Role of TcPARG in <i>Trypanosoma cruzi</i> epimastigotes proliferation and cell cycle progression.

<p>A) Effect of the PARG inhibitors ADP-HPD and DEA on <i>T. cruzi</i> growth and survival was determined by incubating epimastigotes at an initial density of 10⁷ parasites/ml in the continuous presence of inhibitors at 1 µM. The number of epimastigotes was determined daily by counting formaldehyde-fixed parasites in a Neubauer chamber. All data points were determined in triplicates and shown as means with standard deviation. The significance of the results versus the control at day 4 was analyzed with t test and indicated in the figure (* p0.05). B) Effect of ADP-HPD at 1 µM concentration on cell cycle progression of epimastigotes was determined by adding the inhibitor at the indicated concentration to the culture media of hydroxyurea synchronized parasites after digitonin permeabilization. Samples were drawn every 2 hours for 14 hours and DNA content was determined by propidium iodide staining followed by flow cytometry analysis. The percentage of epimastigotes in each cell cycle phase was determined by setting gates according to the DNA content in the 0 hs of the control sample and maintained for all other samples. The data were analyzed using the Cyflogic software.</p

Amino acid sequence alignment of the PARG signature from different organisms.

<p>The multiple alignment of the PARG signature amino acid sequences corresponding to <i>T. cruzi</i> PARG (accession number ABG73229); <i>T. brucei</i> PARG (GeneDB Systematic Name: Tb09.211.3760); <i>C. elegans</i>_1 PARG (accession number NP_501496) and <i>C. elegans</i>_2 PARG (accession number NP_501508); <i>T</i><i>. thermophila</i> (accession number EAR94344); <i>A. thaliana</i>_1 PARG (accession number NP_973578); <i>A. thaliana</i>_2 PARG (accession number AAK72256); <i>D. discoideum</i> PARG (accession number XP_642024); <i>D. melanogaster</i> PARG (accession number NP_477321); <i>C</i><i>. quinquefasciatus</i> PARG (accession number XP_001853435); <i>A. aegypti</i> PARG (accession number XP_001659301); <i>D. rerio</i> PARG (accession number XP_001338257); <i>X. laevis</i> PARG (accession number NP_001089602); <i>G. gallus</i> PARG (accession number XP_421502); <i>B. taurus</i> PARG (accession number NP_776563); <i>R. norvegicus</i> PARG (accession number NP_112629); <i>M. musculus</i> PARG (accession number NP_036090); <i>H. sapiens</i> PARG (accession number NP_003622); <i>P. abel</i>ii PARG (accession number NP_001125086); <i>P</i><i>. troglodytes</i> PARG (accession number XP_001139727) was generated with the ClustalW2 program and edited with the BOXSHADE (3.21) software. Colors used for amino acids background are as follow: white for different residues, black for identical residues, gray for similar and conserved residues. Asterisk: essential acidic residues D-E-E, underlined: key residues, G and two consecutive E, and black diamond: important Y residue.</p

Effect of PARG inhibitors on <i>T. cruzi</i> infection on Vero or A549 host cells.

<p><i>T. cruzi</i> trypomastigotes were purified from the supernatant of previously infected cells and preincubated for 30 min in the respective culture medium in the absence (Control) or presence of 1 µM PARG inhibitor (DEA). Twenty-four hours Vero, A549 wild type or shPARG (hPARG silenced) cell monolayers were infected with 50 trypomastigotes/cell. The infection process was followed by microscopic direct visualization. At the indicated days (A and C) or at day 6 post-infection (B and D), percentage of infected cells and number of amastigotes intracellular were determined on May-Grünwald Giemsa stained samples. Amastigotes and cells were counted using the ImageJ software in at least 7 fields. The number of trypomastigotes/ml in the supernatant of infected cell cultures was determined by counting unfixed trypomastigotes in a Neubauer chamber at the indicated days (E) or at day 9 post-infection (F). All points were determined in triplicates and shown as means with standard deviation. Significance of the result versus the Control (***p0.001; two way ANOVA) or Wild Type Control (***p0.001; **, p0.01; two way ANOVA) is indicated.</p

Expression of TcPARG throughout the <i>Trypanosoma cruzi</i> life-cycle.

