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Sample records for brucei trypanosoma cruzi

  1. Sulfhydryl reagent susceptibility in proteins with high sequence similarity--triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi and Leishmania mexicana.

    Science.gov (United States)

    Garza-Ramos, G; Cabrera, N; Saavedra-Lira, E; Tuena de Gómez-Puyou, M; Ostoa-Saloma, P; Pérez-Montfort, R; Gómez-Puyou, A

    1998-05-01

    The amino acid sequence of triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68%. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei triosephosphate isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi triosephosphate isomerase was more than 100-fold more sensitive. The sensitivity of wild type triosephosphate isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8-anilino-1-napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half-maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana triosephosphate isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accessibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi triosephosphate isomerase which makes its dimer interface more susceptible for perturbation.

  2. Secondary metabolites from Vietnamese marine invertebrates with activity against Trypanosoma brucei and T. cruzi.

    Science.gov (United States)

    Thao, Nguyen Phuong; No, Joo Hwan; Luyen, Bui Thi Thuy; Yang, Gyongseon; Byun, Soo Young; Goo, Junghyun; Kim, Kyung Tae; Cuong, Nguyen Xuan; Nam, Nguyen Hoai; Van Minh, Chau; Schmidt, Thomas J; Kang, Jong Seong; Kim, Young Ho

    2014-06-11

    Marine-derived natural products from invertebrates comprise an extremely diverse and promising source of the compounds from a wide variety of structural classes. This study describes the discovery of five marine natural products with activity against Trypanosoma species by natural product library screening using whole cell in vitro assays. We investigated the anti-trypanosomal activity of the extracts from the soft corals and echinoderms living in Vietnamese seas. Of the samples screened, the methanolic extracts of several marine organisms exhibited potent activities against cultures of Trypanosoma brucei and T. cruzi (EC50 activity against T. brucei with EC50 values ranging from 1.57 ± 0.14 to 14.6 ± 1.36 μM, relative to the positive control, pentamidine (EC50 = 0.015 ± 0.003 μM). Laevigatol B (1) and 5α-cholest-8(14)-ene-3β,7α-diol (5) exhibited also significant inhibitory effects on T. cruzi. The cytotoxic activity of the pure compounds on mammalian cells was also assessed and found to be insignificant in all cases. This is the first report on the inhibitory effects of marine organisms collected in Vietnamese seas against Trypanosoma species responsible for neglected tropical diseases.

  3. Secondary Metabolites from Vietnamese Marine Invertebrates with Activity against Trypanosoma brucei and T. cruzi

    Directory of Open Access Journals (Sweden)

    Nguyen Phuong Thao

    2014-06-01

    Full Text Available Marine-derived natural products from invertebrates comprise an extremely diverse and promising source of the compounds from a wide variety of structural classes. This study describes the discovery of five marine natural products with activity against Trypanosoma species by natural product library screening using whole cell in vitro assays. We investigated the anti-trypanosomal activity of the extracts from the soft corals and echinoderms living in Vietnamese seas. Of the samples screened, the methanolic extracts of several marine organisms exhibited potent activities against cultures of Trypanosoma brucei and T. cruzi (EC50 < 5.0 μg/mL. Among the compounds isolated from these extracts, laevigatol B (1 from Lobophytum crassum and L. laevigatum, (24S-ergost-4-ene-3-one (2 from Sinularia dissecta, astropectenol A (3 from Astropecten polyacanthus, and cholest-8-ene-3β,5α,6β,7α-tetraol (4 from Diadema savignyi showed inhibitory activity against T. brucei with EC50 values ranging from 1.57 ± 0.14 to 14.6 ± 1.36 μM, relative to the positive control, pentamidine (EC50 = 0.015 ± 0.003 μM. Laevigatol B (1 and 5α-cholest-8(14-ene-3β,7α-diol (5 exhibited also significant inhibitory effects on T. cruzi. The cytotoxic activity of the pure compounds on mammalian cells was also assessed and found to be insignificant in all cases. This is the first report on the inhibitory effects of marine organisms collected in Vietnamese seas against Trypanosoma species responsible for neglected tropical diseases.

  4. Structural Characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei Bound to the Antifungal Drugs Posaconazole and Fluconazole

    Science.gov (United States)

    Chen, Chiung-Kuang; Leung, Siegfried S. F.; Guilbert, Christophe; Jacobson, Matthew P.; McKerrow, James H.; Podust, Larissa M.

    2010-01-01

    Background Chagas Disease is the leading cause of heart failure in Latin America. Current drug therapy is limited by issues of both efficacy and severe side effects. Trypansoma cruzi, the protozoan agent of Chagas Disease, is closely related to two other major global pathogens, Leishmania spp., responsible for leishmaniasis, and Trypansoma brucei, the causative agent of African Sleeping Sickness. Both T. cruzi and Leishmania parasites have an essential requirement for ergosterol, and are thus vulnerable to inhibitors of sterol 14α-demethylase (CYP51), which catalyzes the conversion of lanosterol to ergosterol. Clinically employed anti-fungal azoles inhibit ergosterol biosynthesis in fungi, and specific azoles are also effective against both Trypanosoma and Leishmania parasites. However, modification of azoles to enhance efficacy and circumvent potential drug resistance has been problematic for both parasitic and fungal infections due to the lack of structural insights into drug binding. Methodology/Principal Findings We have determined the crystal structures for CYP51 from T. cruzi (resolutions of 2.35 Å and 2.27 Å), and from the related pathogen T. brucei (resolutions of 2.7 Å and 2.6 Å), co-crystallized with the antifungal drugs fluconazole and posaconazole. Remarkably, both drugs adopt multiple conformations when binding the target. The fluconazole 2,4-difluorophenyl ring flips 180° depending on the H-bonding interactions with the BC-loop. The terminus of the long functional tail group of posaconazole is bound loosely in the mouth of the hydrophobic substrate binding tunnel, suggesting that the major contribution of the tail to drug efficacy is for pharmacokinetics rather than in interactions with the target. Conclusions/Significance The structures provide new insights into binding of azoles to CYP51 and mechanisms of potential drug resistance. Our studies define in structural detail the CYP51 therapeutic target in T. cruzi, and offer a starting point for

  5. In vitro investigation of Brazilian Cerrado plant extract activity against Plasmodium falciparum, Trypanosoma cruzi and T. brucei gambiense.

    Science.gov (United States)

    Charneau, Sébastien; de Mesquita, Mariana Laundry; Bastos, Izabela Marques Dourado; Santana, Jaime Martins; de Paula, José Elias; Grellier, Philippe; Espindola, Laila Salmen

    2016-06-01

    The threatened Brazilian Cerrado biome is an important biodiversity hotspot but still few explored that constitutes a potential reservoir of molecules to treat infectious diseases. We selected eight Cerrado plant species for screening against the erythrocytic stages of Plasmodium falciparum, human intracellular stages of Trypanosoma cruzi and bloodstream forms of T. brucei gambiense, and for their cytotoxicity upon the rat L6-myoblast cell line. Bioassays were performed with 37 hexane, ethyl acetate and ethanol extracts prepared from different plant organs. Activities against parasites were observed for 24 extracts: 9 with anti-P. falciparum, 4 with anti-T. cruzi and 11 with anti-T. brucei gambiense activities. High anti-protozoal activity (IC50 values < 10 μg/mL) without obvious cytotoxicity to L6 cells was observed for eight extracts from plants: Connarus suberosus, Blepharocalyx salicifolius, Psidium laruotteanum and Myrsine guianensis. Overall, studies of plant extracts will contribute to increase the biodiversity knowledge essential for Cerrado conservation and sustainable development.

  6. Taxonomy Icon Data: Trypanosoma brucei [Taxonomy Icon

    Lifescience Database Archive (English)

    Full Text Available Trypanosoma brucei Trypanosoma brucei Trypanosoma_brucei_L.png Trypanosoma_brucei_NL.png Trypanosoma_bruce...i_S.png Trypanosoma_brucei_NS.png http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Trypanosoma+bruce...i&t=L http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Trypanosoma+brucei&t=NL http://bioscie...ncedbc.jp/taxonomy_icon/icon.cgi?i=Trypanosoma+brucei&t=S http://biosciencedbc.jp.../taxonomy_icon/icon.cgi?i=Trypanosoma+brucei&t=NS http://togodb.biosciencedbc.jp/togodb/view/taxonomy_icon_comment_en?species_id=121 ...

  7. The acidocalcisome vacuolar transporter chaperone 4 catalyzes the synthesis of polyphosphate in insect-stages of Trypanosoma brucei and T. cruzi.

    Science.gov (United States)

    Ulrich, Paul N; Lander, Noelia; Kurup, Samarchith P; Reiss, Laura; Brewer, Jessica; Soares Medeiros, Lia C; Miranda, Kildare; Docampo, Roberto

    2014-01-01

    Polyphosphate is a polymer of inorganic phosphate found in both prokaryotes and eukaryotes. Polyphosphate typically accumulates in acidic, calcium-rich organelles known as acidocalcisomes, and recent research demonstrated that vacuolar transporter chaperone 4 catalyzes its synthesis in yeast. The human pathogens Trypanosoma brucei and T. cruzi possess vacuolar transporter chaperone 4 homologs. We demonstrate that T. cruzi vacuolar transporter chaperone 4 localizes to acidocalcisomes of epimastigotes by immunofluorescence and immuno-electron microscopy and that the recombinant catalytic region of the T. cruzi enzyme is a polyphosphate kinase. RNA interference of the T. brucei enzyme in procyclic form parasites reduced short chain polyphosphate levels and resulted in accumulation of pyrophosphate. These results suggest that this trypanosome enzyme is an important component of a polyphosphate synthase complex that utilizes ATP to synthesize and translocate polyphosphate to acidocalcisomes in insect stages of these parasites. © 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.

  8. Trypanosoma cruzi

    Science.gov (United States)

    Ramírez-Toloza, Galia; Ferreira, Arturo

    2017-01-01

    American Trypanosomiasis is an important neglected reemerging tropical parasitism, infecting about 8 million people worldwide. Its agent, Trypanosoma cruzi , exhibits multiple mechanisms to evade the host immune response and infect host cells. An important immune evasion strategy of T. cruzi infective stages is its capacity to inhibit the complement system activation on the parasite surface, avoiding opsonizing, immune stimulating and lytic effects. Epimastigotes, the non-infective form of the parasite, present in triatomine arthropod vectors, are highly susceptible to complement-mediated lysis while trypomastigotes, the infective form, present in host bloodstream, are resistant. Thus T. cruzi susceptibility to complement varies depending on the parasite stage (amastigote, trypomastigotes or epimastigote) and on the T. cruzi strain. To avoid complement-mediated lysis, T. cruzi trypomastigotes express on the parasite surface a variety of complement regulatory proteins, such as glycoprotein 58/68 (gp58/68), T. cruzi complement regulatory protein (TcCRP), trypomastigote decay-accelerating factor (T-DAF), C2 receptor inhibitor trispanning (CRIT) and T. cruzi calreticulin (TcCRT). Alternatively, or concomitantly, the parasite captures components with complement regulatory activity from the host bloodstream, such as factor H (FH) and plasma membrane-derived vesicles (PMVs). All these proteins inhibit different steps of the classical (CP), alternative (AP) or lectin pathways (LP). Thus, TcCRP inhibits the CP C3 convertase assembling, gp58/68 inhibits the AP C3 convertase, T-DAF interferes with the CP and AP convertases assembling, TcCRT inhibits the CP and LP, CRIT confers ability to resist the CP and LP, FH is used by trypomastigotes to inhibit the AP convertases and PMVs inhibit the CP and LP C3 convertases. Many of these proteins have similar molecular inhibitory mechanisms. Our laboratory has contributed to elucidate the role of TcCRT in the host-parasite interplay

  9. INFECTED WITH TRYPANOSOMA BRUCEI BRUCEI

    African Journals Online (AJOL)

    v brucei infection where for example tant components of extra-cellular the parasite infectivity potential and toxicological effects have been sues in animal. They are ... homeostasis (1,2, 3). With a unique ability to main- tain the cellular contents and ions stable concentrations, Trypano- soma b. brucei, to which humans.

  10. Crassiflorone derivatives that inhibit Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) and Trypanosoma cruzi trypanothione reductase (TcTR) and display trypanocidal activity.

    Science.gov (United States)

    Uliassi, Elisa; Fiorani, Giulia; Krauth-Siegel, R Luise; Bergamini, Christian; Fato, Romana; Bianchini, Giulia; Carlos Menéndez, J; Molina, Maria Teresa; López-Montero, Eulogio; Falchi, Federico; Cavalli, Andrea; Gul, Sheraz; Kuzikov, Maria; Ellinger, Bernhard; Witt, Gesa; Moraes, Carolina B; Freitas-Junior, Lucio H; Borsari, Chiara; Costi, Maria Paola; Bolognesi, Maria Laura

    2017-12-01

    Crassiflorone is a natural product with anti-mycobacterial and anti-gonorrhoeal properties, isolated from the stem bark of the African ebony tree Diospyros crassiflora. We noticed that its pentacyclic core possesses structural resemblance to the quinone-coumarin hybrid 3, which we reported to exhibit a dual-targeted inhibitory profile towards Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) and Trypanosoma cruzi trypanothione reductase (TcTR). Following this basic idea, we synthesized a small library of crassiflorone derivatives 15-23 and investigated their potential as anti-trypanosomatid agents. 19 is the only compound of the series showing a balanced dual profile at 10 μM (% inhibition TbGAPDH  = 64% and % inhibition TcTR  = 65%). In phenotypic assay, the most active compounds were 18 and 21, which at 5 μM inhibited Tb bloodstream-form growth by 29% and 38%, respectively. Notably, all the newly synthesized compounds at 10 μM did not affect viability and the status of mitochondria in human A549 and 786-O cell lines, respectively. However, further optimization that addresses metabolic liabilities including solubility, as well as cytochromes P450 (CYP1A2, CYP2C9, CYP2C19, and CYP2D6) inhibition, is required before this class of natural product-derived compounds can be further progressed. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  11. SUMOylation in Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Cornelia Andrea Klein

    2013-10-01

    Full Text Available Small ubiquitin like modifier (SUMO proteins are involved in many processes in eukaryotes. We here show that Trypanosoma brucei SUMO (Tb927.5.3210 modifies many proteins. The levels of SUMOylation were unaffected by temperature changes but were increased by severe oxidative stress. We obtained evidence that trypanosome homologues of the SUMO conjugating enzyme Ubc9 (Tb927.2.2460 and the SUMO-specific protease SENP (Tb927.9.2220 are involved in SUMOylation and SUMO removal, respectively.

  12. Non-radioactive method for labelling Trypanosoma brucei brucei ...

    African Journals Online (AJOL)

    It was observed that (i) the biological activities of the T.brucei brucei like the motility, viability, and growth were not affected adversely by the biotinylation; (ii) as little as 100ug Sulfo-NHS-LC-Biotin was enough to label about 6x106 Trypanosoma Brucei Brucei; (iii) high temperature appears to have adverse effect on the ...

  13. Regulation and spatial organization of PCNA in Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Kaufmann, Doris; Gassen, Alwine [University of Munich (LMU), Department Biology I, Genetics, Grosshaderner Str. 2-4, 82152 Martinsried (Germany); Maiser, Andreas; Leonhardt, Heinrich [University of Munich (LMU), Department Biology II, Grosshaderner Str. 2-4, 82152 Martinsried (Germany); Janzen, Christian J., E-mail: christian.janzen@uni-wuerzburg.de [University of Munich (LMU), Department Biology I, Genetics, Grosshaderner Str. 2-4, 82152 Martinsried (Germany)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Characterization of the proliferating cell nuclear antigen in Trypanosoma brucei (TbPCNA). Black-Right-Pointing-Pointer TbPCNA is a suitable marker to detect replication in T. brucei. Black-Right-Pointing-Pointer TbPCNA distribution and regulation is different compared to closely related parasites T. cruzi and Leishmania donovani. -- Abstract: As in most eukaryotic cells, replication is regulated by a conserved group of proteins in the early-diverged parasite Trypanosoma brucei. Only a few components of the replication machinery have been described in this parasite and regulation, sub-nuclear localization and timing of replication are not well understood. We characterized the proliferating cell nuclear antigen in T. brucei (TbPCNA) to establish a spatial and temporal marker for replication. Interestingly, PCNA distribution and regulation is different compared to the closely related parasites Trypanosoma cruzi and Leishmania donovani. TbPCNA foci are clearly detectable during S phase of the cell cycle but in contrast to T. cruzi they are not preferentially located at the nuclear periphery. Furthermore, PCNA seems to be degraded when cells enter G2 phase in T. brucei suggesting different modes of replication regulation or functions of PCNA in these closely related eukaryotes.

  14. Nuclear structure of Trypanosoma cruzi.

    Science.gov (United States)

    Schenkman, Sergio; Pascoalino, Bruno dos Santos; Nardelli, Sheila C

    2011-01-01

    The presence of nucleus in living organisms characterizes the Eukaryote domain. The nucleus compartmentalizes the genetic material surrounded by a double membrane called nuclear envelope. The nucleus has been observed since the advent of the light microscope, and sub-compartments such as nucleoli, diverse nuclear bodies and condensed chromosomes have been later recognized, being part of highly organized and dynamic structure. The significance and function of such organization has increased with the understanding of transcription, replication, DNA repair, recombination processes. It is now recognized as consequence of adding complexity and regulation in more complex eukaryotic cells. Here we provide a description of the actual stage of knowledge of the nuclear structure of Trypanosoma cruzi. As an early divergent eukaryote, it presents unique and/or reduced events of DNA replication, transcription and repair as well as RNA processing and transport to the cytosol. Nevertheless, it shows peculiar structure changes accordingly to the cell cycle and stage of differentiation. T. cruzi proliferates only as epimastigote and amastigote stages, and when these forms differentiate in trypomastigote forms, their cell cycle is arrested. This arrested stage is capable of invading mammalian cells and of surviving harsh conditions, such as the gut of the insect vector and mammalian macrophages. Transcription and replication decrease during transformation in trypomastigotes implicating large alterations in the nuclear structure. Recent evidences also suggest that T. cruzi nucleus respond to oxidative and nutritional stresses. Due to the phylogenetic proximity with other well-known trypanosomes, such as Trypanosoma brucei and Leishmania major, they are expected to have similar nuclear organization, although differences are noticed due to distinct life cycles, cellular organizations and the specific adaptations for surviving in different host environments. Therefore, the general

  15. Malleable mitochondrion of Trypanosoma brucei.

    Science.gov (United States)

    Verner, Zdeněk; Basu, Somsuvro; Benz, Corinna; Dixit, Sameer; Dobáková, Eva; Faktorová, Drahomíra; Hashimi, Hassan; Horáková, Eva; Huang, Zhenqiu; Paris, Zdeněk; Peña-Diaz, Priscila; Ridlon, Lucie; Týč, Jiří; Wildridge, David; Zíková, Alena; Lukeš, Julius

    2015-01-01

    The importance of mitochondria for a typical aerobic eukaryotic cell is undeniable, as the list of necessary mitochondrial processes is steadily growing. Here, we summarize the current knowledge of mitochondrial biology of an early-branching parasitic protist, Trypanosoma brucei, a causative agent of serious human and cattle diseases. We present a comprehensive survey of its mitochondrial pathways including kinetoplast DNA replication and maintenance, gene expression, protein and metabolite import, major metabolic pathways, Fe-S cluster synthesis, ion homeostasis, organellar dynamics, and other processes. As we describe in this chapter, the single mitochondrion of T. brucei is everything but simple and as such rivals mitochondria of multicellular organisms. Copyright © 2015. Published by Elsevier Inc.

  16. Endocytosis in Trypanosoma cruzi

    OpenAIRE

    Silva, Narcisa Leal da Cunha e; Sant’Anna, Celso; Pereira, Miria Gomes; Souza, Wanderley de

    2010-01-01

    Endocytic activity is particularly intense in Trypanosoma cruzi epimastigotes, while in amastigotes and trypomastigotes it is untraceable. Cargo molecules enters through the cytostome or flagellar pocket at the parasite anterior region, goes along a branched early endosomal network of tubules and vesicles spread from nuclear periphery to the posterior pole, until delivery to reservosomes, the final compartment. Reservosomes are acid compartments that store protein and lipid cargo and also acc...

  17. Identification of Trypanosoma evansi, Trypanosoma equiperdum and Trypanosoma brucei brucei using repetitive DNA probes.

    Science.gov (United States)

    Zhang, Z Q; Baltz, T

    1994-06-01

    The phylogenetic relatedness of 15 stocks of Trypanosoma evansi, three stocks of Trypanosoma equiperdum and one stock of Trypanosoma brucei brucei was determined using Southern blot analysis of restriction enzyme digested DNA, probed with two repetitive DNA sequences from T. b. brucei. A dendrogram derived by cluster analysis of restriction fragment length polymorphism (RFLP) revealed three groups of related stocks. Group 1 included 14 stocks of T. evansi and one stock of T. equiperdum. Group 2 included two stocks of T. equiperdum and one stock of T. evansi. Group 3 included the one stock of T. brucei brucei. Group 2 is more closely related to Group 3 than Group 1, by analysis of the banding patterns. Further analysis of the T. evansi in Group 1 revealed that the patterns of isolates from different provinces in China were identical, but differed from T. evansi isolated from Africa, South America and the Philippines. These results provide insight into the origins of T. evansi and suggest that RFLP may be a useful means of distinguishing closely related trypanosomes.

  18. Anti-Trypanosoma cruzi activity of nicotinamide.

    Science.gov (United States)

    Soares, Milena B P; Silva, Cinara V; Bastos, Tanira M; Guimarães, Elisalva T; Figueira, Claudio P; Smirlis, Despina; Azevedo, Walter F

    2012-05-01

    Inhibition of Trypanosoma brucei and Leishmania spp. sirtuins has shown promising antiparasitic activity, indicating that these enzymes may be used as targets for drug discovery against trypanosomatid infections. In the present work we carried out a virtual screening focused on the C pocket of Sir2 from Trypanosoma cruzi. Using this approach, the best ligand found was nicotinamide. In vitro tests confirmed the anti-T. cruzi activity of nicotinamide on epimastigote and trypomastigote forms. Moreover, treatment of T. cruzi-infected macrophages with nicotinamide caused a significant reduction in the number of amastigotes. In addition, alterations in the mitochondria and an increase in the vacuolization in the cytoplasm were observed in epimastigotes treated with nicotinamide. Analysis of the complex of Sir2 and nicotinamide revealed the details of the possible ligand-target interaction. Our data reveal a potential use of TcSir2 as a target for anti-T. cruzi drug discovery. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. What happens when Trypanosoma brucei leaves Africa.

    Science.gov (United States)

    Jensen, Robert E; Simpson, Larry; Englund, Paul T

    2008-10-01

    Julius Lukes and co-workers evaluated the evolutionary origin of Trypanosoma equiperdum and Trypanosoma evansi, parasites that cause horse and camel diseases. Although similar to T. brucei, the sleeping-sickness parasite, these trypanosomes do not cycle through the tsetse fly and have been able to spread beyond Africa. Transmission occurs sexually, or via blood-sucking flies or vampire bats. They concluded that these parasites, which resemble yeast petite mutants, are T. brucei sub-species, which have evolved recently through changes in mitochondrial DNA.

  20. Haematology of experimental Trypanosoma brucei rhodesiense ...

    African Journals Online (AJOL)

    Haematological aberrations associated with human infective trypanosomes were investigated in the vervet monkey model of the Rhodesian sleeping sickness. Four monkeys were infected intravenously with 104 Trypanosoma brucei rhodesiense and monitored for changes in the blood profile using a haematological ...

  1. Characterization of Trypanosoma brucei gambiense stocks isolated ...

    African Journals Online (AJOL)

    Characterization of Trypanosoma brucei gambiense stocks isolated from humans by RAPD fingerprinting in Côte d'Ivoire: another evidence for multiple infections. ... a given isoenzyme profile (zymodeme), confirming that this fingerprinting method has a higher discriminative power (faster molecular clock) than isoenzymes.

  2. Nucleoside analysis of DNA from Trypanosoma brucei and Trypanosoma equiperdum.

    Science.gov (United States)

    Crozatier, M; De Brij, R J; Den Engelse, L; Johnson, P J; Borst, P

    1988-11-01

    We have digested trypanosome DNA with a combination of pancreatic DNase I, nuclease P1 and bovine alkaline phosphatase and fractionated the resulting nucleosides on a Supelcosil LC-18-S column by high pressure liquid chromatography. We find less than 0.1% unusual nucleosides, both in Trypanosoma brucei and in a Trypanosoma equiperdum stock, in contrast to a previous report of an unusual nucleoside replacing dC at 1.3% of total nucleosides in T. equiperdum. Our results agree with previous suggestions that the modification of inactive telomeric expression sites for variant-specific surface glycoprotein genes in T. brucei only affects a very small fraction of the total DNA.

  3. The genome of the African trypanosome Trypanosoma brucei.

    Science.gov (United States)

    Berriman, Matthew; Ghedin, Elodie; Hertz-Fowler, Christiane; Blandin, Gaëlle; Renauld, Hubert; Bartholomeu, Daniella C; Lennard, Nicola J; Caler, Elisabet; Hamlin, Nancy E; Haas, Brian; Böhme, Ulrike; Hannick, Linda; Aslett, Martin A; Shallom, Joshua; Marcello, Lucio; Hou, Lihua; Wickstead, Bill; Alsmark, U Cecilia M; Arrowsmith, Claire; Atkin, Rebecca J; Barron, Andrew J; Bringaud, Frederic; Brooks, Karen; Carrington, Mark; Cherevach, Inna; Chillingworth, Tracey-Jane; Churcher, Carol; Clark, Louise N; Corton, Craig H; Cronin, Ann; Davies, Rob M; Doggett, Jonathon; Djikeng, Appolinaire; Feldblyum, Tamara; Field, Mark C; Fraser, Audrey; Goodhead, Ian; Hance, Zahra; Harper, David; Harris, Barbara R; Hauser, Heidi; Hostetler, Jessica; Ivens, Al; Jagels, Kay; Johnson, David; Johnson, Justin; Jones, Kristine; Kerhornou, Arnaud X; Koo, Hean; Larke, Natasha; Landfear, Scott; Larkin, Christopher; Leech, Vanessa; Line, Alexandra; Lord, Angela; Macleod, Annette; Mooney, Paul J; Moule, Sharon; Martin, David M A; Morgan, Gareth W; Mungall, Karen; Norbertczak, Halina; Ormond, Doug; Pai, Grace; Peacock, Chris S; Peterson, Jeremy; Quail, Michael A; Rabbinowitsch, Ester; Rajandream, Marie-Adele; Reitter, Chris; Salzberg, Steven L; Sanders, Mandy; Schobel, Seth; Sharp, Sarah; Simmonds, Mark; Simpson, Anjana J; Tallon, Luke; Turner, C Michael R; Tait, Andrew; Tivey, Adrian R; Van Aken, Susan; Walker, Danielle; Wanless, David; Wang, Shiliang; White, Brian; White, Owen; Whitehead, Sally; Woodward, John; Wortman, Jennifer; Adams, Mark D; Embley, T Martin; Gull, Keith; Ullu, Elisabetta; Barry, J David; Fairlamb, Alan H; Opperdoes, Fred; Barrell, Barclay G; Donelson, John E; Hall, Neil; Fraser, Claire M; Melville, Sara E; El-Sayed, Najib M

    2005-07-15

    African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei. The 26-megabase genome contains 9068 predicted genes, including approximately 900 pseudogenes and approximately 1700 T. brucei-specific genes. Large subtelomeric arrays contain an archive of 806 variant surface glycoprotein (VSG) genes used by the parasite to evade the mammalian immune system. Most VSG genes are pseudogenes, which may be used to generate expressed mosaic genes by ectopic recombination. Comparisons of the cytoskeleton and endocytic trafficking systems with those of humans and other eukaryotic organisms reveal major differences. A comparison of metabolic pathways encoded by the genomes of T. brucei, T. cruzi, and Leishmania major reveals the least overall metabolic capability in T. brucei and the greatest in L. major. Horizontal transfer of genes of bacterial origin has contributed to some of the metabolic differences in these parasites, and a number of novel potential drug targets have been identified.

  4. Trypanosoma brucei: two steps to spread out from Africa.

    Science.gov (United States)

    Lun, Zhao-Rong; Lai, De-Hua; Li, Feng-Jun; Lukes, Julius; Ayala, Francisco J

    2010-09-01

    Trypanosoma brucei equiperdum and Trypanosoma brucei evansi are typically considered separate species, although a recent study suggested that these organisms can be classified as subspecies of Trypanosoma brucei, which we also favor. Here we present a scenario that attempts to explain the continuing evolution of the dyskinetoplastic and akinetoplastic strains, as a consequence of loss of selective pressure(s) leading to the loss of kinetoplast DNA. Copyright 2010 Elsevier Ltd. All rights reserved.

  5. Detection of Trypanosoma brucei gambiense and T. b. rhodesiense ...

    African Journals Online (AJOL)

    Detection of Trypanosoma brucei gambiense and T. b. rhodesiense in Glossina fuscipes fuscipes ( Diptera: Glossinidae ) and Stomoxys flies using the polymerase chain reaction (PCR) technique in southern Sudan.

  6. Pathogenicity of Trypanosoma brucei in African giant rats ...

    African Journals Online (AJOL)

    Pathogenicity of Trypanosoma brucei in African giant rats ( Cricetomys gambianus , Water House) ... The course of trypanosomosis was investigated over a period of two weeks in six African giant rats (Cricetomys gambianus) experimentally infected with Trypanosoma brucei. Six other rats served as uninfected control.

  7. A Protein Complex Map of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Vahid H Gazestani

    2016-03-01

    Full Text Available The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org.

  8. Cloning, expression, purification and characterization of triosephosphate isomerase from Trypanosoma cruzi.

    Science.gov (United States)

    Ostoa-Saloma, P; Garza-Ramos, G; Ramírez, J; Becker, I; Berzunza, M; Landa, A; Gómez-Puyou, A; Tuena de Gómez-Puyou, M; Pérez-Montfort, R

    1997-03-15

    The gene that encodes for triosephosphate isomerase from Trypanosoma cruzi was cloned and sequenced. In T. cruzi, there is only one gene for triosephosphate isomerase. The enzyme has an identity of 72% and 68% with triosephosphate isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric triosephosphate isomerase from T. brucei, 29 are conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that triosephosphate isomerase from T. cruzi is a homodimer. Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T. brucei and L. mexicana. Its circular dichroism spectrum was almost identical to that of triosephosphate isomerase from T. brucei. Its kinetic properties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3-phosphate as substrate. The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2-SMe) was determined; the sensitivity of the T. cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T. brucei and L. mexicana, respectively. Triosephosphate isomerase from T. cruzi and L. mexicana have the three cysteine residues that exist in the T. brucei enzyme (positions 14, 39, 126, using the numbering of the T. brucei enzyme); however, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulfhydryl reagent. The disposition of the cysteine in triosephosphate isomerase from T. cruzi appears to

  9. Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

    Directory of Open Access Journals (Sweden)

    S Andrea Moreno

    2015-06-01

    Full Text Available Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF of T. b. brucei: (i fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme in glycosomes, (ii enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes and a GAPDH isoenzyme in the cytosol, (iii malate dehydrogenase in cytosol and (iv glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

  10. (Berenil(B)) in the Treatment of Experimental Trypanosoma brucei ...

    African Journals Online (AJOL)

    Dr Olaleye

    Animal welfare was observed in accordance with. International guidelines for the use of laboratory animals for biomedical research (EC, 1996). Trypanosomes: Trypanosoma brucei brucei (Federe strain) obtained from NITR, Vom, was used and the parasites were maintained by serial passages in rats. One millilitre of.

  11. The Effect of Garlic Extracts on Experimental Trypanosoma brucei ...

    African Journals Online (AJOL)

    The anti-trypanosomal effect of aqueous and methanolic extracts of garlic were studied in Trypanosoma brucei brucei infected rabbits. With the establishment of infection, parasitaemia, anaemia, leucopenia, neutropenia and lymphocytosis developed. There was decrease in total serum protein, albumin and increase in ...

  12. Non-cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei

    NARCIS (Netherlands)

    Bienen, E. J.; Maturi, R. K.; Pollakis, G.; Clarkson, A. B.

    1993-01-01

    The life cycle of Trypanosoma brucei brucei involves a series of differentiation steps characterized by marked changes in mitochondrial development and function. The bloodstream forms of this parasite completely lack cytochromes and have not been considered to have any Krebs cycle function. It has

  13. Mechanisms Mediating Antigenic Variation in Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Rudenko Gloria

    1999-01-01

    Full Text Available Antigenic variation in Trypanosoma brucei is a highly sophisticated survival strategy involving switching between the transcription of one of an estimated thousand variant surface glycoprotein (VSG genes. Switching involves either transcriptional control, resulting in switching between different VSG expression sites; or DNA rearrangement events slotting previously inactive VSG genes into an active VSG expression site. In recent years, considerable progress has been made in techniques allowing us to genetically modify infective bloodstream form trypanosomes. This is allowing us to reengineer VSG expression sites, and look at the effect on the mechanisms subsequently used for antigenic variation. We can now begin a dissection of a highly complicated survival strategy mediated by many different mechanisms operating simultaneously.

  14. Evaluation of In Vitro Activity of Essential Oils against Trypanosoma brucei brucei and Trypanosoma evansi

    Directory of Open Access Journals (Sweden)

    Nathan Habila

    2010-01-01

    Full Text Available Essential oils (EOs from Cymbopogon citratus (CC, Eucalyptus citriodora (EC, Eucalyptus camaldulensis (ED, and Citrus sinensis (CS were obtained by hydrodistillation process. The EOs were evaluated in vitro for activity against Trypanosoma brucei brucei (Tbb and Trypanosoma evansi (T. evansi. The EOs were found to possess antitrypanosomal activity in vitro in a dose-dependent pattern in a short period of time. The drop in number of parasite over time was achieved doses of 0.4 g/ml, 0.2 g/mL, and 0.1 g/mL for all the EOs. The concentration of 0.4 g/mL CC was more potent at 3 minutes and 2 minutes for Tbb and T. evansi, respectively. The GC-MS analysis of the EOs revealed presence of Cyclobutane (96.09% in CS, 6-octenal (77.11% in EC, Eucalyptol (75% in ED, and Citral (38.32% in CC among several other organic compounds. The results are discussed in relation to trypanosome chemotherapy.

  15. The diversity and expansion of the trans-sialidase gene family is a common feature in Trypanosoma cruzi clade members.

    Science.gov (United States)

    Chiurillo, Miguel Angel; Cortez, Danielle R; Lima, Fábio M; Cortez, Caroline; Ramírez, José Luis; Martins, Andre G; Serrano, Myrna G; Teixeira, Marta M G; da Silveira, José Franco

    2016-01-01

    Trans-sialidase (TS) is a polymorphic protein superfamily described in members of the protozoan genus Trypanosoma. Of the eight TS groups recently described, TS group I proteins (some of which have catalytic activity) are present in the distantly related Trypanosoma brucei and Trypanosoma cruzi phylogenetic clades, whereas other TS groups have only been described in some species belonging to the T. cruzi clade. In the present study we analyzed the repertoire, distribution and phylogenetic relationships of TS genes among species of the T. cruzi clade based on sequence similarity, multiple sequence alignment and tree-reconstruction approaches using TS sequences obtained with the aid of PCR-based strategies or retrieved from genome databases. We included the following representative isolates of the T. cruzi clade from South America: T. cruzi, T. cruzi Tcbat, Trypanosoma cruzi marinkellei, Trypanosoma dionisii, Trypanosoma rangeli and Trypanosoma conorhini. The cloned sequences encoded conserved TS protein motifs Asp-box and VTVxNVxLYNR but lacked the FRIP motif (conserved in TS group I). The T. conorhini sequences were the most divergent. The hybridization patterns of TS probes with chromosomal bands confirmed the abundance of these sequences in species in the T. cruzi clade. Divergence and relationship analysis placed most of the TS sequences in the groups defined in T. cruzi. Further examination of members of TS group II, which includes T. cruzi surface glycoproteins implicated in host cell attachment and invasion, showed that sequences of T. cruzi Tcbat grouped with those of T. cruzi genotype TcI. Our analysis indicates that different members of the T. cruzi clade, with different vertebrate hosts, vectors and pathogenicity, share the extensive expansion and sequence diversification of the TS gene family. Altogether, our results are congruent with the evolutionary history of the T. cruzi clade and represent a contribution to the understanding of the molecular

  16. Immunization with recombinant actin from Trypanosoma evansi induces protective immunity against T. evansi, T. equiperdum and T. b. brucei infection.

    Science.gov (United States)

    Li, San-Qiang; Yang, Wu-Biao; Lun, Zhao-Rong; Ma, Ling-Jun; Xi, Shou-Min; Chen, Qun-Li; Song, Xiao-Wei; Kang, Jian; Yang, Lan-Ze

    2009-01-01

    Actin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi actin shows 100%, 98.7%, and 93.1%, homology with Trypanosoma equiperdum, Trypanosoma brucei brucei, and Trypanosoma cruzi. Recombinant actin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant actin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818, and T. b. brucei STIB 940, showing 63.3%, 56.7%, and 53.3% protection, respectively. Serum collected from the rabbit immunized with recombinant actin inhibited the growth of T. evansi, T. equiperdum, and T. b. brucei in vitro cultivation. Serum from mice and rabbits immunized with recombinant actin only recognized T. evansi actin but not mouse actin. The results of this study suggest that the recombinant T. evansi actin induces protective immunity against T. evansi, T. equiperdum, and T. b. brucei infection and may be useful in the development of a vaccine with other cytoskeletal proteins to prevent animal trypanosomiasis caused by these three trypanosome species.

  17. Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Alloatti, Andres [Instituto de Biologia Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Santa Fe (Argentina); Gupta, Shreedhara; Gualdron-Lopez, Melisa; Nguewa, Paul A. [Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Universite Catholique de Louvain, Brussels (Belgium); Altabe, Silvia G. [Instituto de Biologia Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Santa Fe (Argentina); Deumer, Gladys; Wallemacq, Pierre [Department of Clinical Chemistry, Cliniques Universitaires Saint-Luc, LTAP, Universite Catholique de Louvain, Brussels (Belgium); Michels, Paul A.M. [Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Universite Catholique de Louvain, Brussels (Belgium); Uttaro, Antonio D., E-mail: toniuttaro@yahoo.com.ar [Instituto de Biologia Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Santa Fe (Argentina)

    2011-08-26

    Highlights: {yields} Inhibiting {Delta}9 desaturase drastically changes T. brucei's fatty-acid composition. {yields} Isoxyl specifically inhibits the {Delta}9 desaturase causing a growth arrest. {yields} RNA interference of desaturase expression causes a similar effect. {yields} Feeding T. brucei-infected mice with Isoxyl decreases the parasitemia. {yields} 70% of Isoxyl-treated mice survived the trypanosome infection. -- Abstract: Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes named desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei's survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC{sub 50}) of PCF was 1.0 {+-} 0.2 {mu}M for Isoxyl and 5 {+-} 2 {mu}M for 10-TS, whereas BSF appeared more susceptible with EC{sub 50} values 0.10 {+-} 0.03 {mu}M (Isoxyl) and 1.0 {+-} 0.6 {mu}M (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents' survival.

  18. Nitroheterocyclic drug resistance mechanisms in Trypanosoma brucei.

    Science.gov (United States)

    Wyllie, Susan; Foth, Bernardo J; Kelner, Anna; Sokolova, Antoaneta Y; Berriman, Matthew; Fairlamb, Alan H

    2016-03-01

    The objective of this study was to identify the mechanisms of resistance to nifurtimox and fexinidazole in African trypanosomes. Bloodstream-form Trypanosoma brucei were selected for resistance to nifurtimox and fexinidazole by stepwise exposure to increasing drug concentrations. Clones were subjected to WGS to identify putative resistance genes. Transgenic parasites modulating expression of genes of interest were generated and drug susceptibility phenotypes determined. Nifurtimox-resistant (NfxR) and fexinidazole-resistant (FxR) parasites shared reciprocal cross-resistance suggestive of a common mechanism of action. Previously, a type I nitroreductase (NTR) has been implicated in nitro drug activation. WGS of resistant clones revealed that NfxR parasites had lost >100 kb from one copy of chromosome 7, rendering them hemizygous for NTR as well as over 30 other genes. FxR parasites retained both copies of NTR, but lost >70 kb downstream of one NTR allele, decreasing NTR transcription by half. A single knockout line of NTR displayed 1.6- and 1.9-fold resistance to nifurtimox and fexinidazole, respectively. Since NfxR and FxR parasites are ∼6- and 20-fold resistant to nifurtimox and fexinidazole, respectively, additional factors must be involved. Overexpression and knockout studies ruled out a role for a putative oxidoreductase (Tb927.7.7410) and a hypothetical gene (Tb927.1.1050), previously identified in a genome-scale RNAi screen. NTR was confirmed as a key resistance determinant, either by loss of one gene copy or loss of gene expression. Further work is required to identify which of the many dozens of SNPs identified in the drug-resistant cell lines contribute to the overall resistance phenotype. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

  19. Purification and Partial characterization of Trypanosoma cruzi triosephosphate isomerase.

    Science.gov (United States)

    Bourguignon, S C; Meirelles, M N; Pacheco, R S; De Simone, S G

    1998-01-01

    The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins.

  20. Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase

    Directory of Open Access Journals (Sweden)

    Bourguignon SC

    1998-01-01

    Full Text Available The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1 was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa with similar isoelectric points (pI 9.3-9.5 which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai. The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins

  1. ( Nigella sativa ) oil on Trypanosoma brucei -infected rats

    African Journals Online (AJOL)

    The effect of black seed oil (Nigella sativa oil) on parasitaemia, some serum and liver enzymes as well as some haematological parameters in Trypanosoma brucei-infected rats were investigated. The results show there was low parasitaemia and extension of life span of rats from 12 days of the infected untreated (control) ...

  2. Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei.

    NARCIS (Netherlands)

    Helfert, S.; Estevez, A.M.; Bakker, B.M.; Michels, P.A.M.; Clayton, C.

    2001-01-01

    Kinetoplastid protozoa compartmentalize the first seven enzymes of glycolysis and two enzymes of glycerol metabolism in a microbody, the glycosome. While in its mammalian host, Trypanosoma brucei depends entirely on glucose for ATP generation. Under aerobic conditions, most of the glucose is

  3. Role of cytokines in Trypanosoma brucei-induced anaemia: A ...

    African Journals Online (AJOL)

    the blood-brain barrier. The subspecies Trypanosoma brucei rhodesiense in eastern and southern Africa generally causes a more acute form of ... The persistence of anaemia beyond the initial wave of parasitaemia (after which low numbers of parasites are in circulation) indicates that anaemia may be directly induced.

  4. Acetylated alpha-tubulin in Trypanosoma cruzi: immunocytochemical localization

    Directory of Open Access Journals (Sweden)

    Thais Souto-Padron

    1993-12-01

    Full Text Available We have used monoclonal antibodies specific for acetylated and non-acetylated alpha-tubulin to localize microtubules containing acetylated alpha-tubulin in all developmental forms of the life cycle of Trypanosoma cruzi. This was demonstrated using immunofluorescence and by transmission electron microscopy of thin sections, negative stained cells, and replicas of whole Triton X-100 extracted cells immunolabeled with antibody-gold complex. The antibody specific for acetylated alpha-tubulin (6-11B-1 binds to the flagellar, as well as to the sub-pellicular microtubules. The extent of labeling of the sub-pellicular microtubules with the monoclonal antibody recognized alpha-acetylated tubulin was smaller than that observed with the antibody which recognizes all tubulin isoforms. In relation to the developmental forms, the extent of labeling of the microtubules with antibody 6-11B-1 was larger in epimastigote and trypomastigote than in amastigote forms. Incubation of the parasites for 1 h at 0º C or in the presence of either colchicine or vinblastine did not interfere with the sub-pellicular microtubules. These observations, in agreement with those reported for Trypanosoma brucei brucei (Schneider et al., 1987; Schulze et al., 1987; Sasse per cent Gull, 1988 indicate that the sub-pellicular microtubules of trypanosomatids represent stable microtubules containing acetylated alpha-tubulin (or the alpha 3-tubulin isotype.

  5. Mechanism of Trypanosoma brucei gambiense resistance to human serum

    DEFF Research Database (Denmark)

    Uzureau, Pierrick; Uzureau, Sophie; Lecordier, Laurence

    2013-01-01

    The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease......GP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According...

  6. Trypanosoma brucei mitochondrial respiratome: Composition and organization in procyclic form

    KAUST Repository

    Acestor, Nathalie

    2011-05-24

    The mitochondrial respiratory chain is comprised of four different protein complexes (I-IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by F oF 1-ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Biological characterization of Trypanosoma cruzi strains

    Directory of Open Access Journals (Sweden)

    Martínez-Díaz Rafael A

    2001-01-01

    Full Text Available Biological parameters of five Trypanosoma cruzi strains from different sources were determined in order to know the laboratory behaviour of natural populations. The parameters evaluated were growth kinetics of epimastigotes, differentiation into metacyclic forms, infectivity in mammalian cells grown in vitro and parasite susceptibility to nifurtimox, benznidazole and gentian violet. Differences in transformation to metacyclic, in the percentage of infected cells as well as in the number of amastigotes per cell were observed among the strains. Regarding to pharmacological assays, Y strain was the most sensitive to the three assayed compounds. These data demonstrate the heterogeneity of natural populations of T. cruzi, the only responsible of infection in humans.

  8. Genetic control of resistance to Trypanosoma brucei brucei infection in mice.

    Directory of Open Access Journals (Sweden)

    Matyáš Síma

    2011-06-01

    Full Text Available Trypanosoma brucei brucei infects livestock, with severe effects in horses and dogs. Mouse strains differ greatly in susceptibility to this parasite. However, no genes controlling these differences were mapped.We studied the genetic control of survival after T. b. brucei infection using recombinant congenic (RC strains, which have a high mapping power. Each RC strain of BALB/c-c-STS/A (CcS/Dem series contains a different random subset of 12.5% genes from the parental "donor" strain STS/A and 87.5% genes from the "background" strain BALB/c. Although BALB/c and STS/A mice are similarly susceptible to T. b. brucei, the RC strain CcS-11 is more susceptible than either of them. We analyzed genetics of survival in T. b. brucei-infected F(2 hybrids between BALB/c and CcS-11. CcS-11 strain carries STS-derived segments on eight chromosomes. They were genotyped in the F(2 hybrid mice and their linkage with survival was tested by analysis of variance.We mapped four Tbbr (Trypanosoma brucei brucei response loci that influence survival after T. b. brucei infection. Tbbr1 (chromosome 3 and Tbbr2 (chromosome 12 have effects on survival independent of inter-genic interactions (main effects. Tbbr3 (chromosome 7 influences survival in interaction with Tbbr4 (chromosome 19. Tbbr2 is located on a segment 2.15 Mb short that contains only 26 genes.This study presents the first identification of chromosomal loci controlling susceptibility to T. b. brucei infection. While mapping in F(2 hybrids of inbred strains usually has a precision of 40-80 Mb, in RC strains we mapped Tbbr2 to a 2.15 Mb segment containing only 26 genes, which will enable an effective search for the candidate gene. Definition of susceptibility genes will improve the understanding of pathways and genetic diversity underlying the disease and may result in new strategies to overcome the active subversion of the immune system by T. b. brucei.

  9. The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.

    Directory of Open Access Journals (Sweden)

    Nikolay G Kolev

    2010-09-01

    Full Text Available The genome of Trypanosoma brucei, the causative agent of African trypanosomiasis, was published five years ago, yet identification of all genes and their transcripts remains to be accomplished. Annotation is challenged by the organization of genes transcribed by RNA polymerase II (Pol II into long unidirectional gene clusters with no knowledge of how transcription is initiated. Here we report a single-nucleotide resolution genomic map of the T. brucei transcriptome, adding 1,114 new transcripts, including 103 non-coding RNAs, confirming and correcting many of the annotated features and revealing an extensive heterogeneity of 5' and 3' ends. Some of the new transcripts encode polypeptides that are either conserved in T. cruzi and Leishmania major or were previously detected in mass spectrometry analyses. High-throughput RNA sequencing (RNA-Seq was sensitive enough to detect transcripts at putative Pol II transcription initiation sites. Our results, as well as recent data from the literature, indicate that transcription initiation is not solely restricted to regions at the beginning of gene clusters, but may occur at internal sites. We also provide evidence that transcription at all putative initiation sites in T. brucei is bidirectional, a recently recognized fundamental property of eukaryotic promoters. Our results have implications for gene expression patterns in other important human pathogens with similar genome organization (Trypanosoma cruzi, Leishmania sp. and revealed heterogeneity in pre-mRNA processing that could potentially contribute to the survival and success of the parasite population in the insect vector and the mammalian host.

  10. Interaction between Trypanosoma brucei and Haemonchus ...

    African Journals Online (AJOL)

    In order to investigate the immunomodulatory influence of concurrent T. brucei and H. contortus infection in West African Dwarf (WAD) goats, 28 infected and 7 uninfected (control) of 8-9 months old male WAD goats were studied. The infected goats were separated into resistant (Class 1) and susceptible (Class 2) Faecal ...

  11. Wild chimpanzees are infected by Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Milan Jirků

    2015-12-01

    Finally, we demonstrated that the mandrill serum was able to efficiently lyse T. b. brucei and T. b. rhodesiense, and to some extent T. b. gambiense, while the chimpanzee serum failed to lyse any of these subspecies.

  12. Adaptations of Trypanosoma brucei to gradual loss of kinetoplast DNA: Trypanosoma equiperdum and Trypanosoma evansi are petite mutants of T. brucei.

    Science.gov (United States)

    Lai, De-Hua; Hashimi, Hassan; Lun, Zhao-Rong; Ayala, Francisco J; Lukes, Julius

    2008-02-12

    Trypanosoma brucei is a kinetoplastid flagellate, the agent of human sleeping sickness and ruminant nagana in Africa. Kinetoplastid flagellates contain their eponym kinetoplast DNA (kDNA), consisting of two types of interlocked circular DNA molecules: scores of maxicircles and thousands of minicircles. Maxicircles have typical mitochondrial genes, most of which are translatable only after RNA editing. Minicircles encode guide RNAs, required for decrypting the maxicircle transcripts. The life cycle of T. brucei involves a bloodstream stage (BS) in vertebrates and a procyclic stage (PS) in the tsetse fly vector. Partial [dyskinetoplastidy (Dk)] or total [akinetoplastidy (Ak)] loss of kDNA locks the trypanosome in the BS form. Transmission between vertebrates becomes mechanical without PS and tsetse mediation, allowing the parasite to spread outside the African tsetse belt. Trypanosoma equiperdum and Trypanosoma evansi are agents of dourine and surra, diseases of horses, camels, and water buffaloes. We have characterized representative strains of T. equiperdum and T. evansi by numerous molecular and classical parasitological approaches. We show that both species are actually strains of T. brucei, which lost part (Dk) or all (Ak) of their kDNA. These trypanosomes are not monophyletic clades and do not qualify for species status. They should be considered two subspecies, respectively T. brucei equiperdum and T. brucei evansi, which spontaneously arose recently. Dk/Ak trypanosomes may potentially emerge repeatedly from T. brucei.

  13. Phenolic Constituents of Medicinal Plants with Activity against Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Ya Nan Sun

    2016-04-01

    Full Text Available Neglected tropical diseases (NTDs affect over one billion people all over the world. These diseases are classified as neglected because they impact populations in areas with poor financial conditions and hence do not attract sufficient research investment. Human African Trypanosomiasis (HAT or sleeping sickness, caused by the parasite Trypanosoma brucei, is one of the NTDs. The current therapeutic interventions for T. brucei infections often have toxic side effects or require hospitalization so that they are not available in the rural environments where HAT occurs. Furthermore, parasite resistance is increasing, so that there is an urgent need to identify novel lead compounds against this infection. Recognizing the wide structural diversity of natural products, we desired to explore and identify novel antitrypanosomal chemotypes from a collection of natural products obtained from plants. In this study, 440 pure compounds from various medicinal plants were tested against T. brucei by in a screening using whole cell in vitro assays. As the result, twenty-two phenolic compounds exhibited potent activity against cultures of T. brucei. Among them, eight compounds—4, 7, 11, 14, 15, 18, 20, and 21—showed inhibitory activity against T. brucei, with IC50 values below 5 µM, ranging from 0.52 to 4.70 μM. Based on these results, we attempt to establish some general trends with respect to structure-activity relationships, which indicate that further investigation and optimization of these derivatives might enable the preparation of potentially useful compounds for treating HAT.

  14. A monoclonal antibody that inhibits Trypanosoma cruzi growth in vitro and its reaction with intracellular triosephosphate isomerase.

    Science.gov (United States)

    Cortés-Figueroa, A A; Pérez-Torres, A; Salaiza, N; Cabrera, N; Escalona-Montaño, A; Rondán, A; Aguirre-García, M; Gómez-Puyou, A; Pérez-Montfort, R; Becker, I

    2008-03-01

    In parasites of the order Kinetoplastida, such as Trypanosoma cruzi and Trypanosoma brucei, glycolysis is carried out by glycolytic enzymes in glycosomes. One of the glycolytic enzymes is triosephosphate isomerase (TIM), which in T. brucei is localized exclusively in glycosomes, whereas in T. cruzi, the localization of TIM has not been fully ascertained. In the present work, we made a monoclonal antibody (mAb 6-11G) against recombinant T. cruzi TIM (rTcTIM). Incubation of T. cruzi epimastigotes with the mAb inhibited parasite survival. Western blotting showed that the mAb recognized rTcTIM and a 27 kDa band in T. cruzi lysates that corresponded to TcTIM. Sera from patients with Chagas disease recognized rTcTIM and cross-reacted with human recombinant TIM. The cross reactivity between parasite and human TIM possibly contributes to the autoimmune pathogenesis of Chagas disease. Electron microscopy of T. cruzi epimastigotes with the mAb showed that TIM was located within glycosomes, in the cytoplasm, the nucleus, and the kinetoplast. Collectively, the data shed new light on T. cruzi TIM and opens perspectives for drug design.

  15. A Flagellar Pocket Membrane Fraction from Trypanosoma brucei rhodesiense: Immunogold Localization and Nonvariant Immunoprotection

    Science.gov (United States)

    1988-01-01

    membrane components. specific glycoproteins from Trypanosoma equiperdum variants. Exp. Parasitol. 64:1-11. FEBS Lett. 82:93-96. 20. Melton, D. W., D. S...Fraction from Trypanosoma brucei rhodesiense: Immunogold Localization and Nonvariant Immunoprotection JOHN G. OLENICK,* RUTH WOLFF,’ ROBERT K. NAUMAN...obtained for each of three distinct variable antigenic types (VATs) of a serodeme of Trypanosoma brucei rhodesiense Wellcome strain. Products of

  16. Trypanosoma cruzi, cancer and the Cold War.

    Science.gov (United States)

    Krementsov, Nikolai

    2009-07-01

    In the summer of 1946, the international community of cancer researchers was inspired by the announcement that two Soviet scientists, Nina Kliueva and Grigorii Roskin, had discovered anticancer properties in culture extracts made from the South American protozoan, Trypanosoma cruzi, and had produced a preparation--named after its discoverers KR--which showed clear therapeutic effects on cancer patients. Research teams from various countries enthusiastically pursued the promising new line of investigation. The story of the rise and fall of interest in the anticancer properties of T. cruzi in different countries suggests that during the second half of the twentieth century, the Cold War competition between the superpowers played an important role in shaping the research agendas of cancer studies.

  17. Knockdown of asparagine synthetase A renders Trypanosoma brucei auxotrophic to asparagine.

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    Inês Loureiro

    Full Text Available Asparagine synthetase (AS catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A, known as strictly ammonia-dependent, and asparagine synthetase B (AS-B, which can use either ammonia or glutamine. The absence of AS-A in humans, and its presence in trypanosomes, suggested AS-A as a potential drug target that deserved further investigation. We report the presence of functional AS-A in Trypanosoma cruzi (TcAS-A and Trypanosoma brucei (TbAS-A: the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP, ammonia and Mg(2+. TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor, but surprisingly, can also use glutamine, a characteristic so far never described for any AS-A. TbAS-A knockdown by RNAi didn't affect in vitro growth of bloodstream forms of the parasite. However, growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine. As expected, mice infections with induced and non-induced T. brucei RNAi clones were similar to those from wild-type parasites. However, when induced T. brucei RNAi clones were injected in mice undergoing asparaginase treatment, which depletes blood asparagine, the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites. Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting, but is unlikely to be suitable as a drug target.

  18. CHARACTERIZATION AND ANTIPARASITIC ACTIVITY OF BENZOPHENONE THIOSEMICARBAZONES ON Trypanosoma brucei brucei

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    Georges C. Accrombessi

    2011-02-01

    Full Text Available The structure of four synthesized thiosemicarbazones, substituted or not, of benzophenone has been confirmed by spectrometrical analysis IR, NMR 1H and 13C. Their anti-trypanosomal activities were evaluated on Trypanosoma brucei brucei. Among these compounds, benzophenone 4 phenyl-3-thiosemicarbazone 4 has the highest activity with the half-inhibitory concentration (IC50 = 8.48 micromolar (µM. Benzophenone 4-methyl-3-thiosemicarbazone 3 and benzophenone thiosemicarbazone 1 showed moderate anti-trypanosomal activity with IC50 values equal to 23.27 µM and 67.17 µM respectively. Benzophenone 2 methyl-3-thiosemicarbazone 2 showed no activity up to IC50 = 371.74 µM.

  19. Immune Evasion Strategies of Trypanosoma cruzi

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    Ana Flávia Nardy

    2015-01-01

    Full Text Available Microbes have evolved a diverse range of strategies to subvert the host immune system. The protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease, provides a good example of such adaptations. This parasite targets a broad spectrum of host tissues including both peripheral and central lymphoid tissues. Rapid colonization of the host gives rise to a systemic acute response which the parasite must overcome. The parasite in fact undermines both innate and adaptive immunity. It interferes with the antigen presenting function of dendritic cells via an action on host sialic acid-binding Ig-like lectin receptors. These receptors also induce suppression of CD4+ T cells responses, and we presented evidence that the sialylation of parasite-derived mucins is required for the inhibitory effects on CD4 T cells. In this review we highlight the major mechanisms used by Trypanosoma cruzi to overcome host immunity and discuss the role of parasite colonization of the central thymic lymphoid tissue in chronic disease.

  20. Further evidence from SSCP and ITS DNA sequencing support Trypanosoma evansi and Trypanosoma equiperdum as subspecies or even strains of Trypanosoma brucei

    OpenAIRE

    Wen, Y; Lun, Z; Zhu, X; Hide, G; Lai, D

    2016-01-01

    The subgenus Trypanozoon includes three species Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum, which are morphologically identical and indistinguishable even using some molecular methods. In this study, PCR-based single strand conformation polymorphism (PCR-SSCP) was used to analyze the ribosomal DNA of the Trypanozoon species. Data indicate different patterns of ITS2 fragments between T. brucei, T. evansi and T. equiperdum by SSCP. Furthermore, analysis of total ITS seque...

  1. Crystal structure of arginine methyltransferase 6 from Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Chongyuan Wang

    Full Text Available Arginine methylation plays vital roles in the cellular functions of the protozoan Trypanosoma brucei. The T. brucei arginine methyltransferase 6 (TbPRMT6 is a type I arginine methyltransferase homologous to human PRMT6. In this study, we report the crystal structures of apo-TbPRMT6 and its complex with the reaction product S-adenosyl-homocysteine (SAH. The structure of apo-TbPRMT6 displays several features that are different from those of type I PRMTs that were structurally characterized previously, including four stretches of insertion, the absence of strand β15, and a distinct dimerization arm. The comparison of the apo-TbPRMT6 and SAH-TbPRMT6 structures revealed the fine rearrangements in the active site upon SAH binding. The isothermal titration calorimetry results demonstrated that SAH binding greatly increases the affinity of TbPRMT6 to a substrate peptide derived from bovine histone H4. The western blotting and mass spectrometry results revealed that TbPRMT6 methylates bovine histone H4 tail at arginine 3 but cannot methylate several T. brucei histone tails. In summary, our results highlight the structural differences between TbPRMT6 and other type I PRMTs and reveal that the active site rearrangement upon SAH binding is important for the substrate binding of TbPRMT6.

  2. Crystal Structure of Triosephosphate Isomerase from Trypanosoma cruzi in Hexane

    National Research Council Canada - National Science Library

    Xiu-Gong Gao; Ernesto Maldonado; Ruy Perez-Montfort; Georgina Garza-Ramos; Marietta Tuena de Gomez-Puyou; Armando Gomez-Puyou; Adela Rodriguez-Romero

    1999-01-01

    ... the starting point for drug design. Here, a crystal of the dimeric enzyme triosephosphate isomerase from the pathogenic parasite Trypanosoma cruzi was soaked and diffracted in hexane and its structure solved at 2- angstrom resolution...

  3. Trypanosoma cruzi CYP51 inhibitor derived from a Mycobacterium tuberculosis screen hit.

    Directory of Open Access Journals (Sweden)

    Chiung-Kuang Chen

    Full Text Available The two front-line drugs for chronic Trypanosoma cruzi infections are limited by adverse side-effects and declining efficacy. One potential new target for Chagas' disease chemotherapy is sterol 14alpha-demethylase (CYP51, a cytochrome P450 enzyme involved in biosynthesis of membrane sterols.In a screening effort targeting Mycobacterium tuberculosis CYP51 (CYP51(Mt, we previously identified the N-[4-pyridyl]-formamide moiety as a building block capable of delivering a variety of chemotypes into the CYP51 active site. In that work, the binding modes of several second generation compounds carrying this scaffold were determined by high-resolution co-crystal structures with CYP51(Mt. Subsequent assays against the CYP51 orthologue in T. cruzi, CYP51(Tc, demonstrated that two of the compounds tested in the earlier effort bound tightly to this enzyme. Both were tested in vitro for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. One of the compounds had potent, selective anti-T. cruzi activity in infected mouse macrophages. Cure of treated host cells was confirmed by prolonged incubation in the absence of the inhibiting compound. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability (phenylalanine versus isoleucine of a single residue at a critical position in the active site.CYP51(Mt-based crystal structure analysis revealed that the functional groups of the two tightly bound compounds are likely to occupy different spaces in the CYP51 active site, suggesting the possibility of combining the beneficial features of both inhibitors in a third generation of compounds to achieve more potent and selective inhibition of CYP51(Tc.

  4. Repertoire, Genealogy and Genomic Organization of Cruzipain and Homologous Genes in Trypanosoma cruzi, T. cruzi-Like and Other Trypanosome Species

    Science.gov (United States)

    Lima, Luciana; Ortiz, Paola A.; da Silva, Flávia Maia; Alves, João Marcelo P.; Serrano, Myrna G.; Cortez, Alane P.; Alfieri, Silvia C.; Buck, Gregory A.; Teixeira, Marta M. G.

    2012-01-01

    Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine

  5. Trypanosoma Cruzi Cyp51 Inhibitor Derived from a Mycobacterium Tuberculosis Screen Hit

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chiung-Kuang; Doyle, Patricia S.; Yermalitskaya, Liudmila V.; Mackey, Zachary B.; Ang, Kenny K.H.; McKerrow, James H.; Podust, Larissa M.; (Vanderbilt); (UCSF)

    2009-02-18

    The two front-line drugs for chronic Trypanosoma cruzi infections are limited by adverse side-effects and declining efficacy. One potential new target for Chagas disease chemotherapy is sterol 14{alpha}-demethylase (CYP51), a cytochrome P450 enzyme involved in biosynthesis of membrane sterols. In a screening effort targeting Mycobacterium tuberculosis CYP51 (CYP51{sub Mt}), we previously identified the N-[4-pyridyl]-formamide moiety as a building block capable of delivering a variety of chemotypes into the CYP51 active site. In that work, the binding modes of several second generation compounds carrying this scaffold were determined by high-resolution co-crystal structures with CYP51{sub Mt}. Subsequent assays against the CYP51 orthologue in T. cruzi, CYP51{sub Tc}, demonstrated that two of the compounds tested in the earlier effort bound tightly to this enzyme. Both were tested in vitro for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. One of the compounds had potent, selective anti-T. cruzi activity in infected mouse macrophages. Cure of treated host cells was confirmed by prolonged incubation in the absence of the inhibiting compound. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability (phenylalanine versus isoleucine) of a single residue at a critical position in the active site. CYP51{sub Mt}-based crystal structure analysis revealed that the functional groups of the two tightly bound compounds are likely to occupy different spaces in the CYP51 active site, suggesting the possibility of combining the beneficial features of both inhibitors in a third generation of compounds to achieve more potent and selective inhibition of CYP51{sub Tc}. Enzyme sterol 14{alpha}-demethylase (CYP51) is a well-established target for anti-fungal therapy and is a prospective target for Chagas disease therapy. We previously identified a

  6. Shelter dogs as sentinels for Trypanosoma cruzi transmission across Texas.

    Science.gov (United States)

    Tenney, Trevor D; Curtis-Robles, Rachel; Snowden, Karen F; Hamer, Sarah A

    2014-08-01

    Chagas disease, an infection with the parasite Trypanosoma cruzi, is increasingly diagnosed among humans in the southern United States. We assessed exposure of shelter dogs in Texas to T. cruzi; seroprevalence across diverse ecoregions was 8.8%. Canine serosurveillance is a useful tool for public health risk assessment.

  7. In or out? On the tightness of glycosomal compartmentalizatin of metabolites and enzymes in Trypanosoma brucei.

    NARCIS (Netherlands)

    Haanstra, J.R.; Bakker, B.M.; Michels, P.A.M.

    2014-01-01

    Trypanosomatids sequester large parts of glucose metabolism inside specialised peroxisomes, called glycosomes. Many studies have shown that correct glycosomal compartmentalization of glycolytic enzymes is essential for bloodstream-form Trypanosoma brucei. The recent finding of pore-forming

  8. 1H, 13C and 15N resonance assignment of Urm1 from Trypanosoma brucei.

    Science.gov (United States)

    Zhang, Wen; Ding, Husheng; Zhang, Jiahai; Tu, Xiaoming

    2008-06-01

    Urm1 (ubiquitin-related modifier), involved in diverse biological processes in yeast, is proved to be a "molecular fossil" in ubiquitin superfamily. Here we report the resonance assignment of Urm1 from Trypanosoma brucei.

  9. Trypanosoma cruzi Polyamine Transporter: Its Role on Parasite Growth and Survival Under Stress Conditions.

    Science.gov (United States)

    Reigada, Chantal; Sayé, Melisa; Vera, Edward Valera; Balcazar, Darío; Fraccaroli, Laura; Carrillo, Carolina; Miranda, Mariana R; Pereira, Claudio A

    2016-08-01

    Trypanosoma cruzi is the etiological agent of Chagas disease, a major health problem in Latin America. Polyamines are polycationic compounds that play a critical role as regulators of cell growth and differentiation. In contrast with other protozoa, T. cruzi is auxotrophic for polyamines because of its inability to synthesize putrescine due to the lack of both, arginine and ornithine decarboxylase; therefore, the intracellular availability of polyamines depends exclusively on transport processes. In this work, the polyamine transporter TcPAT12 was overexpressed in T. cruzi epimastigotes demonstrating that growth rates at different concentrations of polyamines strongly depend on the regulation of the polyamine transport. In addition, parasites overexpressing TcPAT12 showed a highly increased resistance to hydrogen peroxide and the trypanocidal drugs nifurtimox and benznidazole, which act by oxidative stress and interfering the synthesis of polyamine derivatives, respectively. Finally, the presence of putative polyamine transporters was analyzed in T. cruzi, Trypanosoma brucei, and Leishmania major genomes identifying 3-6 genes in these trypanosomatids.

  10. Differential gene expression during Trypanosoma cruzi metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Marco Aurelio Krieger

    1999-09-01

    Full Text Available The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE. The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells, while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.

  11. Flagellar Motility of Trypanosoma cruzi Epimastigotes

    Directory of Open Access Journals (Sweden)

    G. Ballesteros-Rodea

    2012-01-01

    Full Text Available The hemoflagellate Trypanosoma cruzi is the causative agent of American trypanosomiasis. Despite the importance of motility in the parasite life cycle, little is known about T. cruzi motility, and there is no quantitative description of its flagellar beating. Using video microscopy and quantitative vectorial analysis of epimastigote trajectories, we find a forward parasite motility defined by tip-to-base symmetrical flagellar beats. This motion is occasionally interrupted by base-to-tip highly asymmetric beats, which represent the ciliary beat of trypanosomatid flagella. The switch between flagellar and ciliary beating facilitates the parasite's reorientation, which produces a large variability of movement and trajectories that results in different distance ranges traveled by the cells. An analysis of the distance, speed, and rotational angle indicates that epimastigote movement is not completely random, and the phenomenon is highly dependent on the parasite behavior and is characterized by directed and tumbling parasite motion as well as their combination, resulting in the alternation of rectilinear and intricate motility paths.

  12. A α-glycerophosphate dehydrogenase is present in Trypanosoma cruzi glycosomes

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    JL Concepcion

    2001-07-01

    Full Text Available α-glycerophosphate dehydrogenase (α-GPDH-EC.1.1.1.8 has been considered absent in Trypanosoma cruzi in contradiction with all other studied trypanosomatids. After observing that the sole malate dehydrogenase can not maintain the intraglycosomal redox balance, GPDH activity was looked for and found, although in very variable levels, in epimastigotes extracts. GPDH was shown to be exclusively located in the glycosome of T. cruzi by digitonin treatment and isopycnic centrifugation. Antibody against T. brucei GPDH showed that this enzyme seemed to be present in an essentially inactive form at the beginning of the epimastigotes growth. GPDH is apparently linked to a salicylhydroxmic-sensitive glycerophosphate reoxidizing system and plays an essential role in the glycosome redox balance.

