Selective Blockade of Trypanosomatid Protein Synthesis by a Recombinant Antibody Anti-Trypanosoma cruzi P2β Protein

The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2β protein (TcP2β) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope

Depletion of Trypanosome CTR9 Leads to Gene Expression Defects

The Paf complex of Opisthokonts and plants contains at least five subunits: Paf1, Cdc73, Rtf1, Ctr9, and Leo1. Mutations in, or loss of Paf complex subunits have been shown to cause defects in histone modification, mRNA polyadenylation, and transcription by RNA polymerase I and RNA polymerase II. We here investigated trypanosome CTR9, which is essential for trypanosome survival. The results of tandem affinity purification suggested that trypanosome CTR9 associates with homologues of Leo1 and Cdc73; genes encoding homologues of Rtf1 and Paf1 were not found. RNAi targeting CTR9 resulted in at least ten-fold decreases in 131 essential mRNAs: they included several that are required for gene expression and its control, such as those encoding subunits of RNA polymerases, exoribonucleases that target mRNA, RNA helicases and RNA-binding proteins. Simultaneously, some genes from regions subject to chromatin silencing were derepressed, possibly as a secondary effect of the loss of two proteins that are required for silencing, ISWI and NLP1

Proteins associated with SF3a60 in T. brucei.

Trypanosoma brucei relies on Spliced leader trans splicing to generate functional messenger RNAs. Trans splicing joins the specialized SL exon from the SL RNA to pre-mRNAs and is mediated by the trans-spliceosome, which is made up of small nuclear ribonucleoprotein particles and non-snRNP factors. Although the trans spliceosome is essential for trypanosomatid gene expression, not all spliceosomal protein factors are known and of these, only a few are completely characterized. In this study, we have characterized the trypanosome Splicing Factor, SF3a60, the only currently annotated SF3a component. As expected, epitope-tagged SF3a60 localizes in the trypanosome nucleus. SF3a60 is essential for cell viability but its depletion seem to have no detectable effect on trans-splicing. In addition, we used SF3a60 as bait in a Yeast-2-hybrid system screen and identified its interacting protein factors. The interactions with SF3a120, SF3a66 and SAP130 were confirmed by tandem affinity purification and mass spectrometry

SF3a60-associated protein preys from a genome-wide Yeast-2-hybrid screen.

1<p>Confidence levels according to PBS scores <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091956#pone.0091956-Formstecher1" target="_blank">[32]</a>.</p>2<p>Domains (superfamily, Pfam) predicted in TritrypDB (<a href="http://tritrypdb.org/tritrypdb/" target="_blank">http://tritrypdb.org/tritrypdb/</a>); a blank space indicates those without known domains.</p>3<p>Functional designation. Functions predicted on the basis of sequence match alone are shown in parentheses.</p

Preparative tandem affinity purification identifies SF3a60 associated proteins.

<p>Proteins were separated on 12% SDS-PAGE and stained with SYPRO Ruby. Marker protein sizes in kilo Daltons are indicated on the left and corresponding protein bands are on the right. Details are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091956#pone-0091956-t001" target="_blank">Table 1</a>.</p

Expression profiling of Trypanosoma congolense genes during development in the tsetse fly vector Glossina morsitans morsitans

