[Show abstract][Hide abstract] ABSTRACT: Background
Clathrin-mediated vesicular trafficking, the mechanism by which proteins and lipids are transported between membrane-bound organelles, accounts for a large proportion of import from the plasma membrane (endocytosis) and transport from the trans-Golgi network towards the endosomal system. Clathrin-mediated events are still poorly understood in the protozoan Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. In this study, clathrin heavy (TcCHC) and light (TcCLC) chain gene expression and protein localization were investigated in different developmental forms of T. cruzi (epimastigotes, trypomastigotes and amastigotes), using both polyclonal and monoclonal antibodies raised against T. cruzi recombinant proteins.
Analysis by confocal microscopy revealed an accumulation of TcCHC and TcCLC at the cell anterior, where the flagellar pocket and Golgi complex are located. TcCLC partially colocalized with the Golgi marker TcRAB7-GFP and with ingested albumin, but did not colocalize with transferrin, a protein mostly ingested via uncoated vesicles at the cytostome/cytopharynx complex.
Clathrin heavy and light chains are expressed in T. cruzi. Both proteins typically localize anterior to the kinetoplast, at the flagellar pocket and Golgi complex region. Our data also indicate that in T. cruzi epimastigotes clathrin-mediated endocytosis of albumin occurs at the flagellar pocket, while clathrin-independent endocytosis of transferrin occurs at the cytostome/cytopharynx complex.
[Show abstract][Hide abstract] ABSTRACT: Gene knockout is a widely used approach to evaluate loss-of-function phenotypes and it can be facilitated by the incorporation of a DNA cassette having a drug-selectable marker. Confirmation of the correct knockout cassette insertion is an important step in gene removal validation and has generally been performed by polymerase chain reaction (PCR) assays following a time-consuming DNA extraction step. Here, we show a rapid procedure for the identification of Trypanosoma cruzi transfectants by PCR directly from liquid culture - without prior DNA extraction. This simple approach enabled us to generate PCR amplifications from different cultures varying from 106-108 cells/mL. We also show that it is possible to combine different primer pairs in a multiplex detection reaction and even to achieve knockout confirmation with an extremely simple interpretation of a real-time PCR result. Using the "culture PCR" approach, we show for the first time that we can assess different DNA sequence combinations by PCR directly from liquid culture, saving time in several tasks for T. cruzi genotype interrogation.
Preview · Article · Jun 2014 · Memórias do Instituto Oswaldo Cruz
[Show abstract][Hide abstract] ABSTRACT: Heteroxenic and monoxenic trypanosomatids were screened for the presence of actin using a mouse polyclonal antibody produced against the entire sequence of the Trypanosoma cruzi actin gene, encoding a 41.9 kDa protein. Western blot analysis showed that this antibody reacted with a polypeptide of approximately 42 kDa in the whole-cell lysates of parasites targeting mammals (T. cruzi, Trypanosoma brucei and Leishmania major), insects (Angomonas deanei, Crithidia fasciculata, Herpetomonas samuelpessoai and Strigomonas culicis) and plants (Phytomonas serpens). A single polypeptide of approximately 42 kDa was detected in the whole-cell lysates of T. cruzi cultured epimastigotes, metacyclic trypomastigotes and amastigotes at similar protein expression levels. Confocal microscopy showed that actin was expressed throughout the cytoplasm of all the tested trypanosomatids. These data demonstrate that actin expression is widespread in trypanosomatids.
Full-text · Article · Aug 2013 · Memórias do Instituto Oswaldo Cruz
[Show abstract][Hide abstract] ABSTRACT: Trypanosoma cruzi is the etiological agent of Chagas disease, an illness that affects about 10 million people, mostly in South America, for which there is no effective treatment or vaccine. In this context, transgenic parasites expressing reporter genes are interesting tools for investigating parasite biology and host-parasite interactions, with a view to developing new strategies for disease prevention and treatment. We describe here the construction of a stably transfected fluorescent T. cruzi clone in which the GFP gene is integrated into the chromosome carrying the ribosomal cistron in T. cruzi Dm28c. This fluorescent T. cruzi produces detectable amounts of GFP only at replicative stages (epimastigote and amastigote), consistent with the larger amounts of GFP mRNA detected in these forms than in the non replicative trypomastigote stages. The fluorescence signal was also strongly correlated with the total number of parasites in T. cruzi cultures, providing a simple and rapid means of determining the growth inhibitory dose of anti-T.cruzi drugs in epimastigotes, by fluorometric microplate screening, and in amastigotes, by the flow cytometric quantification of T. cruzi-infected Vero cells. This fluorescent T. cruzi clone is, thus, an interesting tool for unbiased detection of the proliferating stages of the parasite, with multiple applications in the genetic analysis of T. cruzi, including analyses of host-parasite interactions, gene expression regulation and drug development.