  13. Rab23 is a flagellar protein in Trypanosoma brucei

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    Field Mark C

    2011-06-01

    Full Text Available Abstract Background Rab small GTPases are important mediators of membrane transport, and orthologues frequently retain similar locations and functions, even between highly divergent taxa. In metazoan organisms Rab23 is an important negative regulator of Sonic hedgehog signaling and is crucial for correct development and differentiation of cellular lineages by virtue of an involvement in ciliary recycling. Previously, we reported that Trypanosoma brucei Rab23 localized to the nuclear envelope 1, which is clearly inconsistent with the mammalian location and function. As T. brucei is unicellular the potential that Rab23 has no role in cell signaling was possible. Here we sought to further investigate the role(s of Rab23 in T. brucei to determine if Rab23 was an example of a Rab protein with divergent function in distinct taxa. Methods/major findings The taxonomic distribution of Rab23 was examined and compared with the presence of flagella/cilia in representative taxa. Despite evidence for considerable secondary loss, we found a clear correlation between a conventional flagellar structure and the presence of a Rab23 orthologue in the genome. By epitope-tagging, Rab23 was localized and found to be present at the flagellum throughout the cell cycle. However, RNAi knockdown did not result in a flagellar defect, suggesting that Rab23 is not required for construction or maintenance of the flagellum. Conclusions The location of Rab23 at the flagellum is conserved between mammals and trypanosomes and the Rab23 gene is restricted to flagellated organisms. These data may suggest the presence of a Rab23-mediated signaling mechanism in trypanosomes.

  14. Telomeric expression sites are highly conserved in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Christiane Hertz-Fowler

    Full Text Available Subtelomeric regions are often under-represented in genome sequences of eukaryotes. One of the best known examples of the use of telomere proximity for adaptive purposes are the bloodstream expression sites (BESs of the African trypanosome Trypanosoma brucei. To enhance our understanding of BES structure and function in host adaptation and immune evasion, the BES repertoire from the Lister 427 strain of T. brucei were independently tagged and sequenced. BESs are polymorphic in size and structure but reveal a surprisingly conserved architecture in the context of extensive recombination. Very small BESs do exist and many functioning BESs do not contain the full complement of expression site associated genes (ESAGs. The consequences of duplicated or missing ESAGs, including ESAG9, a newly named ESAG12, and additional variant surface glycoprotein genes (VSGs were evaluated by functional assays after BESs were tagged with a drug-resistance gene. Phylogenetic analysis of constituent ESAG families suggests that BESs are sequence mosaics and that extensive recombination has shaped the evolution of the BES repertoire. This work opens important perspectives in understanding the molecular mechanisms of antigenic variation, a widely used strategy for immune evasion in pathogens, and telomere biology.

  15. Effects of Morinda lucida leaf extract on Trypanosoma brucei brucei infection in mice.

    Science.gov (United States)

    Asuzu, I U; Chineme, C N

    1990-10-01

    The dried leaves of Morinda lucida were extracted with 50% methanol and the extract was recovered in a 9.7% w/w yield. Acute toxicity tests were performed in mice and the intraperitoneal LD50 of the extract was 2000 mg/kg. The extract induced purgation in mice from the first hour after oral administration and reached its peak between the third and fourth hour. The purgation was not dose-dependent. M. lucida leaf extract i.p. significantly suppressed the level of parasitemia after Trypanosoma brucei infection in mice. Suppression of existing parasitemia appeared dose-dependent with 1000 mg/kg i.p. producing the maximum effect. The best trypanocidal activity was obtained when treatment with M. lucida extract commenced simultaneously with trypanosome inoculation.

  16. Sexual transmission of Trypanosoma cruzi in murine model.

    Science.gov (United States)

    Ribeiro, Marcelle; Nitz, Nadjar; Santana, Camilla; Moraes, Aline; Hagström, Luciana; Andrade, Rafael; Rios, Adriano; Sousa, Alessandro; Dallago, Bruno; Gurgel-Gonçalves, Rodrigo; Hecht, Mariana

    2016-03-01

    Trypanosoma cruzi is mainly transmitted by blood-sucking triatomines, but other routes also have epidemiological importance, such as blood transfusion and congenital transmission. Although the possibility of sexual transmission of T. cruzi has been suggested since its discovery, few studies have been published on this subject. We investigated acquisition of T. cruzi by sexual intercourse in an experimental murine model. Male and female mice in the chronic phase of Chagas disease were mated with naive partners. Parasitological, serological and molecular tests demonstrated the parasites in tissues and blood of partners. These results confirm the sexual transmission of T. cruzi in mice. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Survival of Trypanosoma cruzi in experimentally contaminated drinks Supervivencia de Trypanosoma cruzi en bebidas experimentalmente contaminadas

    Directory of Open Access Journals (Sweden)

    Diana Carolina Suárez

    2011-09-01

    Full Text Available

    Introduction. Trypanosoma cruzi is the causative agent of Chagas disease, transmitted primarily by triatomine insects. However, in 2005, oral transmission was documented in countries where the disease is endemic for Chagas disease. This trend may also occur in Colombia, a situation that motivated epidemiological alerts and the necessity for exploring the risk level of oral, human-to-human infection by T. cruzi.
    Objective. Survival times were established for the T. cruzi strain DS using juices involved in the outbreak of Lebrija County (Cesar, Colombia in 2008.
    Materials and methods. Survival of the T. cruzi strain was evaluated as defined by vitality (forward movement and viability (growth in isolation medium Novy, McNeal and Nicolle/liver infusion tryptose. This strain was molecularly characterized as TCLA, isolated from a patient associated with an outbreak in Aguachica County (Santander, very near Lebrija. Its survival was tested in tangerine juice, guava, soursop (guanábana, water and sugar water.
    Results. The T. cruzi strain DS remained vital in mandarin at room temperature for 72 hr, at refrigerated temperatures for 36 hr;, the soursop (guanábana for 48 hr at room temperature and 384 hr under refrigeration; and guava at both temperatures 24 hr. This strain was viable 2 and 24 hours post-infection in each of the other juices at the two temperature conditions.
    Conclusions: The DS T. cruzi strain survived in all drinks for more than 24 hours post-infection, with a survival time of 384 hr in the juice of soursop (guanábana under refrigeration.

    Introducción. Trypanosoma cruzi es el agente causal de la enfermedad de Chagas, el cual puede ser transmitido por diferentes vías. A partir de 2005 la transmisión oral se incrementó en aquellos países donde la enfermedad es considerada endémica por transmisión vectorial. Colombia no se aparta de esta tendencia, situación que motivó la alerta epidemiol

  18. Differential virulence and tsetse fly transmissibility of Trypanosoma congolense and Trypanosoma brucei strains

    Directory of Open Access Journals (Sweden)

    Purity K. Gitonga

    2017-01-01

    Full Text Available African animal trypanosomiasis causes significant economic losses in sub-Saharan African countries because of livestock mortalities and reduced productivity. Trypanosomes, the causative agents, are transmitted by tsetse flies (Glossina spp.. In the current study, we compared and contrasted the virulence characteristics of five Trypanosoma congolense and Trypanosoma brucei isolates using groups of Swiss white mice (n = 6. We further determined the vectorial capacity of Glossina pallidipes, for each of the trypanosome isolates. Results showed that the overall pre-patent (PP periods were 8.4 ± 0.9 (range, 4–11 and 4.5 ± 0.2 (range, 4–6 for T. congolense and T. brucei isolates, respectively (p < 0.01. Despite the longer mean PP, T. congolense–infected mice exhibited a significantly (p < 0.05 shorter survival time than T. brucei–infected mice, indicating greater virulence. Differences were also noted among the individual isolates with T. congolense KETRI 2909 causing the most acute infection of the entire group with a mean ± standard error survival time of 9 ± 2.1 days. Survival time of infected tsetse flies and the proportion with mature infections at 30 days post-exposure to the infective blood meals varied among isolates, with subacute infection–causing T. congolense EATRO 1829 and chronic infection–causing T. brucei EATRO 2267 isolates showing the highest mature infection rates of 38.5% and 23.1%, respectively. Therefore, our study provides further evidence of occurrence of differences in virulence and transmissibility of eastern African trypanosome strains and has identified two, T. congolense EATRO 1829 and T. brucei EATRO 2267, as suitable for tsetse infectivity and transmissibility experiments.

  19. Shotgun sequencing analysis of Trypanosoma cruzi I Sylvio X10/1 and comparison with T. cruzi VI CL Brener.

    Directory of Open Access Journals (Sweden)

    Oscar Franzén

    Full Text Available Trypanosoma cruzi is the causative agent of Chagas disease, which affects more than 9 million people in Latin America. We have generated a draft genome sequence of the TcI strain Sylvio X10/1 and compared it to the TcVI reference strain CL Brener to identify lineage-specific features. We found virtually no differences in the core gene content of CL Brener and Sylvio X10/1 by presence/absence analysis, but 6 open reading frames from CL Brener were missing in Sylvio X10/1. Several multicopy gene families, including DGF, mucin, MASP and GP63 were found to contain substantially fewer genes in Sylvio X10/1, based on sequence read estimations. 1,861 small insertion-deletion events and 77,349 nucleotide differences, 23% of which were non-synonymous and associated with radical amino acid changes, further distinguish these two genomes. There were 336 genes indicated as under positive selection, 145 unique to T. cruzi in comparison to T. brucei and Leishmania. This study provides a framework for further comparative analyses of two major T. cruzi lineages and also highlights the need for sequencing more strains to understand fully the genomic composition of this parasite.

  20. Shotgun sequencing analysis of Trypanosoma cruzi I Sylvio X10/1 and comparison with T. cruzi VI CL Brener.

    Science.gov (United States)

    Franzén, Oscar; Ochaya, Stephen; Sherwood, Ellen; Lewis, Michael D; Llewellyn, Martin S; Miles, Michael A; Andersson, Björn

    2011-03-08

    Trypanosoma cruzi is the causative agent of Chagas disease, which affects more than 9 million people in Latin America. We have generated a draft genome sequence of the TcI strain Sylvio X10/1 and compared it to the TcVI reference strain CL Brener to identify lineage-specific features. We found virtually no differences in the core gene content of CL Brener and Sylvio X10/1 by presence/absence analysis, but 6 open reading frames from CL Brener were missing in Sylvio X10/1. Several multicopy gene families, including DGF, mucin, MASP and GP63 were found to contain substantially fewer genes in Sylvio X10/1, based on sequence read estimations. 1,861 small insertion-deletion events and 77,349 nucleotide differences, 23% of which were non-synonymous and associated with radical amino acid changes, further distinguish these two genomes. There were 336 genes indicated as under positive selection, 145 unique to T. cruzi in comparison to T. brucei and Leishmania. This study provides a framework for further comparative analyses of two major T. cruzi lineages and also highlights the need for sequencing more strains to understand fully the genomic composition of this parasite.

  1. Compositional compartmentalization of the nuclear genomes of Trypanosoma brucei and Trypanosoma equiperdum.

    Science.gov (United States)

    Isacchi, A; Bernardi, G; Bernardi, G

    1993-12-06

    High molecular weight DNA preparations from Trypanosoma brucei and Trypanosoma equiperdum were fractionated by preparative centrifugation in a Cs2SO4 density gradient in the presence of BAMD, bis(acetatomercurimethyl)dioxane, a sequence-specific DNA ligand. Analytical centrifugation in CsCl of the DNA fractions so obtained showed that both DNAs had a bimodal distribution with two major peaks banding at 1.702-1.703 and 1.708 g/cm3 and representing 1/3 and 2/3 of total DNA, respectively. Several minor components were also detected. These results indicate that a compositional compartmentalization is not only found in the genome of vertebrates and plants, as already described, but also in those of protozoa such as Trypanosomes.

  2. Rational development of 4-aminopyridyl-based inhibitors targeting Trypanosoma cruzi CYP51 as anti-chagas agents.

    Science.gov (United States)

    Choi, Jun Yong; Calvet, Claudia M; Gunatilleke, Shamila S; Ruiz, Claudia; Cameron, Michael D; McKerrow, James H; Podust, Larissa M; Roush, William R

    2013-10-10

    A new series of 4-aminopyridyl-based lead inhibitors targeting Trypanosoma cruzi CYP51 (TcCYP51) has been developed using structure-based drug design as well as structure-property relationship (SPR) analyses. The screening hit starting point, LP10 (KD ≤ 42 nM; EC50 = 0.65 μM), has been optimized to give the potential leads 14t, 27i, 27q, 27r, and 27t, which have low-nanomolar binding affinity to TcCYP51 and significant activity against T. cruzi amastigotes cultured in human myoblasts (EC50 = 14-18 nM for 27i and 27r). Many of the optimized compounds have improved microsome stability, and most are selective against human CYPs 1A2, 2D6, and 3A4 (Trypanosoma brucei CYP51 (TbCYP51) orthologue has been characterized by X-ray structure analysis.

  3. Immunomodulatory activity of Buchholzia coriacea seed methanol extract on Trypanosoma brucei brucei infected mice.

    Science.gov (United States)

    Eze, James I; Ekelozie, Chioma F; Nweze, Nwakego E

    2017-12-01

    The seeds of Buchholzia coriacea Engler (Capparaceae) are used in Eastern Nigeria to treat feverish conditions, and to treat malaria and sleeping sickness that cause fever. The current study assesses the immunomodulatory activity of Buchholzia coriacea seed extract on Trypanosoma brucei brucei infected mice. Delayed hypersensitivity reaction, humoral antibody response and in-vivo leucocyte mobilization tests were assessed in three different experiments to determine the effect of the extract on immune response. Seventy-five (75) mice (25 mice per experiment) were used for the study and were each infected with 1.00 × 106 trypanosomes intra-peritoneally. Groups A, B and C were given 250, 500 and 1000 mg/kg of the extract, respectively, group D received 7.5 mg/kg body weight of levamisole and group E was the control. Sheep RBCs were used as antigen. The acute toxicity tests did not cause clinical signs or death within 24 h post treatment at all the doses tested. The extract inhibited delayed hypersensitivity reaction by 20.9 and 20.8% at 250 and 500 mg/kg, respectively, while at 1000 mg/kg, the paw size increased (-101.9%) when compared with the control. The extract elevated the antibody titre from 1.60 ± 0.40 for control to 8.00 ± 3.58 for 500 mg/kg group. The extract increased in total leucocytes counts. The extract has a very wide safety margin and was able to improve immune response. The results of the present study showed that Buchholzia coriacea seed methanol extract possesses immunostimulatory activity on trypanosome-infected mice.

  4. Role of iron in Trypanosoma cruzi infection of mice.

    OpenAIRE

    Lalonde, R G; Holbein, B E

    1984-01-01

    The role of iron in experimental infection of mice with Trypanosoma cruzi was investigated. B6 mice had a transient parasitemia and a transient anemia, both of maximal intensity 28 d after the inoculation of T. cruzi. There was a biphasic hypoferremic host response to infection with T. cruzi with the peak hypoferremia also occurring 28 d after inoculation of the parasite. The mortality rate from infection was increased from 23% in phosphate-buffered saline-treated B6 mice to 50% in a group of...

  5. High-throughput Gene Tagging in Trypanosoma brucei.

    Science.gov (United States)

    Dyer, Philip; Dean, Samuel; Sunter, Jack

    2016-08-12

    Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

  6. The Phosphoproteome of Bloodstream Form Trypanosoma brucei, Causative Agent of African Sleeping Sickness

    OpenAIRE

    Nett, Isabelle R. E.; Martin, David M. A.; Miranda-Saavedra, Diego; Lamont, Douglas; Barber, Jonathan D.; Mehlert, Angela; Ferguson, Michael A. J.

    2009-01-01

    The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of severa...

  7. Steroid Alkaloids from Holarrhena africana with Strong Activity against Trypanosoma brucei rhodesiense

    OpenAIRE

    Charles Okeke Nnadi; Ngozi Justina Nwodo; Marcel Kaiser; Reto Brun; Thomas J. Schmidt

    2017-01-01

    In our continued search for natural compounds with activity against Trypanosoma brucei, causative agent of human African trypanosomiasis (HAT, “sleeping sickness”), we have investigated extracts from the leaves and bark of the West African Holarrhena africana (syn. Holarrhena floribunda; Apocynaceae). The extracts and their alkaloid-enriched fractions displayed promising in vitro activity against bloodstream forms of T. brucei rhodesiense (Tbr; East African HAT). Bioactivity-guided chromatogr...

  8. Validation of N-myristoyltransferase as Potential Chemotherapeutic Target in Mammal-Dwelling Stages of Trypanosoma cruzi.

    Science.gov (United States)

    Herrera, Linda J; Brand, Stephen; Santos, Andres; Nohara, Lilian L; Harrison, Justin; Norcross, Neil R; Thompson, Stephen; Smith, Victoria; Lema, Carolina; Varela-Ramirez, Armando; Gilbert, Ian H; Almeida, Igor C; Maldonado, Rosa A

    2016-04-01

    Trypanosoma cruzi causes Chagas disease, an endemic and debilitating illness in Latin America. Lately, owing to extensive population movements, this neglected tropical disease has become a global health concern. The two clinically available drugs for the chemotherapy of Chagas disease have rather high toxicity and limited efficacy in the chronic phase of the disease, and may induce parasite resistance. The development of new anti-T. cruzi agents is therefore imperative. The enzyme N-myristoyltransferase (NMT) has recently been biochemically characterized, shown to be essential in Leishmania major, Trypanosoma brucei, and T. cruzi¸ and proposed as promising chemotherapeutic target in these trypanosomatids. Here, using high-content imaging we assayed eight known trypanosomatid NMT inhibitors, against mammal-dwelling intracellular amastigote and trypomastigote stages and demonstrated that three of them (compounds 1, 5, and 8) have potent anti-proliferative effect at submicromolar concentrations against T. cruzi, with very low toxicity against human epithelial cells. Moreover, metabolic labeling using myristic acid, azide showed a considerable decrease in the myristoylation of proteins in parasites treated with NMT inhibitors, providing evidence of the on-target activity of the inhibitors. Taken together, our data point out to the potential use of NMT inhibitors as anti-T. cruzi chemotherapy.

  9. Validation of N-myristoyltransferase as Potential Chemotherapeutic Target in Mammal-Dwelling Stages of Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Linda J Herrera

    2016-04-01

    Full Text Available Trypanosoma cruzi causes Chagas disease, an endemic and debilitating illness in Latin America. Lately, owing to extensive population movements, this neglected tropical disease has become a global health concern. The two clinically available drugs for the chemotherapy of Chagas disease have rather high toxicity and limited efficacy in the chronic phase of the disease, and may induce parasite resistance. The development of new anti-T. cruzi agents is therefore imperative. The enzyme N-myristoyltransferase (NMT has recently been biochemically characterized, shown to be essential in Leishmania major, Trypanosoma brucei, and T. cruzi¸ and proposed as promising chemotherapeutic target in these trypanosomatids.Here, using high-content imaging we assayed eight known trypanosomatid NMT inhibitors, against mammal-dwelling intracellular amastigote and trypomastigote stages and demonstrated that three of them (compounds 1, 5, and 8 have potent anti-proliferative effect at submicromolar concentrations against T. cruzi, with very low toxicity against human epithelial cells. Moreover, metabolic labeling using myristic acid, azide showed a considerable decrease in the myristoylation of proteins in parasites treated with NMT inhibitors, providing evidence of the on-target activity of the inhibitors.Taken together, our data point out to the potential use of NMT inhibitors as anti-T. cruzi chemotherapy.

  10. Trypanosoma cruzi: avirulence of the PF strain to Callithrix marmosets

    Directory of Open Access Journals (Sweden)

    Humberto Menezes

    1981-06-01

    Full Text Available Callithrix jacchus geoffroy marmosets (HumBol. 1812 were injected once subcutaneously with 10.000 parasites/g body weight and followed for a period of six months. The PF strain of Trypanosoma cruzi was used. Follow-up was done through blood cultures, xenodiagnosis, serological tests, and ECG. A small number of normaI animais served as control.

  11. Effective gene delivery to Trypanosoma cruzi epimastigotes through nucleofection.

    Science.gov (United States)

    Pacheco-Lugo, Lisandro; Díaz-Olmos, Yirys; Sáenz-García, José; Probst, Christian Macagnan; DaRocha, Wanderson Duarte

    2017-06-01

    New opportunities have raised to study the gene function approaches of Trypanosoma cruzi after its genome sequencing in 2005. Functional genomic approaches in Trypanosoma cruzi are challenging due to the reduced tools available for genetic manipulation, as well as to the reduced efficiency of the transient transfection conducted through conventional methods. The Amaxa nucleofector device was systematically tested in the present study in order to improve the electroporation conditions in the epimastigote forms of T. cruzi. The transfection efficiency was quantified using the green fluorescent protein (GFP) as reporter gene followed by cell survival assessment. The herein used nucleofection parameters have increased the survival rates (>90%) and the transfection efficiency by approximately 35%. The small amount of epimastigotes and DNA required for the nucleofection can turn the method adopted here into an attractive tool for high throughput screening (HTS) applications, and for gene editing in parasites where genetic manipulation tools remain relatively scarce. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. The Effectiveness of Natural Diarylheptanoids against Trypanosoma cruzi: Cytotoxicity, Ultrastructural Alterations and Molecular Modeling Studies.

    Directory of Open Access Journals (Sweden)

    Vitor Sueth-Santiago

    Full Text Available Curcumin (CUR is the major constituent of the rhizomes of Curcuma longa and has been widely investigated for its chemotherapeutic properties. The well-known activity of CUR against Leishmania sp., Trypanosoma brucei and Plasmodium falciparum led us to investigate its activity against Trypanosoma cruzi. In this work, we tested the cytotoxic effects of CUR and other natural curcuminoids on different forms of T. cruzi, as well as the ultrastructural changes induced in epimastigote form of the parasite. CUR was verified as the curcuminoid with more significant trypanocidal properties (IC50 10.13 μM on epimastigotes. Demethoxycurcumin (DMC was equipotent to CUR (IC50 11.07 μM, but bisdemethoxycurcumin (BDMC was less active (IC50 45.33 μM and cyclocurcumin (CC was inactive. In the experiment with infected murine peritoneal macrophages all diarylheptanoids were more active than the control in the inhibition of the trypomastigotes release. The electron microscopy images showed ultrastructural changes associated with the cytoskeleton of the parasite, indicating tubulin as possible target of CUR in T. cruzi. The results obtained by flow cytometry analysis of DNA content of the parasites treated with natural curcuminoids suggested a mechanism of action on microtubules related to the paclitaxel`s mode of action. To better understand the mechanism of action highlighted by electron microscopy and flow cytometry experiments we performed the molecular docking of natural curcuminoids on tubulin of T. cruzi in a homology model and the results obtained showed that the observed interactions are in accordance with the IC50 values found, since there CUR and DMC perform similar interactions at the binding site on tubulin while BDMC do not realize a hydrogen bond with Lys163 residue due to the absence of methoxyl groups. These results indicate that trypanocidal properties of CUR may be related to the cytoskeletal alterations.

  13. Further evidence from SSCP and ITS DNA sequencing support Trypanosoma evansi and Trypanosoma equiperdum as subspecies or even strains of Trypanosoma brucei.

    Science.gov (United States)

    Wen, Yan-Zi; Lun, Zhao-Rong; Zhu, Xing-Quan; Hide, Geoff; Lai, De-Hua

    2016-07-01

    The subgenus Trypanozoon includes three species Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum, which are morphologically identical and indistinguishable even using some molecular methods. In this study, PCR-based single strand conformation polymorphism (PCR-SSCP) was used to analyze the ribosomal DNA of the Trypanozoon species. Data indicate different patterns of ITS2 fragments between T. brucei, T. evansi and T. equiperdum by SSCP. Furthermore, analysis of total ITS sequences within these three members of the subgenus Trypanozoon showed a high degree of homology using phylogenetic analysis but were polyphyletic in haplotype networks. These data provide novel nuclear evidence to further support the notion that T. evansi and T. equiperdum should be subspecies or even strains of T. brucei. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Trypanosoma cruzi surface mucins: host-dependent coat diversity.

    Science.gov (United States)

    Buscaglia, Carlos A; Campo, Vanina A; Frasch, Alberto C C; Di Noia, Javier M

    2006-03-01

    The surface of the protozoan parasite Trypanosoma cruzi is covered in mucins, which contribute to parasite protection and to the establishment of a persistent infection. Their importance is highlighted by the fact that the approximately 850 mucin-encoding genes comprise approximately 1% of the parasite genome and approximately 6% of all predicted T. cruzi genes. The coordinate expression of a large repertoire of mucins containing variable regions in the mammal-dwelling stages of the T. cruzi life cycle suggests a possible strategy to thwart the host immune response. Here, we discuss the expression profiling of T. cruzi mucins, the mechanisms leading to the acquisition of mucin diversity and the possible consequences of a mosaic surface coat in the interplay between parasite and host.

  15. Particle-bound enzymes in the bloodstream form of Trypanosoma brucei

    NARCIS (Netherlands)

    Opperdoes, F. R.; Borst, P.; Spits, H.

    1977-01-01

    We have screened the bloodstream form of Trypanosoma brucei for the presence of enzymes that could serve as markers for the microbodies and the highly repressed mitochondrion of this organism. None of seven known microbody enzymes were detected at all, but glycerol-3-phosphate oxidase, ATPase,

  16. Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation

    NARCIS (Netherlands)

    Weelden, van S.W.H.; Fast, B.; Vogt, A.; Meer, van der P.; Saas, J.; Hellemond, van J.J.; Tielens, A.G.M.; Boshart, M.

    2003-01-01

    The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene

  17. Contribution of glucose transport to the control of the glycolytic flux in Trypanosoma brucei.

    NARCIS (Netherlands)

    Bakker, B.M.; Walsh, M.C.; ter Kuile, B.; Mensonides, F.I.C.; Michels, P.A.M.; Opperdoes, F.R.; Westerhoff, H.V.

    1999-01-01

    The rate of glucose transport across the plasma membrane of the bloodstream form of Trypanosoma brucei was modulated by titration of the hexose transporter with the inhibitor phloretin, and the effect on the glycolytic flux was measured. A rapid glucose uptake assay was developed to measure the

  18. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei

    NARCIS (Netherlands)

    Zomerdijk, J. C.; Ouellette, M.; ten Asbroek, A. L.; Kieft, R.; Bommer, A. M.; Clayton, C. E.; Borst, P.

    1990-01-01

    The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site

  19. Isolation and characterization of kinetoplast DNA from the bloodstream form of Trypanosoma brucei.

    NARCIS (Netherlands)

    A.H. Fairlamb; P.O. Weislogel; J.H.J. Hoeijmakers (Jan); P. Borst (Piet)

    1978-01-01

    textabstractWe have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in kinetoplast DNA (kDNA) networks of Trypanosoma brucei (East African

  20. Deletion of the Trypanosoma brucei Superoxide Dismutase Gene sodb1 Increases Sensitivity to Nifurtimox and Benznidazole▿

    Science.gov (United States)

    Prathalingham, S. Radhika; Wilkinson, Shane R.; Horn, David; Kelly, John M.

    2007-01-01

    It has been more than 25 years since it was first reported that nifurtimox and benznidazole promote superoxide production in trypanosomes. However, there has been no direct evidence of an association between the drug-induced free radicals and trypanocidal activity. Here, we identify a superoxide dismutase required to protect Trypanosoma brucei from drug-generated superoxide. PMID:17145786

  1. Deletion of the Trypanosoma brucei superoxide dismutase gene sodb1 increases sensitivity to nifurtimox and benznidazole.

    Science.gov (United States)

    Prathalingham, S Radhika; Wilkinson, Shane R; Horn, David; Kelly, John M

    2007-02-01

    It has been more than 25 years since it was first reported that nifurtimox and benznidazole promote superoxide production in trypanosomes. However, there has been no direct evidence of an association between the drug-induced free radicals and trypanocidal activity. Here, we identify a superoxide dismutase required to protect Trypanosoma brucei from drug-generated superoxide.

  2. Trypanosoma brucei Infection in a herd of sedentary cattle in Danja ...

    African Journals Online (AJOL)

    Trypanosoma brucei Infection in a herd of sedentary cattle in Danja Local Government Area, Katsina State, Northern Nigeria– A possible resurgence of Tsetse flies ... resulted in advocacy for pastoralists to settle down into productive cattle production compared to the nomadic behavior which does not enhance productivity.

  3. Novel molecular mechanism for targeting the parasite Trypanosoma brucei with snake venom toxins

    DEFF Research Database (Denmark)

    Martos Esteban, Andrea; Laustsen, Andreas Hougaard; Carrington, Mark

    Trypanosoma brucei is a parasitic protozoan species capable to infecting insect vectors whose bite further produces African sleeping sickness inhuman beings. During parasites’extracellular lives in the mammalian host, its outer coat, mainly composedof Variable surface glycoproteins (VSGs)[2], und...

  4. Protective effect of humus extract against Trypanosoma brucei infection in mice.

    Science.gov (United States)

    Kodama, Hiroshi; Denso; Okazaki, Fumi; Ishida, Saeko

    2008-11-01

    Humic substances are formed during the decomposition of organic matter in humus, and are found in many natural environments in which organic materials and microorganisms are present. Oral administration of humus extract to mice successfully induced effective protection against experimental challenge by the two subspecies, Trypanosoma brucei brucei and T. brucei gambiense. Mortality was most reduced among mice who received a 3% humus extract for 21 days in drinking water ad libitum. Spleen cells from humus-administered mice exhibited significant non-specific cytotoxic activity against L1210 mouse leukemia target cells. Also, spleen cells produced significantly higher amounts of Interferon-gamma when stimulated in vitro with Concanavalin A than cells from normal controls. These results clearly show that administration to mice of humus extract induced effective resistance against Trypanosoma infection. Enhancement of the innate immune system may be involved in host defense against trypanosomiasis.

  5. Can sexual transmission support the enzootic cycle of Trypanosoma cruzi?

    Science.gov (United States)

    Rios, Adriano; Ribeiro, Marcelle; Sousa, Alessandro; Pimentel, Fernando; Hagström, Luciana; Andrade, Rafael; Alves, Rozeneide M; Rosa, Ana de Cássia; Teixeira, Antônio Rl; Nitz, Nadjar; Hecht, Mariana M

    2018-01-01

    Trypanosoma cruzi circulates in sylvatic habitats, mainly through blood-feeding triatomines, although other routes also contribute to its dispersion. Sexual transmission of T. cruzi is an understudied topic, especially among wild mammals. Because of the difficulties inherent to field work, experimentally infected mice are frequently used to evaluate the transmission of T. cruzi. This study aimed to evaluate the sexual transmission of T. cruzi in acutely infected mice. Male and female mice in the acute phase of Chagas disease were mated with naïve partners. Then, parasitological tests, immunohistochemistry, serological assays, and polymerase chain reaction (PCR) assays were used to detect infection. Parasitological analysis showed trypomastigotes in the blood of 20% of the naïve mice after mating with infected partners. Serological assays detected anti-T. cruzi antibodies in all naïve females mated with infected males and in 60% of naïve males mated with infected females. PCR showed T. cruzi nDNA bands for all naïve mice mated with infected partners. The possibility of sexual transmission was also confirmed by visualisation of amastigotes in the testes. Our results demonstrate that sexual transmission of T. cruzi is an ordinary event that may contribute to maintenance of the parasite's enzootic cycle.

  6. Can sexual transmission support the enzootic cycle of Trypanosoma cruzi?

    Directory of Open Access Journals (Sweden)

    Adriano Rios

    Full Text Available BACKGROUND Trypanosoma cruzi circulates in sylvatic habitats, mainly through blood-feeding triatomines, although other routes also contribute to its dispersion. Sexual transmission of T. cruzi is an understudied topic, especially among wild mammals. Because of the difficulties inherent to field work, experimentally infected mice are frequently used to evaluate the transmission of T. cruzi. OBJECTIVE This study aimed to evaluate the sexual transmission of T. cruzi in acutely infected mice. METHODS Male and female mice in the acute phase of Chagas disease were mated with naïve partners. Then, parasitological tests, immunohistochemistry, serological assays, and polymerase chain reaction (PCR assays were used to detect infection. FINDINGS Parasitological analysis showed trypomastigotes in the blood of 20% of the naïve mice after mating with infected partners. Serological assays detected anti-T. cruzi antibodies in all naïve females mated with infected males and in 60% of naïve males mated with infected females. PCR showed T. cruzi nDNA bands for all naïve mice mated with infected partners. The possibility of sexual transmission was also confirmed by visualisation of amastigotes in the testes. MAIN CONCLUSIONS Our results demonstrate that sexual transmission of T. cruzi is an ordinary event that may contribute to maintenance of the parasite's enzootic cycle.

  7. Interferon-Gamma Promotes Infection of Astrocytes by Trypanosoma cruzi

    Science.gov (United States)

    Silva, Rafael Rodrigues; Mariante, Rafael M.; Silva, Andrea Alice; dos Santos, Ana Luiza Barbosa; Roffê, Ester; Santiago, Helton; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli

    2015-01-01

    The inflammatory cytokine interferon-gamma (IFNγ) is crucial for immunity against intracellular pathogens such as the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (CD). IFNγ is a pleiotropic cytokine which regulates activation of immune and non-immune cells; however, the effect of IFNγ in the central nervous system (CNS) and astrocytes during CD is unknown. Here we show that parasite persists in the CNS of C3H/He mice chronically infected with the Colombian T. cruzi strain despite the increased expression of IFNγ mRNA. Furthermore, most of the T. cruzi-bearing cells were astrocytes located near IFNγ+ cells. Surprisingly, in vitro experiments revealed that pretreatment with IFNγ promoted the infection of astrocytes by T. cruzi increasing uptake and proliferation of intracellular forms, despite inducing increased production of nitric oxide (NO). Importantly, the effect of IFNγ on T. cruzi uptake and growth is completely blocked by the anti-tumor necrosis factor (TNF) antibody Infliximab and partially blocked by the inhibitor of nitric oxide synthesis L-NAME. These data support that IFNγ fuels astrocyte infection by T. cruzi and critically implicate IFNγ-stimulated T. cruzi-infected astrocytes as sources of TNF and NO, which may contribute to parasite persistence and CNS pathology in CD. PMID:25695249

  8. Trypanosoma cruzi and Chagas' Disease in the United States

    Science.gov (United States)

    Bern, Caryn; Kjos, Sonia; Yabsley, Michael J.; Montgomery, Susan P.

    2011-01-01

    Summary: Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and causes potentially life-threatening disease of the heart and gastrointestinal tract. The southern half of the United States contains enzootic cycles of T. cruzi, involving 11 recognized triatomine vector species. The greatest vector diversity and density occur in the western United States, where woodrats are the most common reservoir; other rodents, raccoons, skunks, and coyotes are also infected with T. cruzi. In the eastern United States, the prevalence of T. cruzi is highest in raccoons, opossums, armadillos, and skunks. A total of 7 autochthonous vector-borne human infections have been reported in Texas, California, Tennessee, and Louisiana; many others are thought to go unrecognized. Nevertheless, most T. cruzi-infected individuals in the United States are immigrants from areas of endemicity in Latin America. Seven transfusion-associated and 6 organ donor-derived T. cruzi infections have been documented in the United States and Canada. As improved control of vector- and blood-borne T. cruzi transmission decreases the burden in countries where the disease is historically endemic and imported Chagas' disease is increasingly recognized outside Latin America, the United States can play an important role in addressing the altered epidemiology of Chagas' disease in the 21st century. PMID:21976603

  9. Effect of diminazene aceturate, levamisole and vitamin C combination therapy in rats experimentally infected with Trypanosoma brucei brucei.

    Science.gov (United States)

    Chekwube, Adieme Ijeoma; Onyema, Ezeh Ikenna; Ikenna, Ugochukwu Emmanuel; Ezeokonkwo, Romanus Chukwuduruo

    2014-06-01

    To investigate the effect of diminazene aceturate (DA) alone or in combination with either levamisole and/or Vitamin C in albino rats experimentally infected with Trypanosoma brucei brucei. Thirty adult male albino rats, randomly assigned into 6 groups (A-F) of 5 rats each were used. They were either infected with 1×10(6) trypanosomes intraperitoneally (groups A-E) or uninfected (group F). The different groups were treated respectively as follows: group A-with 3.5 mg/kg DA; group B-3.5 mg/kg DA and 7.5 mg/kg levamisole; group C-3.5 mg/kg DA and 100 mg/kg vitamin C; and group D-3.5 mg/kg DA and 7.5 mg/kg levamisole and 100 mg/kg vitamin C. Group E was left untreated. Parameters assessed include: rectal temperature, body weight changes, packed cell volume (PCV), Haemoglobin concentration (Hb), total leucocyte count (TLC) differential leucocyte count (DLC), parasitaemia, clinical signs and survivability. Average pre-patent period of 5 days was recorded. Parasites in the blood were cleared in all treated groups (A-D) within 48 hours post treatment (PT). Untreated rats in group E died between 25 and 32 days post infection (PI). Relapse was not recorded in all the treated groups (A-D). The initial reduction in PCV, Hb, TLC and increases in rectal temperature following infection were reversed by the treatments. The rats that received drug combinations (groups B, C and D) showed faster and higher recovery rates than the uninfected control and group A. Levamisole and/or Vitamin C combination with DA were more effective in the treatment of rats infected with Trypanosoma brucei brucei. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  10. Surface electrical charge of bloodstream trypomastigotes of Trypanosoma cruzi strains

    Directory of Open Access Journals (Sweden)

    Maria Auxiliadora de Sousa

    1983-12-01

    Full Text Available Bloodstream trypomastigotes of some Trypanosoma cruzi strains were processed through DEAE-cellulose columns under standardized conditions. The results obtained suggest mainly that these strains present different surface charges, that there are subpopulations of bloodstream trypomastigotes as regards electrical charges and that the broad forms are less negative than the slender ones.Tripomastigotas sanguíneos de algumas cepas de Trypanosoma cruzi foram processadas em colunas de DEAE-celulose sob condições padronizadas. Os resultados obtidos sugerem principalmente que estas cepas possuem cargas superficiais diferentes, que em relação a este aspecto existem subpopulações de tripomastigotas e que as formas largas são menos negativas do que as finas.

  11. Vaccination with Trypanosoma rangeli induces resistance of guinea pigs to virulent Trypanosoma cruzi.

    Science.gov (United States)

    Basso, B; Moretti, E; Fretes, R

    2014-01-15

    Chagas' disease, endemic in Latin America, is spread in natural environments through animal reservoirs, including marsupials, mice and guinea pigs. Farms breeding guinea pigs for food are located in some Latin-American countries with consequent risk of digestive infection. The aim of this work was to study the effect of vaccination with Trypanosoma rangeli in guinea pigs challenged with Trypanosoma cruzi. Animals were vaccinated with fixated epimastigotes of T. rangeli, emulsified with saponin. Controls received only PBS. Before being challenged with T. cruzi, parasitemia, survival rates and histological studies were performed. The vaccinated guinea pigs revealed significantly lower parasitemia than controls (pguinea pigs and dogs. The development of vaccines for use in animals, like domestic dogs and guinea pigs in captivity, opens up new opportunities for preventive tools, and could reduce the risk of infection with T. cruzi in the community. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Betulinic acid induces cell death by necrosis in Trypanosoma cruzi.

    Science.gov (United States)

    Sousa, Paloma Leão; Souza, Racquel Oliveira da Silva; Tessarolo, Louise Donadello; de Menezes, Ramon Róseo Paula Pessoa Bezerra; Sampaio, Tiago Lima; Canuto, Jader Almeida; Martins, Alice Maria Costa

    2017-10-01

    Chagas' disease is a neglected disease caused by the protozoan parasite Trypanosoma cruzi and constitutes a serious health problem worldwide. The treatment is limited, with variable efficacy of benznidazole and nifurtimox. Betulinic Acid (BA), a triterpene, can be found in medicinal herbs and has a wide variety of biological and pharmacological activities. The objective was to evaluate betulinic acid effects on the cell death mechanism in Trypanosoma cruzi strain Y. BA inhibited the growth of epimastigotes in periods of 24h (IC 50 =73.43μM), 48h (IC 50 =119.8μM) and 72h (IC 50 =212.2μM) of incubation; of trypomastigotes (IC 50 =51.88μM) in periods of 24h and intracellular amastigotes (IC 50 =25.94μM) in periods of 24 and 48h of incubation, no toxicity on LLC-MK 2 cells at the concentrations used. Analysis of the possible mechanism of parasite cell death showed alterations in mitochondrial membrane potential, alterations in cell membrane integrity, an increase in the formation of reactive oxygen species and increase swelling of the reservosomes. In conclusion, betulinic acid was be able to inhibition all developmental forms of Trypanosoma cruzi Y strain with necrotic mechanism and involvement of mitochondrial membrane potential alteration and increase in reactive oxygen species. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis

    DEFF Research Database (Denmark)

    Queiroz, Rayner M L; Charneau, Sebastien; Mandacaru, Samuel C

    2014-01-01

    Chagas disease is a tropical neglected disease endemic in Latin America and it is caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote and amastigote. The differentiation from infective...... this well-established differentiation protocol to perform a comprehensive quantitative proteomic and phosphoproteomic analysis of the T. cruzi amastigogenesis. Samples from fully differentiated forms and two biologically relevant intermediate time points were Lys-C/trypsin digested, i...... induced by incubation in acidic medium were also evinced. To our knowledge, this work is the most comprehensive quantitative proteomics study of the T. cruzi amastigogenesis and this data will provide trustworthy basis for future studies and possibly for new potential drug targets....

  14. Pentamidine exerts in vitro and in vivo anti Trypanosoma cruzi activity and inhibits the polyamine transport in Trypanosoma cruzi.

    Science.gov (United States)

    Díaz, María V; Miranda, Mariana R; Campos-Estrada, Carolina; Reigada, Chantal; Maya, Juan D; Pereira, Claudio A; López-Muñoz, Rodrigo

    2014-06-01

    Pentamidine is an antiprotozoal and fungicide drug used in the treatment of leishmaniasis and African trypanosomiasis. Despite its extensive use as antiparasitic drug, little evidence exists about the effect of pentamidine in Trypanosoma cruzi, the etiological agent of Chagas' disease. Recent studies have shown that pentamidine blocks a polyamine transporter present in Leishmania major; consequently, its might also block these transporters in T. cruzi. Considering that T. cruzi lacks the ability to synthesize putrescine de novo, the inhibition of polyamine transport can bring a new therapeutic target against the parasite. In this work, we show that pentamidine decreases, not only the viability of T. cruzi trypomastigotes, but also the parasite burden of infected cells. In T. cruzi-infected mice pentamidine decreases the inflammation and parasite burden in hearts from infected mice. The treatment also decreases parasitemia, resulting in an increased survival rate. In addition, pentamidine strongly inhibits the putrescine and spermidine transport in T. cruzi epimastigotes and amastigotes. Thus, this study points to reevaluate the utility of pentamidine and introduce evidence of a potential new action mechanism. In the quest of new therapeutic strategies against Chagas disease, the extensive use of pentamidine in human has led to a well-known clinical profile, which could be an advantage over newly synthesized molecules that require more comprehensive trials prior to their clinical use. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Trypanosoma cruzi: Transporte de metabolitos esenciales obtenidos del hospedador Trypanosoma cruzi: Transport of essential metabolites acquired from the host

    Directory of Open Access Journals (Sweden)

    Claudio A. Pereira

    2008-10-01

    Full Text Available El Trypanosoma cruzi es el agente causal de la enfermedad de Chagas, endémica en Argentina y en toda América Latina. Presenta numerosas características metabólicas diferenciales respecto a sus hospedadores insectos y mamíferos. Algunas de estas diferencias fueron consecuencia de millones de años de adaptación al parasitismo en los cuales estos organismos protozoarios reemplazaron, a lo largo de su evolución, muchas rutas metabólicas de biosíntesis por sistemas de transporte de metabolitos desde el hospedador. En esta revisión se describen los avances en el conocimiento de los sistemas de transporte tanto bioquímicos como también de las moléculas involucradas en dichos procesos. Se aborda con especial énfasis los transportadores de aminoácidos y poliaminas de T. cruzi de la familia AAAP (Amino Acid/Auxin Permeases ya que parece ser exclusiva de los tripanosomátidos. Teniendo en cuenta que estas moléculas se encuentran completamente ausentes en mamíferos podrían ser consideradas como potenciales blancos contra el Trypanosoma cruzi.Trypanosoma cruzi is the etiological agent of Chagas disease, a disease endemic not only in Argentina but also in all of Latinamerica. T. cruzi presents several metabolic characteristics which are completely absent in its insect vectors and in mammalian hosts. Some of these differences were acquired after millions of years of adaptation to parasitism, during which this protozoan replaced many biosynthetic routes for transport systems. In the present review, we describe the advances in the knowledge of T. cruzi transport processes and the molecules involved. In particular, we focus on aminoacid and polyamine transporters from the AAAP family (Amino Acid/Auxin Permeases, because they seem to be exclusive transporters from trypanosomatids. Taking into account that these permeases are completely absent in mammals, they could be considered as a potential target against Trypanosoma cruzi.

  16. Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies.

    Science.gov (United States)

    Arakaki, Tracy L; Buckner, Frederick S; Gillespie, J Robert; Malmquist, Nicholas A; Phillips, Margaret A; Kalyuzhniy, Oleksandr; Luft, Joseph R; Detitta, George T; Verlinde, Christophe L M J; Van Voorhis, Wesley C; Hol, Wim G J; Merritt, Ethan A

    2008-04-01

    Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluorouracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection. Specifically, a combination therapy including a T. brucei-specific DHODH inhibitor plus 5-FU may prove to be an effective therapeutic strategy. We also show that this trypanosomal enzyme is inhibited by known inhibitors of bacterial Class 1A DHODH, in distinction to the sensitivity of DHODH from human and other higher eukaryotes. This selectivity is supported by the crystal structure of the T. brucei enzyme, which is reported here at a resolution of 1.95 A. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH.

  17. DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli.

    Science.gov (United States)

    Naves, Lucila Langoni; da Silva, Marcos Vinícius; Fajardo, Emanuella Francisco; da Silva, Raíssa Bernardes; De Vito, Fernanda Bernadelli; Rodrigues, Virmondes; Lages-Silva, Eliane; Ramírez, Luis Eduardo; Pedrosa, André Luiz

    2017-01-01

    Trypanosoma cruzi, a human protozoan parasite, is the causative agent of Chagas disease. Currently the species is divided into six taxonomic groups. The genome of the CL Brener clone has been estimated to be 106.4-110.7 Mb, and DNA content analyses revealed that it is a diploid hybrid clone. Trypanosoma rangeli is a hemoflagellate that has the same reservoirs and vectors as T. cruzi; however, it is non-pathogenic to vertebrate hosts. The haploid genome of T. rangeli was previously estimated to be 24 Mb. The parasitic strains of T. rangeli are divided into KP1(+) and KP1(-). Thus, the objective of this study was to investigate the DNA content in different strains of T. cruzi and T. rangeli by flow cytometry. All T. cruzi and T. rangeli strains yielded cell cycle profiles with clearly identifiable G1-0 (2n) and G2-M (4n) peaks. T. cruzi and T. rangeli genome sizes were estimated using the clone CL Brener and the Leishmania major CC1 as reference cell lines because their genome sequences have been previously determined. The DNA content of T. cruzi strains ranged from 87,41 to 108,16 Mb, and the DNA content of T. rangeli strains ranged from 63,25 Mb to 68,66 Mb. No differences in DNA content were observed between KP1(+) and KP1(-) T. rangeli strains. Cultures containing mixtures of the epimastigote forms of T. cruzi and T. rangeli strains resulted in cell cycle profiles with distinct G1 peaks for strains of each species. These results demonstrate that DNA content analysis by flow cytometry is a reliable technique for discrimination between T. cruzi and T. rangeli isolated from different hosts.

  18. Trypanosoma cruzi: Inhibition of infection of human monocytes by aspirin.

    Science.gov (United States)

    Carvalho de Freitas, Rafael; Lonien, Sandra Cristina Heim; Malvezi, Aparecida Donizette; Silveira, Guilherme Ferreira; Wowk, Pryscilla Fanini; da Silva, Rosiane Valeriano; Yamauchi, Lucy Megumi; Yamada-Ogatta, Sueli Fumie; Rizzo, Luiz Vicente; Bordignon, Juliano; Pinge-Filho, Phileno

    2017-11-01

    Cell invasion by Trypanosoma cruzi and its intracellular replication are essential for progression of the parasite life cycle and development of Chagas disease. Prostaglandin E2 (PGE 2 ) and other eicosanoids potently modulate host response and contribute to Chagas disease progression. In this study, we evaluated the effect of aspirin (ASA), a non-selective cyclooxygenase (COX) inhibitor on the T. cruzi invasion and its influence on nitric oxide and cytokine production in human monocytes. The pretreatment of monocytes with ASA or SQ 22536 (adenylate-cyclase inhibitor) induced a marked inhibition of T. cruzi infection. On the other hand, the treatment of monocytes with SQ 22536 after ASA restored the invasiveness of T. cruzi. This reestablishment was associated with a decrease in nitric oxide and PGE 2 production, and also an increase of interleukin-10 and interleukin-12 by cells pre-treated with ASA. Altogether, these results reinforce the idea that the cyclooxygenase pathway plays a fundamental role in the process of parasite invasion in an in vitro model of T. cruzi infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Selective blockade of trypanosomatid protein synthesis by a recombinant antibody anti-Trypanosoma cruzi P2β protein.

    Science.gov (United States)

    Ayub, Maximiliano Juri; Nyambega, Benson; Simonetti, Leandro; Duffy, Tomas; Longhi, Silvia A; Gómez, Karina A; Hoebeke, Johan; Levin, Mariano J; Smulski, Cristian R

    2012-01-01

    The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.

  20. Selective blockade of trypanosomatid protein synthesis by a recombinant antibody anti-Trypanosoma cruzi P2β protein.

    Directory of Open Access Journals (Sweden)

    Maximiliano Juri Ayub

    Full Text Available The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5 directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.

  1. Secretome analysis of Trypanosoma cruzi by proteomics studies

    Science.gov (United States)

    Gulin, Julián Ernesto Nicolás; Bisio, Margarita Maria Catalina; Chapelle, Manuel; Marinach-Patrice, Carine; Bordessoules, Mallaury; Palazon Ruiz, George; Vion, Jeremy; Paris, Luc; Altcheh, Jaime; Mazier, Dominique

    2017-01-01

    Background Chagas disease is a debilitating often fatal disease resulting from infection by the protozoan parasite Trypanosoma cruzi. Chagas disease is endemic in 21 countries of the Americas, and it is an emerging disease in other countries as a result of migration. Given the chronic nature of the infection where intracellular parasites persist for years, the diagnosis of T. cruzi by direct detection is difficult, whereas serologic tests though sensitive may yield false-positive results. The development of new rapid test based on the identification of soluble parasitic antigens in serum would be a real innovation in the diagnosis of Chagas disease. Methods To identify new soluble biomarkers that may improve diagnostic tests, we investigated the proteins secreted by T. cruzi using mass spectrometric analyses of conditioned culture media devoid of serum collected during the emergence of trypomastigotes from infected Vero cells. In addition, we compared the secretomes of two T. cruzi strains from DTU Tc VI (VD and CL Brener). Results Analysis of the secretome collected during the emergence of trypomastigotes from Vero cells led to the identification of 591 T. cruzi proteins. Three hundred sixty three proteins are common to both strains and most belong to different multigenic super families (i.e. TcS, GP63, MASP, and DGF1). Ultimately we have established a list of 94 secreted proteins, common to both DTU Tc VI strains that do not belong to members of multigene families. Conclusions This study provides the first comparative analysis of the secretomes from two distinct T. cruzi strains of DTU TcVI. This led us to identify a subset of common secreted proteins that could potentially serve as serum markers for T. cruzi infection. Their potential could now be evaluated, with specific antibodies using sera collected from patients and residents from endemic regions. PMID:28972996

  2. Sympatry influence in the interaction of Trypanosoma cruzi with triatomine.

    Science.gov (United States)

    Dworak, Elaine Schultz; Araújo, Silvana Marques de; Gomes, Mônica Lúcia; Massago, Miyoko; Ferreira, Érika Cristina; Toledo, Max Jean de Ornelas

    2017-01-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is widely distributed in nature, circulating between triatomine bugs and sylvatic mammals, and has large genetic diversity. Both the vector species and the genetic lineages of T. cruzi present a varied geographical distribution. This study aimed to verify the influence of sympatry in the interaction of T. cruzi with triatomines. Methods: The behavior of the strains PR2256 (T. cruzi II) and AM14 (T. cruzi IV) was studied in Triatoma sordida (TS) and Rhodnius robustus (RR). Eleven fifth-stage nymphs were fed by artificial xenodiagnosis with 5.6 × 103 blood trypomastigotes/0.1mL of each T. cruzi strain. Every 20 days, their excreta were examined for up to 100 days, and every 30 days, the intestinal content was examined for up to 120 days, by parasitological (fresh examination and differential count with Giemsa-stained smears) and molecular (PCR) methods. Rates of infectivity, metacyclogenesis and mortality, and mean number of parasites per insect and of excreted parasites were determined. Sympatric groups RR+AM14 and TS+PR2256 showed higher values of the four parameters, except for mortality rate, which was higher (27.3%) in the TS+AM14 group. General infectivity was 72.7%, which was mainly proven by PCR, showing the following decreasing order: RR+AM14 (100%), TS+PR2256 (81.8%), RR+PR2256 (72.7%) and TS+AM14 (36.4%). Our working hypothesis was confirmed once higher infectivity and vector capacity (flagellate production and elimination of infective metacyclic forms) were recorded in the groups that contained sympatric T. cruzi lineages and triatomine species.

  3. Phospholipase A1: a novel virulence factor in Trypanosoma cruzi.

    Science.gov (United States)

    Belaunzarán, María Laura; Wilkowsky, Silvina Elizabeth; Lammel, Estela María; Giménez, Guadalupe; Bott, Emanuel; Barbieri, Manuel Alejandro; de Isola, Elvira Luisa Durante

    2013-02-01

    Phospholipase A1 (PLA1) has been described in the infective stages of Trypanosoma cruzi as a membrane-bound/secreted enzyme that significantly modified host cell lipid profile with generation of second lipid messengers and concomitant activation of protein kinase C. In the present work we determined higher levels of PLA1 expression in the infective amastigotes and trypomastigotes than in the non-infective epimastigotes of lethal RA strain. In addition, we found similar expression patterns but distinct PLA1 activity levels in bloodstream trypomastigotes from Cvd and RA (lethal) and K98 (non-lethal) T. cruzi strains, obtained at their corresponding parasitemia peaks. This fact was likely due to the presence of different levels of anti-T. cruzi PLA1 antibodies in sera of infected mice, that modulated the enzyme activity. Moreover, these antibodies significantly reduced in vitro parasite invasion indicating the participation of T. cruzi PLA1 in the early events of parasite-host cell interaction. We also demonstrated the presence of lysophospholipase activity in live infective stages that could account for self-protection against the toxic lysophospholipids generated by T. cruzi PLA1 action. At the genome level, we identified at least eight putative genes that codify for T. cruzi PLA1 with high amino acid sequence variability in their amino and carboxy-terminal regions; a putative PLA1 selected gene was cloned and expressed as a recombinant protein that possessed PLA1 activity. Collectively, the results presented here point out at T. cruzi PLA1 as a novel virulence factor implicated in parasite invasion. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Population genetic structure and cladistic analysis of Trypanosoma brucei isolates

    NARCIS (Netherlands)

    Agbo, E.C.; Clausen, P.H.; Buscher, P.; Majiwa, P.A.O.; Pas, te M.F.W.; Claassen, E.

    2003-01-01

    Using a novel multilocus DNA marker analysis method, we studied the population genetic structure of Trypansoma brucei stocks and derived clones isolated from animal and rhodesiense sleeping sickness patients during a national sleeping sickness control program in Mukono district, Uganda. We then

  5. Aparasitemic serological suspects in Trypanosoma brucei gambiense human African trypanosomiasis : a potential human reservoir of parasites ?

    OpenAIRE

    Koffi, Mathurin; Solano, Philippe; Denizot, M.; Courtin, D.; Garcia, André; Lejon, V.; Buscher, P.; Cuny, Gérard; Jamonneau, Vincent

    2006-01-01

    The serological and parasitological tests used for Trypanosoma brucei gambiense human African trypanosomiasis (HAT) diagnosis have low specificity and sensitivity, respectively, and in the field, control program teams are faced with subjects with positive serology but negative parasitology who remain untreated. The aim of this work was to explore, using PCR tool, the significance of these aparasitemic serological suspects. Since discordant PCR results have been observed earlier with different...

  6. Role of centrins 2 and 3 in organelle segregation and cytokinesis in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Angamuthu Selvapandiyan

    Full Text Available Centrins are calcium binding proteins involved in cell division in eukaryotes. Previously, we have shown that depletion of centrin1 in Trypanosoma brucei (T. brucei displayed arrested organelle segregation resulting in loss of cytokinesis. In this study we analyzed the role of T. brucei centrin2 (TbCen2 and T. brucei 3 (TbCen3 in the early events of T. brucei procyclic cell cycle. Both the immunofluorescence assay and electron microscopy showed that TbCen2 and 3-deficient cells were enlarged in size with duplicated basal bodies, multinuclei and new flagella that are detached along the length of the cell body. In both TbCen2 and TbCen3 depleted cells segregation of the organelles i.e. basal bodies, kinetoplast and nucleus was disrupted. Further analysis of the cells with defective organelle segregation identified three different sub configurations of organelle mis-segregations (Type 1-3. In addition, in majority of the TbCen2 depleted cells and in nearly half of the TbCen3 depleted cells, the kinetoplasts were enlarged and undivided. The abnormal segregations ultimately led to aborted cytokinesis and hence affected growth in these cells. Therefore, both centrin2 and 3 are involved in organelle segregation similar to centrin1 as was previously observed. In addition, we identified their role in kinetoplast division which may be also linked to overall mis-segregation.

  7. Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model.

    Science.gov (United States)

    Dauchy, Frédéric-Antoine; Bonhivers, Mélanie; Landrein, Nicolas; Dacheux, Denis; Courtois, Pierrette; Lauruol, Florian; Daulouède, Sylvie; Vincendeau, Philippe; Robinson, Derrick R

    2016-11-01

    Trypanosoma brucei gambiense is the main causative agent of Human African Trypanosomiasis (HAT), also known as sleeping sickness. Because of limited alternatives and treatment toxicities, new therapeutic options are urgently needed for patients with HAT. Sterol 14alpha-demethylase (CYP51) is a potential drug target but its essentiality has not been determined in T. brucei. We used a tetracycline-inducible RNAi system to assess the essentiality of CYP51 in T. brucei bloodstream form (BSF) cells and we evaluated the effect of posaconazole, a well-tolerated triazole drug, within a panel of virulent strains in vitro and in a murine model. Expression of CYP51 in several T. brucei cell lines was demonstrated by western blot and its essentiality was demonstrated by RNA interference (CYP51RNAi) in vitro. Following reduction of TbCYP51 expression by RNAi, cell growth was reduced and eventually stopped compared to WT or non-induced cells, showing the requirement of CYP51 in T. brucei. These phenotypes were rescued by addition of ergosterol. Additionally, CYP51RNAi induction caused morphological defects with multiflagellated cells (pnifurtimox-eflornithine combinations showed similar improvement in mice survival (p≤0.001). Our results provide support for a CYP51 targeting based treatment in HAT. Thus posaconazole used in combination may represent a therapeutic alternative for trypanosomiasis.

  8. The role of IL-12 in experimental Trypanosoma cruzi infection

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    J.S. Silva

    1998-01-01

    Full Text Available Host resistance to Trypanosoma cruzi infection is dependent on both natural and acquired immune responses. During the early acute phase of infection in mice, natural killer (NK cell-derived IFN-g is involved in controlling intracellular parasite replication, mainly through the induction of nitric oxide biosynthesis by activated macrophages. We have shown that IL-12, a powerful inducer of IFN-g production by NK cells, is synthesized soon after trypomastigote-macrophage interaction. The role of IL-12 in the control of T. cruzi infection in vivo was determined by treating infected mice with anti-IL-12 monoclonal antibody (mAb and analyzing both parasitemia and mortality during the acute phase of infection. The anti-IL-12 mAb-treated mice had higher levels of parasitemia and mortality compared to control mice. Also, treatment of infected mice with mAb specific for IFN-g or TNF-a inhibited the protective effect of exogenous IL-12. On the other hand, TGF-ß and IL-10 produced by infected macrophages inhibited the induction and effects of IL-12. Therefore, while IL-12, TNF-a and IFN-g correlate with resistance to T. cruzi infection, TGF-ß and IL-10 promote susceptibility. These results provide support for a role of innate immunity in the control of T. cruzi infection. In addition to its protective role, IL-12 may also be involved in the modulation of T. cruzi-induced myocarditis, since treatment of infected mice with IL-12 or anti-IL-12 mAb leads to an enhanced or decreased inflammatory infiltrate in the heart, respectively. Understanding the role of the cytokines produced during the acute phase of T. cruzi infection and their involvement in protection and pathogenesis would be essential to devise new vaccines or therapies.

  9. Occurrence of Trypanosoma cruzi in Maryland

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    Herman, C.M.; Bruce, J.I.

    1962-01-01

    During 1954-1960, 2005 mammals of 18 species collected at the Patuxent Wildlife Research Center, Maryland, were examined for trypanosomes. T. cruzi was found in 10 raccoons between October 31 and November 30. Infection occurred in 2 percent of all raccoons sampled, and in 11.3 percent of the 80 raccoons sampled in November. Examination was by direct smears, stained smears and cultures of heart blood. Although, in previous studies, at least two experimentally infected raccoons exhibited extended parasitemia (14 and 8 weeks), no such continuing parasitemia was observed in the natural infections. No trypanosomes were found in any of the other mammals examined.

  10. Electron Microscopy Analysis of the Nucleolus of Trypanosoma cruzi

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    López-Velázquez, Gabriel; Hernández, Roberto; López-Villaseñor, Imelda; Reyes-Vivas, Horacio; Segura-Valdez, María De L.; Jiménez-García, Luis F.

    2005-08-01

    The nucleolus is the main site for synthesis and processing of ribosomal RNA in eukaryotes. In mammals, plants, and yeast the nucleolus has been extensively characterized by electron microscopy, but in the majority of the unicellular eukaryotes no such studies have been performed. Here we used ultrastructural cytochemical and immunocytochemical techniques as well as three-dimensional reconstruction to analyze the nucleolus of Trypanosoma cruzi, which is an early divergent eukaryote of medical importance. In T. cruzi epimastigotes the nucleolus is a spherical intranuclear ribonucleoprotein organelle localized in a relatively central position within the nucleus. Dense fibrillar and granular components but not fibrillar centers were observed. In addition, nuclear bodies resembling Cajal bodies were observed associated to the nucleolus in the surrounding nucleoplasm. Our results provide additional morphological data to better understand the synthesis and processing of the ribosomal RNA in kinetoplastids.

  11. Proteomic Analysis of Trypanosoma cruzi Epimastigotes Subjected to Heat Shock

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    Deyanira Pérez-Morales

    2012-01-01

    Full Text Available Trypanosoma cruzi is exposed to sudden temperature changes during its life cycle. Adaptation to these variations is crucial for parasite survival, reproduction, and transmission. Some of these conditions may change the pattern of genetic expression of proteins involved in homeostasis in the course of stress treatment. In the present study, the proteome of T. cruzi epimastigotes subjected to heat shock and epimastigotes grow normally was compared by two-dimensional gel electrophoresis followed by mass spectrometry for protein identification. Twenty-four spots differing in abundance were identified. Of the twenty-four changed spots, nineteen showed a greater intensity and five a lower intensity relative to the control. Several functional categories of the identified proteins were determined: metabolism, cell defense, hypothetical proteins, protein fate, protein synthesis, cellular transport, and cell cycle. Proteins involved in the interaction with the cellular environment were also identified, and the implications of these changes are discussed.

  12. Early Trypanosoma cruzi Infection Reprograms Human Epithelial Cells

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    María Laura Chiribao

    2014-01-01

    Full Text Available Trypanosoma cruzi, the causative agent of Chagas disease, has the peculiarity, when compared with other intracellular parasites, that it is able to invade almost any type of cell. This property makes Chagas a complex parasitic disease in terms of prophylaxis and therapeutics. The identification of key host cellular factors that play a role in the T. cruzi invasion is important for the understanding of disease pathogenesis. In Chagas disease, most of the focus is on the response of macrophages and cardiomyocytes, since they are responsible for host defenses and cardiac lesions, respectively. In the present work, we studied the early response to infection of T. cruzi in human epithelial cells, which constitute the first barrier for establishment of infection. These studies identified up to 1700 significantly altered genes regulated by the immediate infection. The global analysis indicates that cells are literally reprogrammed by T. cruzi, which affects cellular stress responses (neutrophil chemotaxis, DNA damage response, a great number of transcription factors (including the majority of NFκB family members, and host metabolism (cholesterol, fatty acids, and phospholipids. These results raise the possibility that early host cell reprogramming is exploited by the parasite to establish the initial infection and posterior systemic dissemination.

  13. Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

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    Víctor T Contreras

    2002-12-01

    Full Text Available Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU medium at 37°C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.

  14. Leishmania major and Trypanosoma cruzi present distinct DNA damage responses.

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    Garcia, Juliana B F; Rocha, João P Vieira da; Costa-Silva, Héllida M; Alves, Ceres L; Machado, Carlos R; Cruz, Angela K

    2016-05-01

    Leishmania major and Trypanosoma cruzi are medically relevant parasites and interesting model organisms, as they present unique biological processes. Despite increasing data regarding the mechanisms of gene expression regulation, there is little information on how the DNA damage response (DDR) occurs in trypanosomatids. We found that L. major presented a higher radiosensitivity than T. cruzi. L. major showed G1 arrest and displayed high mortality in response to ionizing radiation as a result of the inefficient repair of double-strand breaks (DSBs). Conversely, T. cruzi exhibited arrest in the S/G2 cell cycle phase, was able to efficiently repair DSBs and did not display high rates of cell death after exposure to gamma irradiation. L. major showed higher resistance to alkylating DNA damage, and only L. major was able to promote DNA repair and growth recovery in the presence of MMS. ASF1c overexpression did not interfere with the efficiency of DNA repair in either of the parasites but did accentuate the DNA damage checkpoint response, thereby delaying cell fate after damage. The observed differences in the DNA damage responses of T. cruzi and L. major may originate from the distinct preferred routes of genetic plasticity of the two parasites, i.e., DNA recombination versus amplification. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Diterpenoids from Azorella compacta (Umbelliferae active on Trypanosoma cruzi

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    Araya Jorge E

    2003-01-01

    Full Text Available The anti-Trypanosoma cruzi activity of natural products isolated from Azorella compacta was evaluated, with particular emphasis on their effect against intracellular amastigotes. Five diterpenoids from A. compacta derived from mulinane and azorellane were isolated and identified. Only two products, named azorellanol (Y-2 and mulin-11,3-dien-20-oic acid (Y-5, showed trypanocidal activity against all stages of T. cruzi including intracellular amastigotes. At 10 µM, these compounds displayed a strong lytic activity. It ranged from 88.4 ± 0.6 to 99.0 ± 1 % for all strains and stages evaluate, with an IC50 /18 h values of 20-84 µM and 41-87 µM, respectively. The development of intracellular amastigotes was also inhibited by nearly 60% at 25 µM. The trypanocidal molecules Y-2 and Y-5 did show different degrees of cytotoxicity depending on the cell line tested, with an IC50 /24 h ranging from 33.2 to 161.2 µM. We evaluated the effect of diterpenoids against intracellular T. cruzi forms by immunofluorescent identification of a specific membrane molecular marker (Ssp-4 antigen of the T. cruzi amastigote forms. The accuracy and reproducibility of the measurements were found to be outstanding when examined by confocal microscopy.

  16. Implication of Apoptosis for the Pathogenesis of Trypanosoma cruzi Infection

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    Débora Decote-Ricardo

    2017-05-01

    Full Text Available Apoptosis is induced during the course of immune response to different infectious agents, and the ultimate fate is the recognition and uptake of apoptotic bodies by neighboring cells or by professional phagocytes. Apoptotic cells expose specific ligands to a set of conserved receptors expressed on macrophage cellular surface, which are the main cells involved in the clearance of the dying cells. These scavenger receptors, besides triggering the production of anti-inflammatory factors, also block the production of inflammatory mediators by phagocytes. Experimental infection of mice with the parasite Trypanosoma cruzi shows many pathological changes that parallels the evolution of human infection. Leukocytes undergoing intense apoptotic death are observed during the immune response to T. cruzi in the mouse model of the disease. T. cruzi replicate intensely and secrete molecules with immunomodulatory activities that interfere with T cell-mediated immune responses and secretion of pro-inflammatory cytokine secretion. This mechanism of immune evasion allows the infection to be established in the vertebrate host. Under inflammatory conditions, efferocytosis of apoptotic bodies generates an immune-regulatory phenotype in phagocytes, which is conducive to intracellular pathogen replication. However, the relevance of cellular apoptosis in the pathology of Chagas’ disease requires further studies. Here, we review the evidence of leukocyte apoptosis in T. cruzi infection and its immunomodulatory mechanism for disease progression.

  17. Effects of astaxanthin in mice acutely infected with Trypanosoma cruzi

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    Contreras-Ortiz José María Eloy

    2017-01-01

    Full Text Available During Trypanosoma cruzi infection, oxidative stress is considered a contributing factor for dilated cardiomyopathy development. In this study, the effects of astaxanthin (ASTX were evaluated as an alternative drug treatment for Chagas disease in a mouse model during the acute infection phase, given its anti-inflammatory, immunomodulating, and anti-oxidative properties. ASTX was tested in vitro in parasites grown axenically and in co-culture with Vero cells. In vivo tests were performed in BALB/c mice (4–6 weeks old infected with Trypanosoma cruzi and supplemented with ASTX (10 mg/kg/day and/or nifurtimox (NFMX; 100 mg/kg/day. Results show that ASTX has some detrimental effects on axenically cultured parasites, but not when cultured with mammalian cell monolayers. In vivo, ASTX did not have any therapeutic value against acute Trypanosoma cruzi infection, used either alone or in combination with NFMX. Infected animals treated with NFMX or ASTX/NFMX survived the experimental period (60 days, while infected animals treated only with ASTX died before day 30 post-infection. ASTX did not show any effect on the control of parasitemia; however, it was associated with an increment in focal heart lymphoplasmacytic infiltration, a reduced number of amastigote nests in cardiac tissue, and less hyperplasic spleen follicles when compared to control groups. Unexpectedly, ASTX showed a negative effect in infected animals co-treated with NFMX. An increment in parasitemia duration was observed, possibly due to ASTX blocking of free radicals, an anti-parasitic mechanism of NFMX. In conclusion, astaxanthin is not recommended during the acute phase of Chagas disease, either alone or in combination with nifurtimox.

