The 3-phosphoinositide–dependent protein kinase 1 is an essential upstream activator of protein kinase A in malaria parasites

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signalling is essential for the proliferation of Plasmodium falciparum malaria blood stage parasites. The mechanisms regulating the activity of the catalytic subunit PfPKAc, however, are only partially understood, and PfPKAc function has not been investigated in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown (cKD) mutant, we confirm the essential role for PfPKAc in erythrocyte invasion by merozoites and show that PfPKAc is involved in regulating gametocyte deformability. We furthermore demonstrate that overexpression of PfPKAc is lethal and kills parasites at the early phase of schizogony. Strikingly, whole genome sequencing (WGS) of parasite mutants selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the parasite orthologue of 3-phosphoinositide–dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we demonstrate that PfPDK1 is required to activate PfPKAc and that T189 in the PfPKAc activation loop is the crucial target residue in this process. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and imply that PfPDK1 acts as a crucial upstream regulator in this pathway and potential new drug target.


Introduction
Malaria is caused by protozoan parasites of the genus Plasmodium. Infections with Plasmodium falciparum are responsible for the vast majority of severe and fatal malaria cases. People get infected through female Anopheles mosquitoes that inject sporozoites into the skin tissue during their blood meal. After reaching the liver, sporozoites infect and multiply inside hepatocytes, generating thousands of merozoites that are released into the blood stream. Merozoites of conditional loss-of-function mutants showed that PfPKAc is essential for the process of merozoite invasion, where it is required for the phosphorylation and timely shedding of the invasion ligand AMA1 from the merozoite surface [5,6,8,9]. Likewise, depletion of cAMP levels through conditional disruption of pfacβ phenocopied the invasion defect observed for the pfpkac mutant [8]. Interestingly, a conditional pfpdeβ null mutant, which displays increased cAMP levels and PfPKAc hyperactivation, also showed a severe merozoite invasion defect that was linked to elevated phosphorylation and premature shedding of AMA1 [27]. These studies highlighted that tight regulation of PfPKAc activity is crucial for successful merozoite invasion and parasite proliferation. In addition, PfPKA seems to have additional functions in blood stage parasites. Global phosphoproteomic studies of pfpdeβ, pfacβ, and pfpkac conditional knockout (KO) cell lines identified 39 proteins as high confidence targets of cAMP/PfPKAdependent phosphorylation [8,27]. These proteins include invasion factors (e.g., AMA1 and coronin) and several proteins with predicted roles in other processes (e.g., chromatin organisation and protein transport) or with unknown functions [8,27]. In addition, cAMP/PfPKAdependent signalling has been implicated in the regulation of ion channel conductance and new permeability pathways (NPPs) in asexual blood stage parasites [28] and gametocytes [29] as shown through the use of pharmacological approaches (PKA/PDE inhibitors, exogenous 8-Bromo-cAMP) and transgenic cell lines (deletion of PfPDEδ, overexpression of PfPKAr) [28,29]. Similar experiments identified a putative role for cAMP/PfPKA-dependent signalling in regulating gametocyte-infected erythrocyte deformability [30].
While several studies demonstrated the importance of cAMP in activating PfPKAc, the role of PfPKAc phosphorylation in regulating PfPKAc activity remains elusive. High-throughput phosphoproteomic approaches identified several phosphorylated residues in PfPKAc, including T189 that corresponds to the PDK1 target residue T197 in the activation segment of mammalian PKAc [31][32][33][34]. However, if and to what extent phosphorylation of T189 is important for PfPKAc activation in P. falciparum and whether T189 phosphorylation is deposited via autophosphorylation or by another kinase, is unknown. Furthermore, besides the well-established role for PfPKAc in parasite invasion, other possible functions of PfPKA in asexual and sexual development are only poorly understood.
Here, we used reverse genetics approaches to study the function of PfPKAc in asexual blood stage parasites, sexual commitment, and gametocytogenesis. Our results confirm the essential role for PfPKAc in merozoite invasion and show that while PfPKAc plays no obvious role in the control of sexual commitment or gametocyte maturation, it contributes to the regulation of gametocyte-infected erythrocyte deformability. We further demonstrate that overexpression of PfPKAc is lethal in asexual blood stage parasites. Intriguingly, whole genome sequencing (WGS) of parasites selected to tolerate PfPKAc overexpression identified mutations exclusively in the gene encoding the P. falciparum orthologue of phosphoinositidedependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we show that the T189 residue is crucial for PfPKAc activity and that activation of PfPKAc is likely dependent on PfPDK1-mediated phosphorylation.

PfPKAc plays no major role in sexual commitment and gametocyte maturation but contributes to the regulation of gametocyte rigidity
To investigate whether PfPKAc activity regulates sexual commitment, we split ring stage parasites (0 to 6 hpi) and cultured them separately under PfPKAc-GFPDD-depleting (-Shield-1/ +GlcN) and PfPKAc-GFPDD-stabilising conditions (+Shield-1/-GlcN). Sexually committed parasites were identified based on PfAP2-G-mScarlet positivity in late schizonts (40 to 46 hpi) using high content imaging. We observed a significant increase in SCRs in PfPKAc-GFPDDdepleted (-Shield-1/+GlcN) compared to control parasites (+Shield-1/-GlcN) (1.56-fold ± 0.13 SD) (S3 Fig). We can exclude that these differences are caused by the Shield-1 compound as we have previously shown that Shield-1 treatment has no effect on SCRs [43]. However, treatment with GlcN also caused a similar increase in SCRs in NF54/AP2-G-mScarlet control parasites (1.44-fold ± 0.14 SD) (S3 Fig). We therefore conclude that PfPKAc plays no major role in regulating sexual commitment and that the increased SCRs observed in PfPKAc-GFPDD-depleted parasites are caused by the presence of 2.5 mM GlcN in the culture medium.
Lastly, we tested if PfPKAc activity is involved in regulating gametocyte rigidity. Immature gametocytes display high cellular rigidity and sequester in the bone marrow and the spleen, whereas stage V gametocytes are more deformable and can reenter the bloodstream to be taken up by feeding mosquitoes [45][46][47]. Experiments employing PKA and PDE inhibitors, a transgenic cell line overexpressing PfPKAr, or treatment with exogenous 8-bromo-cAMP to increase cellular cAMP levels provided evidence for a potential role for PfPKAc in maintaining the rigidity of immature gametocyte-infected erythrocytes [30]. To test if PfPKAc is indeed involved in controlling this process, we measured the deformability status of immature stage III and mature stage V gametocytes using microsphiltration experiments [46]. Microsphiltration exploits the fact that differences in cellular rigidity correlate with cell retention rates in a microsphere-based artificial spleen system [46,47]. We observed a slight but significant decrease in the retention rates of PfPKAc-GFPDD-depleted gametocytes (-Shield-1) compared to the control (+Shield-1), both in immature stage III (day 6) (76.0% ± 5.7 SD versus 82.2% ± 8.0 SD) and mature stage V (day 11) gametocytes (13.9% ± 16.8 SD versus 27.6% ± 14.5 SD) (Fig 1E). By contrast, NF54 WT gametocytes cultured in the presence or absence of Shield-1 showed no difference in retention rates (S4 Fig). In summary, our results demonstrate that PfPKAc plays no major role in the regulation of sexual commitment, gametocyte maturation, or male gametogenesis but that gametocyte-iRBC rigidity is at least partially regulated by PfPKAc. Furthermore, we discovered that the presence of 2.5 mM GlcN in the culture medium affects both sexual commitment and ExRs, which needs to be taken into account when studying these processes in conditional mutants employing the glmS riboswitch system.

