175 tyrosine phosphorylation. P175 was undetectable in noninvasive epimastigotes. The phosphorylation-inducing activity of HeLa cell extract was abrogated by adsorption with metacyclic trypomastigotes but not with epimastigotes or when it was mixed with recombinant protein J18, which contains the entire peptide sequence of gp82, a metacyclic stage-specific surface glycoprotein implicated in target cell invasion. These data suggest that, in metacyclic trypomastigotes, gp82 is the signaling receptor that mediates protein tyrosine phosphorylation necessary for host cell invasion.

Cell surface characterization of amastigotes of Trypanosoma cruzi obtained from different sources.

The present study analyses the morphology and the exposition of surface carbohydrates and the Ssp4 antigen of amastigote forms of Trypanosoma cruzi (Y strain) obtained from three different sources: (a) intracellular, isolated from infected Vero cells 3 days after infection, (b) extracellular, isolated from the supernatant of Vero cells 15 days after infection, and (c) axenic, obtained by incubation of tissue culture trypomastigotes in LIT medium, at 37 degrees C for 4 days. No morphological differences were observed by light microscopy among these amastigotes. Transmission electron microscopy of thin sections showed a thick cell coat easily observed on the plasma membrane of axenic amastigotes. Carbohydrate-containing sites on the surface of the three different amastigotes were analysed using lectins, agglutination assays and flow cytometry. Mannose and/or glucose residues were found on the surface of all populations, but intracellular amastigotes showed the highest number. A small group of cells from the different populations expressed galactose and N-acetyl-glucosamine residues. The presence and distribution of the Ssp4 antigen in the different amastigote populations were evaluated using FITC and gold-labelled antibodies, and observed with an electronic programmable individual cell sorter and transmission electron microscopy. Ssp4 antigen was present on the membrane lining the flagellar pocket and on the cell surface, as well as inside the cytoplasmic vesicles of the host cell. Flow cytometry analysis of different amastigote populations showed that intracellular amastigotes presented the highest percentage of Ssp4-expressing cells.

In vitro trypanocidal activity of tetraethylthiuram disulfide and sodium diethylamine-N-carbodithioate on Trypanosoma cruzi.

Tetraethylthiuram disulfide (disulfiram) (TETD) and sodium diethylamine-N-carbodithioate (DECD) were examined for their in vitro trypanocidal activity against Trypanosoma cruzi. Benznidazole (BNZ), the drug used clinically for the chemotherapy of Chagas' disease, was used as a positive control. Inhibition assays included evaluation against the epimastigote, trypomastigote, and amastigote forms. Tetraethylthiuram disulfide and its reductive metabolite DECD inhibited 64.6% and 69.7%, respectively, of epimastigotes at a concentration of 50 micrograms/ml after 72 hr and BNZ caused 69.1% inhibition at the same concentration. Both TETD and DECD were not as effective against tissue culture trypomastigotes as BNZ (TETD = 47.7%,; DECD = 46.1%; BNZ = 88.7%) at 50 micrograms/ml after 24 hr. However, TETD and DECD treatment of amastigote-infected 3T3 fibroblasts yielded 60 and 67% inhibition, respectively, as compared to BNZ with an inhibition of 62%. A possible mechanism of action of TETD and DECD is via interference with the essential metal metabolism of T. cruci. Since toxicity data for both TETD and DECD are available and both drugs are active against the parasite, these drugs are candidates for further study to determine if they are of potential clinical interest as a prophylactic or therapeutic agent against Chagas' disease.

Reactivity of Trypanosoma cruzi strains with peanut agglutinin (PNA) correlates with number of in vitro infected host cells

Reactivities of 4 lectins with intact trypomastigote forms derived from 8 different Trypanosoma cruzi strains were compared with their capacity to infect in vitro cultured LLC-MK 2 cells. A sensitive and reproducible titration method for lectin binding sites (ELLA: Enzyme Linked Lectin Assay) was employed, in which reactivities were scored through optical densities in an ELISA reader. Tissue culture trypomastigotes from the strains Y, CL, SC4, SC24, SC25, SC28, SC32 and SC33 were investigated for expression of different cell surface carbohydrate residues using Concanavalin A (ConA), Peanut agglutinin (PNA), Soybean agglutinin (SBA) and Wheat germ agglutinin (WGA) conjugated to peroxidase. The reactivity of the strains to PNA lectin was SC28>SC32>SC33>SC25>SC24>Y>CL>SC4. The optical density values obtained were highly correlated (r 2 = 0.986, p < 10 −4) with the number of parasitized LLC-MK 2 cells 24 hours after infection by trypomastigotes from each corresponding strain. We concluded that galactose and N-acetyl-D-galactosamine residues that are present on the surface of trypomastigotes are important in host-cell recognition.