  18. Antitrypanosomal effect of methanolic extract of Zingiber officinale (ginger on Trypanosoma brucei brucei-infected Wistar mice

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    P. I. Kobo

    2014-10-01

    Full Text Available Aim: The study was carried out to determine the in vivo antitrypanosomal effect of methanolic extract of Zingiber officinale (ginger in Trypanosoma brucei brucei-infected mice. Materials and Methods: Twenty-five mice were randomly allocated into five groups of five animals each. Group I and II were given Tween 80 (1 ml/kg and diminazene aceturate (3.5 mg/kg to serve as untreated and treated controls, respectively. Groups III-V received the extract at 200, 400 and 800 mg/kg body weight, respectively. All treatments were given for 6 consecutive days and through the oral route. The mean body weight, mean survival period and daily level of parasitaemia were evaluated. Results: Acute toxicity showed the extract to be relatively safe. There was an insignificant increase in body weight and survival rate of mice treated with the extract. The level of parasitaemia in the extract treated groups was decreased. Conclusion: This study shows the in vivo potential of methanolic extract of Z. officinale in the treatment of trypanosomiasis.

  19. The Complement System: A Prey of Trypanosoma cruzi

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    Lidani, Kárita C. F.; Bavia, Lorena; Ambrosio, Altair R.; de Messias-Reason, Iara J.

    2017-01-01

    Trypanosoma cruzi is a protozoan parasite known to cause Chagas disease (CD), a neglected sickness that affects around 6–8 million people worldwide. Originally, CD was mainly found in Latin America but more recently, it has been spread to countries in North America, Asia, and Europe due the international migration from endemic areas. Thus, at present CD represents an important concern of global public health. Most of individuals that are infected by T. cruzi may remain in asymptomatic form all lifelong, but up to 40% of them will develop cardiomyopathy, digestive mega syndromes, or both. The interaction between the T. cruzi infective forms and host-related immune factors represents a key point for a better understanding of the physiopathology of CD. In this context, the complement, as one of the first line of host defense against infection was shown to play an important role in recognizing T. cruzi metacyclic trypomastigotes and in controlling parasite invasion. The complement consists of at least 35 or more plasma proteins and cell surface receptors/regulators, which can be activated by three pathways: classical (CP), lectin (LP), and alternative (AP). The CP and LP are mainly initiated by immune complexes or pathogen-associated molecular patterns (PAMPs), respectively, whereas AP is spontaneously activated by hydrolysis of C3. Once activated, several relevant complement functions are generated which include opsonization and phagocytosis of particles or microorganisms and cell lysis. An important step during T. cruzi infection is when intracellular trypomastigotes are release to bloodstream where they may be target by complement. Nevertheless, the parasite uses a sequence of events in order to escape from complement-mediated lysis. In fact, several T. cruzi molecules are known to interfere in the initiation of all three pathways and in the assembly of C3 convertase, a key step in the activation of complement. Moreover, T. cruzi promotes secretion of plasma

  20. Immunization with recombinant beta-tubulin from Trypanosoma evansi induced protection against T. evansi, T. equiperdum and T. b. brucei infection in mice.

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    Li, S-Q; Fung, M-C; Reid, S A; Inoue, N; Lun, Z-R

    2007-04-01

    The beta-tubulin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi beta-tubulin shows 100%, 99.8%, 99.1%, and 98.6% homology with T. equiperdum, T. b. brucei, T. cruzi and T. danilewskyi, respectively, but is diverse from that of T. cyclops, showing only 51.6% of homology. Recombinant beta-tubulin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant beta-tubulin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818 and T. b. brucei STIB 940, showing 83.3%, 70% and 76.7% protection, respectively. Serum collected from the rabbit immunized with recombinant beta-tubulin inhibited the growth of T. evansi, T. equiperdum and T. b. brucei in vitro. Serum from mice and rabbits immunized with recombinant beta-tubulin recognized only T. evansi beta-tubulin and not mouse beta-tubulin. The results of this study demonstrated that the recombinant T. evansi beta-tubulin is a potential candidate for the development of a vaccine to prevent animal trypanosomiasis caused by these three trypanosome species.

  1. The regulation of autophagy differentially affects Trypanosoma cruzi metacyclogenesis.

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    María Cristina Vanrell

    2017-11-01

    Full Text Available Autophagy is a cellular process required for the removal of aged organelles and cytosolic components through lysosomal degradation. All types of eukaryotic cells from yeasts to mammalian cells have the machinery to activate autophagy as a result of many physiological and pathological situations. The most frequent stimulus of autophagy is starvation and the result, in this case, is the fast generation of utilizable food (e.g. amino acids and basic nutrients to maintain the vital biological processes. In some organisms, starvation also triggers other associated processes such as differentiation. The protozoan parasite Trypanosoma cruzi undergoes a series of differentiation processes throughout its complex life cycle. Although not all autophagic genes have been identified in the T. cruzi genome, previous works have demonstrated the presence of essential autophagic-related proteins. Under starvation conditions, TcAtg8, which is the parasite homolog of Atg8/LC3 in other organisms, is located in autophagosome-like vesicles. In this work, we have characterized the autophagic pathway during T. cruzi differentiation from the epimastigote to metacyclic trypomastigote form, a process called metacyclogenesis. We demonstrated that autophagy is stimulated during metacyclogenesis and that the induction of autophagy promotes this process. Moreover, with exception of bafilomycin, other classical autophagy modulators have similar effects on T. cruzi autophagy. We also showed that spermidine and related polyamines can positively regulate parasite autophagy and differentiation. We concluded that both polyamine metabolism and autophagy are key processes during T. cruzi metacyclogenesis that could be exploited as drug targets to avoid the parasite cycle progression.

  2. Trypanosoma cruzi maxicircle heterogeneity in Chagas disease patients from Brazil.

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    Carranza, Julio César; Valadares, Helder M S; D'Avila, Daniella A; Baptista, Rodrigo P; Moreno, Margoth; Galvão, Lúcia M C; Chiari, Egler; Sturm, Nancy R; Gontijo, Eliane D; Macedo, Andrea M; Zingales, Bianca

    2009-07-15

    The majority of individuals in the chronic phase of Chagas disease are asymptomatic (indeterminate form, IF). Each year, approximately 3% of them develop lesions in the heart or gastrointestinal tract. Cardiomyopathy (CCHD) is the most severe manifestation of Chagas disease. The factors that determine the outcome of the infection are unknown, but certainly depend on complex interactions amongst the genetic make-up of the parasite, the host immunogenetic background and environment. In a previous study we verified that the maxicircle gene NADH dehydrogenase (mitochondrial complex I) subunit 7 (ND7) from IF isolates had a 455 bp deletion compared with the wild type (WT) ND7 gene from CCHD strains. We proposed that ND7 could constitute a valuable target for PCR assays in the differential diagnosis of the infective strain. In the present study we evaluated this hypothesis by examination of ND7 structure in parasites from 75 patients with defined pathologies, from Southeast Brazil. We also analysed the structure of additional mitochondrial genes (ND4/CR4, COIII and COII) since the maxicircle is used for clustering Trypanosoma cruzi strains into three clades/haplogroups. We conclude that maxicircle genes do not discriminate parasite populations which induce IF or CCHD forms. Interestingly, the great majority of the analysed isolates belong to T. cruzi II (discrete typing unit, (DTU) IIb) genotype. This scenario is at variance with the prevalence of hybrid (DTU IId) human isolates in Bolivia, Chile and Argentina. The distribution of WT and deleted ND7 and ND4 genes in T. cruzi strains suggests that mutations in the two genes occurred in different ancestrals in the T. cruzi II cluster, allowing the identification of at least three mitochondrial sub-lineages within this group. The observation that T. cruzi strains accumulate mutations in several genes coding for complex I subunits favours the hypothesis that complex I may have a limited activity in this parasite.

  3. Targeted Screening Strategies to Detect Trypanosoma cruzi Infection in Children

    Science.gov (United States)

    Levy, Michael Z.; Kawai, Vivian; Bowman, Natalie M.; Waller, Lance A.; Cabrera, Lilia; Pinedo-Cancino, Viviana V.; Seitz, Amy E.; Steurer, Frank J.; Cornejo del Carpio, Juan G.; Cordova-Benzaquen, Eleazar; Maguire, James H.; Gilman, Robert H.; Bern, Caryn

    2007-01-01

    Background Millions of people are infected with Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. Anti-trypanosomal drug therapy can cure infected individuals, but treatment efficacy is highest early in infection. Vector control campaigns disrupt transmission of T. cruzi, but without timely diagnosis, children infected prior to vector control often miss the window of opportunity for effective chemotherapy. Methods and Findings We performed a serological survey in children 2–18 years old living in a peri-urban community of Arequipa, Peru, and linked the results to entomologic, spatial and census data gathered during a vector control campaign. 23 of 433 (5.3% [95% CI 3.4–7.9]) children were confirmed seropositive for T. cruzi infection by two methods. Spatial analysis revealed that households with infected children were very tightly clustered within looser clusters of households with parasite-infected vectors. Bayesian hierarchical mixed models, which controlled for clustering of infection, showed that a child's risk of being seropositive increased by 20% per year of age and 4% per vector captured within the child's house. Receiver operator characteristic (ROC) plots of best-fit models suggest that more than 83% of infected children could be identified while testing only 22% of eligible children. Conclusions We found evidence of spatially-focal vector-borne T. cruzi transmission in peri-urban Arequipa. Ongoing vector control campaigns, in addition to preventing further parasite transmission, facilitate the collection of data essential to identifying children at high risk of T. cruzi infection. Targeted screening strategies could make integration of diagnosis and treatment of children into Chagas disease control programs feasible in lower-resource settings. PMID:18160979

  4. The regulation of autophagy differentially affects Trypanosoma cruzi metacyclogenesis.

    Science.gov (United States)

    Vanrell, María Cristina; Losinno, Antonella Denisse; Cueto, Juan Agustín; Balcazar, Darío; Fraccaroli, Laura Virginia; Carrillo, Carolina; Romano, Patricia Silvia

    2017-11-01

    Autophagy is a cellular process required for the removal of aged organelles and cytosolic components through lysosomal degradation. All types of eukaryotic cells from yeasts to mammalian cells have the machinery to activate autophagy as a result of many physiological and pathological situations. The most frequent stimulus of autophagy is starvation and the result, in this case, is the fast generation of utilizable food (e.g. amino acids and basic nutrients) to maintain the vital biological processes. In some organisms, starvation also triggers other associated processes such as differentiation. The protozoan parasite Trypanosoma cruzi undergoes a series of differentiation processes throughout its complex life cycle. Although not all autophagic genes have been identified in the T. cruzi genome, previous works have demonstrated the presence of essential autophagic-related proteins. Under starvation conditions, TcAtg8, which is the parasite homolog of Atg8/LC3 in other organisms, is located in autophagosome-like vesicles. In this work, we have characterized the autophagic pathway during T. cruzi differentiation from the epimastigote to metacyclic trypomastigote form, a process called metacyclogenesis. We demonstrated that autophagy is stimulated during metacyclogenesis and that the induction of autophagy promotes this process. Moreover, with exception of bafilomycin, other classical autophagy modulators have similar effects on T. cruzi autophagy. We also showed that spermidine and related polyamines can positively regulate parasite autophagy and differentiation. We concluded that both polyamine metabolism and autophagy are key processes during T. cruzi metacyclogenesis that could be exploited as drug targets to avoid the parasite cycle progression.

  5. Benznidazole induces in vitro anaerobic metabolism in Trypanosoma cruzi epimastigotes

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    Marina Clare Vinaud

    2017-11-01

    Full Text Available Objective: To determine the biochemical alterations of the energetic metabolism of Trypanosoma cruzi epimastigotes in vitro exposed to different concentrations of benzinidazole. Methods: Biochemical analyses were performed at 3, 6 (log phase, 9 and 12 (stationary phase days of culture. Parasites were exposed to five concentrations of benzinidazole. Glycolysis, tricarboxilic acid cycle and fatty acids oxidation pathways were quantified through chromatography. Glucose, urea and creatinine were quantified through spectrophotometric analysis. Results: Anaerobic fermentation and fatty acids oxidation were increased in the stationary phase of the culture. Benzinidazole at high concentrations induced anaerobic metabolism in the log phase of the culture while the parasites exposed to the lower concentrations preferred the citric acid cycle as energy production pathway. Benzinidazole did not influence on the proteins catabolism. Conclusions: It is possible to conclude that there are metabolic differences between evolutive forms of Trypanosoma cruzi and the main drug used for its treatment induces the anaerobic metabolism in the parasite, possibly impairing the mitochondrial pathways.

  6. Megazol and its bioisostere 4H-1,2,4-triazole: comparing the trypanocidal, cytotoxic and genotoxic activities and their in vitro and in silico interactions with the Trypanosoma brucei nitroreductase enzyme

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    Alcione Silva de Carvalho

    2014-06-01

    Full Text Available Megazol (7 is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8 in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7 for nitrogen (in the triazole in 8, the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7.

  7. The Trypanosoma cruzi nucleic acid binding protein Tc38 presents changes in the intramitochondrial distribution during the cell cycle

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    Nardelli Sheila C

    2009-02-01

    Full Text Available Abstract Background Tc38 of Trypanosoma cruzi has been isolated as a single stranded DNA binding protein with high specificity for the poly [dT-dG] sequence. It is present only in Kinetoplastidae protozoa and its sequence lacks homology to known functional domains. Tc38 orthologues present in Trypanosoma brucei and Leishmania were proposed to participate in quite different cellular processes. To further understand the function of this protein in Trypanosoma cruzi, we examined its in vitro binding to biologically relevant [dT-dG] enriched sequences, its expression and subcellular localization during the cell cycle and through the parasite life stages. Results By using specific antibodies, we found that Tc38 protein from epimastigote extracts participates in complexes with the poly [dT-dG] probe as well as with the universal minicircle sequence (UMS, a related repeated sequence found in maxicircle DNA, and the telomeric repeat. However, we found that Tc38 predominantly localizes into the mitochondrion. Though Tc38 is constitutively expressed through non-replicating and replicating life stages of T. cruzi, its subcellular localization in the unique parasite mitochondrion changes according to the cell cycle stage. In epimastigotes, Tc38 is found only in association with kDNA in G1 phase. From the S to G2 phase the protein localizes in two defined and connected spots flanking the kDNA. These spots disappear in late G2 turning into a diffuse dotted signal which extends beyond the kinetoplast. This later pattern is more evident in mitosis and cytokinesis. Finally, late in cytokinesis Tc38 reacquires its association with the kinetoplast. In non-replicating parasite stages such as trypomastigotes, the protein is found only surrounding the entire kinetoplast structure. Conclusions The dynamics of Tc38 subcellular localization observed during the cell cycle and life stages support a major role for Tc38 related to kDNA replication and maintenance.

  8. Nitric oxide-releasing polymeric nanoparticles against Trypanosoma cruzi

    Science.gov (United States)

    Seabra, A. B.; Kitice, N. A.; Pelegrino, M. T.; Lancheros, C. A. C.; Yamauchi, L. M.; Pinge-Filho, P.; Yamada-Ogatta, S. F.

    2015-05-01

    Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite, Trypanosoma cruzi (T. cruzi), and the disease remains a major health problem in many Latin American countries. Several papers report that the killing of the parasite is dependent on the production of nitric oxide (NO). The endogenous free radical NO is an important cellular signalling molecule that plays a key role in the defense against pathogens, including T. cruzi. As T. cruzi is able to compromise host macrophages decreasing endogenous NO production, the administration of exogenous NO donors represents an interesting strategy to combat Chagas disease. Thus, the aims of this study were to prepare and evaluate the antimicrobial activity of NO-releasing polymeric nanoparticles against T. cruzi. Biocompatible polymeric nanoparticles composed of chitosan/sodium tripolyphosphate(TPP) were prepared and used to encapsulate mercaptosuccinic acid (MSA), which is a thiol-containing molecule. Nitrosation of free thiols (SH) groups of MSA were performed by the addition of equimolar amount of sodium nitrite (NaNO2), leading to the formation of S-nitroso-MSA-containing nanoparticles. These polymeric nanoparticles act as spontaneous NO donors, with free NO release. The results show the formation of nanoparticles with average hydrodynamic diameter ranging from 270 to 500 nm, average of polydispersity index of 0.35, and encapsulation efficiency in the range of 99%. The NO release kinetics from the S-nitroso-MSA-containing nanoparticles showed sustained and controlled NO release over several hours. The microbicidal activity of S-nitroso-MSA-containing nanoparticles was evaluated by incubating NO-releasing nanoparticles (200 - 600 μg/mL) with replicative and non-infective epimastigote, and non-replicative and infective trypomastigote forms of T. cruzi. In addition, a significant decrease in the percentage of macrophage-infected (with amastigotes) and

  9. Biosynthesis of SUMOylated Proteins in Bacteria Using the Trypanosoma brucei Enzymatic System

    Science.gov (United States)

    Iribarren, Paula Ana; Berazategui, María Agustina; Cazzulo, Juan José; Alvarez, Vanina Eder

    2015-01-01

    Post-translational modification with the Small Ubiquitin-like Modifier (SUMO) is conserved in eukaryotic organisms and plays important regulatory roles in proteins affecting diverse cellular processes. In Trypanosoma brucei, member of one of the earliest branches in eukaryotic evolution, SUMO is essential for normal cell cycle progression and is likely to be involved in the epigenetic control of genes crucial for parasite survival, such as those encoding the variant surface glycoproteins. Molecular pathways modulated by SUMO have started to be discovered by proteomic studies; however, characterization of functional consequences is limited to a reduced number of targets. Here we present a bacterial strain engineered to produce SUMOylated proteins, by transferring SUMO from T. brucei together with the enzymes essential for its activation and conjugation. Due to the lack of background in E. coli, this system is useful to express and identify SUMOylated proteins directly in cell lysates by immunoblotting, and SUMOylated targets can be eventually purified for biochemical or structural studies. We applied this strategy to describe the ability of TbSUMO to form chains in vitro and to detect SUMOylation of a model substrate, PCNA both from Saccharomyces cerevisiae and from T. brucei. To further validate targets, we applied an in vitro deconjugation assay using the T. brucei SUMO-specific protease capable to revert the pattern of modification. This system represents a valuable tool for target validation, mutant generation and functional studies of SUMOylated proteins in trypanosomatids. PMID:26258470

  10. Phosphatidylinositol 4-kinase III-beta is required for Golgi maintenance and cytokinesis in Trypanosoma brucei.

    Science.gov (United States)

    Rodgers, Melissa J; Albanesi, Joseph P; Phillips, Margaret A

    2007-07-01

    The parasitic protozoan Trypanosoma brucei contains two type III phosphatidylinositol 4-kinases (alpha and beta). We have cloned the gene encoding the T. brucei type III phosphatidylinositol 4-kinase beta (TbPI4KIII-beta), expressed the protein in COS-7 cells, and confirmed that the protein catalyzes the phosphorylation of phosphatidylinositol. Depletion of TbPI4KIII-beta in procyclic T. brucei by RNA interference (RNAi) resulted in inhibition of cell growth and a distorted cellular morphology. RNAi cells had a distorted Golgi apparatus, and lysosomal and flagellar pocket proteins were mislocalized. Ultrastructural analysis revealed the internal accumulation of a heterogeneous population of vesicles, abnormal positioning of organelles, and a loss of cell polarity. Scanning electron microcopy revealed a twisted phenotype, and dividing cells often exhibited a detached daughter flagellum and lacked a cleavage furrow. Cell cycle analysis confirmed that cells depleted of TbPI4KIII-beta have a postmitotic cytokinesis block that occurs after a single round of mitosis, suggestive of a specific cell cycle block. In summary, TbPI4KIII-beta is an essential protein in procyclic T. brucei, required for maintenance of Golgi structure, protein trafficking, normal cellular shape, and cytokinesis.

  11. The Flagellar Arginine Kinase in Trypanosoma brucei Is Important for Infection in Tsetse Flies.

    Directory of Open Access Journals (Sweden)

    Cher-Pheng Ooi

    Full Text Available African trypanosomes are flagellated parasites that cause sleeping sickness. Parasites are transmitted from one mammalian host to another by the bite of a tsetse fly. Trypanosoma brucei possesses three different genes for arginine kinase (AK including one (AK3 that encodes a protein localised to the flagellum. AK3 is characterised by the presence of a unique amino-terminal insertion that specifies flagellar targeting. We show here a phylogenetic analysis revealing that flagellar AK arose in two independent duplication events in T. brucei and T. congolense, the two species of African trypanosomes that infect the tsetse midgut. In T. brucei, AK3 is detected in all stages of parasite development in the fly (in the midgut and in the salivary glands as well as in bloodstream cells, but with predominance at insect stages. Genetic knockout leads to a slight reduction in motility and impairs parasite infectivity towards tsetse flies in single and competition experiments, both phenotypes being reverted upon expression of an epitope-tagged version of AK3. We speculate that this flagellar arginine kinase is important for T. brucei infection of tsetse, especially in the context of mixed infections and that its flagellar targeting relies on a system equivalent to that discovered for calflagins, a family of trypanosome flagellum calcium binding proteins.

  12. The Flagellar Arginine Kinase in Trypanosoma brucei Is Important for Infection in Tsetse Flies.

    Science.gov (United States)

    Ooi, Cher-Pheng; Rotureau, Brice; Gribaldo, Simonetta; Georgikou, Christina; Julkowska, Daria; Blisnick, Thierry; Perrot, Sylvie; Subota, Ines; Bastin, Philippe

    2015-01-01

    African trypanosomes are flagellated parasites that cause sleeping sickness. Parasites are transmitted from one mammalian host to another by the bite of a tsetse fly. Trypanosoma brucei possesses three different genes for arginine kinase (AK) including one (AK3) that encodes a protein localised to the flagellum. AK3 is characterised by the presence of a unique amino-terminal insertion that specifies flagellar targeting. We show here a phylogenetic analysis revealing that flagellar AK arose in two independent duplication events in T. brucei and T. congolense, the two species of African trypanosomes that infect the tsetse midgut. In T. brucei, AK3 is detected in all stages of parasite development in the fly (in the midgut and in the salivary glands) as well as in bloodstream cells, but with predominance at insect stages. Genetic knockout leads to a slight reduction in motility and impairs parasite infectivity towards tsetse flies in single and competition experiments, both phenotypes being reverted upon expression of an epitope-tagged version of AK3. We speculate that this flagellar arginine kinase is important for T. brucei infection of tsetse, especially in the context of mixed infections and that its flagellar targeting relies on a system equivalent to that discovered for calflagins, a family of trypanosome flagellum calcium binding proteins.

  13. Maf1 is a negative regulator of transcription in Trypanosoma brucei.

    Science.gov (United States)

    Romero-Meza, Gabriela; Vélez-Ramírez, Daniel E; Florencio-Martínez, Luis E; Román-Carraro, Fiordaliso C; Manning-Cela, Rebeca; Hernández-Rivas, Rosaura; Martínez-Calvillo, Santiago

    2017-02-01

    RNA polymerase III (Pol III) produces small RNA molecules that play essential roles in mRNA processing and translation. Maf1, originally described as a negative regulator of Pol III transcription, has been studied from yeast to human. Here we characterized Maf1 in the parasitic protozoa Trypanosoma brucei (TbMaf1), representing the first report to analyse Maf1 in an early-diverged eukaryote. While Maf1 is generally encoded by a single-copy gene, the T. brucei genome contains two almost identical TbMaf1 genes. The TbMaf1 protein has the three conserved sequences and is predicted to fold into a globular structure. Unlike in yeast, TbMaf1 localizes to the nucleus in procyclic forms of T. brucei under normal growth conditions. Cell lines that either downregulate or overexpress TbMaf1 were generated, and growth curve analysis with them suggested that TbMaf1 participates in the regulation of cell growth of T. brucei. Nuclear run-on and chromatin immunoprecipitation analyses demonstrated that TbMaf1 represses Pol III transcription of tRNA and U2 snRNA genes by associating with their promoters. Interestingly, 5S rRNA levels do not change after TbMaf1 ablation or overexpression. Notably, our data also revealed that TbMaf1 regulates Pol I transcription of procyclin gene and Pol II transcription of SL RNA genes. © 2016 John Wiley & Sons Ltd.

  14. Aquaglyceroporins Are the Entry Pathway of Boric Acid in Trypanosoma brucei.

    Science.gov (United States)

    Marsiccobetre, Sabrina; Rodríguez-Acosta, Alexis; Lang, Florian; Figarella, Katherine; Uzcátegui, Néstor L

    2017-05-01

    The boron element possesses a range of different effects on living beings. It is essential to beneficial at low concentrations, but toxic at excessive concentrations. Recently, some boron-based compounds have been identified as promising molecules against Trypanosoma brucei, the causative agent of sleeping sickness. However, until now, the boron metabolism and its access route into the parasite remained elusive. The present study addressed the permeability of T. brucei aquaglyceroporins (TbAQPs) for boric acid, the main natural boron species. To this end, the three TbAQPs were expressed in Saccharomyces cerevisiae and Xenopus laevis oocytes. Our findings in both expression systems showed that all three TbAQPs are permeable for boric acid. Especially TbAQP2 is highly permeable for this compound, displaying one of the highest conductances reported for a solute in these channels. Additionally, T. brucei aquaglyceroporin activities were sensitive to pH. Taken together, these results establish that TbAQPs are channels for boric acid and are highly efficient entry pathways for boron into the parasite. Our findings stress the importance of studying the physiological functions of boron and their derivatives in T. brucei, as well as the pharmacological implications of their uptake by trypanosome aquaglyceroporins. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Biosynthesis of SUMOylated Proteins in Bacteria Using the Trypanosoma brucei Enzymatic System.

    Directory of Open Access Journals (Sweden)

    Paula Ana Iribarren

    Full Text Available Post-translational modification with the Small Ubiquitin-like Modifier (SUMO is conserved in eukaryotic organisms and plays important regulatory roles in proteins affecting diverse cellular processes. In Trypanosoma brucei, member of one of the earliest branches in eukaryotic evolution, SUMO is essential for normal cell cycle progression and is likely to be involved in the epigenetic control of genes crucial for parasite survival, such as those encoding the variant surface glycoproteins. Molecular pathways modulated by SUMO have started to be discovered by proteomic studies; however, characterization of functional consequences is limited to a reduced number of targets. Here we present a bacterial strain engineered to produce SUMOylated proteins, by transferring SUMO from T. brucei together with the enzymes essential for its activation and conjugation. Due to the lack of background in E. coli, this system is useful to express and identify SUMOylated proteins directly in cell lysates by immunoblotting, and SUMOylated targets can be eventually purified for biochemical or structural studies. We applied this strategy to describe the ability of TbSUMO to form chains in vitro and to detect SUMOylation of a model substrate, PCNA both from Saccharomyces cerevisiae and from T. brucei. To further validate targets, we applied an in vitro deconjugation assay using the T. brucei SUMO-specific protease capable to revert the pattern of modification. This system represents a valuable tool for target validation, mutant generation and functional studies of SUMOylated proteins in trypanosomatids.

  16. Trypanosoma cruzi infection in neotropical wild carnivores (Mammalia: Carnivora): at the top of the T. cruzi transmission chain.

    Science.gov (United States)

    Rocha, Fabiana Lopes; Roque, André Luiz Rodrigues; de Lima, Juliane Saab; Cheida, Carolina Carvalho; Lemos, Frederico Gemesio; de Azevedo, Fernanda Cavalcanti; Arrais, Ricardo Corassa; Bilac, Daniele; Herrera, Heitor Miraglia; Mourão, Guilherme; Jansen, Ana Maria

    2013-01-01

    Little is known on the role played by Neotropical wild carnivores in the Trypanosoma cruzi transmission cycles. We investigated T. cruzi infection in wild carnivores from three sites in Brazil through parasitological and serological tests. The seven carnivore species examined were infected by T. cruzi, but high parasitemias detectable by hemoculture were found only in two Procyonidae species. Genotyping by Mini-exon gene, PCR-RFLP (1f8/Akw21I) and kDNA genomic targets revealed that the raccoon (Procyon cancrivorus) harbored TcI and the coatis (Nasua nasua) harbored TcI, TcII, TcIII-IV and Trypanosoma rangeli, in single and mixed infections, besides four T. cruzi isolates that displayed odd band patterns in the Mini-exon assay. These findings corroborate the coati can be a bioaccumulator of T. cruzi Discrete Typing Units (DTU) and may act as a transmission hub, a connection point joining sylvatic transmission cycles within terrestrial and arboreal mammals and vectors. Also, the odd band patterns observed in coatis' isolates reinforce that T. cruzi diversity might be much higher than currently acknowledged. Additionally, we assembled our data with T. cruzi infection on Neotropical carnivores' literature records to provide a comprehensive analysis of the infection patterns among distinct carnivore species, especially considering their ecological traits and phylogeny. Altogether, fifteen Neotropical carnivore species were found naturally infected by T. cruzi. Species diet was associated with T. cruzi infection rates, supporting the hypothesis that predator-prey links are important mechanisms for T. cruzi maintenance and dispersion in the wild. Distinct T. cruzi infection patterns across carnivore species and study sites were notable. Musteloidea species consistently exhibit high parasitemias in different studies which indicate their high infectivity potential. Mesocarnivores that feed on both invertebrates and mammals, including the coati, a host that can be

  17. Trypanosoma cruzi Infection in Neotropical Wild Carnivores (Mammalia: Carnivora): At the Top of the T. cruzi Transmission Chain

    Science.gov (United States)

    Rocha, Fabiana Lopes; Roque, André Luiz Rodrigues; de Lima, Juliane Saab; Cheida, Carolina Carvalho; Lemos, Frederico Gemesio; de Azevedo, Fernanda Cavalcanti; Arrais, Ricardo Corassa; Bilac, Daniele; Herrera, Heitor Miraglia; Mourão, Guilherme; Jansen, Ana Maria

    2013-01-01

    Little is known on the role played by Neotropical wild carnivores in the Trypanosoma cruzi transmission cycles. We investigated T. cruzi infection in wild carnivores from three sites in Brazil through parasitological and serological tests. The seven carnivore species examined were infected by T. cruzi, but high parasitemias detectable by hemoculture were found only in two Procyonidae species. Genotyping by Mini-exon gene, PCR-RFLP (1f8/Akw21I) and kDNA genomic targets revealed that the raccoon (Procyon cancrivorus) harbored TcI and the coatis (Nasua nasua) harbored TcI, TcII, TcIII-IV and Trypanosoma rangeli, in single and mixed infections, besides four T. cruzi isolates that displayed odd band patterns in the Mini-exon assay. These findings corroborate the coati can be a bioaccumulator of T. cruzi Discrete Typing Units (DTU) and may act as a transmission hub, a connection point joining sylvatic transmission cycles within terrestrial and arboreal mammals and vectors. Also, the odd band patterns observed in coatis’ isolates reinforce that T. cruzi diversity might be much higher than currently acknowledged. Additionally, we assembled our data with T. cruzi infection on Neotropical carnivores’ literature records to provide a comprehensive analysis of the infection patterns among distinct carnivore species, especially considering their ecological traits and phylogeny. Altogether, fifteen Neotropical carnivore species were found naturally infected by T. cruzi. Species diet was associated with T. cruzi infection rates, supporting the hypothesis that predator-prey links are important mechanisms for T. cruzi maintenance and dispersion in the wild. Distinct T. cruzi infection patterns across carnivore species and study sites were notable. Musteloidea species consistently exhibit high parasitemias in different studies which indicate their high infectivity potential. Mesocarnivores that feed on both invertebrates and mammals, including the coati, a host that can be

  18. Trypanosoma cruzi infection in neotropical wild carnivores (Mammalia: Carnivora: at the top of the T. cruzi transmission chain.

    Directory of Open Access Journals (Sweden)

    Fabiana Lopes Rocha

    Full Text Available Little is known on the role played by Neotropical wild carnivores in the Trypanosoma cruzi transmission cycles. We investigated T. cruzi infection in wild carnivores from three sites in Brazil through parasitological and serological tests. The seven carnivore species examined were infected by T. cruzi, but high parasitemias detectable by hemoculture were found only in two Procyonidae species. Genotyping by Mini-exon gene, PCR-RFLP (1f8/Akw21I and kDNA genomic targets revealed that the raccoon (Procyon cancrivorus harbored TcI and the coatis (Nasua nasua harbored TcI, TcII, TcIII-IV and Trypanosoma rangeli, in single and mixed infections, besides four T. cruzi isolates that displayed odd band patterns in the Mini-exon assay. These findings corroborate the coati can be a bioaccumulator of T. cruzi Discrete Typing Units (DTU and may act as a transmission hub, a connection point joining sylvatic transmission cycles within terrestrial and arboreal mammals and vectors. Also, the odd band patterns observed in coatis' isolates reinforce that T. cruzi diversity might be much higher than currently acknowledged. Additionally, we assembled our data with T. cruzi infection on Neotropical carnivores' literature records to provide a comprehensive analysis of the infection patterns among distinct carnivore species, especially considering their ecological traits and phylogeny. Altogether, fifteen Neotropical carnivore species were found naturally infected by T. cruzi. Species diet was associated with T. cruzi infection rates, supporting the hypothesis that predator-prey links are important mechanisms for T. cruzi maintenance and dispersion in the wild. Distinct T. cruzi infection patterns across carnivore species and study sites were notable. Musteloidea species consistently exhibit high parasitemias in different studies which indicate their high infectivity potential. Mesocarnivores that feed on both invertebrates and mammals, including the coati, a host that

  19. Heterogeneous infectiousness in guinea pigs experimentally infected with Trypanosoma cruzi.

    Science.gov (United States)

    Castillo-Neyra, Ricardo; Borrini Mayorí, Katty; Salazar Sánchez, Renzo; Ancca Suarez, Jenny; Xie, Sherrie; Náquira Velarde, Cesar; Levy, Michael Z

    2016-02-01

    Guinea pigs are important reservoirs of Trypanosoma cruzi, the causative parasite of Chagas disease, and in the Southern Cone of South America, transmission is mediated mainly by the vector Triatoma infestans. Interestingly, colonies of Triatoma infestans captured from guinea pig corrals sporadically have infection prevalence rates above 80%. Such high values are not consistent with the relatively short 7-8 week parasitemic period that has been reported for guinea pigs in the literature. We experimentally measured the infectious periods of a group of T. cruzi-infected guinea pigs by performing xenodiagnosis and direct microscopy each week for one year. Another group of infected guinea pigs received only direct microscopy to control for the effect that inoculation by triatomine saliva may have on parasitemia in the host. We observed infectious periods longer than those previously reported in a number of guinea pigs from both the xenodiagnosis and control groups. While some guinea pigs were infectious for a short time, other "super-shedders" were parasitemic up to 22 weeks after infection, and/or positive by xenodiagnosis for a year after infection. This heterogeneity in infectiousness has strong implications for T. cruzi transmission dynamics and control, as super-shedder guinea pigs may play a disproportionate role in pathogen spread. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  20. Trypanosoma cruzi strain TcIV infects raccoons from Illinois

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    Cailey Vandermark

    Full Text Available BACKGROUND The northern limits of Trypanosoma cruzi across the territory of the United States remain unknown. The known vectors Triatoma sanguisuga and T. lecticularia find their northernmost limits in Illinois; yet, earlier screenings of those insects did not reveal the presence of the pathogen, which has not been reported in vectors or reservoir hosts in this state. OBJECTIVES Five species of medium-sized mammals were screened for the presence of T. cruzi. METHODS Genomic DNA was isolated from heart, spleen and skeletal muscle of bobcats (Lynx rufus, n = 60, raccoons (Procyon lotor, n = 37, nine-banded armadillos (Dasypus novemcinctus, n = 5, Virginia opossums (Didelphis virginiana, n = 3, and a red fox (Vulpes vulpes. Infections were detected targeting DNA from the kinetoplast DNA minicircle (kDNA and satellite DNA (satDNA. The discrete typing unit (DTU was determined by amplifying two gene regions: the Spliced Leader Intergenic Region (SL, via a multiplex polymerase chain reaction, and the 24Sα ribosomal DNA via a heminested reaction. Resulting sequences were used to calculate their genetic distance against reference DTUs. FINDINGS 18.9% of raccoons were positive for strain TcIV; the rest of mammals tested negative. MAIN CONCLUSIONS These results confirm for the first time the presence of T. cruzi in wildlife from Illinois, suggesting that a sylvatic life cycle is likely to occur in the region. The analyses of sequences of SL suggest that amplicons resulting from a commonly used multiplex reaction may yield non-homologous fragments.

  1. Geographical Distribution of Trypanosoma cruzi Genotypes in Venezuela

    Science.gov (United States)

    Carrasco, Hernán J.; Segovia, Maikell; Llewellyn, Martin S.; Morocoima, Antonio; Urdaneta-Morales, Servio; Martínez, Cinda; Martínez, Clara E.; Garcia, Carlos; Rodríguez, Marlenes; Espinosa, Raul; de Noya, Belkisyolé A.; Díaz-Bello, Zoraida; Herrera, Leidi; Fitzpatrick, Sinead; Yeo, Matthew; Miles, Michael A.; Feliciangeli, M. Dora

    2012-01-01

    Chagas disease is an endemic zoonosis native to the Americas and is caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The parasite is also highly genetically diverse, with six discrete typing units (DTUs) reported TcI – TcVI. These DTUs broadly correlate with several epidemiogical, ecological and pathological features of Chagas disease. In this manuscript we report the most comprehensive evaluation to date of the genetic diversity of T. cruzi in Venezuela. The dataset includes 778 samples collected and genotyped over the last twelve years from multiple hosts and vectors, including nine wild and domestic mammalian host species, and seven species of triatomine bug, as well as from human sources. Most isolates (732) can be assigned to the TcI clade (94.1%); 24 to the TcIV group (3.1%) and 22 to TcIII (2.8%). Importantly, among the 95 isolates genotyped from human disease cases, 79% belonged to TcI - a DTU common in the Americas, however, 21% belonged to TcIV- a little known genotype previously thought to be rare in humans. Furthermore, were able to assign multiple oral Chagas diseases cases to TcI in the area around the capital, Caracas. We discuss our findings in the context of T. cruzi DTU distributions elsewhere in the Americas, and evaluate the impact they have on the future of Chagas disease control in Venezuela. PMID:22745843

  2. High throughput selection of effective serodiagnostics for Trypanosoma cruzi infection.

    Directory of Open Access Journals (Sweden)

    Gretchen Cooley

    2008-10-01

    Full Text Available Diagnosis of Trypanosoma cruzi infection by direct pathogen detection is complicated by the low parasite burden in subjects persistently infected with this agent of human Chagas disease. Determination of infection status by serological analysis has also been faulty, largely due to the lack of well-characterized parasite reagents for the detection of anti-parasite antibodies.In this study, we screened more than 400 recombinant proteins of T. cruzi, including randomly selected and those known to be highly expressed in the parasite stages present in mammalian hosts, for the ability to detect anti-parasite antibodies in the sera of subjects with confirmed or suspected T. cruzi infection.A set of 16 protein groups were identified and incorporated into a multiplex bead array format which detected 100% of >100 confirmed positive sera and also documented consistent, strong and broad responses in samples undetected or discordant using conventional serologic tests. Each serum had a distinct but highly stable reaction pattern. This diagnostic panel was also useful for monitoring drug treatment efficacy in chronic Chagas disease.These results substantially extend the variety and quality of diagnostic targets for Chagas disease and offer a useful tool for determining treatment success or failure.

  3. Imidazolium compounds are active against all stages of Trypanosoma cruzi.

    Science.gov (United States)

    Faral-Tello, Paula; Liang, Mary; Mahler, Graciela; Wipf, Peter; Robello, Carlos

    2014-03-01

    Imidazolium salts are best known for their applications in organic synthesis as room-temperature ionic liquids, or as precursors of stable carbenes, but they also show important biological properties such as anti-oxidative effects, induction of mitochondrial membrane permeabilisation and inhibition of the infection cycle of Plasmodium falciparum. For these reasons, and since chemotherapy for Chagas disease is inefficient, the aim of this study was to test the use of imidazolium compounds against the kinetoplastid haemoflagellate aetiological agent for this disease, namely Trypanosoma cruzi. The results show that five of the tested compounds are more effective than the reference drug benznidazole against the epimastigote and trypomastigote forms of T. cruzi. Moreover, intracellular amastigotes were also affected by the compounds, which showed lower toxicity in host cells. Transmission electron microscopy analysis demonstrated that the tested agents induced alterations of the kinetoplast and particularly of the mitochondria, leading to extraordinary swelling of the organelle. These results further demonstrate that the test agents with the best profile are those bearing symmetrical bulky substituents at N(1) and N(3), displaying promising activity against all forms of T. cruzi, interesting selectivity indexes and exceptional activity at low doses. Accordingly, these agents represent promising candidates for the treatment of Chagas disease. Copyright © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  4. Synthesis and anti-Trypanosoma cruzi activity of diaryldiazepines.

    Science.gov (United States)

    Menezes, Júlio César L; Vaz, Luana Beatriz A; de Abreu Vieira, Paula Melo; da Silva Fonseca, Kátia; Carneiro, Cláudia Martins; Taylor, Jason G

    2014-12-23

    Chagas disease is a so-called "neglected disease" and endemic to Latin America. Nifurtimox and benznidazole are drugs that have considerable efficacy in the treatment of the acute phase of the disease but cause many significant side effects. Furthermore, in the Chronic Phase its efficiency is reduced and their therapeutic effectiveness is dependent on the type of T. cruzi strain. For this reason, the present work aims to drive basic research towards the discovery of new chemical entities to treat Chagas disease. Differently substituted 5,7-diaryl-2,3-dihydro-1,4-diazepines were synthesized by cyclocondensation of substituted flavones with ethylenediamine and tested as anti-Trypanosoma cruzi candidates. Epimastigotes of the Y strain from T. cruzi were used in this study and the number of parasites was determined in a Neubauer chamber. The most potent diaryldiazepine that reduced epimastigote proliferation exhibited an IC50 value of 0.25 μM, which is significantly more active than benznidazole.

  5. Synthesis and Anti-Trypanosoma cruzi Activity of Diaryldiazepines

    Directory of Open Access Journals (Sweden)

    Júlio César L. Menezes

    2014-12-01

    Full Text Available Chagas disease is a so-called “neglected disease” and endemic to Latin America. Nifurtimox and benznidazole are drugs that have considerable efficacy in the treatment of the acute phase of the disease but cause many significant side effects. Furthermore, in the Chronic Phase its efficiency is reduced and their therapeutic effectiveness is dependent on the type of T. cruzi strain. For this reason, the present work aims to drive basic research towards the discovery of new chemical entities to treat Chagas disease. Differently substituted 5,7-diaryl-2,3-dihydro-1,4-diazepines were synthesized by cyclocondensation of substituted flavones with ethylenediamine and tested as anti-Trypanosoma cruzi candidates. Epimastigotes of the Y strain from T. cruzi were used in this study and the number of parasites was determined in a Neubauer chamber. The most potent diaryldiazepine that reduced epimastigote proliferation exhibited an IC50 value of 0.25 μM, which is significantly more active than benznidazole.

  6. Trypanosoma cruzi: effect of parasite subpopulation on murine pregnancy outcome.

    Science.gov (United States)

    Solana, María Elisa; Celentano, Ana María; Tekiel, Valeria; Jones, Marta; González Cappa, Stella Maris

    2002-02-01

    C3H/HeN female mice infected with distinct Trypanosoma cruzi subpopulations (RA strain [pantropic/reticulotropic] and K98 clone of the CA-I strain [myotropic]) show differences both in inflammatory compromise of the genital tract and in the outcome of pregnancy. The group of mice infected with the K98 clone show lymphomononuclear infiltrates in pelvian fat and in uterus interstitium, coexisting with the presence of T. cruzi DNA, and show moderate oophoritis, perioophoritis, and vasculitis. However, neither parasite DNA nor inflammatory foci were detected in the uterus, and only mild oophoritis was observed among RA-infected mice at mating time. Independently from the parasite subpopulation, females developed estrous 30 days postinoculation (PI), and at the same time, parasite counts were similar for K98 and for RA-infected mice. However, fertility was significantly diminished in K98-infected females. On day 14 of gestation, fetal resorptions increased in this group and cannot be attributed to hormonal disbalance because similar serum progesterone levels were found in all groups. At this time (44 days PI), parasitemia was higher in K98- than in RA-infected mice. However, resorptions were not triggered by massive infection because polymerase chain reaction failed to prove parasite DNA in resorbing fetuses. In contrast with K98 females, RA-infected mice delivered T. cruzi-infected newborns.

  7. Antimicrobial activity of synthetic bornyl benzoates against Trypanosoma cruzi

    Science.gov (United States)

    Corrêa, P R C; Miranda, R R S; Duarte, L P; Silva, G D F; Filho, S A Vieira; Okuma, A A; Carazza, F; Morgado-Díaz, J A; Pinge-Filho, P; Yamauchi, L M; Nakamura, C V; Yamada-Ogatta, S F

    2012-01-01

    We report here for the first time the in vitro effects of (1S,2R,4S)-1,7,7-trimethyl-bicyclo[2.2.1]heptan-2-yl-3′,4′,5′-trimethoxy benzoate (1) and (1S,2R,4S)-1,7,7-trimethyl-bicyclo[2.2.1]heptan-2-yl benzoate (2) on the growth and ultrastructure of Trypanosoma cruzi. These two synthetic compounds exerted an antiproliferative effect on the epimastigote forms of the parasite. The ICs50/72h of two synthetic L-bornyl benzoates, 1 and 2, was 10.1 and 12.8 μg/ml, respectively. Both compounds were more selective against epimastigotes than HEp-2 cells. Ultrastructural analysis revealed intense cytoplasmic vacuolization and the appearance of cytoplasmic materials surrounded by membranes. The treatment of peritoneal macrophages with compounds 1 and 2 caused a significant decrease in the number of T. cruzi-infected cells. L-Bornyl benzoate derivatives may serve as a potential source for the development of more effective and safer chemotherapeutic agents against T. cruzi infections. PMID:22943546

  8. High throughput screening for anti-Trypanosoma cruzi drug discovery.

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    Julio Alonso-Padilla

    2014-12-01

    Full Text Available The discovery of new therapeutic options against Trypanosoma cruzi, the causative agent of Chagas disease, stands as a fundamental need. Currently, there are only two drugs available to treat this neglected disease, which represents a major public health problem in Latin America. Both available therapies, benznidazole and nifurtimox, have significant toxic side effects and their efficacy against the life-threatening symptomatic chronic stage of the disease is variable. Thus, there is an urgent need for new, improved anti-T. cruzi drugs. With the objective to reliably accelerate the drug discovery process against Chagas disease, several advances have been made in the last few years. Availability of engineered reporter gene expressing parasites triggered the development of phenotypic in vitro assays suitable for high throughput screening (HTS as well as the establishment of new in vivo protocols that allow faster experimental outcomes. Recently, automated high content microscopy approaches have also been used to identify new parasitic inhibitors. These in vitro and in vivo early drug discovery approaches, which hopefully will contribute to bring better anti-T. cruzi drug entities in the near future, are reviewed here.

  9. Circulating levels of cyclooxygenase metabolites in experimental Trypanosoma cruzi infections

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    Rita L. Cardoni

    2004-01-01

    Full Text Available TRYPANOSOMA cruzi induces inflammatory reactions in several tissues. The production of prostaglandin F2α, 6-keto-prostaglandin F1α and thromboxane B2, known to regulate the immune response and to participate in inflammatory reactions, was studied in mice experimentally infected with T. cruzi. The generation of nitric oxide (NO, which could be regulated by cyclooxygenase metabolites, was also evaluated. In the acute infection the extension of inflammatory infiltrates in skeletal muscle as well as the circulating levels of cyclooxygenase metabolites and NO were higher in resistant C3H mice than in susceptible BALB/c mice. In addition, the spontaneous release of NO by spleen cells increased earlier in the C3H mouse strain. In the chronic infections, the tissue inflammatory reaction was still prominent in both groups of mice, but a moderate increase of thromboxane B2 concentration and in NO released by spleen cells was observed only in C3H mice. This comparative study shows that these mediators could be mainly related to protective mechanisms in the acute phase, but seem not to be involved in its maintenance in the chronic T. cruzi infections.

  10. Altered Cardiomyocyte Function and Trypanosoma cruzi Persistence in Chagas Disease.

    Science.gov (United States)

    Cruz, Jader Santos; Santos-Miranda, Artur; Sales-Junior, Policarpo Ademar; Monti-Rocha, Renata; Campos, Paula Peixoto; Machado, Fabiana Simão; Roman-Campos, Danilo

    2016-05-04

    Chagas disease, caused by the triatominae Trypanosoma cruzi, is one of the leading causes of heart malfunctioning in Latin America. The cardiac phenotype is observed in 20-30% of infected people 10-40 years after their primary infection. The cardiac complications during Chagas disease range from cardiac arrhythmias to heart failure, with important involvement of the right ventricle. Interestingly, no studies have evaluated the electrical properties of right ventricle myocytes during Chagas disease and correlated them to parasite persistence. Taking advantage of a murine model of Chagas disease, we studied the histological and electrical properties of right ventricle in acute (30 days postinfection [dpi]) and chronic phases (90 dpi) of infected mice with the Colombian strain of T. cruzi and their correlation to parasite persistence. We observed an increase in collagen deposition and inflammatory infiltrate at both 30 and 90 dpi. Furthermore, using reverse transcriptase polymerase chain reaction, we detected parasites at 90 dpi in right and left ventricles. In addition, we observed action potential prolongation and reduced transient outward K(+) current and L-type Ca(2+) current at 30 and 90 dpi. Taking together, our results demonstrate that T. cruzi infection leads to important modifications in electrical properties associated with inflammatory infiltrate and parasite persistence in mice right ventricle, suggesting a causal role between inflammation, parasite persistence, and altered cardiomyocyte function in Chagas disease. Thus, arrhythmias observed in Chagas disease may be partially related to altered electrical function in right ventricle. © The American Society of Tropical Medicine and Hygiene.

  11. Prevalence of Trypanosoma cruzi/HIV coinfection in southern Brazil

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    Dulce Stauffert

    2017-03-01

    Full Text Available Chagas disease reactivation has been a defining condition for acquired immune deficiency syndrome in Brazil for individuals coinfected with Trypanosoma cruzi and HIV since 2004. Although the first coinfection case was reported in the 1980s, its prevalence has not been firmly established. In order to know coinfection prevalence, a cross-sectional study of 200 HIV patients was performed between January and July 2013 in the city of Pelotas, in southern Rio Grande do Sul, an endemic area for Chagas disease. Ten subjects were found positive for T. cruzi infection by chemiluminescence microparticle immunoassay and indirect immunofluorescence. The survey showed 5% coinfection prevalence among HIV patients (95% CI: 2.0–8.0, which was 3.8 times as high as that estimated by the Ministry of Health of Brazil. Six individuals had a viral load higher than 100,000 copies per μL, a statistically significant difference for T. cruzi presence. These findings highlight the importance of screening HIV patients from Chagas disease endemic areas.

  12. Molecular characterization of Trypanosoma cruzi Mexican strains and their behavior in the mouse experimental model

    National Research Council Canada - National Science Library

    Gómez-Hernández, César; Rezende-Oliveira, Karine; Nascentes, Gabriel Antônio Nogueira; Batista, Lara Rocha; Kappel, Henrique Borges; Martinez-Ibarra, José Alejandro; Trujillo Contreras, Francisco; Lages-Silva, Eliane; Ramírez, Luis Eduardo

    2011-01-01

    ... of Chagas disease in Mexico, this study aimed to identify the molecular and biological characterization of Trypanosoma cruzi isolates from different endemic areas of this country, especially of the State of Jalisco...

  13. In vitro evaluation of the activity of aromatic nitrocompounds against Trypanosoma cruzi

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    Renata B Oliveira

    2003-01-01

    Full Text Available Fourteen compounds were evaluated for their activity against Trypanosoma cruzi blood stream forms at the concentration of 500 µg/ml. Six compounds were active and re-tested at lower concentrations.

  14. Alkaloids Induce Programmed Cell Death in Bloodstream Forms of Trypanosomes (Trypanosoma b. brucei

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    Michael Wink

    2008-10-01

    Full Text Available The potential induction of a programmed cell death (PCD in Trypanosoma b. brucei by 55 alkaloids of the quinoline, quinolizidine, isoquinoline, indole, terpene, tropane, steroid, and piperidine type was studied by measuring DNA fragmentation and changes in mitochondrial membrane potential. For comparison, the induction of apoptosis by the same alkaloids in human leukemia cells (Jurkat APO-S was tested. Several alkaloids of the isoquinoline, quinoline, indole and steroidal type (berberine, chelerythrine, emetine, sanguinarine, quinine, ajmalicine, ergotamine, harmine, vinblastine, vincristine, colchicine, chaconine, demissidine and veratridine induced programmed cell death, whereas quinolizidine, tropane, terpene and piperidine alkaloids were mostly inactive. Effective PCD induction (EC50 below 10 µM was caused in T. brucei by chelerythrine, emetine, sanguinarine, and chaconine. The active alkaloids can be characterized by their general property to inhibit protein biosynthesis, to intercalate DNA, to disturb membrane fluidity or to inhibit microtubule formation.

  15. Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi.

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    Carolina Furtado

    Full Text Available The oxidative lesion 8-oxoguanine (8-oxoG is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1. This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1, the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1(-/- (CD138 to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H(2O(2. Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H(2O(2 of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER.

  16. Protein 3-nitrotyrosine formation during Trypanosoma cruzi infection in mice

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    M. Naviliat

    2005-12-01

    Full Text Available Nitric oxide (·NO is a diffusible messenger implicated in Trypanosoma cruzi resistance. Excess production of ·NO and oxidants leads to the generation of nitrogen dioxide (·NO2, a strong nitrating agent. Tyrosine nitration is a post-translational modification resulting from the addition of a nitro (-NO2 group to the ortho-position of tyrosine residues. Detection of protein 3-nitrotyrosine is regarded as a marker of nitro-oxidative stress and is observed in inflammatory processes. The formation and role of nitrating species in the control and myocardiopathy of T. cruzi infection remain to be studied. We investigated the levels of ·NO and protein 3-nitrotyrosine in the plasma of C3H and BALB/c mice and pharmacologically modulated their production during the acute phase of T. cruzi infection. We also looked for protein 3-nitrotyrosine in the hearts of infected animals. Our results demonstrated that C3H animals produced higher amounts of ·NO than BALB/c mice, but their generation of peroxynitrite was not proportionally enhanced and they had higher parasitemias. While N G-nitro-arginine methyl ester treatment abolished ·NO production and drastically augmented the parasitism, mercaptoethylguanidine and guanido-ethyl disulfide, at doses that moderately reduced the ·NO and 3-nitrotyrosine levels, paradoxically diminished the parasitemia in both strains. Nitrated proteins were also demonstrated in myocardial cells of infected mice. These data suggest that the control of T. cruzi infection depends not only on the capacity to produce ·NO, but also on its metabolic fate, including the generation of nitrating species that may constitute an important element in parasite resistance and collateral myocardial damage.

  17. Trypanosoma cruzi, cancer and the Cold War Trypanosoma cruzi, câncer e a Guerra Fria

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    Nikolai Krementsov

    2009-07-01

    Full Text Available In the summer of 1946, the international community of cancer researchers was inspired by the announcement that two Soviet scientists, Nina Kliueva and Grigorii Roskin, had discovered anticancer properties in culture extracts made from the South American protozoan, Trypanosoma cruzi, and had produced a preparation - named after its discoverers KR - which showed clear therapeutic effects on cancer patients. Research teams from various countries enthusiastically pursued the promising new line of investigation. The story of the rise and fall of interest in the anticancer properties of T. cruzi in different countries suggests that during the second half of the twentieth century, the Cold War competition between the superpowers played an important role in shaping the research agendas of cancer studies.No verão de 1946, a comunidade internacional que desenvolve pesquisas sobre o câncer, inspirou-se no anúncio de que dois cientistas soviéticos, Nina Kliueva e Grigorii Roskin, descobriram propriedades anticancerígenas em cultura extraída do protozoário existente na América Latina, o Trypanosoma cruzi e produziram um preparado que foi denominado com as iniciais KR - em sua homenagem. Grupos de pesquisadores de diversos países buscaram com entusiasmo as promessas dessa nova linha de investigação. A história da ascensão e queda do interesse nas propriedades anticâncer do T. cruzzi em diferentes países sugere que durante a segunda metade do século 20, a Guerra Fria teve um papel importante na definição das agendas de pesquisas sobre o câncer.

  18. Trypanosoma cruzi-Trypanosoma rangeli co-infection ameliorates negative effects of single trypanosome infections in experimentally infected Rhodnius prolixus.

    Science.gov (United States)

    Peterson, Jennifer K; Graham, Andrea L; Elliott, Ryan J; Dobson, Andrew P; Triana Chávez, Omar

    2016-08-01

    Trypanosoma cruzi, causative agent of Chagas disease, co-infects its triatomine vector with its sister species Trypanosoma rangeli, which shares 60% of its antigens with T. cruzi. Additionally, T. rangeli has been observed to be pathogenic in some of its vector species. Although T. cruzi-T. rangeli co-infections are common, their effect on the vector has rarely been investigated. Therefore, we measured the fitness (survival and reproduction) of triatomine species Rhodnius prolixus infected with just T. cruzi, just T. rangeli, or both T. cruzi and T. rangeli. We found that survival (as estimated by survival probability and hazard ratios) was significantly different between treatments, with the T. cruzi treatment group having lower survival than the co-infected treatment. Reproduction and total fitness estimates in the T. cruzi and T. rangeli treatments were significantly lower than in the co-infected and control groups. The T. cruzi and T. rangeli treatment group fitness estimates were not significantly different from each other. Additionally, co-infected insects appeared to tolerate higher doses of parasites than insects with single-species infections. Our results suggest that T. cruzi-T. rangeli co-infection could ameliorate negative effects of single infections of either parasite on R. prolixus and potentially help it to tolerate higher parasite doses.

  19. A genomic scale map of genetic diversity in Trypanosoma cruzi

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    Ackermann Alejandro A

    2012-12-01

    Full Text Available Abstract Background Trypanosoma cruzi, the causal agent of Chagas Disease, affects more than 16 million people in Latin America. The clinical outcome of the disease results from a complex interplay between environmental factors and the genetic background of both the human host and the parasite. However, knowledge of the genetic diversity of the parasite, is currently limited to a number of highly studied loci. The availability of a number of genomes from different evolutionary lineages of T. cruzi provides an unprecedented opportunity to look at the genetic diversity of the parasite at a genomic scale. Results Using a bioinformatic strategy, we have clustered T. cruzi sequence data available in the public domain and obtained multiple sequence alignments in which one or two alleles from the reference CL-Brener were included. These data covers 4 major evolutionary lineages (DTUs: TcI, TcII, TcIII, and the hybrid TcVI. Using these set of alignments we have identified 288,957 high quality single nucleotide polymorphisms and 1,480 indels. In a reduced re-sequencing study we were able to validate ~ 97% of high-quality SNPs identified in 47 loci. Analysis of how these changes affect encoded protein products showed a 0.77 ratio of synonymous to non-synonymous changes in the T. cruzi genome. We observed 113 changes that introduce or remove a stop codon, some causing significant functional changes, and a number of tri-allelic and tetra-allelic SNPs that could be exploited in strain typing assays. Based on an analysis of the observed nucleotide diversity we show that the T. cruzi genome contains a core set of genes that are under apparent purifying selection. Interestingly, orthologs of known druggable targets show statistically significant lower nucleotide diversity values. Conclusions This study provides the first look at the genetic diversity of T. cruzi at a genomic scale. The analysis covers an estimated ~ 60% of the genetic diversity present in the

  20. Recent, independent and anthropogenic origins of Trypanosoma cruzi hybrids.

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    Michael D Lewis

    2011-10-01

    Full Text Available The single celled eukaryote Trypanosoma cruzi, a parasite transmitted by numerous species of triatomine bug in the Americas, causes Chagas disease in humans. T. cruzi generally reproduces asexually and appears to have a clonal population structure. However, two of the six major circulating genetic lineages, TcV and TcVI, are TcII-TcIII inter-lineage hybrids that are frequently isolated from humans in regions where chronic Chagas disease is particularly severe. Nevertheless, a prevalent view is that hybridisation events in T. cruzi were evolutionarily ancient and that active recombination is of little epidemiological importance. We analysed genotypes of hybrid and non-hybrid T. cruzi strains for markers representing three distinct evolutionary rates: nuclear GPI sequences (n = 88, mitochondrial COII-ND1 sequences (n = 107 and 28 polymorphic microsatellite loci (n = 35. Using Maximum Likelihood and Bayesian phylogenetic approaches we dated key evolutionary events in the T. cruzi clade including the emergence of hybrid lineages TcV and TcVI, which we estimated to have occurred within the last 60,000 years. We also found evidence for recent genetic exchange between TcIII and TcIV and between TcI and TcIV. These findings show that evolution of novel recombinants remains a potential epidemiological risk. The clearly distinguishable microsatellite genotypes of TcV and TcVI were highly heterozygous and displayed minimal intra-lineage diversity indicative of even earlier origins than sequence-based estimates. Natural hybrid genotypes resembled typical meiotic F1 progeny, however, evidence for mitochondrial introgression, absence of haploid forms and previous experimental crosses indicate that sexual reproduction in T. cruzi may involve alternatives to canonical meiosis. Overall, the data support two independent hybridisation events between TcII and TcIII and a recent, rapid spread of the hybrid progeny in domestic transmission cycles

  1. Variation of G-rich mitochondrial transcripts among stocks of Trypanosoma brucei.

    Science.gov (United States)

    Jasmer, D P; Feagin, J E; Payne, M; Stuart, K

    1987-01-15

    We have compared maxicircle transcripts from eight stocks of subspecies of Trypanosoma brucei. Transcripts from the rRNA and protein genes have a constant size among stocks and exhibit only minor variation in abundance. In contrast, four of the G+C rich sequences encode multiple transcripts that very markedly in size or abundance. Maxicircle nucleotide sequence comparison of three stocks shows very limited sequence divergence suggesting that sequence divergence may not explain the transcript variability. These results suggest that the G-rich transcripts do not encode proteins and that their variability among stocks may result from posttranscriptional processing events.

  2. Comprehensive glycoprofiling of the epimastigote and trypomastigote stages of Trypanosoma cruzi

    DEFF Research Database (Denmark)

    Alves, Maria Julia Manso; Kawahara, Rebeca; Viner, Rosa

    2017-01-01

    Trypanosoma cruzi, the protozoan that causes Chagas disease, has a complex life cycle involving insect and mammalian hosts and distinct developmental stages. During T. cruzi developmental stages, glycoproteins play important role in the host-parasite interaction, such as cellular recognition, host...... the significant T. cruzi stage-specific expression of glycoproteins that can help to better understand the T. cruzi phenotype and response caused by the interaction with different hosts during its complex life cycle. BIOLOGICAL SIGNIFICANCE: Chagas disease caused by the protozoan Trypanosoma cruzi is a neglected...... cell invasion and adhesion, and immune evasion. In this study, comprehensive glycoprofiling analysis was performed in the epimastigote and trypomastigote stages of T. cruzi using two glycopeptide enrichment strategies, lectin-based and hydrophilic interaction liquid chromatography, followed by high...

  3. The phosphoproteome of bloodstream form Trypanosoma brucei, causative agent of African sleeping sickness.

    Science.gov (United States)

    Nett, Isabelle R E; Martin, David M A; Miranda-Saavedra, Diego; Lamont, Douglas; Barber, Jonathan D; Mehlert, Angela; Ferguson, Michael A J

    2009-07-01

    The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of several T. brucei kinases, very few specific phosphorylation sites have been determined in this organism. Using a gel-free, phosphopeptide enrichment-based proteomics approach we performed the first large scale phosphorylation site analyses for T.brucei. Serine, threonine, and tyrosine phosphorylation sites were determined for a cytosolic protein fraction of the bloodstream form of the parasite, resulting in the identification of 491 phosphoproteins based on the identification of 852 unique phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in this study are predicted from their genome annotations to participate in a wide variety of biological processes, including signal transduction, processing of DNA and RNA, protein synthesis, and degradation and to a minor extent in metabolic pathways. The analysis of phosphopeptides and phosphorylation sites was facilitated by in-house developed software, and this automated approach was validated by manual annotation of spectra of the kinase subset of proteins. Analysis of the cytosolic bloodstream form T. brucei kinome revealed the presence of 44 phosphorylated protein kinases in our data set that could be classified into the major eukaryotic protein kinase groups by applying a multilevel hidden Markov model library of the kinase catalytic domain. Identification of the kinase phosphorylation sites showed conserved phosphorylation sequence motifs in several kinase activation segments, supporting the view that

  4. Relationship between biological behaviour and randomly amplified polymorphic DNA profiles of Trypanosoma cruzi strains

    Directory of Open Access Journals (Sweden)

    Rafael A Martínez-Díaz

    2001-02-01

    Full Text Available Once known some biological characteristics of six Trypanosoma cruzi strains, randomly amplified polymorphic DNA (RAPD analysis was made. Cluster analysis by UPGMA (unweighted pair group method analysis was then applied both to biological parameters and RAPD profiles. Inspection of the UPGMA phenograms indicates identical clusters, so supporting that usefulness of biological parameters to characterization of T. cruzi strains still remains.

  5. Risk Factors and Screening for Trypanosoma cruzi Infection of Dutch Blood Donors

    NARCIS (Netherlands)

    Slot, Ed; Hogema, Boris M.; Molier, Michel; Bart, Aldert; Zaaijer, Hans L.

    2016-01-01

    Blood donors unaware of Trypanosoma cruzi infection may donate infectious blood. Risk factors and the presence of T. cruzi antibodies in at-risk Dutch blood donors were studied to assess whether specific blood safety measures are warranted in the Netherlands. Birth in a country endemic for Chagas

  6. Comprehensive proteomic analysis of Trypanosoma cruzi epimastigote cell surface proteins by two complementary methods

    DEFF Research Database (Denmark)

    Queiroz, Rayner M L; Charneau, Sébastien; Motta, Flávia N

    2013-01-01

    Trypanosoma cruzi is a protozoan that causes Chagas' disease, a neglected infectious illness that affects millions of people, mostly in Latin America. Here, the cell surface subproteome of the T. cruzi epimastigote life form was characterized. In order to prepare samples enriched in epimastigote...... of the labeled proteins. Both T. cruzi subproteomes were analyzed by LC-MS/MS. The results showed that the methodologies offered comprehensive and complementary information about the parasite's plasma membrane subproteome....

  7. Human Galectin-3 Promotes Trypanosoma cruzi Adhesion to Human Coronary Artery Smooth Muscle Cells

    OpenAIRE

    Kleshchenko, Yuliya Y.; Moody, Tapria N.; Furtak, Vyacheslav A.; Ochieng, Josiah; Lima, Maria F.; Villalta, Fernando

    2004-01-01

    Human galectin-3 binds to the surface of Trypanosoma cruzi trypomastigotes and human coronary artery smooth muscle (CASM) cells. CASM cells express galectin-3 on their surface and secrete it. Exogenous galectin-3 increased the binding of T. cruzi to CASM cells. Trypanosome binding to CASM cells was enhanced when either T. cruzi or CASM cells were preincubated with galectin-3. Cells stably transfected with galectin-3 antisense show a dramatic decrease in galectin-3 expression and very little T...

  8. Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model.

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    Frédéric-Antoine Dauchy

    2016-11-01

    Full Text Available Trypanosoma brucei gambiense is the main causative agent of Human African Trypanosomiasis (HAT, also known as sleeping sickness. Because of limited alternatives and treatment toxicities, new therapeutic options are urgently needed for patients with HAT. Sterol 14alpha-demethylase (CYP51 is a potential drug target but its essentiality has not been determined in T. brucei. We used a tetracycline-inducible RNAi system to assess the essentiality of CYP51 in T. brucei bloodstream form (BSF cells and we evaluated the effect of posaconazole, a well-tolerated triazole drug, within a panel of virulent strains in vitro and in a murine model. Expression of CYP51 in several T. brucei cell lines was demonstrated by western blot and its essentiality was demonstrated by RNA interference (CYP51RNAi in vitro. Following reduction of TbCYP51 expression by RNAi, cell growth was reduced and eventually stopped compared to WT or non-induced cells, showing the requirement of CYP51 in T. brucei. These phenotypes were rescued by addition of ergosterol. Additionally, CYP51RNAi induction caused morphological defects with multiflagellated cells (p<0.05, suggesting cytokinesis dysfunction. The survival of CYP51RNAi Doxycycline-treated mice (p = 0.053 and of CYP51RNAi 5-day pre-induced Doxycycline-treated mice (p = 0.008 were improved compared to WT showing a CYP51 RNAi effect on trypanosomal virulence in mice. The posaconazole concentrations that inhibited parasite growth by 50% (IC50 were 8.5, 2.7, 1.6 and 0.12 μM for T. b. brucei 427 90-13, T. b. brucei Antat 1.1, T. b. gambiense Feo (Feo/ITMAP/1893 and T. b. gambiense Biyamina (MHOM/SD/82, respectively. During infection with these last three virulent strains, posaconazole-eflornithine and nifurtimox-eflornithine combinations showed similar improvement in mice survival (p≤0.001. Our results provide support for a CYP51 targeting based treatment in HAT. Thus posaconazole used in combination may represent a therapeutic

  9. Parasite Genome Projects and the Trypanosoma cruzi Genome Initiative

    Directory of Open Access Journals (Sweden)

    Wim Degrave

    1997-11-01

    Full Text Available Since the start of the human genome project, a great number of genome projects on other "model" organism have been initiated, some of them already completed. Several initiatives have also been started on parasite genomes, mainly through support from WHO/TDR, involving North-South and South-South collaborations, and great hopes are vested in that these initiatives will lead to new tools for disease control and prevention, as well as to the establishment of genomic research technology in developing countries. The Trypanosoma cruzi genome project, using the clone CL-Brener as starting point, has made considerable progress through the concerted action of more than 20 laboratories, most of them in the South. A brief overview of the current state of the project is given

  10. Beta-interferon inhibits cell infection by Trypanosoma cruzi

    Science.gov (United States)

    Kierszenbaum, F.; Sonnenfeld, G.

    1984-01-01

    Beta interferon has been shown to inhibit the capacity of bloodstream forms of the flagellate Trypanosoma cruzi, the causative agent of Chagas' disease, to associate with and infect mouse peritoneal macrophages and rat heart myoblasts. The inhibitory effect was abrogated in the presence of specific antibodies to the interferon. Pretreatment of the parasites with interferon reduced their infectivity for untreated host cells, whereas pretreament of either type of host cell did not affect the interaction. The effect of interferon on the trypanosomes was reversible; the extent of the inhibitory effect was significantly reduced afer 20 min, and was undetectable after 60 min when macrophages were used as host cells. For the myoblasts, 60 min elapsed before the inhibitory effect began to subside and 120 min elapsed before it became insignificant or undetectable.