PfPKAc overexpression in asexual blood stage parasites is lethal
The merozoite invasion and post-invasion developmental defects observed for pfpdeβ KO parasites had been linked to increased cAMP levels and PfPKAc hyperactivity [27]. To further study the consequences of PfPKAc overexpression, we generated a PfPKAc conditional overexpression (cOE) line using CRISPR/Cas-9-based gene editing. To this end, we inserted an ectopic pfpkac-gfp transgene cassette into the nonessential glp3 (cg6, Pf3D7_0709200) locus in NF54 WT parasites (NF54/PKAc cOE) (S5 Fig). Here, the constitutive calmodulin (PF3D7_1434200) promoter and a glmS ribozyme element [48] control expression of the pfpkac-gfp gene. Since the initial transgenic NF54/PKAc cOE population still contained some parasites carrying the WT glp3 locus, we obtained clonal lines by limiting dilution cloning [49]. In 2 clones (NF54/PKAc cOE M1 and M2), correct integration of the inducible PfPKAc-GFP OE expression cassette and absence of WT parasites was confirmed by PCR on gDNA (S5 Fig). Live cell fluorescence imaging and western blot analysis confirmed the efficient induction of PfPKAc-GFP OE upon GlcN removal in both clones (Figs 2A and S6). Interestingly, PfPKAc-GFP OE (-GlcN) resulted in a complete block in parasite development half way through the IDC (Fig 2B). To study this growth defect in more detail, we quantified the number of nuclei per parasite in late schizont stages (40 to 46 hpi), 40 hours after triggering PfPKAc-GFP OE (-GlcN) in young ring stage parasites (0 to 6 hpi). This experiment revealed that NF54/PKAc cOE M1 parasites overexpressing PfPKAc-GFP did not develop beyond the late trophozoite/early schizont stage as most parasites contained only 1 or 2 nuclei as opposed to the control population (+GlcN) that progressed normally through several rounds of nuclear division (Fig 2C). To test whether these parasites were still able to produce progeny, we performed parasite multiplication assays. NF54/PKAc cOE M1 ring stage parasites were split at 0 to 6 hpi, cultured separately in the presence or absence of GlcN and parasite multiplication was quantified over 3 generations. PfPKAc-GFP OE (-GlcN) completely failed to multiply as no increase in parasitaemia was observed, in contrast to the control population (+GlcN) that multiplied normally (Figs 2D and S6). Notably, however, viable PfPKAc-GFP OE parasites emerged approximately 2 weeks after maintaining NF54/PKAc cOE M1 parasites constantly in culture medium lacking GlcN. We termed these parasites "PfPKAc OE survivors" (NF54/ PKAc cOE S1). Importantly, NF54/PKAc cOE S1 parasites still overexpressed PfPKAc-GFP in absence of GlcN (Figs 2E and S6). Furthermore, Sanger sequencing confirmed that neither the endogenous nor the ectopic pfpkac genes in the NF54/PKAc cOE S1 survivor population carried any mutations. Quantifying the number of nuclei per schizont and parasite multiplication assays revealed that NF54/PKAc cOE S1 parasites were completely tolerant to PfPKAc-GFP OE and developed and multiplied identically irrespective of whether PfPKAc-GFP OE was induced (-GlcN) or not (+GlcN) (Figs 2F, 2G and S6).
In conclusion, PfPKAc OE causes a lethal phenotype in asexual parasites by preventing parasite development beyond the late trophozoite/early schizont stage. However, parasites tolerant to PfPKAc-GFP OE can be selected for, and these parasites show no defect in intraerythrocytic development and parasite multiplication.

Parasites tolerant to PfPKAc overexpression carry mutations in the gene encoding P. falciparum 3-phosphoinositide-dependent protein kinase-1 (PfPDK1)
The above findings suggested that genetic mutations in the NF54/PKAc cOE S1 survivor population might cause their tolerance to elevated PfPKAc-GFP expression levels. To address this hypothesis, we performed WGS of the 2 parental NF54/PKAc cOE clones M1 and M2 and the 6 independently grown survivor populations (NF54/PKAc cOE S1-S6), 3 each originating from the M1 and M2 clones, respectively. Intriguingly, we found that all 6 NF54/PKAc cOE survivors, but not the parental M1 and M2 clones, carried missense mutations in the gene encoding a putative serine/threonine protein kinase (Pf3D7_1121900) (Fig 3A, S1 Data). No other mutations were identified in the NF54/PKAc cOE survivor populations, consistent with the Sanger sequencing results, neither the endogenous nor the ectopic pfpkac gene carried mutations in any of the 6 survivors. The Plasmodium vivax orthologue of Pf3D7_1121900 (PVX_091715) is annotated as putative 3-phosphoinositide dependent protein kinase-1 (PDK1) (www.plasmodb.org), and a multiple sequence alignment suggested that Pf3D7_1121900 is indeed an orthologue of PDK1, a kinase widely conserved among eukaryotes and known as a master regulator of AGC kinases including PKA [17,50] (S7 Fig). However, similar to PDK1 enzymes from most fungi, nonvascular plants, and other alveolates, Pf3D7_1121900 and its P. vivax orthologue lack the carboxyl-terminal phospholipid-binding pleckstrin homology (PH) domain that is found in PDK1s from animals and vascular plants and important to localise PDK1 to the plasma membrane for PKB activation in response to the PI3K-dependent production of phosphatidylinositol bis-/trisphosphates [17,50,51]. Construction of a homology model of the Pf3D7_1121900 protein kinase domain based on the human PDK1 crystallographic structure (PDB ID 1UU9) [52] allowed us to visualise the location of the amino acids mutated in the 6 NF54/PKAc cOE survivors ( Fig 3B). None of these mutations affected the putative PIF-binding pocket. Rather, all mutated amino acids were located at or in the periphery of the predicted ATP-binding cleft, although only one mutation (N45S) may influence ATP coordination directly (Figs 3B and S7). We hence surmise that all identified mutations alter the catalytic activity but not the protein interaction preferences of PfPDK1.
In conclusion, we identified missense mutations in the Pf3D7_1121900 gene in 6 independently obtained NF54/PKAc cOE survivor parasite lines, which likely confer tolerance to PfPKAc OE. Bioinformatic analysis indicates that this gene encodes PfPDK1, the P. falciparum orthologue of PDK1, and modelling of the PfPDK1 structure predicts that the acquired mutations may affect its catalytic activity. The raw data are available in the source data file (S2 Data). (G) Increase in parasitaemia in NF54/PKAc cOE S1 survivor parasites over 3 generations under PfPKAc-GFP OE-inducing (-GlcN) and control (+GlcN) conditions. Parasites were cultured as described in panel D. Open squares represent data points for individual replicates and the means and SD (error bars) of 3 biological replicates are shown. The raw data are available in the source data file (S2 Data). cOE, conditional overexpression; DIC, differential interference contrast; GlcN, glucosamine.
https://doi.org/10.1371/journal.pbio.3001483.g002 Conditional depletion of PfPDK1 has no major effect on parasite multiplication, sexual commitment, and gametocytogenesis To gain further insight into the function of PfPDK1, we attempted to generate a pfpdk1 KO line by gene disruption using a CRISPR/Cas-9-based single plasmid approach [53]. However, consistent with the results obtained in previous kinome-or genome-wide KO screens in P. falciparum and Plasmodium berghei [31,54,55], we failed to obtain a pfpdk1 null mutant, indicating that the gene is essential in asexual parasites. We therefore engineered the PfPDK1 conditional knockdown line NF54/PDK1 cKD by tagging the endogenous pfpdk1 gene with gfp fused to the dd sequence in NF54 WT parasites (S8 Fig). Correct editing of the pfpdk1 gene and absence of the WT locus was confirmed by PCR on gDNA. Donor plasmid integration downstream of the pfpdk1 gene was detected in a subset of parasites (S8 Fig), but this is not expected to compromise pfpdk1-gfpdd expression since the 551 bp 3 0 homology region (HR) used for homology-directed repair includes the native terminator as based on published RNA sequencing (RNA-seq) data [56]. Live cell fluorescence imaging and western blot analysis revealed that PfPDK1-GFPDD is expressed in the cytosol and nucleus throughout the IDC with highest expression in schizonts, consistent with published gene expression data [56,57] (Figs 4A and S9). Furthermore, PfPDK1-GFPDD expression was efficiently reduced after Shield-1 removal compared to parasites grown in the presence of Shield-1 (Figs 4B and S9). Surprisingly, PfPDK1-GFPDD-depleted parasites (-Shield-1) showed slightly higher multiplication rates compared to the control (+Shield-1) (Fig 4C). This increased multiplication rate cannot be attributed to the removal of Shield-1 itself, since NF54 WT parasites cultured in the presence or absence of Shield-1 multiplied equally (S9 Fig). We also tested whether PfPDK1-GFPDD plays a role in gametocytogenesis but did not detect any differences in SCRs, gametocyte morphology or ExRs when comparing PfPDK1-GFPDD-depleted (-Shield-1) with control parasites (+Shield-1) (S10 Fig).
Taken together, we show that PfPDK1 is expressed in the parasite nucleus and cytosol throughout asexual development, reaching peak expression in schizonts. While PfPDK1 is likely essential, the results obtained with the PfPDK1 cKD mutant suggest that residual PfPDK1 expression levels are sufficient to sustain parasite viability. Furthermore, PfPDK1 seems to play no major role in regulating sexual commitment, gametocytogenesis or male gametogenesis, but it is again conceivable that residual PfPDK1 expression in the cKD mutant may have been sufficient to maintain these processes.