Role of nitric oxide in resistance and histopathology during experimental infection with Trypanosoma cruzi

We analyzed the biological role of nitric oxide (NO) during murine Trypanosoma cruzi infection. Infection of mice with T. cruzi markedly increased NO synthesis. Administration of N-nitro- l-arginine methyl-esther ( l-NAME) intraperitoneally or intragastrically diminished endogenous NO synthesis and resistance of mice to acute infection with three biologically different strains of T. cruzi. Mice protected against challenge with T. cruzi by transfer of T-cell-enriched populations from chronically infected animals, showed higher serum nitrate levels than controls non-transferred, or transferred, with T cells from non-immune mice. Administration of l-NAME abrogated transfer of resistance, suggesting NO participation in this process. Depletion of T cells from the transferred population abolished both protection and NO 3 − increase. On the contrary, mice chronically infected with T. cruzi showed no increased parasitemia or death upon treatment with l-NAME. The NO donor drug S-nitroso-acetyl-penicillamine was able to kill tissue culture or bloodstream trypomastigotes in vitro at biologically relevant concentrations. Conversely, NO appeared not to play a role in formation of inflammatory foci during T. cruzi infection, since infected mice treated with l-NAME showed no reduced inflammation.

Trypanosoma cruzi: Characterization of Two Recombinant Antigens with Potential Application in the Diagnosis of Chagas′ Disease

A genomic library of Trypanosoma cruzi, constructed in the vector λgt11, was screened with a hyperimmune rabbit antiserum against tissue culture trypomastigotes. Two clones, B12 and B13, containing inserts of 350 and 600 bp, respectively, were isolated. Sequencing data indicated that both clones present a pattern of tandemly repeated nucleotide units of 60 bp for B 12 and 36 bp for B13. Southern blot analysis suggests that both corresponding genes exist as a single copy. The inserts of both recombinants were subcloned in the vector pMSgt11, in phase with the lacZ gene. Recombinant proteins were affinity purified on pAPTG–agarose columns and employed to immunize rabbits, as well as to immunoselect human chagasic antibodies. By Western blot, antibodies to B12 reacted with bands of 230 kDa in trypomastigotes and 200 kDa in epimastigotes, while those to B13 identified bands of 140 and 116 kDa in trypomastigotes and epimastigotes. Immunoprecipitation of radioiodinated parasites revealed that the 140-kDa antigen recognized by antibodies to B13 is located on the membrane of trypomastigotes but not epimastigotes. The potential application of either recombinant antigen in the serological diagnosis of Chagas" disease was evaluated initially by RIA. It was observed that B13 presents a very good performance with sensitivity of 97%. For B12, the corresponding value was 82%. The reactivity to B13 was also evaluated by ELISA tests run in parallel with conventional serological reactions for Chagas" disease. Analysis of 209 serum samples indicates that B13 presents similar or even better performance in relation to the use of total epimastigote antigens, making it a promising candidate for the diagnosis of Chagas" disease.

Stage-specific phospholipase C-mediated release of Trypanosoma cruzi neuraminidase

Several studies indicate that the neuraminidase from Trypanosoma cruzi (TCNA) [1 ] plays an important role in infection. Trypomastigotes remove sialic acid from rat myocardial and human endothelial cells in vitro [2], and from mouse [1] and human [3] erythrocytes during infection with T. cruzi in vivo. The extent of desialylation of host cells correlates with the number of parasites and TCNA activity [3]. Inhibition of enzyme activity by polyclonal [4] and monoclonal [5] antibodies and by high [6] and low-density lipoproteins [7] augment invasion of host cells by trypomastigotes suggesting that the enzyme exerts a negative control on T. cruzi infection (reviewed in ref. 8). We have previously shown that TCNA is spontaneously released into condition medium when tissue culture trypomastigotes are incubated at 4°C [4] or 37°C [5] for several hours or days, suggesting that the enzyme is anchored to the cell surface by a glycophosphatidylinositol (GPI) linkage. Release of membrane-bound proteins by PI-PLC is now regarded as convincing evidence for the involvement of GPI in membrane anchoring [9]. We therefore tested whether TCNA could be specifically removed from the surface of the Silvio X10/4

Structural and functional identification of GP57/51 antigen of Trypanosoma cruzi as a cysteine proteinase

