Characterization of Apicomplexan Amino Acid Transporters (ApiATs) in the Malaria Parasite Plasmodium falciparum

ABSTRACT During the symptomatic human blood phase, malaria parasites replicate within red blood cells. Parasite proliferation relies on the uptake of nutrients, such as amino acids, from the host cell and blood plasma, requiring transport across multiple membranes. Amino acids are delivered to the parasite through the parasite-surrounding vacuolar compartment by specialized nutrient-permeable channels of the erythrocyte membrane and the parasitophorous vacuole membrane (PVM). However, further transport of amino acids across the parasite plasma membrane (PPM) is currently not well characterized. In this study, we focused on a family of Apicomplexan amino acid transporters (ApiATs) that comprises five members in Plasmodium falciparum. First, we localized four of the P. falciparum ApiATs (PfApiATs) at the PPM using endogenous green fluorescent protein (GFP) tagging. Next, we applied reverse genetic approaches to probe into their essentiality during asexual replication and gametocytogenesis. Upon inducible knockdown and targeted gene disruption, a reduced asexual parasite proliferation was detected for PfApiAT2 and PfApiAT4. Functional inactivation of individual PfApiATs targeted in this study had no effect on gametocyte development. Our data suggest that individual PfApiATs are partially redundant during asexual in vitro proliferation and fully redundant during gametocytogenesis of P. falciparum parasites. IMPORTANCE Malaria parasites live and multiply inside cells. To facilitate their extremely fast intracellular proliferation, they hijack and transform their host cells. This also requires the active uptake of nutrients, such as amino acids, from the host cell and the surrounding environment through various membranes that are the consequence of the parasite’s intracellular lifestyle. In this paper, we focus on a family of putative amino acid transporters termed ApiAT. We show expression and localization of four transporters in the parasite plasma membrane of Plasmodium falciparum-infected erythrocytes that represent one interface of the pathogen to its host cell. We probed into the impact of functional inactivation of individual transporters on parasite growth in asexual and sexual blood stages of P. falciparum and reveal that only two of them show a modest but significant reduction in parasite proliferation but no impact on gametocytogenesis, pointing toward dispensability within this transporter family.

ABSTRACT During the symptomatic human blood phase, malaria parasites replicate within red blood cells. Parasite proliferation relies on the uptake of nutrients, such as amino acids, from the host cell and blood plasma, requiring transport across multiple membranes. Amino acids are delivered to the parasite through the parasite-surrounding vacuolar compartment by specialized nutrient-permeable channels of the erythrocyte membrane and the parasitophorous vacuole membrane (PVM). However, further transport of amino acids across the parasite plasma membrane (PPM) is currently not well characterized. In this study, we focused on a family of Apicomplexan amino acid transporters (ApiATs) that comprises five members in Plasmodium falciparum. First, we localized four of the P. falciparum ApiATs (PfApiATs) at the PPM using endogenous green fluorescent protein (GFP) tagging. Next, we applied reverse genetic approaches to probe into their essentiality during asexual replication and gametocytogenesis. Upon inducible knockdown and targeted gene disruption, a reduced asexual parasite proliferation was detected for PfApiAT2 and PfApiAT4. Functional inactivation of individual PfApiATs targeted in this study had no effect on gametocyte development. Our data suggest that individual PfApiATs are partially redundant during asexual in vitro proliferation and fully redundant during gametocytogenesis of P. falciparum parasites. IMPORTANCE Malaria parasites live and multiply inside cells. To facilitate their extremely fast intracellular proliferation, they hijack and transform their host cells. This also requires the active uptake of nutrients, such as amino acids, from the host cell and the surrounding environment through various membranes that are the consequence of the parasite's intracellular lifestyle. In this paper, we focus on a family of putative amino acid transporters termed ApiAT. We show expression and localization of four transporters in the parasite plasma membrane of Plasmodium falciparum-infected erythrocytes that represent one interface of the pathogen to its host cell. We probed into the impact of functional inactivation of individual transporters on parasite growth in asexual and sexual blood stages of P. falciparum and reveal that only two of them show a modest but significant reduction in parasite proliferation but no impact on gametocytogenesis, pointing toward dispensability within this transporter family.