Expression of WT PfPDK1 is incompatible with PfPKAc overexpression, whereas expression of the M51R PfPDK1 mutant causes PfPKAc overexpression tolerance
To test if PfPDK1 is indeed involved in regulating PfPKAc activity, we used CRISPR/Cas-9based targeted mutagenesis to change the PfPDK1 M51R mutation back to the WT sequence in the NF54/PKAc cOE S1 survivor line (NF54/PKAc cOE S1/PDK1_wt). Sanger sequencing independently grown survivors (S1-S6). Missense mutations and their positions within the pfpdk1 coding sequence as well as the corresponding amino acid substitutions in the PfPDK1 protein sequence are shown. (B) Predicted PfPDK1 structure shown in orthogonal (top left versus top right) or opposing (top left versus lower left) views. PfPDK1 was modelled on the crystallographic structure of human PDK1 (PDB ID 1UU9) [52]. PfPDK1 segments with no correspondence in human PDK1 (amino acids 1 to 27, 188 to 307, and 423 to 525) were omitted from modelling. The ATP substrate (sticks), mutated amino acids (substitution in parenthesis; red spheres), and residues forming the PIFbinding pocket [22] (green sticks) are indicated. The PIF-binding pocket is shown in surface representation in the lower right view. c., cDNA; cOE, conditional overexpression; PIF, PDK1-interacting fragment; WGS, whole genome sequencing.
These data collectively confirm the importance of the pfpdk1 mutations identified in NF54/PKAc cOE survivor parasites in conferring resistance to PfPKAc OE and suggest that PfPDK1 is the kinase that phosphorylates and activates PfPKAc. They furthermore imply that the PfPDK1 M51R mutant kinase can still phosphorylate PfPKAc with reduced efficiency and therefore sustain parasite viability in the presence of elevated PfPKAc levels.

The PfPKAc activation loop residue T189 is essential for PfPKAc activity
Previous research in human cell lines and yeast identified a specific threonine residue in the PKAc activation loop (T197 in mammals) as the target of PDK1-dependent phosphorylation [15,18,23]. In PfPKAc, T189 likely represents the activation loop phosphorylation site corresponding to T197 in human PKAc. Hence, we tested whether the T189 residue is indeed important for PfPKAc activity. To achieve this, we employed the same approach as already used to obtain NF54/PKAc cOE parasites (S5 Fig) to generate the NF54/PKAcT189V cOE line that conditionally overexpresses a mutated version of PfPKAc in which T189 has been parasitaemia (left) and parasite multiplication rates (right) under PfPDK1-GFPDD-depleting (-Shield-1) and control (+Shield-1) conditions. Synchronous parasites (0 to 6 hpi) were split (±Shield-1) 18 hours before the first measurement in generation 1. Open squares represent data points for individual replicates and the means and SD (error bars) of 3 biological replicates are shown. Differences in multiplication rates have been compared using a paired 2-tailed Student t test (statistical significance cutoff: p < 0.05). The raw data are available in the source data file (S2 Data). cKD, conditional PfPKAc knockdown; DIC, differential interference contrast.
https://doi.org/10.1371/journal.pbio.3001483.g004 Live cell fluorescence microscopy and western blot analysis confirmed that PfPKAcT189V-GFP OE was efficiently induced upon removal of GlcN (Figs 6A and S13). Strikingly, in contrast to the lethal effect provoked by OE of PfPKAc-GFP, OE of the PfPKAcT189V-GFP mutant (-GlcN) had no effect on intraerythrocytic parasite development, multiplication, and survival when compared to the control population (+GlcN) (Fig 6B). Hence, these results suggest that PfPKAc activity is strictly dependent on phosphorylation of T189 in the activation loop segment. In combination with the findings obtained through the mutational analysis of PfPDK1, they further imply that PfPDK1 is directly or indirectly responsible for T189 phosphorylation and thus activation of PfPKAc. Increase in parasitaemia (left) and parasite multiplication rates (right) of NF54/PKAcT189V cOE parasites over 3 generations under PfPKAcT189V-GFP OE-inducing (-GlcN) and control conditions (+GlcN). Synchronous parasites (0 to 6 hpi) were split (±GlcN) 18 hours before the first measurement in generation 1. Open squares represent data points for individual replicates and the means and SD (error bars) of 3 biological replicates are shown. Differences in multiplication rates have been compared using a paired 2-tailed Student t test (statistical significance cutoff: p < 0.05). The raw data are available in the source data file (S2 Data). DIC, differential interference contrast. https://doi.org/10.1371/journal.pbio.3001483.g006

Kinase inhibitors targeting human PDK1 are active against asexual blood stage parasites
Many PDK1-dependent AGC kinases (e.g., AKT/PKB, RSK, PKC, S6K, and SGK) are downstream effectors in the PI3K/protein kinase B (AKT) or mitogen-activated protein kinase (MAPK) growth factor signalling pathways and are aberrantly activated in various types of cancer in humans [60]. In addition, PDK1 expression itself is augmented in many tumours [60]. For these reasons, human PDK1 is pursued as a potential drug target for cancer therapy, and a large number of inhibitors targeting human PDK1 have been developed and patented over the past 15 years [61][62][63]. For instance, BX-795 and BX-912, 2 related aminopyrimidine compounds, inhibit recombinant human PDK1 activity with half-maximal inhibitory concentrations (IC 50 ) of 11 nM and 26 nM, respectively [64], and the aminopyrimidine-aminoindazole GSK2334470 has similar in vitro potency against PDK1 (15 nM IC 50 ) [65,66]. While BX-795 was shown to also inhibit several other human kinases in vitro [67], GSK2334470 displayed high specificity for PDK1 over a large panel of other recombinant human kinases [65]. In cell-based assays, all 3 compounds inhibited the PDK1-dependent phosphorylation of several AGC kinase substrates at submicromolar concentrations [64][65][66]. Here, we tested these 3 commercially available ATP-competitive inhibitors of human PDK1 for their potential to kill P. falciparum asexual blood stage parasites using a [ 3 H]-hypoxanthine incorporation assay [68]. We found that BX-795, BX-912, and GSK2334470 all inhibited parasite proliferation with IC 50 values of 1.83 μM (± 0.23 SD), 1.31 μM (± 0.24 SD), and 1.83 μM (± 0.11 SD), respectively (S14 Fig). In an attempt to test whether the lethal effect of these molecules is due to the specific inhibition of PfPDK1, we repeated the dose response assays on NF54/PDK1 cKD parasites cultured in the presence (control) or absence of Shield-1 (PfPDK1 depleted). However, we observed no reduction in IC 50 values for PfPDK1 depleted (−Shield-1) compared to NF54/PDK1 cKD control parasites (+Shield-1) or compared to NF54 WT parasites cultured in the presence or absence of Shield-1 (S14 Fig), suggesting that all 3 inhibitors are not specific for PfPDK1 but likely target additional/other essential parasite kinases. To investigate the effects these compounds have on intraerythrocytic parasite development, we assessed the morphology of drugtreated parasites by visual inspection of Giemsa-stained thin blood smears. Analysis of NF54 WT early ring stage parasites treated with each of the 3 PDK1 inhibitors over a period of 60 hours revealed that parasites were unable to progress beyond the ring stage and became pyknotic thereafter, in contrast to untreated control parasites that progressed through schizogony and gave rise to ring stage progeny as expected (S14 Fig). Furthermore, additional results obtained from separate 18-hour treatments of early ring stages, early trophozoites or early schizonts suggest that all 3 inhibitors are active against all intraerythrocytic stages, as in all cases the morphology of drugtreated parasites was reminiscent of dying or pyknotic forms and viable trophozoites, schizonts, and ring stage progeny were not observed (S14 Fig). Hence, based on the promising activities of these molecules against blood stage parasites, attempts to identify their target(s) as well as the screening of extended PDK1 inhibitor libraries and experimental validation of PfPDK1 as a drug target would be worthwhile activities to be pursued in future research.

