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1.
Matthias A. Fügi Kapila Gunasekera Torsten Ochsenreiter Xueli Guan Markus R. Wenk Pascal M?ser 《Journal of lipid research》2014,55(5):929-938
Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering drugs are blockbusters in human health, and inhibitors of ergosterol biosynthesis are widely used as antifungals. Inhibitors of ergosterol synthesis are also being developed for Chagas’s disease, caused by Trypanosoma cruzi. Here we develop an in silico pipeline to globally evaluate sterol metabolism and perform comparative genomics. We generate a library of hidden Markov model-based profiles for 42 sterol biosynthetic enzymes, which allows expressing the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally groups eukaryote proteomes and reveals convergent evolution, in particular metabolic reduction in obligate endoparasites. We experimentally explore sterol metabolism by testing a set of sterol biosynthesis inhibitors against trypanosomatids, Plasmodium falciparum, Giardia, and mammalian cells, and by quantifying the expression levels of sterol biosynthetic genes during the different life stages of T. cruzi and Trypanosoma brucei. The phenotypic data correlate with genomic makeup for simvastatin, which showed activity against trypanosomatids. Other findings, such as the activity of terbinafine against Giardia, are not in agreement with the genotypic profile. 相似文献
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Ana Cristina Nogueira de Melo Camila Guarany Ramalho Elias Marta Helena Branquinha Flávia Coelho Garcia dos Reis Ana Paula Cabral de Araujo Lima Maria Nazareth Leal Meirelles 《Experimental parasitology》2010,125(3):256-263
In this study, the cell-associated and extracellular peptidases of Trypanosoma cruzi grown in modified Roitman’s complex (MRC) medium were analyzed by measuring peptidase activity in gelatin-containing zymograms. Our results showed that the cell-associated peptidases as well as peptidases extracellularly released by T. cruzi displayed two distinct proteolytic classes: cysteine and metallopeptidase activities. The major cysteine peptidase, cruzipain, synthesized by T. cruzi cells was detected in cellular parasite content, as a 50 kDa reactive polypeptide, after probing with anti-cruzipain antibody. In addition, metallo-type peptidases belonging to the matrix metallopeptidase-9 (MMP-9) family were revealed, after Western blotting, as a 97 kDa protein band in cellular extract and an 85 kDa polypeptide in both cellular and secreted parasite extracts. The MMP-9-like activity present in cells and spent culture medium was immunoprecipitated by an anti-MMP-9 polyclonal antibody. The surface location of MMP-9-like proteins in T. cruzi was also evidenced by means of flow cytometry analysis. Furthermore, doxycycline that has direct MMP-9 inhibiting properties in vitro, inhibited MMP-9-like activities in gel zymography, immunoprecipitation and flow cytometry analyses. This is the first report of the presence of MMP-9-like molecules in T. cruzi. The presence of a matrix extracellular-degrading enzyme may play a role in the T. cruzi-host cell interaction, making this enzyme a potential target for future drug development against this pathogenic trypanosomatid. 相似文献
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Yael Garcia-Marchan Felipe Sojo Noraida Zerpa Ivan Galindo-Castro Gustavo Benaim 《Experimental parasitology》2009,123(4):326-333
We have cloned and expressed calmodulin (CaM) from Trypanosoma cruzi, for the first time, to obtain large amounts of protein. CaM is a very well conserved protein throughout evolution, sharing 100% amino acid sequence identity between different vertebrates and 99% between trypanosomatids. However, there is 89% amino acid sequence identity between T. cruzi and vertebrate CaMs. The results demonstrate significant differences between calmodulin from T. cruzi and mammals. First, a polyclonal antibody developed in an egg-yolk system to the T. cruzi CaM recognizes the autologous CaM but not the CaM from rat. Second, it undergoes a larger increase in the α-helix content upon binding with Ca2+, when compared to CaM from vertebrates. Finally, two classic CaM antagonists, calmidazolium and trifluoperazine, capable of inhibiting the action of CaM in mammals when assayed on the plasma membrane Ca2+ pump, showed a significant loss of activity when assayed upon stimulation with the T. cruzi CaM. 相似文献
