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

Cyclic AMP (cAMP) signalling is crucial for the propagation of asexual malaria blood stage parasites. Recent work on Plasmodium falciparum demonstrated that phosphorylation of the invasion ligand AMA1 by the catalytic subunit of cAMP-dependent protein kinase A (PfPKAc) is an essential step during parasite invasion into red blood cells. However, the exact mechanisms regulating PfPKAc activity are only partially understood and PfPKAc function has not been extensively studied in gametocytes, the sexual blood stage forms that are essential for malaria transmission. By studying a conditional PfPKAc knockdown mutant, we confirm the essential role for PfPKAc in erythrocyte invasion and demonstrate that PfPKAc is involved in regulating gametocyte deformability. Interestingly, we observed that the conditional overexpression of PfPKAc also caused a profound lethal phenotype by preventing intra-erythrocytic parasite multiplication. Whole genome sequencing of parasites selected to tolerate increased PfPKAc expression levels identified missense mutations exclusively in the gene encoding the putative parasite orthologue of 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we show that PfPDK1 is essential for PfPKAc activation, most likely by phosphorylating T189 in the PfPKAc activation loop. In summary, our results corroborate the importance of tight regulation of PfPKA signalling for parasite survival and identify PfPDK1 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 P. 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 invade red blood cells (RBCs) and develop intracellularly through the ring stage into a trophozoite and finally a schizont stage parasite, which undergoes four to five rounds of nuclear division followed by cytokinesis to produce up to 32 new merozoites. Upon rupture of the infected RBC (iRBC), the released merozoites infect new erythrocytes to initiate another intra-erythrocytic developmental cycle (IDC). The consecutive rounds of RBC invasion and intra-3 erythrocytic parasite proliferation are responsible for all malaria-related pathology and deaths.
Importantly, during each round of replication, a small subset of trophozoites commits to sexual development and their ring stage progeny differentiates over the course of 10-12 days and five distinct morphological stages (stage I-V) into mature male or female gametocytes. Sexual commitment occurs in response to environmental triggers that activate expression of the transcription factor PfAP2-G, the master regulator of sexual conversion [1][2][3] . When taken up by an Anopheles mosquito, mature stage V gametocytes develop into gametes and undergo fertilization. The resulting zygote develops into an ookinete that migrates through the midgut wall and transforms into an oocyst, generating thousands of infectious sporozoites ready to be injected into another human host.
Erythrocyte invasion by merozoites is a highly regulated multi-step process starting with the initial attachment of the merozoite to the RBC surface, followed by parasite reorientation and formation of a so-called tight junction 4 . The tight junction is the intimate contact area between the merozoite and RBC membranes that moves along the merozoite surface during the actin-myosin motor-driven invasion process 4 . Alongside the secreted rhoptry neck proteins, the micronemal transmembrane protein apical membrane antigen 1 (AMA1) is an integral component of the tight junction 4 . The cytoplasmic domain of AMA1 bears an essential role in merozoites during RBC invasion [5][6][7] . In particular, the phosphorylation of residues in the AMA1 cytoplasmic tail (S610 and T613) is essential for AMA1 function in RBC invasion [5][6][7] . Recent research has shown that the P. falciparum cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PfPKA) is responsible for AMA1 5 experiments identified a putative role for cAMP/PfPKA-dependent signalling in regulating gametocyteinfected 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 auto-phosphorylation or by another kinase, is unknown. 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 phosphoinositide-dependent protein kinase-1 (PfPDK1). Using targeted mutagenesis, we show that the T189 residue is crucial for PfPKAc activity and that activation of PfPKAc is strictly dependent on PfPDK1-mediated phosphorylation.