Discussion
PfPKA is essential for the proliferation of asexual blood stage parasites, and the phosphorylation of the invasion ligand AMA1 is one of its crucial functions [5,6,8,9]. In addition, recent research described important roles for the PDE PfPDEβ and the AC PfACβ in regulating cAMP levels and hence PfPKA activity [8,27]. Here, we studied the function and regulation of the catalytic PfPKA subunit PfPKAc to obtain further insight into PfPKA-dependent signalling in P. falciparum blood stage parasites.
Using the NF54/AP2-G-mScarlet/PKAc cKD parasite line, we confirmed the previously described essential role for PfPKAc in merozoite invasion [8,9]. We also demonstrated that PfPKAc plays no major role in regulating sexual commitment. The dispensability of PfPKAc in the sexual commitment pathway seems rather surprising since early studies performed over 3 decades ago claimed a potential involvement of cAMP signalling in regulating sexual commitment [69,70]. However, these studies only indirectly suggested an involvement of cAMP/ PfPKA-signalling in this process. For instance, Kaushal and colleagues determined the effect of high exogenous cAMP concentrations (1 mM) on sexual commitment and reported that under static culture conditions (high parasitaemia without addition of uninfected red blood cells [uRBCs]) nearly all parasites developed into gametocytes [69]. Rather than reflecting the true induction of sexual commitment by cAMP signalling, we suspect that observations may have been related to the selective killing of asexual stages by high cAMP concentrations (as indeed reported in their study) and/or the stimulation of high SCRs due to LysoPC depletion from the growth medium at high parasitaemia [3]. Our results further suggest that PfPKAc is not required for the morphological maturation of gametocytes and for male gametogenesis. However, even though our cKD system allowed for efficient depletion of PfPKAc expression, we cannot exclude the possibility that residual PfPKAc expression levels still supported normal sexual development. At this point, we would also like to reiterate that our experiments conducted with the NF54/AP2-G-mScarlet/PKAc cKD line showed that GlcN (2.5 mM), but not Shield-1 (675 nM), acts as a confounding factor when studying sexual commitment and male gametocyte exflagellation. We therefore advise to use the FKBP/DD-Shield-1 [36,37] or DiCre/rapamycin [71][72][73] conditional expression systems when studying these processes.
The cellular rigidity of immature P. falciparum gametocytes is linked to a dynamic reorganisation of the RBC spectrin and actin networks [45] as well as the presence of parasiteencoded STEVOR proteins at the iRBC membrane [47,74]. By contrast, the increased deformability gained by stage V gametocytes is accompanied by the reversal of these cytoskeletal rearrangements [45] and dissociation of STEVOR from the iRBC membrane [47,74]. Interestingly, results obtained from experiments using pharmacological agents to increase cellular cAMP levels or to inhibit PKA activity demonstrated that gametocyte rigidity is positively regulated by cAMP/PKA-dependent signalling [30,74]. While potential PKA substrates involved in this process are largely unknown, the PKA-dependent phosphorylation of the cytoplasmic tail of STEVOR (S324) is important to maintain cellular rigidity of immature gametocytes, and dephosphorylation of this residue is linked to the increased deformability of stage V gametocytes [74]. Given that PfPKA is not known to be exported into the iRBC cytosol, however, PKA-dependent phosphorylation events in the RBC compartment are likely exerted by human rather than parasite PKA. Notably though, overexpression of the regulatory subunit PfPKAr, which is expected to lower PfPKAc activity, caused increased deformability of stage III gametocytes [30]. Consistent with these data, we demonstrated that PfPKAc depletion caused a significant, yet only moderate, increase in stage III and V gametocyte deformability. While these results provide direct evidence for a role of PfPKAc-dependent phosphorylation in regulating the biomechanical properties of gametocyte-iRBCs, they also suggest that cAMP signalling through PfPKA is not the only driver of this process. We envisage that PfPKAc activity may regulate the expression, trafficking, or function of parasite-encoded proteins destined for export into the iRBC or of proteins of the inner membrane complex and/or microtubular and actin networks underneath the parasite plasma membrane that play important roles in determining cellular shape during gametocytogenesis [75][76][77]. Comparative phosphoproteomic analyses of the conditional PfPKAc mutants generated here and elsewhere [8,9] may be a promising approach to test this hypothesis and identify the actual substrates involved.
Three recent studies employing DiCre-inducible KO parasites for PfPKAc [8,9], the PDE PfPDEβ [27], and the AC PfACβ [8] highlighted the importance for tight regulation of PfPKAc activity in asexual blood stage parasites. In these studies, induction of the corresponding gene KOs in ring stage parasites caused no immediate defects in intraerythrocytic parasite development but resulted in a complete or severe block in RBC invasion by newly released merozoites due to prevention of PfPKAc activity (PfPKAc and PfACβ KOs) [8,9] or PfPKAc hyperactivation (PfPDEβ KO) [27], respectively. These findings are consistent with the specific expression pattern of all 3 cAMP signalling components in late schizont stages [78]. Interestingly, however, Flueck and colleagues showed that some PfPDEβ KO merozoites successfully invaded RBCs but were then unable to develop into ring stage parasites [27], providing compelling evidence for a lethal effect of PfPKAc hyperactivity also on early intraerythrocytic parasite development. Similarly, we showed that the OE of PfPKAc through a constitutively active heterologous promoter blocked parasite progression through schizogony. We believe this detrimental effect is due to the incapacity of endogenous PfPKAr to complex and inactivate the excess of PfPKAc enzymes, resulting in illegitimate activity of free PfPKAc and hence untimely phosphorylation of substrates prior to the intrinsic PfPKA activity window in late schizonts. While we did not engage in further explorations towards identifying the molecular mechanisms underlying the lethal consequences of PfPKAc overexpression, we discovered another kinase that is likely required for PfPKAc activation. We identified this function by selecting for "PfPKAc OE survivor" parasites able to tolerate PfPKAc OE. All 6 independently selected NF54/PKAc cOE survivor populations carried mutations in the same gene encoding a putative serine/threonine kinase (Pf3D7_1121900). Bioinformatic analyses and structural modelling suggested this kinase is an orthologue of the eukaryotic phosphoinositide-dependent protein kinase 1 (PDK1), hence termed PfPDK1.
Interestingly, all PfPDK1 mutations identified in the various NF54/PKAc cOE survivors are positioned proximal to the ATP-binding cleft and do not coincide with the PIF-binding pocket, suggesting that these mutations impair the catalytic efficiency of PfPDK1 rather than its capacity to interact with substrates. It therefore seems that the most straightforward manner for the parasite to overcome the lethal effect of PfPKAc OE was to acquire mutations reducing PfPKAc activation through PfPDK1-mediated phosphorylation. We confirmed this scenario by (1) reverting the PfPDK1 M51R mutation in the NF54/PKAc cOE S1 survivor, which rendered these parasites again sensitive to PfPKAc OE; and (2) by introducing the PfPDK1 M51R mutation into the PfPKAc OE-sensitive clone M1, which rendered these parasites resistant to PfPKAc OE. Notably, we could also show that OE of the PfPKAcT189V mutant form of PfPKAc, which carries a nonphosphorylatable valine residue instead of the target threonine in the activation loop, had no negative effect on intraerythrocytic parasite development and multiplication. Together, these striking results imply that in addition to the cAMP-mediated release of PfPKAc from the regulatory subunit PfPKAr, phosphorylation of the T189 residue is essential for PfPKAc activity and provide compelling evidence that PfPDK1 is the kinase that targets this residue. While this hypothesis is entirely consistent with the evolutionary conserved role for PDK1 in activating PfPKAc in other eukaryotes [14][15][16][17][18], further experiments will be required to confirm that PfPDK1 indeed interacts with and activates endogenous PfPKAc via T189 phosphorylation in vivo.
Conditional depletion of PfPDK1 did not result in any obvious multiplication or developmental defects in asexual and sexual blood stage parasites, showing that largely diminished PfPDK1 protein levels are still sufficient to sustain parasite viability and proliferation. However, several lines of evidence strongly argue for an essential role for PfPDK1 in asexual parasites and that at least one of its vital functions is to activate PfPKAc. First, previous studies [31,54] and our own attempts failed to obtain PfPDK1 null mutants via gene disruption approaches. Second, none of the different mutations identified in the 6 NF54/PKAc cOE survivors introduced a nonsense loss-of-function mutation into the pfpdk1 open reading frame. This observation again supports the notion that PfPDK1 function is vital and that the PfPDK1 mutant enzymes retain residual kinase activity. Third, we were only able to introduce the M51R PfPDK1 mutation into parasites overexpressing PfPKAc but not into WT parasites, suggesting that parasites expressing functionally compromised PfPDK1 mutants can only survive if impaired PfPDK1-dependent PfPKAc activation is compensated for by elevated PfPKAc expression levels. Given that PDK1 is widely conserved in eukaryotes and required to activate AGC kinases at large [17], it is conceivable that PfPDK1 may also regulate the activity of PfPKG or PfPKB, the only other 2 known members of the AGC family in P. falciparum, which are both essential in blood stage parasites [31,79]. Furthermore, the manifestation of the PfPKAc OE phenotype in late trophozoites/early schizonts implies that PfPDK1 is active and phosphorylates other substrates already at this stage, hours prior to the expression window of endogenous PfPKAc in late schizonts. Importantly, however, the fact that the NF54/PKAc cOE survivor parasites expressing functionally impaired PfPDK1 mutant enzymes are fully viable suggests that the PfPDK1-dependent phosphorylation of other substrates is either not essential or can still be executed at functionally relevant baseline levels by mutated PfPDK1.
In summary, we provide unprecedented functional insight into the cAMP/PfPKA signalling pathway in the malaria parasite P. falciparum. Our results complement earlier studies highlighting the importance of tight regulation of PfPKA activity for parasite survival, showing that diminished as well as augmented PfPKAc expression levels are lethal for asexual blood stage parasites. In addition to the well-established roles for the regulatory subunit PfPKAr, the AC PfACβ and the PDE PfPDEβ in regulating PfPKAc activity via cAMP levels, we provide compelling evidence that PfPDK1 is required to activate PfPKAc, most likely through activation loop phosphorylation at T189. In light of the essential role for PfPDK1 in this and possibly other parasite AGC kinase-dependent signalling pathways, and the promising anti-parasite activity of PDK1 kinase inhibitors, PfPDK1 represents an attractive candidate for further functional and structural studies and to be explored as a possible new antimalarial drug target.
All primers used for cloning of the described transfection constructs are listed in S1 Table. Transfection and transgenic cell lines P. falciparum ring stage parasite transfection was performed as described [53]. A total of 100 μg plasmid DNA was used to transfect NF54/AP2-G-mScarlet and NF54 WT parasites (100 μg of the SLI_PKAc_cKD plasmid; 50 μg each of all described CRISPR/Cas-9 suicide and donor plasmids). Moreover, 24 hours after transfection of the SLI_PKAc_cKD plasmid, parasites were cultured on 1.5 μM DSM1 until a stably growing parasite population was obtained. This culture was subsequently treated with 2.5 μg/mL BSD-S-HCl to select for parasites in which the pfpkac gene was successfully tagged. Similarly, 24 hours after transfection of CRISPR/Cas-9-based plasmids, the cultures were treated with 2.5 μg/mL BSD-S-HCl (for 10 subsequent days) or 4 nM WR99210 (for 6 subsequent days) depending on the resistance cassette encoded by the suicide plasmid. NF54/AP2-G-mScarlet/PKAc cKD and NF54/PDK1 cKD parasites were constantly cultured on 675 nM Shield-1 (+Shield-1) to stabilise the PfPKAc-GFPDD or PfPDK1-GFPDD protein, respectively. NF54/PKAc cOE, NF54/ PKAcT189V cOE, NF54/PKAc cOE M1/PDK1_mut, and NF54/PKAc cOE S1/PDK1_wt parasites were constantly cultured on 2.5 mM GlcN to block OE of PfPKAc or PfPKAcT189V. About 2 to 3 weeks after transfection, stably growing parasite cultures were obtained and diagnostic PCRs on gDNA were used to confirm correct genome editing. Primers used to verify for correct gene editing are listed in S2 Table. Correct targeted mutagenesis of the pfpdk1 gene in NF54/PKAc cOE M1/PDK1_mut and NF54/PKAc cOE S1/PDK1_wt parasites was confirmed by Sanger sequencing. Sanger sequencing data analysis and visualisation was performed using SnapGene software 4.1.6 (Insightful Science, San Diego, CA, USA).

