MOESM1 of Differential induction of malaria liver pathology in mice infected with Plasmodium chabaudi AS or Plasmodium berghei NK65

Additional file 1. ALT and AST determination in mice infected with P. berghei NK65 or P. chabaudi AS. C57BL/6J mice were injected intraperitoneally with 104 erythrocytes infected with P. berghei NK65 or P. chabaudi AS. Serum levels of AST (panel A), ALT (panel B) and the AST/ALT ratio (panel C) were determined at day 8 and 10 post infection according to manufacturer’s protocol (Teco Diagnostics, California, USA). n = 3-6 mice for each time point and strain, additional data can be found in [16]. *p < 0.05; **p < 0.01 versus control

<i>Pf</i>PKA-mediated phosphorylation contributes to immature GIE stiffness.

<p><b>A.</b> Retention in microsphilters of stage III GIEs from the B10 clone pre-incubated 30 min to 1 h at 37°C with 10 μM H89, 10 μM KT5720, 10 μM PKI-m, 10 μM compound 2, 10 μM GGTI-298 or 0,1% DMSO (Control). Error bars denote the standard error of the mean. *** and ** Highly significant differences in retention rates (*** <i>P</i> < 0.001; **<i>P <</i> 0.01); ns: non-significant differences in retention rates compared to control; n: number of experiments. Outliers are shown as open circles. <b>B.</b> Retention in microsphilters of stages III GIEs from the B10 clone and the pHL-<i>pfpka-r</i> clone cultivated with and without pyrimethamine for 15 generations. The pHL-<i>pfpka-r</i> clone was pre-incubated 15 min at 37°C with 100μM 8Br-cAMP. Error bars denote the standard error of the mean. ***Highly significant differences in retention rates (<i>P <</i> 0.001); ns: non-significant differences in retention rates; n: number of experiments. Outliers are shown as open circles. <b>C</b>. Western-blot analysis of <i>Pf</i>PKA-R expression in stage III GIE from the B10 clone and the pHL-<i>pfpka-r</i> clone cultivated in presence (+ pyr) or absence (- pyr) of pyrimethamine. Immunoblots were probed with rabbit polyclonal antibodies directed against <i>Pf</i>PKA-R and with a mAb directed against <i>Pf</i>HSP70 to normalize expression. Black star indicates the expected size for <i>Pf</i>PKA-R (50.8 kDa); Red star indicates <i>Pf</i>PKA-R with post-translational modifications. The experiment has been performed seven times. Error bars denote the standard error of the mean. *Significant differences in phosphorylation signal (*<i>P</i> < 0.05); ns: non-significant differences in phosphorylation signal. <b>D</b>. Quantitation of signal intensities in panel C using Quantity One software (BioRad). Analysis shows a 1.6-fold increase in <i>Pf</i>PKA-R expression in the pHL-<i>pfpka-r</i> clone (+ pyr) compared to B10. Decrease of <i>Pf</i>PKA-R expression in absence of pyrimethamine (- pyr) indicates a loss of episomal expression of the <i>Pf</i>PKA-R protein. <b>E.</b> Immunofluorescence analysis of stage III GIE from the B10 clone and the pHL-<i>pfpka-r</i> clone cultured for 15 generations in the presence (+ pyr) or absence (- pyr) of pyrimethamine. Infected erythrocytes were stained with anti-<i>Pf</i>PKA-R antibodies followed by anti-rabbit Alexa 594-conjugated IgG. Pictures were taken under identical exposure conditions. The bars represent 2 μm.</p

Sildenafil impairs mature GIE filterability.