Generation of a conditional PfPKAc loss-of-function mutant
To study PfPKAc function, we generated a conditional PfPKAc knockdown (cKD) line using a selection-linked integration (SLI)-based gene editing approach 35 . Successful engineering tags the pfpkac gene with the fluorescent marker gene gfp fused to the fkbp destabilization domain (dd) sequence 36,37 , followed by a sequence encoding the 2A skip peptide 38,39 , the blasticidin S deaminase (bsd) marker gene and finally the glmS ribozyme element 40 (Supplementary Fig. 1). The FKBP/DD system allows 6 for protein destabilization in the absence of the small molecule ligand Shield-1 36,37 , whereas the glmS ribozyme in the 3' untranslated region of the mRNA causes transcript degradation in the presence of glucosamine (GlcN) 40 . To be able to easily quantify sexual commitment rates, we modified the pfpkac gene in NF54/AP2-G-mScarlet parasites (Brancucci et al., manuscript in preparation), which allows identifying sexually committed cells by visualising expression of fluorophore-tagged PfAP2-G 3,41 .

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 (0-6 hpi) and cultured them separately under PfPKAc-GFPDD-depleting (-Shield-1/+GlcN) andstabilizing conditions (+Shield-1/-GlcN). Sexually committed parasites were identified based on PfAP2-G-mScarlet positivity in late schizonts (40-46 hpi) using high content imaging. We observed a significant increase in sexual commitment rates (SCRs) in PfPKAc-GFPDD-depleted (-Shield-1/+GlcN) compared to control parasites (+Shield-1/-GlcN) (1.56-fold ±0.13 SD) ( Supplementary Fig.   3). 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 42 . However, treatment with GlcN also caused a similar increase in SCRs in NF54/AP2-G-mScarlet control parasites (1.44-fold ±0.14 SD) ( Supplementary Fig. 3). 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 re-enter the bloodstream to be taken up by feeding mosquitoes [44][45][46] . Experiments employing PKA and PDE inhibitors, a transgenic cell line overexpressing 8 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 45 . Microsphiltration exploits the fact that differences in cellular rigidity correlate with cell retention rates in a microsphere-based artificial spleen system 45,46 . We observed a 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 vs 82.2% ±8.0 SD) and mature stage V (day 11) gametocytes (13.9% ±16.8 SD vs 27.6% ±14.5 SD) (Fig. 1e). In contrast, NF54 WT gametocytes cultured in the presence or absence of Shield-1 showed no difference in retention rates ( Supplementary Fig. 4).
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-infected RBC 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 exflagellation rates, 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 pfpde 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 CRIPSR/Cas9-based gene editing. To this end, we inserted an ectopic pfpkac-gfp transgene cassette into the non-essential glp3 (cg6, Pf3D7_0709200) locus in NF54 WT parasites (NF54/PKAc cOE) ( Supplementary Fig. 5). Here, the constitutive calmodulin (PF3D7_1434200) promoter and a glmS ribozyme element 47 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 48 . In two clones (NF54/PKAc cOE M1 and M2), correct integration of the 9 inducible PfPKAc-GFP OE expression cassette and absence of WT parasites was confirmed by PCR on gDNA ( Supplementary Fig. 5). Live cell fluorescence imaging and Western blot analysis confirmed the efficient induction of PfPKAc-GFP OE upon GlcN removal in both clones ( Fig. 2a and Supplementary   Fig. 6). 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 (40-46 hpi), 40 hours after triggering PfPKAc-GFP OE (-GlcN) in young ring stage parasites (0-6 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 one or two 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 0-6 hpi, cultured separately in the presence or absence of GlcN and parasite multiplication was quantified over three 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 ( Fig. 2d and Supplementary Fig. 6). Notably, however, viable PfPKAc-GFP OE parasites emerged approximately two 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 ( Fig. 2e and Supplementary Fig. 6). 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 Supplementary Fig. 6).
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 intra-erythrocytic development and parasite multiplication.

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 whole genome sequencing (WGS) of the two unselected NF54/PKAc cOE clones M1 and M2 and the six independently grown survivor populations (NF54/PKAc cOE S1-S6), three each originating from the M1 and M2 clones, respectively. Intriguingly, we found that all six NF54/PKAc cOE survivors, but not the unselected M1 and M2 clones, carried missense mutations in the gene encoding a putative serine/threonine protein kinase (Pf3D7_1121900) (   (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 ( Fig. 3b and Supplementary Fig.   7). 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 six 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.