Limiting dilution cloning
Limiting dilution cloning was performed as previously described [49]. In brief, synchronous ring stage parasite cultures were diluted with fresh PCM and RBCs to a haematocrit of 0.75% and a parasitaemia of 0.0006% (= parasite cell suspension). Each well of a flat-bottom 96-well microplate (Costar #3596) was filled with 200 μL PCM/0.75% haematocrit (= RBC suspension). In each well of row A, 100 μL of the parasite cell suspension was mixed with the 200 μL RBC suspension (1/3 dilution), resulting in a parasitaemia of 0.0002%, which equals approximately 30 parasites per well. Subsequently, 100 μL of the row A parasite cell suspensions were mixed with 200 μL RBC suspension in the wells of row B resulting again in a 1/3 dilution (approximately 10 parasites/well). This serial dilution was continued until the last row of the plate was reached. The 96-well microplate was kept in a gassed airtight container at 37˚C for 11 to 14 days without medium change. Subsequently, using the Perfection V750 Pro scanner (Epson, Nagano, Japan), the 96-well microplate was imaged to visualise plaques in the RBC layer. The content of wells containing a single plaque was then transferred individually into 5-mL cell culture plates and cultured using PCM until a stably growing parasite culture was obtained.

Fluorescence microscopy
Live cell fluorescence imaging was performed to visualise protein expression as described [85]. Parasite nuclei were stained using 5 μg/ml Hoechst (Merck, Buchs, Switzerland) and Vectashield (Vector Laboratories, Burlingame, CA, USA) was used to mount the microscopy slides. Live cell fluorescence microscopy was performed using a Leica DM5000 B fluorescence microscope (20×, 40×, and 63× objectives), and images were acquired using the Leica application suite (LAS) software Version 4.9.0 and the Leica DFC345 FX camera. Images were processed using Adobe Photoshop CC 2018, and for each experiment identical settings for both image, acquisition and processing were used for all samples analysed. For the quantification of the number of nuclei per schizont, synchronous NF54/PfPKAc cOE M1 and S1 ring stage parasites (0 to 6 hpi) were split and cultured either in presence or absence GlcN. Moreover, 40 hours later (40 to 46 hpi), schizonts were stained using 5 μg/ml Hoechst and Vectashield-mounted slides visually inspected using the Leica fluorescence microscope and the LAS software. Three biological replicate experiments were performed and for each replicate the number of nuclei in 100 schizonts was determined by manual counting.
To quantify SCRs of NF54/PDK1 cKD parasites, GlcNAc assays were performed [44]. For this purpose, synchronous parasites (0 to 6 hpi) were split (±Shield-1) and 18 hours later (18 to 24 hpi) the culture medium was replaced with standardised-SerM/CC medium (2% parasitaemia, 5% haematocrit). Upon reinvasion, the ring stage parasitaemia was quantified from Giemsa-stained thin blood smears prepared at 18 to 24 hpi. This parasitaemia corresponds to the cumulative counts of asexual ring stages and sexual ring stages (day 1 of gametocytogenesis). From 24 to 30 hpi onwards, parasites were cultured in +SerM medium supplemented with 50 mM GlcNAc (Sigma) to eliminate asexual parasites [44]. On day 4 of gametocytogenesis (stage II gametocytes), the parasitaemia was again quantified from Giemsastained thin blood smears. SCRs were determined as the percentage of the day 4 parasitaemia (stage II gametocytes) compared the total parasitaemia observed on day 1.

Gametocyte cultures
Synchronous gametocyte cultures were used to study gametocyte morphology, to extract protein samples and to perform microsphiltration and exflagellation assays. Sexual commitment was induced at 18 to 24 hpi using-SerM medium. Upon reinvasion (0 to 6 hpi) (asexual and sexual ring stages; day 1 of gametocytogenesis), parasites were cultured in +SerM medium. Another 24 hours later (24 to 30 hpi) (trophozoites and stage I gametocytes, day 2 of gametocytogenesis), 50 mM GlcNAc was added (+SerM/GlcNAc) to eliminate asexual parasites [44]. The +SerM/GlcNAc medium was changed daily for 6 consecutive days and thereafter gametocytes were cultured in +SerM medium that was replaced daily on a 37˚C heating plate to prevent gametocyte activation.

Microsphiltration experiments
Synchronous NF54/AP2-G-mScarlet/PKAc cKD and NF54 WT parasites were split at 0 to 6 hpi and cultured separately in presence and absence of Shield-1 (±Shield-1). Subsequently, sexual commitment was induced at 18 to 24 hpi using-SerM medium, and after reinvasion, gametocytes were cultured using +SerM/GlcNAc and +SerM medium as described above. On day 6 (stage III) and 11 (mature stage V) of gametocytogenesis, microsphiltration experiments were conducted as described previously [46]. Per sample and condition, either 1 (NF54 WT) or 2 (NF54/AP2-G-mScarlet/PKAc cKD) independent biological replicate experiments with 6 technical replicates each were conducted. The experiment starts by transferring gametocyte culture aliquots into 15 mL Falcon tubes and lowering the haematocrit to 1.5% by addition of PCM. Six microsphere filters (technical replicates) were loaded per sample and condition. After injection of 600 μL cell suspension, filters were washed with 5 mL +SerM medium at a speed of 60 mL per hour using a medical grade pump (Syramed μSP6000, Acromed, Switzerland). Gametocytaemia before ("UP") and after ("DOWN") the microsphiltration process were determined from Giemsa-stained thin blood smears by counting at least 1,000 RBCs. The "UP" gametocytaemia was determined as the mean gametocytaemia calculated from 2 independent Giemsa-stained thin blood smears. The "DOWN" gametocytaemia was determined for each filter separately. Gametocyte retention rates were calculated as 1-("DOWN" gametocytaemia divided by "UP" gametocytaemia). Samples were kept at 37˚C whenever possible to prevent gametocyte activation and lack thereof was confirmed by visual inspection of Giemsastained thin blood smears.

Exflagellation assays
On day 14 of gametocytogenesis (mature stage V), exflagellation assays were performed as described previously [87]. Briefly, in a Neubauer chamber gametocytes were activated using 100 μM xanthurenic acid (XA) and a drop in temperature (from 37˚C to 22˚C). After 15 minutes of activation, the total number of RBCs per mL of culture and the number of exflagellation centres by activated male gametocytes were quantified by bright-field microscopy (40× objective). The gametocytaemia before activation was determined from Giemsa-stained thin blood smears. ExRs were calculated as the proportion of exflagellating gametocytes among all gametocytes. At least 3 biological replicates were performed per experiment.

Illumina whole genome sequencing
To perform WGS, gDNA of the NF54/PKAc cOE clones M1 and M2 and the 6 independently grown NF54/PKAc cOE survivors (S1-S6) was isolated using a phenol/chloroform-based extraction protocol as described [88]. To avoid an amplification bias due to the high AT-content of P. falciparum gDNA, DNA sequencing libraries were prepared using the PCR-free KAPA HyperPrep Kit (Roche). Libraries were sequenced on an Illumina NextSeq 500 and the quality of the raw sequencing reads was analysed with FastQC (version 0.11.4) [89]. The raw reads were mapped to the P. falciparum 3D7 reference genome (PlasmoDB version 39) complemented with the corresponding transfection plasmid sequences using the Burrows-Wheeler Aligner (version 0.7.17) [90] with default parameters. The alignment files in SAM format were converted to binary BAM files with SAMtools (version 1.7) [91], and the BAM files were coordinate sorted and indexed, and read groups were added with Picard (version 2.6.0) [92]. Sequence variants (SNP and Indels) were directly called with the Genome Analysis Toolkit's (GATK, version 4.0.7.0) HaplotypeCaller in the GVCF mode to allow multisample analysis [93]. The resulting g.vcf files of the different samples were combined into one file and genotyped using GATK [93]. To predict the consequences of the obtained variants, they were annotated with SnpEff (version 4.3T) [94] using the SnpEff database supplemented manually with the 3D7 genome annotation (PlasmoDB version 39). The detected variants were filtered for (i) "HIGH" or "MODERATE" impact (thus nonsynonymous variants); (ii) absence in NF54/ PKAc cOE clones M1 and M2; and (iii) an allele frequency of the alternative allele of >40% in at least one of the NF54/PKAc cOE survivors (S1-S6). The obtained list of 83 candidate variants was first screened manually (i) for variants present in all NF54/PKAc cOE survivors; and (ii) for genes mutated in all NF54/PKAc cOE survivors, leaving the variants identified in Pf3D7_1121900 as only candidates. Additionally, all 83 original candidate variants were inspected visually with the Integrative Genomics Viewer (version 2.7.0) [95]. Variants were excluded if they were suspected to be false positives because they were (i) only supported by a very small number of reads, and (iia) also detected in reads of the mother clones and not called because of low allele frequencies or (iib) insertions and deletions after/before large homopolymers or repeat tracts. This again left the variants in Pf3D7_1121900 as only candidates.
To analyse PfPKAc-GFP cOE cassette copy numbers, the sequencing coverage over the whole genome was determined in 50-nucleotide windows using the software igvtools (version 2.5.3) [95] and the mean coverage of (i) the whole genome; (ii) the endogenous pfpkac locus; and (iii) the ectopic pfpkac-gfp cOE cassettes were calculated in RStudio (R version 3.6.2, RStudio release 1.2.5033). The mean coverage of the endogenous pfpkac locus and ectopic pfpkacgfp cOE cassettes was summed up, as both sequences are identical and reads derived from the ectopic pfpkac-gfp cOE cassettes were mapped to both alleles. pfpkac coverage was normalised to the mean genome-wide coverage (assuming the copy number of the genome is 1) and to the pfpkac coverage of WT parasites. Finally, the endogenous pfpkac was subtracted (−1) to obtain the approximate copy number of ectopic PfPKAc-GFP cOE cassettes.