RESULTS
P. falciparum ApiATs localize at the parasite plasma membrane. The five members of the ApiAT family in P. falciparum show different gene expression patterns and mRNA levels during the intraerythrocytic developmental cycle (IDC) (37). While PfApiAT2 has its maximum transcript level in early ring stage parasites (8 h postinfection [hpi]) and PfApiAT4 and PfApiAT10 mRNA levels peak in late ring stage parasites (16 hpi), PfApiAT8 shows a maximum of transcripts in late stage schizonts (48 hpi) and mRNA of PfApiAT9 is almost absent during the IDC (Fig. 1A). Overall, PfApiAT2 and PfApiAT4 are most abundantly expressed on the mRNA level.
To determine their protein expression and localization, we tagged each of the five members of the PfApiAT family endogenously with GFP using the selection-linked integration (SLI) system (38). Correct integration of the corresponding targeting plasmids into the respective genomic loci was verified by PCR (see Fig. S1A in the supplemental material). Except for 3D7-ApiAT9-GFP, all generated transgenic cell lines expressed the full-length fusion protein (Fig. S1B) to a sufficient level that allowed its subcellular localization. All of them are localized at the parasite periphery (Fig. 1B). Subsequent colocalization with the episomally expressed PPM marker Lyn (39)-mCherry (38) reveals PPM localization that becomes particularly evident in free merozoites, where PPM-and PVM-localized proteins can clearly be separated (Fig. 1C).
In contrast to published data (36), we also found endogenously GFP-tagged PfApiAT10 localizing at the PPM. To reprobe into the apparent PPM localization of endogenously GFP-tagged PfApiAT10, we also overexpressed this gene as a GFP and mCherry fusion protein using two different promoters (crt [45] or ama1 [46]). This allowed us to assess the influence of the tags as well as differential expression profiles on PfApiAT10 protein localization. All cell lines showed PPM localization of the PfApiAT10 fusion proteins (Fig. 1B to E and Fig. S1). Of note, functional inactivation of PfApiAT10 did not result in conferral of drug resistance to atovaquone (Fig. S2B).
Individual PfApiATs are not essential for asexual blood stage development. To probe into the essentiality of the ApiAT family for asexual parasite proliferation, we first targeted the most abundantly expressed PfApiATs, PfApiAT2 and PfApiAT4 by conditional knockdown. Downregulation was achieved by introducing a glmS ribozyme sequence (40) before the 39 untranslated region (39UTR) of either the apiat2 or apiat4 genomic locus (Fig. S1). The ribozyme was activated by the addition of 2.5 mM D-(1)-glucosamine hydrochloride (GLCN), which resulted in degradation of mRNA and therefore decreased protein levels. This was assessed and quantified by live-cell fluorescence microscopy after two cycles. GLCN treatment resulted in a decreased GFP fluorescence of 85.9% 6 0.9% (mean 6 standard deviation [SD]) for 3D7-PfApiAT2-GFP or 74.8% 6 14.0% for 3D7-PfApiAT4-GFP ( Fig. 2A to D) and led to a moderate, but significant, reduction of parasite growth of 14.0 to 20.7% compared to 3D7 parasites cultured with GLCN (Fig. 2E). These data indicate that PfApiAT2 and PfApiAT4 play a role in efficient blood cell proliferation but imply that they might be nonessential. Therefore, we targeted these genes with deletion constructs using the SLI system (38) that led to the expression of severely truncated versions of the ApiATs. In this targeted gene disruption (TGD) approach, we also included PfApiAT8 and PfApiAT10 (Fig. S1C).
Imaging of the TGD cell lines revealed a more diffuse but still membrane-associated GFP signal (Fig. 3A to D). This might be due to the remaining transmembrane domains of the truncated PfApiAT mutants; however, our approach deleted at least three quarters of their predicted transmembrane (TM) domains and thus most likely abolished transporter activity. In concordance with the inducible knockdown data, functional inactivation by truncation of PfApiAT2 and PfApiAT4 in the corresponding transgenic cell lines (3D7-ApiAT2-TGD and 3D7-ApiAT4-TGD) led to a moderate decrease of parasite proliferation of 20.2% 6 3.2% and 19.8% 6 8.6% after two parasite replication cycles (Fig. 3E). No significant reduction of growth was observed upon disruption of PfApiAT8 and PfApiAT10 (Fig. 3E). Interestingly, cultivation in amino acid-depleted medium (approximately 90% reduced concentration) did not indicate a higher susceptibility of any of the TGD cell lines to low amino acid concentrations compared to wild-type parasites (Fig. 3F).