<p>(A) Microarray expression data for TcPARG over the course of <i>T. cruzi</i> life-cycle. TcPARG mRNA relative abundance was evaluated by using the transcriptome analysis of different <i>T. cruzi</i> stages available at Gene Expression Omnibus database (<a href="http://www.ncbi.nlm.nih.gov/geo" target="_blank">www.ncbi.nlm.nih.gov/geo</a>, DataSets: GSE14641). Shown are mean microarray log₂ ratios (stage/reference) for TS significantly regulated in <i>Trypanosoma cruzi</i> amastigotes (AMA), trypomastigotes (TRYP), epimastigotes (EPI), and metacyclic trypomastigotes (META). (B) Western blot analysis of the three life-cycle stages of <i>T. cruzi</i>. Protein extracts (35 µg) of amastigote, epimastigote or trypomastigote stages of <i>T. cruzi</i> were solved in a 10% polyacrylamide gel, transfer to a nitrocellulose membrane and revealed with an anti-TcPARG (1:10000) specific antiserum. β-tubulin was used as loading control.</p

Immunolocalization of PARG on <i>Trypanosoma cruzi</i>, CL Brener epimastigotes.

<p>The parasites were fixed for 25 min with 3.8% (W/V) formaldehyde in PBS at 4°C, permeabilized with fresh PBS - 0,1% Triton X-100 and blocked for 1 h at room temperature with 5% (W/V) BSA in PBS. (A) Differential interference contrast (DIC). (B) Cells were counterstained with DAPI to identify nuclear DNA and kinetoplastid DNA. (C) PARG was detected with 1:500 mouse polyclonal TcPARG antibody followed by 1:600 Alexa Fluor 488 goat anti-mouse IgG antibody. (D) Merge of PARG and DNA signals show the nuclear localization of this enzyme. Bar: 10 µm. (E–F) For electron microscopy, epimastigotes were fixed in PBS 2.5% glutaraldehyde, 4% formaldehyde, embedded in epoxy resin and PARG detected with 1:50 mouse polyclonal TcPARG antibody followed by 1:100 anti-mouse antibody conjugated with 10-nm gold particle. N: nucleus; K: kinetoplast. Bar: 0.2 µm.</p

Effects of TbWee1 knockdown on the procyclic form of <i>T. brucei</i> cells.

<p>(A) Cells of strain 29-13 harboring the TbWee1-RNAi construct were incubated in culture medium with (+ Tet) or without (-Tet) 2.5 µg/ml tetracycline at 28°C. The cell growth rate was monitored daily, and the cell number was plotted in a logarithmic scale. The insets show the intracellular mRNA level after 3 days of RNAi as monitored by Northern blot. RNAr was used as loading control. Western blot of extracts of induced and non-induced cells were analyzed with anti-TbWee1 antibody (Right inset). (B) Time course of RNAi-induced <i>T. brucei</i> procyclic-form. Cells were stained with propidium iodide and subjected to FACS analysis to measure DNA content. The percentages of cells in G1, S and G2/M phases were determined with the ModFitLT software and plotted on the right panel. </p

Amplification and purification of TbWee1-6xHis fusion protein to produce anti-TbWee1 polyclonal antibodies.