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/Cas9-based single plasmid approach 52 . However, consistent with the results obtained in previous kinome-or genome-wide KO screens in P. falciparum and P. berghei 31,53,54 , 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 ( Supplementary Fig.   8). Correct editing of the pfpdk1 gene and absence of the wild type locus was confirmed by PCR on gDNA. Donor plasmid integration downstream of the pfpdk1 gene was detected in a subset of parasites ( Supplementary Fig. 8), but this is not expected to compromise pfpdk1-gfpdd expression since the 551 bp 3' homology region (HR) used for homology-directed repair includes the native terminator as based on published RNA-seq data 55 . 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 55,56 (Fig. 4a and Supplementary Fig. 9).
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 confirm the specific function of PfPDK1 in regulating PfPKAc activity, we used CRISPR/Cas9based 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 verified the successful reversion of the PfPDK1 M51R mutation ( Supplementary Fig. 11), and live cell fluorescence imaging and Western Blot analysis confirmed that GlcN removal still triggered efficient PfPKAc-GFP OE in NF54/PKAc cOE S1/PDK1_wt parasites (  Fig. 11). However, in contrast to NF54/PKAc cOE S1 parasites, which carry the same M51R PfPDK1 mutation, the multiplication rates of NF54/PKAc cOE M1/PDK1_mut parasites overexpressing PfPKAc-GFP (-GlcN) reached only 60% to 80% compared to the matching control (+GlcN) ( Fig. 5d and Supplementary Fig. 11). This observation suggested that an additional selection step had taken place in NF54/PKAc cOE S1 parasites that conferred full tolerance to PfPKAc-GFP OE (see Fig. 3g). Indeed, based on the WGS data the parental NF54/PKAc cOE M1 clone carries eight copies of the PfPKAc cOE transgene cassette, whereas the NF54/PKAc cOE S1 survivor population carries only two copies ( Supplementary Fig. 12). We therefore believe that due to the negative impact of continuous PfPKAc-GFP OE on parasite viability, NF54/PfPKAc cOE S1 parasites carrying fewer PfPKAc cOE cassettes and hence lower overall PfPKAc expression levels had a comparative advantage during the selection process for PfPKAc-GFP OE tolerance.
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 ( Supplementary   Fig. 5) to generate the NF54/PKAcT189V cOE line that conditionally overexpresses a mutated version of PfPKAc in which T189 has been substituted with a non-phosphorylatable valine residue 57,58 ( Supplementary Fig. 13). Correct insertion of the PfPKAcT189V cOE cassette into the glp3 locus was verified by PCR on gDNA ( Supplementary Fig. 13). Live cell fluorescence microscopy and Western blot analysis confirmed that PfPKAcT189V-GFP OE was efficiently induced upon removal of GlcN ( Fig. 6a and Supplementary Fig. 13). Strikingly, in contrast to the lethal effect provoked by OE of PfPKAc-GFP, OE of the PfPKAcT189V-GFP mutant (-GlcN) had no effect on intra-erythrocytic 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.  64,65 . While BX-795 was shown to also inhibit several other human kinases in vitro 66 , GSK2334470 displayed high specificity for PDK1 over a large panel of other recombinant human kinases 64 . In cellbased assays, all three compounds inhibited the PDK1-dependent phosphorylation of several AGC kinase substrates at submicromolar concentrations [63][64][65] . Here, we tested these three 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 67 . We found that BX-795, BX-912 and GSK2334470 all inhibited parasite proliferation with IC50 values of 1.83 µM (± 0.23 SD), 1.31 µM (± 0.24 SD) and 1.83 µM (± 0.11 SD), respectively ( Supplementary Fig. 14). Whether the lethal effect of these compounds is due to the specific inhibition of PfPDK1 or whether they target additional/other parasite kinases is unknown at this stage. However, based on the promising activities of these molecules against blood stage parasites and the expected essentiality of PfPDK1 for parasite survival, the screening of extended PDK1 inhibitor libraries as well as 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 due to its indispensable role in phosphorylating the invasion ligand AMA1 5,6,8,9 . In addition, recent research described important roles for the phosphodiesterase PfPDEβ and the adenylyl cyclase 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 three decades ago claimed a potential involvement of cAMP signalling in regulating sexual commitment 68,69 . However, these studies only indirectly suggested an involvement of cAMP/PfPKA-signalling in this process. For instance, Kaushal et al. 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 uRBCs) nearly all parasites developed into gametocytes 68 . 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 sexual commitment rates 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 [70][71][72] conditional expression systems when studying these processes.