To probe into potential transcriptional perturbations within this gene family due to functional inactivation of a single member, quantitative real-time PCR (qPCR) analysis was performed using RNA from four different time points during asexual blood stage replication. However, no consistent upregulation of RNA levels of other PfApiAT family members was observed in individual PfApiAT TGDs (Fig. S3).
PfApiATs are dispensable during gametocyte development. Previous data (30,35) indicated a role of ApiAT8 during gametocyte development of the rodent malaria parasite P. berghei. Therefore, we reengineered the GFP-tagged gene knockdown (Fig. S4) and deletion cell lines (Fig. S5) for PfApiAT2, PfApiAT4, PfApiAT8, and PfApiAT10 in an inducible gametocyte-producing parasite line (3D7-iGP-GDV1GFP-DD [41]) using the same SLI approach. The resulting parasite lines allowed a robust, efficient, and synchronized induction of gametocytogenesis by expression of GDV1 upon addition of Shield-1 and therefore a solid basis for phenotypic analysis. First, using the C-terminal GFP tag, we confirmed expression of all four PfApiATs in gametocytes. As expected, most PfApiATs remain PPM localized during gametocytogenesis, which was additionally confirmed by the colocalization with the episomally expressed PPM marker Lyn (39)-mCherry (38) (Fig. 4A to F). The exception was PfApiAT9, which showed only a faint background staining in all gametocyte stages in the 3D7-ApiAT9-GFP line (Fig. 4D). The observed prominent GFP signal at the food vacuole in gametocytes is most likely an unspecific hemozoin signal and not derived from GFP fusion proteins, as also observed in 3D7-iGP and 3D7 wild-type control parasites (Fig. S4C). Of note, PfApiAT2 was observed to be strongest expressed in early stage gametocytes and weaker in late stages ( Fig. 5B and D; Fig. S4D), while PfApiAT4 showed strongest expression in late stage gametocytes ( Fig. 5C and E). Next, we investigated the consequence of glmS-based conditional knockdown for PfApiAT2 and PfApiAT4 (Fig. 5A). Although 75 to 80% knockdown of PfApiAT2 or PfApiAT4 expression was achieved ( Fig. 5B to E), no significant reduction in gametocytemia or aberrant gametocyte development could be detected (Fig. 5F). This was reinvestigated by targeted gene disruption. Likewise, deletion of these two genes as well as of PfApiAT8 and PfApiAT10 did not result in any measurable impairment of gametocyte development or morphology, indicating the dispensability of these individual PfApiATs for the sexual stage development of the parasite ( Fig. 6 and Fig. S5).

DISCUSSION
We localized four putative amino acid transporters (PfApiAT2, PfApiAT4, PfApiAT8, and PfApiAT10) of the ApiAT family to the PPM in asexual blood stage parasites and gametocytes. Due to the low expression of PfApiAT9-GFP-in agreement with the transcript levels of apiat9 in these stages (37,42,43)-no conclusive localization could be delivered. The observed PPM localization of the investigated ApiATs is in concordance with the following published data. (i) The P. berghei ApiAT8 homologue was shown to be a general cationic amino acid transporter of the PPM (29,30). (ii) PfApiAT8 has recently been localized to the PPM using an overexpression approach (31). (iii) Several T. gondii ApiATs (TgApiAT1, TgApiAT2, TgApiAT3-1, TgApiAT3-2, TgApiAT3-3, TgApiAT5-3, TgApiAT6-1, and TgApiAT6-3) have been located at the PPM as well (25,29,32,33,44).   (82). P values displayed were determined using a two-tailed unpaired t test with Welch's correction. (E) Growth of parasites treated with or without 2.5 mM GLCN after two and four parasite replication cycles (Continued on next page) Recent work using episomally overexpressed PfApiAT10-GFP implied an association of this transporter with the mitochondrial membrane (36) in P. falciparum Dd2 (PfDd2) parasites. This localization differs from the observed PPM localization of endogenously GFP-tagged PfApiAT10 in both 3D7 as well as 3D7-iGP parasites ( Fig. 1B and 4C; see also Fig. S2A in the supplemental material) reported in this study. We confirmed the PPM localization of PfApiAT10 by its overexpression either as a GFP or mCherry fusion using two different promoters (crt [45] and ama1 [46]). In line with that, the reported reduced sensitivity to the mitochondrial electron transport chain inhibitor atovaquone (47) upon knockout of PfApiAT10 (36) was not observed upon targeted gene disruption in our study (Fig. S2B). It is possible but appears unlikely that the reported mitochondrial association as well as reduced sensitivity to atovaquone upon overexpression is due to the different parasite strains (PfDd2 [48] versus 3D7 [this study]), given that PfDd2_apiat10 (PFDd2_030017500) has only one silent mutation at position G1080A compared to Pf3D7_apiat10 (49). Of note, PfApiAT10 is also not part of the recently published Plasmodium mitochondrial proteome (50).
