The protein family TcTASV-C is a novel Trypanosoma cruzi virulence factor secreted in extracellular vesicles by trypomastigotes and highly expressed in bloodstream forms.

TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage of Trypanosoma cruzi. We have previously shown that TcTASV-C is located at the parasite surface and secreted to the medium. Here we report that the expression of different TcTASV-C genes occurs simultaneously at the trypomastigote stage and while some secreted and parasite-associated products are found in both fractions, others are different. Secreted TcTASV-C are mainly shedded through trypomastigote extracellular vesicles, of which they are an abundant constituent, despite its scarce expression on culture-derived trypomastigotes. In contrast, TcTASV-C is highly expressed in bloodstream trypomastigotes; its upregulation in bloodstream parasites was observed in different T. cruzi strains and was specific for TcTASV-C, suggesting that some host-molecules trigger TcTASV-C expression. TcTASV-C is also strongly secreted by bloodstream parasites. A DNA prime-protein boost immunization scheme with TcTASV-C was only partially effective to control the infection in mice challenged with a highly virulent T. cruzi strain. Vaccination triggered a strong humoral response that delayed the appearance of bloodstream trypomastigotes at the early phase of the infection. Linear epitopes recognized by vaccinated mice were mapped within the TcTASV-C family motif, suggesting that blockade of secreted TcTASV-C impacts on the settlement of infection. Furthermore, although experimental and naturally T. cruzi-infected hosts did not react with antigens from extracellular vesicles, vaccinated and challenged mice recognized not only TcTASV-C but also other vesicle-antigens. We hypothesize that TcTASV-C is involved in the establishment of the initial T. cruzi infection in the mammalian host. Altogether, these results point towards TcTASV-C as a novel secreted virulence factor of T. cruzi trypomastigotes

TbRRM1 is essential for parasite survival.

<p><b>(A)</b> Cumulative growth of TbRRM1 RNAi trypanosomes in the absence (TET-) or presence (TET+) of tetracycline for the indicated times. Error bars indicate the standard deviations around the means of three independent experiments. <b>(B)</b> Northern blot analysis of TbRRM1 mRNA after silencing. A 28S rRNA probe was used as loading control. <b>(C)</b> Western blot analysis of TbRRM1 over time following induction of TbRRM1 RNAi in both 5´UTR and ORF cell lines. Either anti-transaldolase (TAL) or anti-enolase (Enol) serum was used as loading control. <b>(D)</b> Immunofluorescence of TbRRM1 in un-induced cultures or 48 h after TET addition. From left to right: phase contrast, DAPI stained cells, anti-TbRRM1 stained cells and merged images. Note the nozzle phenotype in TET+ cells.</p

TbRRM1 is involved in the regulation of TbNOP86.

<p>Change in mRNA abundance after 24 h of TbRRM1 RNAi induction assayed by RTqPCR. Values are expressed as mRNA relative abundance from TET+ / TET- assays. Data were obtained from at least three independent experiments.</p

TbRRM1 depletion induced impairment of DNA synthesis.

<p><b>(A)</b> BrdU incorporation assay over time obtained by immunofluorescence (IIF, circles) or FACS (triangles). DNA synthesis impairment was determined by the TET+/TET- ratio, calculated as the percentage of BrdU-positive cells from both 24 h and 48 h TET+ cultures relative to the percentage of BrdU-positive cells from 24 h and 48 h TET- cultures, respectively, multiplied by 100. Error bars indicate the standard deviations around the means of the TET+/TET- ratio from either four independent experiments for FACS assay or three for IIF. <b>(B)</b> FACS histograms of un-induced or TET+ treated parasites labeled with BrdU (FL-1). Histograms representative of quadruplicate experiments are shown. <b>(C)</b> Graphic showing the index of variation (IV) obtained by the equation (TM-CM)/CM, where TM is the mean of FL-1 fluorescence for TET+ parasites and CM is that of the non-treated cells. Horizontal line shows the IV mean for the biological replicates. The table indicates the number of BrdU positive cells analyzed in each independent experiment.</p

TbRRM1 silencing induced aberrant nucleus-kinetoplast (N-K) configurations and abnormal phenotypes.

<p><b>(A)</b> Distribution of normal and aberrant phenotypes (nozzle, lollipop, zoid and indeterminate cells) after RNAi induction. <b>(B)</b> N-K configuration graphics showing the percentage of cells presenting xNxK arrangement at different time points after RNAi induction. Inset: Percentage of N-K configuration of the nozzled cells from 24 h to 72 h after TET addition. For reference, 1N1K* indicates cells with one nucleus—one elongated kinetoplast; 2N2K# represents parasites with abnormal N-K distribution. Results were obtained from more than 200 cells at each time point of three independent experiments. Data was analyzed by Student t test compared to 24 h TET- values. *p<0.05; **p<0.01; ***p<0.001.</p

TbRRM1 deficient cells exhibited a nozzle phenotype.

<p><b>(A)</b> Upper: Graphic bars showing the length from nucleus to kinetoplast (N-K; gray bars) or from kinetoplast to posterior end (K-P; black bars) at 0, 24, 48 and 72 h post RNAi induction. Results are expressed as mean ± standard deviation from more than 200 cells at each time point of three independent experiments (Student t test. *p<0,05; **p<0,01). Bottom: DAPI stained PC. <b>(B)</b> Cytoskeleton analysis of 48 h <i>TbRRM1</i> silenced cells by immunofluorescence (anti-tyrosinated tubulin YL1/2). From left to right: phase contrast, DAPI stained cells, YL1/2 stained cells and merged images. Scale bar: 3 μm.</p

TcTASV-C is secreted in EVs in different <i>T</i>. <i>cruzi</i> strains.

<p>Purified EVs from SylvioX10 (TcI), 173 (TcI), Y (TcII), and RA (TcVI) strains were processed as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006475#pntd.0006475.g003" target="_blank">Fig 3</a>. Tryp: trypomastigote; V2: large EVs; V16: small EVs; VF: vesicle-free fraction. The amount of proteins in EVs of different strains was similar, as determined by micro-BCA assay. One representative of three independent experiment is shown.</p