[Show abstract][Hide abstract] ABSTRACT: Protein tyrosine phosphatases (PTPs) play an essential role in the regulation of cell differentiation in pathogenic trypanosomatids. In this study, we describe a PTP expressed by the non-pathogenic protozoan Trypanosoma rangeli (TrPTP2). The gene for this PTP is orthologous to the T. brucei TbPTP1 and Trypanosoma cruzi (TcPTP2) genes. Cloning and expression of the TrPTP2 and TcPTP2 proteins allowed anti-PTP2 monoclonal antibodies to be generated in BALB/c mice. When expressed by T. rangeli epimastigotes and trypomastigotes, native TrPTP2 is detected as a ~65 kDa protein associated with the parasite's flagellum. Given that the flagellum is an important structure for cell differentiation in trypanosomatids, the presence of a protein responsible for tyrosine dephosphorylation in the T. rangeli flagellum could represent an interesting mechanism of regulation in this structure.
Full-text · Article · Sep 2012 · Memórias do Instituto Oswaldo Cruz
[Show abstract][Hide abstract] ABSTRACT: Trypanosomes are parasitic protozoa in which gene expression is primarily controlled through the regulation of mRNA stability and translation. This post-transcriptional control is mediated by various families of RNA-binding proteins, including those with zinc finger CCCH motifs. CCCH zinc finger proteins have been shown to be essential to differentiation events in trypanosomatid parasites. Here, we functionally characterise TcZFP2 as a predicted post-transcriptional regulator of differentiation in Trypanosoma cruzi. This protein was detected in cell culture-derived amastigotes and trypomastigotes, but it was present in smaller amounts in metacyclic trypomastigote forms of T. cruzi. We use an optimised recombinant RNA immunopreciptation followed by microarray analysis assay to identify TcZFP2 target mRNAs. We further demonstrate that TcZFP2 binds an A-rich sequence in which the adenosine residue repeats are essential for high-affinity recognition. An analysis of the expression profiles of the genes encoding the TcZFP2-associated mRNAs throughout the parasite life cycle by microarray hybridisation showed that most of the associated mRNAs were upregulated in the metacyclic trypomastigote forms, also suggesting a role for TcZFP2 in metacyclic trypomastigote differentiation. Knockdown of the orthologous Trypanosoma brucei protein levels showed ZFP2 to be a positive regulator of specific target mRNA abundance.
Full-text · Article · Sep 2012 · Memórias do Instituto Oswaldo Cruz
[Show abstract][Hide abstract] ABSTRACT: Proteasomes are large protein complexes, whose main function is to degrade unnecessary or damaged proteins. The inhibition of proteasome activity in Trypanosoma cruzi blocks parasite replication and cellular differentiation. We demonstrate that proteasome-dependent proteolysis occurs during the cellular differentiation of T. cruzi from replicative non-infectious epimastigotes to non-replicative and infectious trypomastigotes (metacyclogenesis). No peaks of ubiquitin-mediated degradation were observed and the profile of ubiquitinated conjugates was similar at all stages of differentiation. However, an analysis of carbonylated proteins showed significant variation in oxidized protein levels at the various stages of differentiation and the proteasome inhibition also increased oxidized protein levels. Our data suggest that different proteasome complexes coexist during metacyclogenesis. The 20S proteasome may be free or linked to regulatory particles (PA700, PA26 and PA200), at specific cell sites and the coordinated action of these complexes would make it possible for proteolysis of ubiquitin-tagged proteins and oxidized proteins, to coexist in the cell.