Purified GP57/51, a Trypanosoma cruzi glycoprotein earlier identified as a major antigen in infected humans, was subjected to N-terminal sequence analysis. Alignment of the first 30 amino acids revealed that its N-terminal region is virtually identical to that reported for a cysteine-proteinase isolated from the Tulahuen strain, including the presence of active site cysteine at position 25. The finding of serine at position 24 of GP57/51 (Y strain) has further increased the homology between this protozoan antigen with other members of the eukaryotic family of cysteine proteases, including human cathepsin L. Functional analysis of GP57/51 indicated that the antigen is indeed an active thiol proteinase, which is active across a wide pH range (5–7.5). This was shown using either human IgG or gelatin substrates co-polymerized into polyacrylamide gels prepared for electrophoresis, and also by enzyme assays performed with the synthetic substrate Z-phe-arg-NMec. The enzyme was activated by thiol containing reagents, and was strongly inhibited by low concentrations of E-64 (IC 50 0.1 μM), cystatin (IC 50 1 μM), leupeptin (IC 50 0.1 μM) and antipain (IC 50 0.1 μM). Monoclonal antibodies directed against distinct epitopes of GP57/51 absorbed the hydrolytic activity from purified preparations, demonstrating that the antigenic and enzymatic activities were indeed expressed by the same molecular entities. The subcellular localization of immunoreactive molecules was investigated by electron microscopy; immunogold staining was conspicuously found in vesicles belonging to the endosomal-lysosomal system, in tissue culture trypomastigotes as well as in epimastigotes. The possibility that this highly antigenic protease is actively secreted and/or leaked out of damaged parasites is under investigation; its release to tissues and to the circulation may contribute to pathology, considering that it (i) can degrade proteins across a wide pH range and (ii) stimulates immune T cells from chronic chagasic patients.

Trypanosoma cruzi: serum antibody reactivity to the parasite antigens in susceptible and resistant mice

The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/10J) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected.

Monoclonal antibodies against Trypanosoma cruzi neuraminidase reveal enzyme polymorphism, recognize a subset of trypomastigotes, and enhance infection in vitro.

The identification and characterization of two murine mAb (TCN-1 and TCN-2) that react with the neuraminidase of Trypanosoma cruzi is reported. The mAb were identified based on their ability to inhibit enzyme activity and recognize neuraminidase in crude enzyme preparations. TCN-1 and TCN-2 recognized Ag in tissue culture trypomastigotes but not in the amastigotes, epimastigotes, or metacyclic trypomastigotes using immunoblot assays and immunofluorescence. In addition, clones Y-H6, MV-13, and Silvio X-10/4 of T. cruzi revealed a unique banding pattern characteristic of each clone. In Silvio X-10/4, the mAb recognized four distinct bands ranging from 121,000 to 203,000 whereas in Y-H6 and MV-13 they identified bands ranging from 138,000 to 222,000. Characterization of neuraminidase by two-dimensional PAGE revealed the polypeptides that make up the enzyme to have isoelectrical points ranging from 6.55 to 7.30. Immunofluorescence and C-mediated lysis assays showed that the mAb reacted with a subset of trypomastigotes representing 28% of the total parasite population. Functional studies showed that the mAb enhanced infection of cultured cells by trypomastigotes. Our experiments confirm previous findings with polyclonal Ab and are in accordance with the hypothesis that neuraminidase modulates infection through a negative control mechanism.

Complement component C1q enhances invasion of human mononuclear phagocytes and fibroblasts by Trypanosoma cruzi trypomastigotes.

Internalization and infectivity of Trypanosoma cruzi trypomastigotes by macrophages is enhanced by prior treatment of parasites with normal human serum. Heating serum or removing C1q from serum abrogates the enhancement, but augmentation of attachment and infectivity is restored by addition of purified C1q to either serum source. Although both noninfective epimastigotes (Epi) and vertebrate-stage tissue culture trypomastigotes (TCT) bind C1q in saturable fashion at 4 degrees C, internalization by monocytes and macrophages of TCT but not Epi-bearing C1q is enhanced in comparison to untreated parasites. Adherence of human monocytes and macrophages to surfaces coated with C1q also induces a marked enhancement of the internalization of native TCT. C1q enhances attachment of both Epi and TCT to human foreskin fibroblasts, but only when C1q is on the parasite and not when the fibroblasts are plated on C1q-coated surfaces. Only TCT coated with C1q show enhanced invasion into fibroblasts. Although trypomastigotes produce an inhibitor of the complement cascade which limits C3 deposition during incubation in normal human serum, C1q binds to the parasite and enhances entry of trypomastigotes into target cells.