Sequence alignments and modelling of protein structure
Alignment of the PfPDK1 (Pf3D7_1121900/UniProt ID Q8IIE7) amino acid sequence with PDK1 from P. vivax (PVX_091715, UniProt ID A5K4N1), Arabidopsis thaliana (UniProt ID Q9XF67), Caenorhabditis elegans (UniProt ID Q9Y1J3), and humans (UniProt ID O15530) was performed using Clustal Omega [96]. A homology model of the PfPDK1 structure was built using SWISS-MODEL [97] based on the human PDK1 crystallographic structure (PDB ID 1UU9) [52]. The mean homology model quality (Global Model Quality Estimation, GMQE) was assessed as 0.49, suggesting a model of average quality (possible GMQE values are 0 to 1 on a linear scale, higher values indicate better quality). A large, predicted disordered loop (amino acids 180 to 310) present in PfPDK1 but not in its homologues was not built in the model. Amino acids of the ATP-binding cleft were defined as those within 4 Å of any ATP atom in the human PDK1 structure. The PIF-binding pocket was defined as suggested by Biondi and colleagues [22].
To investigate the effect of human PDK1 inhibitors on intraerythrocytic parasite development, synchronous parasite populations were exposed to BX-795, BX-912, or GSK2334470 at a concentration 10 times the IC 50 (10x IC 50 ) and parasite morphology was assessed on Giemsastained thin blood smears prepared at subsequent time points by bright-field light microscopy (100x objective). In a first assay, synchronous young ring stages (0 to 4 hpi) were exposed to the drugs for 60 hours and Giemsa-stained blood smears prepared every 12 hours. In a second assay, synchronous young ring stages (0 to 4 hpi), late ring stages/early trophozoites (20 to 24 hpi), or late trophozoites/early schizonts (30 to 34 hpi) were exposed to the drugs for 18 hours, after which the drugs were washed out by washing the cells in 2.5 volumes of culture medium and placing them back into culture using drug-free medium for another 24 hours. Giemsastained blood smears were prepared 18, 30, and 42 hours after the start of the assay.

