Abstract
Membrane transport proteins perform crucial roles in cell physiology. The obligate intracellular parasite Plasmodium falciparum, an agent of human malaria, relies on membrane transport proteins for the uptake of nutrients from the host, disposal of metabolic waste, exchange of metabolites between organelles and generation and maintenance of transmembrane electrochemical gradients for its growth and replication within human erythrocytes. Despite their importance for Plasmodium cellular physiology, the functional roles of a number of membrane transport proteins remain unclear, which is particularly true for orphan membrane transporters that have no or limited sequence homology to transporter proteins in other evolutionary lineages. Therefore, in the current study, we applied endogenous tagging, targeted gene disruption, conditional knockdown and knockout approaches to investigate the subcellular localization and essentiality of six membrane transporters during intraerythrocytic development of P. falciparum parasites. They are localized at different subcellular structures – the food vacuole, the apicoplast, and the parasite plasma membrane – and four out of the six membrane transporters are essential during asexual development. Additionally, the plasma membrane resident transporter 1 (PMRT1, PF3D7_1135300), a unique Plasmodium-specific plasma membrane transporter, was shown to be essential for gametocytogenesis and functionally conserved within the genus Plasmodium. Overall, we reveal the importance of four orphan transporters to blood stage P. falciparum development, which have diverse intracellular localizations and putative functions.
Importance Plasmodium falciparum-infected erythrocytes possess multiple compartments with designated membranes. Transporter proteins embedded in these membranes do not only facilitate movement of nutrients, metabolites and other molecules between these compartments, but are common therapeutic targets and can also confer antimalarial drug resistance. Orphan membrane transporter in P. falciparum without sequence homology to transporters in other evolutionary lineages and divergent to host transporters may constitute attractive targets for novel intervention approaches. Here, we localized six of these putative transporters at different subcellular compartments and probed into their importance during asexual parasite growth using reverse genetic approaches. In total, only two candidates turned out to be dispensable for the parasite, highlighting four candidates as putative targets for therapeutic interventions. This study reveals the importance of several orphan transporters to blood stage P. falciparum development.
Introduction
Plasmodium spp. malaria parasites inhabit diverse intracellular niches and need to import nutrients and export waste across both, host-cell and parasite membranes. Despite this, there are less than 150 putative membrane transporters encoded in the genome of Plasmodium falciparum, the most virulent malaria parasite, making up only 2.5% of all encoded genes (P. falciparum 3D7 v3.2: 5280 genes) (1–8), which is reduced compared to other unicellular organisms of similar genome size. The loss of redundant transporters is a typical feature of many intracellular parasites (9) and, as a result, the proportion of transporters that are indispensable for parasite survival increases (2), some of which have been shown to be critical for the uptake of several anti-Plasmodial compounds and/or to be involved in drug resistance (10–23). Moreover, the parasite’s intracellular lifestyle resulted in the evolution of additional specialized transporters without human homologues (1). During its intraerythrocytic development, the parasite relies on the uptake of nutrients, such as amino acids, pantothenate or fatty acids, from its host erythrocyte as well as from the extracellular blood plasma (24–27). As P. falciparum resides in a parasitophorous vacuole (PV) in the host erythrocyte, nutrients acquired from the extracellular milieu must traverse multiple membranes: the erythrocyte plasma membrane (EPM), the parasitophorous vacuole membrane (PVM), the parasite plasma membrane (PPM) and eventually membranes of intracellular organelles, such as those of the apicoplast or mitochondria (24, 28–30). The unique requirements of malaria parasite survival have led to the evolution of a number of orphan transporters, whose localization or function cannot be predicted based on sequence homology to transporters in other organisms (4, 31). Despite the likely importance of uniquely adapted transporters to P. falciparum survival, subcellular localization, essentiality, function and substrate specificity for most P. falciparum transporters has not been directly determined (2, 24, 29). The best functional evidence available for many Plasmodium-specific transporters comes from a recent knockout screen of these orphan transporters in the rodent malaria parasite Plasmodium berghei (31). However, whether observations for different transporters in the P. berghei model are directly transferrable to P. falciparum have yet to be examined. Therefore, in this study, we explored the localization and essentiality of four predicted orphan transporters that had been partially characterised in P. berghei and included two additional transporters with no experimental characterization available.
Results
To date, the predicted ‘transportome’ of P. falciparum consists of 117 putative transport systems (encoded by 144 genes) classified as channels (n=19), carriers (n=69), and pumps (n=29) (2). Functions of the vast majority of transporter genes were inferred from sequence homology to model organisms, however, given their lack of homology, 39 gene products could not be associated with any functional or subcellular localization and were categorized as orphan transporters accordingly (4). A subset of orphan transporters characterized in the P. berghei malaria model was selected for further characterization in P. falciparum. The four transporters selected were reported to be important at different stages of rodent malaria parasite growth with i) P. berghei drug/metabolite transporter 2 (PfDMT2: PF3D7_0716900) found to be essential for asexual blood stage development, ii) P. berghei zinc transporter 1 (PfZIP1: PF3D7_0609100) was essential across transmission stages but not blood stages, where there was only a slight growth defect, iii) P. berghei cation diffusion facilitator family protein (PfCDF: PF3D7_0715900) knockout parasites had a defect during transmission stages but not during asexual stages, and iv) P. berghei major facilitator superfamily domain-containing protein (PfMFS6: PF3D7_1440800) was found to be essential for parasite transmission from mosquitos to a new host, with a growth defect observed at asexual and gametocyte stages but not during mosquito stage parasite growth (31, 32). In order to confirm expression of these four, initially selected, transporters in P. falciparum asexual stages, we searched the list of “Genes coding for transport proteins” included in the Malaria Parasite Metabolic Pathways (MPMP) database (1, 33) for proteins with i) RNA-seq (34, 35) and ii) proteomics evidence (36, 37) in asexual blood stages. During our initial searches of the MPMP database but also including PlasmoDB (38) and the most recent P. falciparum 3D7 genome (v3.2) and annotations, we identified two additional putative transporters in P. falciparum (PF3D7_0523800, PF3D7_1135300), whose P. berghei homologs were not targeted and functionally characterized by Kenthirapalan et al. (31) or investigated in any other experimental model. Given their obvious lack of sequence homology to transporter proteins in other evolutionary lineages and clear classification as orphan membrane transporter, both proteins were subsequently included in our characterization of P. falciparum orphan transporters, and named as ‘food vacuole resident transporter 1’ (FVRT1: PF3D7_0523800) and as ‘plasma membrane resident transporter 1’ (PMRT1: PF3D7_1135300) based on their subcellular localization. AlphaFold-based structure predictions (39) and results from structure homology search (40) of all six selected transporters are provided in Figure S1.