  11. Trypanosoma cruzi: genetic group with peculiar biochemical and biological behavior

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    Mônica Lúcia Gomes

    2003-07-01

    Full Text Available Thirty-two Trypanosoma cruzi strains, isolated from chronic chagasic patients in the northwest of the state of Paraná (Brazil, were analyzed using molecular, biochemical and biological characteristics. Genotypic analysis using randomly amplified polymorphic DNA and simple sequence repeat-anchored polymerase chain reaction amplified profiles showed a large, genetically well-correlated group that contained the majority of the strains and a divergent group that included the PR-150 strain. For glycoconjugate composition, the PR-150 strain was different from the other strains considering the absence or presence of specific bands in aqueous or detergent phases. This strain was also totally different from the others in one out of the six parameters related to in vitro and in vivo biological behavior. We highlight the fact that the PR-150 was totally resistant to benznidazole. For the other biological parameters this strain was not totally distinct from the others, but it showed a peculiar behavior.

  12. Diverse inhibitor chemotypes targeting Trypanosoma cruzi CYP51.

    Directory of Open Access Journals (Sweden)

    Shamila S Gunatilleke

    Full Text Available Chagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority.The similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon Trypanosoma cruzi, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14α-demethylase (CYP51 for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the T. cruzi CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of ∼104,000 small molecules to 185 hits with estimated nanomolar K(D values, while cross-validation against T. cruzi-infected skeletal myoblast cells yielded 57 active hits with EC(50 <10 µM. Two pools of hits partially overlapped. The top hit inhibited T. cruzi with EC(50 of 17 nM and was trypanocidal at 40 nM.The hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5, fatty acid ω-hydroxylases (CYP4, 17α-hydroxylase/17,20-lyase (CYP17 and aromatase (CYP19. Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP51 by sequence or structure. Lead compounds developed by pharmaceutical

  13. Vaccination of dogs with Trypanosoma rangeli induces antibodies against Trypanosoma cruzi in a rural area of Córdoba, Argentina

    Science.gov (United States)

    Basso, Beatriz; Marini, Vanina; Gauna, Diego; Frias, Maria

    2016-01-01

    Dogs play a major role in the domestic cycle of Trypanosoma cruzi, acting as reservoirs. In a previous work we have developed a model of vaccination of dogs in captivity with nonpathogenic Trypanosoma rangeli epimastigotes, resulting in the production of protective antibodies against T. cruzi, with dramatic decrease of parasitaemia upon challenge with 100,000 virulent forms of this parasite. The aim of this work was to evaluate the immunogenicity of this vaccine in dogs living in a rural area. Domestic dogs, free from T. cruziinfection, received three immunisations with fixed T. rangeliepimastigotes. Dogs were not challenged with T. cruzi, but they were left in their environment. This immunisation induced antibodies againstT. cruzi for more than three years in dogs in their natural habitat, while control dogs remained serologically negative. PMID:27074257

  14. In vitro effects of citral on trypanosoma cruzi metacyclogenesis.

    Science.gov (United States)

    Cardoso, Josiane; Soares, Maurilio José

    2010-12-01

    Citral, the main constituent of lemongrass (Cymbopogon citratus) essential oil, was added to Trypanosoma cruzi cultures grown in TAU3AAG medium to observe the effect on the epimastigote-to-trypomastigote differentiation process (metacyclogenesis). Our results showed that citral (20 μg/mL) did not affect epimastigote viability or inhibit the differentiation process. Concentrations higher than 60 μg/mL, however, led to 100% cell death (both epimastigote and trypomastigote forms). Although epimastigotes incubated with 30 μg/mL citral were viable and able to adhere to the substrate, we observed around 50% inhibition in metacyclogenesis, with a calculated concentration that inhibited metacyclogenesis by 50% after 24 h (IC50/24 h) of about 31 μg/mL. Treatment with 30 μg/mL citral did not hinder epimastigote multiplication because epimastigote growth resumed when treated cells were transferred to a drug-free liver infusion tryptose culture medium. Metacyclogenesis was almost totally abolished at 40 μg/mL after 24 h of incubation. Furthermore, the metacyclic trypomastigotes obtained in vitro were similarly susceptible to citral, with an IC50/24 h, concentration that killed 50% of the cells after 24 h, of about 24.5 μg/mL. Therefore, citral appears to be a good candidate as an inhibitory drug for further studies analyzing the T. cruzi metacyclogenesis process.

  15. In vitro effects of citral on Trypanosoma cruzi metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Josiane Cardoso

    2010-12-01

    Full Text Available Citral, the main constituent of lemongrass (Cymbopogon citratus essential oil, was added to Trypanosoma cruzi cultures grown in TAU3AAG medium to observe the effect on the epimastigote-to-trypomastigote differentiation process (metacyclogenesis. Our results showed that citral (20 μg/mL did not affect epimastigote viability or inhibit the differentiation process. Concentrations higher than 60 μg/mL, however, led to 100% cell death (both epimastigote and trypomastigote forms. Although epimastigotes incubated with 30 μg/mL citral were viable and able to adhere to the substrate, we observed around 50% inhibition in metacyclogenesis, with a calculated concentration that inhibited metacyclogenesis by 50% after 24 h (IC50/24 h of about 31 μg/mL. Treatment with 30 μg/mL citral did not hinder epimastigote multiplication because epimastigote growth resumed when treated cells were transferred to a drug-free liver infusion tryptose culture medium. Metacyclogenesis was almost totally abolished at 40 μg/mL after 24 h of incubation. Furthermore, the metacyclic trypomastigotes obtained in vitro were similarly susceptible to citral, with an IC50/24 h, concentration that killed 50% of the cells after 24 h, of about 24.5 μg/mL. Therefore, citral appears to be a good candidate as an inhibitory drug for further studies analyzing the T. cruzi metacyclogenesis process.

  16. Retrospective distribution of Trypanosoma cruzi I genotypes in Colombia.

    Science.gov (United States)

    León, Cielo M; Hernández, Carolina; Montilla, Marleny; Ramírez, Juan David

    2015-05-01

    Trypanosoma cruzi is the aetiological agent of Chagas disease, which affects approximately eight million people in the Americas. This parasite exhibits genetic variability, with at least six discrete typing units broadly distributed in the American continent. T. cruzi I (TcI) shows remarkable genetic diversity; a genotype linked to human infections and a domestic cycle of transmission have recently been identified, hence, this strain was named TcIDom. The aim of this work was to describe the spatiotemporal distribution of TcI subpopulations across humans, insect vectors and mammalian reservoirs in Colombia by means of molecular typing targeting the spliced leader intergenic region of mini-exon gene. We analysed 101 TcI isolates and observed a distribution of sylvatic TcI in 70% and TcIDom in 30%. In humans, the ratio was sylvatic TcI in 60% and TcIDom in 40%. In mammal reservoirs, the distribution corresponded to sylvatic TcI in 96% and TcIDom in 4%. Among insect vectors, sylvatic TcI was observed in 48% and TcIDom in 52%. In conclusion, the circulation of TcIDom is emerging in Colombia and this genotype is still adapting to the domestic cycle of transmission. The epidemiological and clinical implications of these findings are discussed herein.

  17. Retrospective distribution of Trypanosoma cruzi I genotypes in Colombia

    Directory of Open Access Journals (Sweden)

    Cielo M León

    2015-05-01

    Full Text Available Trypanosoma cruzi is the aetiological agent of Chagas disease, which affects approximately eight million people in the Americas. This parasite exhibits genetic variability, with at least six discrete typing units broadly distributed in the American continent. T. cruzi I (TcI shows remarkable genetic diversity; a genotype linked to human infections and a domestic cycle of transmission have recently been identified, hence, this strain was named TcIDom. The aim of this work was to describe the spatiotemporal distribution of TcI subpopulations across humans, insect vectors and mammalian reservoirs in Colombia by means of molecular typing targeting the spliced leader intergenic region of mini-exon gene. We analysed 101 TcI isolates and observed a distribution of sylvatic TcI in 70% and TcIDom in 30%. In humans, the ratio was sylvatic TcI in 60% and TcIDom in 40%. In mammal reservoirs, the distribution corresponded to sylvatic TcI in 96% and TcIDom in 4%. Among insect vectors, sylvatic TcI was observed in 48% and TcIDom in 52%. In conclusion, the circulation of TcIDom is emerging in Colombia and this genotype is still adapting to the domestic cycle of transmission. The epidemiological and clinical implications of these findings are discussed herein.

  18. Are dead Triatoma infestans a competent vector of Trypanosoma cruzi?

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    S. N. Asin

    1991-09-01

    Full Text Available After Triatoma infestans death, Trypanosoma cruzi survived several days, maintaining the ability to infect a vertebrate host. Dead bugs from an endemic area collected during an official spraying comapign showed mobile rectal tripanosomes up to 14 days after vector death. Two days after vector death2, 760 tripomastigotes were found alive in its rectal material. However, the number of mobile tripomastigotes decreased significantly from the 5th day after death. Laboratory proofs with third and fifth nymphal stage showed similar results. Living tripanosomes were found in their rectal material at 10 days in third stage and even at 30 days in fifth nymphal stage. The mean number of tripomastigotes had no changes up to 10 days in third nymphal stage and increased significantly from 1 to 10 days in the fifth stage. Conjuctival instillation as well as intraperitoneal innoculation to mice, of metacyclic forms from dead T. infestans produced infection in the vertebrate host. Present results show that human contact with dead vector highly probable in summer and living and infective T. cruzi are available for transmision in the vector.

  19. Polyclonal antibodies for the detection of Trypanosoma cruzi circulating antigens.

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    Edith S Málaga-Machaca

    2017-11-01

    Full Text Available Detection of Trypanosoma cruzi antigens in clinical samples is considered an important diagnostic tool for Chagas disease. The production and use of polyclonal antibodies may contribute to an increase in the sensitivity of immunodiagnosis of Chagas disease.Polyclonal antibodies were raised in alpacas, rabbits, and hens immunized with trypomastigote excreted-secreted antigen, membrane proteins, trypomastigote lysate antigen and recombinant 1F8 to produce polyclonal antibodies. Western blot analysis was performed to determine specificity of the developed antibodies. An antigen capture ELISA of circulating antigens in serum, plasma and urine samples was developed using IgY polyclonal antibodies against T. cruzi membrane antigens (capture antibody and IgG from alpaca raised against TESA. A total of 33 serum, 23 plasma and 9 urine samples were analyzed using the developed test. Among serum samples, compared to serology, the antigen capture ELISA tested positive in 55% of samples. All plasma samples from serology positive subjects were positive in the antigen capture ELISA. All urine positive samples had corresponding plasma samples that were also positive when tested by the antigen capture ELISA.Polyclonal antibodies are useful for detection of circulating antigens in both the plasma and urine of infected individuals. Detection of antigens is direct evidence of the presence of the parasite, and could be a better surrogate of current infection status.

  20. Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation.

    Science.gov (United States)

    van Weelden, Susanne W H; Fast, Beate; Vogt, Achim; van der Meer, Pieter; Saas, Joachim; van Hellemond, Jaap J; Tielens, Aloysius G M; Boshart, Michael

    2003-04-11

    The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitro differentiation from wild type and mutant (Delta aco::NEO/Delta aco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO(2). Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.

  1. Mapping of branch sites in trans-spliced pre-mRNAs of Trypanosoma brucei.

    Science.gov (United States)

    Patzelt, E; Perry, K L; Agabian, N

    1989-01-01

    The process of trans splicing is essential to the maturation of all mRNAs in the Trypanosomatidae, a family of protozoan parasites, and to specific mRNAs in several species of nematode. In Trypanosoma brucei, a 39-nucleotide (nt) leader sequence originating from a small, 139-nt donor RNA (the spliced leader [SL] RNA) is spliced to the 5' end of mRNAs. An intermediate in this trans-splicing process is a Y structure which contains the 3' 100 nt of the SL RNA covalently linked to the pre-mRNA via a 2'-5' phosphodiester bond at the branch point residue. We mapped the branch points in T. brucei alpha- and beta-tubulin pre-mRNAs. The primary branch acceptors for the alpha- and beta-tubulins are 44 and 56 nt upstream of the 3' splice sites, respectively, and are A residues. Minor branch acceptors were detected 42 and 49 nt upstream of the alpha-tubulin splice site and 58 nt upstream of the splice site in beta-tubulin. The regions surrounding these branch points lack homology to the consensus sequences determined for mammalian cells and yeasts; there is also no conservation among the sequences themselves. Thus, the identified sequences suggest that the mechanism of branch point recognition in T. brucei differs from the mechanism of recognition by U2 RNA that has been proposed for other eucaryotes. Images PMID:2479824

  2. The inositol pyrophosphate synthesis pathway in Trypanosoma brucei is linked to polyphosphate synthesis in acidocalcisomes.

    Science.gov (United States)

    Cordeiro, Ciro D; Saiardi, Adolfo; Docampo, Roberto

    2017-10-01

    Inositol pyrophosphates are novel signaling molecules possessing high-energy pyrophosphate bonds and involved in a number of biological functions. Here, we report the correct identification and characterization of the kinases involved in the inositol pyrophosphate biosynthetic pathway in Trypanosoma brucei: inositol polyphosphate multikinase (TbIPMK), inositol pentakisphosphate 2-kinase (TbIP5K) and inositol hexakisphosphate kinase (TbIP6K). TbIP5K and TbIP6K were not identifiable by sequence alone and their activities were validated by enzymatic assays with the recombinant proteins or by their complementation of yeast mutants. We also analyzed T. brucei extracts for the presence of inositol phosphates using polyacrylamide gel electrophoresis and high-performance liquid chromatography. Interestingly, we could detect inositol phosphate (IP), inositol 4,5-bisphosphate (IP2 ), inositol 1,4,5-trisphosphate (IP3 ), and inositol hexakisphosphate (IP6 ) in T. brucei different stages. Bloodstream forms unable to produce inositol pyrophosphates, due to downregulation of TbIPMK expression by conditional knockout, have reduced levels of polyphosphate and altered acidocalcisomes. Our study links the inositol pyrophosphate pathway to the synthesis of polyphosphate in acidocalcisomes, and may lead to better understanding of these organisms and provide new targets for drug discovery. © 2017 John Wiley & Sons Ltd.

  3. Molecular variation of Trypanosoma brucei subspecies as revealed by AFLP fingerprinting.

    Science.gov (United States)

    Agbo, E E C; Majiwa, P A O; Claassen, H J H M; te Pas, M F W

    2002-04-01

    Genetic analysis of Trypanosoma spp. depends on the detection of variation between strains. We have used the amplified fragment length polymorphism (AFLP) technique to develop a convenient and reliable method for genetic characterization of Trypanosome (sub)species. AFLP accesses multiple independent sites within the genome and would allow a better definition of the relatedness of different Trypanosome (sub)species. Nine isolates (3 from each T. brucei subspecies) were tested with 40 AFLP primer combinations to identify the most appropriate pairs of restriction endonucleases and selective primers. Primers based on the recognition sequences of EcoRI and BglII were chosen and used to analyse 31 T. brucei isolates. Similarity levels calculated with the Pearson correlation coefficient ranged from 15 to 98%, and clusters were determined using the unweighted pair-group method using arithmetic averages (UPGMA). At the intraspecific level, AFLP fingerprints were grouped by numerical analysis in 2 main clusters, allowing a clear separation of T. b. gambiense (cluster I) from T. b. brucei and T. b. rhodesiense isolates (cluster II). Interspecies evaluation of this customized approach produced heterogeneous AFLP patterns, with unique genetic markers, except for T. evansi and T. equiperdum, which showed identical patterns and clustered together.

  4. NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei.

    Science.gov (United States)

    Narayanan, Mani Shankar; Kushwaha, Manish; Ersfeld, Klaus; Fullbrook, Alexander; Stanne, Tara M; Rudenko, Gloria

    2011-03-01

    Trypanosoma brucei mono-allelically expresses one of approximately 1500 variant surface glycoprotein (VSG) genes while multiplying in the mammalian bloodstream. The active VSG is transcribed by RNA polymerase I in one of approximately 15 telomeric VSG expression sites (ESs). T. brucei is unusual in controlling gene expression predominantly post-transcriptionally, and how ESs are mono-allelically controlled remains a mystery. Here we identify a novel transcription regulator, which resembles a nucleoplasmin-like protein (NLP) with an AT-hook motif. NLP is key for ES control in bloodstream form T. brucei, as NLP knockdown results in 45- to 65-fold derepression of the silent VSG221 ES. NLP is also involved in repression of transcription in the inactive VSG Basic Copy arrays, minichromosomes and procyclin loci. NLP is shown to be enriched on the 177- and 50-bp simple sequence repeats, the non-transcribed regions around rDNA and procyclin, and both active and silent ESs. Blocking NLP synthesis leads to downregulation of the active ES, indicating that NLP plays a role in regulating appropriate levels of transcription of ESs in both their active and silent state. Discovery of the unusual transcription regulator NLP provides new insight into the factors that are critical for ES control.

  5. NIMA-related kinase TbNRKC is involved in basal body separation in Trypanosoma brucei.

    Science.gov (United States)

    Pradel, Lydie C; Bonhivers, Mélanie; Landrein, Nicolas; Robinson, Derrick R

    2006-05-01

    The NIMA-related kinase 2 (NEK 2) has important cell cycle functions related to centriole integrity and splitting. Trypanosoma brucei does not possess centrioles, however, cytokinesis is coupled to basal body separation events. Here we report the first functional characterisation of a T. brucei basal body-cytoskeletal NIMA-related kinase (NRK) protein, TbNRKC. The TbNRKC kinase domain has high amino acid identity with the human NEK1 kinase domain (50%) but also shares 42% identity with human NEK2. TbNRKC is expressed in bloodstream and procyclic cells and functions as a bona fide kinase in vitro. Remarkably, RNAi knockdown of TbNRKC and overexpression of kinase-dead TbNRKC in procyclic forms induces the accumulation of cells with four basal bodies, whereas overexpression of active protein produces supernumary basal bodies and blocks cytokinesis. TbNRKC is located on mature and immature basal bodies and is the first T. brucei NRK to be found associated with the basal body cytokinesis pathway.

  6. Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization.

    Science.gov (United States)

    Lobo-Rojas, Ángel E; González-Marcano, Eglys B; Valera-Vera, Edward A; Acosta, Héctor R; Quiñones, Wilfredo A; Burchmore, Richard J S; Concepción, Juan L; Cáceres, Ana J

    2016-10-01

    Two different putative galactokinase genes, found in the genome database of Trypanosoma cruzi were cloned and sequenced. Expression of the genes in Escherichia coli resulted for TcGALK-1 in the synthesis of a soluble and active enzyme, and in the case of TcGALK-2 gene a less soluble protein, with predicted molecular masses of 51.9kDa and 51.3kDa, respectively. The Km values determined for the recombinant proteins were for galactose 0.108mM (TcGALK-1) and 0.091mM (TcGALK-2) and for ATP 0.36mM (TcGALK-1) and 0.1mM (TcGALK-2). Substrate inhibition by ATP (Ki 0.414mM) was only observed for TcGALK-2. Gel-filtration chromatography showed that natural TcGALKs and recombinant TcGALK-1 are monomeric. In agreement with the possession of a type-1 peroxisome-targeting signal by both TcGALKs, they were found to be present inside glycosomes using two different methods of subcellular fractionation in conjunction with mass spectrometry. Both genes are expressed in epimastigote and trypomastigote stages since the respective proteins were immunodetected by western blotting. The T. cruzi galactokinases present their highest (52-47%) sequence identity with their counterpart from Leishmania spp., followed by prokaryotic galactokinases such as those from E. coli and Lactococcus lactis (26-23%). In a phylogenetic analysis, the trypanosomatid galactokinases form a separate cluster, showing an affiliation with bacteria. Epimastigotes of T. cruzi can grow in glucose-depleted LIT-medium supplemented with 20mM of galactose, suggesting that this hexose, upon phosphorylation by a TcGALK, could be used in the synthesis of UDP-galactose and also as a possible carbon and energy source. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. Congenital Trypanosoma cruzi Transmission in Santa Cruz, Bolivia

    Science.gov (United States)

    Bern, Caryn; Verastegui, Manuela; Gilman, Robert H.; LaFuente, Carlos; Galdos-Cardenas, Gerson; Calderon, Maritza; Pacori, Juan; Abastoflor, Maria del Carmen; Aparicio, Hugo; Brady, Mark F.; Ferrufino, Lisbeth; Angulo, Noelia; Marcus, Sarah; Sterling, Charles; Maguire, James H.

    2017-01-01

    Background We conducted a study of congenital Trypanosoma cruzi infection in Santa Cruz, Bolivia. Our objective was to apply new tools to identify weak points in current screening algorithms, and find ways to improve them. Methods Women presenting for delivery were screened by rapid and conventional serological tests. For infants of infected mothers, blood specimens obtained on days 0, 7, 21, 30, 90, 180, and 270 were concentrated and examined microscopically; serological tests were performed for the day 90, 180, and 270 specimens. Maternal and infant specimens, including umbilical tissue, were tested by polymerase chain reaction (PCR) targeting the kinetoplast minicircle and by quantitative PCR. Results Of 530 women, 154 (29%) were seropositive. Ten infants had congenital T. cruzi infection. Only 4 infants had positive results of microscopy evaluation in the first month, and none had positive cord blood microscopy results. PCR results were positive for 6 (67%) of 9 cord blood and 7 (87.5%) of 8 umbilical tissue specimens. PCR-positive women were more likely to transmit T. cruzi than were seropositive women with negative PCR results (P < .05). Parasite loads determined by quantitative PCR were higher for mothers of infected infants than for seropositive mothers of uninfected infants (P < .01). Despite intensive efforts, only 58% of at-risk infants had a month 9 specimen collected. Conclusions On the basis of the low sensitivity of microscopy in cord blood and high rate of loss to follow-up, we estimate that current screening programs miss one-half of all infected infants. Molecular techniques may improve early detection. PMID:19877966

  8. Structural and functional insight into ADF/cofilin from Trypanosoma brucei.

    Science.gov (United States)

    Dai, Kun; Liao, Shanhui; Zhang, Jiahai; Zhang, Xuecheng; Tu, Xiaoming

    2013-01-01

    The ADF/cofilin family has been characterized as a group of actin-binding proteins critical for controlling the assembly of actin within the cells. In this study, the solution structure of the ADF/cofilin from Trypanosoma brucei (TbCof) was determined by NMR spectroscopy. TbCof adopts the conserved ADF/cofilin fold with a central β-sheet composed of six β-strands surrounded by five α-helices. Isothermal titration calorimetry experiments denoted a submicromolar affinity between TbCof and G-actin, and the affinity between TbCof and ADP-G-actin was five times higher than that between TbCof and ATP-G-actin at low ionic strength. The results obtained from electron microscopy and actin filament sedimentation assays showed that TbCof depolymerized but did not co-sediment with actin filaments and its ability of F-actin depolymerization was pH independent. Similar to actin, TbCof was distributed throughout the cytoplasm. All our data indicate a structurally and functionally conserved ADF/cofilin from Trypanosoma brucei.

  9. Investigating the Chaperone Properties of a Novel Heat Shock Protein, Hsp70.c, from Trypanosoma brucei

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    Adélle Burger

    2014-01-01

    Full Text Available The neglected tropical disease, African Trypanosomiasis, is fatal and has a crippling impact on economic development. Heat shock protein 70 (Hsp70 is an important molecular chaperone that is expressed in response to stress and Hsp40 acts as its co-chaperone. These proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. A novel cytosolic Hsp70, from Trypanosoma brucei, TbHsp70.c, contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. The ability of a cytosolic Hsp40 from Trypanosoma brucei J protein 2, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective was to functionally characterize TbHsp70.c to further expand our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile MDH and chemically denatured rhodanese. ATPase assays revealed a 2.8-fold stimulation of the ATPase activity of TbHsp70.c by Tbj2. TbHsp70.c and Tbj2 both demonstrated chaperone activity and Tbj2 functions as a co-chaperone of TbHsp70.c. In vivo heat stress experiments indicated upregulation of the expression levels of TbHsp70.c.

  10. A Pre-clinical Animal Model of Trypanosoma brucei Infection Demonstrating Cardiac Dysfunction.

    Directory of Open Access Journals (Sweden)

    Charlotte S McCarroll

    2015-05-01

    Full Text Available African trypanosomiasis (AT, caused by Trypanosoma brucei species, results in both neurological and cardiac dysfunction and can be fatal if untreated. Research on the pathogenesis and treatment of the disease has centred to date on the characteristic neurological symptoms, whereas cardiac dysfunction (e.g. ventricular arrhythmias in AT remains largely unstudied. Animal models of AT demonstrating cardiac dysfunction similar to that described in field cases of AT are critically required to transform our understanding of AT-induced cardiac pathophysiology and identify future treatment strategies. We have previously shown that T. brucei can interact with heart muscle cells (cardiomyocytes to induce ventricular arrhythmias in ex vivo adult rat hearts. However, it is unknown whether the arrhythmias observed ex vivo are also present during in vivo infection in experimental animal models. Here we show for the first time the characterisation of ventricular arrhythmias in vivo in two animal models of AT infection using electrocardiographic (ECG monitoring. The first model utilised a commonly used monomorphic laboratory strain, Trypanosoma brucei brucei Lister 427, whilst the second model used a pleomorphic laboratory strain, T. b. brucei TREU 927, which demonstrates a similar chronic infection profile to clinical cases. The frequency of ventricular arrhythmias and heart rate (HR was significantly increased at the endpoint of infection in the TREU 927 infection model, but not in the Lister 427 infection model. At the end of infection, hearts from both models were isolated and Langendorff perfused ex vivo with increasing concentrations of the β-adrenergic agonist isoproterenol (ISO. Interestingly, the increased frequency of arrhythmias observed in vivo in the TREU 927 infection model was lost upon isolation of the heart ex vivo, but re-emerged with the addition of ISO. Our results demonstrate that TREU 927 infection modifies the substrate of the myocardium

  11. 1H, 13C and 15N resonance assignments for a putative ADF/Cofilin from Trypanosoma brucei.

    Science.gov (United States)

    Dai, Kun; Yuan, Guangfa; Liao, Shanhui; Zhang, Jiahai; Tu, Xiaoming

    2011-10-01

    Actin-depolymerizing factor (ADF)/cofilin proteins are a family of actin-binding proteins expressed in almost all eukaryotic cells, and play a significant role in regulating actin-filament dynamics. Here we report the resonance assignments of a putative ADF/cofilin from Trypanosoma brucei for further understanding of the relationship between its structure and function.

  12. Variations in maxi-circle and mini-circle sequences in kinetoplast DNAs from different Trypanosoma brucei strains.

    NARCIS (Netherlands)

    P. Borst (Piet); F. Fase-Fowler; J.H.J. Hoeijmakers (Jan); A.C.C. Frasch

    1980-01-01

    textabstractWe have compared a total of 30 recognition sites for eight restriction endonucleases on the 20-kilobase-pair maxi-circle of kinetoplast DNAs from five different Trypanosoma brucei strains. In addition to three polymorphic sites were have found a 5 kilobase-pair region that is not cleaved

  13. THE CYTOSOLIC AND GLYCOSOMAL GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM TRYPANOSOMA-BRUCEI - KINETIC-PROPERTIES AND COMPARISON WITH HOMOLOGOUS ENZYMES

    NARCIS (Netherlands)

    LAMBEIR, AM; LOISEAU, AM; KUNTZ, DA; VELLIEUX, FM; MICHELS, PAM; OPPERDOES, FR

    1991-01-01

    The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like

  14. A tropical tale: how Naja nigricollis venom beats Trypanosoma brucei

    DEFF Research Database (Denmark)

    Martos Esteban, Andrea; Laustsen, Andreas Hougaard; Carrington, Mark

    Trypanosoma brucei is a parasitic protozoan species capable to infecting insect vectors whose bite further produces African sleeping sickness inhuman beings [1]. During the parasite’s extracellular life in the mammalian host,its outer coat, mainly composed of Variable Surface Glycoproteins (VSGs...

  15. Chemical characterisation of Nigerian red propolis and its biological activity against Trypanosoma Brucei.

    Science.gov (United States)

    Omar, Ruwida M K; Igoli, John; Gray, Alexander I; Ebiloma, Godwin Unekwuojo; Clements, Carol; Fearnley, James; Ebel, Ru Angeli Edrada; Zhang, Tong; De Koning, Harry P; Watson, David G

    2016-01-01

    A previous study showed the unique character of Nigerian red propolis from Rivers State, Nigeria (RSN), with regards to chemical composition and activity against Trypanosoma brucei in comparison with other African propolis. To carry out fractionation and biological testing of Nigerian propolis in order to isolate compounds with anti-trypanosomal activity. To compare the composition of the RSN propolis with the composition of Brazilian red propolis. Profiling was carried out using HPLC-UV-ELSD and HPLC-Orbitrap-FTMS on extracts of two samples collected from RSN with data extraction using MZmine software. Isolation was carried out by normal phase and reversed phase MPLC. Elucidation of the compounds with a purity > 95% was performed by 1D/2D NMR HRMS and HRLC-MS(n) . Ten phenolic compounds were isolated or in the case of liquiritigenin partially purified. Data for nine of these correlated with literature reports of known compounds i.e. one isoflavanone, calycosin (1); two flavanones, liquiritigenin (2) and pinocembrin (5); an isoflavan, vestitol (3); a pterocarpan, medicarpin (4); two prenylflavanones, 8-prenylnaringenin (7) and 6-prenylnaringenin (8); and two geranyl flavonoids, propolin D (9) and macarangin (10). The tenth was elucidated as a previously undescribed dihydrobenzofuran (6). The isolated compounds were tested against Trypanosoma brucei and displayed moderate to high activity. Some of the compounds tested had similar activity against wild type T. brucei and two strains displaying pentamidine resistance. Nigerian propolis from RSN has some similarities with Brazilian red propolis. The propolis displayed anti-trypanosomal activity at a potentially useful level. Copyright © 2015 John Wiley & Sons, Ltd.

  16. An Atypical Mitochondrial Carrier That Mediates Drug Action in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Juan P de Macêdo

    2015-05-01

    Full Text Available Elucidating the mechanism of action of trypanocidal compounds is an important step in the development of more efficient drugs against Trypanosoma brucei. In a screening approach using an RNAi library in T. brucei bloodstream forms, we identified a member of the mitochondrial carrier family, TbMCP14, as a prime candidate mediating the action of a group of anti-parasitic choline analogs. Depletion of TbMCP14 by inducible RNAi in both bloodstream and procyclic forms increased resistance of parasites towards the compounds by 7-fold and 3-fold, respectively, compared to uninduced cells. In addition, down-regulation of TbMCP14 protected bloodstream form mitochondria from a drug-induced decrease in mitochondrial membrane potential. Conversely, over-expression of the carrier in procyclic forms increased parasite susceptibility more than 13-fold. Metabolomic analyses of parasites over-expressing TbMCP14 showed increased levels of the proline metabolite, pyrroline-5-carboxylate, suggesting a possible involvement of TbMCP14 in energy production. The generation of TbMCP14 knock-out parasites showed that the carrier is not essential for survival of T. brucei bloodstream forms, but reduced parasite proliferation under standard culture conditions. In contrast, depletion of TbMCP14 in procyclic forms resulted in growth arrest, followed by parasite death. The time point at which parasite proliferation stopped was dependent on the major energy source, i.e. glucose versus proline, in the culture medium. Together with our findings that proline-dependent ATP production in crude mitochondria from TbMCP14-depleted trypanosomes was reduced compared to control mitochondria, the study demonstrates that TbMCP14 is involved in energy production in T. brucei. Since TbMCP14 belongs to a trypanosomatid-specific clade of mitochondrial carrier family proteins showing very poor similarity to mitochondrial carriers of mammals, it may represent an interesting target for drug

  17. Trypanosoma cruzi: antigen-receptor mediated endocytosis of antibody

    Directory of Open Access Journals (Sweden)

    Judith Abelha

    1981-06-01

    Full Text Available Trypanomastigote forms of Trypanosoma cruzi were derived from tissue culture and incubated with immune and non-immune human sera. All immune sera showed high titers of specific humoral antibodies of the IgM or the IgG type. Agglutination and swelling of parasites were observed after incubation at 37ºC, but many trypomastigotes remained free-swimming in the sera for two to three days. The quantitiy of immune serum capable of lysing a maximum of 10 x 10 [raised to the power of 6] sensitized red cells was not capable of lysing 4 x 10 [raised to the power of 3] tripomastigotes. Typically, the parasites underwent cyclical changes with the formation of clumps of amastigotes and the appearance of epimastigote forms. Multiplication of the parasites was observed in immune sera. Further, the infectivity of the parasites to susceptible mice was not lost. All sera used produced similar general effects on the growth of the parasite. The antibody bound to T. cruzi appeard to enter cells by antigen-receptor mediated endocytosis. The ferritin-conjugated antibody was internalized and delivered to phagolysosomes where they might be completely degraded to amino-acids. This seemed to be a coupled process by which the immunoglobulin is first bound to specific parasite surface receptor and then rapidly endocytosed by the cell.Formas tripomastigotas de Trypanosoma cruzi derivadas de cultura de tecido foram encubadas com soros humanos imunes e não-imunes.Todos os soros humanos usados tinham títulos elevados de anticorpos das classes IgM ou IgG. Aglutinação e entumescimento dos parasitos eram observados apos encubação a 37ºC mas muitos tripomastigotas permaneceram circulando livremente nos soros por dois a três dias. A quantidade de soro imune capaz de lisar um máximo de 10 x 10 [elevado a 6] hemácias sensibilizadas não foi capaz de lisar 4 x 10 [elevado a 3] tripomastigotas. Tipicamente, os parasitos apresentavam alterações cíclicas com formação de

  18. The nucleotide sequence of the variable region in Trypanosoma brucei completes the sequence analysis of the maxicircle component of mitochondrial kinetoplast DNA

    NARCIS (Netherlands)

    Sloof, P.; de Haan, A.; Eier, W.; van Iersel, M.; Boel, E.; van Steeg, H.; Benne, R.

    1992-01-01

    The nucleotide sequence of two non-contiguous DNA fragments of 4.0 and 2.2 kb, respectively, of the kinetoplast maxicircle of Trypanosoma brucei brucei EATRO strain 427 has been determined, completing the sequence analysis of the so-called variable region (see also de Vries et al., 1988, Mol.

  19. Identification of contractile vacuole proteins in Trypanosoma cruzi.

    Science.gov (United States)

    Ulrich, Paul N; Jimenez, Veronica; Park, Miyoung; Martins, Vicente P; Atwood, James; Moles, Kristen; Collins, Dalis; Rohloff, Peter; Tarleton, Rick; Moreno, Silvia N J; Orlando, Ron; Docampo, Roberto

    2011-03-18

    Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism

  20. Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.

    Directory of Open Access Journals (Sweden)

    Andrés B Lantos

    2016-04-01

    Full Text Available Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully

  1. Immunotherapy of Trypanosoma cruzi infection with DNA vaccines in mice.

    Science.gov (United States)

    Dumonteil, Eric; Escobedo-Ortegon, Javier; Reyes-Rodriguez, Norma; Arjona-Torres, Arletty; Ramirez-Sierra, Maria Jesus

    2004-01-01

    The mechanisms involved in the pathology of chronic chagasic cardiomyopathy are still debated, and the controversy has interfered with the development of new treatments and vaccines. Because of the potential of DNA vaccines for immunotherapy of chronic and infectious diseases, we tested if DNA vaccines could control an ongoing Trypanosoma cruzi infection. BALB/c mice were infected with a lethal dose (5 x 10(4) parasites) as a model of acute infection, and then they were treated with two injections of 100 microg of plasmid DNA 1 week apart, beginning on day 5 postinfection. Control mice had high levels of parasitemia and mortality and severe cardiac inflammation, while mice treated with plasmid DNA encoding trypomastigote surface antigen 1 or Tc24 had reduced parasitemia and mild cardiac inflammation and >70% survived the infection. The efficacy of the immunotherapy also was significant when it was delayed until days 10 and 15 after infection. Parasitological analysis of cardiac tissue of surviving mice indicated that most mice still contained detectable parasite kinetoplast DNA but fewer mice contained live parasites, suggesting that there was efficient but not complete parasite elimination. DNA vaccine immunotherapy was also evaluated in CD1 mice infected with a low dose (5 x 10(2) parasites) as a model of chronic infection. Immunotherapy was initiated on day 70 postinfection and resulted in improved survival and reduced cardiac tissue inflammation. These results suggest that DNA vaccines have strong potential for the immunotherapy of T. cruzi infection and may provide new alternatives for the control of Chagas' disease.

  2. Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology

    Science.gov (United States)

    Lantos, Andrés B.; Carlevaro, Giannina; Araoz, Beatriz; Ruiz Diaz, Pablo; Camara, María de los Milagros; Buscaglia, Carlos A.; Bossi, Mariano; Yu, Hai; Chen, Xi; Bertozzi, Carolyn R.; Mucci, Juan; Campetella, Oscar

    2016-01-01

    Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form. PMID

  3. Characterization of a Novel Class I Transcription Factor A (CITFA) Subunit That Is Indispensable for Transcription by the Multifunctional RNA Polymerase I of Trypanosoma brucei

    KAUST Repository

    Nguyen, T. N.

    2012-10-26

    Trypanosoma brucei is the only organism known to have evolved a multifunctional RNA polymerase I (pol I) system that is used to express the parasite\\'s ribosomal RNAs, as well as its major cell surface antigens, namely, the variant surface glycoprotein (VSG) and procyclin, which are vital for establishing successful infections in the mammalian host and the tsetse vector, respectively. Thus far, biochemical analyses of the T. brucei RNA pol I transcription machinery have elucidated the subunit structure of the enzyme and identified the class I transcription factor A (CITFA). CITFA binds to RNA pol I promoters, and its CITFA-2 subunit was shown to be absolutely essential for RNA pol I transcription in the parasite. Tandem affinity purification (TAP) of CITFA revealed the subunits CITFA-1 to -6, which are conserved only among kinetoplastid organisms, plus the dynein light chain DYNLL1. Here, by tagging CITFA-6 instead of CITFA-2, a complex was purified that contained all known CITFA subunits, as well as a novel proline-rich protein. Functional studies carried out in vivo and in vitro, as well as a colocalization study, unequivocally demonstrated that this protein is a bona fide CITFA subunit, essential for parasite viability and indispensable for RNA pol I transcription of ribosomal gene units and the active VSG expression site in the mammalian-infective life cycle stage of the parasite. Interestingly, CITFA-7 function appears to be species specific, because expression of an RNA interference (RNAi)-resistant CITFA-7 transgene from Trypanosoma cruzi could not rescue the lethal phenotype of silencing endogenous CITFA-7.

  4. Essential Assembly Factor Rpf2 Forms Novel Interactions within the 5S RNP in Trypanosoma brucei.

    Science.gov (United States)

    Kamina, Anyango D; Jaremko, Daniel; Christen, Linda; Williams, Noreen

    2017-01-01

    Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. During this process, there are several assembly steps that function as regulators to ensure proper ribosome formation. One of these steps is the assembly of the 5S ribonucleoprotein particle (5S RNP) in the central protuberance of the 60S ribosomal subunit. In eukaryotes, the 5S RNP is composed of 5S rRNA, ribosomal proteins L5 and L11, and assembly factors Rpf2 and Rrs1. Our laboratory previously showed that in Trypanosoma brucei, the 5S RNP is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37. In this study, we characterize an additional component of the 5S RNP, the T. brucei homolog of Rpf2. This is the first study to functionally characterize interactions mediated by Rpf2 in an organism other than fungi. T. brucei Rpf2 (TbRpf2) was identified from tandem affinity purification using extracts prepared from protein A-tobacco etch virus (TEV)-protein C (PTP)-tagged L5, P34, and P37 cell lines, followed by mass spectrometry analysis. We characterized the binding interactions between TbRpf2 and the previously characterized members of the T. brucei 5S RNP. Our studies show that TbRpf2 mediates conserved binding interactions with 5S rRNA and L5 and that TbRpf2 also interacts with trypanosome-specific proteins P34 and P37. We performed RNA interference (RNAi) knockdown of TbRpf2 and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation. These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in T. brucei. IMPORTANCETrypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of the

  5. Genome-wide analysis of chromatin structures in Trypanosoma brucei using high-resolution MNase-ChIP-seq.

    Science.gov (United States)

    Wedel, Carolin; Siegel, T Nicolai

    2017-09-01

    Specific DNA-protein interactions are the basis for many important cellular mechanisms like the regulation of gene expression or replication. Knowledge about the precise genomic locations of DNA-protein interactions is important because it provides insight into the regulation of these processes. Recently, we have adapted an approach that combines micrococcal nuclease (MNase) digestion of chromatin with chromatin immunoprecipitation in Trypanosoma brucei. Here, we describe in detail how this method can be used to map the genome-wide distribution of nucleosomes or other DNA-binding proteins at high resolution in T. brucei. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Trypanosoma cruzi trans-sialidase in complex with a neutralizing antibody: structure/function studies towards the rational design of inhibitors.

    Directory of Open Access Journals (Sweden)

    Alejandro Buschiazzo

    2012-01-01

    Full Text Available Trans-sialidase (TS, a virulence factor from Trypanosoma cruzi, is an enzyme playing key roles in the biology of this protozoan parasite. Absent from the mammalian host, it constitutes a potential target for the development of novel chemotherapeutic drugs, an urgent need to combat Chagas' disease. TS is involved in host cell invasion and parasite survival in the bloodstream. However, TS is also actively shed by the parasite to the bloodstream, inducing systemic effects readily detected during the acute phase of the disease, in particular, hematological alterations and triggering of immune cells apoptosis, until specific neutralizing antibodies are elicited. These antibodies constitute the only known submicromolar inhibitor of TS's catalytic activity. We now report the identification and detailed characterization of a neutralizing mouse monoclonal antibody (mAb 13G9, recognizing T. cruzi TS with high specificity and subnanomolar affinity. This mAb displays undetectable association with the T. cruzi superfamily of TS-like proteins or yet with the TS-related enzymes from Trypanosoma brucei or Trypanosoma rangeli. In immunofluorescence assays, mAb 13G9 labeled 100% of the parasites from the infective trypomastigote stage. This mAb also reduces parasite invasion of cultured cells and strongly inhibits parasite surface sialylation. The crystal structure of the mAb 13G9 antigen-binding fragment in complex with the globular region of T. cruzi TS was determined, revealing detailed molecular insights of the inhibition mechanism. Not occluding the enzyme's catalytic site, the antibody performs a subtle action by inhibiting the movement of an assisting tyrosine (Y₁₁₉, whose mobility is known to play a key role in the trans-glycosidase mechanism. As an example of enzymatic inhibition involving non-catalytic residues that occupy sites distal from the substrate-binding pocket, this first near atomic characterization of a high affinity inhibitory molecule

  7. Trypanosoma brucei FKBP12 differentially controls motility and cytokinesis in procyclic and bloodstream forms.

    Science.gov (United States)

    Brasseur, Anaïs; Rotureau, Brice; Vermeersch, Marjorie; Blisnick, Thierry; Salmon, Didier; Bastin, Philippe; Pays, Etienne; Vanhamme, Luc; Pérez-Morga, David

    2013-02-01

    FKBP12 proteins are able to inhibit TOR kinases or calcineurin phosphatases upon binding of rapamycin or FK506 drugs, respectively. The Trypanosoma brucei FKBP12 homologue (TbFKBP12) was found to be a cytoskeleton-associated protein with specific localization in the flagellar pocket area of the bloodstream form. In the insect procyclic form, RNA interference-mediated knockdown of TbFKBP12 affected motility. In bloodstream cells, depletion of TbFKBP12 affected cytokinesis and cytoskeleton architecture. These last effects were associated with the presence of internal translucent cavities limited by an inside-out configuration of the normal cell surface, with a luminal variant surface glycoprotein coat lined up by microtubules. These cavities, which recreated the streamlined shape of the normal trypanosome cytoskeleton, might represent unsuccessful attempts for cell abscission. We propose that TbFKBP12 differentially affects stage-specific processes through association with the cytoskeleton.

  8. Sec16 determines the size and functioning of the Golgi in the protist parasite, Trypanosoma brucei.

    Science.gov (United States)

    Sealey-Cardona, Marco; Schmidt, Katy; Demmel, Lars; Hirschmugl, Tatjana; Gesell, Tanja; Dong, Gang; Warren, Graham

    2014-06-01

    The Sec16 homologue in Trypanosoma brucei has been identified and characterized. TbSec16 colocalizes with COPII components at the single endoplasmic reticulum exit site (ERES), which is next to the single Golgi stack in the insect (procyclic) form of this organism. Depletion of TbSec16 reduces the size of the ERES and the Golgi, and slows growth and transport of a secretory marker to the cell surface; conversely, overexpression of TbSec16 increases the size of the ERES and Golgi but has no effect on growth or secretion. Together these data suggest that TbSec16 regulates the size of the ERES and Golgi and this size is set for optimal growth of the organism. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Enhanced succinic acid production in Aspergillus saccharolyticus by heterologous expression of fumarate reductase from Trypanosoma brucei

    DEFF Research Database (Denmark)

    Yang, Lei; Lübeck, Mette; Ahring, Birgitte K.

    2015-01-01

    production medium as well as the complete medium, but the measured enzyme activities were different depending on the media. Furthermore, a soluble NADH-dependent fumarate reductase gene (frd) from Trypanosoma brucei was inserted and expressed in A. saccharolyticus. The expression of the frd gene led......Aspergillus saccharolyticus exhibits great potential as a cell factory for industrial production of dicarboxylic acids. In the analysis of the organic acid profile, A. saccharolyticus was cultivated in an acid production medium using two different pH conditions. The specific activities...... on the pattern and the amount of organic acids produced by A. saccharolyticus. The wild-type strain produced higher amount of malic acid and succinic acid in the pH buffered condition (pH 6.5) compared with the pH non-buffered condition. The enzyme assays showed that the rTCA branch was active in the acid...

  10. Extracellular Vesicles from Trypanosoma brucei Mediate Virulence Factor Transfer and Cause Host Anemia.

    Science.gov (United States)

    Szempruch, Anthony J; Sykes, Steven E; Kieft, Rudo; Dennison, Lauren; Becker, Allison C; Gartrell, Anzio; Martin, William J; Nakayasu, Ernesto S; Almeida, Igor C; Hajduk, Stephen L; Harrington, John M

    2016-01-14

    Intercellular communication between parasites and with host cells provides mechanisms for parasite development, immune evasion, and disease pathology. Bloodstream African trypanosomes produce membranous nanotubes that originate from the flagellar membrane and disassociate into free extracellular vesicles (EVs). Trypanosome EVs contain several flagellar proteins that contribute to virulence, and Trypanosoma brucei rhodesiense EVs contain the serum resistance-associated protein (SRA) necessary for human infectivity. T. b. rhodesiense EVs transfer SRA to non-human infectious trypanosomes, allowing evasion of human innate immunity. Trypanosome EVs can also fuse with mammalian erythrocytes, resulting in rapid erythrocyte clearance and anemia. These data indicate that trypanosome EVs are organelles mediating non-hereditary virulence factor transfer and causing host erythrocyte remodeling, inducing anemia. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Pyrimidine Salvage Enzymes Are Essential for De Novo Biosynthesis of Deoxypyrimidine Nucleotides in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Christopher Leija

    2016-11-01

    Full Text Available The human pathogenic parasite Trypanosoma brucei possess both de novo and salvage routes for the biosynthesis of pyrimidine nucleotides. Consequently, they do not require salvageable pyrimidines for growth. Thymidine kinase (TK catalyzes the formation of dTMP and dUMP and is one of several salvage enzymes that appear redundant to the de novo pathway. Surprisingly, we show through analysis of TK conditional null and RNAi cells that TK is essential for growth and for infectivity in a mouse model, and that a catalytically active enzyme is required for its function. Unlike humans, T. brucei and all other kinetoplastids lack dCMP deaminase (DCTD, which provides an alternative route to dUMP formation. Ectopic expression of human DCTD resulted in full rescue of the RNAi growth phenotype and allowed for selection of viable TK null cells. Metabolite profiling by LC-MS/MS revealed a buildup of deoxypyrimidine nucleosides in TK depleted cells. Knockout of cytidine deaminase (CDA, which converts deoxycytidine to deoxyuridine led to thymidine/deoxyuridine auxotrophy. These unexpected results suggested that T. brucei encodes an unidentified 5'-nucleotidase that converts deoxypyrimidine nucleotides to their corresponding nucleosides, leading to their dead-end buildup in TK depleted cells at the expense of dTTP pools. Bioinformatics analysis identified several potential candidate genes that could encode 5'-nucleotidase activity including an HD-domain protein that we show catalyzes dephosphorylation of deoxyribonucleotide 5'-monophosphates. We conclude that TK is essential for synthesis of thymine nucleotides regardless of whether the nucleoside precursors originate from the de novo pathway or through salvage. Reliance on TK in the absence of DCTD may be a shared vulnerability among trypanosomatids and may provide a unique opportunity to selectively target a diverse group of pathogenic single-celled eukaryotes with a single drug.

  12. Seroprevalence of Trypanosoma cruzi in stray and pet dogs in Grenada, West Indies.

    Science.gov (United States)

    Chikweto, A; Kumthekar, S; Chawla, P; Tiwari, K P; Perea, L M; Paterson, T; Sharma, R N

    2014-06-01

    American trypanosomiasis (Chagas disease) caused by the protozoan parasite Trypanosoma cruzi is endemic to parts of South America and the Caribbean. Infected dogs are important in the epidemiology of the parasite as they can play a role in the transmission of the parasite to humans. A total of 399 dog sera (242 stray and 157 pet dogs) were examined for T. cruzi infection; using a qualitative immunochromatographic dipstick test, based on recombinant antigens specific for American trypanosomiasis (Trypanosoma detect rapid test; InBios international, Inc., Seattle, Washington). Overall seroprevalence for T. cruzi was estimated at 10.5% (95% confidence interval: 7.5% to 13.5%); with stray dogs being significantly more affected (pdogs in Grenada are moderately exposed to T. cruzi compared to other areas in the region.

  13. Population genetics of Trypanosoma brucei rhodesiense: clonality and diversity within and between foci.

    Directory of Open Access Journals (Sweden)

    Craig W Duffy

    2013-11-01

    Full Text Available African trypanosomes are unusual among pathogenic protozoa in that they can undergo their complete morphological life cycle in the tsetse fly vector with mating as a non-obligatory part of this development. Trypanosoma brucei rhodesiense, which infects humans and livestock in East and Southern Africa, has classically been described as a host-range variant of the non-human infective Trypanosoma brucei that occurs as stable clonal lineages. We have examined T. b. rhodesiense populations from East (Uganda and Southern (Malawi Africa using a panel of microsatellite markers, incorporating both spatial and temporal analyses. Our data demonstrate that Ugandan T. b. rhodesiense existed as clonal populations, with a small number of highly related genotypes and substantial linkage disequilibrium between pairs of loci. However, these populations were not stable as the dominant genotypes changed and the genetic diversity also reduced over time. Thus these populations do not conform to one of the criteria for strict clonality, namely stability of predominant genotypes over time, and our results show that, in a period in the mid 1990s, the previously predominant genotypes were not detected but were replaced by a novel clonal population with limited genetic relationship to the original population present between 1970 and 1990. In contrast, the Malawi T. b. rhodesiense population demonstrated significantly greater diversity and evidence for frequent genetic exchange. Therefore, the population genetics of T. b. rhodesiense is more complex than previously described. This has important implications for the spread of the single copy T. b. rhodesiense gene that allows human infectivity, and therefore the epidemiology of the human disease, as well as suggesting that these parasites represent an important organism to study the influence of optional recombination upon population genetic dynamics.

  14. Structural and Functional Association of Trypanosoma brucei MIX Protein with Cytochrome c Oxidase Complex ▿ †

    Science.gov (United States)

    Zíková, Alena; Panigrahi, Aswini K.; Uboldi, Alessandro D.; Dalley, Rachel A.; Handman, Emanuela; Stuart, Kenneth

    2008-01-01

    A mitochondrial inner membrane protein, designated MIX, seems to be essential for cell viability. The deletion of both alleles was not possible, and the deletion of a single allele led to a loss of virulence and aberrant mitochondrial segregation and cell division in Leishmania major. However, the mechanism by which MIX exerts its effect has not been determined. We show here that MIX is also expressed in the mitochondrion of Trypanosoma brucei, and using RNA interference, we found that its loss leads to a phenotype that is similar to that described for Leishmania. The loss of MIX also had a major effect on cytochrome c oxidase activity, on the mitochondrial membrane potential, and on the production of mitochondrial ATP by oxidative phosphorylation. Using a tandem affinity purification tag, we found that MIX is associated with a multiprotein complex that contains subunits of the mitochondrial cytochrome c oxidase complex (respiratory complex IV), the composition of which was characterized in detail. The specific function of MIX is unknown, but it appears to be important for the function of complex IV and for mitochondrial segregation and cell division in T. brucei. PMID:18776036

  15. Base J and H3.V Regulate Transcriptional Termination in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Danae Schulz

    2016-01-01

    Full Text Available Trypanosoma brucei is a protozoan parasite that lacks many transcription factors found in other eukaryotes, such as those whose binding demarcates enhancers. T. brucei retains histone variants and modifications, however, and it is hypothesized that it relies on epigenetic marks to define transcription-related boundaries. The histone H3 variant (H3.V and an alternate nucleotide, base J (ß-D-glucosyl-hydroxymethyluracil, are two chromatin marks found at both transcription termination sites (TTSs and telomeres. Here, we report that the absence of both base J and H3.V result in transcription readthrough and the appearance of antisense transcripts near TTSs. Additionally, we find that maintaining the transcriptional silencing of pol I-transcribed telomeric Variant Surface Glycoprotein (VSG genes appears to be dependent on deposition of H3.V alone. Our study reveals that gene expression depends on different epigenetic cues depending on chromosomal location and on the transcribing polymerase. This work provides insight into how these signals may have evolved into the more nuanced and fine-tuned gene regulatory mechanisms observed in other model systems.

  16. Tail characteristics of Trypanosoma brucei mitochondrial transcripts are developmentally altered in a transcript-specific manner.

    Science.gov (United States)

    Gazestani, Vahid H; Hampton, Marshall; Shaw, Aubie K; Salavati, Reza; Zimmer, Sara L

    2018-02-01

    The intricate life cycle of Trypanosoma brucei requires extensive regulation of gene expression levels of the mtRNAs for adaptation. Post-transcriptional gene regulatory programs, including unencoded mtRNA 3' tail additions, potentially play major roles in this adaptation process. Intriguingly, T. brucei mitochondrial transcripts possess two distinct unencoded 3' tails, each with a differing functional role; i.e., while one type is implicated in RNA stability (in-tails), the other type appears associated with translation (ex-tails). We examined the degree to which tail characteristics differ among cytochrome c oxidase subunits I and III (CO1 and CO3), and NADH dehydrogenase subunit 1 (ND1) transcripts, and to what extent these characteristics differ developmentally. We found that CO1, CO3 and ND1 transcripts possess longer in-tails in the mammalian life stage. By mathematically modelling states of in-tail and ex-tail addition, we determined that the typical length at which an in-tail is extended to become an ex-tail differs by transcript and, in the case of ND1, by life stage. To the best of our knowledge, we provide the first evidence that developmental differences exist in tail length distributions of mtRNAs, underscoring the potential involvement of in-tail and ex-tail populations in mitochondrial post-transcriptional regulation mechanisms. Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  17. KREX2 is not essential for either procyclic or bloodstream form Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Jason Carnes

    Full Text Available BACKGROUND: Most mitochondrial mRNAs in Trypanosoma brucei require RNA editing for maturation and translation. The edited RNAs primarily encode proteins of the oxidative phosphorylation system. These parasites undergo extensive changes in energy metabolism between the insect and bloodstream stages which are mirrored by alterations in RNA editing. Two U-specific exonucleases, KREX1 and KREX2, are both present in protein complexes (editosomes that catalyze RNA editing but the relative roles of each protein are not known. METHODOLOGY/PRINCIPAL FINDINGS: The requirement for KREX2 for RNA editing in vivo was assessed in both procyclic (insect and bloodstream form parasites by methods that use homologous recombination for gene elimination. These studies resulted in null mutant cells in which both alleles were eliminated. The viability of these cells demonstrates that KREX2 is not essential in either life cycle stage, despite certain defects in RNA editing in vivo. Furthermore, editosomes isolated from KREX2 null cells require KREX1 for in vitro U-specific exonuclease activity. CONCLUSIONS: KREX2 is a U-specific exonuclease that is dispensable for RNA editing in vivo in T. brucei BFs and PFs. This result suggests that the U deletion activity, which is required for RNA editing, is primarily mediated in vivo by KREX1 which is normally found associated with only one type of editosome. The retention of the KREX2 gene implies a non-essential role or a role that is essential in other life cycle stages or conditions.

  18. An Overview of Trypanosoma brucei Infections: An Intense Host-Parasite Interaction.

    Science.gov (United States)

    Ponte-Sucre, Alicia

    2016-01-01

    Trypanosoma brucei rhodesiense and T. brucei gambiense, the causative agents of Human African Trypanosomiasis, are transmitted by tsetse flies. Within the vector, the parasite undergoes through transformations that prepares it to infect the human host. Sequentially these developmental stages are the replicative procyclic (in which the parasite surface is covered by procyclins) and trypo-epimastigote forms, as well as the non-replicative, infective, metacyclic form that develops in the vector salivary glands. As a pre-adaptation to their life in humans, metacyclic parasites begin to express and be densely covered by the Variant Surface Glycoprotein (VSG). Once the metacyclic form invades the human host the parasite develops into the bloodstream form. Herein the VSG triggers a humoral immune response. To avoid this humoral response, and essential for survival while in the bloodstream, the parasite changes its cover periodically and sheds into the surroundings the expressed VSG, thus evading the consequences of the immune system activation. Additionally, tools comparable to quorum sensing are used by the parasite for the successful parasite transmission from human to insect. On the other hand, the human host promotes clearance of the parasite triggering innate and adaptive immune responses and stimulating cytokine and chemokine secretion. All in all, the host-parasite interaction is extremely active and leads to responses that need multiple control sites to develop appropriately.

  19. Comparative genomic analysis of human infective Trypanosoma cruzi lineages with the bat-restricted subspecies T. cruzi marinkellei

    Directory of Open Access Journals (Sweden)

    Franzén Oscar

    2012-10-01

    Full Text Available Abstract Background Trypanosoma cruzi marinkellei is a bat-associated parasite of the subgenus Schizotrypanum and it is regarded as a T. cruzi subspecies. Here we report a draft genome sequence of T. c. marinkellei and comparison with T. c. cruzi. Our aims were to identify unique sequences and genomic features, which may relate to their distinct niches. Results The T. c. marinkellei genome was found to be ~11% smaller than that of the human-derived parasite T. c. cruzi Sylvio X10. The genome size difference was attributed to copy number variation of coding and non-coding sequences. The sequence divergence in coding regions was ~7.5% between T. c. marinkellei and T. c. cruzi Sylvio X10. A unique acetyltransferase gene was identified in T. c. marinkellei, representing an example of a horizontal gene transfer from eukaryote to eukaryote. Six of eight examined gene families were expanded in T. c. cruzi Sylvio X10. The DGF gene family was expanded in T. c. marinkellei. T. c. cruzi Sylvio X10 contained ~1.5 fold more sequences related to VIPER and L1Tc elements. Experimental infections of mammalian cell lines indicated that T. c. marinkellei has the capacity to invade non-bat cells and undergo intracellular replication. Conclusions Several unique sequences were identified in the comparison, including a potential subspecies-specific gene acquisition in T. c. marinkellei. The identified differences reflect the distinct evolutionary trajectories of these parasites and represent targets for functional investigation.

  20. Comparative genomic analysis of human infective Trypanosoma cruzi lineages with the bat-restricted subspecies T. cruzi marinkellei.

    Science.gov (United States)

    Franzén, Oscar; Talavera-López, Carlos; Ochaya, Stephen; Butler, Claire E; Messenger, Louisa A; Lewis, Michael D; Llewellyn, Martin S; Marinkelle, Cornelis J; Tyler, Kevin M; Miles, Michael A; Andersson, Björn

    2012-10-05

    Trypanosoma cruzi marinkellei is a bat-associated parasite of the subgenus Schizotrypanum and it is regarded as a T. cruzi subspecies. Here we report a draft genome sequence of T. c. marinkellei and comparison with T. c. cruzi. Our aims were to identify unique sequences and genomic features, which may relate to their distinct niches. The T. c. marinkellei genome was found to be ~11% smaller than that of the human-derived parasite T. c. cruzi Sylvio X10. The genome size difference was attributed to copy number variation of coding and non-coding sequences. The sequence divergence in coding regions was ~7.5% between T. c. marinkellei and T. c. cruzi Sylvio X10. A unique acetyltransferase gene was identified in T. c. marinkellei, representing an example of a horizontal gene transfer from eukaryote to eukaryote. Six of eight examined gene families were expanded in T. c. cruzi Sylvio X10. The DGF gene family was expanded in T. c. marinkellei. T. c. cruzi Sylvio X10 contained ~1.5 fold more sequences related to VIPER and L1Tc elements. Experimental infections of mammalian cell lines indicated that T. c. marinkellei has the capacity to invade non-bat cells and undergo intracellular replication. Several unique sequences were identified in the comparison, including a potential subspecies-specific gene acquisition in T. c. marinkellei. The identified differences reflect the distinct evolutionary trajectories of these parasites and represent targets for functional investigation.

  1. Murine models susceptibility to distinct Trypanosoma cruzi I genotypes infection.

    Science.gov (United States)

    León, Cielo M; Montilla, Marleny; Vanegas, Ricardo; Castillo, Maria; Parra, Edgar; Ramírez, Juan David

    2017-04-01

    Chagas disease is a complex zoonosis that affects around 8 million people worldwide. This pathology is caused by Trypanosoma cruzi, a kinetoplastid parasite that shows tremendous genetic diversity evinced in six distinct Discrete Typing Units (TcI-TcVI) including a recent genotype named as TcBat and associated with anthropogenic bats. TcI presents a broad geographical distribution and has been associated with chronic cardiomyopathy. Recent phylogenetic studies suggest the existence of two genotypes (Domestic (TcIDom) and sylvatic TcI) within TcI. The understanding of the course of the infection in different mouse models by these two genotypes is not yet known. Therefore, we infected 126 animals (ICR-CD1, National Institute of Health (NIH) and Balb/c) with two TcIDom strains and one sylvatic strain for a follow-up period of 60 days. We quantified the parasitaemia, immune response and histopathology observing that the maximum day of parasitaemia was achieved at day 21 post-infection. Domestic strains showed higher parasitaemia than the sylvatic strain in the three mouse models; however in the survival curves Balb/c mice were less susceptible to infection compared with NIH and ICR-CD1. Our results suggest that the genetic background plays a fundamental role in the natural history of the infection and the sympatric TcI genotypes have relevant implications in disease pathogenesis.

  2. Crystal Structure of Triosephosphate Isomerase from Trypanosoma cruzi in Hexane

    Science.gov (United States)

    Gao, Xiu-Gong; Maldonado, Ernesto; Perez-Montfort, Ruy; Garza-Ramos, Georgina; Tuena de Gomez-Puyou, Marietta; Gomez-Puyou, Armando; Rodriguez-Romero, Adela

    1999-08-01

    To gain insight into the mechanisms of enzyme catalysis in organic solvents, the x-ray structure of some monomeric enzymes in organic solvents was determined. However, it remained to be explored whether the structure of oligomeric proteins is also amenable to such analysis. The field acquired new perspectives when it was proposed that the x-ray structure of enzymes in nonaqueous media could reveal binding sites for organic solvents that in principle could represent the starting point for drug design. Here, a crystal of the dimeric enzyme triosephosphate isomerase from the pathogenic parasite Trypanosoma cruzi was soaked and diffracted in hexane and its structure solved at 2- angstrom resolution. Its overall structure and the dimer interface were not altered by hexane. However, there were differences in the orientation of the side chains of several amino acids, including that of the catalytic Glu-168 in one of the monomers. No hexane molecules were detected in the active site or in the dimer interface. However, three hexane molecules were identified on the surface of the protein at sites, which in the native crystal did not have water molecules. The number of water molecules in the hexane structure was higher than in the native crystal. Two hexanes localized at <4 angstrom from residues that form the dimer interface; they were in close proximity to a site that has been considered a potential target for drug design.

  3. Specific antibodies induce apoptosis in Trypanosoma cruzi epimastigotes.

    Science.gov (United States)

    Fernández-Presas, Ana María; Tato, Patricia; Becker, Ingeborg; Solano, Sandra; Copitin, Natalia; Kopitin, Natalia; Berzunza, Miriam; Willms, Kaethe; Hernández, Joselin; Molinari, José Luis

    2010-05-01

    The susceptibility of Trypanosoma cruzi epimastigotes to lysis by normal or immune sera in a complement-dependent reaction has been reported. Mouse immune sera depleted complement-induced damage in epimastigotes characterized by morphological changes and death. The purpose of this work was to study the mechanism of death in epimastigotes exposed to decomplemented mouse immune serum. Epimastigotes were maintained in RPMI medium. Immune sera were prepared in mice by immunization with whole crude epimastigote extracts. Viable epimastigotes were incubated with decomplemented normal or immune sera at 37 degrees C. By electron microscopy, agglutinated parasites showed characteristic patterns of membrane fusion between two or more parasites; this fusion also produced interdigitation of the subpellicular microtubules. Apoptosis was determined by flow cytometry using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and annexin V assays. Nuclear features were examined by 4'-,6-diamidino-2'-phenylindole diHCI cytochemistry that demonstrated apoptotic nuclear condensation. Caspase activity was also measured. TUNEL results showed that parasites incubated with decomplemented immune sera took up 26% of specific fluorescence as compared to 1.3% in parasites incubated with decomplemented normal sera. The Annexin-V-Fluos staining kit revealed that epimastigotes incubated with decomplemented immune sera exposed phosphatidylserine on the external leaflet of the plasma membrane. The incubation of parasites with immune sera showed caspase 3 activity. We conclude that specific antibodies are able to induce agglutination and apoptosis in epimastigotes, although the pathway is not elucidated.

  4. First Case of Natural Infection in Pigs: Review of Trypanosoma cruzi Reservoirs in Mexico

    Directory of Open Access Journals (Sweden)

    Paz María Salazar-Schettino

    1997-07-01

    Full Text Available An epidemiological research project was performed in the State of Morelos including collection of samples for blood smears and culture, serological tests, and xenodiagnoses from a total of 76 domestic and peridomestic mammals. Two strains of Trypanosoma cruzi were isolated by haemocultures; one from a pig (Sus scrofa, the first case of natural infection reported in Mexico, and the other from a dog (Canis familiaris. This study summarizes current information in Mexico concerning confirmed reservoirs of T. cruzi

  5. In vitro activity of Etanidazole against the protozoan parasite Trypanosoma cruzi

    OpenAIRE

    Petray, Patricia B; Morilla, María J; Corral, Ricardo S; Romero, Eder L

    2004-01-01

    We investigated the in vitro action of an hydrosoluble 2-nitroimidazole, Etanidazole (EZL), against Trypanosoma cruzi, the etiologic agent of Chagas disease. EZL displayed lethal activity against isolated trypomastigotes as well as amastigotes of T. cruzi (RA strain) growing in Vero cells or J774 macrophages, without affecting host cell viability. Although not completely equivalent to Benznidazole (BZL), the reference drug for Chagas chemotherapy, EZL takes advantage in exertingits anti-T. cr...

  6. Molecular characterization of Trypanosoma cruzi Mexican strains and their behavior in the mouse experimental model

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    César Gómez-Hernández

    2011-12-01

    Full Text Available INTRODUCTION: For a long time, the importance of Chagas disease in Mexico, where many regarded it as an exotic malady, was questioned. Considering the great genetic diversity among isolates of Trypanosoma cruzi, the importance of this biological characterization, and the paucity of information on the clinical and biological aspects of Chagas disease in Mexico, this study aimed to identify the molecular and biological characterization of Trypanosoma cruzi isolates from different endemic areas of this country, especially of the State of Jalisco. METHODS: Eight Mexican Trypanosoma cruzi strains were biologically and genetically characterized (PCR specific for Trypanosoma cruzi, multiplex-PCR, amplification of space no transcript of the genes of the mini-exon, amplification of polymorphic regions of the mini-exon, classification by amplification of intergenic regions of the spliced leader genes, RAPD - (random amplified polymorphic DNA. RESULTS: Two profiles of parasitaemia were observed, patent (peak parasitaemia of 4.6×10(6 to 10(7 parasites/mL and subpatent. In addition, all isolates were able to infect 100% of the animals. The isolates mainly displayed tropism for striated (cardiac and skeletal muscle. PCR amplification of the mini-exon gene classified the eight strains as TcI. The RAPD technique revealed intraspecies variation among isolates, distinguishing strains isolated from humans and triatomines and according to geographic origin. CONCLUSIONS: The Mexican T. cruzi strains are myotrophic and belong to group TcI.

  7. Insight into the exoproteome of the tissue-derived trypomastigote form of trypanosoma cruzi

    DEFF Research Database (Denmark)

    Queiroz, Rayner M L; Ricart, Carlos A O; Machado, Mara O

    2016-01-01

    The protozoan parasite Trypanosoma cruzi causes Chagas disease, one of the major neglected infectious diseases. It has the potential to infect any nucleated mammalian cell. The secreted/excreted protein repertoire released by T. cruzi trypomastigotes is crucial in host-pathogen interactions...... in the identification of 540 proteins, the largest set of released proteins identified to date in Trypanosoma spp. Bioinformatic analysis predicted most identified proteins as secreted, predominantly by non-classical pathways, and involved in host-cell infection. Some proteins possess predicted GPI-anchor signals...

