Parasite development in the mosquito.

<p>A) Exflagellation in wild type (WT-GFP) and transgenic Pb<i>^pfpkg</i> parasites. The mean number of exflagellation centres was 7.0 for wild type and 6.8 for Pb<i>^pfpkg</i> parasites. Bar, mean ± SEM (Mann-Whitney U test: ns, not significant, p>0.05 compared to WT-GFP). B) Ookinete conversion in wild type (WT-GFP) and transgenic Pb<i>^pfpkg</i> parasites. Wild type conversion was 63% and Pb<i>^pfpkg</i> conversion was 10%. Data shown as mean ± SEM (Mann-Whitney U test: ***, p<0.001 compared to WT-GFP). C) Gut oocyst numbers in wild type (WT-GFP) and transgenic Pb<i>^pfpkg</i> parasites. Wild type infected guts contained 100 oocysts and Pb<i>^pfpkg</i> infected guts with less than 10 oocysts. Bar, mean ± SEM (Mann-Whitney U test: ***, p<0.001 compared to WT-GFP).</p

Generation of Pb<i>^pfpkg</i> parasites.

<p>A) Schematic diagram of the endogenous <i>pbpkg</i> locus, the targeting construct and the transgenic <i>pb^pfpkg</i> locus. Areas of 5′UTR and 3′UTR cloned into the targeting vector are indicated, S = spacer. Arrows 1–6 indicate binding sites for primers used in diagnostic PCR. Primers 1 and 2 were used to detect 5′ integration. Primers 3 and 4 were used to determine 3′ integration. Primers 5 and 6 bind specifically to the endogenous <i>pbpkg</i> and are used to confirm absence of the endogenous gene in the transgenic line. The area homologous to the probe used in Southern blotting and <i>Bcl</i>I restriction sites used for diagnostic digest are indicated. B) Diagnostic PCR used to determine integration of the targeting construct into the Pb<i>^pfpkg</i> transgenic line. C) Southern blot following <i>Bcl</i>I digest shows integration of the targeting construct as a specific 3.9 kb band and absence of the endogenous 5.1 kb band in the transgenic line (Pb<i>^pfpkg</i>) in comparison to wild type (WT-GFP). D) PFGE of wild type (WT-GFP) and mutant parasite (Pb<i>^pfpkg</i>) confirms integration into the correct chromosome. E) Western blot of asexual blood and ookinete stages confirm expression of PfPKG in the transgenic line (the transgenic PfPKG bands are 25.8% and 21.8% of the PbPKG band in the WT-GFP line in asexual blood and ookinete stages respectively).</p

Subcellular location of PfPKG in mature schizonts.

<p>Dual immunofluorescent detection of PfPKG-HA in fixed smears of early and late schizonts of the PfPKG-HA-3A clone together with (<b>A</b>) PfGAPDH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Daubenberger1" target="_blank">[30]</a>, (<b>B</b>) PfBiP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-vanDooren1" target="_blank">[32]</a>, (<b>C</b>) PfPMV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Klemba1" target="_blank">[31]</a>, (<b>D</b>) PfRab11A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-AgopNersesian1" target="_blank">[33]</a> and (<b>E</b>) PfGAP45 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Yeoman1" target="_blank">[34]</a>. Representative images are shown for each antibody, together with bright field images (first column) and parasite nuclei stained with DAPI (in the merged image). Bars ∼5 µM. To quantify co-localisation, Pearson coefficients <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Manders1" target="_blank">[36]</a> of the individual stains were calculated using Imaris image analysis software (Bitplane).</p

Parasite morphology and global protein phosphorylation pattern of PKG inhibitor-treated <i>P. falciparum</i> schizonts.