Localization of putative P. falciparum transporters
To determine subcellular localization, we tagged the six putative transporters endogenously with GFP using the selection-linked integration (SLI) system (41) (Figure 1A). Additionally, a glmS ribozyme sequence was included in the 3’UTR, which enabled conditional gene knockdown upon addition of glucosamine (42). Correct integration of the plasmid into the respective genomic locus was verified by PCR and expression of the GFP-fusion protein was confirmed by Western blot for each generated cell line (Figure S2A, B).
All transgenic cell lines expressed the GFP-fusion protein, demonstrating that these transporters are expressed in asexual blood stage parasites (Figure 1B-G, S2A). Expression levels were sufficient to allow determination of subcellular localization (Figure 1B–G): (i) PF3D7_0523800-GFP localized to the food vacuole, (ii) PfDMT2-GFP and PfMFS6-GFP apicoplast localization, and (iii) PfZIP1-GFP and PF3D7_1135300-GFP parasite plasma membrane (PPM) localization. However, PfCDF-GFP showed an obscure staining pattern with a weak spot within the parasite cytosol in ring and trophozoite state parasites, but multiple foci in schizont stages (Figure 1D). To pinpoint this localization, an additional cell line with endogenously 3xHA-tagged PfCDF was generated, confirming the focal localization of PfCDF in asexual stages (Figure S2C).
Except for PfCDF, the observed localizations of the other five transporters were confirmed by co-localization studies using appropriate episomally expressed marker proteins: P40PXmCherry (43, 44) for the food vacuole, ACP-mCherry (45, 46) for apicoplast and LynmCherry (41, 47) for PPM. The focal distribution of PfCDF-GFP was co-localized with a rhoptry (ARO-mCherry (48, 49)) and a micronemes (AMA1-mCherry (50, 51)) marker, but PfCDF-GFP did not colocalize with either marker (Figure 1H). Additionally, for PfZIP and PF3D7_1135300 the PPM localization was further confirmed in free merozoites (Figure S2D, E) and by confocal microscopy-based co-localization of PF3D7_1135300-GFP with the PPM marker Lyn-mCherry (Figure S2F). Accordingly, as noted above, we named PF3D7_0523800 as ‘food vacuole resident transporter 1’ (FVRT1) and PF3D7_1135300 as ‘plasma membrane resident transporter 1’ (PMRT1).
Targeted-gene disruption (TGD), conditional knockdown and conditional knockout of putative transporters
In order to test whether the putative transporters are essential for P. falciparum during its intraerythrocytic cycle, we first tried to functionally inactivate them by targeted gene disruption (TGD) using the SLI system (41) (Figure S3A). TGD cell lines were successfully obtained for PfZIP1 and PfCDF (Figure S3B, C). For PfZIP1-TGD, the correct integration of the plasmid into the genomic locus and absence of wildtype locus was verified by PCR and subsequent growth experiments revealed no growth defect compared to P. falciparum 3D7 wildtype parasites (Figure S2B), suggesting its redundancy during asexual parasite proliferation. For PfCDF-TGD the correct integration of the plasmid into the genomic locus was also verified, but wildtype DNA was still detectable and remained even upon prolonged culturing under G418/WR selection and limited dilution cloning (Figure S3C). In contrast, six (PfPMRT1, PfDMT2) or eight (PfFVRT1, PfMFS6) independent attempts to obtain TGD cell lines for the other four transporters with the respective plasmids failed, indicating that these genes have an indispensable role in blood stage parasite growth.
To probe into the function of the putative transporters where we were unable to generate gene-disruptions, we utilized the glmS ribozyme sequence. The corresponding sequence was integrated into the 3’UTR of the targeted genes. This enabled the induction of conditional degradation of respective mRNAs upon addition of glucosamine (42) and the assessment of the phenotypic consequences. Upon addition of 2.5 mM glucosamine to young ring stage parasites we found a 76.8% (+/- SD 3.7) reduction in GFP fluorescence intensity in PfDMT2GFP parasites, 72.7% (+/- SD 9.4) reduction in PfMFS6-GFP and a 77.7% (+/- SD 6.1) reduction in PfPMRT1-GFP in schizonts of the same cycle (Figure 2A–C, S4A–C). No measurable reduction in fluorescence intensity could be detected for PfFVRT1-GFP or PfCDF-GFP expressing parasite lines (Figure S4D–F). Presence of the glmS cassette in both plasmids was confirmed by PCR (Figure S4H). For parasite cell lines with a significant reduction in the expression of the endogenously tagged protein, proliferation was analyzed in the absence and presence of 2.5 mM glucosamine (Figure 2D, S4G). While no significant effect on growth was observed for PfMFS6, a growth reduction of 68.5 % (+/- SD 2.1) over two cycles was observed upon knockdown of PfDMT2. For PfPMRT1, a minor growth delay was measurable, which resulted in a significantly reduced parasitemia at day 3 upon knockdown using 2.5 mM glucosamine (two tailed Wilcoxon rank sum test, W = 15, n1 = 5, n2 = 3, P = 0.03), but was not significant when using 5 mM glucosamine (two tailed Wilcoxon rank sum test, W = 10, n1 = 4, n2 = 3, P = 0.16) (Figure 2E). Additionally, significantly fewer newly formed ring stage parasites were observed at 84 hours post invasion (hpi) (Figure 2F), and multiple pairwise post-hoc comparisons using the Conover-Iman rank sum test and Benjamini-Hochberg method to control the false discovery rates showed significant step-wise reductions of ring stage parasites after induction of GlmS-based knockdown of PfPMRT1 using both, 2.5 mM glucosamine (adjusted P = 0.0078) and 5 mM glucosamine (adjusted P = 0.0005) in comparison to untreated control cell cultures.