The cellular rigidity of immature P. falciparum gametocytes is linked to a dynamic reorganization of the RBC spectrin and actin networks 44 as well as the presence of parasite-encoded STEVOR proteins at the iRBC membrane 46,73 . In contrast, the increased deformability gained by stage V gametocytes is accompanied by the reversal of these cytoskeletal rearrangements 44 and dissociation of STEVOR from the iRBC membrane 46,73 . 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 30,73 . 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 73 .
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-infected RBCs, 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 [74][75][76] . 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 phosphodiesterase PfPDEβ 27 , and the adenylyl cyclase 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 intra-erythrocytic 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 hyper-activation (PfPDEβ KO) 27 , respectively. These findings are consistent with the specific expression pattern of all three cAMP signaling components in late schizont stages 77 . 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 hyper-activity also on early intra-erythrocytic 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 illegitimate activity of 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 the upstream kinase that is required for PfPKAc activation. We identified this function by selecting for "PfPKAc OE survivor" parasites able to tolerate PfPKAc OE. All six 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 18 form of PfPKAc, which carries a non-phosphorylatable valine residue instead of the target threonine in the activation loop, had no negative effect on intra-erythrocytic parasite development and multiplication.
Together, these striking results demonstrate 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.
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, 53 and our own attempts failed to obtain PfPDK1 null mutants via gene disruption approaches. Second, none of the different mutations identified in the six 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 PfPDK1dependent 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 two known members of the AGC family in P. falciparum, which are both essential in blood stage parasites 31,78 .
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 adenylyl cyclase PfACβ and the phosphodiesterase PfPDEβ in regulating PfPKAc activity via cAMP levels, we identified PfPDK1 as the upstream kinase activating 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.
The CRISPR/Cas9 gene editing system employed here is based on co-transfection of suicide and donor plasmids 52 . The pBF-gC-or pHF-gC-derived suicide plasmids encode the Cas9 enzyme, the single guide RNA (sgRNA) cassette and either the blasticidin deaminase (BSD; conferring resistance to BSD-S-HCl) or the human dihydrofolate reductase (hDHFR; conferring resistance to WR99210) resistance markers fused to the negative selection marker yeast cytosine deaminase/uridyl phosphoribosyl transferase (BSD-yFCU or hDHFR-yFCU) 52 . The pD-derived donor plasmid 52 contains the sequence assembly essential for homology-directed repair of the DNA double-strand break induced by Cas9. To obtain the NF54/PKAc cOE parasite line, the pD_pkac_cOE donor plasmid was generated in several cloning steps using Gibson assembly reactions. First, a pD_cOE_DD precursor plasmid was generated using a two-fragment Gibson assembly joining (1) the plasmid backbone including the glp3-specific 5' and 3' HRs amplified from pD_cg6_cam-gdv1-gfp-glmS 82 (primers glp3_F and glp3_R) and (2) the cam 5'-gfpdd-hrp2 3' sequence amplified from pHcamGFP-DD 52 (primers cam_F and hrp2_R).