Statistical analysis
All data from assays quantifying parasite multiplication, number of nuclei per parasite, SCRs, gametocyte deformability, ExRs, and IC 50 values are represented as means with error bars defining the standard deviation. All data were derived from at least 3 biological replicate experiments. Statistical significance (p < 0.05) was determined using paired or unpaired Student t  Fig 2A. (C) Full size western blot shows expression of PfPKAc-GFP in NF54/PKAc cOE S1 parasites under overexpression-inducing (-GlcN) and control (+GlcN) conditions. Parasites were cultured and samples prepared as described in panel A. The membrane was first probed with α-GFP followed by α-GAPDH control antibodies. MW PfPKAc-GFPDD = 79.8 kDa, MW PfGAPDH = 36.6 kDa. Dashed lines mark the blot sections shown in Fig 2E. MW PfPKAc-GFP = 67.3 kDa, MW PfGAPDH = 36.6 kDa. (D) Parasite multiplication rates of NF54/PKAc cOE M1 (left) and S1 survivor parasites (right) under overexpression-inducing (-GlcN) and control conditions (+GlcN) over 2 generations. Open squares represent data points for individual replicates and the means and SD (error bars) of 3 biological replicates are shown. Differences in multiplication rates have been compared using a paired 2-tailed Student t test (statistical significance cutoff: p < 0.05). Note that the same data is presented as an increase in parasitaemia over time in Fig 2D and 2G. The raw data are available in the source data file (S2 Data). cOE, conditional overexpression; DIC, differential interference contrast; hpi, hours postinvasion. (TIF) S7 Fig. Sequence alignment of PDK1 orthologs. Clustal Omega [96] multiple sequence alignment of PfPDK1 (Pf3D7_1121900/UniProt ID Q8IIE7) and PvPDK1 (PVX_091715/UniProt ID A5K4N1) with well-characterised PDK1 homologues from Arabidopsis thaliana (UniProt ID Q9XF67), Caenorhabditis elegans (UniProt ID Q9Y1J3), and humans (UniProt ID O15530). Residues are highlighted in blue gradient depending on fractional conservation. The kinase catalytic domain spans residues 82 to 342 in human PDK1 [52] (purple section underneath the sequences), which also includes a carboxyl-terminal PH domain spanning residues 446-548 [51] (green section). Arrowheads denote residues forming the PIF-binding pocket in human PDK1 [22], asterisks denote residues mutated in PfPKAc OE survivors identified in this study and red bars denote residues that form part of the ATP-binding cleft. PH, pleckstrin homology. stages of NF54/AP2-G-mScarlet/PDK1 cKD parasites under protein-stabilising (+Shield-1) conditions as assessed by western blot analysis. Lysates derived from equal numbers of parasites were loaded per lane. The membrane was first probed with α-GFP followed by α-GAPDH control antibodies. MW PfPDK1-GFPDD = 101.1 kDa, MW PfGAPDH = 36.6 kDa. The full size western blot is shown. (B) Full size western blot shows expression of PfPDK1-GFPDD in NF54/AP2-G-mScarlet/PDK1 cKD parasites under protein-depleting (-Shield-1) and control (+Shield-1) conditions. Synchronous parasites (0 to 8 hpi) were split (±Shield-1) 40 hours before collection of the samples. Lysates derived from equal numbers of parasites were loaded per lane. The membrane was first probed with α-GFP followed by α-GAPDH control antibodies. MW PfPDK1-GFPDD = 101.1 kDa, MW PfGAPDH = 36.6 kDa. Dashed lines mark the blot sections shown in Fig 4B. (C) Increase in parasitaemia (left) and corresponding parasite multiplication rates (right) of NF54 WT parasites cultured in presence (+Shield-1) and absence of Shield-1 (-Shield-1). Synchronous parasites (0 to 6 hpi) were split (±Shield-1) 18 hours before the first measurement in generation 1. Open squares represent data points for individual replicates and the means and SD (error bars) of 3 biological replicates are shown. Differences in multiplication rates have been compared using a paired 2-tailed Student t test (statistical significance cutoff: p < 0.05). The raw data are available in the source data file (S2 Data). hpi, hours postinvasion; WT, wild-type. (TIF) S10 Fig. SCRs, gametocytogenesis, and male gametogenesis of NF54/PDK1 cKD parasites. (A) SCRs of NF54/PDK1 cKD parasites cultured in the presence (+Shield-1) or absence of Shield-1 (-Shield-1). Open squares represent data points for individual replicates and the means and SD (error bars) of 3 biological replicate experiments are shown. Differences in SCRs have been compared using a paired 2-tailed Student t test (statistical significance cutoff: p < 0.05). The raw data are available in the source data file (S2 Data). (B) Representative images captured from Giemsa-stained thin blood smears showing the distinct morphology of stage I to V gametocytes cultured under PfPDK1-GFPDD-depleting (-Shield-1) and control (+Shield-1) conditions over 11 days of maturation. Synchronous parasites were split (±Shield-1) as sexual/asexual ring stage parasites 24 hours after the induction of sexual commitment in the preceding IDC. To eliminate asexual parasites, gametocytes were cultured in +SerM supplemented with 50 mM GlcNAc from day 1 to 6 of gametocytogenesis. Scale bar = 5 μm. dgd, day of gametocyte development. (C) Relative ExRs of mature NF54/PDK1 cKD stage V gametocytes (day 14) cultured in presence (+Shield-1) and absence of Shield-1 (-Shield-1). Synchronous parasites were split (±Shield-1) and cultured as described in panel B. Open squares represent data points for individual replicates and the means and SD (error bars) of 3 biological replicate experiments are shown. Differences in ExRs have been compared using an unpaired 2-tailed Student t test (statistical significance cutoff: p < 0.05). The raw data are available in the source data file (S2 Data). ExR, exflagellation rate; IDC, intraerythrocytic developmental cycle; SCR, sexual commitment rate. (TIF) S11 Fig. CRISPR/Cas-9-based engineering and characterisation of the NF54/PKAc cOE M1/PDK1_mut and NF54/PKAc cOE S1/PDK1_wt lines. (A) Top: Scheme depicting the pfpdk1 locus of NF54/PKAc cOE S1 or M1 parasites, the donor (pD_S1rev or pD_M1mut) and the suicide (pHF_gC_S1rev or pHF_gC_M1mut) constructs transfected into either NF54/ PKAc cOE S1 or NF54/PKAc cOE M1 parasites to generate the NF54/PKAc cOE S1/PDK1_wt and NF54/PKAc cOE M1/PDK1_mut parasite line, respectively, and the edited pfpdk1 locus. Bottom: Sanger sequencing results of modified pfpdk1 genes after targeted mutagenesis