<p>(A) SDS–PAGE analysis of lysates obtained from <i>Escherichia coli</i> IPTG.induced (I) and non-induced (NI) cell cultures transformed with pDEST17-TbWee1. Upper panel: Coomassie Brilliant Blue staining. Lower panel: Western-blot analysis with anti-histidine tag antibody (1:5000). The arrow indicates the position of the ~70 kDa recombinant protein. (B) Purification steps of rTbwee1-6xHis fusion protein were monitored by 12 % SDS-PAGE analysis. The lysate was loaded onto a Ni-agarose column. Flow-through (F), washes (W, 1–3), and eluted fractions (250 mM imidazol, E 1–3) were analyzed by 12% SDS-PAGE stained with Coomassie Brilliant Blue. (C) Western blot analysis of rTbWee1 expression with polyclonal anti-TbWee1 antibody. Protein extracts of the <i>T. brucei</i> bloodstream form (lane 1), the <i>T. brucei</i> procyclic form (lane 2) and the recombinant TbWee1-6xHis (lane 3) were separated by 10 % SDS-PAGE and electroblotted on nitrocellulose membrane. Blots were incubated with anti-TbWee1 polyclonal antibody (1:1000) and revealed by chemiluminescence.</p

Rescue of a <i>Schyzosaccharomyces pombe</i> Wee1 mutant by TbWee1.

<p>(A) <i>S. pombe</i> ∆Wee1 mutants were transformed with the pREP3x vector or with pREP3x in which <i>S. pombe</i> Wee1 or TbWee1 was cloned. Fission yeast were cultured on solid media in the presence (+thia) or absence (-thia) of 15 µM thiamine. Lower panel: <i>S. pombe</i> cells expressing wild type Wee1. (B) DAPI staining of <i>S. pombe</i> cells transformed with pREP3x TbWee1 grown in the absence of thiamine. Average cell size in <i>S. pombe</i> yeasts complemented with TbWee1 was 16,3 µm, with 55% of the population in the 10-15 µm range. Average cell size in control <i>S. pombe</i> yeasts was 10,4 µm, with 57% of the population in the 5-10 µm range. n=100. Bar= 100 µm.</p

Sequence comparison between TbWee1 and homolog proteins in other species.

<p>(A) Multiple sequence alignment of the catalytic domains of the putative <i>Trypanosoma brucei</i> Wee1 protein with other Wee1-like kinases. Amino acid sequences were aligned using the ClustalW2 program (<a href="http://www.ebi.ac.uk/clustalw" target="_blank">http://www.ebi.ac.uk/clustalw</a>). Identities are indicated by asterisks below the sequence. Conserved substitutions are marked with two vertical dots and semi-conserved substitutions are marked with a single dot. Dashes represent gaps introduced for optimal alignment. The 11 conserved subdomains are designated by Roman numerals [45,46]. The catalytic and activation segments are indicated with a blue and pink box respectively. A black box indicates the conserved EGD motif. Triangles indicate amino acids that are conserved in all known members of the Wee1 kinase family, but not in other eukaryotic protein kinases. Sequences shown are for Wee1A kinase of <i>Trypanosoma </i><i>brucei</i> (TbWee1, JN083854), humans (HsWee1, NP003381.1), mice (MmWee1, NP033542.2), <i>Schizosaccharomyces </i><i>pombe</i> (SpWee1, NP587933.1), <i>Saccharomyces </i><i>cerevisiae</i> (ScSwe1, NP012348.1), <i>Arabidopsis </i><i>thaliana</i> (AtWee1, NP171796.1), <i>Xenopus laevis</i> (XlWee1, NP001081784.1), and <i>Trypanosoma cruzi</i> (TcWee90, JN573306; TcWee570, JN257712). (B) Comparison of TbWee1 with other protein kinases. The position of the putative protein kinase domain is shown in black and numbers represent the percent amino acid identity with this region of the predicted TbWee1.</p

Morphological phenotypes of Wee1-deficient <i>T. brucei</i> procyclic-form cells.

<p>Samples of the TbWee1-depleted cells taken at different times were stained with DAPI and examined by fluorescence microscopy. (A) Analysis of the numbers of nuclei and kinetoplasts as determined by DAPI staining. Data are presented as the mean percentages ±S.E. of the total population counted (> 200 cells in each of three independent experiments). (B) Wee1-deficient cells viewed by phase-contrast and fluorescence microscopy. N: nucleus, K: kinetoplast.</p