Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Contreras, Víctor; Navarro, Marian; Lima, Ana Rita de; Arteaga, Rosa; Duran, Francy; Askue, José; Franco, Yunaimy

doi:10.1590/s0074-02762002000800025

Cited by 21 publications

(21 citation statements)

References 25 publications

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“…Cultures were prolonged for 2-4 days until more than 80% of parasites were transformed. The amastigote forms were obtained by culturing metacyclic forms at 37 ºC for 3-4 days under chemically defined conditions (MEM-TAU3AAG medium) as previously reported (Contreras et al, 2002). Total RNA was extracted from cell pellets with the Trizol reagent (Invitrogen) and processed according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Cloning, characterization and subcellular localization of a Trypanosoma cruzi argonaute protein defining a new subfamily distinctive of trypanosomatids

Silva

Tosar

Frugier

et al. 2010

Gene

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Section: Methodsmentioning

confidence: 99%

Cloning, characterization and subcellular localization of a Trypanosoma cruzi argonaute protein defining a new subfamily distinctive of trypanosomatids

Silva

Tosar

Frugier

et al. 2010

Gene

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“…Epimastigote forms were maintained at 28°C in liver infusion tryptose medium supplemented with 10% heat-inactivated fetal bovine serum. Metacyclic trypomastigotes and extracellular amastigotes were prepared as described by Contreras et al (19,20).…”

Section: Methodsmentioning

confidence: 99%

Functional Genomic Characterization of mRNAs Associated with TcPUF6, a Pumilio-like Protein from Trypanosoma cruzi

Dallagiovanna

Correa

Probst

et al. 2008

Journal of Biological Chemistry

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Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. The PUF (Pumilio/ FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypanosomatids. We used a ribonomic approach to identify the putative target mRNAs associated with TcPUF6, a member of the T. cruzi PUF family. TcPUF6 is expressed in discrete sites in the cytoplasm at various stages of the parasite life cycle and is not associated with the translation machinery. The overexpression of a tandem affinity purification-tagged TcPUF6 protein allowed the identification of associated mRNAs by affinity purification assays and microarray hybridization yielding nine putative target mRNAs. Whole expression analysis of transfected parasites showed that the mRNAs associated with TcPUF6 were down-regulated in populations overexpressing TcPUF6. The association of TcPUF6 with the TcDhh1 helicase in vivo and the cellular co-localization of these proteins in epimastigote forms suggest that TcPUF6 promotes degradation of its associated mRNAs through interaction with RNA degradation complexes. Analysis of the mRNA levels of the putative TcPUF6-regulated genes during the parasite life cycle showed that their transcripts were up-regulated in metacyclic trypomastigotes. In these infective forms no co-localization between TcPUF6 and TcDhh1 was observed. Our results suggest that TcPUF6 regulates the half-lives of its associated transcripts via differential association with mRNA degradation complexes throughout its life cycle.

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“…For instance, Contreras and collaborators developed a protocol in which metacyclic trypomastigotes can be differentiated to amastigotes by means of incubation in MEMTAU media, a combination of MEM and TAU3AAA medium, enriched with FBS at 37°C (17). In fact, the production of axenic amastigotes by means of incubation of tissue-culture-derived trypomastigotes in acidic media has been widely employed since the first report by Kanbara and collaborators (18).…”

mentioning

confidence: 99%

Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis

Queiroz

Charneau

Mandacaru

et al. 2014

Molecular & Cellular Proteomics

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Chagas disease is a tropical neglected disease endemic in Latin America caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote, and amastigote. The differentiation from infective trypomastigotes into replicative amastigotes, called amastigogenesis, takes place in vivo inside mammalian host cells after a period of incubation in an acidic phagolysosome. This differentiation process can be mimicked in vitro by incubating tissue-culture-derived trypomastigotes in acidic DMEM. Here we used this well-established differentiation protocol to perform a comprehensive quantitative proteomic and phosphoproteomic analysis of T. cruzi amastigogenesis. Samples from fully differentiated forms and two biologically relevant intermediate time points were Lys-C/trypsin digested, iTRAQ-labeled, and multiplexed. Subsequently, phosphopeptides were enriched using a TiO 2 matrix. Non-phosphorylated peptides were fractionated via hydrophilic interaction liquid chromatography prior to LC-MS/MS analysis. LC-MS/MS and bioinformatics procedures were used for protein and phosphopeptide quantitation, identification, and phosphorylation site assignment. We were able to identify regulated proteins and pathways involved in coordinating amastigogenesis. We also observed that a significant proportion of the regulated proteins were membrane proteins. Modulated phosphorylation events coordinated by protein kinases and phosphatases that are part of the signaling cascade induced by incubation in acidic medium were also evinced. To our knowledge, this work is the most comprehensive quantitative proteomics study of T. cruzi amastigogenesis, and these data will serve as a trustworthy basis for future studies, and possibly for new potential drug targets. Molecular & Cellular Proteomics 13:

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Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Cited by 21 publications

References 25 publications

Cloning, characterization and subcellular localization of a Trypanosoma cruzi argonaute protein defining a new subfamily distinctive of trypanosomatids

Cloning, characterization and subcellular localization of a Trypanosoma cruzi argonaute protein defining a new subfamily distinctive of trypanosomatids

Functional Genomic Characterization of mRNAs Associated with TcPUF6, a Pumilio-like Protein from Trypanosoma cruzi

Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis

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