GFP expression does not affect the growth, viability and metacyclogenesis of <i>T. cruzi</i>.

<p>(A) Growth curves of wild-type (Dm28c) and pBEX/GFP cultured epimastigotes showing no significant difference in growth; cell density was determined with an electronic particle counter (Z2 Coulter Particle Count and Size Analyzer, Beckman Coulter®). (B) Cell viability analysis of epimastigote cultures by vital dye (propidium iodide - PI) staining and flow cytometry quantification of PI-positive cells. (C) Metacyclogenesis efficiency in wild-type and pBEX/GFP cultures; after metacyclogenesis induction (details in methods) the density of metacyclic forms in the culture supernatant was obtained daily, by direct counting in a Neubauer chamber. For all plots, each experimental point represents the mean and standard deviation of triplicate experiments.</p

Cellular localization of tagged Hel45 in <i>Trypanosoma cruzi</i>.

<p>(A) Detection of exogenous Hel45 and a Hel45 NES deletion mutant (Hel45ΔNES) (both tagged with PTP at the NT) by indirect immunofluorescence microscopy with an anti-ProtA antibody. DAPI = DNA stained with DAPI. Hel45 =  localization of tagged Hel45 or Hel45ΔNES. MERGE = merged images for DAPI staining and Hel45 localization. N = nucleus. K = kinetoplast. Arrows = parasites with nuclear accumulation of tagged Hel45. Bar = 5 µm. (B and C) Western blot of total extract from wild-type epimastigotes (WT) and epimastigotes expressing recombinant Hel45 (B) or Hel45ΔNES (C) tagged with a PTP at the N-terminus (NT). Lane 1 =  detection with anti-Hel45 antibodies. Lane 2 =  detection with anti-ProtA antibodies.</p

Primers used for plasmid construction.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067441#pone-0067441-t001" target="_blank">Table 1</a> legend: NEO, neomycin phosphotransferase gene; IRA: intergenic region A; IRB: intergenic region B; IRC: intergenic region C. The restriction enzyme cleavage sites are shown in bold.</p

Flow cytometry analysis of GFP fluorescence in all major stages of the <i>T. cruzi</i> pBEX/GFP life cycle.

<p>(A) Overlay histograms of GFP fluorescence in various forms of <i>T. cruzi</i> (specified in the box). Note that only the replicative forms (epimastigotes and amastigotes) of the parasite display GFP fluorescence. % of Max is a normalization of the total number of events in the overlaid histograms; each sample is scaled to the percentage of its maximum signal. (B) Overlay histograms of GFP fluorescence after various numbers of days of epimastigote culture; exponential growth phase (3 days), stationary phase (5 days) and late stationary phase with the presence of metacyclic forms (7 days). Cell culture was initiated with 1×10⁶ cells ml⁻¹ (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067441#pone-0067441-g002" target="_blank">Figure 2A</a>). (C) Percentage of epimastigotes in each phase of the cell cycle (G1, S and G2/M), as determined by flow cytometric analysis of DNA content with PI staining, during epimastigote culture. (D) Flow cytometry density plot showing the correlation of GFP fluorescence with the cell cycle of epimastigotes of <i>T. cruzi</i> pBEX/GFP after 3 days and 7 days of culture (indicated above the plot). (E) Median GFP fluorescence in each phase of the cell cycle during the culture of pBEX/GFP epimastigotes. The intensity of GFP expression is higher in cultures displaying active replication of DNA (S phase) and is highly dependent on culture time. In the plots, wild-type epimastigotes (Dm28c) were used as a negative control for the normalization of GFP fluorescence level. The results shown in (C), (D) and (E) are representative of duplicate experiments with coefficients of variation of less than 5%.</p

Localization of Hel45 after actinomycin D treatment in <i>T. cruzi</i>.