To insert a new cloning site and generate the next precursor plasmid pD_cOE, another Gibson assembly reaction was performed joining (1) the pD_cOE_glmS plasmid digested using BamHI and NotI and (2) annealed complementary oligonucleotides clon_F and clon_R. The final pD_pkac_cOE plasmid was generated by assembling two Gibson fragments: (1) the BamHI-and SpeI-digested pD_cOE plasmid and (2) the pfpkac sequence amplified from NF54 gDNA using primers pka_F and pka_R. The pBF_gC-cg6 suicide plasmid that was co-transfected with pD_pkac_cOE to generate this NF54/PKAc cOE parasite line encodes the sgRNA targeting the glp3 locus 82 . To obtain the NF54/PKAcT189V cOE parasite line, the pD_pkacT189V_cOE donor plasmid was generated in a Gibson assembly joining three fragments: (1) the BamHI-and SpeI-digested pD_cOE plasmid; (2) the 5' fragment of the pfpkac sequence containing the single point mutation resulting in the T189V amino acid change (primers pka_F and T189V_R); and (3) the 3'fragment of the pfpkac sequence overlapping with the 5' fragment and coding for the same amino acid change (primers T189V_F and pka_R). As described above, the glp3specific suicide plasmid pBF_gC-cg6 was used for co-transfection. The NF54/PDK1 cKD parasite line was obtained by co-transfection of pHF_gC_pdk1-gfpdd (encoding sgRNA_pdk1) and pD_pdk1-gfpdd. The previously published suicide mother plasmid pHF-gC 52 was used to insert the sgRNA sequence targeting the 3' end of pfpdk1 (sgt_pdk1). For this purpose, complementary oligonucleotides were annealed and the resulting double-stranded fragment was ligated into the BsaI-digested pHF-gC plasmid using T4 DNA ligase generating the pHF_gC_pdk1-gfpdd suicide plasmid. The pD_pdk1-gfpdd plasmid was generated by performing a four-fragment Gibson assembly joining (1) the plasmid backbone amplified from pUC19 (primers PCRA_F and PCRA_R) as previously described 52 ; (2) the 5' HR amplified from NF54 gDNA (primers hr1KD_F and hr1KD_R); (3) the gfpdd sequence (primers gfpdd_F and gfpdd_R) amplified from pHcamGDV1-GFP-DD 52 ; and (4) the 3' HR amplified from NF54 gDNA (hr2KD_F and hr2KD_R). The parasite lines NF54/PKAc cOE M1/PDK1_mut and NF54/PKAc cOE S1/PDK1_wt were generated as follows. Plasmids pHF_gC_S1rev and pHF_gC_M1mut were generated by inserting annealed complementary oligonucleotides encoding the respective sgRNAs (sgRNA_S1rev or sgRNA_M1mut) into the BsaI-digested pHF_gC suicide vector 52 . The donor plasmids pD_S1rev and pD_M1mut were generated in a three-fragment Gibson assembly joining (1) the plasmid backbone amplified from pUC19 (primers PCRA_F and PCRA_R) as previously 22 described 52 ; (2) the corresponding 5' HRs amplified from NF54 gDNA (primers hr1_F and either rev_R or mut_R); and (3) the respective 3' HRs amplified from NF54 gDNA (primers hr2_R and either rev_F or mut_F). The primers rev_F/rev_R or mut_F/mut_R encode the WT (M) or mutated (R) amino acid number 51 of PfPDK1, respectively.
All primers used for cloning of the described transfection constructs are listed in Supplementary Table   1.
Transfection and transgenic cell lines. P. falciparum ring stage parasite transfection was performed as described 52 . 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/Cas9 suicide and donor plasmids). 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/Cas9-based plasmids, the cultures were treated with 2.5 μg/mL BSD-S-HCl (for ten subsequent days) or 4 nM WR99210 (for six 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 stabilize 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 two to three 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 Supplementary Table 2. 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).
200,000 RBCs were measured per sample. The measurement was repeated for two (day 3 and 5) or three (day 3, 5 and 7) subsequent generations. The FlowJo_v10.6.1 software was used to analyse the flow cytometry data. The measurements were gated to remove small debris (smaller than cell size) and doublets (two cells in a single measurement). Using an uninfected RBC (uRBC) control sample, uRBCs were separated from iRBCs based on their SYBR Green intensity. Representative plots showing the gating strategy used for all flow cytometry data are presented in Supplementary Fig. 2. Fluorescence microscopy. Live cell fluorescence imaging was performed to visualize protein expression as described 83 . Parasite nuclei were stained using 5 µg/ml Hoechst (Merck) and Vectashield (Vector Laboratories) was used to mount the microscopy slides. Live cell fluorescence microscopy was performed using a Leica DM5000 B fluorescence microscope (20x, 40x and 63x 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 (0-6 hpi) were split and cultured either in presence or absence GlcN. 40 hours later (40-46 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.