  8. Isolation and analysis of the genetic diversity of repertoires of VSG expression site containing telomeres from Trypanosoma brucei gambiense, T. b. brucei and T. equiperdum

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    Becker Marion

    2008-08-01

    Full Text Available Abstract Background African trypanosomes (including Trypanosoma brucei are unicellular parasites which multiply in the mammalian bloodstream. T. brucei has about twenty telomeric bloodstream form Variant Surface Glycoprotein (VSG expression sites (BESs, of which one is expressed at a time in a mutually exclusive fashion. BESs are polycistronic transcription units, containing a variety of families of expression site associated genes (ESAGs in addition to the telomeric VSG. These polymorphic ESAG families are thought to play a role in parasite-host adaptation, and it has been proposed that ESAG diversity might be related to host range. Analysis of the genetic diversity of these telomeric gene families has been confounded by the underrepresentation of telomeric sequences in standard libraries. We have previously developed a method to selectively isolate sets of trypanosome BES containing telomeres using Transformation associated recombination (TAR cloning in yeast. Results Here we describe the isolation of repertoires of BES containing telomeres from three trypanosome subspecies: Trypanosoma brucei gambiense DAL 972 (causative agent of West-African trypanosomiasis, T. b. brucei EATRO 2340 (a nonhuman infective strain and T. equiperdum STIB 818 (which causes a sexually transmitted disease in equines. We have sequenced and analysed the genetic diversity at four BES loci (BES promoter region, ESAG6, ESAG5 and ESAG2 from these three trypanosome BES repertoires. Conclusion With the exception of ESAG2, the BES sequence repertoires derived from T. b. gambiense are both less diverse than and nearly reciprocally monophyletic relative to those from T. b. brucei and T. equiperdum. Furthermore, although we find evidence for adaptive evolution in all three ESAG repertoires in T. b. brucei and T. equiperdum, only ESAG2 appears to be under diversifying selection in T. b. gambiense. This low level of variation in the T. b. gambiense BES sequence repertoires is

  9. Isolation and analysis of the genetic diversity of repertoires of VSG expression site containing telomeres from Trypanosoma brucei gambiense, T. b. brucei and T. equiperdum.

    Science.gov (United States)

    Young, Rosanna; Taylor, Jesse E; Kurioka, Ayako; Becker, Marion; Louis, Edward J; Rudenko, Gloria

    2008-08-12

    African trypanosomes (including Trypanosoma brucei) are unicellular parasites which multiply in the mammalian bloodstream. T. brucei has about twenty telomeric bloodstream form Variant Surface Glycoprotein (VSG) expression sites (BESs), of which one is expressed at a time in a mutually exclusive fashion. BESs are polycistronic transcription units, containing a variety of families of expression site associated genes (ESAGs) in addition to the telomeric VSG. These polymorphic ESAG families are thought to play a role in parasite-host adaptation, and it has been proposed that ESAG diversity might be related to host range. Analysis of the genetic diversity of these telomeric gene families has been confounded by the underrepresentation of telomeric sequences in standard libraries. We have previously developed a method to selectively isolate sets of trypanosome BES containing telomeres using Transformation associated recombination (TAR) cloning in yeast. Here we describe the isolation of repertoires of BES containing telomeres from three trypanosome subspecies: Trypanosoma brucei gambiense DAL 972 (causative agent of West-African trypanosomiasis), T. b. brucei EATRO 2340 (a nonhuman infective strain) and T. equiperdum STIB 818 (which causes a sexually transmitted disease in equines). We have sequenced and analysed the genetic diversity at four BES loci (BES promoter region, ESAG6, ESAG5 and ESAG2) from these three trypanosome BES repertoires. With the exception of ESAG2, the BES sequence repertoires derived from T. b. gambiense are both less diverse than and nearly reciprocally monophyletic relative to those from T. b. brucei and T. equiperdum. Furthermore, although we find evidence for adaptive evolution in all three ESAG repertoires in T. b. brucei and T. equiperdum, only ESAG2 appears to be under diversifying selection in T. b. gambiense. This low level of variation in the T. b. gambiense BES sequence repertoires is consistent both with the relatively narrow host range

  10. Histopathologic identification of Trypanosoma cruzi (Chagas' encephalitis in an AIDS patient

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    Dimath Alyemni

    2017-03-01

    Full Text Available Trypanosoma cruzi (Chagas' encephalitis is an uncommon manifestation of T. cruzi infection, typically seen in immunocompromised patients. Encephalitis results from the reactivation of chronic infection predominately in individuals from endemic areas. Increased awareness of this complication is essential especially with increased migration of patients from endemic areas with concomitant HIV infection. Here we report a case of Chagas' encephalitis in an AIDS patient from Mexico in which there was no evidence of acute serologic, CSF, or blood infection by T. cruzi trypomastigotes.

  11. 5-Nitro-2-furyl derivative actives against Trypanosoma cruzi: preliminary in vivo studies.

    Science.gov (United States)

    Cabrera, Eliana; Murguiondo, Mariana González; Arias, Marelina González; Arredondo, Carolina; Pintos, Cristina; Aguirre, Gabriela; Fernández, Marcelo; Basmadjián, Yester; Rosa, Raquel; Pacheco, José Pedro; Raymondo, Stella; Di Maio, Rossanna; González, Mercedes; Cerecetto, Hugo

    2009-10-01

    Ten 5-nitro-2-furyl derivatives, with good to excellent in vitro anti-Trypanosoma cruzi activity, and nifurtimox were tested oral and intraperitoneally on healthy animals for its acute toxicity on murine models. According to animals' survival percentage, organ histological results, biochemical and haematological findings, three new derivatives, with toxicity like nifurtimox, were selected to test in vivo as antichagasic agents. Clearly, dependences between chemical structure and both acute toxicity and in vivo anti-T. cruzi activity were observed. 4-Hexyl-1-[3-(5-nitro-2-furyl)-2-propenylidene]semicarbazide displayed good profile as anti-T. cruzi agent and better acute toxicity profile than nifurtimox.

  12. Diagnosis of human trypanosomiasis, due to Trypanosoma brucei gambiense in central Africa, by the polymerase chain reaction

    OpenAIRE

    Penchenier, Laurent; Simo, G.; Grébaut, Pascal; Nkinin, S.; Laveissière, Claude; Herder, Stéphane

    2000-01-01

    During a mass screening of sleeping sickness conducted in 1998 and 1999, and involving 27,932 persons in Cameroon and the Central African Republic, we tested the polymerase chain reaction (PCR) on whole blood for the diagnosis of human African trypanosomiasis due to #Trypanosoma brucei gambiense$. The 1858 samples obtained were from 4 groups : 155 infected patients, 1432 serological suspects detected by the card agglutination test for trypanosomiasis (CATT), 222 negative controls living in th...

  13. Apocytochrome b and other mitochondrial DNA sequences are differentially expressed during the life cycle of Trypanosoma brucei.

    OpenAIRE

    Feagin, J E; Jasmer, D P; Stuart, K

    1985-01-01

    Cytochromes and Krebs cycle enzymes are not detected in bloodstream forms of Trypanosoma brucei but are present in procyclic forms. We have analyzed transcription of mitochondrial sequences which contain the apocytochrome b gene and several other open reading frames (ORFs). Multiple transcripts map to individual DNA sequences located on both DNA strands. Larger low abundance transcripts map to multiple ORFs and may be precursor RNAs. Small abundant transcripts map to G + C rich sequences that...

  14. Novel drug design for Chagas disease via targeting Trypanosoma cruzi tubulin: Homology modeling and binding pocket prediction on Trypanosoma cruzi tubulin polymerization inhibition by naphthoquinone derivatives.

    Science.gov (United States)

    Ogindo, Charles O; Khraiwesh, Mozna H; George, Matthew; Brandy, Yakini; Brandy, Nailah; Gugssa, Ayele; Ashraf, Mohammad; Abbas, Muneer; Southerland, William M; Lee, Clarence M; Bakare, Oladapo; Fang, Yayin

    2016-08-15

    Chagas disease, also called American trypanosomiasis, is a parasitic disease caused by Trypanosoma cruzi (T. cruzi). Recent findings have underscored the abundance of the causative organism, (T. cruzi), especially in the southern tier states of the US and the risk burden for the rural farming communities there. Due to a lack of safe and effective drugs, there is an urgent need for novel therapeutic options for treating Chagas disease. We report here our first scientific effort to pursue a novel drug design for treating Chagas disease via the targeting of T. cruzi tubulin. First, the anti T. cruzi tubulin activities of five naphthoquinone derivatives were determined and correlated to their anti-trypanosomal activities. The correlation between the ligand activities against the T. cruzi organism and their tubulin inhibitory activities was very strong with a Pearson's r value of 0.88 (P value cruzi tubulin polymerization inhibition. Subsequent molecular modeling studies were carried out to understand the mechanisms of the anti-tubulin activities, wherein, the homology model of T. cruzi tubulin dimer was generated and the putative binding site of naphthoquinone derivatives was predicted. The correlation coefficient for ligand anti-tubulin activities and their binding energies at the putative pocket was found to be r=0.79, a high correlation efficiency that was not replicated in contiguous candidate pockets. The homology model of T. cruzi tubulin and the identification of its putative binding site lay a solid ground for further structure based drug design, including molecular docking and pharmacophore analysis. This study presents a new opportunity for designing potent and selective drugs for Chagas disease. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Broad patterns in domestic vector-borne Trypanosoma cruzi transmission dynamics: synanthropic animals and vector control.

    Science.gov (United States)

    Peterson, Jennifer K; Bartsch, Sarah M; Lee, Bruce Y; Dobson, Andrew P

    2015-10-22

    Chagas disease (caused by Trypanosoma cruzi) is the most important neglected tropical disease (NTD) in Latin America, infecting an estimated 5.7 million people in the 21 countries where it is endemic. It is one of the NTDs targeted for control and elimination by the 2020 London Declaration goals, with the first goal being to interrupt intra-domiciliary vector-borne T. cruzi transmission. A key question in domestic T. cruzi transmission is the role that synanthropic animals play in T. cruzi transmission to humans. Here, we ask, (1) do synanthropic animals need to be targeted in Chagas disease prevention policies?, and (2) how does the presence of animals affect the efficacy of vector control? We developed a simple mathematical model to simulate domestic vector-borne T. cruzi transmission and to specifically examine the interaction between the presence of synanthropic animals and effects of vector control. We used the model to explore how the interactions between triatomine bugs, humans and animals impact the number and proportion of T. cruzi-infected bugs and humans. We then examined how T. cruzi dynamics change when control measures targeting vector abundance are introduced into the system. We found that the presence of synanthropic animals slows the speed of T. cruzi transmission to humans, and increases the sensitivity of T. cruzi transmission dynamics to vector control measures at comparable triatomine carrying capacities. However, T. cruzi transmission is amplified when triatomine carrying capacity increases with the abundance of syntathoropic hosts. Our results suggest that in domestic T. cruzi transmission scenarios where no vector control measures are in place, a reduction in synanthropic animals may slow T. cruzi transmission to humans, but it would not completely eliminate transmission. To reach the 2020 goal of interrupting intra-domiciliary T. cruzi transmission, it is critical to target vector populations. Additionally, where vector control measures

  16. Molecular epidemiology of domestic and sylvatic Trypanosoma cruzi infection in rural northwestern Argentina

    Science.gov (United States)

    Cardinal, Marta V.; Lauricella, Marta A.; Ceballos, Leonardo A.; Lanati, Leonardo; Marcet, Paula L.; Levin, Mariano J.; Kitron, Uriel; Gürtler, Ricardo E.; Schijman, Alejandro G.

    2011-01-01

    Genetic diversity of Trypanosoma cruzi populations and parasite transmission dynamics have been well documented throughout the Americas, but few studies have been conducted in the Gran Chaco ecoregion, one of the most highly endemic areas for Chagas disease, caused by T. cruzi. In this study we assessed the distribution of T. cruzi lineages (identified by PCR strategies) in Triatoma infestans, domestic dogs, cats, humans and sylvatic mammals from two neighboring rural areas with different histories of transmission and vector control in northern Argentina. Lineage II predominated among the 99 isolates characterized and lineage I among the six isolates obtained from sylvatic mammals. Trypanosoma cruzi lineage IIe predominated in domestic habitats; it was found in 87% of 54 isolates from Tr. infestans, in 82% of 33 isolates from dogs, and in the four cats found infected. Domestic and sylvatic cycles overlapped in the study area in the late 1980s, when intense domestic transmission occurred, and still overlap marginally. The introduction of T. cruzi from sylvatic into domestic habitats is likely to occur very rarely in the current epidemiological context. The household distribution of T. cruzi lineages showed that Tr. infestans, dogs and cats from a given house compound shared the same parasite lineage in most cases. Based on molecular evidence, this result lends further support to the importance of dogs and cats as domestic reservoir hosts of T. cruzi. We believe that in Argentina, this is the first time that lineage IIc has been isolated from naturally-infected domestic dogs and Tr. infestans. PMID:18585717

  17. Dimeric flavonoids from Arrabidaea brachypoda and assessment of their anti-Trypanosoma cruzi activity.

    Science.gov (United States)

    da Rocha, Cláudia Quintino; Queiroz, Emerson Ferreira; Meira, Cássio Santana; Moreira, Diogo Rodrigo Magalhães; Soares, Milena Botelho Pereira; Marcourt, Laurence; Vilegas, Wagner; Wolfender, Jean-Luc

    2014-06-27

    The nonpolar fraction of an aqueous ethanol extract of the roots of Arrabidaea brachypoda, a Brazilian medicinal plant, demonstrated significant in vitro activity against Trypanosoma cruzi, the parasite responsible for Chagas disease. Targeted isolation of the active constituents led to the isolation of three new dimeric flavonoids (1-3), and their structures were elucidated using UV, NMR, and HRMS analysis, as well as by chemical derivatization. The anti-T. cruzi activity and cytotoxicity toward mammalian cells were determined for these substances. Compound 1 exhibited no activity toward T. cruzi, while flavonoids 2 and 3 exhibited selective activity against these trypomastigotes. Compounds 2 and 3 inhibited the parasite invasion process and its intracellular development in host cells with similar potencies to benznidazole. In addition, compound 2 reduced the blood parasitemia of T. cruzi-infected mice. This study has revealed that these two dimeric flavonoids represent potential anti-T. cruzi lead compounds for further drug development.

  18. Astrocyte Apoptosis and HIV Replication Are Modulated in Host Cells Coinfected with Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Javier M. Urquiza

    2017-08-01

    Full Text Available The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease. In immunosuppressed individuals, as it occurs in the coinfection with human immunodeficiency virus (HIV, the central nervous system may be affected. In this regard, reactivation of Chagas disease is severe and often lethal, and it accounts for meningoencephalitis. Astrocytes play a crucial role in the environment maintenance of healthy neurons; however, they can host HIV and T. cruzi. In this report, human astrocytes were infected in vitro with both genetically modified-pathogens to express alternative fluorophore. As evidenced by fluorescence microscopy and flow cytometry, HIV and T. cruzi coexist in the same astrocyte, likely favoring reciprocal interactions. In this context, lower rates of cell death were observed in both T. cruzi monoinfected-astrocytes and HIV-T. cruzi coinfection in comparison with those infected only with HIV. The level of HIV replication is significantly diminished under T. cruzi coinfection, but without affecting the infectivity of the HIV progeny. This interference with viral replication appears to be related to the T. cruzi multiplication rate or its increased intracellular presence but does not require their intracellular cohabitation or infected cell-to-cell contact. Among several Th1/Th2/Th17 profile-related cytokines, only IL-6 was overexpressed in HIV-T. cruzi coinfection exhibiting its cytoprotective role. This study demonstrates that T. cruzi and HIV are able to coinfect astrocytes thus altering viral replication and apoptosis.

  19. Astrocyte Apoptosis and HIV Replication Are Modulated in Host Cells Coinfected with Trypanosoma cruzi.

    Science.gov (United States)

    Urquiza, Javier M; Burgos, Juan M; Ojeda, Diego S; Pascuale, Carla A; Leguizamón, M Susana; Quarleri, Jorge F

    2017-01-01

    The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease. In immunosuppressed individuals, as it occurs in the coinfection with human immunodeficiency virus (HIV), the central nervous system may be affected. In this regard, reactivation of Chagas disease is severe and often lethal, and it accounts for meningoencephalitis. Astrocytes play a crucial role in the environment maintenance of healthy neurons; however, they can host HIV and T. cruzi. In this report, human astrocytes were infected in vitro with both genetically modified-pathogens to express alternative fluorophore. As evidenced by fluorescence microscopy and flow cytometry, HIV and T. cruzi coexist in the same astrocyte, likely favoring reciprocal interactions. In this context, lower rates of cell death were observed in both T. cruzi monoinfected-astrocytes and HIV-T. cruzi coinfection in comparison with those infected only with HIV. The level of HIV replication is significantly diminished under T. cruzi coinfection, but without affecting the infectivity of the HIV progeny. This interference with viral replication appears to be related to the T. cruzi multiplication rate or its increased intracellular presence but does not require their intracellular cohabitation or infected cell-to-cell contact. Among several Th1/Th2/Th17 profile-related cytokines, only IL-6 was overexpressed in HIV-T. cruzi coinfection exhibiting its cytoprotective role. This study demonstrates that T. cruzi and HIV are able to coinfect astrocytes thus altering viral replication and apoptosis.

  20. Astrocyte Apoptosis and HIV Replication Are Modulated in Host Cells Coinfected with Trypanosoma cruzi

    Science.gov (United States)

    Urquiza, Javier M.; Burgos, Juan M.; Ojeda, Diego S.; Pascuale, Carla A.; Leguizamón, M. Susana; Quarleri, Jorge F.

    2017-01-01

    The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease. In immunosuppressed individuals, as it occurs in the coinfection with human immunodeficiency virus (HIV), the central nervous system may be affected. In this regard, reactivation of Chagas disease is severe and often lethal, and it accounts for meningoencephalitis. Astrocytes play a crucial role in the environment maintenance of healthy neurons; however, they can host HIV and T. cruzi. In this report, human astrocytes were infected in vitro with both genetically modified-pathogens to express alternative fluorophore. As evidenced by fluorescence microscopy and flow cytometry, HIV and T. cruzi coexist in the same astrocyte, likely favoring reciprocal interactions. In this context, lower rates of cell death were observed in both T. cruzi monoinfected-astrocytes and HIV-T. cruzi coinfection in comparison with those infected only with HIV. The level of HIV replication is significantly diminished under T. cruzi coinfection, but without affecting the infectivity of the HIV progeny. This interference with viral replication appears to be related to the T. cruzi multiplication rate or its increased intracellular presence but does not require their intracellular cohabitation or infected cell-to-cell contact. Among several Th1/Th2/Th17 profile-related cytokines, only IL-6 was overexpressed in HIV-T. cruzi coinfection exhibiting its cytoprotective role. This study demonstrates that T. cruzi and HIV are able to coinfect astrocytes thus altering viral replication and apoptosis. PMID:28824880

  1. The steady-state transcriptome of the four major life-cycle stages of Trypanosoma cruzi.

    Science.gov (United States)

    Minning, Todd A; Weatherly, D Brent; Atwood, James; Orlando, Ron; Tarleton, Rick L

    2009-08-07

    Chronic chagasic cardiomyopathy is a debilitating and frequently fatal outcome of human infection with the protozoan parasite, Trypanosoma cruzi. Microarray analysis of gene expression during the T. cruzi life-cycle could be a valuable means of identifying drug and vaccine targets based on their appropriate expression patterns, but results from previous microarray studies in T. cruzi and related kinetoplastid parasites have suggested that the transcript abundances of most genes in these organisms do not vary significantly between life-cycle stages. In this study, we used whole genome, oligonucleotide microarrays to globally determine the extent to which T. cruzi regulates mRNA relative abundances over the course of its complete life-cycle. In contrast to previous microarray studies in kinetoplastids, we observed that relative transcript abundances for over 50% of the genes detected on the T. cruzi microarrays were significantly regulated during the T. cruzi life-cycle. The significant regulation of 25 of these genes was confirmed by quantitative reverse-transcriptase PCR (qRT-PCR). The T. cruzi transcriptome also mirrored published protein expression data for several functional groups. Among the differentially regulated genes were members of paralog clusters, nearly 10% of which showed divergent expression patterns between cluster members. Taken together, these data support the conclusion that transcript abundance is an important level of gene expression regulation in T. cruzi. Thus, microarray analysis is a valuable screening tool for identifying stage-regulated T. cruzi genes and metabolic pathways.

  2. The steady-state transcriptome of the four major life-cycle stages of Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Orlando Ron

    2009-08-01

    Full Text Available Abstract Background Chronic chagasic cardiomyopathy is a debilitating and frequently fatal outcome of human infection with the protozoan parasite, Trypanosoma cruzi. Microarray analysis of gene expression during the T. cruzi life-cycle could be a valuable means of identifying drug and vaccine targets based on their appropriate expression patterns, but results from previous microarray studies in T. cruzi and related kinetoplastid parasites have suggested that the transcript abundances of most genes in these organisms do not vary significantly between life-cycle stages. Results In this study, we used whole genome, oligonucleotide microarrays to globally determine the extent to which T. cruzi regulates mRNA relative abundances over the course of its complete life-cycle. In contrast to previous microarray studies in kinetoplastids, we observed that relative transcript abundances for over 50% of the genes detected on the T. cruzi microarrays were significantly regulated during the T. cruzi life-cycle. The significant regulation of 25 of these genes was confirmed by quantitative reverse-transcriptase PCR (qRT-PCR. The T. cruzi transcriptome also mirrored published protein expression data for several functional groups. Among the differentially regulated genes were members of paralog clusters, nearly 10% of which showed divergent expression patterns between cluster members. Conclusion Taken together, these data support the conclusion that transcript abundance is an important level of gene expression regulation in T. cruzi. Thus, microarray analysis is a valuable screening tool for identifying stage-regulated T. cruzi genes and metabolic pathways.

  3. Combinations of alkaloids affecting different molecular targets with the saponin digitonin can synergistically enhance trypanocidal activity against Trypanosoma brucei brucei

    National Research Council Canada - National Science Library

    Krstin, Sonja; Peixoto, Herbenya Silva; Wink, Michael

    2015-01-01

    .... In this study, the potential synergism of mutual combinations of bioactive alkaloids and alkaloids with a membrane-active steroidal saponin, digitonin, was explored with regard to their effect on T. b. brucei...

  4. Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Ludmila R.P. Ferreira

    2008-03-01

    Full Text Available The differentiation of proliferating epimastigote forms of Trypanosoma cruzi , the protozoan parasite that causes Chagas’ disease, into the infective and non-proliferating metacyclic forms can be reproduced in the laboratory by incubating the cells in a chemically-defined medium that mimics the urine of the insect vector. Epimastigotes have a spherical nucleus, a flagellum protruding from the middle of the protozoan cell, and a disk-shaped kinetoplast - an organelle that corresponds to the mitochondrial DNA. Metacyclic trypomastigotes have an elongated shape with the flagellum protruding from the posterior portion of the cell and associated with a spherical kinetoplast. Here we describe the morphological events of this transformation and characterize a novel intermediate stage by three-dimensional reconstruction of electron microscope serial sections. This new intermediate stage is characterized by a kinetoplast compressing an already elongated nucleus, indicating that metacyclogenesis involves active movements of the flagellar structure relative to the cell body. As transcription occurs more intensely in proliferating epimastigotes than in metacyclics, we also examined the presence of RNA polymerase II and measured transcriptional activity during the differentiation process. Both the presence of the enzyme and transcriptional activity remain unchanged during all steps of metacyclogenesis. RNA polymerase II levels and transcriptional activity only decrease after metacyclics are formed. We suggest that transcription is required during the epimastigote-to-metacyclic trypomastigote differentiation process, until the kinetoplast and flagellum reach the posterior position of the parasites in the infective form.A diferenciação de formas epimastigotas (proliferativas do Trypanosoma cruzi, parasita protozoário causador da doença de Chagas, em formas metacíclicas tripomastigotas (infectivas e não proliferativas, pode ser reproduzida em laborat

  5. Chemopreventive effect of methanolic extract of Azadirachta indica on experimental Trypanosoma brucei induced oxidative stress in dogs.

    Science.gov (United States)

    Omobowale, Temidayo O; Oyagbemi, Ademola A; Oyewunmi, Oyefunbi A; Adejumobi, Olumuyiwa A

    2015-01-01

    The medicinal properties of Azadirachta indica have been harnessed for many years in the treatment of many diseases in both humans and animals. Twenty-five apparently healthy dogs weighing between 3 and 8 kg were randomly divided into five groups with five dogs in each group. Ameliorative effect of A. indica on erythrocyte antioxidant status and markers of oxidative stress were assessed. Liver and kidney function tests were also performed. Pre-treatment with methanolic extract of Azadirachta indica (MEAI) at different doses did not significantly alter the values of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activity in Trypanosoma brucei infection. Although, serum creatinine significantly (P indica, after 2 weeks of T. brucei infection. However, the reduced glutathione (GSH) content of the erythrocyte increased significantly in animals pre-treated with 50 mg/kg and 200 mg/kg of A. indica respectively. Markers of oxidative stress such as malondialdehyde and hydrogen peroxide generated were higher in animals infected with T. brucei with no significant (P >0.05) difference compared to the values obtained in pre-treated animals. Pre-treatment with 100 mg/kg and 200 mg/kg of A. indica significantly (P < 0.05) decreased serum myeloperoxidase activity at 2 weeks post-infection with T. brucei. From this study, MEAI showed significant ability to attenuate oxidative stress and inflammation during experimental T. brucei infection.

  6. Maxi-circles and mini-circles in kinetoplast DNA from Trypanosoma cruzi.

    NARCIS (Netherlands)

    W. Leon; A.C.C. Frasch; J.H.J. Hoeijmakers (Jan); F. Fase-Fowler; P. Borst (Piet); F. Brunel; J. Davison

    1980-01-01

    textabstractMaxi-circles are a minor component of kinetoplast DNAs from all trypanosomatids studied, but they have not previously been found in Trypanosoma cruzi; We have spread intact kinetoplast DNA from the epimastigotes of strain Y in protein monolayers and analysed the mini-circle networks by

  7. Kinetic properties and inhibition of Trypanosoma cruzi 3-hydroxy-3-methylglutaryl CoA reductase

    DEFF Research Database (Denmark)

    Hurtado-Guerrrero, Ramón; Pena Diaz, Javier; Montalvetti, Andrea

    2002-01-01

    A detailed kinetic analysis of the recombinant soluble enzyme 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) from Trypanosoma cruzi has been performed. The enzyme catalyzes the normal anabolic reaction and the reductant is NADPH. It also catalyzes the oxidation of mevalonate but at a lower...

  8. The effect of the diterpene 5-epi-icetexone on the cell cycle of Trypanosoma cruzi.

    NARCIS (Netherlands)

    Lozano, E.; Barrera, P.; Tonn, C.; Nieto, M.; Sartor, T.; Sosa, M.A.

    2012-01-01

    Numerous natural compounds have been used against Trypanosoma cruzi, the causative agent of Chagas' disease. Here, we studied the effect of the diterpene 5-epi-icetexone on growth and morphology of parasites synchronized with hydroxyurea, at different periods of time after removal of the nucleotide.

  9. Enzyme catalysed production of sialylated human milk oligosaccharides and galactooligosaccharides by Trypanosoma cruzi trans-sialidase

    DEFF Research Database (Denmark)

    Holck, Jesper; Larsen, Dorte Møller; Michalak, Malwina

    2014-01-01

    A Trypanosoma cruzi trans-sialidase (E.C. 3.2.1.18) was cloned into Pichia pastoris and expressed. The pH and temperature optimum of the enzyme was determined as pH 5.7 and 30°C. Using casein glycomacropeptide (CGMP) and lactose as sialyl-donor and acceptor respectively, the optimal donor...

  10. Galactonolactone oxidoreductase from Trypanosoma cruzi employs a FAD cofactor for the synthesis of vitamin C.

    NARCIS (Netherlands)

    Kudryashova, E.V.; Leferink, N.G.H.; Slot, I.G.M.; Berkel, van W.J.H.

    2011-01-01

    Trypanosoma cruzi, the aetiological agent of Chagas' disease, is unable to salvage vitamin C (l-ascorbate) from its environment and relies on de novo synthesis for its survival. Because humans lack the capacity to synthesize ascorbate, the trypanosomal enzymes involved in ascorbate biosynthesis are

  11. Efficacy of some essential oils in mice infected with Trypanosoma cruzi

    African Journals Online (AJOL)

    Purpose: To evaluate the efficacy of orally administered Cymbopogon citratus, Zingiber officinale and Syzygium aromaticum essential oils (EOs) in mice infected with Trypanosoma cruzi. Methods: Three experiments were conducted with 48 Swiss mice each. The animals were inoculated with 2 x 106 metacyclic ...

  12. Chagas' disease: humoral response to subcellular fraction of Trypanosoma cruzi in symptomatic and asymptomatic patients.

    Science.gov (United States)

    de Titto, E H; Moreno, M; Braun, M; Segura, E L

    1987-09-01

    The capacity of antibodies in serum from individuals with chronic Chagas' disease to react with antigens in different subcellular fractions of Trypanosoma cruzi varied according to the clinical status of the patients. Antibodies in serum of asymptomatic patients were directed mostly against antigens in the citosol of the parasite, whereas in overtly cardiopathic patients antibodies were directed mostly against antigens in the microsomal fractions.

  13. Aspects of resistance to experimental infection with Trypanosoma cruzi; Aspectos da resistencia a infecao experimental com Trypanosoma cruzi

    Energy Technology Data Exchange (ETDEWEB)

    Dias, Viviane Liotti

    2010-07-01

    Chagas disease, a zoonosis caused by the protozoan Trypanosoma cruzi, has a wide distribution in Latin America and extends from the southern part of the United States to Argentina. A number of 10 million of infected people is estimated and another 25 million exposed to the risk. Although discovered over a century, Chagas disease is still a serious infection that causes great socioeconomic impact, with no effective treatment at the chronic phase and in which, a lack of scientific knowledge can be observed. The main goal of this work was that obtaining and using consomic strain of mice, the resistance could be investigated. Consomic strains were produced by programmed mating, in which the animals were monitored with DNA polymorphic markers, and one of his chromosomes was replaced by his homologue from another strain. As parental, were used, the inbred strains C57BL/6/J Unib with resistant phenotype (donor) and as receiver, the A/JUnib strain, that has a susceptible phenotype. These models were used to produce five consomic strains: for the chromosomes 7 (CSs7), 11 (CSs11), 14 (CSs14), 17 (CSs17) and 19 (CSs19), described by Passos et al. (2003) as important in controlling infection caused by the Y strain of T. cruzi. In experimental testing, the consomics were inoculated intraperitoneally at doses of 10{sup 1}, 10{sup 2}, 10{sup 3} and 10{sup 4} using as control, animals from both parental lines. In all consomics, resistance was higher than that observed in the susceptible parental. In a second protocol, the consomics were mated with scheduled associations and the progenies were challenged with inocula employing increasing doses of trypomastigotes. The resistance observed in this group was also higher than that observed in the parental with susceptible phenotype. The observed results demonstrate that the use of the consomic strains that were produced order to assess the contribution of each chromosome in the resistance, as well as the effects of association between

  14. The isolation and identification of Trypanosoma cruzi from raccoons in Maryland

    Science.gov (United States)

    Walton, B.C.; Bauman, P.M.; Diamond, L.S.; Herman, C.M.

    1958-01-01

    Five raccoons trapped at Patuxent Research Refuge, Laurel, Maryland, were found to have trypanosomes in the blood which were morphologically indistinguishable from Trypanosoma cruzi on stained smears. The organism grew well in culture. It developed and reproduced in Triatoma protracta, T. infestans, T. phyllosoma, and Rhodnius prolixus. Experimental infections were produced in raccoons, opossums, mice, rats, and monkeys by inoculation of blood, culture, and triatome forms. Typical leishmaniform bodies were found in tissue sections of cardiac muscle fibers from naturally and experimentally infected animals. Cross agglutinations carried out with Iiving cultural forms and rabbit antisera demonstrated a close antigenic relationship between the raccoon trypanosome and T. cruzi (Brazil strain). On the basis of (1) morphology, (2) presence of leishmaniform tissue stages, (3) development in triatomes, (4) infectivity to a variety of mammals, (5) culture characteristics, and (6) cross reactions in serological tests, this parasite is considered conspecific with Trypanosoma cruzi (Chagas, 1909), the causative agent of American human trypanosomiasis.

  15. Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Abdulsalam A.M. Alkhaldi

    2016-04-01

    Full Text Available Lipophilic bisphosphonium salts are among the most promising antiprotozoal leads currently under investigation. As part of their preclinical evaluation we here report on their mode of action against African trypanosomes, the etiological agents of sleeping sickness. The bisphosphonium compounds CD38 and AHI-9 exhibited rapid inhibition of Trypanosoma brucei growth, apparently the result of cell cycle arrest that blocked the replication of mitochondrial DNA, contained in the kinetoplast, thereby preventing the initiation of S-phase. Incubation with either compound led to a rapid reduction in mitochondrial membrane potential, and ATP levels decreased by approximately 50% within 1 h. Between 4 and 8 h, cellular calcium levels increased, consistent with release from the depolarized mitochondria. Within the mitochondria, the Succinate Dehydrogenase complex (SDH was investigated as a target for bisphosphonium salts, but while its subunit 1 (SDH1 was present at low levels in the bloodstream form trypanosomes, the assembled complex was hardly detectable. RNAi knockdown of the SDH1 subunit produced no growth phenotype, either in bloodstream or in the procyclic (insect forms and we conclude that in trypanosomes SDH is not the target for bisphosphonium salts. Instead, the compounds inhibited ATP production in intact mitochondria, as well as the purified F1 ATPase, to a level that was similar to 1 mM azide. Co-incubation with azide and bisphosphonium compounds did not inhibit ATPase activity more than either product alone. The results show that, in T. brucei, bisphosphonium compounds do not principally act on succinate dehydrogenase but on the mitochondrial FoF1 ATPase.

  16. Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei

    Science.gov (United States)

    Alkhaldi, Abdulsalam A.M.; Martinek, Jan; Panicucci, Brian; Dardonville, Christophe; Zíková, Alena; de Koning, Harry P.

    2015-01-01

    Lipophilic bisphosphonium salts are among the most promising antiprotozoal leads currently under investigation. As part of their preclinical evaluation we here report on their mode of action against African trypanosomes, the etiological agents of sleeping sickness. The bisphosphonium compounds CD38 and AHI-9 exhibited rapid inhibition of Trypanosoma brucei growth, apparently the result of cell cycle arrest that blocked the replication of mitochondrial DNA, contained in the kinetoplast, thereby preventing the initiation of S-phase. Incubation with either compound led to a rapid reduction in mitochondrial membrane potential, and ATP levels decreased by approximately 50% within 1 h. Between 4 and 8 h, cellular calcium levels increased, consistent with release from the depolarized mitochondria. Within the mitochondria, the Succinate Dehydrogenase complex (SDH) was investigated as a target for bisphosphonium salts, but while its subunit 1 (SDH1) was present at low levels in the bloodstream form trypanosomes, the assembled complex was hardly detectable. RNAi knockdown of the SDH1 subunit produced no growth phenotype, either in bloodstream or in the procyclic (insect) forms and we conclude that in trypanosomes SDH is not the target for bisphosphonium salts. Instead, the compounds inhibited ATP production in intact mitochondria, as well as the purified F1 ATPase, to a level that was similar to 1 mM azide. Co-incubation with azide and bisphosphonium compounds did not inhibit ATPase activity more than either product alone. The results show that, in T. brucei, bisphosphonium compounds do not principally act on succinate dehydrogenase but on the mitochondrial FoF1 ATPase. PMID:27054061

  17. Protein functional links in Trypanosoma brucei, identified by gene fusion analysis

    Directory of Open Access Journals (Sweden)

    Trimpalis Philip

    2011-07-01

    Full Text Available Abstract Background Domain or gene fusion analysis is a bioinformatics method for detecting gene fusions in one organism by comparing its genome to that of other organisms. The occurrence of gene fusions suggests that the two original genes that participated in the fusion are functionally linked, i.e. their gene products interact either as part of a multi-subunit protein complex, or in a metabolic pathway. Gene fusion analysis has been used to identify protein functional links in prokaryotes as well as in eukaryotic model organisms, such as yeast and Drosophila. Results In this study we have extended this approach to include a number of recently sequenced protists, four of which are pathogenic, to identify fusion linked proteins in Trypanosoma brucei, the causative agent of African sleeping sickness. We have also examined the evolution of the gene fusion events identified, to determine whether they can be attributed to fusion or fission, by looking at the conservation of the fused genes and of the individual component genes across the major eukaryotic and prokaryotic lineages. We find relatively limited occurrence of gene fusions/fissions within the protist lineages examined. Our results point to two trypanosome-specific gene fissions, which have recently been experimentally confirmed, one fusion involving proteins involved in the same metabolic pathway, as well as two novel putative functional links between fusion-linked protein pairs. Conclusions This is the first study of protein functional links in T. brucei identified by gene fusion analysis. We have used strict thresholds and only discuss results which are highly likely to be genuine and which either have already been or can be experimentally verified. We discuss the possible impact of the identification of these novel putative protein-protein interactions, to the development of new trypanosome therapeutic drugs.

  18. In vitro drug susceptibility of two strains of the wildlife trypanosome, Trypanosoma copemani: A comparison with Trypanosoma cruzi.

    Science.gov (United States)

    Botero, Adriana; Keatley, Sarah; Peacock, Christopher; Thompson, R C Andrew

    2017-04-01

    Trypanosomes are blood protozoan parasites that are capable of producing illness in the vertebrate host. Within Australia, several native Trypanosoma species have been described infecting wildlife. However, only Trypanosoma copemani has been associated with pathological lesions in wildlife hosts and more recently has been associated with the drastic decline of the critically endangered woylie (Bettongia penicillata). The impact that some trypanosomes have on the health of the vertebrate host has led to the development of numerous drug compounds that could inhibit the growth or kill the parasite. This study investigated and compared the in vitro susceptibility of two strains of T. copemani (G1 and G2) and one strain of Trypanosoma cruzi (10R26) against drugs that are known to show trypanocidal activity (benznidazole, posaconazole, miltefosine and melarsoprol) and against four lead compounds, two fenarimols and two pyridine derivatives (EPL-BS1937, EPL-BS2391, EPL-BS0967, and EPL-BS1246), that have been developed primarily against T.cruzi. The in vitro cytotoxicity of all drugs against L6 rat myoblast cells was also assessed. Results showed that both strains of T. copemani were more susceptible to all drugs and lead compounds than T. cruzi, with all IC50 values in the low and sub-μM range for both species. Melarsoprol and miltefosine exhibited the highest drug activity against both T. copemani and T. cruzi, but they also showed the highest toxicity in L6 cells. Interestingly, both fenarimol and pyridine derivative compounds were more active against T. copemani and T. cruzi than the reference drugs benznidazole and posaconazole. T. copemani strains exhibited differences in susceptibility to all drugs demonstrating once again considerable differences in their biological behaviour. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  19. A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi

    DEFF Research Database (Denmark)

    Pena Diaz, Javier; Montalvetti, A; Camacho, A

    1997-01-01

    We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease. The protozoan gene encoded for a smaller polypeptide than the rest...... sensitive to proteolytic inactivation. Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c. Thus Trypanosoma cruzi HMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG...... cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts. Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly...

  20. CRISPR/Cas9-mediated endogenous C-terminal Tagging of Trypanosoma cruzi Genes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor.

    Science.gov (United States)

    Lander, Noelia; Chiurillo, Miguel A; Storey, Melissa; Vercesi, Anibal E; Docampo, Roberto

    2016-12-02

    Methods for genetic manipulation of Trypanosoma cruzi, the etiologic agent of Chagas disease, have been highly inefficient, and no endogenous tagging of genes has been reported to date. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for endogenously tagging genes in this parasite. The utility of the method was established by tagging genes encoding proteins of known localization such as TcFCaBP (flagellar calcium binding protein) and TcVP1 (vacuolar proton pyrophosphatase), and two proteins of undefined or disputed localization, the TcMCU (mitochondrial calcium uniporter) and TcIP 3 R (inositol 1,4,5-trisphosphate receptor). We confirmed the flagellar and acidocalcisome localization of TcFCaBP and TcVP1 by co-localization with antibodies to the flagellum and acidocalcisomes, respectively. As expected, TcMCU was co-localized with the voltage-dependent anion channel to the mitochondria. However, in contrast to previous reports and our own results using overexpressed TcIP 3 R, endogenously tagged TcIP 3 R showed co-localization with antibodies against VP1 to acidocalcisomes. These results are also in agreement with our previous reports on the localization of this channel to acidocalcisomes of Trypanosoma brucei and suggest that caution should be exercised when overexpression of tagged genes is done to localize proteins in T. cruzi. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Trypanosoma cruzi over the ocean: Insular zones of Chile with presence of infected vector Mepraia species.

    Science.gov (United States)

    Rives-Blanchard, Ninette; Torres-Pérez, Fernando; Ortiz, Silvia; Solari, Aldo; Campos-Soto, Ricardo

    2017-08-01

    Chagas disease is one of the main zoonosis mediated by vectors in America. The etiologic agent Trypanosoma cruzi infects different mammals and is transmitted principally by the subfamily Triatominae. Mepraia is a genus endemic to Chile, responsible for transmitting T. cruzi in the sylvatic cycle. Mepraia includes three species: M. gajardoi and M. parapatrica inhabit coastal areas, while M. spinolai inhabits coastal and interior valleys. Previous studies reported the occurrence of Mepraia in Pan de Azucar Island, currently classified as M. parapatrica, but T. cruzi has not been reported in these insects. It is suggested that this could be due to infrequent insect feeding on mammalian hosts. In order to detect T. cruzi in insects from coastal islands, specimens from Pan de Azucar and Santa Maria Islands were examined. A region of kDNA of T. cruzi was amplified by PCR and hybridization assays were performed for T. cruzi genotyping of insect feces. The presence of infected insect and mixed T. cruzi infections was demonstrated. This is the first report of infected Triatominae in coastal islands in Chile. We discuss T. cruzi detection in insular zones, and the presumptive reservoirs that may participate in maintaining its transmission cycle in this habitat. Mixed and unidentified infections suggest that there are complex and unknown reservoir interactions in these habitats. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Canine Macrophage DH82 Cell Line As a Model to Study Susceptibility to Trypanosoma cruzi Infection

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    Pedro Henrique Braz Mendonça

    2017-05-01

    Full Text Available Trypanosoma cruzi is an obligatory intracellular protozoan parasite, and it is the etiological agent of Chagas’ disease that is endemic in the Americas. In addition to humans, a wide spectrum of mammals can be infected by T. cruzi, including dogs. Dogs develop acute and chronic disease, similar to human infection. T. cruzi can infect almost all cell types and after cell invasion, the metacyclics trypomastigotes localize in the cytoplasm, where they transform into amastigotes, the replicative form of T. cruzi in mammals. After amastigote multiplication and differentiation, parasites lyse host cells and spread through the body by blood circulation. In this work, we evaluated the in vitro ability of T. cruzi to infect a canine macrophage cell line DH82 compared with RAW264.7, a murine tissue culture macrophage. Our results have shown that the T. cruzi is able to infect, replicate and differentiate in DH82 cell line. We observed that following treatment with LPS and IFN-γ DH82 cells were more resistant to infection and that resistance was not related reactive oxygen species production in our system. In this study, we also found that DH82 cells became more susceptible to T. cruzi infection when cocultured with apoptotic cells. The analysis of cytokine production has showed elevated levels of the TGF-β, IL-10, and TNF-α produced by T. cruzi-infected canine macrophages. Additionally, we demonstrated a reduced expression of the MHC class II and CD80 by infected DH82 cell line.

  3. Channel-forming activities in the glycosomal fraction from the bloodstream form of Trypanosoma brucei.

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    Melisa Gualdron-López

    Full Text Available BACKGROUND: Glycosomes are a specialized form of peroxisomes (microbodies present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear. METHODS/PRINCIPAL FINDINGS: We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T. brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70-80 pA, 20-25 pA, and 8-11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte. All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20-25 pA is anion-selective (P(K+/P(Cl-∼0.31, while the other two types of channels are slightly selective for cations (P(K+/P(Cl- ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively. The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel's pore. CONCLUSIONS/SIGNIFICANCE: These results indicate that the membrane of glycosomes

  4. Semisolid liver infusion tryptose supplemented with human urine allows growth and isolation of Trypanosoma cruzi and Trypanosoma rangeli clonal lineages

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    Emanuella Francisco Fajardo

    2016-06-01

    Full Text Available Abstract: INTRODUCTION This work shows that 3% (v/v human urine (HU in semisolid Liver Infusion Tryptose (SSL medium favors the growth of Trypanosoma cruzi and T. rangeli. METHODS Parasites were plated as individual or mixed strains on SSL medium and on SSL medium with 3% human urine (SSL-HU. Isolate DNA was analyzed using polymerase chain reaction (PCR and pulsed-field gel electrophoresis (PFGE. RESULTS SSL-HU medium improved clone isolation. PCR revealed that T. cruzi strains predominate on mixed-strain plates. PFGE confirmed that isolated parasites share the same molecular karyotype as parental cell lines. CONCLUSIONS SSL-HU medium constitutes a novel tool for obtaining T. cruzi and T. rangeli clonal lineages.

  5. Interactions Between Trypanosoma cruzi the Chagas Disease Parasite and Naturally Infected Wild Mepraia Vectors of Chile.

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    Campos-Soto, Ricardo; Ortiz, Sylvia; Cordova, Ivan; Bruneau, Nicole; Botto-Mahan, Carezza; Solari, Aldo

    2016-03-01

    Chagas disease, which ranks among the world's most neglected diseases, is a chronic, systemic, parasitic infection caused by the protozoan Trypanosoma cruzi. Mepraia species are the wild vectors of this parasite in Chile. Host-parasite interactions can occur at several levels, such as co-speciation and ecological host fitting, among others. Thus, we are exploring the interactions between T. cruzi circulating in naturally infected Mepraia species in all areas endemic of Chile. We evaluated T. cruzi infection rates of 27 different haplotypes of the wild Mepraia species and identified their parasite genotypes using minicircle PCR amplification and hybridization tests with genotype-specific DNA probes. Infection rates were lower in northern Chile where Mepraia gajardoi circulates (10-35%); in central Chile, Mepraia spinolai is most abundant, and infection rates varied in space and time (0-55%). T. cruzi discrete typing units (DTUs) TcI, TcII, TcV, and Tc VI were detected. Mixed infections with two or more DTUs are frequently found in highly infected insects. T. cruzi DTUs have distinct, but not exclusive, ecological and epidemiological associations with their hosts. T. cruzi infection rates of M. spinolai were higher than in M. gajardoi, but the presence of mixed infection with more than one T. cruzi DTU was the same. The same T. cruzi DTUs (TcI, TcII, TcV, and TcVI) were found circulating in both vector species, even though TcI was not equally distributed. These results suggest that T. cruzi DTUs are not associated with any of the two genetically related vector species nor with the geographic area. The T. cruzi vectors interactions are discussed in terms of old and recent events. By exploring T. cruzi DTUs present in Mepraia haplotypes and species from northern to central Chile, we open the analysis on these invertebrate host-parasite interactions.

  6. Transcriptome Remodeling in Trypanosoma cruzi and Human Cells during Intracellular Infection

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    Li, Yuan; Shah-Simpson, Sheena; Okrah, Kwame; Belew, A. Trey; Choi, Jungmin; Caradonna, Kacey L.; Padmanabhan, Prasad; Ndegwa, David M.; Temanni, M. Ramzi; Corrada Bravo, Héctor; El-Sayed, Najib M.; Burleigh, Barbara A.

    2016-01-01

    Intracellular colonization and persistent infection by the kinetoplastid protozoan parasite, Trypanosoma cruzi, underlie the pathogenesis of human Chagas disease. To obtain global insights into the T. cruzi infective process, transcriptome dynamics were simultaneously captured in the parasite and host cells in an infection time course of human fibroblasts. Extensive remodeling of the T. cruzi transcriptome was observed during the early establishment of intracellular infection, coincident with a major developmental transition in the parasite. Contrasting this early response, few additional changes in steady state mRNA levels were detected once mature T. cruzi amastigotes were formed. Our findings suggest that transcriptome remodeling is required to establish a modified template to guide developmental transitions in the parasite, whereas homeostatic functions are regulated independently of transcriptomic changes, similar to that reported in related trypanosomatids. Despite complex mechanisms for regulation of phenotypic expression in T. cruzi, transcriptomic signatures derived from distinct developmental stages mirror known or projected characteristics of T. cruzi biology. Focusing on energy metabolism, we were able to validate predictions forecast in the mRNA expression profiles. We demonstrate measurable differences in the bioenergetic properties of the different mammalian-infective stages of T. cruzi and present additional findings that underscore the importance of mitochondrial electron transport in T. cruzi amastigote growth and survival. Consequences of T. cruzi colonization for the host include dynamic expression of immune response genes and cell cycle regulators with upregulation of host cholesterol and lipid synthesis pathways, which may serve to fuel intracellular T. cruzi growth. Thus, in addition to the biological inferences gained from gene ontology and functional enrichment analysis of differentially expressed genes in parasite and host, our

  7. Transcriptome Remodeling in Trypanosoma cruzi and Human Cells during Intracellular Infection.

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    Yuan Li

    2016-04-01

    Full Text Available Intracellular colonization and persistent infection by the kinetoplastid protozoan parasite, Trypanosoma cruzi, underlie the pathogenesis of human Chagas disease. To obtain global insights into the T. cruzi infective process, transcriptome dynamics were simultaneously captured in the parasite and host cells in an infection time course of human fibroblasts. Extensive remodeling of the T. cruzi transcriptome was observed during the early establishment of intracellular infection, coincident with a major developmental transition in the parasite. Contrasting this early response, few additional changes in steady state mRNA levels were detected once mature T. cruzi amastigotes were formed. Our findings suggest that transcriptome remodeling is required to establish a modified template to guide developmental transitions in the parasite, whereas homeostatic functions are regulated independently of transcriptomic changes, similar to that reported in related trypanosomatids. Despite complex mechanisms for regulation of phenotypic expression in T. cruzi, transcriptomic signatures derived from distinct developmental stages mirror known or projected characteristics of T. cruzi biology. Focusing on energy metabolism, we were able to validate predictions forecast in the mRNA expression profiles. We demonstrate measurable differences in the bioenergetic properties of the different mammalian-infective stages of T. cruzi and present additional findings that underscore the importance of mitochondrial electron transport in T. cruzi amastigote growth and survival. Consequences of T. cruzi colonization for the host include dynamic expression of immune response genes and cell cycle regulators with upregulation of host cholesterol and lipid synthesis pathways, which may serve to fuel intracellular T. cruzi growth. Thus, in addition to the biological inferences gained from gene ontology and functional enrichment analysis of differentially expressed genes in parasite and

  8. Regulation and use of the extracellular matrix by Trypanosoma cruzi during early infection

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    Pius N. Nde

    2012-11-01

    Full Text Available Chagas disease, which was once thought to be confined to endemic regions of Latin America, has now gone global becoming a new worldwide challenge. For more than a century since its discovery, it has remained neglected with no effective drugs or vaccines. The mechanisms by which Trypanosoma cruzi regulates and uses the extracellular matrix to invade cells and cause disease are just beginning to be understood. Here we critically review and discuss the regulation of the extracellular matrix (ECM interactome by T. cruzi, the use of the ECM by T. cruzi and analyze the molecular ECM/T. cruzi interphase during the early process of infection. It has been shown that invasive trypomastigote forms of T. cruzi use and modulate components of the ECM during the initial process of infection. Infective trypomastigotes up-regulate the expression of laminin γ-1 (LAMC1 and thrombospondin (THBS1 to facilitate the recruitment of trypomastigotes to enhance cellular infection. Silencing the expression of LAMC1 and THBS1 by stable RNAi dramatically reduces trypanosome infection. T. cruzi gp83, a ligand that mediates the attachment of trypanosomes to cells to initiate infection, up-regulates LAMC1 expression to enhance cellular infection. Infective trypomastigotes use Tc85 to interact with laminin, p45 mucin to interact with LAMC1 through galectin-3 (LGALS3, a human lectin, and calreticulin (TcCRT to interact with TSB1 to enhance cellular infection. Silencing the expression of LGALS3 also reduces cellular infection. Despite the role of the ECM in T. cruzi infection, almost nothing is known about the ECM interactome networks operating in the process of T. cruzi infection and its ligands. Here, we present the first elucidation of the human ECM interactome network regulated by T. cruzi and its gp83 ligand that facilitates cellular infection. The elucidation of the human ECM interactome regulated by T. cruzi and the dissection of the molecular ECM/T. cruzi interphase using

  9. Mechanism of Trypanosoma cruzi Placenta Invasion and Infection: The Use of Human Chorionic Villi Explants

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    Ricardo E. Fretes

    2012-01-01

    Full Text Available Congenital Chagas disease, a neglected tropical disease, endemic in Latin America, is associated with premature labor and miscarriage. During vertical transmission the parasite Trypanosoma cruzi (T. cruzi crosses the placental barrier. However, the exact mechanism of the placental infection remains unclear. We review the congenital transmission of T. cruzi, particularly the role of possible local placental factors that contribute to the vertical transmission of the parasite. Additionally, we analyze the different methods available for studying the congenital transmission of the parasite. In that context, the ex vivo infection with T. cruzi trypomastigotes of human placental chorionic villi constitutes an excellent tool for studying parasite infection strategies as well as possible local antiparasitic mechanisms.

  10. A Brief View of the Surface Membrane Proteins from Trypanosoma cruzi

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    Pech-Canul, Ángel de la Cruz

    2017-01-01

    Trypanosoma cruzi is the causal agent of Chagas' disease which affects millions of people around the world mostly in Central and South America. T. cruzi expresses a wide variety of proteins on its surface membrane which has an important role in the biology of these parasites. Surface molecules of the parasites are the result of the environment to which the parasites are exposed during their life cycle. Hence, T. cruzi displays several modifications when they move from one host to another. Due to the complexity of this parasite's cell surface, this review presents some membrane proteins organized as large families, as they are the most abundant and/or relevant throughout the T. cruzi membrane. PMID:28656101

  11. Nanoemulsions of sulfonamide carbonic anhydrase inhibitors strongly inhibit the growth of Trypanosoma cruzi.

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    Vermelho, Alane Beatriz; da Silva Cardoso, Verônica; Ricci Junior, Eduardo; Dos Santos, Elisabete Pereira; Supuran, Claudiu T

    2018-12-01

    Sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors targeting the α-class enzyme from the protozoan pathogen Trypanosoma cruzi, responsible of Chagas disease, were recently reported. Although many such derivatives showed low nanomolar activity in vitro, they were inefficient anti-T. cruzi agents in vivo. Here, we show that by formulating such sulfonamides as nanoemulsions in clove (Eugenia caryophyllus) oil, highly efficient anti-protozoan effects are observed against two different strains of T. cruzi. These effects are probably due to an enhanced permeation of the enzyme inhibitor through the nanoemulsion formulation, interfering in this way with the life cycle of the pathogen either by inhibiting pH regulation or carboxylating reactions in which bicarbonate/CO 2 are involved. This type of formulation of sulfonamides with T. cruzi CA inhibitory effects may lead to novel therapeutic approaches against this orphan disease.

  12. The Role of Heme and Reactive Oxygen Species in Proliferation and Survival of Trypanosoma cruzi

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    Marcia Cristina Paes

    2011-01-01

    Full Text Available Trypanosoma cruzi, the protozoan responsible for Chagas disease, has a complex life cycle comprehending two distinct hosts and a series of morphological and functional transformations. Hemoglobin degradation inside the insect vector releases high amounts of heme, and this molecule is known to exert a number of physiological functions. Moreover, the absence of its complete biosynthetic pathway in T. cruzi indicates heme as an essential molecule for this trypanosomatid survival. Within the hosts, T. cruzi has to cope with sudden environmental changes especially in the redox status and heme is able to increase the basal production of reactive oxygen species (ROS which can be also produced as byproducts of the parasite aerobic metabolism. In this regard, ROS sensing is likely to be an important mechanism for the adaptation and interaction of these organisms with their hosts. In this paper we discuss the main features of heme and ROS susceptibility in T. cruzi biology.

  13. The phosphoarginine energy-buffering system of trypanosoma brucei involves multiple arginine kinase isoforms with different subcellular locations.

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    Frank Voncken

    Full Text Available Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3. Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide 'SNL'. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90% of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed.

  14. The Phosphoarginine Energy-Buffering System of Trypanosoma brucei Involves Multiple Arginine Kinase Isoforms with Different Subcellular Locations

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    Wadforth, Cath; Harley, Maggie; Colasante, Claudia

    2013-01-01

    Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3). Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide ‘SNL’. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90%) of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed. PMID:23776565

  15. Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty.

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    Jason Carnes

    2015-01-01

    Full Text Available Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA. In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.

  16. Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty.

    Science.gov (United States)

    Carnes, Jason; Anupama, Atashi; Balmer, Oliver; Jackson, Andrew; Lewis, Michael; Brown, Rob; Cestari, Igor; Desquesnes, Marc; Gendrin, Claire; Hertz-Fowler, Christiane; Imamura, Hideo; Ivens, Alasdair; Kořený, Luděk; Lai, De-Hua; MacLeod, Annette; McDermott, Suzanne M; Merritt, Chris; Monnerat, Severine; Moon, Wonjong; Myler, Peter; Phan, Isabelle; Ramasamy, Gowthaman; Sivam, Dhileep; Lun, Zhao-Rong; Lukeš, Julius; Stuart, Ken; Schnaufer, Achim

    2015-01-01

    Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.

  17. Chimerization at the AQP2–AQP3 locus is the genetic basis of melarsoprol–pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates

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    Fabrice E. Graf

    2015-08-01

    Full Text Available Aquaglyceroporin-2 is a known determinant of melarsoprol–pentamidine cross-resistance in Trypanosoma brucei brucei laboratory strains. Recently, chimerization at the AQP2–AQP3 tandem locus was described from melarsoprol–pentamidine cross-resistant Trypanosoma brucei gambiense isolates from sleeping sickness patients in the Democratic Republic of the Congo. Here, we demonstrate that reintroduction of wild-type AQP2 into one of these isolates fully restores drug susceptibility while expression of the chimeric AQP2/3 gene in aqp2–aqp3 null T. b. brucei does not. This proves that AQP2–AQP3 chimerization is the cause of melarsoprol–pentamidine cross-resistance in the T. b. gambiense isolates.

  18. Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins.

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    Cornelia Klein

    Full Text Available We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites.

  19. Dynamic modelling under uncertainty: the case of Trypanosoma brucei energy metabolism.

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    Fiona Achcar

    2012-01-01

    Full Text Available Kinetic models of metabolism require detailed knowledge of kinetic parameters. However, due to measurement errors or lack of data this knowledge is often uncertain. The model of glycolysis in the parasitic protozoan Trypanosoma brucei is a particularly well analysed example of a quantitative metabolic model, but so far it has been studied with a fixed set of parameters only. Here we evaluate the effect of parameter uncertainty. In order to define probability distributions for each parameter, information about the experimental sources and confidence intervals for all parameters were collected. We created a wiki-based website dedicated to the detailed documentation of this information: the SilicoTryp wiki (http://silicotryp.ibls.gla.ac.uk/wiki/Glycolysis. Using information collected in the wiki, we then assigned probability distributions to all parameters of the model. This allowed us to sample sets of alternative models, accurately representing our degree of uncertainty. Some properties of the model, such as the repartition of the glycolytic flux between the glycerol and pyruvate producing branches, are robust to these uncertainties. However, our analysis also allowed us to identify fragilities of the model leading to the accumulation of 3-phosphoglycerate and/or pyruvate. The analysis of the control coefficients revealed the importance of taking into account the uncertainties about the parameters, as the ranking of the reactions can be greatly affected. This work will now form the basis for a comprehensive Bayesian analysis and extension of the model considering alternative topologies.

  20. Functional analysis of TOEFAZ1 uncovers protein domains essential for cytokinesis in Trypanosoma brucei.

    Science.gov (United States)

    Sinclair-Davis, Amy N; McAllaster, Michael R; de Graffenried, Christopher L

    2017-10-09

    The parasite Trypanosoma brucei is highly polarized, including a flagellum that is attached along the cell surface by the flagellum attachment zone (FAZ). During cell division, the new FAZ positions the cleavage furrow, which ingresses from the anterior tip of the cell towards the posterior. We recently identified Tip Of the Extending FAZ protein 1 (TOEFAZ1) as an essential protein in trypanosome cytokinesis. We analyzed the localization and function of TOEFAZ1 domains using overexpression and RNAi complementation. TOEFAZ1 comprises three domains with separable functions: an N-terminal α-helical domain that may be involved in FAZ recruitment, a central intrinsically disordered domain that retains the morphogenic kinase TbPLK at the new FAZ tip, and a C-terminal zinc finger domain necessary for TOEFAZ1 oligomerization. Both the N-terminal and C-terminal domains are essential for TOEFAZ1 function, but TbPLK retention at the FAZ is not necessary for cytokinesis. The feasibility of alternate cytokinetic pathways that do not employ TOEFAZ1 are also assessed. Our results show that TOEFAZ1 is a multimeric scaffold for recruiting proteins that control the timing and location of cleavage furrow ingression. © 2017. Published by The Company of Biologists Ltd.

  1. Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei.

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    Daniel Nilsson

    2010-08-01

    Full Text Available Trans-splicing of leader sequences onto the 5'ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5'splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT. The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5' splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.

  2. Isothermal microcalorimetry, a new tool to monitor drug action against Trypanosoma brucei and Plasmodium falciparum.

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    Tanja Wenzler

    Full Text Available Isothermal microcalorimetry is an established tool to measure heat flow of physical, chemical or biological processes. The metabolism of viable cells produces heat, and if sufficient cells are present, their heat production can be assessed by this method. In this study, we investigated the heat flow of two medically important protozoans, Trypanosoma brucei rhodesiense and Plasmodium falciparum. Heat flow signals obtained for these pathogens allowed us to monitor parasite growth on a real-time basis as the signals correlated with the number of viable cells. To showcase the potential of microcalorimetry for measuring drug action on pathogenic organisms, we tested the method with three antitrypanosomal drugs, melarsoprol, suramin and pentamidine and three antiplasmodial drugs, chloroquine, artemether and dihydroartemisinin, each at two concentrations on the respective parasite. With the real time measurement, inhibition was observed immediately by a reduced heat flow compared to that in untreated control samples. The onset of drug action, the degree of inhibition and the time to death of the parasite culture could conveniently be monitored over several days. Microcalorimetry is a valuable element to be added to the toolbox for drug discovery for protozoal diseases such as human African trypanosomiasis and malaria. The method could probably be adapted to other protozoan parasites, especially those growing extracellularly.

  3. Flux Analysis of the Trypanosoma brucei Glycolysis Based on a Multiobjective-Criteria Bioinformatic Approach

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    Amine Ghozlane

    2012-01-01

    Full Text Available Trypanosoma brucei is a protozoan parasite of major of interest in discovering new genes for drug targets. This parasite alternates its life cycle between the mammal host(s (bloodstream form and the insect vector (procyclic form, with two divergent glucose metabolism amenable to in vitro culture. While the metabolic network of the bloodstream forms has been well characterized, the flux distribution between the different branches of the glucose metabolic network in the procyclic form has not been addressed so far. We present a computational analysis (called Metaboflux that exploits the metabolic topology of the procyclic form, and allows the incorporation of multipurpose experimental data to increase the biological relevance of the model. The alternatives resulting from the structural complexity of networks are formulated as an optimization problem solved by a metaheuristic where experimental data are modeled in a multiobjective function. Our results show that the current metabolic model is in agreement with experimental data and confirms the observed high metabolic flexibility of glucose metabolism. In addition, Metaboflux offers a rational explanation for the high flexibility in the ratio between final products from glucose metabolism, thsat is, flux redistribution through the malic enzyme steps.

  4. A transcription-independent epigenetic mechanism is associated with antigenic switching in Trypanosoma brucei.

    Science.gov (United States)

    Aresta-Branco, Francisco; Pimenta, Silvia; Figueiredo, Luisa M

    2016-04-20

    Antigenic variation in Trypanosoma brucei relies on periodic switching of variant surface glycoproteins (VSGs), which are transcribed monoallelically by RNA polymerase I from one of about 15 bloodstream expression sites (BES). Chromatin of the actively transcribed BES is depleted of nucleosomes, but it is unclear if this open conformation is a mere consequence of a high rate of transcription, or whether it is maintained by a transcription-independent mechanism. Using an inducible BES-silencing reporter strain, we observed that chromatin of the active BES remains open for at least 24 hours after blocking transcription. This conformation is independent of the cell-cycle stage, but dependent upon TDP1, a high mobility group box protein. For two days after BES silencing, we detected a transient and reversible derepression of several silent BESs within the population, suggesting that cells probe other BESs before commitment to one, which is complete by 48 hours. FACS sorting and subsequent subcloning confirmed that probing cells are switching intermediates capable of returning to the original BES, switch to the probed BES or to a different BES. We propose that regulation of BES chromatin structure is an epigenetic mechanism important for successful antigenic switching. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Duplicated paralogous genes subject to positive selection in the genome of Trypanosoma brucei.

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    Richard D Emes

    2008-05-01

    Full Text Available Whole genome studies have highlighted duplicated genes as important substrates for adaptive evolution. We have investigated adaptive evolution in this class of genes in the human parasite Trypanosoma brucei, as indicated by the ratio of non-synonymous (amino-acid changing to synonymous (amino acid retaining nucleotide substitution rates.We have identified duplicated genes that are most rapidly evolving in this important human parasite. This is the first attempt to investigate adaptive evolution in this species at the codon level. We identify 109 genes within 23 clusters of paralogous gene expansions to be subject to positive selection.Genes identified include surface antigens in both the mammalian and insect host life cycle stage suggesting that competitive interaction is not solely with the adaptive immune system of the mammalian host. Also surface transporters related to drug resistance and genes related to developmental progression are detected. We discuss how adaptive evolution of these genes may highlight lineage specific processes essential for parasite survival. We also discuss the implications of adaptive evolution of these targets for parasite biology and control.

  6. Antichagasic Activity of Komaroviquinone Is Due to Generation of Reactive Oxygen Species Catalyzed by Trypanosoma cruzi Old Yellow Enzyme

    OpenAIRE

    Uchiyama, Nahoko; Kabututu, Zakayi; Kubata, Bruno K.; Kiuchi, Fumiyuki; Ito, Michiho; Nakajima-Shimada, Junko; Aoki, Takashi; Ohkubo, Kei; Fukuzumi, Shunichi; Martin, Samuel K.; Honda, Gisho; Urade, Yoshihiro

    2005-01-01

    A novel potent trypanocidal diterpene, komaroviquinone, was reduced by Trypanosoma cruzi old yellow enzyme (TcOYE) to its semiquinone radical. The reductase activity in trypanosome lysates was completely immunoabsorbed by anti-TcOYE antibody. Since TcOYE is expressed throughout the T. cruzi life cycle, komaroviquinone is an interesting candidate for developing new antichagasic drugs.

  7. Aspirin Modulates Innate Inflammatory Response and Inhibits the Entry of Trypanosoma cruzi in Mouse Peritoneal Macrophages

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    Aparecida Donizette Malvezi

    2014-01-01

    Full Text Available The intracellular protozoan parasite Trypanosoma cruzi causes Chagas disease, a serious disorder that affects millions of people in Latin America. Cell invasion by T. cruzi and its intracellular replication are essential to the parasite’s life cycle and for the development of Chagas disease. Here, we present evidence suggesting the involvement of the host’s cyclooxygenase (COX enzyme during T. cruzi invasion. Pharmacological antagonist for COX-1, aspirin (ASA, caused marked inhibition of T. cruzi infection when peritoneal macrophages were pretreated with ASA for 30 min at 37°C before inoculation. This inhibition was associated with increased production of IL-1β and nitric oxide (NO∙ by macrophages. The treatment of macrophages with either NOS inhibitors or prostaglandin E2 (PGE2 restored the invasive action of T. cruzi in macrophages previously treated with ASA. Lipoxin ALX-receptor antagonist Boc2 reversed the inhibitory effect of ASA on trypomastigote invasion. Our results indicate that PGE2, NO∙, and lipoxins are involved in the regulation of anti-T. cruzi activity by macrophages, providing a better understanding of the role of prostaglandins in innate inflammatory response to T. cruzi infection as well as adding a new perspective to specific immune interventions.

  8. Interactions between Trypanosoma cruzi secreted proteins and host cell signaling pathways

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    Renata Watanabe Costa

    2016-03-01

    Full Text Available Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6-7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi (T. cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion.

  9. Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays

    DEFF Research Database (Denmark)

    Wiggers, Henrik; Cheleski, J; Zottis, A

    2007-01-01

    OH), in the isothermal titration calorimetry (ITC) kinetic assays for the catalyzed reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi. The solvent effects on T. cruzi GAPDH had not yet been studied. This enzyme was shown here to be affected by the organic solvents content up to 5...... by standard spectrophotometric assays to establish a behavioral performance of T. cruzi GAPDH when used for screening of potential inhibitors. Udgivelsesdato: 2007-Nov-1...

  10. Antiparasitic evaluation of betulinic acid derivatives reveals effective and selective anti-Trypanosoma cruzi inhibitors.

    Science.gov (United States)

    Meira, Cássio Santana; Barbosa-Filho, José Maria; Lanfredi-Rangel, Adriana; Guimarães, Elisalva Teixeira; Moreira, Diogo Rodrigo Magalhães; Soares, Milena Botelho Pereira

    2016-07-01

    Betulinic acid is a pentacyclic triterpenoid with several biological properties already described, including antiparasitic activity. Here, the anti-Trypanosoma cruzi activity of betulinic acid and its semi-synthetic amide derivatives (BA1-BA8) was investigated. The anti-Trypanosoma cruzi activity and selectivity were enhanced in semi-synthetic derivatives, specially on derivatives BA5, BA6 and BA8. To understand the mechanism of action underlying betulinic acid anti-T. cruzi activity, we investigated ultrastructural changes by electron microscopy. Ultrastructural studies showed that trypomastigotes incubated with BA5 had membrane blebling, flagella retraction, atypical cytoplasmic vacuoles and Golgi cisternae dilatation. Flow cytometry analysis showed that parasite death is mainly caused by necrosis. Treatment with derivatives BA5, BA6 or BA8 reduced the invasion process, as well as intracellular parasite development in host cells, with a potency and selectivity similar to that observed in benznidazole-treated cells. More importantly, the combination of BA5 and benznidazole revealed synergistic effects on trypomastigote and amastigote forms of T. cruzi. In conclusion, we demonstrated that BA5 compound is an effective and selective anti-T. cruzi agent. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Active penetration of Trypanosoma cruzi into host cells: historical considerations and current concepts

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    Tecia Maria Ulisses Carvalho

    2013-01-01

    Full Text Available A significant number of scientific groups working on several countries have made efforts to better understand the process of invasion of several types of host cells by Trypanosoma cruzi, the etiologic agent of Chagas disease. In this mini-review we analyze the two mechanisms of invasion considered to be relevant: active penetration and endocytosis. The term active penetration is considered in view of its original description by Dvorak and co-workers. Taking into consideration all results obtained we conclude that endocytosis, with its many variations, is the only mechanism used by T. cruzi to invade host cells.