<p>Detection of exogenous Hel45 (A) tagged with PTP at the NT by indirect immunofluorescence with an anti-ProtA antibody and of mRNA (B) by fluorescence <i>in situ</i> hybridization (FISH) with a digoxigenin-conjugated oligo(dT) probe in <i>T. cruzi</i> after treatment with 50 µg/ml actinomycin D (ACTD) for 24 hours. Probe detection was carried out by indirect immunofluorescence with anti-DIG mouse monoclonal antibodies (Sigma-Aldrich, 1∶300 dilution) followed by secondary Alexa Fluor 488-conjugated antibodies (1∶600 dilution). As a control, 100 µg/ml RNase A was incubated with the parasites before probe hybridization (RNase A). DAPI = DNA stained with DAPI. Hel45 =  localization of tagged Hel45. MERGE = merged images for DAPI staining and Hel45 or mRNA localization. N = nucleus. K = kinetoplast. Arrows = parasites with nuclear accumulation of tagged Hel45. Bar = 5 µm.</p

Hel45 is a component of ribonucleoprotein complexes in the cytoplasm.

<p>Polysome fractionation by sucrose density gradient. The fractions (1–22) were collected after the sedimentation of cytoplasmic extract from <i>T. cruzi</i> treated with 100 µg/ml cycloheximide (A), 2 mM puromycin (B), or 500 U/ml micrococcal nuclease in the presence of 2 mM CaCl₂ (C). The 40S and 60S ribosomal subunits, the 80S ribosome monomer and polysomes are indicated. A western blot was performed with an anti-Hel45 antibody for each fraction. S7, a small ribosomal subunit protein, was used as a control. (D) mRNP isolation assay. Western-blot analysis with anti-Hel45 and anti-S7 antibodies and mRNPs obtained from the <i>T. cruzi</i> cytoplasmic fraction after elution from oligo(dT)-conjugated magnetic beads (El). As a control, cytoplasmic extract was treated with 10 µg/ml RNaseA before mRNP capture. FT = flow-through from cytoplasmic extract not bound to the oligo(dT). El = eluted fraction.</p

Localization of Hel45 after leptomycin B treatment in <i>T. cruzi</i> and localization of the ortholog of Hel45 (TbHel46) in <i>T. brucei</i> after Mex67 RNAi induction.

<p>(A) Detection of exogenous Hel45 tagged with PTP at the NT by indirect immunofluorescence with an anti-ProtA antibody. <i>T. cruzi</i> parasites were treated with 500 ng/ml leptomycin B (LMB) for 24 hours or were untreated (control). (B) Growth curve of <i>T. cruzi</i> parasites after treatment with 500 ng/ml leptomycin B (LMB). (C) Growth curve of <i>T. brucei</i> parasites after the induction of RNAi against Mex67 with 2 µg/ml tetracycline (RNAi-induced). (B) and (C) represent graphics of a biological replica which the density of cells in the culture was determined by counting in triplicate with a particle counter (Beckman Coulter). (D) Western-blot analysis of total protein extracts from parasites 48 (I_{48 h}) or 72 (I_{72 h}) hours after the induction of Mex67 RNAi. Non-induced (NI) parasites are shown as a control. The assay was carried out with anti-Mex67 antibodies. Anti-GAPDH antibodies were used as loading control. (E.1) Cellular localization of mRNA by fluorescence <i>in situ</i> hybridization (FISH) with a digoxigenin-conjugated oligo(dT) probe and localization of TbHel46 by indirect immunofluorescence Cells were fixed 48 hours after the induction of Mex67 RNAi (+TET). Images were processed by deconvolution software Leica AF6000. DAPI = DNA stained with DAPI. Hel45 =  localization of tagged Hel45. TbHel46 =  endogenous TbHel46 localized with anti-Hel45 antibodies. MERGE = merged images for DAPI staining and Hel45 localization (A) or DAPI staining, FISH and TbHel46 localization (E.1). N = nucleus. K = kinetoplast. (E.2 and E.3) Graphs show the quantification of fluorescence intensity of DAPI (blue), FISH (green), and TbHel46 (red) labelling that was detected across the dotted line in E.1. Fluorescence intensity was plotted in the y-axis for Mex67 RNAi-induced parasites (+TET, Figure E.2) and non-induced parasites (−TET, Figure E.3).</p

Flow cytometry assay for intracellular anti-amastigote activity.