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Maximiliano Juri Ayub James Atwood Rick Tarleton 《Biochemical and biophysical research communications》2009,382(1):30-867
Trypanosoma cruzi is a parasite responsible for Chagas disease. The identification of new targets for chemotherapy is a major challenge for the control of this disease. Several lines of evidences suggest that the translational system in trypanosomatids show important differences compared to other eukaryotes. However, there little is known information about this. We have performed a detailed data mining search for ribosomal protein genes in T. cruzi genome data base combined with mass spectrometry analysis of purified T. cruzi ribosomes. Our results show that T. cruzi ribosomal proteins have ∼50% sequence identity to yeast ones. Nevertheless, some parasite proteins are longer due to the presence of several N- or C-terminal extensions, which are exclusive of trypanosomatids. In particular, L19 and S21 show C-terminal extensions of 168 and 164 amino acids, respectively. In addition, we detected two 60S subunit proteins that had not been previously detected in the T. cruzi total proteome; namely, L22 and L42. 相似文献
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Gradia DF Rau K Umaki AC de Souza FS Probst CM Correa A Holetz FB Avila AR Krieger MA Goldenberg S Fragoso SP 《International journal for parasitology》2009,39(1):49-58
We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185 bp long and encodes a 44.3 kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes. 相似文献
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Ana María Cevallos Yayoi X. Segura-Kato Horacio Merchant-Larios Rebeca Manning-Cela Luis Alberto Hernández-Osorio Claudia Márquez-Dueñas Javier R. Ambrosio Olivia Reynoso-Ducoing Roberto Hernández 《Experimental parasitology》2011,(1):249-259
The expression and biological role of actin during the Trypanosoma cruzi life cycle remains largely unknown. Polyclonal antibodies against a recombinant T. cruzi actin protein were used to confirm its expression in epimastigotes, trypomastigotes, and amastigotes. Although the overall levels of expression were similar, clear differences in the subcellular distribution of actin among the developmental stages were identified. The existence of five actin variants in each developmental stage with distinct patterns of expression were uncovered by immunoblotting of protein extracts separated 2D-SDS gels. The isoelectric points of the actin variants in epimastigotes ranged from 4.45 to 4.9, whereas they ranged from 4.9 to 5.24 in trypomastigotes and amastigotes. To determine if the actin variants found could represent previously unidentified actins, we performed a genomic survey of the T. cruzi GeneDB database and found 12 independent loci encoding for a diverse group of actins and actin-like proteins that are conserved among trypanosomatids. 相似文献
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Danielle Kian César Armando Contreras Lancheros Igor Alexandre Campos Damiani Tamiris Zanforlin Olmos Fernandes Phileno Pinge-Filho Márcia Regina Machado dos Santos José Franco da Silveira Celso Vataru Nakamura Jo?o Santana da Silva Sueli Fumie Yamada-Ogatta Lucy Megumi Yamauchi 《The Korean journal of parasitology》2015,53(4):483-488
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Wanderley de Souza Celso Sant’Anna Narcisa L. Cunha-e-Silva 《Progress in histochemistry and cytochemistry》2009,44(2):67-124
Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoa it is also important for evading from host immune defenses and to support intense proliferation characteristic of some life cycle stages. Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosis in Trichomonas, Giardia, Entamoeba, Plasmodium, and trypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania. 相似文献
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Paulo H. M. Calixto Mainá Bitar Keila A. M. Ferreira Odonírio Abrahão Jr. Eliane Lages-Silva Glória R. Franco Luis E. Ramírez André L. Pedrosa 《Journal of molecular modeling》2013,19(8):3053-3064