  12. Cell surface proteome analysis of human-hosted Trypanosoma cruzi life stages

    DEFF Research Database (Denmark)

    Queiroz, Rayner M L; Charneau, Sébastien; Bastos, Izabela M D

    2014-01-01

    Chagas' disease is a neglected infectious illness, caused by the protozoan Trypanosoma cruzi. It remains a challenging health issue in Latin America, where it is endemic, and so far there is no immunoprophylatic vaccine or satisfactory chemotherapic treatment for its chronic stage. The present work...... addressed the analysis of the plasma membrane (PM) subproteome from T. cruzi human-hosted life stages, trypomastigote and axenic amastigote, by two complementary PM protein enrichment techniques followed by identification using an LC-MS/MS approach. The results revealed an extensive repertoire of proteins...

  13. Medicinal plants of Chile: evaluation of their anti-Trypanosoma cruzi activity.

    Science.gov (United States)

    Muñoz, Orlando M; Maya, Juan D; Ferreira, Jorge; Christen, Philippe; San Martin, José; López-Muñoz, Rodrigo; Morello, Antonio; Kemmerling, Ulrike

    2013-01-01

    The extracts of several plants of Central Chile exhibited anti-Trypanosoma cruzi trypomastigotes activity. Most active extracts were those obtained from Podanthus ovatifolius, Berberis microphylla, Kageneckia oblonga, and Drimys winteri. The active extract of Drimys winteri (IC50 51.2 microg/mL) was purified and three drimane sesquiterpenes were obtained: polygodial, drimenol, and isodrimenin. Isodrimenin and drimenol were found to be active against the trypomastigote form of T. cruzi with IC50 values of 27.9 and 25.1 microM, respectively.

  14. AFAP-1L1-mediated actin filaments crosslinks hinder Trypanosoma cruzi cell invasion and intracellular multiplication.

    Science.gov (United States)

    de Araújo, Karine Canuto Loureiro; Teixeira, Thaise Lara; Machado, Fabrício Castro; da Silva, Aline Alves; Quintal, Amanda Pifano Neto; da Silva, Claudio Vieira

    2016-10-01

    Host actin cytoskeleton polymerization has been shown to play an important role during Trypanosoma cruzi internalization into mammalian cell. The structure and dynamics of the actin cytoskeleton in cells are regulated by a vast number of actin-binding proteins. Here we aimed to verify the impact of AFAP-1L1, during invasion and multiplication of T. cruzi. Knocking-down AFAP-1L1 increased parasite cell invasion and intracellular multiplication. Thus, we have shown that the integrity of the machinery formed by AFAP-1L1 in actin cytoskeleton polymerization is important to hinder parasite infection. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Steroid Alkaloids from Holarrhena africana with Strong Activity against Trypanosoma brucei rhodesiense.

    Science.gov (United States)

    Nnadi, Charles Okeke; Nwodo, Ngozi Justina; Kaiser, Marcel; Brun, Reto; Schmidt, Thomas J

    2017-07-06

    In our continued search for natural compounds with activity against Trypanosoma brucei, causative agent of human African trypanosomiasis (HAT, "sleeping sickness"), we have investigated extracts from the leaves and bark of the West African Holarrhenaafricana (syn. Holarrhena floribunda; Apocynaceae). The extracts and their alkaloid-enriched fractions displayed promising in vitro activity against bloodstream forms of T. brucei rhodesiense (Tbr; East African HAT). Bioactivity-guided chromatographic fractionation of the alkaloid-rich fractions resulted in the isolation of 17 steroid alkaloids, one nitrogen-free steroid and one alkaloid-like non-steroid. Impressive activities (IC50 in µM) against Tbr were recorded for 3β-holaphyllamine (0.40 ± 0.28), 3α-holaphyllamine (0.37 ± 0.16), 3β-dihydroholaphyllamine (0.67 ± 0.03), N-methylholaphyllamine (0.08 ± 0.01), conessimine (0.17 ± 0.08), conessine (0.42 ± 0.09), isoconessimine (0.17 ± 0.11) and holarrhesine (0.12 ± 0.08) with selectivity indices ranging from 13 to 302. Based on comparison of the structures of this congeneric series of steroid alkaloids and their activities, structure-activity relationships (SARs) could be established. It was found that a basic amino group at position C-3 of the pregnane or pregn-5-ene steroid nucleus is required for a significant anti-trypanosomal activity. The mono-methylated amino group at C-3 represents an optimum for activity. ∆(5,6) unsaturation slightly increased the activity while hydrolysis of C-12β ester derivatives led to a loss of activity. An additional amino group at C-20 engaged in a pyrrolidine ring closed towards C-18 significantly increased the selectivity index of the compounds. Our findings provide useful empirical data for further development of steroid alkaloids as a novel class of anti-trypanosomal compounds which represent a promising starting point towards new drugs to combat human African trypanosomiasis.

  16. Steroid Alkaloids from Holarrhena africana with Strong Activity against Trypanosoma brucei rhodesiense

    Directory of Open Access Journals (Sweden)

    Charles Okeke Nnadi

    2017-07-01

    Full Text Available In our continued search for natural compounds with activity against Trypanosoma brucei, causative agent of human African trypanosomiasis (HAT, “sleeping sickness”, we have investigated extracts from the leaves and bark of the West African Holarrhena africana (syn. Holarrhena floribunda; Apocynaceae. The extracts and their alkaloid-enriched fractions displayed promising in vitro activity against bloodstream forms of T. brucei rhodesiense (Tbr; East African HAT. Bioactivity-guided chromatographic fractionation of the alkaloid-rich fractions resulted in the isolation of 17 steroid alkaloids, one nitrogen-free steroid and one alkaloid-like non-steroid. Impressive activities (IC50 in µM against Tbr were recorded for 3β-holaphyllamine (0.40 ± 0.28, 3α-holaphyllamine (0.37 ± 0.16, 3β-dihydroholaphyllamine (0.67 ± 0.03, N-methylholaphyllamine (0.08 ± 0.01, conessimine (0.17 ± 0.08, conessine (0.42 ± 0.09, isoconessimine (0.17 ± 0.11 and holarrhesine (0.12 ± 0.08 with selectivity indices ranging from 13 to 302. Based on comparison of the structures of this congeneric series of steroid alkaloids and their activities, structure-activity relationships (SARs could be established. It was found that a basic amino group at position C-3 of the pregnane or pregn-5-ene steroid nucleus is required for a significant anti-trypanosomal activity. The mono-methylated amino group at C-3 represents an optimum for activity. ∆5,6 unsaturation slightly increased the activity while hydrolysis of C-12β ester derivatives led to a loss of activity. An additional amino group at C-20 engaged in a pyrrolidine ring closed towards C-18 significantly increased the selectivity index of the compounds. Our findings provide useful empirical data for further development of steroid alkaloids as a novel class of anti-trypanosomal compounds which represent a promising starting point towards new drugs to combat human African trypanosomiasis.

  17. Diagnostic accuracy of loopamp Trypanosoma brucei detection kit for diagnosis of human African trypanosomiasis in clinical samples.

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    Patrick Mitashi

    Full Text Available BACKGROUND: Molecular methods have great potential for sensitive parasite detection in the diagnosis of human African trypanosomiasis (HAT, but the requirements in terms of laboratory infrastructure limit their use to reference centres. A recently developed assay detects the Trypanozoon repetitive insertion mobile element (RIME DNA under isothermal amplification conditions and has been transformed into a ready-to-use kit format, the Loopamp Trypanosoma brucei. In this study, we have evaluated the diagnostic performance of the Loopamp Trypanosoma brucei assay (hereafter called LAMP in confirmed T.b. gambiense HAT patients, HAT suspects and healthy endemic controls from the Democratic Republic of the Congo (DRC. METHODOLOGY/PRINCIPAL FINDINGS: 142 T.b. gambiense HAT patients, 111 healthy endemic controls and 97 HAT suspects with unconfirmed status were included in this retrospective evaluation. Reference standard tests were parasite detection in blood, lymph or cerebrospinal fluid. Archived DNA from blood of all study participants was analysed in duplicate with LAMP. Sensitivity of LAMP in parasitologically confirmed cases was 87.3% (95% CI 80.9-91.8% in the first run and 93.0% (95% CI 87.5-96.1% in the second run. Specificity in healthy controls was 92.8% (95% CI 86.4-96.3% in the first run and 96.4% (95% CI 91.1-98.6% in the second run. Reproducibility was excellent with a kappa value of 0.81. CONCLUSIONS/SIGNIFICANCE: In this laboratory-based study, the Loopamp Trypanosoma brucei Detection Kit showed good diagnostic accuracy and excellent reproducibility. Further studies are needed to assess the feasibility of its routine use for diagnosis of HAT under field conditions.

  18. Trypanosoma cruzi utilizes the host low density lipoprotein receptor in invasion.

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    Fnu Nagajyothi

    2011-02-01

    Full Text Available Trypanosoma cruzi, an intracellular protozoan parasite that infects humans and other mammalian hosts, is the etiologic agent in Chagas disease. This parasite can invade a wide variety of mammalian cells. The mechanism(s by which T. cruzi invades its host cell is not completely understood. The activation of many signaling receptors during invasion has been reported; however, the exact mechanism by which parasites cross the host cell membrane barrier and trigger fusion of the parasitophorous vacuole with lysosomes is not understood.In order to explore the role of the Low Density Lipoprotein receptor (LDLr in T. cruzi invasion, we evaluated LDLr parasite interactions using immunoblot and immunofluorescence (IFA techniques. These experiments demonstrated that T. cruzi infection increases LDLr levels in infected host cells, inhibition or disruption of LDLr reduces parasite load in infected cells, T. cruzi directly binds recombinant LDLr, and LDLr-dependent T. cruzi invasion requires PIP2/3. qPCR analysis demonstrated a massive increase in LDLr mRNA (8000 fold in the heart of T. cruzi infected mice, which is observed as early as 15 days after infection. IFA shows a co-localization of both LDL and LDLr with parasites in infected heart.These data highlight, for the first time, that LDLr is involved in host cell invasion by this parasite and the subsequent fusion of the parasitophorous vacuole with the host cell lysosomal compartment. The model suggested by this study unifies previous models of host cell invasion for this pathogenic protozoon. Overall, these data indicate that T. cruzi targets LDLr and its family members during invasion. Binding to LDL likely facilitates parasite entry into host cells. The observations in this report suggest that therapeutic strategies based on the interaction of T. cruzi and the LDLr pathway should be pursued as possible targets to modify the pathogenesis of disease following infection.

  19. Trypanosoma cruzi utilizes the host low density lipoprotein receptor in invasion.

    Science.gov (United States)

    Nagajyothi, Fnu; Weiss, Louis M; Silver, David L; Desruisseaux, Mahalia S; Scherer, Philipp E; Herz, Joachim; Tanowitz, Herbert B

    2011-02-01

    Trypanosoma cruzi, an intracellular protozoan parasite that infects humans and other mammalian hosts, is the etiologic agent in Chagas disease. This parasite can invade a wide variety of mammalian cells. The mechanism(s) by which T. cruzi invades its host cell is not completely understood. The activation of many signaling receptors during invasion has been reported; however, the exact mechanism by which parasites cross the host cell membrane barrier and trigger fusion of the parasitophorous vacuole with lysosomes is not understood. In order to explore the role of the Low Density Lipoprotein receptor (LDLr) in T. cruzi invasion, we evaluated LDLr parasite interactions using immunoblot and immunofluorescence (IFA) techniques. These experiments demonstrated that T. cruzi infection increases LDLr levels in infected host cells, inhibition or disruption of LDLr reduces parasite load in infected cells, T. cruzi directly binds recombinant LDLr, and LDLr-dependent T. cruzi invasion requires PIP2/3. qPCR analysis demonstrated a massive increase in LDLr mRNA (8000 fold) in the heart of T. cruzi infected mice, which is observed as early as 15 days after infection. IFA shows a co-localization of both LDL and LDLr with parasites in infected heart. These data highlight, for the first time, that LDLr is involved in host cell invasion by this parasite and the subsequent fusion of the parasitophorous vacuole with the host cell lysosomal compartment. The model suggested by this study unifies previous models of host cell invasion for this pathogenic protozoon. Overall, these data indicate that T. cruzi targets LDLr and its family members during invasion. Binding to LDL likely facilitates parasite entry into host cells. The observations in this report suggest that therapeutic strategies based on the interaction of T. cruzi and the LDLr pathway should be pursued as possible targets to modify the pathogenesis of disease following infection.

  20. Antiprotozoal drug nitazoxanide enhances parasitemia, tissue lesions and mortality caused by Trypanosoma cruzi in murine model.

    Science.gov (United States)

    Valle-Reyes, Juan Salvador; Melnikov, Valery; Dobrovinskaya, Oxana; Rodriguez-Hernández, Alejandrina; Wookee-Zea, Cristina; Pimientel-Rodrigez, Víctor; Rueda-Valdovinos, Gabriela; Delgado-Enciso, Iván; López-Lemus, Uriel A; Espinoza-Gómez, Francisco

    2017-01-01

    Chagas' disease is caused by unicellular parasite Trypanosoma cruzi (T. cruzi). It is endemic throughout Latin America, but nowadays has become a global challenge due to tourism and migration. Non-treated infection may result in health-threatening complications and lead to death. Current medications for this infection are nifurtimox (NFT) and benznidazol. Both drugs may cause side effects and are ineffective in the chronic phase. Therefore, new antichagasic compounds are urgently required. Nitazoxanide (NTZ) is a broad spectrum antiparasitic drug, proposed recently as a potential candidate to be added to the list of essential medicines for integrated neglected tropical disease control and elimination. Although the effect of NTZ against T. cruzi epimastigotes in vitro was reported, the corresponding experiments in animal models of T. cruzi infection have never been undertaken. The present work was designed to fill this gap and evaluate the effect of NTZ on experimental murine trypanosomiasis, in comparison with classical antichagasic agent NFT. Highly sensitive to T. cruzi BALB/c mice were infected using Albarrada T. cruzi strain, recently isolated in Mexico. Experimental groups were either left untreated, or otherwise treated with NFT, NTZ (100 and 1000 mg/kg), or with both drugs simultaneously. The severity of the infection was estimated based on criteria such as parasitemia, lesions in target tissues (heart, muscles and lungs) and mortality. Despite the expected protective effect, NTZ drastically aggravates the course of T. cruzi infection. Namely, parasitemia, tissue lesions and mortality caused by T. cruzi infection were significantly higher in NTZ-treated mice groups, even in comparison with untreated infected animals. NTZ by itself no produced mortality o tissue damage, and NFT showed an expected protective effect. Our results indicate that NTZ cannot be considered for Chagas' disease treatment. Moreover, NTZ should be used with caution in patients

  1. Modulation of host central carbon metabolism and in situ glucose uptake by intracellular Trypanosoma cruzi amastigotes.

    Science.gov (United States)

    Shah-Simpson, Sheena; Lentini, Gaelle; Dumoulin, Peter C; Burleigh, Barbara A

    2017-11-01

    Obligate intracellular pathogens satisfy their nutrient requirements by coupling to host metabolic processes, often modulating these pathways to facilitate access to key metabolites. Such metabolic dependencies represent potential targets for pathogen control, but remain largely uncharacterized for the intracellular protozoan parasite and causative agent of Chagas disease, Trypanosoma cruzi. Perturbations in host central carbon and energy metabolism have been reported in mammalian T. cruzi infection, with no information regarding the impact of host metabolic changes on the intracellular amastigote life stage. Here, we performed cell-based studies to elucidate the interplay between infection with intracellular T. cruzi amastigotes and host cellular energy metabolism. T. cruzi infection of non-phagocytic cells was characterized by increased glucose uptake into infected cells and increased mitochondrial respiration and mitochondrial biogenesis. While intracellular amastigote growth was unaffected by decreased host respiratory capacity, restriction of extracellular glucose impaired amastigote proliferation and sensitized parasites to further growth inhibition by 2-deoxyglucose. These observations led us to consider whether intracellular T. cruzi amastigotes utilize glucose directly as a substrate to fuel metabolism. Consistent with this prediction, isolated T. cruzi amastigotes transport extracellular glucose with kinetics similar to trypomastigotes, with subsequent metabolism as demonstrated in 13C-glucose labeling and substrate utilization assays. Metabolic labeling of T. cruzi-infected cells further demonstrated the ability of intracellular parasites to access host hexose pools in situ. These findings are consistent with a model in which intracellular T. cruzi amastigotes capitalize on the host metabolic response to parasite infection, including the increase in glucose uptake, to fuel their own metabolism and replication in the host cytosol. Our findings enrich

  2. Molecular profiles of Trypanosoma brucei, T. evansi and T. equiperdum stocks revealed by the random amplified polymorphic DNA method.

    Science.gov (United States)

    Lun, Zhao-Rong; Li, An-Xing; Chen, Xiao-Guang; Lu, Li-Xin; Zhu, Xing-Quan

    2004-03-01

    A total of 20 random primers (10-mers) were used to amplify RAPD markers from the genomic DNA of four Trypanosoma brucei stocks from East and West Africa, four T. evansi stocks from Africa, Asia and South America and one T. equiperdum stock from Asia. Between 65 and 88 reproducible fragments ranging from 0.25 to 2.15 kb were generated from these stocks depending on the stock/primer combination. The similarity coefficient (SC) among the stocks of T. brucei from Kenya, Nigeria, Tanzania and Zambia ranged from 62.9% to 74.0% (average: 67.6%). The SC among the stocks of T. evansi from Kenya, China and Brazil was 76.4%-95.5% (average: 86.4%), while the SC between T. evansi stock from China and Brazil was 95.5%. For T. evansi and T. equiperdum, the SC among the stocks ranged from 81.2% to 94.4% (average: 87.6%). As for the SC among the stocks of T. brucei and T. evansi, it was found to be from 54.7% to 80.3% (average: 68.0%) and the SC among stocks of T. brucei and T. equiperdum was from 59.4% to 76.9% (average: 68.1%). Our results indicate that the stocks of T. evansi from China and from Brazil are more closely related to the stock of T. equiperdum from China than to the stocks of T. evansi isolated from Kenya and to the stocks of T. brucei. In addition, our results further support the hypothesis that T. evansi stocks from China and Brazil could have arisen from a single lineage. The possible evolution of T. evansi and T. equiperdum is also discussed.

  3. ATG24 Represses Autophagy and Differentiation and Is Essential for Homeostasy of the Flagellar Pocket in Trypanosoma brucei.

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    Ana Brennand

    Full Text Available We have previously identified homologs for nearly half of the approximately 30 known yeast Atg's in the genome database of the human sleeping sickness parasite Trypanosoma brucei. So far, only a few of these homologs have their role in autophagy experimentally confirmed. Among the candidates was the ortholog of Atg24 that is involved in pexophagy in yeast. In T. brucei, the peroxisome-like organelles named glycosomes harbor core metabolic processes, especially glycolysis. In the autotrophic yeast, autophagy is essential for adaptation to different nutritional environments by participating in the renewal of the peroxisome population. We hypothesized that autophagic turnover of the parasite's glycosomes plays a role in differentiation during its life cycle, which demands adaptation to different host environments and associated dramatic changes in nutritional conditions. We therefore characterized T. brucei ATG24, the T. brucei ortholog of yeast Atg24 and mammalian SNX4, and found it to have a regulatory role in autophagy and differentiation as well as endocytic trafficking. ATG24 partially localized on endocytic membranes where it was recruited via PI3-kinase III/VPS34. ATG24 silencing severely impaired receptor-mediated endocytosis of transferrin, but not adsorptive uptake of a lectin, and caused a major enlargement of the flagellar pocket. ATG24 silencing approximately doubled the number of autophagosomes, suggesting a role in repressing autophagy, and strongly accelerated differentiation, in accordance with a role of autophagy in parasite differentiation. Overexpression of the two isoforms of T. brucei ATG8 fused to GFP slowed down differentiation, possibly by a dominant-negative effect. This was overcome by ATG24 depletion, further supporting its regulatory role.

  4. Preparation, crystallization and preliminary crystallographic analysis of old yellow enzyme from Trypanosoma cruzi

    Energy Technology Data Exchange (ETDEWEB)

    Sugiyama, Shigeru [Maruwa Foods Co. Ltd, Tsutsui-cho 170-1, Yamatokoriyama, Nara 639-1123 (Japan); Tokuoka, Keiji [Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-Oka, Suita, Osaka 565-0871 (Japan); Uchiyama, Nahoko [Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Osaka 565-0874 (Japan); Okamoto, Naoki; Okano, Yousuke; Matsumura, Hiroyoshi [Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-Oka, Suita, Osaka 565-0871 (Japan); Inaka, Koji [Maruwa Foods Co. Ltd, Tsutsui-cho 170-1, Yamatokoriyama, Nara 639-1123 (Japan); Urade, Yoshihiro [Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Osaka 565-0874 (Japan); Inoue, Tsuyoshi, E-mail: inouet@chem.eng.osaka-u.ac.jp [Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-Oka, Suita, Osaka 565-0871 (Japan); Maruwa Foods Co. Ltd, Tsutsui-cho 170-1, Yamatokoriyama, Nara 639-1123 (Japan)

    2007-10-01

    Old yellow enzyme from Trypanosoma cruzi, has been crystallized using the hanging-drop vapour-diffusion method. Old yellow enzyme (OYE) is an NADPH oxidoreductase that contains a flavin mononucleotide as a prosthetic group. The OYE from Trypanosoma cruzi, which produces prostaglandin F{sub 2α}, a potent mediator of various physiological and pathological processes, from prostaglandin H2. The protein was recombinantly expressed and purified from Escherichia coli and was crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 56.3, b = 78.8, c = 78.8 Å, β = 93.4° and two molecules per asymmetric unit. The crystals were suitable for X-ray crystallographic studies and diffracted to 1.70 Å resolution. A Patterson search method is in progress using the structure of OYE from Pseudomonas putida as a starting model.

  5. Inhibition of Trypanosoma cruzi growth in vitro by Solanum alkaloids: a comparison with ketoconazole.

    Science.gov (United States)

    Chataing, B; Concepción, J L; Lobatón, R; Usubillaga, A

    1998-02-01

    The glycoalkaloids alpha-chaconine, alpha-solamargine, alpha-solanine, solasonine, sycophantine, and tomatine, as well as the aglycones demissidine, solanidine, solanocapsine, solasodine, tomatidine, and veratrine were tested as growth inhibitors of Trypanosoma cruzi, strain EP, in LIT medium. Their activity was compared with the antifungal ketoconazole. Glycoalkaloids containing alpha-chacotriose showed trypanolytic activity against the epimastigote form and trypanocidal activity against the bloodstream and metacyclic trypomastigote form of Trypanosoma cruzi in culture medium in micromolar concentrations. Ketoconazole showed a lower activity, at the same concentrations of alpha-chaconine and alpha-solamargine. The observations indicate that the initial target of the compound is at the membrane level with a concomitant change in the parasite morphology. Moreover, internal compartments of the parasites were observed to be affected by the drugs, revealing the dissolution of some organelles as mitocondrias and glycosomes.

  6. The trans-sialidase, the major Trypanosoma cruzi virulence factor: Three decades of studies.

    Science.gov (United States)

    Freire-de-Lima, L; Fonseca, L M; Oeltmann, T; Mendonça-Previato, L; Previato, J O

    2015-11-01

    Chagas' disease is a potentially life-threatening disease caused by the protozoan parasite Trypanosoma cruzi. Since the description of Chagas'disease in 1909 extensive research has identified important events in the disease in order to understand the biochemical mechanism that modulates T. cruzi-host cell interactions and the ability of the parasite to ensure its survival in the infected host. Exactly 30 years ago, we presented evidence for the first time of a trans-sialidase activity in T. cruzi (T. cruzi-TS). This enzyme transfers sialic acid from the host glycoconjugates to the terminal β-galactopyranosyl residues of mucin-like molecules on the parasite's cell surface. Thenceforth, many articles have provided convincing data showing that T. cruzi-TS is able to govern relevant mechanisms involved in the parasite's survival in the mammalian host, such as invasion, escape from the phagolysosomal vacuole, differentiation, down-modulation of host immune responses, among others. The aim of this review is to cover the history of the discovery of T. cruzi-TS, as well as some well-documented biological effects encompassed by this parasite's virulence factor, an enzyme with potential attributes to become a drug target against Chagas disease. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Studying nanotoxic effects of CdTe quantum dots in Trypanosoma cruzi

    Science.gov (United States)

    Stahl, C. V.; Almeida, D. B.; de Thomaz, A. A.; Fontes, A.; Menna-Barreto, R. F. S.; Santos-Mallet, J. R.; Cesar, C. L.; Gomes, S. A. O.; Feder, D.

    2010-02-01

    Many studies have been done in order to verify the possible nanotoxicity of quantum dots in some cellular types. Protozoan pathogens as Trypanosoma cruzi, etiologic agent of Chagas1 disease is transmitted to humans either by blood-sucking triatomine vectors, blood transfusion, organs transplantation or congenital transmission. The study of the life cycle, biochemical, genetics, morphology and others aspects of the T. cruzi is very important to better understand the interactions with its hosts and the disease evolution on humans. Quantum dot, nanocrystals, highly luminescent has been used as tool for experiments in in vitro and in vivo T. cruzi life cycle development in real time. We are now investigating the quantum dots toxicity on T. cruzi parasite cells using analytical methods. In vitro experiments were been done in order to test the interference of this nanoparticle on parasite development, morphology and viability (live-death). Ours previous results demonstrated that 72 hours after parasite incubation with 200 μM of CdTe altered the development of T. cruzi and induced cell death by necrosis in a rate of 34%. QDs labeling did not effect: (i) on parasite integrity, at least until 7 days; (ii) parasite cell dividing and (iii) parasite motility at a concentration of 2 μM CdTe. This fact confirms the low level of cytotoxicity of these QDs on this parasite cell. In summary our results is showing T. cruzi QDs labeling could be used for in vivo cellular studies in Chagas disease.

  8. Interactions between Trypanosoma cruzi Secreted Proteins and Host Cell Signaling Pathways

    Science.gov (United States)

    Watanabe Costa, Renata; da Silveira, Jose F.; Bahia, Diana

    2016-01-01

    Chagas disease is one of the prevalent neglected tropical diseases, affecting at least 6–7 million individuals in Latin America. It is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to vertebrate hosts by blood-sucking insects. After infection, the parasite invades and multiplies in the myocardium, leading to acute myocarditis that kills around 5% of untreated individuals. T. cruzi secretes proteins that manipulate multiple host cell signaling pathways to promote host cell invasion. The primary secreted lysosomal peptidase in T. cruzi is cruzipain, which has been shown to modulate the host immune response. Cruzipain hinders macrophage activation during the early stages of infection by interrupting the NF-kB P65 mediated signaling pathway. This allows the parasite to survive and replicate, and may contribute to the spread of infection in acute Chagas disease. Another secreted protein P21, which is expressed in all of the developmental stages of T. cruzi, has been shown to modulate host phagocytosis signaling pathways. The parasite also secretes soluble factors that exert effects on host extracellular matrix, such as proteolytic degradation of collagens. Finally, secreted phospholipase A from T. cruzi contributes to lipid modifications on host cells and concomitantly activates the PKC signaling pathway. Here, we present a brief review of the interaction between secreted proteins from T. cruzi and the host cells, emphasizing the manipulation of host signaling pathways during invasion. PMID:27065960

  9. Bioenergetic profiling of Trypanosoma cruzi life stages using Seahorse extracellular flux technology.

    Science.gov (United States)

    Shah-Simpson, Sheena; Pereira, Camila F A; Dumoulin, Peter C; Caradonna, Kacey L; Burleigh, Barbara A

    2016-08-01

    Energy metabolism is an attractive target for the development of new therapeutics against protozoan pathogens, including Trypanosoma cruzi, the causative agent of human Chagas disease. Despite emerging evidence that mitochondrial electron transport is essential for the growth of intracellular T. cruzi amastigotes in mammalian cells, fundamental knowledge of mitochondrial energy metabolism in this parasite life stage remains incomplete. The Clark-type electrode, which measures the rate of oxygen consumption, has served as the traditional tool to study mitochondrial energetics and has contributed to our understanding of it in T. cruzi. Here, we evaluate the Seahorse XF(e)24 extracellular flux platform as an alternative method to assess mitochondrial bioenergetics in isolated T. cruzi parasites. We report optimized assay conditions used to perform mitochondrial stress tests with replicative life cycle stages of T. cruzi using the XF(e)24 instrument, and discuss the advantages and potential limitations of this methodology, as applied to T. cruzi and other trypanosomatids. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. In vitro susceptibility of Trypanosoma cruzi strains from Santander, Colombia, to hexadecylphosphocholine (miltefosine), nifurtimox and benznidazole.

    Science.gov (United States)

    Luna, Katherine Paola; Hernández, Indira Paola; Rueda, César Mauricio; Zorro, María Magdalena; Croft, Simon L; Escobar, Patricia

    2009-09-01

    The current chemotherapy for Chagas disease is unsatisfactory with only two drugs available for treatment. Research to discover new drugs for Chagas disease is urgent. Hexadecyl-phosphocholine (HPC, miltefosine) has been demonstrated to have in vitro activity against Trypanosoma cruzi parasites, but its activity on different Colombian T. cruzi strains is not known. To evaluate the in vitro susceptibility of T. cruzi strains isolated from humans and vectors in Santander, Colombia. to miltefosine, nifurtimox and benznidazole. Eight T. cruzi Colombian strains and three reference strains (Esmeraldo, SilvioX10 and Y) were studied. Drug activities against extracellular epimastigotes and intracellular amastigotes were determined by microscopic counting. The results were expressed as the concentrations that inhibited 50% and 90% growth (IC50 and IC90). For miltefosine a similar range of drug activity was observed against all the Colombian strains, all parasites being more susceptible to miltefosine than to the reference drugs. The intracellular amastigotes were more susceptible to miltefosine (IC50 0.08 to 0.63 microM and IC90 0.21 to 2.21 microM) than extracellular forms (IC50 nifurtimox than to benznidazole and some differences in activity of benznidazole between T. cruzi strains was observed. The results showed the significant in vitro activity of miltefosine against T. cruzi stages, and the expected results for the reference drugs. Further in vivo studies with miltefosine are planned.

  11. Trypanosoma cruzi uses macropinocytosis as an additional entry pathway into mammalian host cell.

    Science.gov (United States)

    Barrias, E S; Reignault, L C; De Souza, W; Carvalho, T M U

    2012-11-01

    Several intracellular pathogens are internalized by host cells via multiple endocytic pathways. It is no different with Trypanosoma cruzi. Evidences indicate that T. cruzi entry may occur by endocytosis/phagocytosis or by an active manner. Although macropinocytosis is largely considered an endocytic process where cells internalize only large amounts of solutes, several pathogens use this pathway to enter into host cells. To investigate whether T. cruzi entry into peritoneal macrophages and LLC-MK2 epithelial cells can be also mediated through a macropinocytosis-like process, we used several experimental strategies presently available to characterize macropinocytosis such as the use of different inhibitors. These macropinocytosis' inhibitors blocked internalization of T. cruzi by host cells. To further support this, immunofluorescence microscopy and scanning electron microscopy techniques were used. Field emission scanning electron microscopy revealed that after treatment, parasites remained attached to the external side of host cell plasma membrane. Proteins such as Rabankyrin 5, tyrosine kinases, Pak1 and actin microfilaments, which participate in macropinosome formation, were localized at T. cruzi entry sites. We also observed co-localization between the parasite and an endocytic fluid phase marker. All together, these results indicate that T. cruzi is able to use multiple mechanisms of penetration into host cell, including macropinocytosis. Copyright © 2012. Published by Elsevier Masson SAS.

  12. Trypanosoma cruzi Infection in an Indigenous Kariña Community in Eastern Venezuela

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    Mariolga Berrizbeitia

    2012-01-01

    Full Text Available We investigated the seroprevalence of Trypanosoma cruzi infection in an indigenous Kariña population in eastern Venezuela. A total of 175 serum samples were collected in the community of Piñantal during February 2009. Interviews targeting socioeconomic and environmental factors associated with the T. cruzi transmission were also conducted. Samples were evaluated using trypomastigote excreted/secreted antigens (TESAs in an ELISA format. TESA-ELISA positive samples were confirmed by indirect haemagglutination (HAI (Wiener. A nonsystematic collection of vectors was also undertaken. T. cruzi seroprevalence was 7.43% according to both assays, and the mean age of infected patients was 48.61±10.40 years (range 34 to 73 years. The vector infection rate was 20.00% (2/10. T. cruzi seropositivity was associated with a history of triatomine bites, the ability to recognize the vector and poor knowledge about Chagas disease, but no associations were found with gender, house type, knowledge of how the disease is transmitted, or the presence of vectors or animals inside dwellings. To our knowledge, this is the first study of the seroprevalence of T. cruzi in an indigenous population in eastern Venezuela. All of the epidemiological variables required for the establishment of active vectorial transmission of T. cruzi were present in this community.

  13. In Vitro and in Vivo Anti-Trypanosoma cruzi Activity of a Novel Nitro-derivative

    OpenAIRE

    Muelas-Serrano, Susana; Le-Senne, Ana; Fernández-Portillo, Carlos; Nogal, Juan José; Ochoa, Carmen; Gómez-Barrio, Alicia

    2002-01-01

    Nitroarylidenemalononitriles and their cyanoacetamide derivatives with remarkable anti-epimastigote properties, were synthesized attempting to obtain new 3,5-diamino-4-(5'-nitroarylidene)-4H-thiadiazine 1,1-dioxide derivatives, which in previous reports had shown anti-Trypanosoma cruzi activity. Tests to evaluate the cytotoxicity of compounds were performed on J774 macrophages. 5-nitro-2-thienyl-malononitrile (5NO2TM), was the only product which maintained a high anti-epimastigote activity at...

  14. Rapid Detection of Trypanosoma cruzi in Human Serum by Use of an Immunochromatographic Dipstick Test ▿

    Science.gov (United States)

    Reithinger, Richard; Grijalva, Mario J.; Chiriboga, Rosa F.; Alarcón de Noya, Belkisyolé; Torres, Jaime R.; Pavia-Ruz, Norma; Manrique-Saide, Pablo; Cardinal, Marta V.; Gürtler, Ricardo E.

    2010-01-01

    We evaluated a commercially available immunochromatographic dipstick test to detect Trypanosoma cruzi infection in 366 human serum samples with known serological results from Argentina, Ecuador, Mexico, and Venezuela. One hundred forty-nine of 366 (40.7%) and 171/366 (46.7%) samples tested positive by dipstick and serology, respectively. Dipstick sensitivity was calculated to be 84.8% (range between countries, 77.5 to 95%), and specificity was 97.9% (95.9 to 100%). PMID:20534801

  15. Trypanosoma cruzi Evades the Complement System as an Efficient Strategy to Survive in the Mammalian Host: The Specific Roles of Host/Parasite Molecules and Trypanosoma cruzi Calreticulin

    Science.gov (United States)

    Ramírez-Toloza, Galia; Ferreira, Arturo

    2017-01-01

    American Trypanosomiasis is an important neglected reemerging tropical parasitism, infecting about 8 million people worldwide. Its agent, Trypanosoma cruzi, exhibits multiple mechanisms to evade the host immune response and infect host cells. An important immune evasion strategy of T. cruzi infective stages is its capacity to inhibit the complement system activation on the parasite surface, avoiding opsonizing, immune stimulating and lytic effects. Epimastigotes, the non-infective form of the parasite, present in triatomine arthropod vectors, are highly susceptible to complement-mediated lysis while trypomastigotes, the infective form, present in host bloodstream, are resistant. Thus T. cruzi susceptibility to complement varies depending on the parasite stage (amastigote, trypomastigotes or epimastigote) and on the T. cruzi strain. To avoid complement-mediated lysis, T. cruzi trypomastigotes express on the parasite surface a variety of complement regulatory proteins, such as glycoprotein 58/68 (gp58/68), T. cruzi complement regulatory protein (TcCRP), trypomastigote decay-accelerating factor (T-DAF), C2 receptor inhibitor trispanning (CRIT) and T. cruzi calreticulin (TcCRT). Alternatively, or concomitantly, the parasite captures components with complement regulatory activity from the host bloodstream, such as factor H (FH) and plasma membrane-derived vesicles (PMVs). All these proteins inhibit different steps of the classical (CP), alternative (AP) or lectin pathways (LP). Thus, TcCRP inhibits the CP C3 convertase assembling, gp58/68 inhibits the AP C3 convertase, T-DAF interferes with the CP and AP convertases assembling, TcCRT inhibits the CP and LP, CRIT confers ability to resist the CP and LP, FH is used by trypomastigotes to inhibit the AP convertases and PMVs inhibit the CP and LP C3 convertases. Many of these proteins have similar molecular inhibitory mechanisms. Our laboratory has contributed to elucidate the role of TcCRT in the host-parasite interplay

  16. Trypanosoma cruzi Evades the Complement System as an Efficient Strategy to Survive in the Mammalian Host: The Specific Roles of Host/Parasite Molecules and Trypanosoma cruzi Calreticulin

    Directory of Open Access Journals (Sweden)

    Galia Ramírez-Toloza

    2017-09-01

    Full Text Available American Trypanosomiasis is an important neglected reemerging tropical parasitism, infecting about 8 million people worldwide. Its agent, Trypanosoma cruzi, exhibits multiple mechanisms to evade the host immune response and infect host cells. An important immune evasion strategy of T. cruzi infective stages is its capacity to inhibit the complement system activation on the parasite surface, avoiding opsonizing, immune stimulating and lytic effects. Epimastigotes, the non-infective form of the parasite, present in triatomine arthropod vectors, are highly susceptible to complement-mediated lysis while trypomastigotes, the infective form, present in host bloodstream, are resistant. Thus T. cruzi susceptibility to complement varies depending on the parasite stage (amastigote, trypomastigotes or epimastigote and on the T. cruzi strain. To avoid complement-mediated lysis, T. cruzi trypomastigotes express on the parasite surface a variety of complement regulatory proteins, such as glycoprotein 58/68 (gp58/68, T. cruzi complement regulatory protein (TcCRP, trypomastigote decay-accelerating factor (T-DAF, C2 receptor inhibitor trispanning (CRIT and T. cruzi calreticulin (TcCRT. Alternatively, or concomitantly, the parasite captures components with complement regulatory activity from the host bloodstream, such as factor H (FH and plasma membrane-derived vesicles (PMVs. All these proteins inhibit different steps of the classical (CP, alternative (AP or lectin pathways (LP. Thus, TcCRP inhibits the CP C3 convertase assembling, gp58/68 inhibits the AP C3 convertase, T-DAF interferes with the CP and AP convertases assembling, TcCRT inhibits the CP and LP, CRIT confers ability to resist the CP and LP, FH is used by trypomastigotes to inhibit the AP convertases and PMVs inhibit the CP and LP C3 convertases. Many of these proteins have similar molecular inhibitory mechanisms. Our laboratory has contributed to elucidate the role of TcCRT in the host

  17. 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction.

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    Johanna L Höög

    2016-01-01

    Full Text Available Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility.We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum.The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life cycle of this human parasite.

  18. Glucose uptake occurs by facilitated diffusion in procyclic forms of Trypanosoma brucei.

    Science.gov (United States)

    Wille, U; Seyfang, A; Duszenko, M

    1996-02-15

    The glucose transporter of Trypanosoma brucei procyclic forms was characterized and compared with its bloodstream form counterpart. Measuring the glucose consumption enzymatically, we determined a saturable uptake process of relatively high affinity (Km = 80 microM, Vmax = 4 nmol min-1 10(-8) cells), which showed substrate inhibition at glucose concentrations above 1.5 mM (Ki = 21 mM). Control experiments measuring deoxy-D-[3H]Glc uptake under zero-trans conditions indicated that substrate inhibition occurred on the level of glycolysis. Temperature-dependent kinetics revealed a temperature quotient of Q10 = 2.33 and an activation energy of Ea = 64 kJ mol-1. As shown by trans-stimulation experiments, glucose uptake was stereospecific for the D isomer, whereas L-glucose was not recognized. Inhibitor studies using either the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (5 microM), the H+/ATPase inhibitor N,N'-dicyclohexylcarbodiimide (20 microM), the ionophor monensin (1 microM), or the Na+/K+-ATPase inhibitor ouabain (1 mM) showed insignificant effects on transport efficiency. The procyclic glucose transporter was subsequently enriched in a plasma-membrane fraction and functionally reconstituted into proteoliposomes. Using Na+-free conditions in the absence of a proton gradient, the specific activity of D-[14C]glucose transport was determined as 2.9 nmol min-1 (mg protein)-1 at 0.2 mM glucose. From these cumulative results, we conclude that glucose uptake by the procyclic insect form of the parasite occurs by facilitated diffusion, similar to the hexose-transport system expressed in bloodstream forms. However, the markedly higher substrate affinity indicates a differential expression of different transporter isoforms throughout the lifecycle.

  19. Regulation of Trypanosoma brucei Total and Polysomal mRNA during Development within Its Mammalian Host.

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    Paul Capewell

    Full Text Available The gene expression of Trypanosoma brucei has been examined extensively in the blood of mammalian hosts and in forms found in the midgut of its arthropod vector, the tsetse fly. However, trypanosomes also undergo development within the mammalian bloodstream as they progress from morphologically 'slender forms' to transmissible 'stumpy forms' through morphological intermediates. This transition is temporally progressive within the first wave of parasitaemia such that gene expression can be monitored in relatively pure slender and stumpy populations as well as during the progression between these extremes. The development also represents the progression of cells from translationally active forms adapted for proliferation in the host to translationally quiescent forms, adapted for transmission. We have used metabolic labelling to quantitate translational activity in slender forms, stumpy forms and in forms undergoing early differentiation to procyclic forms in vitro. Thereafter we have examined the cohort of total mRNAs that are enriched throughout development in the mammalian bloodstream (slender, intermediate and stumpy forms, irrespective of strain, revealing those that exhibit consistent developmental regulation rather than sample specific changes. Transcripts that cosediment with polysomes in stumpy forms and slender forms have also been enriched to identify transcripts that escape translational repression prior to transmission. Combined, the expression and polysomal association of transcripts as trypanosomes undergo development in the mammalian bloodstream have been defined, providing a resource for trypanosome researchers. This facilitates the identification of those that undergo developmental regulation in the bloodstream and therefore those likely to have a role in the survival and capacity for transmission of stumpy forms.

  20. 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction

    Science.gov (United States)

    Höög, Johanna L.; Lacomble, Sylvain; Bouchet-Marquis, Cedric; Briggs, Laura; Park, Kristin; Hoenger, Andreas; Gull, Keith

    2016-01-01

    Background Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC) is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility. Methodology/Principal Findings We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum. Conclusions/Significance The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life

  1. Virulence and pathogenicity of three Trypanosoma brucei rhodesiense stabilates in a Swiss white mouse model

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    Christopher Kariuki

    2015-05-01

    Full Text Available Background: A key objective in basic research on human African trypanosomiasis (HAT is developing a cheap and reliable experimental model of the disease for use in pathogenesis and drug studies.Objective: With a view to improving current models, a study was undertaken to characterise the virulence and pathogenicity of three Trypanosoma brucei rhodesiense stabilates, labelled as International Livestock Research Institute (ILRI-2918, ILRI-3953, and Institute of Primate Research (IPR-001, infected into Swiss white mice.Methods: Swiss white mice were infected intraperitoneally with trypanosomes and observedfor parasitaemia using wet blood smears obtained by tail snipping. Induction of late-stagedisease was undertaken using diminazene aceturate (40 mg/kg, Berenil with curativetreatment done using melarsoprol (3.6 mg/kg, Arsobal.Results: The prepatent period for the stabilates ranged from three to four days with mean peak parasitaemia ranging from Log10 6.40 to 8.36. First peak parasitaemia for all stabilates varied between six and seven days post infection (DPI followed by secondary latency in ILRI-2918 (15–17 DPI and IPR-001 (17–19 DPI. Survival times ranged from six DPI (ILRI-3953 to 86 DPI (IPR-001. Hindleg paresis was observed in both ILRI-3953 (at peak parasitaemia and ILRI-2918 (after relapse parasitaemia. Mice infected with IPR-001 survived until 54 DPI when curative treatment was undertaken.Conclusions: This study demonstrated that the stabilates ILRI-2918 and ILRI-3953 were unsuitable for modelling late-stage HAT in mice. The stabilate IPR-001 demonstrated the potential to induce chronic trypanosomiasis in Swiss white mice for use in development of a late-stage model of HAT.

  2. Structural Insights into Inhibition of Sterol 14[alpha]-Demethylase in the Human Pathogen Trypanosoma cruzi

    Energy Technology Data Exchange (ETDEWEB)

    Lepesheva, Galina I.; Hargrove, Tatiana Y.; Anderson, Spencer; Kleshchenko, Yuliya; Furtak, Vyacheslav; Wawrzak, Zdzislaw; Villalta, Fernando; Waterman, Michael R. (Vanderbilt); (NWU); (Meharry)

    2010-09-02

    Trypanosoma cruzi causes Chagas disease (American trypanosomiasis), which threatens the lives of millions of people and remains incurable in its chronic stage. The antifungal drug posaconazole that blocks sterol biosynthesis in the parasite is the only compound entering clinical trials for the chronic form of this infection. Crystal structures of the drug target enzyme, Trypanosoma cruzi sterol 14{alpha}-demethylase (CYP51), complexed with posaconazole, another antifungal agent fluconazole and an experimental inhibitor, (R)-4{prime}-chloro-N-(1-(2,4-dichlorophenyl)-2-(1H-imid-azol-1-yl)ethyl)biphenyl-4-carboxamide (VNF), allow prediction of important chemical features that enhance the drug potencies. Combined with comparative analysis of inhibitor binding parameters, influence on the catalytic activity of the trypanosomal enzyme and its human counterpart, and their cellular effects at different stages of the Trypanosoma cruzi life cycle, the structural data provide a molecular background to CYP51 inhibition and azole resistance and enlighten the path for directed design of new, more potent and selective drugs to develop an efficient treatment for Chagas disease.

  3. Mammalian cellular culture models of Trypanosoma cruzi infection: a review of the published literature

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    Duran-Rehbein Gabriel Alberto

    2014-01-01

    Full Text Available Cellular culture infection with Trypanosoma cruzi is a tool used to dissect the biological mechanisms behind Chagas disease as well as to screen potential trypanocidal compounds. Data on these models are highly heterogeneous, which represents a challenge when attempting to compare different studies. The purpose of this review is to provide an overview of the cell culture infectivity assays performed to date. Scientific journal databases were searched for articles in which cultured cells were infected with any Trypanosoma cruzi strain or isolate regardless of the study’s goal. From these articles the cell type, parasite genotype, culture conditions and infectivity results were extracted. This review represents an initial step toward the unification of infectivity model data. Important differences were detected when comparing the pathophysiology of Chagas disease with the experimental conditions used in the analyzed studies. While Trypanosoma cruzi preferentially infects stromal cells in vivo, most of the assays employ epithelial cell lines. Furthermore, the most commonly used parasite strain (Tulahuen-TcVI is associated with chagasic cardiomyopathy only in the Southern Cone of South America. Suggestions to overcome these discrepancies include the use of stromal cell lines and parasite genotypes associated with the known characteristics of the natural history of Chagas disease.

  4. Mammalian cellular culture models of Trypanosoma cruzi infection: a review of the published literature.

    Science.gov (United States)

    Duran-Rehbein, Gabriel Alberto; Vargas-Zambrano, Juan Camilo; Cuéllar, Adriana; Puerta, Concepción Judith; Gonzalez, John Mario

    2014-01-01

    Cellular culture infection with Trypanosoma cruzi is a tool used to dissect the biological mechanisms behind Chagas disease as well as to screen potential trypanocidal compounds. Data on these models are highly heterogeneous, which represents a challenge when attempting to compare different studies. The purpose of this review is to provide an overview of the cell culture infectivity assays performed to date. Scientific journal databases were searched for articles in which cultured cells were infected with any Trypanosoma cruzi strain or isolate regardless of the study's goal. From these articles the cell type, parasite genotype, culture conditions and infectivity results were extracted. This review represents an initial step toward the unification of infectivity model data. Important differences were detected when comparing the pathophysiology of Chagas disease with the experimental conditions used in the analyzed studies. While Trypanosoma cruzi preferentially infects stromal cells in vivo, most of the assays employ epithelial cell lines. Furthermore, the most commonly used parasite strain (Tulahuen-TcVI) is associated with chagasic cardiomyopathy only in the Southern Cone of South America. Suggestions to overcome these discrepancies include the use of stromal cell lines and parasite genotypes associated with the known characteristics of the natural history of Chagas disease. © G.A. Duran-Rehbein et al., published by EDP Sciences, 2014.

  5. PCR approach for the detection of Trypanosoma brucei and T. equiperdum and their differentiation from T. evansi based on maxicircle kinetoplast DNA.

    Science.gov (United States)

    Li, Feng-Jun; Gasser, Robin B; Lai, De-Hua; Claes, Filip; Zhu, Xing-Quan; Lun, Zhao-Rong

    2007-02-01

    The goal of this study was to develop a PCR approach based on the sequence of maxicircle kinetoplast DNA (kDNA) of Trypanosoma brucei to distinguish T. brucei/T. equiperdum from T. evansi and to evaluate its diagnostic use for their detection in blood samples. Primers derived from the sequence of the maxicircle kDNA of T. brucei, encoding the NADH dehydrogenase subunit 5 (nad5) gene, were used to test the PCR-amplification from T. brucei (including T. b. brucei and T. b. rhodesiense), T. equiperdum, T. evansi, T. vivax and T. congolense. A primer pair to a nuclear DNA region incorporated into a separate PCR was employed to control for the presence of amplifiable genomic DNA (representing the subgenus Trypanozoon) in each sample subjected to the PCR. Products of approximately 395bp were amplified from all T. brucei and T. equiperdum samples tested using the nad5-PCR, but not from T. evansi DNA samples or any of the control samples representing T. vivax, T. congolense, or host. The current PCR approach allows the rapid differentiation of T. brucei/T.equiperdum from T. evansi and can detect the equivalent of 20-25 cells of T. brucei or T. equiperdum in purified genomic DNA or infected blood samples.

  6. Population genetic analysis of Colombian Trypanosoma cruzi isolates revealed by enzyme electrophoretic profiles

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    Manuel Ruiz-Garcia

    2001-01-01

    Full Text Available Although Colombia presents an enormous biological diversity, few studies have been conducted on the population genetics of Trypanosoma cruzi. This study was carried out with 23 Colombian stocks of this protozoa analyzed for 13 isoenzymatic loci. The Hardy-Weinberg equilibrium, the genetic diversity and heterogeneity, the genetic relationships and the possible spatial structure of these 23 Colombian stocks of T. cruzi were estimated. The majority of results obtained are in agreement with a clonal population structure. Nevertheless, two aspects expected in a clonal structure were not discovered in the Colombian T. cruzi stocks. There was an absence of given zymodemes over-represented from a geographical point of view and the presumed temporal stabilizing selective phenomena was not observed either in the Colombian stocks sampled several times through the years of the study. Some hypotheses are discussed in order to explain the results found.

  7. Trypanosoma cruzi P21: a potential novel target for chagasic cardiomyopathy therapy

    Science.gov (United States)

    Teixeira, Thaise Lara; Machado, Fabrício Castro; Alves da Silva, Aline; Teixeira, Samuel Cota; Borges, Bruna Cristina; dos Santos, Marlus Alves; Martins, Flávia Alves; Brígido, Paula Cristina; Rodrigues, Adele Aud; Notário, Ana Flávia Oliveira; Ferreira, Bruno Antônio; Servato, João Paulo Silva; Deconte, Simone Ramos; Lopes, Daiana Silva; Ávila, Veridiana Melo Rodrigues; Araújo, Fernanda de Assis; Tomiosso, Tatiana Carla; Silva, Marcelo José Barbosa; da Silva, Claudio Vieira

    2015-01-01

    Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of cardiomyopathy in Latin America. It is estimated that 10%–30% of all infected individuals will acquire chronic chagasic cardiomyopathy (CCC). The etiology of CCC is multifactorial and involves parasite genotype, host genetic polymorphisms, immune response, signaling pathways and autoimmune progression. Herein we verified the impact of the recombinant form of P21 (rP21), a secreted T. cruzi protein involved in host cell invasion, on progression of inflammatory process in a polyester sponge-induced inflammation model. Results indicated that rP21 can recruit immune cells induce myeloperoxidase and IL-4 production and decrease blood vessels formation compared to controls in vitro and in vivo. In conclusion, T. cruzi P21 may be a potential target for the development of P21 antagonist compounds to treat chagasic cardiomyopathy. PMID:26574156

  8. Infection by Trypanosoma cruzi in mammals in Yucatan, Mexico: a serological and parasitological study.

    Science.gov (United States)

    Zavala-Velázquez, J; Barrera-Pérez, M; Rodríguez-Félix, M E; Guzmán-Marín, E; Ruíz-Piña, H

    1996-01-01

    In order to determine Trypanosoma cruzi infection among mammals in Yucatan, Mexico, 372 animals, both wild and synanthropic including carnivores, marsupials and rodents were studied. Serological studies by indirect haemagglutination (IHA) were carried out to detect antibodies to T. cruzi and a parasitological study was also performed (blood smear and histopathology). Of all the animals tested 18.54% were serologically positive, with a significantly higher frequency among the wild ones (33.33%) compared to the synanthropic ones (17.79%). To determine T. cruzi in positive animals, blood was inoculated into a white mouse (webster type) to prove myocardium colonization. The serological and parasitological positivity of these animals, as well as their behavior in the environment, taken together with the socioeconomic and cultural characteristics of the population, suggest that in Yucatan, Mexico, Canis familiaris, Didelphis marsupialis and Rattus rattus act as a link with the wild cycle.

  9. Circulation of Tc Ia discrete type unit Trypanosoma cruzi in Yucatan Mexico.

    Science.gov (United States)

    Monteón, Victor; Triana-Chávez, Omar; Mejía-Jaramillo, Ana; Pennignton, Pamela; Ramos-Ligonio, Ángel; Acosta, Karla; Lopez, Ruth

    2016-06-01

    The etiologic agent Trypanosoma cruzi (Tc) has been grouped into six discrete type units (DTU I-VI); within DTU-I exists four subgroups defined Ia-Id. In Colombia, the genotype Ia is associated with human infection and domiciliated Rhodnius vector. In the Yucatan Peninsula of Mexico, the main vector involved in T. cruzi transmission is Triatoma dimidiata predominantly via sylvatic and peridomiciliated cycles. In this study, multiple sequence analysis of mini-exon intergenic regions of T. cruzi isolates obtained from T. dimidiata in the Yucatan Peninsula of Mexico revealed they belonged to Tc Ia DTU along with two additional Mexican strains located 1,570 km away from Yucatan. In conclusion Tc Ia circulates in the Yucatan peninsula in T. dimidiata vector and likewise in the northwest region of Mexico.

  10. Role of Trypanosoma cruzi Trans-sialidase on the Escape from Host Immune Surveillance

    Science.gov (United States)

    Nardy, Ana F. F. R.; Freire-de-Lima, Celio G.; Pérez, Ana R.; Morrot, Alexandre

    2016-01-01

    Chagas disease is caused by the flagellate protozoan Trypanosoma cruzi, affecting millions of people throughout Latin America. The parasite dampens host immune response causing modifications in diverse lymphoid compartments, including the thymus. T. cruzi trans-sialidase (TS) seems to play a fundamental role in such immunopathological events. This unusual enzyme catalyses the transference of sialic acid molecules from host glycoconjugates to acceptor molecules placed on the parasite surface. TS activity mediates several biological effects leading to the subversion of host immune system, hence favoring both parasite survival and the establishment of chronic infection. This review summarizes current findings on the roles of TS in the immune response during T. cruzi infection. PMID:27047464

  11. In vitro activity of Etanidazole against the protozoan parasite Trypanosoma cruzi

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    Patricia B Petray

    2004-03-01

    Full Text Available We investigated the in vitro action of an hydrosoluble 2-nitroimidazole, Etanidazole (EZL, against Trypanosoma cruzi, the etiologic agent of Chagas disease. EZL displayed lethal activity against isolated trypomastigotes as well as amastigotes of T. cruzi (RA strain growing in Vero cells or J774 macrophages, without affecting host cell viability. Although not completely equivalent to Benznidazole (BZL, the reference drug for Chagas chemotherapy, EZL takes advantage in exertingits anti-T. cruzi activity for longer periods without serious toxic side effects, as those recorded in BZL-treated patients. Our present results encourage further experiments to study in depth the trypanocidal properties of this drug already licensed for use in human cancers.

  12. JVG9, a benzimidazole derivative, alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes.

    Science.gov (United States)

    Díaz-Chiguer, Dylan L; Hernández-Luis, Francisco; Nogueda-Torres, Benjamín; Castillo, Rafael; Reynoso-Ducoing, Olivia; Hernández-Campos, Alicia; Ambrosio, Javier R

    2014-09-01

    Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of microtubules and actin microfilaments. Therefore, it is an excellent target for the development of new anti-parasitic drugs. Benzimidazole 2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown to inhibit the in vitro growth of many protozoa. Therefore, to find efficient anti-trypanosomal (trypanocidal) drugs, our group has designed and synthesised several benzimidazole derivatives. One, named JVG9 (5-chloro-1H-benzimidazole-2-thiol), has been found to be effective against T. cruzi bloodstream trypomastigotes under both in vitro and in vivo conditions. Here, we present the in vitro effects observed by laser scanning confocal and scanning electron microscopy on T. cruzi trypomastigotes. Changes in the surface and the distribution of the cytoskeletal proteins are consistent with the hypothesis that the trypanocidal activity of JVG9 involves the cytoskeleton as a target.

  13. JVG9, a benzimidazole derivative, alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes

    Directory of Open Access Journals (Sweden)

    Dylan L Díaz-Chiguer

    2014-09-01

    Full Text Available Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of microtubules and actin microfilaments. Therefore, it is an excellent target for the development of new anti-parasitic drugs. Benzimidazole 2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown to inhibit the in vitro growth of many protozoa. Therefore, to find efficient anti-trypanosomal (trypanocidal drugs, our group has designed and synthesised several benzimidazole derivatives. One, named JVG9 (5-chloro-1H-benzimidazole-2-thiol, has been found to be effective against T. cruzi bloodstream trypomastigotes under both in vitro and in vivo conditions. Here, we present the in vitro effects observed by laser scanning confocal and scanning electron microscopy on T. cruzi trypomastigotes. Changes in the surface and the distribution of the cytoskeletal proteins are consistent with the hypothesis that the trypanocidal activity of JVG9 involves the cytoskeleton as a target.

  14. New 1,3-thiazole derivatives and their biological and ultrastructural effects on Trypanosoma cruzi.

    Science.gov (United States)

    de Moraes Gomes, Paulo André Teixeira; de Oliveira Barbosa, Miria; Farias Santiago, Edna; de Oliveira Cardoso, Marcos Veríssimo; Capistrano Costa, Natáli Tereza; Hernandes, Marcelo Zaldini; Moreira, Diogo Rodrigo Magalhães; da Silva, Aline Caroline; Dos Santos, Thiago André Ramos; Pereira, Valéria Rêgo Alves; Brayner Dos Santosd, Fábio André; do Nascimento Pereira, Glaécia Aparecida; Ferreira, Rafaela Salgado; Leite, Ana Cristina Lima

    2016-10-04

    In previous studies, the compound 3-(bromopropiophenone) thiosemicarbazone was described as a potent anti-Trypanosoma cruzi and cruzain inhibitor. In view to optimize this activity, 1,3-thiazole core was used as building-block strategy to access new lead generation of anti T. cruzi agents. In this way a series of thiazole derivatives were synthesized and most of these derivatives exhibited antiparasitic activity similar to benznidazole (Bzd). Among them, compounds (1c) and (1g) presented better selective index (SI) than Bzd. In addition, compounds showed inhibitory activity against the cruzain protease. As observed by electron microscopy, compound (1c) treatment caused irreversible and specific morphological changes on ultrastructure organization of T. cruzi, demonstrating that this class of compounds is killing parasites. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. Dialkylimidazole inhibitors of Trypanosoma cruzi sterol 14α-demethylase as anti-Chagas disease agents.

    Science.gov (United States)

    Suryadevara, Praveen Kumar; Racherla, Kishore Kumar; Olepu, Srinivas; Norcross, Neil R; Tatipaka, Hari Babu; Arif, Jennifer A; Planer, Joseph D; Lepesheva, Galina I; Verlinde, Christophe L M J; Buckner, Frederick S; Gelb, Michael H

    2013-12-01

    New dialkylimidazole based sterol 14α-demethylase inhibitors were prepared and tested as potential anti-Trypanosoma cruzi agents. Previous studies had identified compound 2 as the most potent and selective inhibitor against parasite cultures. In addition, animal studies had demonstrated that compound 2 is highly efficacious in the acute model of the disease. However, compound 2 has a high molecular weight and high hydrophobicity, issues addressed here. Systematic modifications were carried out at four positions on the scaffold and several inhibitors were identified which are highly potent (EC50 cruzi in culture. The halogenated derivatives 36j, 36k, and 36p, display excellent activity against T. cruzi amastigotes, with reduced molecular weight and lipophilicity, and exhibit suitable physicochemical properties for an oral drug candidate. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. JVG9, a benzimidazole derivative, alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes

    Science.gov (United States)

    Díaz-Chiguer, Dylan L; Hernández-Luis, Francisco; Nogueda-Torres, Benjamín; Castillo, Rafael; Reynoso-Ducoing, Olivia; Hernández-Campos, Alicia; Ambrosio, Javier R

    2014-01-01

    Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of microtubules and actin microfilaments. Therefore, it is an excellent target for the development of new anti-parasitic drugs. Benzimidazole 2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown to inhibit the in vitro growth of many protozoa. Therefore, to find efficient anti-trypanosomal (trypanocidal) drugs, our group has designed and synthesised several benzimidazole derivatives. One, named JVG9 (5-chloro-1H-benzimidazole-2-thiol), has been found to be effective against T. cruzi bloodstream trypomastigotes under both in vitro and in vivo conditions. Here, we present the in vitro effects observed by laser scanning confocal and scanning electron microscopy on T. cruzi trypomastigotes. Changes in the surface and the distribution of the cytoskeletal proteins are consistent with the hypothesis that the trypanocidal activity of JVG9 involves the cytoskeleton as a target. PMID:25317703

  17. Chagas disease (Trypanosoma cruzi and HIV co-infection in Colombia

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    Carolina Hernández

    2014-09-01

    Full Text Available Chagas disease is a complex zoonotic pathology caused by the kinetoplastid Trypanosoma cruzi. This parasite presents remarkable genetic variability and has been grouped into six discrete typing units (DTUs. The association between the DTUs and clinical outcome remains unknown. Chagas disease and co-infection with HIV/AIDS has been reported widely in Brazil and Argentina. Herein, we present the molecular analyses from a Chagas disease patient with HIV/AIDS co-infection in Colombia who presented severe cardiomyopathy, pleural effusion, and central nervous system involvement. A mixed infection by T. cruzi genotypes was detected. We suggest including T. cruzi in the list of opportunistic pathogens for the management of HIV patients in Colombia. The epidemiological implications of this finding are discussed.

  18. [Seroprevalence of Trypanosoma cruzi infection in the rural population of Sucre State, Venezuela].

    Science.gov (United States)

    García-Jordán, Noris; Berrizbeitia, Mariolga; Rodríguez, Jessicca; Concepción, Juan Luis; Cáceres, Ana; Quiñones, Wilfredo

    2017-10-26

    The current study aimed to determine the seroprevalence of Trypanosoma cruzi infection in Sucre State, Venezuela, and its association with epidemiological risk factors. The cluster sampling design allowed selecting 96 villages and 576 dwellings in the State's 15 municipalities. A total of 2,212 serum samples were analyzed by ELISA, HAI, and IFI. Seroprevalence in Sucre State was 3.12%. Risk factors associated with T. cruzi infection were: accumulated garbage, flooring and wall materials, type of dwelling, living in a house with wattle and daub walls and/or straw roofing, living in a house with risky walls and roofing, risky buildings and wattle and daub outbuildings, poultry inside the human dwelling, and presence of firewood. Infection was associated with individual age, and three seropositive cases were found in individuals less than 15 years of age. Sucre State has epidemiological factors that favor the risk of acquiring T. cruzi infection.

  19. Molecular epidemiology of Trypanosoma cruzi and Triatoma dimidiata in costal Ecuador.

    Science.gov (United States)

    Wong, Yim Yan; Sornosa Macias, Karen Jeniffer; Guale Martínez, Doris; Solorzano, Luis F; Ramirez-Sierra, Maria Jesus; Herrera, Claudia; Dumonteil, Eric

    2016-07-01

    Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. In Ecuador, Triatoma dimidiata and Rhodnius ecuadoriensis are the main vector species, responsible for over half of the cases of T. cruzi infection in the country. T. dimidiata is believed to have been introduced in Ecuador during colonial times, and its elimination from the country is thus believed to be feasible. We investigated here the molecular ecology of T. dimidiata and T. cruzi in costal Ecuador to further guide control efforts. Analysis of the Internal Transcribed Spacer 2 (ITS-2) of 23 specimens from Progreso, Guayas, unambiguously supported the likely importation of T. dimidiata from Central America to Ecuador. The observation of a very high parasite infection rate (54%) and frequent feeding on humans (3/5) confirmed a continued risk of transmission to humans. All genotyped parasites corresponded to TcI DTU and Trypanosoma rangeli was not detected in T. dimidiata. TcI subgroups corresponded to TcIa (25%), and mixed infections with TcIa and TcId (75%). Further studies should help clarify T. cruzi genetic structure in the country, and the possible impact of the introduction of T. dimidiata on the circulating parasite strains. The elevated risk posed by this species warrants continuing efforts for its control, but its apparent mobility between peridomestic and domestic habitats may favor reinfestation following insecticide spraying. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Trypanosoma cruzi: strain selection by diferent schedules of mouse passage of an initially mixed infection

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    Maria P. Deane

    1984-12-01

    Full Text Available From an initial double infection in mice, established by simultaneous and equivalent inocula of bloodstream forms of strains Y and F of Trypanosoma cruzi, two lines were derived by subinoculations: one (W passaged every week, the other (M every month. Through biological and biochemical methods only the Y strain was identified at the end of the 10th and 16th passages of line W and only the F strain at the 2nd and 4th passages of line M. The results illustrate strain selection through laboratory manipulation of initially mixed populations of T. cruzi.De uma infecção inicialmente dupla em camundongo, estabelecida por inóculo simultaneo e equivalente de formas sanguíneas das cepas Y e F de Trypanosoma cruzi, duas linhagens foram originadas por subinoculações: uma (W passada casa semana, a outra (M cada mês. Por métodos biológicos e bioquímicos apenas a cepa Y foi identificada ao fim a 10a. e 16a. passagens da linhagem W e apenas a cepa F na 2a. e 4a.passagens de linhagem M. Os resultados demonstram a seleção de cepas através de manipulação em laboratorio de populações inicialmente mistas de T. cruzi.

  1. Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

    Energy Technology Data Exchange (ETDEWEB)

    Buarque, Diego S.; Spindola, Leticia M.N. [Department of Biochemistry, Universidade Federal de Sao Paulo, Escola Paulista de Medicina, 04044-020 Sao Paulo, SP (Brazil); Martins, Rafael M. [Biology of Host Parasite Interactions Unit, Institute Pasteur, 75015 Paris (France); Braz, Gloria R.C. [Department of Biochemistry, Instituto de Quimica, Universidade Federal do Rio de Janeiro, 21941-909 Rio de Janeiro (Brazil); Tanaka, Aparecida S., E-mail: Tanaka.bioq@epm.br [Department of Biochemistry, Universidade Federal de Sao Paulo, Escola Paulista de Medicina, 04044-020 Sao Paulo, SP (Brazil)

    2011-09-23

    Highlights: {yields} Tigutcystatin inhibits Trypanosoma cruzi cysteine proteases with high specificity. {yields} Tigutcystatin expression is up-regulated in response to T. cruzi infection. {yields} It is the first cysteine proteases inhibitor characterized from a triatomine insect. -- Abstract: The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS-PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K{sub i} = 3.29 nM) and human cathepsin L (K{sub i} = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.

  2. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development.

    Science.gov (United States)

    Cueto, Juan Agustín; Vanrell, María Cristina; Salassa, Betiana Nebaí; Nola, Sébastien; Galli, Thierry; Colombo, María Isabel; Romano, Patricia Silvia

    2017-06-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N-ethylmaleimide-sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI-VAMP/VAMP7 and cellubrevin/VAMP3, two v-SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI-VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development. © 2016 John Wiley & Sons Ltd.

  3. Inhibition of HIV-1 replication in human monocyte-derived macrophages by parasite Trypanosoma cruzi.

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    Guadalupe Andreani

    Full Text Available BACKGROUND: Cells of monocyte/macrophage lineage are one of the major targets of HIV-1 infection and serve as reservoirs for viral persistence in vivo. These cells are also the target of the protozoa Trypanosoma cruzi, the causative agent of Chagas disease, being one of the most important endemic protozoonoses in Latin America. It has been demonstrated in vitro that co-infection with other pathogens can modulate HIV replication. However, no studies at cellular level have suggested an interaction between T. cruzi and HIV-1 to date. METHODOLOGY/PRINCIPAL FINDINGS: By using a fully replicative wild-type virus, our study showed that T. cruzi inhibits HIV-1 antigen production by nearly 100% (p99% being stronger than HIV-T. cruzi (approximately 90% for BaL and approximately 85% for VSV-G infection. In MDM with established HIV-1 infection, T. cruzi significantly inhibited luciferate activity (p<0.01. By quantifying R-U5 and U5-gag transcripts by real time PCR, our study showed the expression of both transcripts significantly diminished in the presence of trypomastigotes (p<0.05. Thus, T. cruzi inhibits viral post-integration steps, early post-entry steps and entry into MDM. Trypomastigotes also caused a approximately 60-70% decrease of surface CCR5 expression on MDM. Multiplication of T. cruzi inside the MDM does not seem to be required for inhibiting HIV-1 replication since soluble factors secreted by trypomastigotes have shown similar effects. Moreover, the major parasite antigen cruzipain, which is secreted by the trypomastigote form, was able to inhibit viral production in MDM over 90% (p<0.01. CONCLUSIONS/SIGNIFICANCE: Our study showed that T. cruzi inhibits HIV-1 replication at several replication stages in macrophages, a major cell target for both pathogens.

