Effect of the PLK-1 inhibitor BI2536 on larval development.

<p>Schistosomula were treated with 100 nM BI2356 (B, D and E) or carrier DMSO alone (A and C). A and B: cultures without hRBC; C, D and E: cultures with hRBC. Images A-D: after culture for 20 days; image E: after culture for 30 days (arrow shows a viable lung-form schistosomulum) (scale bar = 100μm). (F) Mean ± SE larval survival based on triplicate cultures.</p

Effect of <i>Sm-</i>Calm suppression on adult schistosomes.

<p>(A) <i>Sm-</i>Calm expression in adult schistosomes 7 days after electroporation with <i>Sm-</i>Calm long dsRNA (CALM) relative to that in untreated control worms (CTR) or worms similarly electorporated with irrelevant dsRNA (IRR). (B-C) Light microscope images of adult worms electroporated 3 weeks earlier with IRR dsRNA (B) or <i>Sm-</i>Calm long dsRNA (C). Note the waves of contraction along the bodies of worms following <i>Sm</i>-Calm knockdown (scale bar = 100μm). Data are representative of 3 independent experiments.</p

Effect of <i>Sm-</i>aPKC suppression in schistosomula.

<p>(A) <i>Sm-</i>aPKC expression in schistosomula 7 days after soaking with <i>Sm</i>-aPKC long dsRNA, irrelevant ds RNA (IRR) or untreated controls (CTR). (B) Light microscope images of schistosomula 4 weeks after the RNAi treatment. Top images:—wells without hRBC; bottom images:—wells with hRBC (scale bar = 100μm). Percent normal development (C) and percent survival (D) in cultures with hRBC (mean±SE triplicate cultures). Data are representative of 3 independent experiments.</p

Effect of the PLK-1 inhibitor BI2536 on egg development.

<p>Unembryonated eggs recovered from <i>ex-vivo</i> adult worms and cultured in vitro for 3 days failed to embryonate when subsequently cultured in 100 nM BI2536 (B) in contrast most eggs cultured with DMSO carrier alone developed normally (A). Thin arrow: free miracidium; thick arrow: embryonated egg containing viable miracidium. Scale bar = 100μm.</p

Effect of <i>Sm</i>-aPKC suppression on adult schistosomes.

<p>(A) <i>Sm-</i>aPKC expression in adult schistosomes 7 days after electroporation with <i>Sm-</i>aPKC long dsRNA (PKC) relative to that in untreated control worms (CTR) or worms similarly electroporated with irrelevant ds RNA (IRR). (B) Light microscope images of adult worms 2 or 8 weeks following electroporation with IRR or <i>Sm</i>-aPKC (PKC) long dsRNA (scale bars = 100μm). (C) Visual score of worm viability 3 weeks after silencing:- 3-healthy, attached; 2-healthy, not attached; 1- darkened/low motility; 0-severely damaged. Data are representative of 3 independent experiments.</p

Effect of RNAi silencing of <i>Sm</i>-MAPK p38.

<p>Graphs showing percentage survival of schistosomula following treatment with long dsRNA corresponding to the <i>Sm</i>-MAPK p38 family member (Smp_133020) (P38) compared with untreated controls (CTR) or controls treated with irrelevant dsRNA (IRR). Schistosomula were cultured in the absence (A) or presence (B) of hRBC.</p

Effect of <i>Sm-</i>PLK1 silencing on schistosomula viability.

<p>(A-B) Survival of parasites treated with <i>Sm</i>-PLK1 long dsRNA compared with untreated controls (CTR) or controls treated with irrelevant dsRNA (IRR) in the absence (A) or presence (B) of hRBC. (C-D) Reduced viability and a lower percentage of blood feeding parasites in the presence of hRBC following <i>Sm</i>-PLK1 knockdown. The photomicrographs (C) are of schistosomula cultured for 3 weeks after RNAi treatment (scale bar = 100μm).</p

Worm recovery following <i>in vivo</i> injection of schistosomula silenced for <i>Sm-</i>aPKC or <i>Sm-</i>PLK1.

<p>Worms recovered by portal perfusion 4 weeks after i.m. injection of 1000 schistosomula treated with <i>Sm</i>-aPKC (aPKC), <i>Sm</i>-PLK1 (PLK1), irrelevant long dsRNA (IRR) or untreated (CTR). Significant differences were assessed by unpaired Student’s t-test; * = P<0.05.</p

ATP:ADP antiporter mimics turbo-state.

<p>(<i>A</i>) Overview of the models used in this figure. Model A and D are from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003371#pcbi-1003371-g001" target="_blank">Figure 1</a>, model A–glyc is model A without glycosomal localization, as described in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003371#pcbi.1003371-Haanstra2" target="_blank">[31]</a>, model A+AAT is model A with an ATP:ADP antiporter. (<i>B–C</i>) Steady-state concentrations of glycosomal Glc-6-P and Fru-1,6-BP are depicted in the various models. (<i>D</i>) Increasing the activity of the ATP:ADP antiporter (V_{max,ATP:ADP antiporter}) in model D leads to a high risk of accumulation of hexose phosphates. The green line indicates the concentration of Fru-1,6-BP in the original model of glycolysis (17.2 mM, panel C, model A). Glc_e in this simulation is 25 mM. (<i>E</i>) Time course simulation of model D at 25 mM Glc_e and various values for the V_{max,ATP:ADP antiporter} parameter. Plotted is the concentration of glycosomal phosphates (ΣP similar as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003371#pcbi-1003371-g002" target="_blank">Figure 2</a>, moiety 5 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003371#pcbi-1003371-t002" target="_blank">Table 2</a>). ATP:ADP antiporter activity values below 1 nmol·min⁻¹·mg protein⁻¹ result in depletion of glycosomal phosphates (cf. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003371#pcbi-1003371-g002" target="_blank">Figure 2</a>). <i>k_TOX</i> = 2 µl·min⁻¹·mg protein⁻¹ in all models. Solid lines indicate medians, shaded areas and error bars show interquartile ranges, as derived from the uncertainty modeling.</p

Simulations of 6PGDH inhibition and 6-PG accumulation.

<p>(<i>A–B</i>) The effects of inhibition of 6PGDH on 6-PG concentrations and metabolic fluxes were simulated by reducing <i>V</i>_max,6PGDH in model C and D at high oxidative stress (<i>k_TOX</i> = 200 µl·min⁻¹·mg protein⁻¹). Simulations at low oxidative stress (<i>k_TOX</i> = 2 µl·min⁻¹·mg protein⁻¹) are shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003371#pcbi.1003371.s009" target="_blank">Figure S6</a>. ATP production flux as steady-state flux through PFK is indicated in red, while trypanothione reductase steady-state flux is indicated in yellow, both plotted on the left y-axis. Steady-state concentration of cytosolic (blue) and glycosomal (green) 6-phosphogluconate are plotted on the right y-axis. Shaded areas indicate interquartile ranges. (<i>C</i>) Steady-state flux through glycolysis as a function of the glycosomal 6-PG concentration in model A. A glycosomal 6-PG concentration of around 500 mM reduces the glycolytic flux by 50%.</p