Abstract Background The tsetse transmitted parasitic flagellate Trypanosoma congolense causes animal African trypanosomosis (AAT) across sub-Saharan Africa. AAT negatively impacts agricultural, economic, nutritional and subsequently, health status of the affected populace. The molecular mechanisms that underlie T. congolense’s developmental program within tsetse are largely unknown due to considerable challenges with obtaining sufficient parasite cells to perform molecular studies. Methods In this study, we used RNA-seq to profile T. congolense gene expression during development in two distinct tsetse tissues, the cardia and proboscis. Indirect immunofluorescent antibody test (IFA) and confocal laser scanning microscope was used to localize the expression of a putative protein encoded by the hypothetical protein (TcIL3000_0_02370). Results Consistent with current knowledge, genes coding several variant surface glycoproteins (including metacyclic specific VSGs), and the surface coat protein, congolense epimastigote specific protein, were upregulated in parasites in the proboscis (PB-parasites). Additionally, our results indicate that parasites in tsetse’s cardia (C-parasites) and PB employ oxidative phosphorylation and amino acid metabolism for energy. Several genes upregulated in C-parasites encoded receptor-type adenylate cyclases, surface carboxylate transporter family proteins (or PADs), transport proteins, RNA-binding proteins and procyclin isoforms. Gene ontology analysis of products of genes upregulated in C-parasites showed enrichment of terms broadly associated with nucleotides, microtubules, cell membrane and its components, cell signaling, quorum sensing and several transport activities, suggesting that the parasites colonizing the cardia may monitor their environment and regulate their density and movement in this tissue. Additionally, cell surface protein (CSP) encoding genes associated with the Fam50 ‘GARP’, ‘iii’ and ‘i’ subfamilies were also significantly upregulated in C-parasites, suggesting that they are important for the long non-dividing trypomastigotes to colonize tsetse’s cardia. The putative products of genes that were upregulated in PB-parasites were linked to nucleosomes, cytoplasm and membrane-bound organelles, which suggest that parasites in this niche undergo cell division in line with prior findings. Most of the CSPs upregulated in PB-parasites were hypothetical, thus requiring further functional characterization. Expression of one such hypothetical protein (TcIL3000_0_02370) was analyzed using immunofluorescence and confocal laser scanning microscopy, which together revealed preferential expression of this protein on the entire surface coat of T. congolense parasite stages that colonize G. m. morsitans’ proboscis. Conclusion Collectively, our results provide insight into T. congolense gene expression profiles in distinct niches within the tsetse vector. Our results show that the hypothetical protein TcIL3000_0_02370, is expressed on the entire surface of the trypanosomes inhabiting tsetse’s proboscis. We discuss our results in terms of their relevance to disease transmission processes

Sequence conservation for putative T. brucei PRP40.

<p><b>A</b>. The WW domains of human FBP11 (O75400.2), yeast PRP40 (NP_012913.3) and the putative Trypanosome FBP11 homologue contain the tryptophans, prolines and a central set of three hydrophobic residues characteristic of WW domains. Evidently, the putative <i>T. brucei</i> FBP11 lacks a second WW domain.</p

S3a60 is essential for trypanosome viability.

<p>(A) Cumulative growth curves of TbSF3a60 depleted trypanosomes. The cells grown with (triangles, solid line) or without (squares, dashed line) tetracycline. Every 24 h, samples were taken for the determination of cell density (cells/ml), and grown cultures were diluted down to 5×10⁵ cells/ml with fresh medium. (B). Effect on <i>SF3a60</i> mRNA. Cells were grown either without <i>Tet</i> (–), or in the presence of 100 ng/ml <i>Tet</i> (+) for 24, 48 and 72 hours. Each lane on the Northern blot contains 10 μg RNA from bloodstream form wild type (WT) or an RNAi cell line. Asterisk (*) denotes dsRNA. Trypanosome SRP and Tubulin (TUB) RNAs are used as loading controls. Arrow indicates SF3a60 mRNA.</p

SF3a60 RNAi has no effect on splicing.

<p>The Y structure <i>trans</i> splicing intermediate is not decreased after two days of RNAi targeting SF3a60. The full-length spliced leader RNA and the Y structure were detected by primer extension (diagram in panel A) followed by denaturing polyacrylamide gel electrophoresis (B) with the U3 snRNA as a loading control. The RNA was prepared from the RNAi cell line grown without tetracycline (panel B, lanes 1 and 4), or with tetracycline for one or two days (lanes 2 and 3). M: markers.</p

Proteins associated with SF3a60 by tandem affinity purification.

<p>Proteins associated with SF3a60 by tandem affinity purification.</p