To better characterize the minor growth phenotype of PfPMRT1-GFP-glmS parasites that might be due to incomplete knockdown, we generated a conditional PfPMRT1 knockout cell line (condΔPMRT1) using the Dimerizable Cre (DiCre) system (52, 53). Again using the SLI system (41), the endogenous PfPMRT1 was disrupted upstream of the region encoding the N-terminal transmembrane domain, but, at the same time introducing a recodonized second functional copy of PfPMRT1 flanked by loxP sites in the genomic locus. This loxP-flanked allelic copy of PfPMRT1 encodes an additional 3x hemagglutinin (HA) tag, which can be conditionally excised upon addition of a rapamycin analog (rapalog) via the enzymatic activity of an episomally expressed DiCre (Figure 3A). First, correct integration of the plasmid into the genomic locus was verified by PCR (Figure 3B). Second, expression and localization of the recodonized HA-tagged protein at the PPM was verified by colocalization with the merozoite plasma membrane marker MSP1 (54) (Figure 3C). Third, excision of the recodonized gene upon rapalog addition was confirmed on genomic level by PCR (Figure 3D) and on protein level by Western blot analysis at 24 hpi and 48 hpi (Figure 3E). To assess the effect of conditional PfPMRT1 knockout on parasite proliferation, we determined growth of the transgenic parasite cell line with and without rapalog over five days (Figure 3F, S5A). In contrast to the glmS-based knockdown experiment, DiCre-based gene excision (induced by the addition of rapalog to young ring stages of condΔPMRT1 parasite cell cultures) abolished growth within the first replication cycle (Figure 3F, S5A). The specificity of the observed growth phenotype was verified by gene complementation. To achieve this, we episomally expressed recodonized PfPMRT1 with TY1-epitope tag either under the constitutive nmd3 or the weaker sf3a2 promoter (55) in the condΔPMRT1 cell line (Figure 3D, F, S5B, C). Correct localization of the TY1-tagged PfPMRT1 at the PPM was verified by immunofluorescence assays (IFA) (Figure 3G). Notably both, complementation of the PfPMRT1 knockout cell line (condΔPMRT1) with recodonized PfPMRT1 either under control of the constitutive nmd3 or the weaker sf3a2 promoter, restored parasite growth (Figure 3F, S5B, C). The level of growth restoration with low level expression of recodonized PfPMRT1 is in line with the results from glmS-knockdown experiments, which showed that a reduction of about 75% in protein expression resulted only in a minor growth perturbation (Figure 2C, D).
Loss of the PPM-localized PfPMRT1 leads to an arrest of parasite development at trophozoite stage and the formation of PPM derived protrusions
To determine, which particular parasite stages are affected by the knockout of PfPMRT1, we added rapalog to tightly synchronized parasites at different time points (4, 20 and 32 hpi) (Figure 4A) and monitored parasite growth by flow cytometry. Additionally, we quantified growth perturbation by microscopy of Giemsa smears at 4, 20, 24, 32, 40, 48, 72 and 96 hpi (Figure 4B, S6A, B). When adding rapalog at 4 hpi, parasite development progressed through ring and early trophozoite stages up to 24 hpi with no visible abnormality. Afterwards, parasites with deformed and enlarged protrusions started to appear and further development occurred to be stalled. At 32 hpi, almost all parasites had developed to late trophozoites/early schizonts in the control, whereas these stages were completely absent in PfPMRT1-deficient parasites. Over 50% of the parasites were pycnotic or possessed large protrusions, the remaining parasites stayed arrested at the trophozoite stage. Quantification of the percentage of parasites with protrusions between 20 hpi and 32 hpi revealed 94.8% (+/- SD 4.0) protrusion-positive parasites (Figure 4C). The activation of gene excision at later time points by adding rapalog at 20 hpi or 32 hpi resulted in no or minor growth perturbation in the first cycle with successful re-invasion, but again led to parasites arresting at the trophozoite stage in the second cycle with an accumulation of protrusions (Figure 4A, S6A, B).
In order to get further insights into the morphological changes in PfPMRT1-deficient parasites, we incubated these parasites with dihydroethidium (DHE) to visualize the parasite cytosol (44). We observed an absence of staining within the protrusions, suggesting they are not filled with parasite cytosol (Figure 4D). Next, we transfected the condΔPMRT1 cell line with a plasmid encoding the PPM marker Lyn-mCherry (41) and observed Lyn-mCherry-positive protrusions upon knockout of PfPMRT1 starting to become visible at 24 hpi, indicating that the protrusions originate from the PPM (Figure 4E). In line with this, protrusion membranes were also stainable with BODIPY TR C5 ceramide in condΔPMRT1 parasites at 32 hpi (Figure 4F).
Depletion of PfPMRT1 results in an early arrest of gametocyte development
RNA-seq data suggest PfPMRT1 is also expressed during other developmental stages, such as gametocytes (56, 57). Therefore, we assessed expression of PfPMRT1-GFP during gametocytogenesis by re-engineering PfPMRT1-GFP-glmS in the inducible gametocyte producer (iGP) ‘3D7-iGP’ (58) parasite line, which allows the robust induction of sexual commitment by conditional expression of gametocyte development 1 protein (GDV1) upon addition of shield-1 (58) (Figure S7A). We show that PfPMRT1 is indeed expressed during all stages of gametocytogenesis and again localizes to the PPM, colocalizing with the PPM-marker Lyn-mCherry (41) (Figure 5A, B). Conditional knockdown of PfPMRT1 via the glmS-ribozyme system (Figure S7B) resulted in a reduction in PfPMRT1-GFP fluorescence intensity of 79.4% (+/- SD 9.2%) at 7 days post induction (dpi) or 75.5% (+/- SD 23.2%) at 10 dpi, without an effect on gametocyte development (Figure S7C–F). In order to exclude that a role of PfPMRT1 in gametocytogenesis is covered by only a partial knockdown resulting in low levels of expressed protein and to determine if PfPMRT1 is essential for gametocytogenesis, we episomally expressed GDV1-GFP-DD in the condΔPMRT1 parasite line, enabling conditional induction of sexual commitment upon addition of shield 1 in these parasites (59). Conditional knockout of PfPMRT1 in these transgenic parasites at day three post gametocyte induction resulted in pycnotic parasites from day 5 onwards, while excision of PfPMRT1 at day 5 post induction had no effect on gametocyte development (Figure 5C, D). Excision of the recodonized gene upon rapalog addition was confirmed at a genomic level by PCR for both conditions (Figure 5E). Quantification of parasite stages at day 10 post induction of GDV1 expression revealed 77.9% (+/- SD 7.7%) gametocytes and 22.1% (+/- SD 7.7%) pycnotic parasites in the control, while 100% of parasites were already pycnotic in the cultures, with induced knockout by addition of rapalog at day 3 post gametocyte induction by GDV1 expression (Figure 5F). This data indicates that PfPMRT1 is important for early gametocyte development.