Trypanosoma rangeli is a hemoflagellate parasite which is able to infect humans. Distinct from Trypanosoma cruzi, the causative agent of Chagas disease, T. rangeli is non-pathogenic to the vertebrate host. The manner by which the T. rangeli interacts with the host is still unknown, but it certainly depends on the surface molecules. Major surface proteins (MSP) are GPI-anchored, zinc-dependent metalloproteases present in the surface of all trypanosomatids studied so far, which are implicated as virulence factors in pathogenic trypanosomatids, such as Leishmania spp and T. cruzi. The aims of this work were to generate the complete sequence of a T. rangeli MSP (TrMSP) gene and to determine the 3D-structure of the predicted protein by homology modeling. The plasmid bearing a complete copy of a TrMSP gene was completely sequenced and the predicted protein was modeled using Modeller software. Results indicate that TrMSP open reading frame (ORF) codes for a predicted 588 amino acid protein and shows all elements required for its posttranslational processing. Multiple sequence alignment of TrMSP with other trypanosomatids’ MSPs showed an extensive conservation of the N-terminal and central regions and a more divergent C-terminal region. Leishmania major MSP (LmMSP), which had its crystal structure previously determined, has an overall 35 % identity with TrMSP. This identity allowed the comparative molecular modeling of TrMSP, which demonstrated a high degree of structural conservation between MSPs from other trypanosomatids (TrypMSPs). All modeled MSPs have a conserved folding pattern, apart from structural divergences in the C-domain and discrete differences of charge and topology in the catalytic cleft, and present the same geometry of the canonical HEXXH zinc-binding motif. The determination of surface charges of the molecules revealed that TrMSP is a predominantly positive protein, whereas LmMSP and Trypanosoma cruzi MSP (TcMSP) are negative proteins, suggesting that substrates recognized by TcMSP and LmMSP could not interact with TrMSP. Moreover, the comparison between TrMSP and TcMSP protein sequences has revealed 45 non-neutral amino acid substitutions, which can be further assessed through protein engineering. The characteristics of TrMSP could explain, at least in part, the lack of pathogenicity of T. rangeli to humans and point to the necessity of identifying the biological targets of this enzyme. Figure
In this study, we performed a comparative analysis of surface charges of Major Surface Proteases from Leishmania major (1LML), Chritidia fasciculata, Trypanosoma brucei, Trypanosoma cruzi and Trypanosoma rangeli. According to our results, TrMSP presents the distribution of surface charges predominantly positive, while the TrypMSPs have a profile electronegative. Solvent-accessible residues are colored according to their charge as described in the scale below each structure (varying from more negative, in red to more positive, in blue). The histogram indicates the amount of charged residues in each protein: H, K and R are basic (positive) residues, while D and E are acid (negative) residues. 相似文献
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Torriceli Souza Thé Renata Siqueira Portella Marcos Lázaro Guerreiro Sonia Gumes Andrade 《Memórias do Instituto Oswaldo Cruz》2013,108(6):691-698
Acute infection with Trypanosoma cruzi results in intense
myocarditis, which progresses to a chronic, asymptomatic indeterminate form. The
evolution toward this chronic cardiac form occurs in approximately 30% of all
cases of T. cruzi infection. Suppression of delayed type
hypersensitivity (DTH) has been proposed as a potential explanation of the
indeterminate form. We investigated the effect of cyclophosphamide (CYCL)
treatment on the regulatory mechanism of DTH and the participation of heart
interstitial dendritic cells (IDCs) in this process using BALB/c mice
chronically infected with T. cruzi. One group was treated with
CYCL (20 mg/kg body weight) for one month. A DTH skin test was performed by
intradermal injection of T. cruzi antigen (3 mg/mL) in the
hind-footpad and measured the skin thickness after 24 h, 48 h and 72 h. The skin
test revealed increased thickness in antigen-injected footpads, which was more
evident in the mice treated with CYCL than in those mice that did not receive
treatment. The thickened regions were characterised by perivascular infiltrates
and areas of necrosis. Intense lesions of the myocardium were present in
three/16 cases and included large areas of necrosis. Morphometric evaluation of
lymphocytes showed a predominance of TCD8 cells. Heart IDCs were immunolabelled
with specific antibodies (CD11b and CD11c) and T. cruzi
antigens were detected using a specific anti-T. cruzi antibody.