Western blot analysis. Parasite pellets were obtained by lysing RBCs using 0.15% saponin in PBS (10 min on ice) followed by centrifugation and washed in ice-cold PBS until the supernatant was clear.
Following protein transfer to a nitrocellulose membrane, the membrane was blocked for 30 min using To quantify SCRs of NF54/PDK1 cKD parasites, N-acetyl-D-glucosamine (GlcNAc) assays were performed 43 . For this purpose, synchronous parasites (0-6 hpi) were split (±Shield-1) and 18 hours later (18-24 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 18-24 hpi. This parasitaemia corresponds to the cumulative counts of asexual ring stages and sexual ring stages (day one of gametocytogenesis). From 24-30 hpi onwards, parasites 26 were cultured in +SerM medium supplemented with 50 mM GlcNAc (Sigma) to eliminate asexual parasites 43 . On day four of gametocytogenesis (stage II gametocytes), the parasitaemia was again quantified from Giemsa-stained thin blood smears. SCRs were determined as the percentage of the day four parasitaemia (stage II gametocytes) compared the total parasitaemia observed on day one.
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 18-24 hpi using -SerM medium. Upon reinvasion (0-6 hpi) (asexual and sexual ring stages; day one of gametocytogenesis), parasites were cultured in +SerM medium. Another 24 hours later (24-30 hpi) (trophozoites and stage I gametocytes, day two of gametocytogenesis), 50 mM GlcNAc was added (+SerM/GlcNAc) to eliminate asexual parasites 43 . The +SerM/GlcNAc medium was changed daily for six consecutive days and thereafter gametocytes were cultured in +SerM medium that was replaced daily on a 37 °C heating plate to prevent gametocyte activation.
Subsequently, sexual commitment was induced at 18-24 hpi using -SerM medium and after reinvasion gametocytes were cultured using +SerM/GlcNAc and +SerM medium as described above. On day six (stage III) and eleven (mature stage V) of gametocytogenesis, microsphiltration experiments were conducted as described previously 45 . Per sample and condition, either one (NF54 WT) or two (NF54/AP2-G-mScarlet/PKAc cKD) independent biological replicate experiments with six 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 AG, 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 two 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 Giemsa-stained thin blood smears.
Exflagellation assays. On day 14 of gametocytogenesis (mature stage V), exflagellation assays were performed as described previously 85 . 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 min of activation, the total number of RBCs per mL of culture and the number of exflagellation centers by activated male gametocytes were quantified by bright-field microscopy (40x objective). The gametocytaemia before activation was determined from Giemsa-stained thin blood smears.
Exflagellation rates were calculated as the proportion of exflagellating gametocytes among all gametocytes. At least three 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 six independently grown NF54/PKAc cOE survivors (S1-S6) was isolated using a phenol/chloroform-based extraction protocol as described 86 . 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) 87 . 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) 88 with default parameters. The alignment files in SAM format were converted to binary BAM files with SAMtools (version 1.7) 89 and the BAM files were coordinate-sorted, indexed and read-groups were added with Picard (version 2.6.0) 90 . 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 multi-sample analysis 91 . The resulting g.vcf files of the different samples were combined into one file and genotyped using GATK 91 . To predict the consequences of the obtained variants, they were annotated with SnpEff (version 4.3T) 92 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 non-synonymous 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) 93 . 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) 93 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 pfpkac-gfp cOE cassettes were 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 normalized 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.

Data availability
Whole genome sequencing data was deposited on the European Nucleotide Archive, accession number PRJEB40033. The source data for all the graphs and charts in the main and Supplementary figures are present in the Source Data file and any other remaining information can be available from the corresponding author upon reasonable request.   Synchronous parasites (0-6 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 three biological replicates are shown. Differences in multiplication rates have been compared using a paired two-tailed Student's t test (statistical significance cut-off: p<0.05).