PMRT1 is unique to the genus Plasmodium and interspecies complementation assays showed partial functional conservation
PfPMRT1 shows a lack of sequence similarities with known or putative transporters and/or conserved domains shared with known transporter families (2, 5). Our phylogenetic analysis revealed that homologs of PfPMRT1 are present across Plasmodium species with amino acid sequence identities of about 90% in the subgenus Laverania, but about 50% outside Laverania (Figure 6A). However, prediction of the protein structure using AlphaFold (39) indicates two bundles of four transmembrane helices with reasonable similarity of the C-terminal bundle with the photosynthetic reaction center Maquette-3 protein (60) (RMSD of 3.12) (Figure 6B, Figure S1B). In order to test for functional conservation, we expressed the PfPMRT1 homologs of P. vivax (PVP01_0936100) and P. knowlesi (PKNH_0933400) episomally as C-terminal Ty-1 fusion proteins under the nmd3 promoter in the condΔPMRT1 parasites. Both fusion proteins are expressed. They were again localized at the PPM as shown by IFA (Figure 6C, Figure S8), and, importantly, were able to partially restore growth after two cycles to 64.8% (+/- SD 9.8%) and 65.1% (+/- SD 7.4%) compared to condΔPMRT1 parasites (Figure 6D, S8). Excision of the recodonized endogenous Pfpmrt1 gene upon rapalog addition was confirmed at a genomic level by PCR (Figure 6E). These data indicate that PMRT1 is functionally conserved within the genus Plasmodium.
Discussion
In this manuscript we functionally described four so called “orphan transporter” (31) in P. falciparum, which were partially characterized in P. berghei, and included two additional so far uncharacterized proteins with transporter sequence signature.
We localized PfFVRT1-GFP – annotated on PlasmoDB (38) as putative divalent metal transporter – at the food vacuole of the parasite, which is in line with a previously predicted food vacuole association (1) and its reported homology (1, 61) to the conserved eukaryotic endosomal/lysosomal natural resistance-associated macrophage protein (NRAMP) transporter (62) in our structure similarity search. Repeated attempts to generate a TGD cell line failed, indicating an important role of this transporter during asexual blood stage development which is in agreement with data from a P. falciparum genome wide essentiality screen (63).
In concordance with recently published data identifying PbDMT2 and PbMFS6 as leaderless apicoplast transporters (32), we localized GFP-fusion proteins of PfDMT2 and PfMFS6 at the apicoplast. Successful knockdown of PfDMT2 resulted in a growth defect in the second cycle after induction, resembling the described delayed death phenotype of other apicoplast genes that were functionally inactivated (32, 64–66). It suggests an essential role of PfDMT2 in apicoplast physiology, as observed by Sayers et al. (32) for the rodent malaria P. berghei. This is further supported by our failed attempts to disrupt this gene using the SLI system.
We also failed to disrupt the PfMFS6 locus, which is in agreement with the gene knockout studies in P. berghei that led to a markedly decreased multiplication rate (31, 32, 67). Nevertheless, glmS-based knock-down, although comparable to PfDMT2-GFP knockdown (72.7% versus 76.8% reduction in GFP fluorescence, respectively) had no effect on parasite proliferation in our study. This might indicate that these reduced levels of PfMFS6, in contrast to reduced levels of PfDMT2, are sufficient for normal asexual replication in vitro.
Another candidate, PfCDF, annotated as putative cation diffusion facilitator family protein, showed multiple cytosolic foci within the parasite with no co-localization with apical organelle markers. The homologue in Toxoplasma gondii, TgZnT (TgGT1_251630) shows a similar cellular distribution (68). It has recently been shown to transport Zn2+, to localize to vesicles at the plant-like vacuole in extracellular tachyzoites and to be present at dispersed vesicles throughout the cytoplasm of intracellular tachyzoites (68). The essentiality of PfCDF for in vitro blood stage growth is debatable. We were not able to generate a clonal wild-type free TGD cell line although correct integration of the plasmid into the genomic locus could be verified (Figure S3C). This points towards its dispensability for in vitro blood stage growth, which is supported by i) its high (1.0) mutagenesis index score in a P. falciparum genome-wide mutagenesis screen (63) and ii) gene deletion experiments in rodent malaria species showing that CDF proteins are non-essential for in vivo blood stage development in P. yoelii (69) and P. berghei (31, 67).
Finally, two putative transporters, PfZIP1 and PfPMRT1, localized to the PPM. We show that PfZIP1 is non-essential for P. falciparum in vitro blood stage development, in line with a high (0.7) mutagenesis index score in a P. falciparum genome-wide mutagenesis screen (63). However, this is in contrast to the reported strong fitness loss in P. berghei (67) knockout mutants and failed knockout attempts in P. yoelli and P. berghei in vivo mouse models (32, 69). These observations may reflect differences between Plasmodium species or differing requirements for in vitro and in vivo growth conditions.
PfPMRT1 is annotated as a conserved Plasmodium membrane protein with unknown function. It has been described as a protein showing structural characteristics of a transporter, without sharing sequence similarities with known or putative transporters and/or conserved domains of known transporter families (2, 5). It encompasses 410 amino acids with eight predicted (70) transmembrane domains (TM) (Figure S1). The N- and C-terminal parts of PfPMRT1 are both predicted (71) to be facing the cytosolic side of the parasite. Surface electrostatics indicate a clear polarity of PfPMRT1 with negative charges facing the parasitophorous vacuole (PV) lumen and positive charges inside the parasite cytosol (Figure S8F). The loops protruding into the PV lumen of PfPMRT1 are generally larger than the cytosolic loops and possess stretches of negatively charged amino acids likely relevant for its transport function. Further functional characterization of PfMRT1 will deliver insight into its transporter capabilities and its physiological role.
Our phylogenetic analysis confirmed PMRT1 as unique for Plasmodium species with high sequence conservation only within the Laverania subgenus (72). In line with data from genome-wide mutagenesis screens (63, 67) and reported failed knockout attempts in P. yoelii (69), we found that PfPMRT1 is essential for parasite growth, as its functional inactivation resulted in growth arrest at the trophozoite stage accompanied by the accumulation of PPM-derived protrusions within the parasite. In contrast, conditional knockdown resulted only in a growth delay, indicating that minor residual PfPMRT1 protein levels appear to be sufficient to promote parasite growth. This finding was validated by episomal expression of an allelic copy under the control of the weak sf3a2 promoter (55) in the PfPMRT1 knockout parasites. Additionally, we found that PfPMRT1 is essential for early gametocytogenesis. Interestingly, the induction of the knockout at stage II–III had no effect on gametocytogenesis. This might be due to sufficient amounts of PfPMRT1 already present at the PPM, but could also indicate that the function of the transporter is not required for later stage gametocyte maturation.