  4. The Ly49E receptor inhibits the immune control of acute Trypanosoma cruzi infection

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    Jessica Filtjens

    2016-11-01

    Full Text Available The protozoan parasite Trypanosoma cruzi (T. cruzi circulates in the blood upon infection and invades a variety of cells. Parasites intensively multiply during the acute phase of infection and persist lifelong at low levels in tissues and blood during the chronic phase. Natural killer (NK and NKT cells play an important role in the immune control of T. cruzi infection, mainly by releasing the cytokine IFN-γ that activates the microbicidal action of macrophages and other cells and shapes a protective type 1 immune response. The mechanisms by which immune cells are regulated to produce IFN-γ during T. cruzi infection are still incompletely understood. Here, we show that urokinase plasminogen activator (uPA is induced early upon T. cruzi infection, and remains elevated until day 20 post inoculation. We previously demonstrated that the inhibitory receptor Ly49E, which is expressed, among others, on NK and NKT cells, is triggered by uPA. Therefore, we compared wild type (WT to Ly49E knockout (KO mice for their control of experimental T. cruzi infection. Our results show that young, i.e. 4- and 6-week-old, Ly49E KO mice control the infection better than WT mice, indicated by a lower parasite load and less cachexia. The beneficial effect of Ly49E depletion is more obvious in 4-week-old male than in female mice and weakens in 8-week-old mice. In young mice, the lower T. cruzi parasitemia in Ly49E KO mice is paralleled by higher IFN-γ production compared to their WT controls. Our data indicate that Ly49E receptor expression inhibits the immune control of T. cruzi infection. This is the first demonstration that the inhibitory Ly49E receptor can interfere with the immune response to a pathogen in vivo.

  5. Interaction between Gallotannin and a Recombinant Form of Arginine Kinase of Trypanosoma brucei: Thermodynamic and Spectrofluorimetric Evaluation

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    O. S. Adeyemi

    2014-01-01

    Full Text Available Current chemotherapies against trypanosomiasis are beset with diverse challenges, a situation which underscores the numerous research efforts aimed at finding newer and effective treatments. Arginine kinase of trypanosome has been validated as target for drug development against trypanosomiasis. The present study investigated the interaction between a recombinant form of the arginine kinase (rTbAK of trypanosome and gallotannin. The interaction between gallotannin and recombinant arginine kinase of Trypanosoma brucei caused significant decrease of enzyme activity. Kinetic analysis revealed the interaction to be of noncompetitive inhibition. Further thermodynamic analysis showed that the interaction between gallotannin and the recombinant arginine kinase was nonspontaneous and involved hydrophobic forces. The Ksv values and the FRET analysis suggest that static quenching of fluorescence intensity by gallotannin was static. Data revealed inhibitory interactions between gallotannin and rTbAK of trypanosome. Although the mechanism of inhibition is not clear yet, molecular docking studies are ongoing to clearly define the inhibitory interactions between the gallotannin and rTbAK. The knowledge of such binding properties would enrich development of selective inhibitors for the arginine kinase of Trypanosoma brucei.

  6. Comprehensive glycoprofiling of the epimastigote and trypomastigote stages of Trypanosoma cruzi.

    Science.gov (United States)

    Alves, Maria Julia Manso; Kawahara, Rebeca; Viner, Rosa; Colli, Walter; Mattos, Eliciane Cevolani; Thaysen-Andersen, Morten; Larsen, Martin Røssel; Palmisano, Giuseppe

    2017-01-16

    Trypanosoma cruzi, the protozoan that causes Chagas disease, has a complex life cycle involving insect and mammalian hosts and distinct developmental stages. During T. cruzi developmental stages, glycoproteins play important role in the host-parasite interaction, such as cellular recognition, host cell invasion and adhesion, and immune evasion. In this study, comprehensive glycoprofiling analysis was performed in the epimastigote and trypomastigote stages of T. cruzi using two glycopeptide enrichment strategies, lectin-based and hydrophilic interaction liquid chromatography, followed by high resolution LC-MS/MS. Following deglycosylation, a total of 1306 N-glycosylation sites in NxS/T/C motifs were identified from 690 T. cruzi glycoproteins. Among them, 170 and 334 glycoproteins were exclusively identified in epimastigotes and trypomastigotes, respectively. Besides, global site-specific characterization of the N- and O-linked glycan heterogeneity in the two life stages of T. cruzi was achieved by intact glycopeptide analysis, revealing 144/466 unique N-linked and 10/97 unique O-linked intact glycopeptides in epimastigotes/trypomastigotes, respectively. Conclusively, this study documents the significant T. cruzi stage-specific expression of glycoproteins that can help to better understand the T. cruzi phenotype and response caused by the interaction with different hosts during its complex life cycle. Chagas disease caused by the protozoan Trypanosoma cruzi is a neglected disease which affects millions of people especially in Latin America. The absence of efficient drugs and vaccines against Chagas disease stimulates the search for novel targets. Glycoproteins are very attractive therapeutic candidate targets since they mediate key processes in the host-parasite interaction, such as cellular recognition, host cell invasion and adhesion, and immune evasion. This study aimed to provide an in depth characterization of the N-linked and O-linked glycoproteome of two T

  7. The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase of Trypanosomatidae and the glycocomal redox balance of insect stages of Trypanosoma brucei and Leishmania spp.

    NARCIS (Netherlands)

    Guerra, D.G.; Decottignies, A.; Bakker, B.M.; Michels, P.A.M.

    2006-01-01

    The genes for the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase were identified in Trypanosoma brucei and Leishmania major genomes. We have expressed the L. major gene in Saccharomyces cerevisiae and confirmed the subcellular localization and activity of the produced enzyme. Using

  8. Trypanosoma brucei TBRGG1, a mitochondrial oligo(U)-binding protein that co-localizes with an in vitro RNA editing activity

    NARCIS (Netherlands)

    Vanhamme, L.; Perez-Morga, D.; Marchal, C.; Speijer, D.; Lambert, L.; Geuskens, M.; Alexandre, S.; Ismaïli, N.; Göringer, U.; Benne, R.; Pays, E.

    1998-01-01

    We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their

  9. Discovery of novel Trypanosoma brucei phosphodiesterase B1 inhibitors by virtual screening against the unliganded TbrPDEB1 crystal structure

    NARCIS (Netherlands)

    Jansen, C.; Wang, H.; Kooistra, A.J.; de Graaf, C.; Orrling, K.M.; Tenor, H.; Seebeck, T.; de Esch, I.J.P.; Ke, H.; Leurs, R.

    2013-01-01

    Trypanosoma brucei cyclic nucleotide phosphodiesterase B1 (TbrPDEB1) and TbrPDEB2 have recently been validated as new therapeutic targets for human African trypanosomiasis by both genetic and pharmacological means. In this study we report the crystal structure of the catalytic domain of the

  10. Immunospecific immunoglobulins and IL-10 as markers for Trypanosoma brucei rhodesiense late stage disease in experimentally infected vervet monkeys

    DEFF Research Database (Denmark)

    Ngotho, Maina; Kagira, J.M.; Jensen, Henrik Michael Elvang

    2009-01-01

    OBJECTIVE: To determine the usefulness of IL-10 and immunoglobulin M (IgM) as biomarkers for staging HAT in vervet monkeys, a useful pathogenesis model for humans. METHODS: Vervet monkeys were infected with Trypanosoma brucei rhodesiense and subsequently given sub-curative and curative treatment 28...

  11. Anotaciones sobre la enfermedad de Chagas en Panamá. Frecuencia y distribución de Trypanosoma cruzi y Trypanosoma rangeli

    OpenAIRE

    Sousa, Octavio E.

    2016-01-01

    In Panama Chagas' disease occurs primarily in the provinces of Panama and Colon, near the Canal Zone. Trypanosoma cruzi infections appear to be of moderate parasitemia and short patency, without inducement of megaviscera. In Panama province the prevalence in the rural population was 2.8% and 1.9% in the province of Colon. In both provinces T. rangeli is 6 to 10 times more frequent than T. cruzi. Positive serologic reactions (complement fixation) were found in almost all the provinces, ranging...

  12. Activity of Novel Tryptophan Analogs against Trypanosoma cruzi ...

    African Journals Online (AJOL)

    Nx 6110

    3Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel St,. London, WC 1E 7HT, United Kingdom. Drugs available for the ... activity against T. cruzi, producing more than 50 % inhibition of multiplication at or below 30 μM concentrations. Four compounds also had significant activity ...

  13. Trypanocidal drugs for chronic asymptomatic Trypanosoma cruzi infection.

    Science.gov (United States)

    Villar, Juan Carlos; Perez, Juan Guillermo; Cortes, Olga Lucia; Riarte, Adelina; Pepper, Micah; Marin-Neto, Jose Antonio; Guyatt, Gordon H

    2014-05-27

    Prevention of chronic chagasic cardiomyopathy (CCC) by treating infected populations with trypanocidal therapy (TT) remains a challenge. Despite a renewed enthusiasm for TT, uncertainty regarding its efficacy, concerns about its safety and limited availability remain barriers for a wider use of conventional drugs. We have updated a previous version of this review. To systematically search, appraise, identify and extract data from eligible studies comparing the outcome of cohorts of seropositive individuals to Trypanosoma cruzi exposed to TT versus placebo or no treatment. We sought eligible studies in electronic databases (Cochrane Central Register of Controlled Trials (CENTRAL), Issue 1, 2014); MEDLINE (Ovid, 1946 to January week 5 2014); EMBASE (Ovid, 1980 to 2014 week 6) and LILACS (up to 6 May 2010)) by combining terms related with the disease and the treatment. The search also included a Google search, handsearch for references in review or selected articles, and search of expert files. We applied no language restrictions. Review authors screened the retrieved references for eligibility (those dealing with human participants treated with TT) and then assessed the pre-selected studies in full for inclusion. We included randomised controlled trials (RCTs) and observational studies that provided data on either mortality or clinical progression of CCC after at least four years of follow-up. Teams of two review authors independently carried out the study selection, data extraction and risk of bias assessment, with a referee resolving disagreement within the pairs. Data collection included study design, characteristics of the population and interventions or exposures and outcome measures. We defined categories of outcome data as parasite-related (positive serology, xenodiagnosis or polymerase chain reaction (PCR) after TT) and participant-related (including efficacy outcomes such as progression towards CCC, all-cause mortality and side effects of TT). We reported

  14. A high-affinity putrescine-cadaverine transporter from Trypanosoma cruzi

    Science.gov (United States)

    Hasne, Marie-Pierre; Coppens, Isabelle; Soysa, Radika; Ullman, Buddy

    2011-01-01

    Summary Whereas mammalian cells and most other organisms can synthesize polyamines from basic amino acids, the protozoan parasite Trypanosoma cruzi is incapable of polyamine biosynthesis de novo and therefore obligatorily relies upon putrescine acquisition from the host to meet its nutritional requirements. The cell surface proteins that mediate polyamine transport into T. cruzi, as well as most eukaryotes, however, have by-in-large eluded discovery at the molecular level. Here we report the identification and functional characterization of two polyamine transporters, TcPOT1.1 and TcPOT1.2, encoded by alleles from two T. cruzi haplotypes. Overexpression of the TcPOT1.1 and TcPOT1.2 genes in T. cruzi epimastigotes revealed that TcPOT1.1 and TcPOT1.2 were high-affinity transporters that recognized both putrescine and cadaverine but not spermidine or spermine. Furthermore, the activities and subcellular locations of both TcPOT1.1 and TcPOT1.2 in intact parasites were profoundly influenced by extracellular putrescine availability. These results establish TcPOT1.1 and TcPOT1.2 as key components of the T. cruzi polyamine transport pathway, an indispensable nutritional function for the parasite that may be amenable to therapeutic manipulation. PMID:20149109

  15. Triatominae (Hemiptera, Reduviidae in the Pantanal region: association with Trypanosoma cruzi, different habitats and vertebrate hosts

    Directory of Open Access Journals (Sweden)

    Filipe Martins Santos

    2015-10-01

    Full Text Available ABSTRACTINTRODUCTION: The transmission cycle of Trypanosoma cruzi in the Brazilian Pantanal region has been studied during the last decade. Although considerable knowledge is available regarding the mammalian hosts infected by T. cruzi in this wetland, no studies have investigated its vectors in this region. This study aimed to investigate the presence of sylvatic triatomine species in different habitats of the Brazilian Pantanal region and to correlate their presence with the occurrences of vertebrate hosts and T. cruzi infection.METHODS: The fieldwork involved passive search by using light traps and Noireau traps and active search by visual inspection. The light traps were placed at five selected points along forested areas for seven nights during each of the nine excursions. At each point where a light trap was set, eight Noireau traps were placed in palm trees and bromeliads.RESULTS: In all, 88 triatomine bugs were collected: two and one individuals from light traps and Noireau traps, respectively; three from peridomestic areas; 23 in coati nests; and 59 in thornbird nests. In this study, active search in microhabitats showed higher efficiency than passive search, since 95% of the triatomine bugs were caught in nests. Further, triatomine bugs were only found to be infected by T. cruzi in coati nests.CONCLUSIONS: Coati nests might act as a point of convergence and dispersion for triatomine bugs and mammal hosts infected by T. cruzi, thereby playing an important role in the sylvatic cycle of T. cruziin the Pantanal region.

  16. Trypanosoma cruzi strains from triatomine collected in Bahia and Rio Grande do Sul, Brazil

    Science.gov (United States)

    Ribeiro, Aline Rimoldi; Mendonça, Vagner José; Alves, Renata Tomé; Martinez, Isabel; de Araújo, Renato Freitas; Mello, Fernanda; da Rosa, João Aristeu

    2014-01-01

    OBJECTIVE Collection of triatomines in domestic, peridomestic and sylvatic environments in states of Bahia and Rio Grande do Sul, Northeastern and Southern Brazil respectively, and isolation of Trypanosoma cruzi strains. METHODS First, the captured triatomines were identified using insect identification keys, then their intestinal content was examined by abdominal compression, and the samples containing trypanosomatid forms were inoculated in LIT medium and Swiss mice. RESULTS Six triatomine species were collected in cities in Bahia, namely Panstrongylus geniculatus (01), Triatoma melanocephala (11), T. lenti (94), T. pseudomaculata (02), T. sherlocki (26) and T. sordida (460), and two in cities in Rio Grande do Sul, namely T. circummaculata (11) and T. rubrovaria (115). Out of the specimens examined, T. cruzi was isolated from 28 triatomine divided into four different species: T. melanocephala (one), T. lenti (one), T. rubrovaria (16) and T. sordida (10). Their index of natural infection by T. cruzi was 6.4%. CONCLUSIONS The isolation of T. cruzi strains from triatomines found in domestic and peridomestic areas shows the potential risk of transmission of Chagas disease in the studied cities. The maintenance of those T. cruzi strains in laboratory is intended to promote studies that facilitate the understanding of the parasite-vector-host relationship. PMID:24897051

  17. Effects of buthionine sulfoximine nifurtimox and benznidazole upon trypanothione and metallothionein proteins in Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    JUAN DIEGO MAYA

    2004-01-01

    Full Text Available Proteins rich in sulfhydryl groups, such as metallothionein, are present in several strains of the parasite Trypanosoma cruzi, the etiological agent of Chagas' disease. Metallothionein-like protein concentrations ranged from 5.1 to 13.2 pmol/mg protein depending on the parasite strain and growth phase. Nifurtimox and benznidazole, used in the treatment of Chagas' disease, decreased metallothionein activity by approximately 70%. T. cruzi metallothionein was induced by ZnCl2. Metallothionein from T. cruzi was partially purified and its monobromobimane derivative showed a molecular weight of approximately 10,000 Da by SDS-PAGE analysis. The concentration of trypanothione, the major glutathione conjugate in T. cruzi, ranged from 3.8 to 10.8 nmol/mg protein, depending on the culture phase. The addition of buthionine sulfoximine to the protozoal culture considerably reduced the concentration of trypanothione and had no effect upon the metallothionein concentration. The possible contribution of metallothionein-like proteins to drug resistance in T. cruzi is discussed.

  18. Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase

    Energy Technology Data Exchange (ETDEWEB)

    Schormann, Norbert; Velu, Sadanandan E.; Murugesan, Srinivasan; Senkovich, Olga; Walker, Kiera; Chenna, Bala C.; Shinkre, Bidhan; Desai, Amar; Chattopadhyay, Debasish (UAB)

    2010-09-17

    Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas disease. We have undertaken a detailed structure-activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme-ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60-70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.

  19. Activity of P536, a UDP-glucose analog, against Trypanosoma cruzi

    Energy Technology Data Exchange (ETDEWEB)

    Alcina, A.; Fresno, M.; Alarcon, B.

    1988-09-01

    P536, a UDP-glucose analog which was previously described as an antiviral agent, has a potent and selective activity against the intracellular and extracellular stages of Trypanosoma cruzi in vitro. It had a 50% inhibitory concentration of less than 5 micrograms/ml for T. cruzi extracellular cultured forms (epimastigote) and of 25 micrograms/ml for T. cruzi intracellular forms (amastigote) growing inside J774G8 macrophage-like cells. In contrast, the 50% inhibitory concentration was 100 micrograms/ml or greater for cultured mammalian cells and 180 micrograms/ml for the proliferation of mouse spleen lymphocytes. Furthermore, the addition of P536 (50 micrograms/ml) to T. cruzi-infected J774G8 cells cured the infected macrophages, making them able to grow and function normally. Studies on the mechanism of action of this drug indicated that it inhibited incorporation of (TVS)methionine, (TH)thymidine, (TH)mannose, ( UC)-N-acetylglucosamine, and (TH)uridine into macromolecules by T. cruzi epimastigotes, the last being the most sensitive.

  20. Molecular Epidemiology of Trypanosomatids and Trypanosoma cruzi in Primates from Peru.

    Science.gov (United States)

    Aysanoa, Esar; Mayor, Pedro; Mendoza, A Patricia; Zariquiey, Carlos M; Morales, E Angelo; Pérez, Jocelyn G; Bowler, Mark; Ventocilla, Julio A; González, Carlos; Baldeviano, G Christian; Lescano, Andrés G

    2017-11-02

    We determined the prevalence rate and risk of infection of Trypanosoma cruzi and other trypanosomatids in Peruvian non-human primates (NHPs) in the wild (n = 126) and in different captive conditions (n = 183). Blood samples were collected on filter paper, FTA cards, or EDTA tubes and tested using a nested PCR protocol targeting the 24Sα rRNA gene. Main risk factors associated with trypanosomatid and T. cruzi infection were genus and the human-animal context (wild vs captive animals). Wild NHPs had higher prevalence of both trypanosomatids (64.3 vs 27.9%, P < 0.001) and T. cruzi (8.7 vs 3.3%, P = 0.057), compared to captive NHPs, suggesting that parasite transmission in NHPs occurs more actively in the sylvatic cycle. In terms of primate family, Pitheciidae had the highest trypanosomatid prevalence (20/22, 90.9%) and Cebidae had the highest T. cruzi prevalence (15/117, 12.8%). T. cruzi and trypanosomatids are common in Peruvian NHPs and could pose a health risk to human and animals that has not been properly studied.

  1. Persistent infections in chronic Chagas' disease patients treated with anti-Trypanosoma cruzi nitroderivatives

    Directory of Open Access Journals (Sweden)

    BRAGA M. Socorro

    2000-01-01

    Full Text Available We used a molecular method and demonstrated that treatment of the chronic human Trypanosoma cruzi infections with nitroderivatives did not lead to parasitological cure. Seventeen treated and 17 untreated chronic Chagas' disease patients, with at least two out of three positive serologic assays for the infection, and 17 control subjects formed the study groups. PCR assays with nested sets of T. cruzi DNA primers monitored the efficacy of treatment. The amplification products were hybridized to their complementary internal sequences. Untreated and treated Chagas' disease patients yielded PCR amplification products with T. cruzi nuclear DNA primers. Competitive PCR was conducted to determine the quantity of parasites in the blood and revealed < 1 to 75 T. cruzi/ml in untreated (means 25.83 ± 26.32 and < 1 to 36 T. cruzi/ml in treated (means 6.45 ± 9.28 Chagas' disease patients. The difference between the means was not statistically significant. These findings reveal a need for precise definition of the role of treatment of chronic Chagas' disease patients with nitrofuran and nitroimidazole compounds.

  2. Trypanosoma cruzi: In vitro effect of aspirin with nifurtimox and benznidazole.

    Science.gov (United States)

    López-Muñoz, Rodrigo; Faúndez, Mario; Klein, Sebastián; Escanilla, Sebastián; Torres, Gloria; Lee-Liu, Dasfne; Ferreira, Jorge; Kemmerling, Ulrike; Orellana, Myriam; Morello, Antonio; Ferreira, Arturo; Maya, Juan D

    2010-02-01

    Nifurtimox and benznidazole are the only active drugs against Trypanosoma cruzi; however, they have limited efficacy and severe side effects. During primoinfection, T. cruzi infected macrophages mount an antiparasitic response, which the parasite evades through an increase of tumor growth factor beta and PGE(2) activation as well as decreased iNOS activity. Thus, prostaglandin synthesis inhibition with aspirin might increase macrophage antiparasitic activity and increase nifurtimox and benznidazole effect. Aspirin alone demonstrated a low effect upon macrophage antiparasitic activity. However, isobolographic analysis of the combined effects of aspirin, nifurtimox and benznidazole indicated a synergistic effect on T. cruzi infection of RAW cells, with combinatory indexes of 0.71 and 0.61, respectively. The observed effect of aspirin upon T. cruzi infection was not related with the PGE(2) synthesis inhibition. Nevertheless, NO() levels were restored by aspirin in T. cruzi-infected RAW cells, contributing to macrophage antiparasitic activity improvement. Thus, the synergy of aspirin with nifurtimox and benznidazole is due to the capability of aspirin to increase antiparasitic activity of macrophages. Copyright 2009 Elsevier Inc. All rights reserved.

  3. Effects of buthionine sulfoximine nifurtimox and benznidazole upon trypanothione and metallothionein proteins in Trypanosoma cruzi.

    Science.gov (United States)

    Maya, Juan Diego; Rodríguez, Andrés; Pino, Laura; Pabón, Adriana; Ferreira, Jorge; Pavani, Mario; Repetto, Yolanda; Morello, Antonio

    2004-01-01

    Proteins rich in sulfhydryl groups, such as metallothionein, are present in several strains of the parasite Trypanosoma cruzi, the etiological agent of Chagas' disease. Metallothionein-like protein concentrations ranged from 5.1 to 13.2 pmol/mg protein depending on the parasite strain and growth phase. Nifurtimox and benznidazole, used in the treatment of Chagas' disease, decreased metallothionein activity by approximately 70%. T. cruzi metallothionein was induced by ZnCl2. Metallothionein from T. cruzi was partially purified and its monobromobimane derivative showed a molecular weight of approximately 10,000 Da by SDS-PAGE analysis. The concentration of trypanothione, the major glutathione conjugate in T. cruzi, ranged from 3.8 to 10.8 nmol/mg protein, depending on the culture phase. The addition of buthionine sulfoximine to the protozoal culture considerably reduced the concentration of trypanothione and had no effect upon the metallothionein concentration. The possible contribution of metallothionein-like proteins to drug resistance in T. cruzi is discussed.

  4. Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo.

    Science.gov (United States)

    Akle, Veronica; Agudelo-Dueñas, Nathalie; Molina-Rodriguez, Maria A; Kartchner, Laurel Brianne; Ruth, Annette Marie; González, John M; Forero-Shelton, Manu

    2017-09-30

    Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique's relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

  5. Activities of Psilostachyin A and Cynaropicrin against Trypanosoma cruzi In Vitro and In Vivo

    Science.gov (United States)

    da Silva, Cristiane França; Batista, Denise da Gama Jaen; De Araújo, Julianna Siciliano; Batista, Marcos Meuser; Lionel, Jessica; de Souza, Elen Mello; Hammer, Erica Ripoll; da Silva, Patricia Bernardino; De Mieri, Maria; Adams, Michael; Zimmermann, Stefanie; Hamburger, Matthias; Brun, Reto; Schühly, Wolfgang

    2013-01-01

    In vitro and in vivo activities against Trypanosoma cruzi were evaluated for two sesquiterpene lactones: psilostachyin A and cynaropicrin. Cynaropicrin had previously been shown to potently inhibit African trypanosomes in vivo, and psilostachyin A had been reported to show in vivo effects against T. cruzi, albeit in another test design. In vitro data showed that cynaropicrin was more effective than psilostachyin A. Ultrastructural alterations induced by cynaropicrin included shedding events, detachment of large portions of the plasma membrane, and vesicular bodies and large vacuoles containing membranous structures, suggestive of parasite autophagy. Acute toxicity studies showed that one of two mice died at a cynaropicrin dose of 400 mg/kg of body weight given intraperitoneally (i.p.). Although no major plasma biochemical alterations could be detected, histopathology demonstrated that the liver was the most affected organ in cynaropicrin-treated animals. Although cynaropicrin was as effective as benznidazole against trypomastigotes in vitro, the treatment (once or twice a day) of T. cruzi-infected mice (up to 50 mg/kg/day cynaropicrin) did not suppress parasitemia or protect against mortality induced by the Y and Colombiana strains. Psilostachyin A (0.5 to 50 mg/kg/day given once a day) was not effective in the acute model of T. cruzi infection (Y strain), reaching 100% animal mortality. Our data demonstrate that although it is very promising against African trypanosomes, cynaropicrin does not show efficacy compared to benznidazole in acute mouse models of T. cruzi infection. PMID:23939901

  6. Risk factors associated with Trypanosoma cruzi exposure in domestic dogs from a rural community in Panama.

    Science.gov (United States)

    Saldaña, Azael; Calzada, José E; Pineda, Vanessa; Perea, Milixa; Rigg, Chystrie; González, Kadir; Santamaria, Ana Maria; Gottdenker, Nicole L; Chaves, Luis F

    2015-11-01

    Chagas disease, caused by Trypanosoma cruzi infection, is a zoonosis of humans, wild and domestic mammals, including dogs. In Panama, the main T. cruzi vector is Rhodnius pallescens, a triatomine bug whose main natural habitat is the royal palm, Attalea butyracea. In this paper, we present results from three T. cruzi serological tests (immunochromatographic dipstick, indirect immunofluorescence and ELISA) performed in 51 dogs from 24 houses in Trinidad de Las Minas, western Panama. We found that nine dogs were seropositive (17.6% prevalence). Dogs were 1.6 times more likely to become T. cruzi seropositive with each year of age and 11.6 times if royal palms where present in the peridomiciliary area of the dog's household or its two nearest neighbours. Mouse-baited-adhesive traps were employed to evaluate 12 peridomestic royal palms. All palms were found infested with R. pallescens with an average of 25.50 triatomines captured per palm. Of 35 adult bugs analysed, 88.6% showed protozoa flagellates in their intestinal contents. In addition, dogs were five times more likely to be infected by the presence of an additional domestic animal species in the dog's peridomiciliary environment. Our results suggest that interventions focused on royal palms might reduce the exposure to T. cruzi infection.

  7. Triatominae (Hemiptera, Reduviidae) in the Pantanal region: association with Trypanosoma cruzi, different habitats and vertebrate hosts.

    Science.gov (United States)

    Santos, Filipe Martins; Jansen, Ana Maria; Mourão, Guilherme de Miranda; Jurberg, José; Nunes, Alessandro Pacheco; Herrera, Heitor Miraglia

    2015-01-01

    The transmission cycle of Trypanosoma cruzi in the Brazilian Pantanal region has been studied during the last decade. Although considerable knowledge is available regarding the mammalian hosts infected by T. cruzi in this wetland, no studies have investigated its vectors in this region. This study aimed to investigate the presence of sylvatic triatomine species in different habitats of the Brazilian Pantanal region and to correlate their presence with the occurrences of vertebrate hosts and T. cruzi infection. The fieldwork involved passive search by using light traps and Noireau traps and active search by visual inspection. The light traps were placed at five selected points along forested areas for seven nights during each of the nine excursions. At each point where a light trap was set, eight Noireau traps were placed in palm trees and bromeliads. In all, 88 triatomine bugs were collected: two and one individuals from light traps and Noireau traps, respectively; three from peridomestic areas; 23 in coati nests; and 59 in thornbird nests. In this study, active search in microhabitats showed higher efficiency than passive search, since 95% of the triatomine bugs were caught in nests. Further, triatomine bugs were only found to be infected by T. cruzi in coati nests. Coati nests might act as a point of convergence and dispersion for triatomine bugs and mammal hosts infected by T. cruzi, thereby playing an important role in the sylvatic cycle of T. cruziin the Pantanal region.

  8. Usefulness of microsatellite typing in population genetic studies of Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    Macedo Andrea M

    2001-01-01

    Full Text Available Through microsatellite analysis of 53 monoclonal populations of Trypanosoma cruzi, we found a remarkable degree of genetic polymorphism with no single multilocus genotype being observed more than once. The microsatellite profile proved to be stable during 70 generations of the CL Brener clone in culture. The microsatellite profiling presented also high diagnostic sensitivity since DNA amplifications could be achieved with less than 100 fg DNA, corresponding to half parasite total DNA content. Based on these technical attributes the microsatellite assay turns out to be an important tool for direct typing T. cruzi in biological samples. By using this approach we were able to type T. cruzi in feces of artificially infected bugs and in single cells sorted by FACS. The microsatellites have shown to be excellent markers for T. cruzi phylogenetic reconstruction. We used maximum parsimony based on the minimum number of mutational steps to build an unrooted Wagner network, which confirms previous conclusions based on the analysis of the D7 domain of the LSU rDNA gene that T. cruzi is composed by two major groups. We also obtained evidence that strains belonging to rRNA group 2 are subdivided into two genetically distant clusters, and that one of these clusters is more related to rRNA group 1/2. These results suggest different origins for these strains.

  9. A target-based high throughput screen yields Trypanosoma brucei hexokinase small molecule inhibitors with antiparasitic activity.

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    Elizabeth R Sharlow

    2010-04-01

    Full Text Available The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK, an enzyme essential to the parasite that transfers the gamma-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay.Exploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were approximately 20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03brucei parasites, Leishmania promastigotes, and mammalian cell lines. Analysis of two structurally related compounds, ebselen and SID 17387000, revealed that both were mixed inhibitors of TbHK1 with respect to ATP. Additionally, both compounds inhibited parasite lysate-derived HK activity. None of the compounds displayed structural similarity to known hexokinase inhibitors or human African trypanosomiasis therapeutics.The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome.

  10. Response to chemotherapy with benznidazole of clones isolated from the 21SF strain of Trypanosoma cruzi (biodeme Type II, Trypanosoma cruzi II Resposta à quimioterapia com benzonidazol de clones isolados da cepa 21SF do Trypanosoma cruzi (biodema Tipo II, Trypanosoma cruzi II

    Directory of Open Access Journals (Sweden)

    Rozália Figueira Campos

    2005-04-01

    Full Text Available Susceptibility to chemotherapy with benznidazole was investigated of 5 clones isolated from the 21 SF strain (biodeme Type II, Trypanosoma cruzi II. Swiss mice were infected with the parental strain for each clone and submitted to chemotherapy with benznidazole (100mg/kg/day during 90 days. Treatment determined negativity of the parasitemia. Cure rates were evaluated by parasitological cure tests. Serology was evaluated for treated animals (titers from negative to 1:640 and untreated controls (1:160 to 1:640. Cure rates varied from 30 to 100% for the 5 clones, and were 25% for the parental strain. Results suggested that the variability of response to treatment of the clonal populations of Trypanosoma cruzi II strains is responsible for the high variation in the response to chemotherapy with benznidazole and nifurtimox by strains of this biodeme.A suscetibilidade à quimioterapia com o benzonidazol, de 5 clones isolados da cepa 21SF (biodema Tipo II, T. cruzi II, foi investigada. Camundongos suíços foram infectados com a cepa parental e com cada clone e submetidos à quimioterapia com benzonidazol (100mg/k/dia durante 90 dias. Os índices de cura foram avaliados pelos testes de cura parasitológicos. A sorologia foi avaliada para os animais tratados e (de negativo a 1: 640 e para os controles não tratados( 1:160 a 1:640. Os índices de cura variaram de 30% a 100% para os 5 clones sendo de 25% para a cepa parental. Os resultados sugerem que a variabilidade de resposta ao tratamento das populações clonais das cepas Trypanosoma cruzi II é responsável pela grande variação na resposta à quimioterapia com benzonidazol e nifurtimox das cepas deste biodema.

  11. Mode of Action of the Sesquiterpene Lactones Psilostachyin and Psilostachyin C on Trypanosoma cruzi.

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    Valeria P Sülsen

    Full Text Available Trypanosoma cruzi is the causative agent of Chagas' disease, which is a major endemic disease in Latin America and is recognized by the WHO as one of the 17 neglected tropical diseases in the world. Psilostachyin and psilostachyin C, two sesquiterpene lactones isolated from Ambrosia spp., have been demonstrated to have trypanocidal activity. Considering both the potential therapeutic targets present in the parasite, and the several mechanisms of action proposed for sesquiterpene lactones, the aim of this work was to characterize the mode of action of psilostachyin and psilostachyin C on Trypanosoma cruzi and to identify the possible targets for these molecules. Psilostachyin and psilostachyin C were isolated from Ambrosia tenuifolia and Ambrosia scabra, respectively. Interaction of sesquiterpene lactones with hemin, the induction of oxidative stress, the inhibition of cruzipain and trypanothione reductase and their ability to inhibit sterol biosynthesis were evaluated. The induction of cell death by apoptosis was also evaluated by analyzing phosphatidylserine exposure detected using annexin-V/propidium iodide, decreased mitochondrial membrane potential, assessed with Rhodamine 123 and nuclear DNA fragmentation evaluated by the TUNEL assay. Both STLs were capable of interacting with hemin. Psilostachyin increased about 5 times the generation of reactive oxygen species in Trypanosoma cruzi after a 4h treatment, unlike psilostachyin C which induced an increase in reactive oxygen species levels of only 1.5 times. Only psilostachyin C was able to inhibit the biosynthesis of ergosterol, causing an accumulation of squalene. Both sesquiterpene lactones induced parasite death by apoptosis. Upon evaluating the combination of both compounds, and additive trypanocidal effect was observed. Despite their structural similarity, both sesquiterpene lactones exerted their anti-T. cruzi activity through interaction with different targets. Psilostachyin

  12. Mode of Action of the Sesquiterpene Lactones Psilostachyin and Psilostachyin C on Trypanosoma cruzi.

    Science.gov (United States)

    Sülsen, Valeria P; Puente, Vanesa; Papademetrio, Daniela; Batlle, Alcira; Martino, Virginia S; Frank, Fernanda M; Lombardo, María E

    2016-01-01

    Trypanosoma cruzi is the causative agent of Chagas' disease, which is a major endemic disease in Latin America and is recognized by the WHO as one of the 17 neglected tropical diseases in the world. Psilostachyin and psilostachyin C, two sesquiterpene lactones isolated from Ambrosia spp., have been demonstrated to have trypanocidal activity. Considering both the potential therapeutic targets present in the parasite, and the several mechanisms of action proposed for sesquiterpene lactones, the aim of this work was to characterize the mode of action of psilostachyin and psilostachyin C on Trypanosoma cruzi and to identify the possible targets for these molecules. Psilostachyin and psilostachyin C were isolated from Ambrosia tenuifolia and Ambrosia scabra, respectively. Interaction of sesquiterpene lactones with hemin, the induction of oxidative stress, the inhibition of cruzipain and trypanothione reductase and their ability to inhibit sterol biosynthesis were evaluated. The induction of cell death by apoptosis was also evaluated by analyzing phosphatidylserine exposure detected using annexin-V/propidium iodide, decreased mitochondrial membrane potential, assessed with Rhodamine 123 and nuclear DNA fragmentation evaluated by the TUNEL assay. Both STLs were capable of interacting with hemin. Psilostachyin increased about 5 times the generation of reactive oxygen species in Trypanosoma cruzi after a 4h treatment, unlike psilostachyin C which induced an increase in reactive oxygen species levels of only 1.5 times. Only psilostachyin C was able to inhibit the biosynthesis of ergosterol, causing an accumulation of squalene. Both sesquiterpene lactones induced parasite death by apoptosis. Upon evaluating the combination of both compounds, and additive trypanocidal effect was observed. Despite their structural similarity, both sesquiterpene lactones exerted their anti-T. cruzi activity through interaction with different targets. Psilostachyin accomplished its antiparasitic

  13. Mode of Action of the Sesquiterpene Lactones Psilostachyin and Psilostachyin C on Trypanosoma cruzi

    Science.gov (United States)

    Papademetrio, Daniela; Batlle, Alcira; Martino, Virginia S.; Frank, Fernanda M.; Lombardo, María E.

    2016-01-01

    Trypanosoma cruzi is the causative agent of Chagas’ disease, which is a major endemic disease in Latin America and is recognized by the WHO as one of the 17 neglected tropical diseases in the world. Psilostachyin and psilostachyin C, two sesquiterpene lactones isolated from Ambrosia spp., have been demonstrated to have trypanocidal activity. Considering both the potential therapeutic targets present in the parasite, and the several mechanisms of action proposed for sesquiterpene lactones, the aim of this work was to characterize the mode of action of psilostachyin and psilostachyin C on Trypanosoma cruzi and to identify the possible targets for these molecules. Psilostachyin and psilostachyin C were isolated from Ambrosia tenuifolia and Ambrosia scabra, respectively. Interaction of sesquiterpene lactones with hemin, the induction of oxidative stress, the inhibition of cruzipain and trypanothione reductase and their ability to inhibit sterol biosynthesis were evaluated. The induction of cell death by apoptosis was also evaluated by analyzing phosphatidylserine exposure detected using annexin-V/propidium iodide, decreased mitochondrial membrane potential, assessed with Rhodamine 123 and nuclear DNA fragmentation evaluated by the TUNEL assay. Both STLs were capable of interacting with hemin. Psilostachyin increased about 5 times the generation of reactive oxygen species in Trypanosoma cruzi after a 4h treatment, unlike psilostachyin C which induced an increase in reactive oxygen species levels of only 1.5 times. Only psilostachyin C was able to inhibit the biosynthesis of ergosterol, causing an accumulation of squalene. Both sesquiterpene lactones induced parasite death by apoptosis. Upon evaluating the combination of both compounds, and additive trypanocidal effect was observed. Despite their structural similarity, both sesquiterpene lactones exerted their anti-T. cruzi activity through interaction with different targets. Psilostachyin accomplished its

  14. Transcriptome and Functional Genomics Reveal the Participation of Adenine Phosphoribosyltransferase in Trypanosoma cruzi Resistance to Benznidazole.

    Science.gov (United States)

    García-Huertas, Paola; Mejía-Jaramillo, Ana María; González, Laura; Triana-Chávez, Omar

    2017-07-01

    Currently, the only available treatments for Trypanosoma cruzi are benznidazole (Bz) and nifurtimox (Nfx). The mechanisms of action and resistance to these drugs in this parasite are not complete known. In order to identify differentially expressed transcripts between sensitive and resistant parasites, a massive pyrosequencing of the T. cruzi transcriptome was carried out. Additionally, the 2D gel electrophoresis profile of sensitive and resistant parasites was analyzed and the data were supported with functional genomics. The results showed 133 differentially expressed genes in resistant parasites. The transcriptome analysis revealed the regulation of different genes with several functions and metabolic pathways, which could suggest that resistance in T. cruzi is a multigenic process. Additionally, using transcriptomics, one gene, adenine phosphoribosyltransferase (APRT), was found to be down-regulated in the resistant parasites and its expression profile was confirmed by 2D electrophoresis analysis. The role of this gene in the resistance to Bz was confirmed overexpressing it in sensitive and resistant parasites. Interestingly, both parasites became more sensitive to Bz and H 2 O 2 . This is the first RNA-seq study to identify regulated genes in T. cruzi associated with Bz resistance and to show the role of APRT in T. cruzi resistance. Although T. cruzi regulation is mainly post-transcriptional, the transcriptome analysis, supported by 2D gel analysis and functional genomic, provides an overall idea of the expression profiles of genes under resistance conditions. These results contribute essential information to further the understanding of the mechanisms of action and resistance to Bz in T. cruzi. J. Cell. Biochem. 118: 1936-1945, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. How Trypanosoma cruzi deals with oxidative stress: Antioxidant defence and DNA repair pathways.

    Science.gov (United States)

    Machado-Silva, Alice; Cerqueira, Paula Gonçalves; Grazielle-Silva, Viviane; Gadelha, Fernanda Ramos; Peloso, Eduardo de Figueiredo; Teixeira, Santuza Maria Ribeiro; Machado, Carlos Renato

    2016-01-01

    Trypanosoma cruzi, the causative agent of Chagas disease, is an obligatory intracellular parasite with a digenetic life cycle. Due to the variety of host environments, it faces several sources of oxidative stress. In addition to reactive oxygen species (ROS) produced by its own metabolism, T. cruzi must deal with high ROS levels generated as part of the host's immune responses. Hence, the conclusion that T. cruzi has limited ability to deal with ROS (based on the lack of a few enzymes involved with oxidative stress responses) seems somewhat paradoxical. Actually, to withstand such variable sources of oxidative stress, T. cruzi has developed complex defence mechanisms. This includes ROS detoxification pathways that are distinct from the ones in the mammalian host, DNA repair pathways and specialized polymerases, which not only protect its genome from the resulting oxidative damage but also contribute to the generation of genetic diversity within the parasite population. Recent studies on T. cruzi's DNA repair pathways as mismatch repair (MMR) and GO system suggested that, besides a role associated with DNA repair, some proteins of these pathways may also be involved in signalling oxidative damage. Recent data also suggested that an oxidative environment might be beneficial for parasite survival within the host cell as it contributes to iron mobilization from the host's intracellular storages. Besides contributing to the understanding of basic aspects of T. cruzi biology, these studies are highly relevant since oxidative stress pathways are part of the poorly understood mechanisms behind the mode of action of drugs currently used against this parasite. By unveiling new peculiar aspects of T. cruzi biology, emerging data on DNA repair pathways and other antioxidant defences from this parasite have revealed potential new targets for a much needed boost in drug development efforts towards a better treatment for Chagas disease. Copyright © 2015. Published by Elsevier B.V.

  16. Trypanosoma cruzi extracts elicit protective immune response against chemically induced colon and mammary cancers.

    Science.gov (United States)

    Ubillos, Luis; Freire, Teresa; Berriel, Edgardo; Chiribao, María Laura; Chiale, Carolina; Festari, María Florencia; Medeiros, Andrea; Mazal, Daniel; Rondán, Mariella; Bollati-Fogolín, Mariela; Rabinovich, Gabriel A; Robello, Carlos; Osinaga, Eduardo

    2016-04-01

    Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, has anticancer effects mediated, at least in part, by parasite-derived products which inhibit growth of tumor cells. We investigated whether immunity to T. cruzi antigens could induce antitumor activity, using two rat models which reproduce human carcinogenesis: colon cancer induced by 1,2-dimethylhydrazine (DMH), and mammary cancer induced by N-nitroso-N-methylurea (NMU). We found that vaccination with T. cruzi epimastigote lysates strongly inhibits tumor development in both animal models. Rats immunized with T. cruzi antigens induce activation of both CD4(+) and CD8(+) T cells and splenocytes from these animals showed higher cytotoxic responses against tumors as compared to rats receiving adjuvant alone. Tumor-associated immune responses included increasing number of CD11b/c(+) His48(-) MHC II(+) cells corresponding to macrophages and/or dendritic cells, which exhibited augmented NADPH-oxidase activity. We also found that T. cruzi lysate vaccination developed antibodies specific for colon and mammary rat cancer cells, which were capable of mediating antibody-dependent cellular cytotoxicity (ADCC) in vitro. Anti-T. cruzi antibodies cross-reacted with human colon and breast cancer cell lines and recognized 41/60 (68%) colon cancer and 38/63 (60%) breast cancer samples in a series of 123 human tumors. Our results suggest that T. cruzi antigens can evoke an integrated antitumor response involving both the cellular and humoral components of the immune response and provide novel insights into the understanding of the intricate relationship between parasite infection and tumor growth. © 2015 UICC.

  17. Prevalence of Trypanosoma cruzi and Other Trypanosomatids in Frequently-Hunted Wild Mammals from the Peruvian Amazon.

    Science.gov (United States)

    Morales, E Angelo; Mayor, Pedro; Bowler, Mark; Aysanoa, Esar; Pérez-Velez, Erika S; Pérez, Jocelyn; Ventocilla, Julio A; Baldeviano, G Christian; Lescano, Andrés G

    2017-11-01

    To better understand the ecology of Trypanosoma cruzi in the northeastern Peruvian Amazon, we evaluated the prevalence of T. cruzi and other trypanosomatids in four orders of wild mammals hunted and consumed by inhabitants of three remote indigenous communities in the Peruvian Amazon. Of 300 wild mammals sampled, 115 (38.3%) were infected with trypanosomatids and 15 (5.0%) with T. cruzi. The prevalence of T. cruzi within each species was as follows: large rodents (Cuniculus paca, 5.5%; Dasyprocta spp., 2.6%), edentates (Dasypus novemcinctus, 4.2%), and carnivores with higher prevalence (Nasua nasua, 18.8%). The high prevalence of T. cruzi and other trypanosomatids in frequently hunted wild mammals suggests a sizeable T. cruzi sylvatic reservoir in remote Amazonian locations.

  18. High Trypanosoma cruzi infection prevalence associated with minimal cardiac pathology among wild carnivores in central Texas

    Directory of Open Access Journals (Sweden)

    Rachel Curtis-Robles

    2016-08-01

    Full Text Available Infection with the zoonotic vector-borne protozoal parasite Trypanosoma cruzi causes Chagas disease in humans and dogs throughout the Americas. Despite the recognized importance of various wildlife species for perpetuating Trypanosoma cruzi in nature, relatively little is known about the development of cardiac disease in infected wildlife. Using a cross-sectional study design, we collected cardiac tissue and blood from hunter-donated wildlife carcasses- including raccoon (Procyon lotor, coyote (Canis latrans, gray fox (Urocyon cinereoargenteus, and bobcat (Lynx rufus – from central Texas, a region with established populations of infected triatomine vectors and increasing diagnoses of Chagas disease in domestic dogs. Based on PCR analysis, we found that 2 bobcats (14.3%, 12 coyotes (14.3%, 8 foxes (13.8%, and 49 raccoons (70.0% were positive for T. cruzi in at least one sample (right ventricle, apex, and/or blood clot. Although a histologic survey of right ventricles showed that 21.1% of 19 PCR-positive hearts were characterized by mild lymphoplasmocytic infiltration, no other lesions and no amastigotes were observed in any histologic section. DNA sequencing of the TcSC5D gene revealed that raccoons were infected with T. cruzi strain TcIV, and a single racoon harbored a TcI/TcIV mixed infection. Relative to other wildlife species tested here, our data suggest that raccoons may be important reservoirs of TcIV in Texas and a source of infection for indigenous triatomine bugs. The overall high level of infection in this wildlife community likely reflects high levels of vector contact, including ingestion of bugs. Although the relationship between the sylvatic cycle of T. cruzi transmission and human disease risk in the United States has yet to be defined, our data suggest that hunters and wildlife professionals should take precautions to avoid direct contact with potentially infected wildlife tissues.

  19. Evaluation of proline analogs as trypanocidal agents through the inhibition of a Trypanosoma cruzi proline transporter.

    Science.gov (United States)

    Sayé, Melisa; Fargnoli, Lucía; Reigada, Chantal; Labadie, Guillermo R; Pereira, Claudio A

    2017-11-01

    Trypanosoma cruzi, the etiological agent of Chagas disease, uses proline as its main carbon source, essential for parasite growth and stage differentiation in epimastigotes and amastigotes. Since proline is involved in many essential biological processes in T. cruzi, its transport and metabolism are interesting drug targets. Four synthetic proline analogues (ITP-1B/1C/1D/1G) were evaluated as inhibitors of proline transport mediated through the T. cruzi proline permease TcAAAP069. The trypanocidal activity of the compounds was also assessed. The compounds ITP-1B and ITP-1G inhibited proline transport mediated through TcAAAP069 permease in a dose-dependent manner. The analogues ITP-1B, -1D and -1G had trypanocidal effect on T. cruzi epimastigotes with IC 50 values between 30 and 40μM. However, only ITP-1G trypanocidal activity was related with its inhibitory effect on TcAAAP069 proline transporter. Furthermore, this analogue strongly inhibited the parasite stage differentiation from epimastigote to metacyclic trypomastigote. Finally, compounds ITP-1B and ITP-1G were also able to inhibit the transport mediated by other permeases from the same amino acid permeases family, TcAAAP. It is possible to design synthetic amino acid analogues with trypanocidal activity. The compound ITP-1G is an interesting starting point for new trypanocidal drug design which is also an inhibitor of transport of amino acids and polyamines mediated by permeases from the TcAAAP family, such as proline transporter TcAAAP069 among others. The Trypanosoma cruzi amino acid transporter family TcAAAP constitutes a multiple and promising therapeutic target for the development of new treatments against Chagas disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Triatomine bugs, their microbiota and Trypanosoma cruzi: asymmetric responses of bacteria to an infected blood meal.

    Science.gov (United States)

    Díaz, Sebastián; Villavicencio, Bianca; Correia, Nathália; Costa, Jane; Haag, Karen L

    2016-12-09

    Triatomine bugs (Hemiptera: Reduviidae) are vectors of the flagellate Trypanosoma cruzi, the causative agent of Chagas disease. The study of triatomine gut microbiota has gained relevance in the last years due to its possible role in vector competence and prospective use in control strategies. The objective of this study is to examine changes in the gut microbiota composition of triatomines in response to a T. cruzi-infected blood meal and identifying key factors determining those changes. We sampled colony-reared individuals from six triatomine vectors (Panstrongylus megistus, Rhodnius prolixus, Triatoma brasiliensis, T. infestans, T. juazeirensis and T. sherlocki) comparing experimentally T. cruzi strain 0354-challenged and non-challenged insects. The microbiota of gut and gonad tissues was characterized using high throughput sequencing of region V3-V4 of bacterial 16S rRNA gene. The triatomine microbiota had a low intra-individual diversity, and a high inter-individual variation within the same host species. Arsenophonous appeared as the dominant triatomine bacterial symbiont in our study (59% of the total 16S coverage), but there were significant differences in the distribution of bacterial genera among vectors. In Rhodnius prolixus the dominant symbiont was Pectobacterium. Trypanosoma cruzi-challenge significantly affects microbiota composition, with challenged vectors harbouring a significantly more diverse bacterial community, both in the gut and the gonads. Our results show that blood-feeding with T. cruzi epimastigotes strongly affects microbiota composition in a species-specific manner. We suggest that triatomine-adapted enterobacteria such as Arsenophonus could be used as stable vectors for genetic transformation of triatomine bugs and control of Chagas disease.

  1. Trypanosome species, including Trypanosoma cruzi, in sylvatic and peridomestic bats of Texas, USA.

    Science.gov (United States)

    Hodo, Carolyn L; Goodwin, Chloe C; Mayes, Bonny C; Mariscal, Jacqueline A; Waldrup, Kenneth A; Hamer, Sarah A

    2016-12-01

    In contrast to other mammalian reservoirs, many bat species migrate long-distances and have the potential to introduce exotic pathogens to new areas. Bats have long been associated with blood-borne protozoal trypanosomes of the Schizotrypanum subgenus, which includes the zoonotic parasite Trypanosoma cruzi, agent of Chagas disease. Another member of the subgenus, Trypanosoma dionisii, infects bats of Europe and South America, and genetic similarities between strains from the two continents suggest transcontinental movement of this parasite via bats. Despite the known presence of diverse trypanosomes in bats of Central and South America, and the presence of T. cruzi-infected vectors and wildlife in the US, the role of bats in maintaining and dispersing trypanosomes in the US has not yet been reported. We collected hearts and blood from 8 species of insectivorous bats from 30 counties across Texas. Using PCR and DNA sequencing, we tested 593 bats for trypanosomes and found 1 bat positive for T. cruzi (0.17%), 9 for T. dionisii (1.5%), and 5 for Blastocrithidia spp. (0.8%), a group of insect trypanosomes. The T. cruzi-infected bat was carrying TcI, the strain type associated with human disease in the US. In the T. dionisii-infected bats, we detected three unique variants associated with the three infected bat species. These findings represent the first report of T. cruzi in a bat in the US, of T. dionisii in North America, and of Blastocrithidia spp. in mammals, and underscore the importance of bats in the maintenance of trypanosomes, including agents of human and animal disease, across broad geographic locales. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  2. Minimum Information Loss Based Multi-kernel Learning for Flagellar Protein Recognition in Trypanosoma Brucei

    KAUST Repository

    Wang, Jim Jing-Yan

    2014-12-01

    Trypanosma brucei (T. Brucei) is an important pathogen agent of African trypanosomiasis. The flagellum is an essential and multifunctional organelle of T. Brucei, thus it is very important to recognize the flagellar proteins from T. Brucei proteins for the purposes of both biological research and drug design. In this paper, we investigate computationally recognizing flagellar proteins in T. Brucei by pattern recognition methods. It is argued that an optimal decision function can be obtained as the difference of probability functions of flagella protein and the non-flagellar protein for the purpose of flagella protein recognition. We propose to learn a multi-kernel classification function to approximate this optimal decision function, by minimizing the information loss of such approximation which is measured by the Kull back-Leibler (KL) divergence. An iterative multi-kernel classifier learning algorithm is developed to minimize the KL divergence for the problem of T. Brucei flagella protein recognition, experiments show its advantage over other T. Brucei flagellar protein recognition and multi-kernel learning methods. © 2014 IEEE.

  3. Genotyping of Trypanosoma cruzi DTUs and Trypanosoma rangeli genetic groups in experimentally infected Rhodnius prolixus by PCR-RFLP.

    Science.gov (United States)

    Sá, Amanda R N; Dias, Greicy B M; Kimoto, Karen Y; Steindel, Mário; Grisard, Edmundo C; Toledo, Max Jean O; Gomes, Mônica L

    2016-04-01

    The specific detection and genetic typing of trypanosomes that infect humans, mammalian reservoirs, and vectors is crucial for diagnosis and epidemiology. We utilized a PCR-RFLP assay that targeted subunit II of cytochrome oxidase and 24Sα-rDNA to simultaneously detect and discriminate six Trypanosoma cruzi discrete typing units (DTUs) and two genetic groups of Trypanosoma rangeli (KP1+/KP1-) in intestinal contents of experimentally infected Rhodnius prolixus. The PCR assays showed that in 23 of 29 (79.4%) mixed infections with the six T. cruzi DTUs and mixed infections with individual DTUs and/or groups KP1+ and KP1-, both parasites were successfully detected. In six mixed infections that involved TcIII, the TcI, TcII, TcV, and TcVI DTUs predominated to the detriment of TcIII, indicating the selection of genetic groups. Interactions between different genetic groups and vectors may lead to genetic selection over TcIII. The elimination of this DTU by the immune system of the vector appears unlikely because TcIII was present in other mixed infections (TcIII/TcIV and TcIII/KP1+). Both molecular markers used in this study were sensitive and specific, demonstrating their usefulness in a wide geographical area where distinct genotypes of these two species are sympatric. Although the cellular and molecular mechanisms that are involved in parasite-vector interactions are still poorly understood, our results indicate a dynamic selection toward specific T. cruzi DTUs in R. prolixus during mixed genotype infections. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Synthesis and Anti-Trypanosoma cruzi Activity of Diaryldiazepines

    OpenAIRE

    Júlio César L. Menezes; Luana Beatriz A. Vaz; Paula Melo de Abreu Vieira; Kátia da Silva Fonseca; Cláudia Martins Carneiro; Jason G. Taylor

    2014-01-01

    Chagas disease is a so-called “neglected disease” and endemic to Latin America. Nifurtimox and benznidazole are drugs that have considerable efficacy in the treatment of the acute phase of the disease but cause many significant side effects. Furthermore, in the Chronic Phase its efficiency is reduced and their therapeutic effectiveness is dependent on the type of T. cruzi strain. For this reason, the present work aims to drive basic research towards the discovery of new chemical entities t...

  5. Trypanosoma brucei: analysis of cytoplasmic Ca2+ during differentiation of bloodstream stages in vitro.

    Science.gov (United States)

    Stojdl, D F; Clarke, M W

    1996-06-01

    The highly regulated intracellular concentration of calcium (Ca2+) is a well-described regulator of diverse cellular events, including cell cycle control. In the present study we have addressed the regulation of cytosolic Ca2+ in differentiation events in the life cycle of the protozoan parasite Trypanosoma brucei. Bloodstream form (BSF) trypanosomes include the mitotically active long slender forms (LS) which differentiate to two nondividing stages--intermediate (INT) which transform into short stumpy (SS) forms. An axenic in vitro culture system was used to cultivate LS to a density greater than 1.0 x 10(6) cells/ml/day. Populations of the intermediate BSF (INT) and SS were derived from cultured LS by treatment with difluoromethyl ornithine (DFMO, 100 microM) for 2 and 4 days, respectively. A semiquantitative reverse transcriptase-coupled polymerase chain reaction protocol (SQ-RT-PCR) was developed to objectively distinguish the three BSF by monitoring the relative levels of stage-specific mRNAs--cytochrome oxidase II (COXII), variant surface glycoprotein, and procyclin during the differentiation of LS to SS, showing an increase in COXII and procyclin mRNA expression during this process of differentiation. Basal cytosolic Ca2+ levels [Ca2+]i of populations of LS, INT, and SS were studied using Indo-1 dual emission fluorometry. [Ca2+]i was maximal in dividing LS cells and was shown to decrease coincidentally with early events in the process of differentiation to INT and SS. Thapsigargin (1 microM), reported to cause the release of Ca2+ from the endoplasmic reticulum, elevated [Ca2+]i by about 30-60 nM in all BSF; however, the total thapsigargin-releasable stores decreased in parallel with the decrease in basal [Ca2+]i. Control treatments verified that elevations in [Ca2+]i in response to thapsigargin were intracellular in origin. These results may reflect the cessation of cytosolic Ca2+ transients involved in the regulation of mitosis as the parasite exits from

  6. The use of yellow fluorescent hybrids to indicate mating in Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Ferris Vanessa

    2008-02-01

    Full Text Available Abstract Background Trypanosoma brucei undergoes genetic exchange in its insect vector, the tsetse fly, by an unknown mechanism. The difficulties of working with this experimental system of genetic exchange have hampered investigation, particularly because the trypanosome life cycle stages involved cannot be cultured in vitro and therefore must be examined in the insect. Searching for small numbers of hybrid trypanosomes directly in the fly has become possible through the incorporation of fluorescent reporter genes, and we have previously carried out a successful cross using a reporter-repressor strategy. However, we could not be certain that all fluorescent trypanosomes observed in that cross were hybrids, due to mutations of the repressor leading to spontaneous fluorescence, and we have therefore developed an alternative strategy. Results To visualize the production of hybrids in the fly, parental trypanosome clones were transfected with a gene encoding Green Fluorescent Protein (GFP or Red Fluorescent Protein (RFP. Co-infection of flies with red and green fluorescent parental trypanosomes produced yellow fluorescent hybrids, which were easily visualized in the fly salivary glands. Yellow trypanosomes were not seen in midgut or proventricular samples and first appeared in the glands as epimastigotes as early as 13 days after fly infection. Cloned progeny originating from individual salivary glands had yellow, red, green or no fluorescence and were confirmed as hybrids by microsatellite, molecular karyotype and kinetoplast (mitochondrial DNA analyses. Hybrid clones showed biparental inheritance of both nuclear and kinetoplast genomes. While segregation and reassortment of the reporter genes and microsatellite alleles were consistent with Mendelian inheritance, flow cytometry measurement of DNA content revealed both diploid and polyploid trypanosomes among the hybrid progeny clones. Conclusion The strategy of using production of yellow hybrids

  7. The Dialogue of the Host-Parasite Relationship: Leishmania spp. and Trypanosoma cruzi Infection

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    Carlos Gustavo Vieira de Morais

    2015-01-01

    Full Text Available The intracellular protozoa Leishmania spp. and Trypanosoma cruzi and the causative agents of Leishmaniasis and Chagas disease, respectively, belong to the Trypanosomatidae family. Together, these two neglected tropical diseases affect approximately 25 million people worldwide. Whether the host can control the infection or develops disease depends on the complex interaction between parasite and host. Parasite surface and secreted molecules are involved in triggering specific signaling pathways essential for parasite entry and intracellular survival. The recognition of the parasite antigens by host immune cells generates a specific immune response. Leishmania spp. and T. cruzi have a multifaceted repertoire of strategies to evade or subvert the immune system by interfering with a range of signal transduction pathways in host cells, which causes the inhibition of the protective response and contributes to their persistence in the host. The current therapeutic strategies in leishmaniasis and trypanosomiasis are very limited. Efficacy is variable, toxicity is high, and the emergence of resistance is increasingly common. In this review, we discuss the molecular basis of the host-parasite interaction of Leishmania and Trypanosoma cruzi infection and their mechanisms of subverting the immune response and how this knowledge can be used as a tool for the development of new drugs.

  8. Aspectos ultra-estruturais da forma epimastigota do Trypanosoma cruzi em meio LIT

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    Wanderley de Souza

    1975-06-01

    Full Text Available E feito um estudo da ultra-estrutura da forma epismastigota do Trypanosoma cruzi mantida em meio de cultivo acelular. O núcleo das formas em divisão apresenta um aspecto homogêneo. Microtúbulos intranucleares são observados durante a divisão. No entanto, a membrana nuclear permanece íntegra. O citoplasma apresenta-se com vacúolos de dimensões e aspectos variados. Com o método do ácido periódico-tiosemicarbazida-proteinato de prata, polissacaríáeos e/ou glicoproteínas foram localizados na membrana celular e na membrana que delimita certos vacúolos citoplasmáticos.In this paper the fine structure of the epimastigotes forms of Trypanosoma cruzi maintained in an acellular culture medium, is described. During division the nucleus shows a homogenous structure owing to the dispersion of its chromatin and nucleolar material. Microtubules similar to those of a mitotic spindle in metazoan cells appear, running frorn one pole to the other. During the whole process of division the nuclear membrane remains intact. The cytoplam shows many vacuoles. With the cytochemical method of acid periodic-thiosemicarbazide-silver proteinate, a polysaccharide surface coat is observed on the whole surface membrane system of T. cruzi. A positive re- action is also observed in the membranes of some vacuoles in the cytoplasm of the parasites.

  9. The Dialogue of the Host-Parasite Relationship: Leishmania spp. and Trypanosoma cruzi Infection

    Science.gov (United States)

    de Morais, Carlos Gustavo Vieira; Castro Lima, Ana Karina; dos Santos, Rosiane Freire; Da-Silva, Silvia Amaral Gonçalves; Dutra, Patrícia Maria Lourenço

    2015-01-01

    The intracellular protozoa Leishmania spp. and Trypanosoma cruzi and the causative agents of Leishmaniasis and Chagas disease, respectively, belong to the Trypanosomatidae family. Together, these two neglected tropical diseases affect approximately 25 million people worldwide. Whether the host can control the infection or develops disease depends on the complex interaction between parasite and host. Parasite surface and secreted molecules are involved in triggering specific signaling pathways essential for parasite entry and intracellular survival. The recognition of the parasite antigens by host immune cells generates a specific immune response. Leishmania spp. and T. cruzi have a multifaceted repertoire of strategies to evade or subvert the immune system by interfering with a range of signal transduction pathways in host cells, which causes the inhibition of the protective response and contributes to their persistence in the host. The current therapeutic strategies in leishmaniasis and trypanosomiasis are very limited. Efficacy is variable, toxicity is high, and the emergence of resistance is increasingly common. In this review, we discuss the molecular basis of the host-parasite interaction of Leishmania and Trypanosoma cruzi infection and their mechanisms of subverting the immune response and how this knowledge can be used as a tool for the development of new drugs. PMID:26090399

  10. Environment, interactions between Trypanosoma cruzi and its host, and health Meio-ambiente, interações entre Trypanosoma cruzi e seu hospedeiro e saúde humana

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    Antonio R. L. Teixeira

    2009-01-01

    Full Text Available An epidemiological chain involving Trypanosoma cruzi is discussed at the environmental level, and in terms of fine molecular interactions in invertebrate and vertebrate hosts dwelling in different ecosystems. This protozoan has a complex, genetically controlled plasticity, which confers adaptation to approximately 40 blood-sucking triatomine species and to over 1,000 mammalian species, fulfilling diverse metabolic requirements in its complex life-cycle. The Tr. cruzi infections are deeply embedded in countless ecotypes, where they are difficult to defeat using the control methods that are currently available. Many more field and laboratory studies are required to obtain data and information that may be used for the control and prevention of Tr. cruzi infections and their various disease manifestations. Emphasis should be placed on those sensitive interactions at cellular and environmental levels that could become selected targets for disease prevention. In the short term, new technologies for social mobilization should be used by people and organizations working for justice and equality through health information and promotion. A mass media directed program could deliver education, information and communication to protect the inhabitants at risk of contracting Tr. cruzi infections.Uma rede epidemiológica envolvendo o Trypanosoma cruzi foi discutida nos níveis ambientais e de interações moleculares nos hospedeiros que habitam em 19 diferentes ecossistemas. O protozoário tem uma enorme plasticidade controlada geneticamente que confere sua adaptação a cerca de quarenta espécies de triatomíneos e mais de mil espécies de mamíferos. Essas infecções estão profundamente embutidas em inúmeros ecótopos, onde elas estão inacessíveis aos métodos de controle utilizados. Muito mais estudos de campo e de laboratório são necessários à obtenção de dados e informação pertinentes ao controle e prevenção das infecções pelo Tr. cruzi e

  11. The in vitro activity of fatty diamines and amino alcohols against mixed amastigote and trypomastigote Trypanosoma cruzi forms

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    Policarpo Ademar Sales Júnior

    2014-06-01

    Full Text Available Four diamines and three amino alcohols derived from 1-decanol, 1-dodecanol and 1,2-dodecanediol were evaluated in an in vitro assay against a mixture of trypomastigote and intracellular amastigote forms of Trypanosoma cruzi. Two of these compounds (6 and 7 showed better activity against both proliferative stages of T. cruzi than the positive control benznidazole, three were of similar potency (1, 2 and 5 and two were less active (3 and 4.

  12. Differentiation of Trypanosoma cruzi I (TcI) and T. cruzi II (TcII) genotypes using genes encoding serine carboxypeptidases.

    Science.gov (United States)

    de Araújo, Catarina Andréa Chaves; Mayer, Christoph; Waniek, Peter Josef; Azambuja, Patricia; Jansen, Ana Maria

    2016-11-01

    The parasite Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae) can be classified based on biochemical and molecular markers, into six lineages or discrete typing units (DTUs), T. cruzi I-VI (TcI-VI), from which TcI and TcII are the parental genotypes. Trying to understand the dispersion of the subpopulations of T. cruzi in nature and its complex transmission cycles, the serine carboxypeptidase genes of T. cruzi were used as a molecular marker in the present study. DTUs of 25 T. cruzi isolates derived from different hosts and from different regions of Brazil were classified. Using specific primers, the complete serine carboxypeptidase open reading frame of 1401 bp was sequenced. The obtained data shows significant differences in the sequences of TcI and TcII. The analysis of the T. cruzi significantly different serine carboxypeptidase genes allowed distinguishing between the parental DTUs TcI to TcII and the hybrid DTU TcVI which grouped within the latter branch. The sequence diversity within the T. cruzi subpopulations was rather low. The analysis using the genes encoding proteases seems to be an interesting approach for the reconstruction of the origin and genotype evolution of T. cruzi.

  13. Should Trypanosoma cruzi be called "cruzi" complex? A review of the parasite diversity and the potential of selecting population after in Vitro culturing and mice infection

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    Devera Rodolfo

    2003-01-01

    Full Text Available Morpho-biological diversity of Trypanosoma cruzi has been known since Chagas' first works in 1909. Several further studies confirmed the morphological differences among the parasite strains, which were isolated from different reservoirs and vectors, as well as from human beings. In the early sixties, antigenic differences were found in the parasite strains from various sources. These differences, coupled to the observation of regional variations of the disease, led to the proposal of the term cruzi complex to designate the taxon T. cruzi. Since then this protozoan has been typed in distinct biodemes, zymodemes and lineages which were consensually grouped into T. cruzi I, T. cruzi II and into non-grouped strains. T. cruzi genotypic characterization, initially carried out by schizodeme analysis and more recently by various other techniques, has shown a great diversity of the parasite strains. In fact, T. cruzi is formed by groups of heterogeneous sub-population, which present specific characteristics, including distinct histotropism. The interaction of the different infecting clones of the cruzi complex and the human host will determine the morbidity of the disease.

  14. Biochemical behavior of Trypanosoma cruzi strains isolated from mice submitted to specific chemotherapy

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    Jesila Pinto M. Marretto

    1994-12-01

    Full Text Available To investigate the influence of chemotherapy on the biochemical beha vior of Trypanosoma cruzi strains, three groups of mice were infected with one of three strains of T. cruzi of different biological and isoenzymic patterns (Peruvian, 21 SF and Colombian strains. Each group was subdivided into subgroups: 1 - treated with nifurtimox; 2 - treated with benznidazole and 3 - untreated infected controls. At the end of treatment, that lasted for 90 days, xenodiagnosis, sub inoculation of blood into new born mice and haemoculture were performed as tests of cure. From the positive tests, 22 samples of T. cruzi were isolated from all subgroups. Electrophoretic analysis of the isoenzymes PGM, GP1, ALAT and AS AT failed to show any difference between parasite strains isolated from treated and untreated mice, which indicates that no detectable clonal selection or parasite genetic markers alterations concerning the isoenzymes analysed have been determined by treatment with drugs of recognized antiparasitic effect, suggesting stability of the phenotypic characteristics of the three biological types of T. cruzi strains.Com o objetivo de investigar a influência da quimioterapia no padrão bioquímico de diferentes cepas do Trypanosoma cruzi, três grupos de camundongos foram infectados respectivamente com as cepas Peruana, 21 SF e Colombiana, que correspondem a diferentes padrões biológicos e isoenzimáticos. Cada grupo foi subdividido em subgrupos: 1 - tratados com nifurtimox; 2 - tratados com benzonidazol; 3- controles infectados não tratados. Ao final do tratamento que durou 90 dias, os animais foram submetidos a testes parasitológicos de cura: xenodiagnóstico, subinoculação do sangue em camundongos recém-nascidos e hemocultura em meio Warren. A partir da positivação destes testes, foram isoladas 22 amostras do T. cruzi dos três subgrupos. A análise eletroforética dos extratos enzimáticos obtidos após cultura para as enzimas PGM, GPI, ALAT e

  15. Unraveling Chagas disease transmission through the oral route: Gateways to Trypanosoma cruzi infection and target tissues.

