An Essential Nuclear Protein in Trypanosomes Is a Component of mRNA Transcription/Export Pathway

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

Knockout confirmation for Hurries: rapid genotype identification of Trypanosoma cruzi transfectants by polymerase chain reaction directly from liquid culture

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

A novel nanoluciferase-based system to monitor <i>Trypanosoma cruzi</i> infection in mice by bioluminescence imaging

<div><p>Chagas disease, caused by the intracellular protozoan <i>Trypanosoma cruzi</i>, affects 8–10 million people worldwide and represents a major public health challenge. There is no effective treatment or vaccine to control the disease that is characterized by a mild acute phase followed by a chronic life-long infection. Approximately 30% of chronically infected individuals develop cardiac and/or digestive pathologies. <i>T</i>. <i>cruzi</i> can invade a wide variety of nucleated cells, but only persists at specific tissues in the host. However, the mechanisms that determine tissue tropism and the progression of the infection have not been fully described. Identification of infection niches in animal models has been difficult due to the limited quantity of parasite-infected cells and their focal distribution in tissues during the chronic phase. To better understand the course of chronic infections and parasite dissemination, we developed a bioluminescence imaging system based on the use of transgenic <i>T</i>. <i>cruzi</i> Colombiana strain parasites expressing nanoluciferase. Swiss Webster mice were infected with luminescent trypomastigotes and monitored for 126 days. Whole animal <i>in vivo</i> imaging showed parasites predominantly distributed in the abdominal cavity and surrounding areas throughout the infection. Bioluminescence signal reached a peak between 14 to 21 days post infection (dpi) and decreased progressively over time. Total animal luminescence could still be measured 126 dpi while parasites remained undetectable in blood by microscopy in most animals. <i>Ex vivo</i> imaging of specific tissues and organs dissected post-mortem at 126 dpi revealed a widespread parasite distribution in the skeletal muscle, heart, intestines and mesenteric fat. Parasites were also detected in lungs and liver. This noninvasive imaging model represents a novel tool to study host-parasite interactions and to identify parasite reservoirs of chronic Chagas Disease.</p></div

Quantification of parasite loads in tissues and organs of chronically TcCOL-NLuc-infected mice at 126 dpi by bioluminescence and qPCR.

<p>(A) Luminescence signal quantified for each individual organ/tissue shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195879#pone.0195879.g005" target="_blank">Fig 5</a> and expressed as radiance (photons/second/cm²/sr). Data represent fold-changes in bioluminescence intensity for organs from infected mice compared to the matching organ from the non-infected control mouse. The detection threshold (grey dashed line) was estimated using the fold-change in radiance for an empty region determined in images taken for the infected mice compared with a matching empty region in images obtained for the infected mouse control. (B) qPCR evaluation of tissue-specific parasite loads. Reactions were conducted using 50 ng of tissue DNA isolated from four TcCOL-NLuc- infected mice and one non-infected mouse. <i>T</i>. <i>cruzi</i>-specific threshold cycle (Ct) values were converted to inferred numbers of parasite equivalents by reference to a standard curve with a range of 1 x10⁴ to 1 x10^-2 parasite equivalents/50 ng of tissue DNA. The IL12 p40 genomic region was used as endogenous control to verify that equal amounts of purified DNA were present in all PCR amplifications. Grey dashed lines indicate the limit of detection (1 x10^-2 parasite equivalents). Data represent the median with interquartile range. Statistical differences among tissues were calculated using one-way ANOVA analysis in GraphPad Prism. *: <i>p</i> < 0.05 indicates significant difference with the heart; ns: no significant difference with the heart.</p