For future work, further functional and pharmacological characterization of this transporter will provide insights into its biological role in different stages of the parasites life cycle, as transcriptomic data indicates – along with expression in blood stages (34, 35) – PfPMRT1 is expressed in oocysts of P. falciparum (73, 74) and P. berghei (75).
Material and methods
Cloning of plasmid constructs for parasite transfection
For endogenous tagging using the SLI system (41) a 889 bp (for PfPMRT1; PF3D7_1135300), 905 bp (PfFVRT1; PF3D7_0523800), 827bp (PfZIP1; PF3D7_0609100), 873 bp (PfDMT2; PF3D7_0716900), 877 bp (PfMFS6; PF3D7_1440800), 785 bp (PfCDF; PF3D7_0715900) long homology region (HR) was amplified using 3D7 gDNA and cloned into pSLI-GFP-glmS (76) (derived from pSLI-GFP (41)) using the NotI/MluI restriction site. In order to generate PfPMRT1-2xFKBP-GFP a 1000 bp long HR was amplified using 3D7 gDNA and cloned into pSLI-2xFKBP-GFP (41).
For SLI-based targeted gene disruption (SLI-TGD) (41) a 501 bp (PfPMRT1), 378 bp (PfFVRT1), 511 bp (PfZIP1), 399 bp (PfDMT2), 396 bp (PfMFS6), 741 bp (PfCDF) long homology region was amplified using 3D7 gDNA and cloned into the pSLI-TGD plasmid (41) using NotI and MluI restriction sites.
For conditional deletion of PfPMRT1, the first 492 bp of the PfPMRT1 gene were PCR amplified to append a first loxP site and a recodonized T2A skip peptide. The recodonized full-length coding region of PfPMRT1 was synthesized (GenScript, Piscataway, NJ, USA) and PCR amplified with primers to add a second loxP site after the gene to obtain a second fragment. Both fragments were cloned into pSLI-3xHA (55), using NotI/SpeI and AvrII/XmaI sites. This resulted in plasmid pSLI-PfPMRT1-loxP and the resulting transgenic cell line after successful genomic modification was transfected with pSkip-Flox (41) using 2 μg/ml Blasticidin S to obtain a line expressing the DiCre fragments (condΔPMRT1).
For complementation constructs, the recodonized PfPMRT1 gene was PCR amplified using primers to append the TY1 sequence and cloned via XhoI and AvrII or KpnI into pEXP1comp (55) containing yDHODH as a resistance marker and different promoters (nmd3 (PF3D7_0729300), sf3a2 (PF3D7_0619900)) driving expression of the expression cassette. This resulted in plasmids c-nmdrPfPMRT1-ty1 and c-sf3a2PfPMRT1-ty1.
PfPMRT1 homologues of P. vivax (PVP01_0936100) (77) and P. knowlesi (PKNH_0933400) (78) were amplified from parasite gDNA and cloned into pnmd3EXP1comp (55) via the XhoI/AvrII restriction site. For co-localization experiments the plasmids pLyn-FRB-mCherry (41), P40PX-mCherry (44), pARL-crtACP-mCherry (46), pARL-ama1ARO-mCherry (49) and pARL-ama1AMA1-mCherry (51) were used. For conditional gametocyte induction yDHODH was amplified by PCR from pARL-ama1AMA1-mCherry-yDHODH (51) and cloned into GDV1-GFP-DD-hDHFR(59)(59) using the XhoI/XhoI restriction site.
Oligonucleotides and plasmids used in this study are listed in Table S1A and S1B.
P. falciparum culture and transfection
Blood stages of P. falciparum 3D7 were cultured in human erythrocytes (O+). Cultures were maintained at 37°C in an atmosphere of 1% O2, 5% CO2 and 94% N2 using RPMI complete medium containing 0.5% Albumax according to standard protocols (79). To maintain synchronized parasites, cultures were treated with 5% sorbitol (80).
Induction of gametocytogenesis was done as previously described (58, 59). Briefly, GDV1-GFP-DD expression was achieved by addition of 4 µM shield-1 to the culture medium and gametocyte cultures were treated with 50 mM N-acetyl-D-glucosamine (GlcNAc) for five days starting 72 hours post shield-1 addition to eliminate asexual parasites(81). Alternatively, asexual ring stage cultures with >10% parasitemia were synchronized with Sorbitol (80) cultured for 24 hours and treated with 50 mM N-acetyl-D-glucosamine (GlcNAc) (81) for five days.
For transfection, Percoll-purified (82) late-schizont-stage parasites were transfected with 50 µg of plasmid DNA using Amaxa Nucleofector 2b (Lonza, Switzerland) as previously described(83). Transfectants were selected either using 4 nM WR99210 (Jacobus Pharmaceuticals), 2 μg/ml Blasticidin S (Life Technologies, USA), or 0.9 μM DSM1 (84) (BEI Resources; https://www.beiresources.org). In order to select for parasites carrying the genomic modification using the SLI system (41), G418 (Sigma-Aldrich, St. Louis, MO) at a final concentration of 400 µg/ml was added to 5% parasitemia culture. The selection process and testing for integration were performed as previously described (41).
For SLI-TGD, a total of six (PfPMRT1, PfDMT2, PfZIP1, PfCDF) or eight (PfFVRT1, PfMFS6) independent 5 ml cultures containing the episomal plasmid were selected under G418 for at least eight weeks.
Imaging and immunofluorescence analysis (IFA)
Fluorescence images of infected erythrocytes were observed and captured using a Zeiss Axioskop 2plus microscope with a Hamamatsu Digital camera (Model C4742-95), a Leica D6B fluorescence microscope equipped with a Leica DFC9000 GT camera and a Leica Plan Apochromat 100x/1.4 oil objective or an Olympus FV3000 with a x100 MPLAPON oil objective (NA 1.4). Confocal microscopy was performed using a Leica SP8 microscope with laser excitation at 405 nm, 490 nm, and 550 nm for DAPI, GFP, and mCherry excitation, respectively. An HC PL APO 63x NA 1.4 oil immersion objective was used and images were acquired with the HyVolution mode of the LASX microscopy software. After recording, images were deconvolved using Huygens (express deconvolution, setting ‘Standard’).