<p><b>A</b>. Stage V GIE were harvested by magnetic isolation and incubated at 37°C for 30 min with 100 μM sildenafil, or 0.1% DMSO (Control). The total intracellular cAMP concentration was measured on aliquots of 6.10⁶ cells in duplicate wells. The assay was carried out five times. Error bars denote the standard error of the mean. **Highly significant differences in retention rates compared to control (DMSO) (<i>P <</i> 0.01). <b>B.</b> Graphical representation for the proportion of GIE showing a regular (light green), or deformed (dark green) shape in a population of paraformaldehyde-fixed GIE, as they flow through the microsphilters after pre-incubation at 37°C for 30 min with 100μM sildenafil, or 0.1% DMSO (Control). <b>C</b>. Differential interference contrast images of paraformaldehyde-fixed GIE, as they flow through the microsphilters. <b>D</b>. Retention rates in microsphilters of stage V GIE (light grey) and uninfected red blood cells (uRBC, dark grey) pre-incubated 30 min at 37°C with different concentrations of sildenafil. Error bars denote the standard error of the mean. Outliers are shown as open circles. *** and **Highly significant differences in retention rates compared to control (**<i>P <</i> 0.01; ***<i>P <</i> 0.001); ns: non-significant differences in retention rates compared to control. n: number of experiments.</p

Viability of 3D7 stage V GIE treated with different inhibitors.

<p>3D7 Stage V GIE were treated with the inhibitors for the indicated times, then washed and their viability evaluated with the colorimetric pLDH assay either immediately (time 0) or after 72 h incubation at 37°C, as described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004815#ppat.1004815.ref056" target="_blank">56</a>]. The data are expressed as percent of untreated controls and are the mean of two experiments in quadruplicate.</p><p>Viability of 3D7 stage V GIE treated with different inhibitors.</p

Interfering with cAMP levels impairs mature GIE filterability.

<p><b>A.</b> Stage V GIE were harvested by magnetic isolation and incubated at 37°C for 30 min with 150 μM forskolin, 65 μM zaprinast, or 0.1% DMSO (Control). The total intracellular cAMP concentration was measured on aliquots of 6.10⁶ cells in duplicate wells. The assay was carried out three times. Error bars denote the standard error of the mean. *Significant differences in retention rates compared to control (<i>P <</i> 0.05). <b>B.</b> Retention in microsphilters of stages V GIEs (light grey), or uninfected red blood cells (uRBCs, dark grey). GIEs were pre-incubated at 37°C for 30 min with 150 μM forskolin, 65 μM zaprinast or 0,1% DMSO (control). ***Highly significant differences in retention rates compared to control (DMSO) (<i>P <</i> 0.001); ns: non-significant differences in retention rates compared to control. n: number of experiments. <b>C.</b> Graphical representation for the proportion of GIE showing a regular (light green), or deformed (dark green) shape in a population of paraformaldehyde-fixed GIE, as they flow through the microsphilters after pre-incubation at 37°C for 30 min with 150 μM forskolin, 65 μM zaprinast or 0.1% DMSO (control).</p

The effect of PDE inhibitors on particulate fractions isolated from <i>P</i>. <i>falciparum</i> parasites.

<p>IC₅₀ values (the concentration of compound that gave 50% inhibition of native PDE activity) are mean ± SEM in μM and were calculated using the Prism software package (GraphPad Software, Inc.). Percentage inhibition data were fitted to a sigmoidal dose-response curve using non-linear regression. Bovine control PDE column displays cAMP dependent activity. All TbPDE1 and most Human values obtained from Kunz, <i>et al</i>.[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004815#ppat.1004815.ref058" target="_blank">58</a>].</p><p>*Value obtained from Tang, <i>et al</i>.[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004815#ppat.1004815.ref059" target="_blank">59</a>].</p><p>>200, sufficient inhibition was not observed for an IC₅₀ calculation.</p><p>Tb: <i>Trypanosama brucei</i>.</p><p>The effect of PDE inhibitors on particulate fractions isolated from <i>P</i>. <i>falciparum</i> parasites.</p

GIE filterability is dependent on cAMP concentration.

<p><b>A.</b> cAMP concentration drops in mature GIE. The total intracellular cAMP concentration was measured in stage III and V GIE using a competitive immunoassay for the quantitative determination of cAMP. GIE were harvested by magnetic isolation and aliquots of 6.10⁶ cells were assayed in duplicate wells. The assay was carried out three times. Error bars denote the standard error of the mean. **Highly significant difference compared to stage III GIE (<i>P <</i> 0.01). <b>B</b>. Western-blot analysis of PKA-R expression in MACS-purified stage III and stage V GIE (5.10e6 parasites/lane). Immunoblots were probed with rabbit polyclonal antibodies directed against <i>Pf</i>PKA-R and with a mAb directed against <i>Pf</i>HSP70 to normalize expression. Quantity One (BioRad) analysis shows that <i>Pf</i>PKA-R levels were not significantly different between stage III and stage V. <b>C</b>. Retention rates in microsphilters of stage III GIE (dark grey), stage V GIE (light grey) and uninfected red blood cells (uRBC, black) pre-incubated 15 min at 37°C with different concentrations of 8Br-cAMP. Error bars denote the standard error of the mean. Outliers are shown as open circles. **Highly significant differences in retention rates compared to control without 8Br-cAMP (<i>P <</i> 0.01); ns: non-significant differences in retention rates compared to control; n: number of experiments.</p