Identification of T. cruzi antigens, sequestered in these cells
using specific anti-T. cruzi antibodies was done, showing a
significant increase in the number of these cells in treated mice. These results
indicate that IDCs participate in the regulatory mechanisms of DTH response to
T. cruzi infection. 相似文献
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Bin Chou Kenji Hiromatsu Hajime Hisaeda Takashi Imai Keiji Tanaka 《Biochemical and biophysical research communications》2010,392(3):277-7419
Cytotoxic CD8+ 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+ 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+ T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4+ 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+ T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi. 相似文献
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Azael Salda?a José E Calzada Vanessa Pineda Milixa Perea Chystrie Rigg Kadir González Ana Maria Santamaria Nicole L Gottdenker Luis F Chaves 《Memórias do Instituto Oswaldo Cruz》2015,110(7):936-944
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 hodnius 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. 相似文献
19.
Adriana Botero Irit Kapeller Crystal Cooper Peta L. Clode Joseph Shlomai R.C Andrew Thompson 《International journal for parasitology》2018,48(9-10):691-700
Kinetoplast DNA (kDNA) is the mitochondrial genome of trypanosomatids. It consists of a few dozen maxicircles and several thousand minicircles, all catenated topologically to form a two-dimensional DNA network. Minicircles are heterogeneous in size and sequence among species. They present one or several conserved regions that contain three highly conserved sequence blocks. CSB-1 (10?bp sequence) and CSB-2 (8?bp sequence) present lower interspecies homology, while CSB-3 (12?bp sequence) or the Universal Minicircle Sequence is conserved within most trypanosomatids. The Universal Minicircle Sequence is located at the replication origin of the minicircles, and is the binding site for the UMS binding protein, a protein involved in trypanosomatid survival and virulence. Here, we describe the structure and organisation of the kDNA of Trypanosoma copemani, a parasite that has been shown to infect mammalian cells and has been associated with the drastic decline of the endangered Australian marsupial, the woylie (Bettongia penicillata). Deep genomic sequencing showed that T. copemani presents two classes of minicircles that share sequence identity and organisation in the conserved sequence blocks with those of Trypanosoma cruzi and Trypanosoma lewisi. A 19,257?bp partial region of the maxicircle of T. copemani that contained the entire coding region was obtained. Comparative analysis of the T. copemani entire maxicircle coding region with the coding regions of T. cruzi and T. lewisi showed they share 71.05% and 71.28% identity, respectively. The shared features in the maxicircle/minicircle organisation and sequence between T. copemani and T. cruzi/T. lewisi suggest similarities in their process of kDNA replication, and are of significance in understanding the evolution of Australian trypanosomes. 相似文献
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Nineteen Trypanosoma cruzi stocks, most of them of wild origin, and four Trypanosoma rangeli stocks from Colombia were analysed by molecular karyotype analysis with cloned DNA cruzipain as the probe. Another 27 cloned stocks of T. cruzi from different geographic areas of South America were used as reference for T. cruzi lineages. Phenetic analysis of chromosome size polymorphism demonstrated a great variability of Colombian T. cruzi stocks, suggesting that most belong to lineage I, although two of them belong to lineage II. The 2 lineage II T. cruzi, 17 T. cruzi lineage I, and 3 T. rangeli stocks from Colombia were studied further by Southern blot analysis with a panel of kinetoplast DNA minicircle probes. Hybridisation results indicate that the two T. cruzi II stocks are genetically distant from each other and from T. cruzi lineages IIb, IId, and IIe from Chile. Finally, T. cruzi minicircle probes do not cross-hybridise in any stringency condition tested with T. rangeli minicircles, a clear indication that these parasites can be easily distinguished by this method. 相似文献