Microscopy of unfixed IEs was performed as previously described (85). Briefly, parasites were incubated in RPMI1640 culture medium with Hoechst-33342 (Invitrogen) for 15 minutes at 37°C prior to imaging. 7 µl of IEs were added on a glass slide and covered with a cover slip. Control images of 3D7 wild type parasites across the IDC are included in Figure S8D, E. BODIPY TR C5 ceramide (Invitrogen) staining was performed by adding the dye to 32 hours post invasion parasites in a final concentration of 2.5 μM in RPMI as previously described (85). For DHE staining of the parasite cytosol (44), 80 µl of resuspended parasite culture were incubated with DHE at a final concentration of 4.5 µg/ml in the dark for 15 minutes prior to imaging.
IFAs were performed as described previously (86). Briefly, IEs were smeared on slides and air-dried. Cells were fixed in 100% ice cold methanol for 3 minutes at -20°C. Afterwards, cells were blocked with 5% milk powder for 30 minutes. Next primary antibodies were diluted in PBS/3% milk powder and incubated for 2 hours, followed by three washing steps in PBS. Secondary antibodies were applied for 2 hours in PBS/3% milk powder containing 1 μg/ml Hoechst-33342 (Invitrogen) or DAPI (Roche) for nuclei staining, followed by 3 washes with PBS. One drop of mounting medium (Mowiol 4-88 (Calbiochem)) was added and the slide sealed with a coverslip for imaging.
To assess the localisation of the endogenously HA-tagged PfPMRT1 IFAs were performed in suspension with Compound 2-stalled schizonts (87) to distinguish protein located at the PPM from that located at the PVM as previously done (55, 88). For this, trophozoite stages were treated with Compound 2 (1 μM) overnight, and arrested schizonts were harvested, washed in PBS, and fixed with 4% paraformaldehyde/0.0075% glutaraldehyde in PBS. Cells were permeabilized with 0.5% Triton X-100 in PBS, blocked with 3% BSA in PBS, and incubated overnight with primary antibodies diluted in 3% BSA in PBS. Cells were washed 3 times with PBS and incubated for 1 hour with Alexa 488 nm or Alexa 594 nm conjugated secondary antibodies specific for human and rat IgG (Invitrogen) diluted 1:2,000 in 3% BSA in PBS and containing 1 μg/ml DAPI. Cells were directly imaged after washing 5 times with PBS
Antisera used: 1:200 mouse anti-GFP clones 7.1 and 13.1 (Roche), 1:500 rat anti-HA clone 3F10 (Roche), 1:1000 human anti-MSP1 (89), 1:10000 mouse anti-TY1 (ThermoFischer Scientific Cat.No: MA5-23513). Contrast and intensities were linear adjusted if necessary and cropped images were assembled as panels using Fiji (90), Corel Photo-Paint X6 and Adobe Photoshop CC 2021.
Immunoblots
For immunoblotting parasites were released from erythrocytes by incubation with 0.03% saponin in PBS for 10 minutes on ice followed by three wash steps with D-PBS. Proteins were then extracted with lysis buffer (4 % SDS, 0.5 % Triton X-100, 0.5x D-PBS in dH2O) in the presence of protease cocktail inhibitor (Roche) and 1 mM PMSF followed by addition of reducing SDS sample buffer and 5 minutes incubation at 55°C. Parasite proteins were separated on a 10% SDS-PAGE gel using standard procedures and transferred to a nitrocellulose membrane (Amersham(tm)Protran(tm) 0.45 µm NC, GE Healthcare) using a transblot device (Bio-Rad) according to manufacturer’s instructions or to a nitrocellulose membrane (Licor) in a tankblot device (Bio-Rad) using transfer buffer (0.192 M glycine, 0.1% SDS, 25 mM Tris-HCl pH = 8.0) with 20% methanol.
Rabbit anti-aldolase (91) and anti-SBP1 (91) antibodies were diluted 1:2,000, mouse anti-GFP clones 7.1 and 13.1 (Roche) antibody was diluted 1:500 or 1:1,000, mouse anti-Ty1 (Sigma) was diluted 1:20000, rabbit anti-BIP (92) was diluted 1:2500 and rat anti-HA clone 3F10 (Roche) antibody was diluted 1:1,000.
The chemiluminescent signal of the HRP-coupled secondary antibodies (Dianova) was visualized using a Chemi Doc XRS imaging system (Bio-Rad) and processed with Image Lab Software 5.2 (Bio-Rad). To perform loading controls and ensure equal loading of parasite material anti-aldolase antibodies were used. The corresponding immunoblots were incubated two times in stripping buffer (0.2 M glycine, 50 mM DTT, 0.05% Tween 20) at 55°C for 1 hour and washed 3 times with TBS for 10 minutes. For Western blots shown in Figure S8C fluorescent signals of secondary goat anti-rabbit IgG coupled to IRDye® 680CW and goat anti-mouse IgG coupled to IRDye® 800CW were visualized using Odyssey Fc Imager by LI-COR Biosciences.
Growth Assay
A flow cytometry-based assay adapted from previously published assays (44, 93) was performed. For this, parasite cultures were resuspended and 20 µl samples were transferred to an Eppendorf tube. 80 µl RPMI containing Hoechst-33342 and dihydroethidium (DHE) was added to obtain final concentrations of 5 µg/ml and 4.5 µg/ml, respectively. Samples were incubated for 20 minutes (protected from UV light) at room temperature, and parasitemia was determined using an LSRII flow cytometer by counting 100,000 events using the FACSDiva software (BD Biosciences) or using an ACEA NovoCyte flow cytometer.
Stage distribution assay
In order to obtain tightly synchronized parasite cultures, percoll purified schizonts (82) were cultured for four hours together with fresh erythrocytes, followed by sorbitol synchronization and resulting in a four-hour age window of parasites. Next, the culture was divided in four dishes and rapalog was added at a final concentration of 250 nM immediately to one dish and at 20 hours post invasion (hpi) and 32 hpi to the respective dishes. Giemsa smears and samples for flow cytometry were collected at the indicated timepoints. The parasitemia was determined using a flow cytometry assay and the stages were determined microscopically counting at least 50 infected erythrocytes per sample and timepoint.
Gametocyte stage distribution assay
Giemsa-stained blood smears 10 days post induction of GDV1 expression were obtained and at least 10 fields of view were recorded using a 63x objective per treatment and time point. Erythrocyte numbers were then determined using the automated Parasitemia software (http://www.gburri.org/parasitemia/) while the number of gametocytes, pycnotic and asexual parasites was determined manually in >1800 erythrocytes per sample. This assay was done blinded.
GlmS-based knockdown
GlmS based knockdown assay was adapted from previously published assays (42, 76). To induce knockdown 2.5 or 5 mM glucosamine was added to highly synchronous early rings stage parasites. As a control, the same amount of glucosamine was also added to 3D7 wildtype parasites. For all analyses, the growth medium was changed daily, and fresh glucosamine were added every day.