[Show abstract][Hide abstract] ABSTRACT: Peptidase activities were determined by fluorimetric quantification of the hydrolysis of various specific fluorogenic substrates: chymotrypsin-like (A), trypsin-like (B), and caspase-like (C) proteases. Black bars represent the activity and white bars represent the inhibition of the activity in each group of parasites analyzed in three independent experiments: three-day-old cultured epimastigotes (Epi 3d), five-day-old cultured epimastigotes (Epi 5d), five-day-old cultured epimastigotes under nutritional stress (Epi ST), adhered epimastigotes after 12 h of differentiation (Adh 12 h), adhered epimastigotes after 24 h of differentiation (Adh 24 h) and metacyclic trypomastigotes (MT). Results are shown as means of three independent experiments ± SD.
[Show abstract][Hide abstract] ABSTRACT: FISH of poliA+mRNA in T. cruzi. Localization of mRNAs by FISH using DIG-labeled oligo d(T)30 in wild-type (WT) and single knockout of TcSub2 (KO) epimastigotes. Cells were stained with DAPI to locate the nuclear and kinetoplast DNA. For control, cells were treated with 100 µg ml−1 of RNAse A before hybridization. Bars = 1 µm.
[Show abstract][Hide abstract] ABSTRACT: Analysis of phosphatidylserine exposure after TbSub2 knockdown by RNAi. The density plots show the non-induced (NI, top row) or induced (I, bottom row) cells co-stained with Annexin-V-FITC and Propidium Iodide after 48 to 96 hours (indicated above the plots) of RNAi induction. The values inside the density plots represent the percentage of cells inside each region.
[Show abstract][Hide abstract] ABSTRACT: Nuclear colocalization of TcSub2 with telomere repeats. FISH of telomeric repeat regions followed by immunofluorescence of TcSub2 was perfomed in epimastigote forms. Telomeric repeats (red); TcSub2 (green). Cells were stained with DAPI to locate the nuclear and kinetoplast DNA. Bars = 1 µm.
[Show abstract][Hide abstract] ABSTRACT: Flow cytometry analysis of cell viability and cell cycle after TbSub2 knockdown by RNAi. (A) Overlay histograms of induced (I) and non-induced (NI) cells stained with Propidium Iodide after 24 to 96 hours (h, indicated inside the graphs) of RNAi induction; the percentage of PI positive cells (“Dead” region) is indicated. (B) Overlay histograms of I and NI cells stained with rhodamine 123 for mitochondrial membrane potential analysis. (C) Analysis after RNAi induction of DNA content by staining of permeabilized cells with propidium iodide; note the gradual increase of cells in G2/S phase after 48 hours of RNAi induction. (D) Cell cycle analysis by Dean-Jett-Fox algorithm of FlowJo software; data of non-induced (left histogram) and induced (right histogram) cells after 48 hours of RNAi induction are shown; the percentage of cells in each phase of cell cycle is indicated inside the graphs.
[Show abstract][Hide abstract] ABSTRACT: In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX) multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei -except the fibrillar center of nucleolus- and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II), but not RNA polymerase I (RNA pol I) or Spliced Leader (SL) transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and decrease of translation levels, reinforcing that Trypanosoma-Sub2 (Tryp-Sub2) is a component of mRNA transcription/export pathway in trypanosomes.
[Show abstract][Hide abstract] ABSTRACT: Cell proliferation and viability analysis. (A) Growth curves of induced (I) and non-induced (NI) cells by direct counting in a Neubauer chamber; each point represents the mean and standard deviations of triplicate experiments. Western blot of TbSub2 during RNAi induction. Antiserum against GAPDH was used as a protein-loading control. (B) Percentage of cells in G2 phase of cell cycle calculated by flow cytometry (details in methods); (C) Percentage of non-viable cells (dead cells) calculated by flow cytometry using propidium iodide staining; (D) Comparison of R123 fluorescence intensity between induced to non induced cells plotted as fold change graph using geometric mean of FL1-H detector intensity. For flow cytometry data (B–D), each experimental point represents the average and respective standard deviation of duplicate experiments.
[Show abstract][Hide abstract] ABSTRACT: FISH of poliA+mRNA in T. brucei. Localization of mRNAs by FISH using DIG-labeled oligo d(T)30 after 48 and 72 hours of RNAi induction. Cells were stained with DAPI to locate the nuclear and kinetoplast in non-induced cells (NI) and induced cells (I). DNA. For control, cells were treated with 100 µg ml−1 RNAse A before hybridization. Bars = 1 µm.
