Artemisinin-resistant K13 mutations rewire Plasmodium falciparum's intra-erythrocytic metabolic program to enhance survival

The emergence and spread of artemisinin resistance, driven by mutations in Plasmodium falciparum K13, has compromised antimalarial efficacy and threatens the global malaria elimination campaign. By applying systems-based quantitative transcriptomics, proteomics, and metabolomics to a panel of isogenic K13 mutant or wild-type P. falciparum lines, we provide evidence that K13 mutations alter multiple aspects of the parasite's intra-erythrocytic developmental program. These changes impact cell-cycle periodicity, the unfolded protein response, protein degradation, vesicular trafficking, and mitochondrial metabolism. K13-mediated artemisinin resistance in the Cambodian Cam3.II line was reversed by atovaquone, a mitochondrial electron transport chain inhibitor. These results suggest that mitochondrial processes including damage sensing and anti-oxidant properties might augment the ability of mutant K13 to protect P. falciparum against artemisinin action by helping these parasites undergo temporary quiescence and accelerated growth recovery post drug elimination

A Putative Homologue of CDC20/CDH1 in the Malaria Parasite Is Essential for Male Gamete Development

Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis

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

Spatiotemporal and functional characterisation of the Plasmodium falciparum cGMP-dependent protein kinase.

Signalling by 3'-5'-cyclic guanosine monophosphate (cGMP) exists in virtually all eukaryotes. In the apicomplexan parasite Plasmodium, the cGMP-dependent protein kinase (PKG) has previously been reported to play a critical role in four key stages of the life cycle. The Plasmodium falciparum isoform (PfPKG) is essential for the initiation of gametogenesis and for blood stage schizont rupture and work on the orthologue from the rodent malaria parasite P. berghei (PbPKG) has shown additional roles in ookinete differentiation and motility as well as liver stage schizont development. In the present study, PfPKG expression and subcellular location in asexual blood stages was investigated using transgenic epitope-tagged PfPKG-expressing P. falciparum parasites. In Western blotting experiments and immunofluorescence analysis (IFA), maximal PfPKG expression was detected at the late schizont stage. While IFA suggested a cytosolic location, a degree of overlap with markers of the endoplasmic reticulum (ER) was found and subcellular fractionation showed some association with the peripheral membrane fraction. This broad localisation is consistent with the notion that PfPKG, as with the mammalian orthologue, has numerous cellular substrates. This idea is further supported by the global protein phosphorylation pattern of schizonts which was substantially changed following PfPKG inhibition, suggesting a complex role for PfPKG during schizogony

Malaria protein kinase CK2 (PfCK2) shows novel mechanisms of regulation

Casein kinase 2 (protein kinase CK2) is a conserved eukaryotic serine/theronine kinase with multiple substrates and roles in the regulation of cellular processes such as cellular stress, cell proliferation and apoptosis. Here we report a detailed analysis of the Plasmodium falciparum CK2, PfCK2, demonstrating that this kinase, like the mammalian orthologue, is a dual specificity kinase able to phosphorylate at both serine and tyrosine. However, unlike the human orthologue that is auto-phosphorylated on tyrosine within the activation loop, PfCK2 shows no activation loop auto-phosphorylation but rather is auto-phosphorylated at threonine 63 within subdomain I. Phosphorylation at this site in PfCK2 is shown here to regulate the intrinsic kinase activity of PfCK2. Furthermore, we generate an homology model of PfCK2 in complex with the known selective protein kinase CK2 inhibitor, quinalizarin, and in so doing identify key co-ordinating residues in the ATP binding pocket that could aid in designing selective inhibitors to PfCK2

PfCK2 auto-phosphorylates <i>in vitro</i> on threonine 63.

<p><b>A</b>: <i>In vitro</i> kinase assay for GST-PfCK2 autophosphorylation, top panel: autoradiograph, bottom panel: Coomassie stain. <b>B</b>: LC-MS/MS trace identifying phosphorylation of PfCK2 at T63; right: Also shown is the hypothetical fragmentation table where the b-ions and y-ions detected in the LC-MS/MS spectra are shown in red and bold, respectively. <b>C</b>: Sequence of PfCK2 showing the phosphopeptide identified in the LC-MS/MS analysis (underlined) and the threonine 63 phosphorylation site (in red).</p

PfCK2 phosphorylates MCM2 on Ser13 and Tyr16 <i>in vitro</i>.

<p><b>A</b>: In vitro kinase assay using a GST fusion protein containing a N-terminal portion of MCM2 (GST-MCM2) or the same fusion protein but where residue Y16 is mutated to an phenylalanine (Y16F) or where residue S13 is mutated to an alanine (S13A) or where both S13 and Y16 are mutated to an alanine and phenylalanine respectively (S13A/Y16F). Top panel: autoradiograph, bottom panel: Coomassie stain. <b>B</b>: LC-MS/MS trace of the fusion protein GST-PfMCM2 containing the S13 to alanine mutation following phosphorylation with PfCK2 indicating the phosphorylation of residue Y16. Also shown is the fragmentation table (detected b-ions and y-ions are represented respectively in bold red and bold blue). <b>C</b>: N-terminal sequence of PfMCM2 protein showing the phospho-peptide identified in the LC-MS/MS analysis that contains the tyrosine phosphorylated residue (in red).</p

Autophosphorylation of PfCK2 regulates kinase activity.

<p>The activity of PfCK2α and a mutant PfCK2α where threonine 63 was mutated to alanine (T63A) was tested in <i>in vitro</i> kinase assays using α-casein as a substrate. <b>A</b>: Example of the <i>in vitro</i> kinase assay with PfCK2α and the T63A mutant. Top panel: autoradiograph, bottom panel: Coomassie stain. <b>B</b>: kinase activity quantification. Date represents the mean ± S.E.M (n = 3) <b>C</b>: LC-MS/MS trace of PfCK2 identifying T63 phosphorylation from a shizont stage lysate of <i>P. falciparum</i>. Indicated are the b-ions and b-ions (−98daltons) that were identified in the LC-MS/MS spectra. Also shown is the hypothetical fragmentation table where the ions that were identified in the LC-MS/MS spectra are shown in red.</p