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

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.  Methodology/Principal Findings: The T. rangeli haploid genome is ,24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heatshock proteins.  Conclusions/Significance: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets

GST-δ-AmastinH specifically binds to HeLa cells in a dose-dependent saturable manner.

<p><b>A. SDS-PAGE showing the purity of recombinant GST-δ-amastinH:</b> 1; Molecular weigh markes, in kiloDaltons; 2: GST alone; 3: purified recombinant GST-δ-AmastinH; 4 total extract of induced <i>E. coli</i>. <b>B: Recombinant GST-δ-AmastinH binds to HeLa cells</b>. Increasing concentrations of GST-AmastinH or GST (negative control) were added to wells in ELISA plates containing adhered and fixed HeLa cells. After washing, cells were sequentially incubated with anti-GST antibodies and anti-rabbit IgG conjugated to peroxidase. The bound enzyme was revealed by <i>o-</i>phenylenediamine as a substrate. Representative results of two independent experiments are shown. *p<0.05.</p

Engineering a single-chain antibody against Trypanosoma cruzi metacyclic trypomastigotes to block cell invasion

Trypanosoma cruzi is a flagellate protozoan pathogen that causes Chagas disease. Currently there is no preventive treatment and the efficiency of the two drugs available is limited to the acute phase. Therefore, there is an unmet need for innovative tools to block transmission in endemic areas. In this study, we engineered a novel recombinant molecule able to adhere to the T. cruzi surface, termed scFv-10D8, that consists of a single-chain variable fragment (scFv) derived from mAb-10D8 that targets gp35/50. The synthetic gene encoding scFv-10D8 was cloned and fused to a 6×His tag and expressed in a prokaryotic expression system. Total periplasmic or 6xHis tag affinity-purified fractions of scFv-10D8 retained the capacity to bind to gp35/50, as shown by Western blot analyses. Pre-incubation of metacyclic trypomastigotes with scFv-10D8 showed a remarkable reduction in cell invasion capacity. Our results suggest that scFv-10D8 can be used in a paratransgenic approach to target parasites in insect vectors, avoiding dissemination of infective forms. Such advances in the development of this functional molecule will surely prompt the improvement of alternative strategies to control Chagas disease by targeting mammalian host stages

Delta-amastin interferes with EA-HeLa cell interaction. A: GST-AmastinH inhibited host cell invasion by <i>T. cruzi</i> EAs.

<p>Prior to invasion, HeLa cells were treated for 1 h with 5 µg/ml of GST (white column) or GST-δ-AmastinH (dark column). The parasites were then added to HeLa cells and the invasion proceeded for 2 h. The number of internalized parasites was counted in a total of at least 300 cells. <b>B: EAs (G strain) overexpressing amastin showed lower infectivity toward HeLa cells.</b> EAs of the G strain expressing high levels of δ-amastin (black column) showed a significant decrease in cell invasion when compared to wild type parasites (clear column) or parasites expressing only GFP (gray column). The invasion proceeded for 2 h. The values are shown as means ± standard deviations of two independent experiments performed in duplicate. *p<0.05.</p

Delta-amastin is more abundant in less infective <i>T. cruzi</i> extracellular amastigotes. A.

<p><b>mRNAs in EAs of the G strain are less abundant when compared to EAs of the CL-Brener clone </b><b><i>T. cruzi</i></b><b>.</b> Transcript levels were determined by quantitative real-time PCR using SYBR® Green I chemistry. qRT-PCR was performed on RNA samples from EAs of G and CL strains. The comparative mRNA levels were determined after normalization with GAPDH amplicons. Standard deviations are derived from three replicates. *p<0.05 <b>B. mRNA corresponding to amastin is preferentially expressed in amastigotes from CL Brener clone.</b> Northern blot analyses of total RNA (10 µg) from <i>T. cruzi</i> epimastigotes (E), trypomastigotes (T) and amastigotes (A) from CL-Brener clone or the G <i>T. cruzi</i> strain was submitted to electrophoresis and blotted on nylon membranes by standard procedures. Each blot was hybridized with amastin probe previously labeled with [α-^32P]-dCTP. To determine equal loading of RNA, the 1.2% agarose/MOPS/formaldehyde gel was stained with ethidium bromide (bottom panel).</p

Engineering a single-chain antibody against Trypanosoma cruzi metacyclic trypomastigotes to block cell invasion.

Trypanosoma cruzi is a flagellate protozoan pathogen that causes Chagas disease. Currently there is no preventive treatment and the efficiency of the two drugs available is limited to the acute phase. Therefore, there is an unmet need for innovative tools to block transmission in endemic areas. In this study, we engineered a novel recombinant molecule able to adhere to the T. cruzi surface, termed scFv-10D8, that consists of a single-chain variable fragment (scFv) derived from mAb-10D8 that targets gp35/50. The synthetic gene encoding scFv-10D8 was cloned and fused to a 6×His tag and expressed in a prokaryotic expression system. Total periplasmic or 6xHis tag affinity-purified fractions of scFv-10D8 retained the capacity to bind to gp35/50, as shown by Western blot analyses. Pre-incubation of metacyclic trypomastigotes with scFv-10D8 showed a remarkable reduction in cell invasion capacity. Our results suggest that scFv-10D8 can be used in a paratransgenic approach to target parasites in insect vectors, avoiding dissemination of infective forms. Such advances in the development of this functional molecule will surely prompt the improvement of alternative strategies to control Chagas disease by targeting mammalian host stages

Schematic proposed model for the role of δ-amastin in <i>T. cruzi</i> virulence.

<p>The model indicates two distinct parasites, EAs expressing high levels of amastin was represented by dotted line membrane of amastigotes (left cell side) whereas the low amastin expression was represented by spaced dots in the amastigotes membrane (right cell side), during EA invasion and differentiation processes of <i>T. cruzi</i> virulence in a hypothetical host cell. <b>1.</b> EAs expressing more amastin (left) show a lower infectivity rate when compared with parasites expressing lower levels of the protein. <b>2.</b> High levels of amastin accelerate the transformation of amastigotes into TCTs (left).</p