<p>(A) Overlaid histograms of GFP fluorescence in Vero cells infected with various ratios of trypomastigotes/Vero cells (indicated within the graph) together with a non infected control. The GFP⁺ Vero cells were considered to be infected with amastigotes. (B) Correlation between the mean number of amastigotes per Vero cell (obtained by manual counting on Giemsa-stained smears) and the median GFP signal of GFP⁺ Vero cells (obtained in flow cytometry experiments). Each experimental point corresponds to Vero infection with a different ratio of trypomastigotes/Vero cells (as in (A)). Note that the results of the two methods of intracellular amastigote quantification display a strong linear correlation (R² = 0.976). (C) Correlation between the % infected cells obtained by microscopy and flow cytometry (R² = 0.996). (D) Comparison of the infection indices in Vero cells obtained by microscopy (IF_M) and flow cytometry (IF_FC), to estimate intracellular amastigote growth. Data are shown as a percentage of maximum growth. The results of the two methods are highly correlated (R² = 0.983). For details of IF_M and IF_FC calculation, see Materials and Methods. (E) Overlaid histograms of infected Vero cells (10 trypomastigotes/Vero cell) treated with different doses of benznidazole (indicated within the graph) together with uninfected and untreated controls. (F) Calculation of the benznidazole IC₅₀/24 h (indicated within the graph) by the flow cytometry method. Each experimental point represents the mean and standard deviation of duplicate drug exposures.</p

Fluorescence confocal microscopy assessing GFP expression in various stages of <i>T. cruzi</i> pBEX/GFP.

<p>(A) Amastigote-infected Vero cells after 48 h of trypomastigote infection. (B) Trypomastigotes produced after 4 days of Vero cell infection. (C) Epimastigotes in the exponential growth phase (upper photos) and purified metacyclic forms (lower photos). (D) Mixture of live epimastigotes and metacyclic forms, showing the absence of GFP fluorescence in trypomastigotes. DIC: differential interference contrast; DNA: staining of DNA with Hoechst 33342 dye (for better visualization in overlay images, Hoechst fluorescence has been artificially converted to red); GFP: fluorescence of GFP; DNA+GFP: overlay images of Hoechst and GFP fluorescence. Bars correspond to 10 µm, except in A (25 µm).</p

Multiple sequence alignment and prediction of the structure of Hel45.

<p>(A) Multiple sequence alignment of the diagnostic conserved region of the DEAD-box helicase family (positions 25–365 according to Hel45). The nine putative conserved motifs (<i>Q</i>, <i>I</i> (WalkerA), <i>Ia, Ib, II</i> (WalkerB), <i>III, IV, V, VI</i>) are marked with orange boxes. Alignment columns displaying 100%, more than 90%, and more than 80% of similarity are highlighted in black, dark grey, and light grey, respectively. Sequences are identified with organism abbreviation and gene name, except Hel45. The organism abbreviations are: Sc: <i>Saccharomyces cerevisiae</i>, Hs: <i>Homo sapiens</i>, Pf: <i>Plasmodium falciparum</i>. The sequences have the following GenBank Identifiers (GIs): Hel45 (71418343), Sc_TIF2 (6322323), Sc_FAL1 (398365053), Sc_DBP5 (6324620), Hs_EIF4A1 (4503529), Hs_EIF4A2 (83700235), Hs_EIF4A3 (7661920), Hs_DDX19A (8922886), Pf_PFD1070w (124505577), Pf_H45 (124810293), Pf_DBP5 (6324620). (B) Schematic representation showing the nine conserved helicase motifs are boxed in orange. The N-terminal domain (NTD) contains the motifs Q, I and II for ATP-binding, Ia and Ib for RNA-binding, and III for ATP hydrolysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109521#pone.0109521-Cordin1" target="_blank">[44]</a>. The C-terminal domain (CTD) contains the motifs IV and V for RNA-binding, and VI for ATPase and unwinding activities <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109521#pone.0109521-Cordin1" target="_blank">[44]</a>. The predicted nuclear export signal (NES) in the LYDTLTI sequence (255–261 position) is shown in yellow. (C) Molecular modeling of Hel45. The nine motifs are highlighted in orange, the predicted NES (yellow) is close to the CT extremity (green). A zoom of this region (box) shows the side chains of amino-acids D257, T258 and D393, and the interactions that maintain the structure at its C-terminal extremity. The organization of the NES in the CT is shown in the inset (upper right corner).</p