<p>(<b>A</b>) Immunofluorescent staining of DMSO/compound 1-treated WT schizonts using antibodies detecting (i) PfGAP45 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Yeoman1" target="_blank">[34]</a>, (ii) PfSUB1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Yeoh1" target="_blank">[14]</a> and (iii) PfAMA1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Harris1" target="_blank">[23]</a>. Representative images are shown for each staining together with parasite nuclei stained with DAPI. Bars ∼5 µM. (<b>B</b>) Metabolic labelling of phosphoproteins in <i>P. falciparum</i> schizonts. Autoradiographs of (i) 3D7 WT and (ii) gatekeeper mutant 3D7 PfPKG_T618Q schizonts, treated with ³²P-orthophosphate and DMSO (−) or compound 2 (+) prior to lysis, ÄKTA anion exchange chromatography (fractions 10–14 are shown) and separation by SDS-PAGE. Rectangular boxes highlight bands that show a differential signal following inhibitor-treatment in WT, but not PfPKG_T618Q schizonts.</p

PfPKG expression peaks in late blood stages and is carbonate-soluble.

<p>(<b>A</b>) Western blots of synchronised cultures of the PfPKG-HA-3A clone and WT parasites (3D7 clone), 24 hours (mostly mid trophozoites), 30 hours (mostly late trophozoites), 41 hours (mostly early schizonts) and 46 hours (mostly late schizonts) post invasion were detected with anti-HA and anti-humanPKG, respectively. Blots were re-probed with an antibody against Pfαtubulin to estimate the relative total protein loading between lanes. (<b>B</b>) Sequential solubilisation of parasite proteins from saponin-released late trophozoites and schizonts. S1: soluble protein fraction (5 mM Tris-HCl, freeze thaw); S2: peripheral membrane fraction (extraction with 100 mM Na₂CO₃); S3: integral membrane fraction (extraction with 4% SDS/0.5% TX-114/0.5×PBS). Equal volumes of the three supernatants were analysed by SDS-PAGE and Western blots were probed for the integral membrane protein PfPMV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Klemba1" target="_blank">[31]</a>, stripped and re-probed simultaneously for PfGAPDH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Daubenberger1" target="_blank">[30]</a> and PfPKG-HA. Densitometric analysis of the scan of the blot presented revealed that 89.3% of PfPKG-HA is present in fraction S1, while fractions S2 and S3 contain 9.5% and 1.2%, respectively. (<b>C</b>) Immunofluorescent anti-HA detection in fixed smears of erythrocytic stages of the PfPKG-HA-3A clone. Representative images of (i) a ring stage parasite, (ii) three early trophozoites, (iii) an early schizont, (iv, v) late schizonts (approximate hours post invasion: (i) 4–10, (ii) 20–26, (iii) 33–39, (iv, v) 45–48) and (vi) a stage III gametocyte are shown together with bright field images (first column) and parasite nuclei stained with DAPI (second column). Bars ∼5 µM.</p

Zaprinast does not prevent invasion.

<p>Cultures containing mature schizonts (∼5% parasitaemia) were incubated in the absence (green) or presence (red and blue plots) of C1 (2.5 µM). After 160 min (arrowed), all cultures were washed and recultured in fresh medium minus C1, but with the addition of zaprinast (75 µM) to one culture (blue). Appearance of rings was monitored with time. Within 30 min following the wash and reculture step, the parasitaemia profiles had merged, demonstrating rapid invasion immediately following release of the C1 block, regardless of the presence of zaprinast. The subsequent relatively slow increase in parasitaemia in the zaprinast-containing culture (blue) likely reflects premature egress of the small number of residual immature schizonts still present at the point of reculture, which would release non-invasive merozoites. Time-points at which statistically different parasitaemia values were obtained for the two cultures which had been subjected to the C1 block are indicated with asterisks (Student's unpaired <i>t</i>-test, two-tailed P value = 0.0024 [225 min time-point] and P value = 0.0045 [285 min time-point]). Data are presented as mean values of triplicate counts ±SD. See also Figure S6 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#ppat.1003344.s005" target="_blank">Text S1</a>.</p

PfPKG inhibitors block discharge of exonemes and micronemes.