Knockdown was quantified by fluorescence live cell microscopy at day 1 and 3 of the growth assay. Parasites with similar size were imaged, and fluorescence was captured with the same acquisition settings to obtain comparable measurements of the fluorescence intensity. Fluorescence intensity (integrated density) was measured with Fiji(90), and background was subtracted in each image. The data were analyzed with Graph Pad Prism version 8.
GlmS based knockdown experiments in gametocytes were performed as described previously (94). Briefly, synchronized ring stage cultures were induced by the addition of shield-1. At day 3 post induction the culture was spilt into two dishes and one dish was cultured in the presence of 2.5 mM glucosamine for the remaining ten days. Knockdown was quantified by fluorescence live cell microscopy at day 7 and 10 post induction, as described above and gametocyte parasitemia was determined at day 10 post induction using the automated Parasitemia software (http://www.gburri.org/parasitemia/).
DiCre mediated conditional knockout
The parasites containing the integrated pSLI-PfPMRT1-loxP construct were transfected with pSkip-Flox (41) using 2 μg/ml Blasticidin S to obtain a line expressing the DiCre fragments. To induce excision, the tightly synchronized parasites (detailed description see growth assay) were split into 2 dishes and rapalog was added to one dish (Clontech, Mountain View, CA) to a final concentration of 250 nM. The untreated dish served as control culture. Excision was verified at genomic level after 24 and 48 hours of cultivation by PCR and on protein level by Western blot using anti-HA antibodies.
Phylogenetic analysis
A blastp search of the PMRT1 sequence (PlasmoDB (38): PF3D7_1135300; UniProt: Q8II12) was performed against the nr database (9 May 2021) using Geneious Prime 2021.2.2 (https://www.geneious.com) and an E-value of 10e-0 (BLOSUM62 substitution matrix). Blast hits were filtered for sequences from taxa represented in the currently favored haemosporidian parasite phylogeny (95). The phylogeny derived from an amino acid alignment using Bayesian framework with a partitioned supermatrix and a relaxed molecular clock (18_amino_acid_partitioned_BEAST_relaxed_clock_no_outgroup.tre; (95)) was visualized with associated data using the R package ggtree v3.3.0.900 (96, 97). A multiple protein sequence alignment of PMRT1 and homologous sequences was performed using MAFFT v7.490 (98) using the G-INS-I algorithm to obtain a highly accurate alignment. Protein statistics were calculated using Geneious Prime 2021.2.2 (https://www.geneious.com) and EMBOSS pepstats v6.6.0.0 (99).
Prediction of protein structures
AlphaFold structure predictions (39) were retrieved from https://alphafold.ebi.ac.uk and the PDB used for DALI protein structure homology search (40). PyMOL Molecular Graphics System, Version 2.5.2 Schrödinger was used for visualization of all structures, generation of figures and the calculation of the root mean square deviation (RMSD) between the predicted crystal structure of PfPMRT1 and the Maquette-3 protein (PDB: 5vjt (60)) by cealign. The Adaptive Poisson-Boltzmann Solver (APBS) within PyMOL was used to predict the surface electrostatics of PfPMRT1.
Parasite icons were generated using BioRender (biorender.com), plasmids and oligonucleotides were designed using ApE (100) and statistical analysis was performed using GraphPad Prism version 8 (GraphPad Software, USA).
Funding
AB and JSW were funded by the German Research Foundation (DFG) grant BA 5213/3-1. Partnership of Universität Hamburg and DESY (PIER) project ID PIF-2018-87 (JSTR, CL, TWG), CSSB Seed grant KIF 2019/002 (TWG), Hospital Research Foundation Fellowship (DW), DAAD/Universities Australia joint research co-operation scheme (TWG, DW, BL). IH and BL were supported by Australian Government Research Training Stipend. JY, SG and GN were funded by DFG Projektnummer 374031971 TRR 240 A04 and 417451587. JSTÄ thanks the Jürgen Manchot Stiftung for funding and PMR, TS acknowledge funding by the DFG (SP1209/4-1)
Author contribution
Conceptualization: JSW, TWG, AB, JSTR
Methodology: GN, SG
Investigation: JSW, PMR, JY, GF; JSTÄ, HVT, AA, IH, BL
Formal Analysis: JSTR
Writing original manuscript: JSW, AB, JSTR, DW
Review & Editing: JSW, PMR, TWG, AB, JSTR, DW
Funding Acquisition: DW, CL, TWG, AB, JSTR
Resources: TWG
Project Administration: TWG, AB, JSTR
Supervision: DW, TS, SG, TWG, AB
All authors read and approved the manuscript.
Figures
Figure S1: Structure predictions and structure homology search of candidate proteins
(A) AlphaFold structure predictions of the six selected orphan transporters visualized in PyMol. (B) Results from protein structure comparison server Dali using the AlphaFold-generated PDB files of the selected transporters as input structure. Shown are the top five non-redundant hits with Z score (significance estimate), msd (difference between the root-mean-square-deviation (rmsd) value associated with a protein structure pair and the rmsd value that would have been observed in the case that the two structures had the same crystallographic resolution), lali (number of aligned positions), nres (number of residues in the matched structure) and %id (the percentage sequence identity in the match).
Figure S2: Validation of generated transgenic cell lines by PCR and Western blot.
(A) Confirmatory PCR of unmodified wildtype (WT) and transgenic knock-in (KI) cell lines (PF3D7_0523800-GFP-glmS (PfFVRT1), PF3D7_0609100-GFP-glmS (PfZIP1), PF3D7_0715900-GFP-glmS (PfCDF), PF3D7_0716900-GFP-glmS (PfDMT2), PF3D7_1440800-GFP-glmS (PfMFS6) and PF3D7_1135300-GFP-glmS (PfPMRT1)) to check for genomic integration at the 3’- and 5’-end of the locus. Position of the primer used are indicated with numbered arrows in Figure 1A. (B) Western Blot analysis of wildtype (3D7) and knock-in (KI) cell lines using mouse anti-GFP to detect the tagged full-length protein (upper panel) and rabbit anti-aldolase to control for equal loading (lower panel). Protein size is indicated in kDa. Expected molecular weight for GFP fusion proteins: PfFVRT1 (107.5 kDa), PfZIP1 (69.0 kDa), PfDMT2 (66.4 kDa), PfMFS6 (98.8 kDa), PfPMRT1 (77.5 kDa), PfCDF (91.6 kDa) (C) Localization of PfCDF-3xHA by IFA in ring, trophozoite and schizont parasites. Nuclei were stained with Hoechst. Diagnostic PCR of unmodified wildtype (WT) and transgenic knock-in (KI) cell line. (D) Localization of PfPMRT1_2xFKBP-GFP across the IDC. Nuclei were stained with DAPI. Scale bar, 2 µm. Diagnostic PCR of unmodified wildtype (WT) and transgenic knock-in (KI) cell line. (E) Localization of PfZIP1-GFP in merozoites. Nuclei were stained with DAPI. Scale bar, 2 µm. (F) Confocal microscopy of PfPMRT1-GFP co-expressing the PPM marker Lyn-mCherry. Scale bar, 1µm. Nuclei were stained with Hoechst.