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    Danielle Silva-Dos-Santos

    2017-04-01

    Full Text Available Oral transmission of Trypanosoma cruzi, the causative agent of Chagas disease, is the most important route of infection in Brazilian Amazon and Venezuela. Other South American countries have also reported outbreaks associated with food consumption. A recent study showed the importance of parasite contact with oral cavity to induce a highly severe acute disease in mice. However, it remains uncertain the primary site of parasite entry and multiplication due to an oral infection. Here, we evaluated the presence of T. cruzi Dm28c luciferase (Dm28c-luc parasites in orally infected mice, by bioluminescence and quantitative real-time PCR. In vivo bioluminescent images indicated the nasomaxillary region as the site of parasite invasion in the host, becoming consistently infected throughout the acute phase. At later moments, 7 and 21 days post-infection (dpi, luminescent signal is denser in the thorax, abdomen and genital region, because of parasite dissemination in different tissues. Ex vivo analysis demonstrated that the nasomaxillary region, heart, mandibular lymph nodes, liver, spleen, brain, epididymal fat associated to male sex organs, salivary glands, cheek muscle, mesenteric fat and lymph nodes, stomach, esophagus, small and large intestine are target tissues at latter moments of infection. In the same line, amastigote nests of Dm28c GFP T. cruzi were detected in the nasal cavity of 6 dpi mice. Parasite quantification by real-time qPCR at 7 and 21 dpi showed predominant T. cruzi detection and expansion in mouse nasal cavity. Moreover, T. cruzi DNA was also observed in the mandibular lymph nodes, pituitary gland, heart, liver, small intestine and spleen at 7 dpi, and further, disseminated to other tissues, such as the brain, stomach, esophagus and large intestine at 21 dpi. Our results clearly demonstrated that oral cavity and adjacent compartments is the main target region in oral T. cruzi infection leading to parasite multiplication at the nasal

  16. Galectin-1 Prevents Infection and Damage Induced by Trypanosoma cruzi on Cardiac Cells

    Science.gov (United States)

    Benatar, Alejandro F.; García, Gabriela A.; Bua, Jacqeline; Cerliani, Juan P.; Postan, Miriam; Tasso, Laura M.; Scaglione, Jorge; Stupirski, Juan C.; Toscano, Marta A.

    2015-01-01

    Background Chronic Chagas cardiomyopathy caused by Trypanosoma cruzi is the result of a pathologic process starting during the acute phase of parasite infection. Among different factors, the specific recognition of glycan structures by glycan-binding proteins from the parasite or from the mammalian host cells may play a critical role in the evolution of the infection. Methodology and Principal Findings Here we investigated the contribution of galectin–1 (Gal–1), an endogenous glycan-binding protein abundantly expressed in human and mouse heart, to the pathophysiology of T. cruzi infection, particularly in the context of cardiac pathology. We found that exposure of HL–1 cardiac cells to Gal–1 reduced the percentage of infection by two different T. cruzi strains, Tulahuén (TcVI) and Brazil (TcI). In addition, Gal–1 prevented exposure of phosphatidylserine and early events in the apoptotic program by parasite infection on HL–1 cells. These effects were not mediated by direct interaction with the parasite surface, suggesting that Gal–1 may act through binding to host cells. Moreover, we also observed that T. cruzi infection altered the glycophenotype of cardiac cells, reducing binding of exogenous Gal–1 to the cell surface. Consistent with these data, Gal–1 deficient (Lgals1 -/-) mice showed increased parasitemia, reduced signs of inflammation in heart and skeletal muscle tissues, and lower survival rates as compared to wild-type (WT) mice in response to intraperitoneal infection with T. cruzi Tulahuén strain. Conclusion/Significance Our results indicate that Gal–1 modulates T. cruzi infection of cardiac cells, highlighting the relevance of galectins and their ligands as regulators of host-parasite interactions. PMID:26451839

  17. Ten-year incidence of Chagas cardiomyopathy among asymptomatic Trypanosoma cruzi-seropositive former blood donors.

    Science.gov (United States)

    Sabino, Ester C; Ribeiro, Antonio L; Salemi, Vera M C; Di Lorenzo Oliveira, Claudia; Antunes, Andre P; Menezes, Marcia M; Ianni, Barbara M; Nastari, Luciano; Fernandes, Fabio; Patavino, Giuseppina M; Sachdev, Vandana; Capuani, Ligia; de Almeida-Neto, Cesar; Carrick, Danielle M; Wright, David; Kavounis, Katherine; Goncalez, Thelma T; Carneiro-Proietti, Anna Barbara; Custer, Brian; Busch, Michael P; Murphy, Edward L

    2013-03-12

    Very few studies have measured disease penetrance and prognostic factors of Chagas cardiomyopathy among asymptomatic Trypanosoma cruzi-infected persons. We performed a retrospective cohort study among initially healthy blood donors with an index T cruzi-seropositive donation and age-, sex-, and period-matched seronegatives in 1996 to 2002 in the Brazilian cities of São Paulo and Montes Claros. In 2008 to 2010, all subjects underwent medical history, physical examination, ECGs, and echocardiograms. ECG and echocardiogram results were classified by blinded core laboratories, and records with abnormal results were reviewed by a blinded panel of 3 cardiologists who adjudicated the outcome of Chagas cardiomyopathy. Associations with Chagas cardiomyopathy were tested with multivariate logistic regression. Mean follow-up time between index donation and outcome assessment was 10.5 years for the seropositives and 11.1 years for the seronegatives. Among 499 T cruzi seropositives, 120 (24%) had definite Chagas cardiomyopathy, and among 488 T cruzi seronegatives, 24 (5%) had cardiomyopathy, for an incidence difference of 1.85 per 100 person-years attributable to T cruzi infection. Of the 120 seropositives classified as having Chagas cardiomyopathy, only 31 (26%) presented with ejection fraction <50%, and only 11 (9%) were classified as New York Heart Association class II or higher. Chagas cardiomyopathy was associated (P<0.01) with male sex, a history of abnormal ECG, and the presence of an S3 heart sound. There is a substantial annual incidence of Chagas cardiomyopathy among initially asymptomatic T cruzi-seropositive blood donors, although disease was mild at diagnosis.

  18. Unraveling Chagas disease transmission through the oral route: Gateways to Trypanosoma cruzi infection and target tissues

    Science.gov (United States)

    Silva-dos-Santos, Danielle; Barreto-de-Albuquerque, Juliana; Guerra, Bárbara; Moreira, Otacilio C.; Berbert, Luiz Ricardo; Ramos, Mariana Tavares; Mascarenhas, Barbara Angelica S.; Britto, Constança; Morrot, Alexandre; Serra Villa-Verde, Déa M.; Garzoni, Luciana Ribeiro; Savino, Wilson; Cotta-de-Almeida, Vinícius; de Meis, Juliana

    2017-01-01

    Oral transmission of Trypanosoma cruzi, the causative agent of Chagas disease, is the most important route of infection in Brazilian Amazon and Venezuela. Other South American countries have also reported outbreaks associated with food consumption. A recent study showed the importance of parasite contact with oral cavity to induce a highly severe acute disease in mice. However, it remains uncertain the primary site of parasite entry and multiplication due to an oral infection. Here, we evaluated the presence of T. cruzi Dm28c luciferase (Dm28c-luc) parasites in orally infected mice, by bioluminescence and quantitative real-time PCR. In vivo bioluminescent images indicated the nasomaxillary region as the site of parasite invasion in the host, becoming consistently infected throughout the acute phase. At later moments, 7 and 21 days post-infection (dpi), luminescent signal is denser in the thorax, abdomen and genital region, because of parasite dissemination in different tissues. Ex vivo analysis demonstrated that the nasomaxillary region, heart, mandibular lymph nodes, liver, spleen, brain, epididymal fat associated to male sex organs, salivary glands, cheek muscle, mesenteric fat and lymph nodes, stomach, esophagus, small and large intestine are target tissues at latter moments of infection. In the same line, amastigote nests of Dm28c GFP T. cruzi were detected in the nasal cavity of 6 dpi mice. Parasite quantification by real-time qPCR at 7 and 21 dpi showed predominant T. cruzi detection and expansion in mouse nasal cavity. Moreover, T. cruzi DNA was also observed in the mandibular lymph nodes, pituitary gland, heart, liver, small intestine and spleen at 7 dpi, and further, disseminated to other tissues, such as the brain, stomach, esophagus and large intestine at 21 dpi. Our results clearly demonstrated that oral cavity and adjacent compartments is the main target region in oral T. cruzi infection leading to parasite multiplication at the nasal cavity. PMID

  19. Evasion of the Immune Response by Trypanosoma cruzi during Acute Infection

    Science.gov (United States)

    Cardoso, Mariana S.; Reis-Cunha, João Luís; Bartholomeu, Daniella C.

    2016-01-01

    Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical disease that affects millions of people mainly in Latin America. To establish a life-long infection, T. cruzi must subvert the vertebrate host’s immune system, using strategies that can be traced to the parasite’s life cycle. Once inside the vertebrate host, metacyclic trypomastigotes rapidly invade a wide variety of nucleated host cells in a membrane-bound compartment known as the parasitophorous vacuole, which fuses to lysosomes, originating the phagolysosome. In this compartment, the parasite relies on a complex network of antioxidant enzymes to shield itself from lysosomal oxygen and nitrogen reactive species. Lysosomal acidification of the parasitophorous vacuole is an important factor that allows trypomastigote escape from the extremely oxidative environment of the phagolysosome to the cytoplasm, where it differentiates into amastigote forms. In the cytosol of infected macrophages, oxidative stress instead of being detrimental to the parasite, favors amastigote burden, which then differentiates into bloodstream trypomastigotes. Trypomastigotes released in the bloodstream upon the rupture of the host cell membrane express surface molecules, such as calreticulin and GP160 proteins, which disrupt initial and key components of the complement pathway, while others such as glycosylphosphatidylinositol-mucins stimulate immunoregulatory receptors, delaying the progression of a protective immune response. After an immunologically silent entry at the early phase of infection, T. cruzi elicits polyclonal B cell activation, hypergammaglobulinemia, and unspecific anti-T. cruzi antibodies, which are inefficient in controlling the infection. Additionally, the coexpression of several related, but not identical, epitopes derived from trypomastigote surface proteins delays the generation of T. cruzi-specific neutralizing antibodies. Later in the infection, the establishment of an anti-T. cruzi

  20. Evidence of meaningful levels of Trypanosoma cruzi in platelet concentrates from seropositive blood donors.

    Science.gov (United States)

    Cancino-Faure, Beatriz; Fisa, Roser; Riera, Cristina; Bula, Ibeth; Girona-Llobera, Enrique; Jimenez-Marco, Teresa

    2015-06-01

    According to the reported cases of transfusion-acquired Trypanosoma cruzi infection, the risk of T. cruzi transfusion transmission appears to be higher with platelet (PLT) products than with other blood components. The aim of this study was to investigate by quantitative real-time polymerase chain reaction (qPCR) the parasitic load detected in leukoreduced plasma and PLT concentrates collected by apheresis from seropositive T. cruzi blood donors and compare them with peripheral whole blood (WB). During 2011 to 2013, a prospective study was carried out in a group of blood donors originating from Chagas-endemic areas but who are now living on the island of Majorca, Spain. Leukoreduced plasma and PLT concentrates were collected by apheresis from seropositive blood donors with detectable parasitemias in peripheral WB. Seropositivity was found in 23 of 1201 donors studied (1.9%), and T. cruzi DNA with less than 1 parasite equivalent/mL was detected in peripheral WB in 60.86% (14 of 23) of these. The study in blood components obtained by apheresis from these donors showed that T. cruzi DNA with a mean ± SD parasitic load of 5.33 ± 6.12 parasite equivalents/mL was detected in 100% of the PLT concentrate samples. Parasite DNA was undetectable in the extract taken from plasma collected from donors with a positive qPCR in peripheral WB. The higher parasitic load found in PLT concentrates compared to plasma and peripheral WB would explain the higher transfusion transmission risk of Chagas disease associated with PLT transfusions described in the reported cases of transfusion-acquired T. cruzi infection. © 2015 AABB.

  1. Biological characterization of a beta-galactosidase expressing clone of Trypanosoma cruzi CL strain

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    Ana Le-Senne

    2002-12-01

    Full Text Available Clone CL B5 of Trypanosoma cruzi is a beta-galactosidase expressing organism that was genetically transfected to be used for in vitro pharmacological screening. Biological parameters were determined, evaluating growth kinetics of epimastigotes, metacyclogenesis, infectivity to mammalian cell lines, parasitemia kinetics in mice and sensibility to nifurtimox and benznidazole. Differences in relation to other strains and CL parental strain were found, the most important being the incapability to produce death to mice in spite of the high inoculum used. However, it possesses the required features to be used for in vitro drug screening. Data obtained demonstrate that heterogeneity of T. cruzi appears even among clones of the same strain, and that these differences found do not prevent the use of clone CL B5 for the purpose that was engineered.

  2. New chemotypes as Trypanosoma cruzi triosephosphate isomerase inhibitors: a deeper insight into the mechanism of inhibition.

    Science.gov (United States)

    Alvarez, Guzmán; Martínez, Jennyfer; Aguirre-López, Beatriz; Cabrera, Nallely; Pérez-Díaz, Leticia; de Gómez-Puyou, Marietta Tuena; Gómez-Puyou, Armando; Pérez-Montfort, Ruy; Garat, Beatriz; Merlino, Alicia; González, Mercedes; Cerecetto, Hugo

    2014-04-01

    Triosephosphate isomerase (TIM) is a ubiquitous enzyme that has been targeted for the discovery of new small molecular weight compounds used against Trypanosoma cruzi, the causative agent of Chagas disease. We have identified phenazine and 1,2,6-thiadiazine chemotypes as novel inhibitors of TIM from T. cruzi (TcTIM). Study the mechanism of TcTIM inhibition by a phenazine derivative and by a 1,2,6-thiadiazine derivative. We performed biochemical and theoretical molecular docking studies to characterize the interaction of the derivatives with wild-type and mutant TcTIM. At low micromolar concentrations, the compounds induce highly selective irreversible inactivation of parasitic TIM. The molecular docking simulations indicate that the phenazine derivative likely interferes with the association of the two monomers of the dimeric enzyme by locating at the dimer interface, while 1,2,6-thiadiazine could act as an inhibitor binding to a region surrounding Cys-118.

  3. Proteomic analysis of Trypanosoma cruzi developmental stages using isotope-coded affinity tag reagents.

    Science.gov (United States)

    Paba, Jaime; Ricart, Carlos A O; Fontes, Wagner; Santana, Jaime M; Teixeira, Antonio R L; Marchese, Jason; Williamson, Brian; Hunt, Tony; Karger, Barry L; Sousa, Marcelo V

    2004-01-01

    Comparative proteome analysis of developmental stages of the human pathogen Trypanosoma cruzi was carried out by isotope-coded affinity tag technology (ICAT) associated with liquid cromatography-mass spectrometry peptide sequencing (LC-MS/MS). Protein extracts of the protozoan trypomastigote and amastigote stages were labeled with heavy (D8) and light (D0) ICAT reagents and subjected to cation exchange and avidin affinity chromatographies followed by LC-MS/MS analysis. High confidence sequence information and expression levels for 41 T. cruzi polypeptides, including metabolic enzymes, paraflagellar rod components, tubulins, and heat-shock proteins were reported. Twenty-nine proteins displayed similar levels of expression in both forms of the parasite, nine proteins presented higher levels in trypomastigotes, whereas three were more expressed in amastigotes.

  4. Genómica del Trypanosoma cruzi. Nuevas oportunidades para tratar el mal de Chagas

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    Jorge A. Huete-Pérez

    2006-12-01

    Full Text Available LA SECUENCIACIÓN DEL GENOMA HUMANO PUBLICADA EN FEBRERO de 2001 ha sido considerada como el hito científico más importante del siglo XX. La secuenciación, cuatro años más tarde, de tres parásitos tripanosmatidas, entre ellos el Trypanosoma cruzi, podría ser también catalogada como uno de los acontecimientos científicos más importantes para la salud publica del continente americano. Aquí se presenta un panorama general sobre los resultados más significativos del estudio geonómico del T. cruzi, se abordan los trabajos realizados por nuestro laboratorio en la Universidad Centroamericana, finalizando con una discusión sobre las perspectivas del uso de la genómica en Nicaragua.

  5. A recombinant protein based on Trypanosoma cruzi P21 enhances phagocytosis.

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    Adele A Rodrigues

    Full Text Available BACKGROUND: P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6 on inflammatory macrophages during phagocytosis. FINDINGS: Our results showed that P21-His(6 acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent onthe PI3-kinase signaling pathway. CONCLUSIONS: Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6 represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.

  6. Indazoles: a new top seed structure in the search of efficient drugs against Trypanosoma cruzi.

    Science.gov (United States)

    Aguilera-Venegas, Benjamín; Olea-Azar, Claudio; Arán, Vicente J; Speisky, Hernán

    2013-10-01

    For years, Chagas disease treatment has been limited to only two drugs of highly questionable and controversial use (Nifurtimox(®) and Benznidazole(®)). In the search of effective drugs, many efforts have been made, but only a few structures have emerged as actual candidates. Heading into this, the multitarget-directed approach appears as the best choice. In this framework, indazoles were shown to be potent Trypanosoma cruzi growth inhibitors, being able to lead both the formation of reactive oxygen species and the inhibition of trypanothione reductase. Herein, we discuss the main structural factors that rule the anti-T. cruzi properties of indazoles, and how they would be involved in the biological properties as well as in the action mechanisms, attempting to make parallels between the old paradigms and current evidences in order to outline what could be the next steps to follow in regard to the future drug design for Chagas disease treatment.

  7. Glycoproteomics of Trypanosoma cruzi trypomastigotes using subcellular fractionation, lectin affinity, and stable isotope labeling.

    Science.gov (United States)

    Atwood, James A; Minning, Todd; Ludolf, Fernanda; Nuccio, Arthur; Weatherly, Daniel B; Alvarez-Manilla, Gerardo; Tarleton, Rick; Orlando, Ron

    2006-12-01

    Herein we detail the first glycoproteomic analysis of a human pathogen. We describe an approach that enables the identification of organelle and cell surface N-linked glycoproteins from Trypanosoma cruzi, the causative agent of Chagas' disease. This approach is based on a subcellular fractionation protocol to produce fractions enriched in either organelle or plasma membrane/cytoplasmic proteins. Through lectin affinity capture of the glycopeptides from each subcellular fraction and stable isotope labeling of the glycan attachment sites with H(2)18O, we unambiguously identified 36 glycosylation sites on 35 glycopeptides which mapped to 29 glycoproteins. We also present the first expression evidence for 11 T. cruzi specific glycoproteins and provide experimental data indicating that the mucin associated surface protein family (MASP) and dispersed gene family (DGF-1) are post-translationally modified by N-linked glycans.

  8. Natural populations of Trypanosoma cruzi, the agent of Chagas disease, have a complex multiclonal structure

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    Tibayrenc, M.; Ward, P.; Moya, A.; Ayala, F.J.

    1986-01-01

    The authors have studied 15 gene loci coding for enzymes in 121 Trypanosoma cruzi stocks from a wide geographic range - from the US and Mexico to Chile and southern Brazil. T.cruzi is diploid but reproduction is basically clonal, with very little if any sexuality remaining at present. They have identified 43 different clones by their genetic composition; the same genetic clone is often found in very distant places and in diverse hosts. There is much genetic heterogeneity among the different clones, and they cannot be readily classified into a few discrete groups that might represent natural taxa. These findings imply that the biological and medical characteristics need to be ascertained separately for each natural clone. The evidence indicates that clonal evolution is very ancient in T.cruzi. The authors propose two alternative hypotheses concerning the relationship between the biochemical diversity and the heterogeneity in other biological and medical characteristics of T. cruzi. One hypothesis is that the degree of diversity between strains simply reflects the time elapsed since their last common ancestor. The second hypothesis is that biological and medical heterogeneity is recent and reflects adaptation to different transmission cycles. A decision between the two hypotheses can be reached with appropriate studies, with important medical consequences.

  9. Trypanosoma cruzi: Maintenance in Culture Modify Gene and Antigenic Expression of Metacyclic Trypomastigotes

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    Víctor T Contreras

    1998-11-01

    Full Text Available In this study we examined whether the maintenance of Trypanosoma cruzi by long-time in axenic culture produces changes in gene expression and antigenic profiles. The studies were made with a Dm30L-clone from a low-virulent strain and a non-cloned virulent EP-strain of T. cruzi. Both parasites were maintained, for at least seven years, by successive alternate passage triatomine/mouse (triatomine condition, or by serial passage in axenic medium (culture condition. The comparison of the [35S]methionine metabolic labeling products of virulent and non-virulent parasites by 2D-SDS-PAGE, clearly indicates that the expression of metacyclic trypomastigotes (but not of epimastigotes proteins have been altered by laboratory maintenance conditions. Western blot analysis of EP and Dm30L-epimastigotes using a serum anti-epimastigotes revealed that although most of antigens are conserved, four antigens are characteristics of triatomine condition parasites and three other are characteristics of culture condition parasites. Anti-metacyclics serum revealed significative differences in EP- and Dm30L-metacyclic trypomastigotes from triatomine condition. However, avirulent metacyclic forms were antigenically very similar. These results suggest that besides a possible selection of avirulent subpopulation from T. cruzi strains genetically heterogeneous when maintained by long time in axenic culture, changes in virulence might be due to post-translational modifications of the antigens induced by the absence of the natural alternability (vertebrate-invertebrate in the life-cycle of T. cruzi

  10. Isolation, mouse pathogenicity, and genotyping of Trypanosoma cruzi from an English Cocker Spaniel from Virginia, USA.

    Science.gov (United States)

    Patel, Jay M; Rosypal, Alexa C; Zimmerman, Kurt L; Monroe, William E; Sriranganathan, Nammalwar; Zajac, Anne M; Yabsley, Michael J; Lindsay, David S

    2012-07-06

    Trypanosoma cruzi was demonstrated in blood smears and heart tissue from a 5-year old, female, English Cocker Spaniel that had never been outside of the state of Virginia, USA. Plasma from the dog was positive in a commercially available immunochromatographic dipstick assay for T. cruzi and negative in an immunochromatographic dipstick assay for visceral Leishmania spp. The plasma from the dog had an indirect immunofluorescent antibody titer of 1:800 against epimastigotes of T. cruzi while the titer was 1:50 against promastigotes of L. infantum. The parasite was isolated from the blood in vitro from the dog (TcVT-1 isolate) and used to experimentally infect female C3H and ICR mice. The parasite was nonpathogenic for experimentally inoculated mice. DNA was isolated from parasites grown in vitro and used to determine that the genotype of T. cruzi present in the dog was genotype TcIV. This genotype is common in raccoons, Procyon lotor, in North America and suggests that raccoons may serve as reservoirs for canine infection. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Immunological Identification of Trypanosoma cruzi Lineages in Human Infection Along the Endemic Area

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    Risso, Marikena G.; Sartor, Paula A.; Burgos, Juan M.; Briceño, Luis; Rodríguez, Eva M.; Guhl, Felipe; Chavez, Omar Triana; Espinoza, Berta; Monteón, Victor M.; Russomando, Graciela; Schijman, Alejandro G.; Bottasso, Oscar A.; Leguizamón, Maria Susana

    2011-01-01

    Genotyping studies show a polarized geographic distribution of Trypanosoma cruzi lineages in humans. Here, we assessed their distribution along Latin America through an immunological approach we designated Western blot (WB) assay with Trypomastigote small-surface antigen (TSSA) I and TSSA II (TSSA-WB). These antigens are expressed by T. cruzi I (TCI; now TcI) and T. cruzi II (TCII; reclassified as TcII to TcVI) parasites. TSSA-WB showed good concordance with genotyping tests. An unexpected frequency of TSSA II recognition was observed in Colombia, Venezuela, and Mexico (northern region of Latin America). In Argentina and Paraguay (southern region), immunophenotyping confirmed the already reported TCII (TcII to TcVI) dominance. The lineage distribution between these regions showed significant difference but not among countries within them (except for Colombia and Venezuela). TSSA-WB shows TCII emergence in the northern region where TCI was reported as dominant or even as the unique T. cruzi lineage infecting humans. PMID:21212206

  12. Anti-Trypanosoma cruzi antibody detection in blood donors in the Southern Brazil

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    A.B. Araújo

    Full Text Available Trypanosoma cruzi, the causal agent of Chagas' Disease, is a widely spread protozoa in America. Blood transfusion is the secondly most important way of acquiring the infection. In blood banks, tests are performed to eliminate potentially infected blood. This study aimed to evaluate the positivity for T. cruzi in blood samples of donor's candidates in Southern Brazil. The study was based on a sampling containing all blood donors of Hemopel - a Pelotas City Blood Center, Rio Grande do Sul State, Brazil, from 2004 to 2005. Serological study was performed using ELISA Chagatest. Sampling containing values ± 20% cut off were evaluated using ELISA Chagatek, ELISA Alka/Adaltis, IHA Chagatest and IIF Imunocruzi. TESA-Blot was used as a confirmatory procedure in situations where blood samples showed conflicting results. From 4,482 samples collected in 2004 and 2005, the reactivity for anti-T. cruzi was 0.96% (43. Among those, 21 cases (0.47% were confirmed as positive - most of them were female, with low school level and averaging 47.2% years old. Interestingly, the blood donors are not aware of being contaminated and this fact makes it difficult for controlling the disease. Chagas' Disease was one of the main reasons for discarding blood bags through serological control in Southern Brazil. Sampling reactivity showed variation among the different techniques used for anti-T. cruzi research. In order to obtaining more secure and conclusive results, more than one diagnostic technique must be used.

  13. Galectin-3: A Friend but Not a Foe during Trypanosoma cruzi Experimental Infection

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    Aline A. da Silva

    2017-11-01

    Full Text Available Trypanosoma cruzi interacts with host cells, including cardiomyocytes, and induces the production of cytokines, chemokines, metalloproteinases, and glycan-binding proteins. Among the glycan-binding proteins is Galectin-3 (Gal-3, which is upregulated after T. cruzi infection. Gal-3 is a member of the lectin family with affinity for β-galactose containing molecules; it can be found in both the nucleus and the cytoplasm and can be either membrane-associated or secreted. This lectin is involved in several immunoregulatory and parasite infection process. Here, we explored the consequences of Gal-3 deficiency during acute and chronic T. cruzi experimental infection. Our results demonstrated that lack of Gal-3 enhanced in vitro replication of intracellular parasites, increased in vivo systemic parasitaemia, and reduced leukocyte recruitment. Moreover, we observed decreased secretion of pro-inflammatory cytokines in spleen and heart of infected Gal-3 knockout mice. Lack of Gal-3 also led to elevated mast cell recruitment and fibrosis of heart tissue. In conclusion, galectin-3 expression plays a pivotal role in controlling T. cruzi infection, preventing heart damage and fibrosis.

  14. Effects of habitat fragmentation on wild mammal infection by Trypanosoma cruzi.

    Science.gov (United States)

    Vaz, V C; D'Andrea, P S; Jansen, A M

    2007-11-01

    Expansion of human activities frequently results in habitat fragmentation, a phenomenon that has been widely recognized in the last decades as one of the major threats to world's biodiversity. The transformation of a continuous forest into a fragmented area results in a hyper-dynamic landscape with unpredictable consequences to overall ecosystem health. The effect of the fragmentation process on Trypanosoma cruzi infection among small wild mammals was studied in an Atlantic Rain Forest landscape. Comparing continous forest to fragmented habitat, marsupials were less abundant than rodents in the continuous landscape. An overall decrease in small wild mammal richness was observed in the smaller fragments. An anti-T. cruzi seroprevalence of 18% (82/440) was deteced by immunofluorescence assay. Moreover, this seroprevalence was higher in the fragmented habitat than in the continuous forest. According to the collected data, 3 main factors seem to modulate infection by T. cruzi in small wild mammals: (i) habitat fragmentation; (ii) biodiversity loss; (iii) increase of marsupial abundance in mammal communities. Furthermore, an extremely mild controlled infection by T. cruzi was detected since no patent parasitaemia could be detected in fresh blood samples, and no parasites were isolated by haemoculture.

  15. Recently differentiated epimastigotes from Trypanosoma cruzi are infective to the mammalian host.

    Science.gov (United States)

    Kessler, Rafael Luis; Contreras, Víctor Tulio; Marliére, Newmar Pinto; Aparecida Guarneri, Alessandra; Villamizar Silva, Luz Helena; Mazzarotto, Giovanny Augusto Camacho Antevere; Batista, Michel; Soccol, Vanete Thomaz; Krieger, Marco Aurelio; Probst, Christian Macagnan

    2017-06-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, has a complex life cycle in which four distinct developmental forms alternate between the insect vector and the mammalian host. It is assumed that replicating epimastigotes present in the insect gut are not infective to mammalian host, a paradigm corroborated by the widely acknowledged fact that only this stage is susceptible to the complement system. In the present work, we establish a T. cruzi in vitro and in vivo epimastigogenesis model to analyze the biological aspects of recently differentiated epimastigotes (rdEpi). We show that both trypomastigote stages of T. cruzi (cell-derived and metacyclic) are able to transform into epimastigotes (processes termed primary and secondary epimastigogenesis, respectively) and that rdEpi have striking properties in comparison to long-term cultured epimastigotes: resistance to complement-mediated lysis and both in vitro (cell culture) and in vivo (mouse) infectivity. Proteomics analysis of all T. cruzi stages reveled a cluster of proteins that were up-regulated only in rdEpi (including ABC transporters and ERO1), suggesting a role for them in rdEpi virulence. The present work introduces a new experimental model for the study of host-parasite interactions, showing that rdEpi can be infective to the mammalian host. © 2017 John Wiley & Sons Ltd.

  16. Differential expression profiles in the midgut of Triatoma infestans infected with Trypanosoma cruzi.

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    Diego S Buarque

    Full Text Available Chagas disease, or American trypanosomiasis, is a parasitic disease caused by the protozoan Trypanosoma cruzi and is transmitted by insects from the Triatominae subfamily. To identify components involved in the protozoan-vector relationship, we constructed and analyzed cDNA libraries from RNA isolated from the midguts of uninfected and T. cruzi-infected Triatoma infestans, which are major vectors of Chagas disease. We generated approximately 440 high-quality Expressed Sequence Tags (ESTs from each T. infestans midgut cDNA library. The sequences were grouped in 380 clusters, representing an average length of 664.78 base pairs (bp. Many clusters were not classified functionally, representing unknown transcripts. Several transcripts involved in different processes (e.g., detoxification showed differential expression in response to T. cruzi infection. Lysozyme, cathepsin D, a nitrophorin-like protein and a putative 14 kDa protein were significantly upregulated upon infection, whereas thioredoxin reductase was downregulated. In addition, we identified several transcripts related to metabolic processes or immunity with unchanged expressions, including infestin, lipocalins and defensins. We also detected ESTs encoding juvenile hormone binding protein (JHBP, which seems to be involved in insect development and could be a target in control strategies for the vector. This work demonstrates differential gene expression upon T. cruzi infection in the midgut of T. infestans. These data expand the current knowledge regarding vector-parasite interactions for Chagas disease.

  17. Biochemical behavior of Trypanosoma cruzi strains isolated from mice submitted to specific chemotherapy

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    Jesila Pinto M. Marretto

    1994-12-01

    Full Text Available To investigate the influence of chemotherapy on the biochemical beha vior of Trypanosoma cruzi strains, three groups of mice were infected with one of three strains of T. cruzi of different biological and isoenzymic patterns (Peruvian, 21 SF and Colombian strains. Each group was subdivided into subgroups: 1 - treated with nifurtimox; 2 - treated with benznidazole and 3 - untreated infected controls. At the end of treatment, that lasted for 90 days, xenodiagnosis, sub inoculation of blood into new born mice and haemoculture were performed as tests of cure. From the positive tests, 22 samples of T. cruzi were isolated from all subgroups. Electrophoretic analysis of the isoenzymes PGM, GP1, ALAT and AS AT failed to show any difference between parasite strains isolated from treated and untreated mice, which indicates that no detectable clonal selection or parasite genetic markers alterations concerning the isoenzymes analysed have been determined by treatment with drugs of recognized antiparasitic effect, suggesting stability of the phenotypic characteristics of the three biological types of T. cruzi strains.

  18. Rhodnius prolixus Life History Outcomes Differ when Infected with Different Trypanosoma cruzi I Strains

    Science.gov (United States)

    Peterson, Jennifer K.; Graham, Andrea L.; Dobson, Andrew P.; Chávez, Omar Triana

    2015-01-01

    The effect of a parasite on the life history of its vector is important for understanding and predicting disease transmission. Chagas disease agent Trypanosoma cruzi is a generalist parasite that is diverse across scales from its genetic diversity to the 100s of mammal and vector species it infects. Its vertebrate hosts show quite variable responses to infection, however, to date there are no studies looking at how T. cruzi variability might result in variable outcomes in its invertebrate host. Therefore, we investigated the effect of different T. cruzi I strains on Rhodnius prolixus survival and development. We found significant variation between insects infected with different strains, with some strains having no effect, as compared with uninfected insects, and others with significantly lower survival and development. We also found that different variables had varying importance between strains, with the effect of time postinfection and the blood:weight ratio of the infective meal significantly affecting the survival of insects infected with some strains, but not others. Our results suggest that T. cruzi can be pathogenic not only to its vertebrate hosts but also to its invertebrate hosts. PMID:26078316

  19. Enhancing effects of gamma interferon on phagocytic cell association with and killing of Trypanosoma cruzi

    Science.gov (United States)

    Wirth, J. J.; Kierszenbaum, F.; Sonnenfeld, G.; Zlotnik, A.

    1985-01-01

    Results are reported from a study of the influence gamma interferon (GIFN) and interleukin 2 (IL2) have on the capability of P388D1 cells and mouse resident peritoneal macrophages (MPM) to attach to the blood-resident parasites Trypanosoma cruzi and kill them. Cultures of trypomastigote forms of the Tulahuen strain of T. cruzi grown in bovine serum were introduced into peritoneal cells of mice, along with P388D1 cells incubated with GIFN, IL2 and both. Control cells were also maintained. Statistical analysis were then performed on data on counts of the number of dead T. Cruzi cells. The GIFN enhanced the interaction of MPM and P388D1 cells with the surface of T. Cruzi, provided the interaction was given over 12 hr to take place. A depression of the cytotoxicity of P388D1 cells was attributed to mediation by H2O2, an effect partially offset by incubation with the lymphokine GIFN.

  20. Genetic immunization based on the ubiquitin-fusion degradation pathway against Trypanosoma cruzi

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    Chou, Bin [Department of Microbiology and Immunology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan); Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan); Hiromatsu, Kenji, E-mail: khiromatsu@fukuoka-u.ac.jp [Department of Microbiology and Immunology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan); Hisaeda, Hajime; Duan, Xuefeng; Imai, Takashi [Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan); Murata, Shigeo; Tanaka, Keiji [Department of Molecular Oncology, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613 (Japan); Himeno, Kunisuke [Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan)

    2010-02-12

    Cytotoxic CD8{sup +} T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8{sup +} T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8{sup +} T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4{sup +} T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8{sup +} T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi.

  1. Fibronectin-degrading activity of Trypanosoma cruzi cysteine proteinase plays a role in host cell invasion.

    Science.gov (United States)

    Maeda, Fernando Yukio; Cortez, Cristian; Izidoro, Mario Augusto; Juliano, Luiz; Yoshida, Nobuko

    2014-12-01

    Trypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix proteins. Such an ability prevails in the parasite forms that circulate in the bloodstream and contributes to host cell invasion. Whether this also applies to the insect-stage metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, is not clear. Using T. cruzi CL strain metacyclic forms, we investigated whether fibronectin bound to the parasites and affected target cell invasion. Fibronectin present in cell culture medium bound to metacyclic forms and was digested by cruzipain, the major T. cruzi cysteine proteinase. G strain, with negligible cruzipain activity, displayed a minimal fibronectin-degrading effect. Binding to fibronectin was mediated by gp82, the metacyclic stage-specific surface molecule implicated in parasite internalization. When exogenous fibronectin was present at concentrations higher than cruzipain can properly digest, or fibronectin expression was stimulated by treatment of epithelial HeLa cells with transforming growth factor beta, the parasite invasion was reduced. Treatment of HeLa cells with purified recombinant cruzipain increased parasite internalization, whereas the treatment of parasites with cysteine proteinase inhibitor had the opposite effect. Metacyclic trypomastigote entry into HeLa cells was not affected by anti-β1 integrin antibody but was inhibited by anti-fibronectin antibody. Overall, our results have indicated that the cysteine proteinase of T. cruzi metacyclic forms, through its fibronectin-degrading activity, is implicated in host cell invasion. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Looking for combination of benznidazole and Trypanosoma cruzi-triosephosphate isomerase inhibitors for Chagas disease treatment

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    Elena Aguilera

    Full Text Available BACKGROUND The current chemotherapy for Chagas disease is based on monopharmacology with low efficacy and drug tolerance. Polypharmacology is one of the strategies to overcome these limitations. OBJECTIVES Study the anti-Trypanosoma cruzi activity of associations of benznidazole (Bnz with three new synthetic T. cruzi-triosephosphate isomerase inhibitors, 2, 3, and 4, in order to potentiate their actions. METHODS The in vitro effect of the drug combinations were determined constructing the corresponding isobolograms. In vivo activities were assessed using an acute murine model of Chagas disease evaluating parasitaemias, mortalities and IgG anti-T. cruzi antibodies. FINDINGS The effect of Bnz combined with each of these compounds, on the growth of epimastigotes, indicated an additive action or a synergic action, when combining it with 2 or 3, respectively, and an antagonic action when combining it with 4. In vivo studies, for the two chosen combinations, 2 or 3 plus one fifth equivalent of Bnz, showed that Bnz can also potentiate the in vivo therapeutic effects. For both combinations a decrease in the number of trypomastigote and lower levels of anti-T. cruzi IgG-antibodies were detected, as well clear protection against death. MAIN CONCLUSIONS These results suggest the studied combinations could be used in the treatment of Chagas disease.

  3. Effects of amiodarone and posaconazole on the growth and ultrastructure of Trypanosoma cruzi.

    Science.gov (United States)

    Veiga-Santos, Phercyles; Barrias, Emile S; Santos, Júlio F C; de Barros Moreira, Thiago Luiz; de Carvalho, Tecia Maria Ulisses; Urbina, Julio A; de Souza, Wanderley

    2012-07-01

    The antifungal posaconazole (PCZ) is the most advanced candidate for the treatment of Chagas disease, having potent anti-Trypanosoma cruzi activity in vitro and in animal models of the disease as well as an excellent safety profile in humans. Amiodarone (AMD) is the antiarrhythmic drug most frequently used for the symptomatic treatment of chronic Chagas disease patients, but it also has specific anti-T. cruzi activity. When used in combination, these drugs exhibit potent synergistic activity against the parasite. In the present work, electron microscopy was used to analyse the effects of both compounds, acting individually or in combination, against T. cruzi. The 50% inhibitory concentration (IC(50)) against epimastigote and amastigote forms was 25 nM and 1.0 nM for PCZ and 8 μM and 5.6 μM for AMD, respectively. The antiproliferative synergism of the drugs (fractional inhibitory concentrationanti-T. cruzi therapy with low side effects. Copyright © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  4. Identification of a hyperendemic area for Trypanosoma cruzi infection in central Veracruz, Mexico.

    Science.gov (United States)

    Ramos-Ligonio, Angel; López-Monteon, Aracely; Guzmán-Gómez, Daniel; Rosales-Encina, José Luis; Limón-Flores, Yairh; Dumonteil, Eric

    2010-07-01

    The state of Veracruz, Mexico, is a well-recognized endemic region for Chagas disease, but the geographic distribution of the disease and its magnitude are still poorly documented. We evaluated the seroprevalence of Trypanosoma cruzi infection in the sanitary jurisdictions of Cordoba and Cosamaloapan in central Veracruz. A total of 654 serum samples from 19 rural localities were tested by using four tests: two enzyme-linked immunosorbent assays, an indirect immunofluorescent, and Western blotting. Overall, 110 (16.8%) of 654 samples were positive for T. cruzi by >/= 2 tests (95% confidence interval = 14.2-19.9%). The municipality of Tezonapa in the jurisdiction of Cordoba was identified as a potential hyperendemic region with seroprevalence rates cruzi transmission dynamics in Tezonapa. The magnitude of T. cruzi infection rate in this region calls for the urgent implementation of extensive epidemiologic surveillance and control programs.

  5. Limited antigenic variation in the Trypanosoma cruzi candidate vaccine antigen TSA-1.

    Science.gov (United States)

    Knight, J M; Zingales, B; Bottazzi, M E; Hotez, P; Zhan, B

    2014-12-01

    Chagas disease (American trypanosomiasis caused by Trypanosoma cruzi) is one of the most important neglected tropical diseases in the Western Hemisphere. The toxicities and limited efficacies of current antitrypanosomal drugs have prompted a search for alternative technologies such as a therapeutic vaccine comprised of T. cruzi antigens, including a recombinant antigen encoding the N-terminal 65 kDa portion of Trypomastigote surface antigen-1 (TSA-1). With at least six known genetically distinct T. cruzi lineages, variability between the different lineages poses a unique challenge for the development of broadly effective therapeutic vaccine. The variability across the major lineages in the current vaccine candidate antigen TSA-1 has not previously been addressed. To assess the variation in TSA-1, we cloned and sequenced TSA-1 from several different T. cruzi strains representing three of the most clinically relevant lineages. Analysis of the different alleles showed limited variation in TSA-1 across the different strains and fit with the current theory for the evolution of the different lineages. Additionally, minimal variation in known antigenic epitopes for the HLA-A 02 allele suggests that interlineage variation in TSA-1 would not impair the range and efficacy of a vaccine containing TSA-1. © 2014 John Wiley & Sons Ltd.

  6. Electrocardiographic alteration among first degree relatives with serologic evidence of Trypanosoma cruzi infection: a sibship study

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    Julio C. Morini

    1994-09-01

    Full Text Available To analyze whether electrocardiographic alterations (ECGA in patients with antibodies to Trypanosoma cruzi showed a patttern of familial aggregation, a sample of 379 young adults (166 men and 213 women distributed in sibships, were assessed for the presence of anti-T.cruzi antibodies, and subjected to a complete clinical examination and a standard resting electrocardiogram (ECG. Positive T. cruzi serology was detected in 165 individuals, 48 of them showing an abnormal ECG (overall prevalence 29 por cento. One hundred and eleven seropositive individuals were distributed in 45 sibships, each of them constituted by more than one seropositive sib, with ECGA being present in 34 out of these patients. Seropositive subjects with ECGA were detected in 27 sibships. Since the index case within each sibship is counted exactly once, affected individuals selected at random as propositi were extracted to calculate the prevalence of ECGA among first degree relatives of probands. Abnormal ECGs were recorded in 7 out of 45 sibs yielding a prevalence that did not differ from estimations registered in the general population or seropositive sibs. Data from the present sample show no familial aggregation for the occurrence of ECGA in patients with T.cruzi infection.

  7. Variation in susceptibility to benznidazole in isolates derived from Trypanosoma cruzi parental strains

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    Veloso VM

    2001-01-01

    Full Text Available In this work, the susceptibility to benznidazole of two parental Trypanosoma cruzi strains, Colombian and Berenice-78, was compared to isolates obtained from dogs infected with these strains for several years. In order to evaluate the susceptibility to benznidazole two groups of mice were infected with one of five distinct populations isolated from dogs as well as the two parental strains of T. cruzi. The first group was treated with benznidazole during the acute phase and the second remained untreated controls. The animals were considered cured when parasitological and serological tests remained persistently negative. Mice infected with the Colombian strain and its isolates Colombian (A and B did not cure after treatment. On the other hand, all animals infected with Berenice-78 were cured by benznidazole treatment. However, 100%, 50% and 70% of cure rates were observed in animals infected with the isolates Berenice-78 B, C and D, respectively. No significant differences were observed in serological profile of infected control groups, with all animals presenting high antibody levels. However, the ELISA test showed differences in serological patterns between mice inoculated with the different T. cruzi isolates and treated with benznidazole. This variability was dependent on the T. cruzi population used and seemed to be associated with the level of resistance to benznidazole.

  8. Ultrastructural damage of Trypanosoma cruzi epimastigotes exposed to decomplemented immune sera.

    Science.gov (United States)

    Fernández-Presas, A M; Zavala, J T; Fauser, I B; Merchant, M T; Guerrero, L R; Willms, K

    2001-08-01

    The susceptibility of Trypanosoma cruzi epimastigotes to lysis by normal or immune sera in a complement-dependent reaction has been reported, but the effects induced directly by immune serum depleted of complement remain unstudied. The aim of this work was to study the ultrastructural alterations induced in T. cruzi epimastigotes by immune mouse or rabbit sera with or without complement. A local isolate of T. cruzi (Queretaro) was used in all experiments. Immune sera were raised in both mouse and rabbit by immunization with T. cruzi epimastigote antigens. Light microscopy showed intense agglutination of epimastigotes when incubated with decomplemented mouse or rabbit immune sera. A distinctive ultrastructural feature of this agglutination pattern was the fusion of plasma membranes and a pattern of intercrossing between subpellicular microtubules. Agglutination was associated with fragmentation of nuclear membranes and swelling of cytoplasm, Golgi cisternae, endoplasmic reticulum, mitochondria and kinetoplast membranes. Agglutinated parasites also incorporated trypan blue stain. Results of [3H]-thymidine incorporation confirmed that epimastigotes exposed to specific antibodies in the absence of complement were incapable of proliferating. Ultrastructural changes observed in epimastigote micrographs incubated with decomplemented immune mouse sera were statistically significant (P<0.001) when compared with results obtained from images after incubation with decomplemented normal mouse sera.

  9. Purinergic ecto-enzymes participate in the thromboregulation in acute in mice infection by Trypanosoma cruzi.

    Science.gov (United States)

    do Carmo, Guilherme M; Doleski, Pedro H; de Sá, Mariângela F; Grando, Thirssa H; Bottari, Nathieli B; Leal, Daniela B R; Gressler, Lucas T; Mendes, Ricardo E; Stefani, Lenita M; Monteiro, Silvia G; Da Silva, Aleksandro S

    2017-08-01

    Coagulation disorders have been described in Chagas disease with thrombocytopenia as an important event. Several mechanisms may be related to this pathogenesis, such as enzymes of the purinergic system, purine, and receptors involved in the regulation and modulation of physiological events related to hemostasis. Therefore, the aim of this study was to evaluate the activities of E-NTPDase, E-5'nucleotidase, and ecto-adenosine deaminase (E-ADA) in platelets of mice experimentally infected by Trypanosoma cruzi. Twelve female mice were used, divided into two groups (n = 6): uninfected and infected. Mice of infected group were intraperitoneally inoculated with 104 trypomastigotes of T. cruzi (strain Y). On day 12 post-infection (PI), blood samples were collected for quantitation and separation of platelets. A significant reduction in the number of platelets of infected mice (P < 0.05) was observed. The activities of E-NTPDase (ATP and ADP substrates), E-5'nucleotidase, and E-ADA in platelets increased significantly (P < 0.05) in mice infected by T. cruzi compared with uninfected animals. A negative correlation (P < 0.01)was observed between the number of platelets and ATP hydrolysis (r = -0.64), and ADP hydrolysis (r = -0.69) by E-NTPDase. Therefore, there is a response from the purinergic system activating ecto-enzymes in platelets of mice T. cruzi infected, as a compensatory effect of thrombocytopenia.

  10. Host cell poly(ADP-ribose glycohydrolase is crucial for Trypanosoma cruzi infection cycle.

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    Salomé C Vilchez Larrea

    Full Text Available Trypanosoma cruzi, etiological agent of Chagas' disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose glycohydrolase in a trypanosomatid (TcPARG. In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl pyrrolidinediol or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas' disease.

  11. Temporal variation of Trypanosoma cruzi discrete typing units in asymptomatic Chagas disease patients.

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    Sánchez, Laura V; Bautista, Diana C; Corredor, Andrés F; Herrera, Victor M; Martinez, Luz X; Villar, Juan Carlos; Ramírez, Juan David

    2013-01-01

    Chagas disease caused by Trypanosoma cruzi is a public health problem in Latin America. This parasite displays a high genetic diversity evidenced in six Discrete Typing Units (DTUs) namely TcI-TcVI. The aim of this study was to observe the temporal variation of the DTUs in asymptomatic patients at three different times (10 days interval). The results showed that intermittence is the rule in the bloodstream of Chagas disease patients. The patients showed different detectable DTUs with short time intervals, which favors the clonal histiotropic model and the multiclonality structure of this parasite. Published by Elsevier Masson SAS.

  12. Structural insights into the catalytic mechanism of Trypanosoma cruzi trans-sialidase.

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    Amaya, María Fernanda; Watts, Andrew G; Damager, Iben; Wehenkel, Annemarie; Nguyen, Tong; Buschiazzo, Alejandro; Paris, Gastón; Frasch, A Carlos; Withers, Stephen G; Alzari, Pedro M

    2004-05-01

    Sialidases are a superfamily of sialic-acid-releasing enzymes that are of significant interest due to their implication as virulence factors in the pathogenesis of a number of diseases. However, extensive studies of viral and microbial sialidases have failed to provide a comprehensive picture of their mechanistic properties, in part because the structures of competent enzyme-substrate complexes and reaction intermediates have never been described. Here we report these structures for the Trypanosoma cruzi trans-sialidase (TcTS), showing that catalysis by sialidases occurs via a similar mechanism to that of other retaining glycosidases, but with some intriguing differences that may have evolved in response to the substrate structure.

  13. Cardiac plexus of dogs experimentally infected with Trypanosoma cruzi: inflammatory lesions and quantitative studies

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    Marcelo V. Caliari

    1995-03-01

    Full Text Available Qualitative and quantitative aspects of the superficial and profound cardiac plexus of dogs experimentally infected with Be-62 and Be-78 strains of Trypanosoma cruzi were studied. Animals were autopsied in the acute phase of infection. The inflammatory process, lesions and number of parasites were more intense and frequent in animals infected with the Be-78 strain than in those infected with Be-62. Despite this, no statistically significant differences could be found between the number of neuron bodies in the ganglia of infected and control dogs.

  14. Studies on the virulence and attenuation of Trypanosoma cruzi using immunodeficient animals

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    Basombrío Miguel Ángel

    2000-01-01

    Full Text Available Tissue invasion and pathology by Trypanosoma cruzi result from an interaction between parasite virulence and host immunity. Successive in vivo generations of the parasite select populations with increasing ability to invade the host. Conversely, prolonged in vitro selection of the parasite produces attenuated sublines with low infectivity for mammals. One such subline (TCC clone has been extensively used in our laboratory as experimental vaccine and tested in comparative experiments with its virulent ancestor (TUL. The experiments here reviewed aimed at the use of immunodeficient mice for testing the infectivity of TCC parasites. It has not been possible to obtain virulent, revertant sublines by prolonged passaged in such mice.

  15. Enantiomers of nifurtimox do not exhibit stereoselective anti-Trypanosoma cruzi activity, toxicity, or pharmacokinetic properties.

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    Moraes, Carolina B; White, Karen L; Braillard, Stéphanie; Perez, Catherine; Goo, Junghyun; Gaspar, Luis; Shackleford, David M; Cordeiro-da-Silva, Anabela; Thompson, R C Andrew; Freitas-Junior, Lucio; Charman, Susan A; Chatelain, Eric

    2015-01-01

    With the aim of improving the available drugs for the treatment of Chagas disease, individual enantiomers of nifurtimox were characterized. The results indicate that the enantiomers are equivalent in their in vitro activity against a panel of Trypanosoma cruzi strains; in vivo efficacy in a murine model of Chagas disease; in vitro toxicity and absorption, distribution, metabolism, and excretion characteristics; and in vivo pharmacokinetic properties. There is unlikely to be any therapeutic benefit of an individual nifurtimox enantiomer over the racemic mixture. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. 2-Pyridyl thiazoles as novel anti-Trypanosoma cruzi agents: structural design, synthesis and pharmacological evaluation.

    Science.gov (United States)

    Cardoso, Marcos Veríssimo de Oliveira; de Siqueira, Lucianna Rabelo Pessoa; da Silva, Elany Barbosa; Costa, Lívia Bandeira; Hernandes, Marcelo Zaldini; Rabello, Marcelo Montenegro; Ferreira, Rafaela Salgado; da Cruz, Luana Faria; Moreira, Diogo Rodrigo Magalhães; Pereira, Valéria Rêgo Alves; de Castro, Maria Carolina Accioly Brelaz; Bernhardt, Paul V; Leite, Ana Cristina Lima

    2014-10-30

    The present work reports on the synthesis, anti-Trypanosoma cruzi activities and docking studies of a novel series of 2-(pyridin-2-yl)-1,3-thiazoles derived from 2-pyridine thiosemicarbazone. The majority of these compounds are potent cruzain inhibitors and showed excellent inhibition on the trypomastigote form of the parasite, and the resulting structure-activity relationships are discussed. Together, these data present a novel series of thiazolyl hydrazones with potential effects against Chagas disease and they could be important leads in continuing development against Chagas disease. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  17. Sialoglycoconjugates in Trypanosoma cruzi-host cell interaction: possible biological model - a review

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    Alane Beatriz Vermelho

    1994-03-01

    Full Text Available A number of glycoconjugates, including glycolipids and glycoproteins, participate in the process of host-cell invasion by Trypanosoma cruzi and one of the most important carbohydrates involved on this interaction is sialic acid. It is known that parasite trans-sialidase participates with sialic acid in a coordinated fashion in the initial stages of invasion. Given the importance of these sialogycoconjugates, this review sets out various possible biological models for the interaction between the parasite and mammalian cells that possess a sialylated receptor/ligand system.

  18. Ultrastructural alterations induced by nifurtimox and another nitro derivative on epimastigotes of Trypanosoma cruzi.

    Science.gov (United States)

    Muelas-Serrano, S; Pérez-Serrano, J; Gómez-Barrio, A; Arán, V J; Rodríguez-Caabeiro, F

    2002-02-01

    We report the ultrastructural alterations induced on epimastigotes by nifurtimox and 5-nitro-2-thienyl-malononitrile (5NO2TM), a novel compound with anti-Trypanosoma cruzi activity. Parasites treated with concentrations of nifurtimox lower than usually employed for this kind of study showed vacuolisation, alterations of the mitochondria, the nucleus and the ribosomes. 5NO2TM caused the same kind of damage, but to a greater degree. This result correlates with the fact that cultures treated with this compound experienced a greater loss of viability.

  19. Anti-Trypanosoma cruzi and cytotoxic activities of Eugenia uniflora L.

    Science.gov (United States)

    Santos, Karla K A; Matias, Edinardo F F; Tintino, Saulo R; Souza, Celestina E S; Braga, Maria F B M; Guedes, Gláucia M M; Rolón, Miriam; Vega, Celeste; de Arias, Antonieta Rojas; Costa, José G M; Menezes, Irwin R A; Coutinho, Henrique D M

    2012-05-01

    Chagas disease is caused by Trypanosoma cruzi, being considered a public health problem. An alternative to combat this pathogen is the use of natural products isolated from fruits such as Eugenia uniflora, a plant used by traditional communities as food and medicine due to its antimicrobial and biological activities. Ethanolic extract from E. uniflora was used to evaluate in vitro anti-epimastigote and cytotoxic activity. This is the first record of anti-Trypanosoma activity of E. uniflora, demonstrating that a concentration presenting 50% of activity (EC(50)) was 62.76 μg/mL. Minimum inhibitory concentration (MIC) was ≤ 1024 μg/mL. Our results indicate that E. uniflora could be a source of plant-derived natural products with anti-epimastigote activity with low toxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Application of core-shell PEGylated CdS/Cd(OH) 2 quantum dots as biolabels of Trypanosoma cruzi parasites

    Science.gov (United States)

    Chaves, C. R.; Fontes, A.; Farias, P. M. A.; Santos, B. S.; de Menezes, F. D.; Ferreira, R. C.; Cesar, C. L.; Galembeck, A.; Figueiredo, R. C. B. Q.

    2008-11-01

    Semiconductor quantum dots are a promising class of materials in the labeling of biological systems. In the present study we show the marking pattern of Trypanosoma cruzi ( T. cruzi) live parasites using PEGylated CdS/Cd(OH) 2 fluorescent nanocrystals. The analysis obtained by confocal fluorescence microscopy and transmission electron microscopy indicates that only the endocytic paths of parasites were labeled. The parasites were alive after the incubation with the CdS/Cd(OH) 2-PEG suspension. Labeling the T. cruzi with quantum dots can help to better understand the endocytosis process and also the cellular differentiation.

  1. Programmed Cell Death in Procyclic Form Trypanosoma brucei rhodesiense - Identification of Differentially Expressed Genes during Con A Induced Death

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    Welburn Susan C

    1999-01-01

    Full Text Available Trypanosoma brucei rhodesiense can be induced to undergo apoptosis after stimulation with Con A. As cell death in these parasites is associated with de novo gene expression we have applied a differential display technique, Randomly Amplified Differential Expressed Sequence-Polymerase Chain Reaction (RADES-PCR to the study of gene expression during Con A induced cell death in these organisms. Twenty-two differentially displayed products have been cloned and sequenced. These represent the first endogenous genes to be identified as implicated in cellular death in trypanosomatids (the most primitive eukaryote in which apoptosis has been described. Evidence for an ancestral death machinery, `proto-apoptosis' in single celled organisms is discussed.

  2. High-confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope-tag organelle enrichment and SILAC proteomics.

    Science.gov (United States)

    Güther, Maria Lucia S; Urbaniak, Michael D; Tavendale, Amy; Prescott, Alan; Ferguson, Michael A J

    2014-06-06

    The glycosome of the pathogenic African trypanosome Trypanosoma brucei is a specialized peroxisome that contains most of the enzymes of glycolysis and several other metabolic and catabolic pathways. The contents and transporters of this membrane-bounded organelle are of considerable interest as potential drug targets. Here we use epitope tagging, magnetic bead enrichment, and SILAC quantitative proteomics to determine a high-confidence glycosome proteome for the procyclic life cycle stage of the parasite using isotope ratios to discriminate glycosomal from mitochondrial and other contaminating proteins. The data confirm the presence of several previously demonstrated and suggested pathways in the organelle and identify previously unanticipated activities, such as protein phosphatases. The implications of the findings are discussed.

  3. Eco-epidemiological aspects of Trypanosoma cruzi, Trypanosoma rangeli and their vector (Rhodnius pallescens in Panama Generalidades do Trypanosoma cruzi, do Trypanosoma rangeli e do seu vetor (Rhodnius pallescens no Panamá

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    Ana Maria de Vasquez

    2004-08-01

    Full Text Available The eco-epidemiology of T. cruzi infection was investigated in the Eastern border of the Panama Canal in Central Panama. Between 1999 and 2000, 1110 triatomines were collected: 1050 triatomines (94.6% from palm trees, 27 (2.4% from periurban habitats and 33 (3.0% inside houses. All specimens were identified as R. pallescens. There was no evidence of vector domiciliation. Salivary glands from 380 R. pallescens revealed a trypanosome natural infection rate of 7.6%, while rectal ampoule content from 373 triatomines was 45%. Isoenzyme profiles on isolated trypanosomes demonstrated that 85.4% (n = 88 were T. cruzi and 14.6% (n = 15 were T. rangeli. Blood meal analysis from 829 R. pallescens demonstrated a zoophilic vector behavior, with opossums as the preferential blood source. Seroprevalence in human samples from both study sites was less than 2%. Our results demonstrate that T. cruzi survives in the area in balanced association with R. pallescens, and with several different species of mammals in their natural niches. However, the area is an imminent risk of infection for its population, consequently it is important to implement a community educational program regarding disease knowledge and control measures.A epidemiologia da infecção do T. cruzi foi investigada na margem oriental do canal do Panamá, na região central da Republica do Panamá. A informação obtida durante o estudo avaliou fatores de risco da doença de Chagas nesta área. Entre 1999 e 2000, 1110 triatomíneos foram coletados: 1050 triatomíneos (94,6% em palmeiras, 27 (2,4% em habitats periurbanos e 33 (3,0% no interior de casas. Todos os espécimens foram identificados como R. pallescens. Não havia nenhuma evidência de domiciliação do vetor. O exame de glândulas salivares de 380 R. pallescens revelaram taxa de infecção natural por Trypanosoma de 7,6%, mas o conteúdo da ampola rectal de 373 triatomíneos mostrou 45% de positividade. Os perfis de isoenzimas em

  4. New functions for parts of the Krebs cycle in procyclic Trypanosoma brucei, a cycle not operating as a cycle.

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    van Weelden, Susanne W H; van Hellemond, Jaap J; Opperdoes, Fred R; Tielens, Aloysius G M

    2005-04-01

    We investigated whether substrate availability influences the type of energy metabolism in procyclic Trypanosoma brucei. We show that absence of glycolytic substrates (glucose and glycerol) does not induce a shift from a fermentative metabolism to complete oxidation of substrates. We also show that glucose (and even glycolysis) is not essential for normal functioning and proliferation of pleomorphic procyclic T. brucei cells. Furthermore, absence of glucose did not result in increased degradation of amino acids. Variations in availability of glucose and glycerol did result, however, in adaptations in metabolism in such a way that the glycosome was always in redox balance. We argue that it is likely that, in procyclic cells, phosphoglycerate kinase is located not only in the cytosol, but also inside glycosomes, as otherwise an ATP deficit would occur in this organelle. We demonstrate that procyclic T. brucei uses parts of the Krebs cycle for purposes other than complete degradation of mitochondrial substrates. We suggest that citrate synthase plus pyruvate dehydrogenase and malate dehydrogenase are used to transport acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, a process we show to occur in proliferating procyclic cells. The part of the Krebs cycle consisting of alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase was used for the degradation of proline and glutamate to succinate. We also demonstrate that the subsequent enzymes of the Krebs cycle, succinate dehydrogenase and fumarase, are most likely used for conversion of succinate into malate, which can then be used in gluconeogenesis.

  5. Trypanosoma brucei TbIF1 inhibits the essential F1-ATPase in the infectious form of the parasite.

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    Brian Panicucci

    2017-04-01

    Full Text Available The mitochondrial (mt FoF1-ATP synthase of the digenetic parasite, Trypanosoma brucei, generates ATP during the insect procyclic form (PF, but becomes a perpetual consumer of ATP in the mammalian bloodstream form (BF, which lacks a canonical respiratory chain. This unconventional dependence on FoF1-ATPase is required to maintain the essential mt membrane potential (Δψm. Normally, ATP hydrolysis by this rotary molecular motor is restricted to when eukaryotic cells experience sporadic hypoxic conditions, during which this compulsory function quickly depletes the cellular ATP pool. To protect against this cellular treason, the highly conserved inhibitory factor 1 (IF1 binds the enzyme in a manner that solely inhibits the hydrolytic activity. Intriguingly, we were able to identify the IF1 homolog in T. brucei (TbIF1, but determined that its expression in the mitochondrion is tightly regulated throughout the life cycle as it is only detected in PF cells. TbIF1 appears to primarily function as an emergency brake in PF cells, where it prevented the restoration of the Δψm by FoF1-ATPase when respiration was chemically inhibited. In vitro, TbIF1 overexpression specifically inhibits the hydrolytic activity but not the synthetic capability of the FoF1-ATP synthase in PF mitochondria. Furthermore, low μM amounts of recombinant TbIF1 achieve the same inhibition of total mt ATPase activity as the FoF1-ATPase specific inhibitors, azide and oligomycin. Therefore, even minimal ectopic expression of TbIF1 in BF cells proved lethal as the indispensable Δψm collapsed due to inhibited FoF1-ATPase. In summary, we provide evidence that T. brucei harbors a natural and potent unidirectional inhibitor of the vital FoF1-ATPase activity that can be exploited for future structure-based drug design.

  6. Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids.

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    Nanavaty, Vishal; Sandhu, Ranjodh; Jehi, Sanaa E; Pandya, Unnati M; Li, Bibo

    2017-06-02

    Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, thereby evading the host's immune response. VSGs are monoallelically expressed from subtelomeric expression sites (ESs), and VSG switching exploits subtelomere plasticity. However, subtelomere integrity is essential for T. brucei viability. The telomeric transcript, TERRA, was detected in T. brucei previously. We now show that the active ES-adjacent telomere is transcribed. We find that TbRAP1, a telomere protein essential for VSG silencing, suppresses VSG gene conversion-mediated switching. Importantly, TbRAP1 depletion increases the TERRA level, which appears to result from longer read-through into the telomere downstream of the active ES. Depletion of TbRAP1 also results in more telomeric RNA:DNA hybrids and more double strand breaks (DSBs) at telomeres and subtelomeres. In TbRAP1-depleted cells, expression of excessive TbRNaseH1, which cleaves the RNA strand of the RNA:DNA hybrid, brought telomeric RNA:DNA hybrids, telomeric/subtelomeric DSBs and VSG switching frequency back to WT levels. Therefore, TbRAP1-regulated appropriate levels of TERRA and telomeric RNA:DNA hybrid are fundamental to subtelomere/telomere integrity. Our study revealed for the first time an important role of a long, non-coding RNA in antigenic variation and demonstrated a link between telomeric silencing and subtelomere/telomere integrity through TbRAP1-regulated telomere transcription. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. In Silico Identification and in Vitro Activity of Novel Natural Inhibitors of Trypanosoma brucei Glyceraldehyde-3-phosphate-dehydrogenase

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    Fabian C. Herrmann

    2015-09-01

    Full Text Available As part of our ongoing efforts to identify natural products with activity against pathogens causing neglected tropical diseases, we are currently performing an extensive screening of natural product (NP databases against a multitude of protozoan parasite proteins. Within this project, we screened a database of NPs from a commercial supplier, AnalytiCon Discovery (Potsdam, Germany, against Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH, a glycolytic enzyme whose inhibition deprives the parasite of energy supply. NPs acting as potential inhibitors of the mentioned enzyme were identified using a pharmacophore-based virtual screening and subsequent docking of the identified hits into the active site of interest. In a set of 700 structures chosen for the screening, 13 (1.9% were predicted to possess significant affinity towards the enzyme and were therefore tested in an in vitro enzyme assay using recombinant TbGAPDH. Nine of these in silico hits (69% showed significant inhibitory activity at 50 µM, of which two geranylated benzophenone derivatives proved to be particularly active with IC50 values below 10 µM. These compounds also showed moderate in vitro activity against T. brucei rhodesiense and may thus represent interesting starting points for further optimization.

  8. Proximity Interactions among Basal Body Components in Trypanosoma brucei Identify Novel Regulators of Basal Body Biogenesis and Inheritance

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    Hung Quang Dang

    2017-01-01

    Full Text Available The basal body shares similar architecture with centrioles in animals and is involved in nucleating flagellar axonemal microtubules in flagellated eukaryotes. The early-branching Trypanosoma brucei possesses a motile flagellum nucleated from the basal body that consists of a mature basal body and an adjacent pro-basal body. Little is known about the basal body proteome and its roles in basal body biogenesis and flagellar axoneme assembly in T. brucei. Here, we report the identification of 14 conserved centriole/basal body protein homologs and 25 trypanosome-specific basal body proteins. These proteins localize to distinct subdomains of the basal body, and several of them form a ring-like structure surrounding the basal body barrel. Functional characterization of representative basal body proteins revealed distinct roles in basal body duplication/separation and flagellar axoneme assembly. Overall, this work identified novel proteins required for basal body duplication and separation and uncovered new functions of conserved basal body proteins in basal body duplication and separation, highlighting an unusual mechanism of basal body biogenesis and inheritance in this early divergent eukaryote.

  9. No gold standard estimation of the sensitivity and specificity of two molecular diagnostic protocols for Trypanosoma brucei spp. in Western Kenya.

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    Barend Mark de Clare Bronsvoort

    2010-01-01

    Full Text Available African animal trypanosomiasis is caused by a range of tsetse transmitted protozoan parasites includingTrypanosoma vivax, Trypanosoma congolense and Trypansoma brucei. In Western Kenya and other parts of East Africa two subspecies of T. brucei, T.b. brucei and the zoonoticT.b. rhodesiense, co-circulate in livestock. A range of polymerase chain reactions (PCR have been developed as important molecular diagnostic tools for epidemiological investigations of T. brucei s.l. in the animal reservoir and of its zoonotic potential. Quantification of the relative performance of different diagnostic PCRs is essential to ensure comparability of studies. This paper describes an evaluation of two diagnostic test systems for T. brucei using a T. brucei s.l. specific PCR [1] and a single nested PCR targeting the Internal Transcribed Spacer (ITS regions of trypanosome ribosomal DNA [2]. A Bayesian formulation of the Hui-Walter latent class model was employed to estimate their test performance in the absence of a gold standard test for detecting T.brucei s.l. infections in ear-vein blood samples from cattle, pig, sheep and goat populations in Western Kenya, stored on Whatman FTA cards. The results indicate that the system employing the T. brucei s.l. specific PCR (Se1=0.760 had a higher sensitivity than the ITS-PCR (Se2=0.640; both have high specificity (Sp1=0.998; Sp2=0.997. The true prevalences for livestock populations were estimated (pcattle=0.091, ppigs=0.066, pgoats=0.005, psheep=0.006, taking into account the uncertainties in the specificity and sensitivity of the two test systems. Implications of test performance include the required survey sample size; due to its higher sensitivity and specificity, the T. brucei s.l. specific PCR requires a consistently smaller sample size than the ITS-PCR for the detection of T. brucei s.l. However the ITS-PCR is able to simultaneously screen samples for other pathogenic trypanosomes and may thus be, overall, a better

  10. Ebi3 PreventsTrypanosoma cruzi-Induced Myocarditis by Dampening IFN-γ-Driven Inflammation.

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