[Show abstract][Hide abstract] ABSTRACT: (A, B) Nuclear colocalization of TcSub2 with transcription sites in different focal planes. Analysis of colocalization of TcSub2 (red) with transcription sites (BrRNA, green) without α-amanitin. Bars = 1 µm.
[Show abstract][Hide abstract] ABSTRACT: The nuclear lamina is a structure that lines the inner nuclear membrane. In metazoans, lamins are the primary structural components of the nuclear lamina and are involved in several processes. Eukaryotes that lack lamins have distinct proteins with homologous functions. Some years ago, a coiled-coil protein in Trypanosoma brucei, NUP-1, was identified as the major filamentous component of its nuclear lamina. However, its precise role has not been determined. We characterized a homologous protein in Trypanosoma cruzi, TcNUP-1, and identified its in vivo DNA binding sites using a chromatin immunoprecipitation assay. We demonstrate for the first time that TcNUP-1 associates with chromosomal regions containing large non-tandem arrays of genes encoding surface proteins. We therefore suggest that TcNUP-1 is a structural protein that plays an essential role in nuclear organization by anchoring T. cruzi chromosomes to the nuclear envelope.
Full-text · Article · Jan 2011 · Experimental Parasitology
[Show abstract][Hide abstract] ABSTRACT: Two species of the genus Trypanosoma infective to humans have been extensively studied at a cell and molecular level, but study of the third, Trypanosoma rangeli, remains in relative infancy. T. rangeli is non-pathogenic, but is frequently mistaken for the related Chagas disease agent Trypanosoma cruzi with which it shares vectors, hosts, significant antigenicity and a sympatric distribution over a wide geographical area. In this study, we present the T. rangeli gene expression profile as determined by the generation of ESTs (Expressed Sequence Tags) and ORESTES (Open Reading Frame ESTs). A total of 4208 unique high quality sequences were analyzed, composed from epimastigote and trypomastigote forms of SC-58 and Choachí strains, representing the two major phylogenetic lineages of this species. Comparative analyses with T. cruzi and other parasitic kinetoplastid species allowed the assignment of putative biological functions to most of the sequences generated and the establishment of an annotated T. rangeli gene expression database. Even though T. rangeli is apathogenic to mammals, genes associated with virulence in other pathogenic kinetoplastids were found. Transposable elements and genes associated mitochondrial gene expression, specifically RNA editing components, are also described for the first time. Our studies confirm the close phylogenetic relationship between T. cruzi and T. rangeli and enable us to make an estimate for the size of the T. rangeli genome repertoire ( approximately 8500 genes).
Full-text · Article · Nov 2010 · Molecular and Biochemical Parasitology
[Show abstract][Hide abstract] ABSTRACT: The sequencing of the complete genome of Anaplasma marginale has enabled the identification of several genes that encode membrane proteins, thereby increasing the chances of identifying candidate immunogens. Little is known regarding the genetic variability of genes that encode membrane proteins in A. marginale isolates. The aim of the present study was to determine the degree of conservation of the predicted amino acid sequences of OMP1, OMP4, OMP5, OMP7, OMP8, OMP10, OMP14, OMP15, SODb, OPAG1, OPAG3, VirB3, VirB9-1, PepA, EF-Tu and AM854 proteins in a Brazilian isolate of A. marginale compared to other isolates. Hence, primers were used to amplify these genes: omp1, omp4, omp5, omp7, omp8, omp10, omp14, omp15, sodb, opag1, opag3, virb3, VirB9-1, pepA, ef-tu and am854. After polimerase chain reaction amplification, the products were cloned and sequenced using the Sanger method and the predicted amino acid sequence were multi-aligned using the CLUSTALW and MEGA 4 programs, comparing the predicted sequences between the Brazilian, Saint Maries, Florida and A. marginale centrale isolates. With the exception of outer membrane protein (OMP) 7, all proteins exhibited 92-100% homology to the other A. marginale isolates. However, only OMP1, OMP5, EF-Tu, VirB3, SODb and VirB9-1 were selected as potential immunogens capable of promoting cross-protection between isolates due to the high degree of homology (over 72%) also found with A. (centrale) marginale.
Full-text · Article · Nov 2010 · Memórias do Instituto Oswaldo Cruz