<p>(A) IFA of wt 3D7 parasites allowed to develop beyond the point of egress in the presence of E64 only (50 µM). Top two rows: segmented schizonts displaying a merozoite surface localisation of PfAMA1 (arrowed; ∼45% of the schizont population) as a result of its discharge from micronemes, exhibit a weak PfSUB1 signal compared to less mature schizonts in which both the PfAMA1 and PfSUB1 signals remain punctate. The bottom row of images in (A) shows a schizont in which the PfAMA1 signal is intermediate between punctate and merozoite surface. (B) Surface translocation of PfAMA1 is associated with loss or redistribution of the PfSUB1-specific IFA signal. The PfSUB1-specific mean fluorescence intensity in individual schizonts relative to that of the PfAMA1-specific signal was acquired as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#s4" target="_blank">Materials and Methods</a>. Individual relative fluorescence intensity values (at least 17 schizonts per group) are shown plotted, with mean and SD indicated. Within the parasites treated with E64 only, values were significantly lower for the schizonts displaying the merozoite surface PfAMA1 phenotype (Student's unpaired <i>t</i>-test, two-tailed P value<0.0001). There was no significant difference between the E64-treated and E64 plus C1-treated values for schizonts with the punctate PfAMA1 phenotype (see panel C). (C) IFA of wt 3D7 parasites allowed to develop beyond the point of egress in the presence of both E64 and C1. No discharge or relocalisation of PfSUB1 or PfAMA1 was evident in counts of >5,000 schizonts from a total of 3 independent experiments. Identical results were obtained with E64 plus C2 (not shown). (D) Western blot of culture supernatants from synchronous wt 3D7 or PfPKG_T618Q schizonts allowed to develop beyond the point of egress in the presence of E64 only (50 µM), C1 only (2.5 µM), C2 only (1.5 µM), or indicated combinations. Cultures were sampled immediately (start) or after incubation for 4 h. The p54 and p47 forms of PfSUB1 usually observed following its maturation are arrowed. The relatively low levels of PfSUB1 in supernatants of the PfPKG_T618Q parasites in the presence of C1 or C2 plus E64 are likely due to the block in egress mediated by E64. See also Figure S3 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#ppat.1003344.s005" target="_blank">Text S1</a>.</p

Structurally distinct PfPKG inhibitors block PfSUB1-mediated proteolytic processing in <i>P. falciparum</i>.

<p>(A) Molecular structures of C1 and C2. (B–D) Mature wild-type (wt 3D7) or PfPKG_T618Q<i>P. falciparum</i> schizonts were supplemented with cysteine protease inhibitor E64 only (50 µM), C1 only (2.5 µM), C2 only (1.5 µM), or combinations indicated, or vehicle only (DMSO, 1% v/v). Cultures were sampled immediately (start) or after further incubation for 4 h. SDS extracts of the residual schizonts (B, C) or culture supernatants (D) were analysed by Western blot, probing with mAbs specific for MSP1 or SERA5. An antibody to erythrocyte spectrin was used as loading control for the schizont extracts. Processing of MSP1 by PfSUB1 converts it to 4 polypeptides that include the MSP1₄₂ fragment <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#ppat.1003344-Koussis1" target="_blank">[4]</a>. In wt parasites, but not in the PfPKG_T618Q clone, both C1 and C2 inhibited PfSUB1-mediated conversion of MSP1 to MSP1₄₂ (B) or conversion of the SERA5 precursor P126 to the processing products P56 and P50 (C), although some background processing is evidence in the presence of the inhibitors, perhaps due to imperfect synchrony of the parasite cultures. The relatively low abundance of processed SERA5 in the PfPKG_T618Q schizont extracts in the presence of C1 or C2 only (C) are likely due to their loss from the schizonts at egress. Note that E64 inhibits conversion of P56 to P50, which is not mediated by PfSUB1 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#ppat.1003344-Yeoh1" target="_blank">[1]</a>. In (D), treatment of wt 3D7 with C1 or C2 resulted in release of only low levels of unprocessed SERA5 P126, whereas the normally-processed P50 form appeared in the PfPKG_T618Q supernatants irrespective of the presence of the PfPKG inhibitors. See also Figure S1 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#ppat.1003344.s005" target="_blank">Text S1</a>.</p

The phosphodiesterase inhibitor zaprinast induces premature egress in a cGMP-dependent manner.