A surface antigen of Trypanosoma cruzi involved in cell invasion (Tc-85) is heterogeneous in expression and molecular constitution.

A monoclonal antibody (McAb), H1A10, causes 46-96% inhibition of the invasion of tissue culture cells by trypomastigotes from different strains and clones of Trypanosoma cruzi. Higher inhibition was observed when axenic medium-derived metacyclic trypomastigotes (72-96%) were employed instead of tissue culture-derived trypomastigotes (46-60%). In contrast to the metacyclic population, in which all individuals reacted with the McAb, part of the tissue-cultured trypomastigote population did not bind the antibody. The molecular masses and isoelectric points of the glycoproteins recognized by the H1A10 McAb differed when tissue culture trypomastigotes of the Y strain and YuYu strain were analyzed. Variability between the strains was also observed when the antigens were metabolically labelled in the presence of tunicamycin. These results suggest that the differences described are probably not due solely to the carbohydrate portion of the molecule.

Terminal β‐d‐galactofuranosyl epitopes recognized by antibodies that inhibit Trypanosoma cruzi internalization into mammalian cells

Polyclonal antisera to 80 - 90-kDa and to 50 - 60-kDa polypeptides of tissue-culture trypomastigotes which inhibit the interiorization of trypomastigotes of the Y strain (up to 70%) and of metacyclic trypomastigotes of the CL-14 clone (up to 50%) in cultured mammalian cells, were obtained. Both sera immunoprecipitate surface polypeptides of 90 kDa, 80 kDa, 72 kDa and 58 kDa in trypomastigotes and of 80 kDa, 77 kDa and 74 kDa in metacyclic trypomastigotes. These antigens are glycoproteins with affinity for concanavalin A. The antibodies (IgG class) of the inhibitory sera are mainly directed against carbohydrate epitopes, which were identified as being beta-D-galactofuranosyl units by radioimmunoinhibition assays. The direct involvement of the beta-D-galactofuranosyl unit in the process of parasite infection was verified using the synthetic disaccharide beta-D-galactofuranose (1----3)-alpha-D-mannopyranose which promoted, at 1 mg/ml concentration, 50% inhibition of internalization.

Lytic rabbit IgG for tissue culture trypomastigotes of Trypanosoma cruzi alters the extent and form of complement deposition

Infective and vertebrate stages of Trypanosoma cruzi are resistant to lysis by the alternative pathway of complement. To further elucidate the mechanism of complement evasion and to study how some immune sera render the infective stage sensitive to lysis, we compared the interaction of complement components C3 and C9 with the surface of complement susceptible, vector stage epimastigotes and vertebrate stage trypomastigotes of T. cruzi. Our studies showed that, upon incubation in human serum, complement resistant tissue culture trypomastigotes (TCT) bound five- to eightfold less C3 or C9 than complement sensitive epimastigotes (Epi). C3 bound to Epi is mainly in the hemolytically active C3b form, while TCT bear predominantly the hemolytically inactive iC3b fragment, which cannot participate in C5 convertase formation or lead to deposition of the lytic C5b-9 complex. Three- to sixfold more C3 and two- to threefold more C9 were deposited on TCT when lytic rabbit immune IgG with broad specificity was used to sensitize the parasites, and nearly one-half of bound C3 was present as C3b. In contrast, a comparison of three different sources of IgG from immune human serum showed a less clear correlation between the titer or specificity of anti- T. cruzi antibody, enhancement of C3 or C9 deposition, change in the form of bound C3, or killing. These results show that lytic rabbit IgG for T. cruzi changes the form and amount of bound complement components in anticipated fashion, but that human immune IgG does not give predictable changes in the extent or form of C3 or C9 deposition.

Biochemical characterization of a factor produced by trypomastigotes of Trypanosoma cruzi that accelerates the decay of complement C3 convertases.

Infective- and vertebrate-stage trypomastigotes of Trypanosoma cruzi resist serum killing by the alternative complement pathway, whereas noninfective vector-stage epimastigotes, from which trypomastigotes derive, are serum-sensitive. This form of developmental preadaption is commonly observed in protozoan parasites, but its mechanisms are poorly understood. We have demonstrated previously that trypomastigotes spontaneously shed molecules which interfere with formation and accelerate the intrinsic decay of complement C3 convertases, a finding which may explain the evasion of complement lysis by trypomastigotes. We now describe the partial purification and characterization of the T. cruzi C3 convertase inhibitor from the supernatant of culture metacyclic and tissue culture trypomastigotes. Decay-accelerating activity for both classical and alternative pathway C3 convertases copurifies on anion-exchange fast protein liquid chromatography and chromatofocusing with 35S-labeled molecules of 87-93 kDa, pI 5.6-5.8. The labeled components are destroyed by papain and retained on concanavalin A-Sepharose, procedures which remove functional decay-accelerating activity from the supernatant. The 87-93-kDa components are immunoprecipitated by sera from patients chronically infected with T. cruzi, but not by antisera to any known regulatory proteins of the human complement cascade. Lytic activity for tissue culture trypomastigotes in chagasic sera is associated with antibody reactivity against the 87-93-kDa 35S-labeled components and with inhibition of decay-accelerating activity. The T. cruzi factor is the first developmentally regulated microbial complement inhibitor to be biochemically characterized.