Phosphorylation events contribute to the switch in deformability.

<p><b>A.</b> Western-blot analysis of Phospho-PKA site (RRXS*/T*) expression in uninfected red blood cells (uRBCs), MACS-purified stage III (stIII) and stage V (stV) GIE from the B10 clone treated (+Calyc) or not (-Calyc) with 50 nM calyculin during 2h at 37°C. Analysis was performed on membrane extracts recovered by centrifugation after 1% Triton X100 treatment. Immunoblot was probed with rabbit monoclonal antibody directed against phospho-PKA sites (RRX*S/*T) and with mouse mAb directed against Band 3 to normalize expression. Red arrows show bands with more intense phosphorylation signal in stage III GIE than in stage V GIE. Green arrow show band with less intense phosphorylation signal in stage III GIE than in stage V GIE. The experiment has been performed three times. Error bars denote the standard error of the mean. ***Highly significant differences in phosphorylation signal (<i>P <</i> 0.001). <b>B.</b> Quantitation of signal intensities in panel D using Quantity One software (BioRad). The strength of the phosphorylation signal in untreated uninfected red blood cells lysate was set to 1 and all other signals are relative to that. <b>C.</b> Retention in microsphilters of stages V GIEs (light grey), or uninfected red blood cells (uRBCs, dark grey). GIEs were pre-incubated at 37°C for 2 h with 50 nM calyculin, or 0.5% DMSO (control). ***Highly significant differences in retention rates compared to control (<i>P <</i> 0.001); ns: non-significant differences in retention rates compared to control; n: number of experiments. <b>D</b>. Graphical representation for the proportion of GIE showing a regular (light green), or deformed (dark green) shape in a population of paraformaldehyde-fixed GIE, as they flow through the microsphilters after pre-incubation at 37°C 2 h with 50 nM calyculin, or 0.5% DMSO (control). <b>E.</b> Differential interference contrast images of paraformaldehyde-fixed GIE, as they flow through the microsphilters. A majority of DMSO-treated GIE (left panel) are twisted and deformed, whereas inhibitor-treated GIE (right panel) keep a regular shape, unlike uninfected erythrocytes (red star).</p

cAMP degradation by phosphodiesterases regulates GIE mechanical properties.

<p><b>A.</b> mRNA levels of four <i>PDE</i>s were determined by real time RT-PCR in asexual blood-stages (asex), stage III GIE (st.III) and stage V GIE (st.V). Relative amounts of transcript were normalized to mRNA levels of the <i>PfHK</i> (<i>PF08_0085)</i>, <i>Pfs48/45</i> (<i>PF13_0247)</i> and <i>Pfs25</i> (<i>PF10_0303)</i> were used as markers of stage III and stage V GIE, respectively. Triplicate PCR reactions were analysed for each sample. <b>B.</b> The total intracellular cAMP concentration was measured in stage III and V GIE from the 3D7 clone and the <i>PfPDEδ-</i>mutant clone 4 using a competitive immunoassay for the quantitative determination of cAMP. GIE were harvested by magnetic isolation and aliquots of 6.10⁶ cells were assayed in triplicate wells. The assay was carried out at least three times for each clone. <b>C.</b> Retention in microsphilters of stages V GIEs from the 3D7 clone and the <i>PfPDEδ-</i>mutant clone 4. Error bars denote the standard error of the mean. Outliers are shown as open circles. n: number of experiments.</p

Antiprotozoal Malaria Box compounds with activity in biological assays and lacking toxicity at therapeutic levels.

<p>Selectivity Index, SI, is toxicity level/activity level; p, probe-like; d, drug-like.</p