<p>(A) Zaprinast induces merozoite egress. Mature schizonts (∼25% segmented) were supplemented with fresh RBCs to achieve a parasitaemia of ∼40%, then either processed at once for microscopic examination of Giemsa-stained thin films (Start), or after further culture for 50 min in the presence of zaprinast or vehicle only (DMSO, 0.5% v/v). (B) Dose-response profile of zaprinast-induced egress. Schizont preparations similar to those in (A), but in protein-free medium and without addition of uninfected RBCs, were incubated for 50 min in the presence of various concentrations of zaprinast. Culture supernatants were then examined by Western blot using mAbs reactive with SERA5, PfAMA1 or MSP1. (C) Quantitation by capture ELISA of shed PfAMA1, and its dependency on zaprinast concentration. Samples from cultures treated as in (B) for 50 min were serially diluted and assayed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#s4" target="_blank">Materials and Methods</a>. Data are presented as mean values of triplicate readings ±SEM, with PfAMA1 concentrations in arbitrary units based on comparison with a standard. Dotted lines indicate [PfAMA1] values in the absence of zaprinast treatment (lower line), or 50% maximal levels (upper line), used to derive an ED₅₀ for zaprinast of ∼25 µM. (D) Zaprinast-induced egress is blocked by PfPKG inhibitors. Western blot of supernatants harvested at different time-points from a schizont culture treated with a single concentration of zaprinast (75 µM), with or without the additional presence of C1 or C2. Blots were probed with the anti-SERA5 mAb 24C6.1F1. (E) Quantitation by ELISA of shed PfAMA1 levels in the supernatant samples probed in (D). Data are presented as in (C). (F) Zaprinast treatment increases cGMP levels in schizonts. The 0 min sample was taken just prior to addition of zaprinast ±C1. (G) BAPTA-AM inhibits egress, and this can be reversed by zaprinast treatment. Schizonts in protein-free medium were incubated in the presence of the indicated reagents for 50 min. BAPTA-AM was used at 100 µM, and zaprinast at 75 µM. See also Figure S4 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003344#ppat.1003344.s005" target="_blank">Text S1</a> and Movies S3 and S4.</p

Model of the role of PKG in egress.

<p>(A) PfSUB1 and PfAMA1 are stored in exonemes and micronemes respectively in developing intracellular merozoites. Rapid turnover of cGMP maintains PfPKG in an ‘off’ state. (B) Upon an endogenous or exogenously-supplied signal (which can be mimicked by zaprinast-mediated inhibition of one or more parasite PDEs), cGMP levels are raised above threshold levels, resulting in activation of PfPKG and phosphorylation of its substrates. This leads to discharge of both PfSUB1 and PfAMA1, perhaps via an increase in cytosolic Ca²⁺ released from intracellular stores. PfAMA1 translocates onto the merozoite surface, whereas PfSUB1 is released into the PV. PVM rupture rapidly ensues, as well as limited permeabilization of the RBC membrane. (C) The Ca²⁺ flux may activate CDPK5 for a downstream role in egress. Eventual rupture of the RBC membrane is mediated by at least one E64-sensitive cysteine protease, allowing egress. PfPKG may rapidly return to an inactive state upon lowering of cGMP levels, whilst PfPKG substrates may be dephosphorylated through the action of endogenous phosphatases.</p