Clathrin expression in Trypanosoma cruzi

Submitted by Luciane Willcox ([email protected]) on 2016-10-03T15:03:33Z No. of bitstreams: 1 Clathrin expression in Trypanosoma cruzi.pdf: 1159100 bytes, checksum: c4095f39954ecf54fd6d8a1509b2d0da (MD5)Approved for entry into archive by Luciane Willcox ([email protected]) on 2016-10-03T15:12:38Z (GMT) No. of bitstreams: 1 Clathrin expression in Trypanosoma cruzi.pdf: 1159100 bytes, checksum: c4095f39954ecf54fd6d8a1509b2d0da (MD5)Made available in DSpace on 2016-10-03T15:12:38Z (GMT). No. of bitstreams: 1 Clathrin expression in Trypanosoma cruzi.pdf: 1159100 bytes, checksum: c4095f39954ecf54fd6d8a1509b2d0da (MD5) Previous issue date: 2014-06-19CAPES, CNPq, FiocruzFundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil. / Universidade Estadual de Ponta Grossa. Departamento de Biologia Geral. Ponta Grossa, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Molecular de Tripanosomas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Celular. Curitiba, PR, Brasil.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. Results: 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. Conclusion: 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

Monitoring the course of TcCOL-NLuc chronic infection in Swiss Webster mice by live bioluminescence imaging.

<p>(A) Representative ventral view images of mouse #1 infected with TcCOL-NLuc tissue culture-derived trypomastigotes (5 x10³ parasites) taken at the indicated time points. NI: non-infected control mouse. Log ₁₀ heat-map scale represents bioluminescence intensity (blue: low; red: high). (B) Total abdominal bioluminescence (total flux) for seven individual mice and their mean (red line) measured in the regions of interest indicated in (A) and over the course of infection. The grey line indicates the detection threshold determined as the mean (solid line) and mean ± 2 s.d. (dashed lines) of background luminescence of the control uninfected mouse. (C) Blood parasitemia (trypomastigotes/ml) determined by microscopy over time for each individual mouse and mean (blue line). Grey line indicates the limit of detection (1.58 x10⁴ parasites/ml blood).</p

<i>Ex vivo</i> bioluminescence imaging of selected organs and tissue samples imaged at 126 dpi with TcCOL-NLuc.

<p>Four randomly selected TcCOL-NLuc-infected mice (M1, M2, M4, and M7) and one non-infected mouse control (NI) were sacrificed 126 dpi. Organs and tissues were excised and transferred to a 24-well plate or Petri dishes. After rinsing with PBS, samples were soaked in a furimazine solution for 5 minutes. Images were acquired using an exposure time from 30 seconds to 2 minutes. Log ₁₀ heat-map scales represent bioluminescence intensity (blue: low; red: high). MF: mesenteric fat.</p

Expression of nanoluciferase in transgenic TcCOL-NLuc parasites.

<p>(A) Luciferase activity was measured in 5 x10³ live TcCOL-NLuc and TcCOL-wt cells using the Nano-Glo^® Live Cell Assay System. (B) Luciferase activity was measured in lysates of 10⁵ TcCOL-NLuc and TcCOL-wt cells using the Nano-Glo^® Luciferase Assay System. Left panel: NLucexpression evaluated in TcCOL-NLuc epimastigotes after passage 1 (P1) and passage 22 (P22) in LIT media containing G418. Right panel: NLuc expression evaluated in tissue culture-derived TcCOL-NLuc trypomastigotes after passage 1 (P1) and passage 5 (P5) in LLC-MK2 cells. (C) Luciferase activity in amastigotes was evaluated in LLC-MK2 cells infected with 5 x10⁴ TcCOL-NLuc or TcCOL-wt trypomastigotes. Four days post-infection, luminescence was assessed in cell lysates using the Nano-Glo^® Luciferase Assay System. (D) Nanoluciferase protein expression in intracellular amastigotes was evaluated in methanol-fixed LLC-MK2 cells infected with 5 x10⁴ TcCOL-NLuc (top panels) or TcCOL-wt (bottom panels) trypomastigotes by immunostaining with rabbit anti-NLuc antibodies (Anti-NLuc). DAPI indicates DNA staining. Values in (A), (B) and (C) represent means ± s.d. of three replicate wells. Statistical differences between groups were calculated using an unpaired t-test with a 95% confidence interval. <i>p</i>-values are indicated. Scale bar: 50 μm.</p