Figure S3: Targeted gene disruption (TGD) of PfZIP1 and PfCDF.
A) Schematic representation of TGD strategy using the selection-linked integration system (SLI). pink, human dihydrofolate dehydrogenase (hDHFR); grey, homology region (HR); green, green fluorescence protein (GFP) tag; dark grey, T2A skip peptide; blue, neomycin resistance cassette. Stars indicate stop codons, and arrows depict primers (P1 to P4) used for the integration check PCR. (B) Localization of PfZIP1-TGD-GFP in ring, trophozoite and schizont parasites. Nuclei were stained with Hoechst-33342. Scale bar, 2 µm. Confirmatory PCR of unmodified wildtype (WT) and transgenic targeted gene disruption (TGD) cell line. Growth curves of PfZIP1-TGD vs. 3D7 parasites monitored over five days by FACS. Three independent growth experiments were performed and a summary is shown as percentage of growth compared to 3D7 parasites. (C) Localization of PfCDF-TGD in ring, trophozoite and schizont parasites. Nuclei were stained with DAPI. Confirmatory PCR of unmodified wildtype (WT) and transgenic targeted gene disruption (TGD) cell line. Scale bar, 1 µm.
Figure S4: Conditional knockdown via glmS system.
Live cell microscopy of (A) PfFVRT1-GFP-glmS, (B) PfCDF-GFP-glmS, (C) PfZIP1-GFP-glmS (D) PfDMT2-GFP-glmS, (E) PfMFS6-GFP-glmS and (F) PfPMRT1-GFP-glmS parasites 40 hours after treatment without (control) or with 2.5 mM Glucosamine. Nuclei were stained with Hoechst-33342. Scale bar, 2 µm. (G) Individual growth curves of the growth assays shown in Figure 2D. (H) PCR using a GFP forward and glmS reverse primer confirming the presence of the GFP and glmS sequence in the pSLI-PfFVRT1-GFP-glmS and PfCDF-GFP-glmS plasmids. pSLI-PF3D7_0631900-GFP (35) was used as negative control.
Figure S5: Conditional knockout of PfPMRT1 via DiCre-based system
Replicates of growth curves of condΔPMRT1, c-nmd3PfPMRT1-ty1 and c-sf3a2PfPMRT1-ty1 parasites +/- rapalog monitored over five days by FACS shown in Figure 3.
Figure S6: Conditional knockout of PfPMRT1
(A) Parasite stage distribution in Giemsa smears displayed as heatmap showing percentage of stages for control, 4 hpi, 20 hpi or 32 hpi rapalog treated 4 hour window synchronized condΔPMRT1parasite cultures over one cycle. (B) Giemsa smears of control and 4 hpi, 20 hpi or 32 hpi rapalog treated parasites at 4, 16, 20, 24, 32, 40 and 48 hpi. Scale bar, 5 µm.
Figure S7: Conditional knockdown of PfPMRT1 has no effect during gametocyte development.
(A) Confirmatory PCR of unmodified wildtype (WT) and transgenic 3D7-iGP-PfPMRT1-GFP-glmS to check for genomic integration at the 3’- and 5’-end of the locus. Position of the primer used are indicated with numbered arrows in Figure 1A. (B) Schematic representation of the experimental setup. (C) Live cell microscopy of 3D7-iGP-PfPMRT1-GFP stage I – V gametocytes. Scale bar, 2 µm. (D) Giemsa smears of stage I – V gametocytes cultured either without (control) or with 2.5 mM glucosamine. Scale bar, 5 µm. (E) Quantification of knockdown by measuring mean fluorescence intensity (MFI) density and size (area) of parasites at day 7 and day 12 post induction of gametocytogenesis cultured either without (control) or with 2.5 mM glucosamine. Scale bar, 2 µm. Statistics are displayed as mean +/- SD of four independent experiments and individual data points are displayed as scatterplot color-coded by experiments according to Superplots guidelines(101)(101). P-values displayed were determined with two-tailed unpaired t-test. (F) For each condition gametocytemia at day 10 post gametocyte induction was determined by counting between 1256-2653 (mean 2147) cells per condition in Giemsa-stained thin blood smears. Displayed are means +/- SD of independent growth experiments with the number of experiments (n) indicated. P-values displayed were determined with two-tailed unpaired t-test.
Figure S8: Individual growth curves of c-nmd3Pk-ty1 (A) and c-nmd3Pv-ty1 (B) parasites +/- rapalog monitored over two IDCs by FACS shown in Figure 6. (C) Western Blot analysis of c-nmd3Pf-ty1, c-nmd3Pk-ty1 and c-nmd3Pv-ty1 cell lines using mouse anti-ty1 to detect the tagged full-length protein (upper panel) and rabbit anti-BIP to control for loading (lower panel). Protein size is indicated in kDa. (D) and (E) 3D7 wild type parasites imaged across the IDC to establish autofluorescence levels with Zeiss Axioskop 2plus microscope (D) or Leica D6B fluorescence microscope (E). (F) Surface electrostatics of the predicted PfPMRT1 structure generated by APBS within PyMol.
Table S1: Oligonucleotides and plasmids used in this study
Acknowledgements
We thank Michael Filarsky for providing the pHcamGDV1-GFP-DD_hDHFR plasmid, Egbert Tannich for providing P. vivax and P. knowlesi gDNA, Mike Blackman for the anti-MSP1 antibody, Jacobus Pharmaceuticals for WR99210, Greg Burri for the parasitemia software and the Advanced Light and Fluorescence Microscopy (ALFM) facility at the Centre for Structural Systems Biology (CSSB), in particular Roland Thuenauer, for support with light microscopy image recording and analysis. DSM1 (MRA-1161) was obtained from MR4/BEI Resources, NIAID, NIH. Furthermore, we thank Maria Rosenthal for her help with the visualization of the predicted protein structures.
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