Evaluation of the rabbit as a model for Chagas' disease. I. Parasitological studies.

In order to investigate the value of the rabbit as an experimental model for Chagas' disease, 72 animals have been inoculated by intraperitoneal and conjunctival route with bloodstream forms, vector-derived metacyclic trypomastigotes and tissue culture trypomastigotes of Trypanosoma cruzi strains Y, CL and Ernane. In 95.6% of the animals trypomastigotes had been detected at the early stages of infection by fresh blood examination. The course of parasitemia at the acute phase was strongly influenced by the parasite strain and route of inoculation. At the chronic phase parasites had been recovered by xenodiagnosis and/or hemoculture in 40% of the examined animals. The xenodiagnosis studies have shown selective interactions between the T. cruzi strains and the four species of vectors used, inducing significant variability in the results. The data herein present are consistent with the parasitological requirements established for a suitable model for chronic Chagas' disease.

Characterization of the mechanism of protein glycosylation and the structure of glycoconjugates in tissue culture trypomastigotes and intracellular amastigotes of Trypanosoma cruzi

Trypomastigote cells of Trypanosoma cruzi incubated with [U-14C]glucose accumulated dolichol-P-P-linked Man2GlcNAc2 and Man9GlcNAc2. Evidence is presented indicating that both oligosaccharides were transferred to asparagine residues in proteins. On the other hand, intracellular amastigotes behaved as epimastigotes, i.e., only Man9GlcNAc2 accumulated and was transferred to proteins under similar incubation conditions. Intracellular amastigotes differed, therefore, from amastigotes obtained from an axenic culture, which behaved as trypomastigotes. A similar processing of protein-linked Man9GlcNAc2 and Man8GlcNAc2 occurred in epimastigotes and trypomastigotes but the structure of the main Man7GlcNAc2 isomer produced by demannosylation of the above mentioned oligosaccharides differed from that of the Man7GlcNAc2 transferred in trypomastigotes and amastigotes from axenic cultures. The infective trypomastigote stage of the parasite showed, therefore, an alteration in the mechanism of protein N-glycosylation when compared to the other stages, namely epimastigote (insect vector stage) and amastigote (mammalian intracellular stage). Complex-type, asparagine-bound oligosaccharides were found to be synthesized in both epimastigotes and trypomastigotes but the amounts of those compounds were extremely low when compared to those of high mannose-type oligosaccharides.

Trypanosoma cruzi interaction with macrophages: differences between tissue culture and bloodstream forms.

Macrófagos obtidos do peritoneo de camundongos após estímulo, com peptona, foram cultivados em lamínulas, infectados com tripomastigotas das cepas F e Y de T. cruzi, obtidos de cultivo de tecidos ou do sangue de camundongos infectados. Os parasitas, obtidos de cultivo de tecidos, tanto da cepa Y como os da cepa F, são interiorizados por macrófagos em proporção muito mais elevada do que os sanguícolas. Parasitas de cultivo de tecidos incubados com soro de camundongos normais, ou soro híperimune específico em diluição sub-aglutinante, comportam-se essencialmente como parasitas não opsonizados. Foram observadas diferenças a nível ultraestrutural na fase inicial de interação entre macrófagos e tripomastigotas das duas origens. Após 30 minutos, tripomastigotas de cultivo de tecidos localizam-se em agrupamentos na área de contato com os macrófagos. Enquanto os tripomastigotas sanguícolas estão na maioria das vezes no interior de vacúolos fagocíticos largos, após 3 horas de interação os tripomastigotas de cultura situam-se em um único vacúolo estreito. Tanto as formas de cultivo de tecidos quanto os tripomastigotas sanguícolas da cepa Y multiplicam-se em macrófagos; os tripomastigotas sanguícolas da cepa F são destruídos no interior da célula hospedeira, enquanto os tripomastigotas de cultivo de tecidos desta cepa são capazes de multiplicar-se.