in NF54/PKAc cOE S1/PDK1_wt and NF54/PKAc cOE M1/PDK1_mut parasites confirms correct editing. The expected sequences after successful editing, the corresponding amino acid changes and sequencing chromatograms are indicated. The asterisk marks the mutated residues (M51R or R51M). Capital letters highlight the synonymous nucleotide substitutions introduced by CRISPR/Cas-9 editing to destroy the sgRNA target site and to introduce the aspired amino acid change. (B) Full size western blots showing expression of PfPKAc-GFP in NF54/PKAc cOE S1/PDK1_wt (left) and NF54/PKAc cOE M1/PDK1_mut (right) parasites under OE-inducing (-GlcN) and control conditions (+GlcN). Synchronous parasites (0 to 8 hpi) were split (±GlcN) 40 hours before sample collection. Lysates derived from equal numbers of parasites were loaded per lane. The membranes were first probed with α-GFP followed by α-GAPDH control antibodies. MW PfPKAc-GFP = 67.3 kDa, MW PfGAPDH = 36.6 kDa. Dashed lines mark the blot sections shown in Fig 5A and 5B. (C) Parasite multiplication rates of NF54/PKAc cOE S1/PDK1_wt (top) and NF54/PKAc cOE M1/PDK1_mut (bottom) parasites under OE-inducing (-GlcN) and control conditions (+GlcN) over 2 generations. Open squares represent data points for individual replicates and the means and SD (error bars) of 3 biological replicates are shown. Differences in multiplication rates have been compared using a paired 2-tailed Student t test (statistical significance cutoff: p < 0.05). Note that the same data is presented as an increase in parasitaemia over time in Fig 5C and 5D. The raw data are available in the source data file (S2 Data). cOE, conditional overexpression; hpi, hours postinvasion; sgRNA, single guide RNA; WT, wild-type. The boxed schematic illustrates the integration of pD_pkac_cOE donor plasmid concatemers into the glp3 locus based on double-crossover recombination of nonadjacent HRs on the concatemer. For reasons of simplicity, the integration of a tandem assembly only is shown. n-1, number of integrated donor plasmids. Estimated mean copy numbers of integrated PfPKAc cOE cassettes (n) are shown for the 2 unselected NF54/PKAc cOE clones (M1, M2) and the 6 independently grown survivor populations (S1-S6), alongside the PfPDK1 mutations identified in the 6 NF54/PKAc cOE survivors (see also Fig 3). Copy numbers of PfPKAc cOE cassettes were calculated from WGS data and the analysis steps are described in the Materials and Methods section. cOE, conditional overexpression; HR, homology region; WGS, whole genome sequencing; WT, wild-type.  A, B) IC 50 values for the 3 human PDK1 inhibitors BX-795, BX-912, and GSK2334470 and the 2 antimalarial control compounds Chloroquine and Artesunate on the multiplication of NF54 WT parasites (A) and NF54/PDK1 cKD and NF54 WT parasites cultured in the absence (−Shield-1) or presence of Shield-1 (+Shield-1) (B). Symbols represent individual IC 50 values calculated from 2 technical replicate dose response assays each. Drug dose response assays were performed in 3 biological replicates, means and SD (error bars) are indicated. The raw data are available in the source data file (S2 Data). (C) Representative images captured from Giemsa-stained thin blood smears of NF54 WT parasites exposed to BX-795, BX-912, and GSK2334470 (10x IC 50 ) and the solvent control (DMSO). Drugs were added to synchronous young ring stage parasites (0 to 4 hpi) and blood smears prepared every 12 hours for 60 hours. The curved arrow marks the time point of merozoite release and invasion into new RBCs in the DMSO control population. (D) Representative images captured from Giemsa-stained thin blood smears of NF54 WT parasites exposed to BX-795, BX-912, and GSK2334470 (10x IC 50 ) and the DMSO solvent control. Drugs were added to synchronous early ring stages (0 to 4 hpi) (left panel), late ring stages/early trophozoites (20 to 24 hpi) (middle panel), or late trophozoites/early schizonts (30 to 34 hpi) (right panel) for 18 hours, followed by drug washout and further culturing in drug-free medium. Blood smears were prepared 18, 30 and, 42 hours after the start of the assay. The curved arrow marks the time point of merozoite release and invasion into new RBCs in the DMSO control population. ER, early ring; ES, early schizont; hpi, hours postinvasion; LR/LR_g2, late rings/late rings generation 2; LR, late ring; LS, late schizont; MR/MR_g2, mid rings/mid rings generation 2; MS, mid schizont; RBC, red blood cell; T, trophozoites; T/T_g2, trophozoites/trophozoites generation 2; WT, wild-type. (TIF) S1 Table. Oligonucleotides used for cloning of transfection constructs. Names and sequences of oligonucleotides, plasmids and cell lines are indicated. Sequences essential for Gibson assembly reactions (Gibson overhangs) or for T4 DNA ligase-dependent cloning of double-stranded sgRNA-encoding fragments (5 0 and 3 0 overhangs) are highlighted with capital letters. Italicised letters highlight the annealed sequences (sgRNAs) and colour-highlighted letters represent introduced sequence mutations. sgRNA, single guide RNA. (PDF) S2 Table. Primers used for diagnostic PCRs on gDNA of transgenic parasite lines. Names and sequences of oligonucleotides and cell lines are indicated. (PDF) S1 Data. Multiple nucleotide sequence alignment of Pf3D7_1121900/pfpdk1 sequences of the NF54/PKAc cOE clones M1 and M2 and the 6 PfPKAc OE-tolerant survivor populations S1-S6 determined by WGS. The pfpdk1 coding sequences of the NF54/PKAc cOE M1 and M2 clones are identical to the Pf3D7_1121900 reference sequence retrieved from Plas-moDB (www.plasmodb.org) (top row). pfpdk1 coding sequences of the NF54/PKAc cOE survivor populations S1-S6 are shown and deviations from the reference sequence are highlighted in green. NF54/PKAc cOE survivor S6 consists of 2 subpopulations with one carrying the c.252A>T mutation and the other one carrying the c.491A>G mutation (as verified by inspection of the sequencing read pairs). cOE, conditional overexpression; WGS, whole genome sequencing. (PDF) S2 Data. Source data for all the graphs and charts presented in the main and Supporting information figures. This file contains separate worksheets. Each worksheet lists the raw data underlying the graphs or charts presented in the main figure panels (Figs 1B, 1E, 2C, 2D, 2F, 2G, 4C, 5C, 5D, and 6B) and Supporting information figures (S3, S4, S6, S9, S10, S11, and S14 Figs). (XLSX)