During the intraerythrocytic development of the parasite, the amino acid requirements are largely covered by degradation of the globin polypeptide (14,51), although -for instance-the import of isoleucine is crucial for the survival of the parasite, as P. falciparum lacks the canonical pathways for its biosynthesis (52) and adult human hemoglobin lacks this amino acid. Dedicated amino acid transporters could fill this gap. Therefore, we tested the impact of functional inactivation of individual PfApiATs on parasite growth in asexual and sexual blood stages of P. falciparum. We observed only a minor but significant reduction in parasite growth upon knockdown or gene disruption of PfApiAT2 and PfApiAT4 in asexual blood stages without compensatory upregulation of other PfApiATs on the transcriptional level, as indicated by qPCR analysis. The phenotypes are in agreement with a previously reported reduced parasite multiplication rate of 36% in PbApiAT4 knockout parasites (35). Moreover, our data are also in line with the finding that PbApiAT8 is not essential for asexual replication (29,30,35,53). Additionally, functional inactivation of individual PfApiAT did not result in parasites more sensitive to reduced amino acid concentrations in the medium. The absence of profound growth effects might be explained by functional redundancy either within the ApiAT family, as recently observed for T. gondii (34), or by the presence of yet unassigned transporters capable of transporting essential amino acids such as isoleucine across the PPM. Like in T. gondii, overlapping substrate specificities and lower transport levels might be sufficient for parasite growth in vitro (34). Of note, in T. gondii, the arginine transporter TgApiAT1 has been shown to be differently regulated on the translational level in dependence of arginine mediated by an upstream open reading frame (uORF) present on the 59 leader sequence of the transcript (26). A similar layer of regulation might also be present in PfApiATs. However, since Toxoplasma and Plasmodium share only one ApiAT, the likely most ancestral ApiAT2 (25), the regulatory elements as well as the general characteristics and substrates of the different PfApiATs remain largely unknown.
Interestingly, a knockout of the ApiAT8 homologue of P. berghei (PBANKA_0208300) resulted in strongly reduced number of mature gametocytes with an aberrant morphology of the remaining parasites (30) and strongly reduced exflagellation (35). In our study, functional inactivation of PfApiAT8 via targeted gene disruption had no impact on gametocyte development and morphology, which might reflect the pronounced differences in ApiATs of the Malaria Parasite Plasmodium falciparum gametocyte development between the rodent-infecting P. berghei and the human-infecting P. falciparum parasites (54). For future work, it will be interesting to target multiple ApiATs by gene disruption in parallel to assess their putative synergy and to probe into PfApiAT function in other fast-replicating stages of P. falciparum such as liver stages, for which the essentiality of several metabolic processes has recently been shown and that primarily rely on the amino acid uptake from their host (55).

MATERIALS AND METHODS
P. falciparum culture. Blood stages of P. falciparum 3D7 (56) were cultured in human red blood cells (O1 or B1). Cultures were maintained at 37°C in an atmosphere of 1% O 2 , 5% CO 2 , and 94% N 2 using RPMI Growth of 3D7-ApiAT-TGD cell lines as a percentage of 3D7 parasite growth, monitored over two intracellular development cycles by flow cytometry. The number of independent growth experiments (n) per 3D7-ApiAT-TGD cell line is indicated. 3D7 wild-type parasites were measured in parallel. Statistical differences were analyzed using a one-sample t test with Benjamini-Hochberg correction accounting for multiple comparisons. (F) Growth of TGD and 3D7 cell lines cultivated in low-amino acid medium relative to their growth in standard medium is shown as percentage of growth after two parasite replication cycles. The number of individual growth experiments (n) performed is indicated for each 3D7-ApiAT-TGD line. Additionally, 3D7 wild-type parasites were analyzed with n = 9. No statistical differences were observed by comparing relative growth of TGD cell lines to 3D7 using a two-tailed unpaired t test with Bonferroni correction. AA, amino acid.
complete medium containing 0.5% Albumax according to standard protocols (57). In order to obtain highly synchronous parasite cultures, late schizonts were isolated by Percoll gradient (58) and cultured with fresh erythrocytes for 4 h. Afterwards sorbitol synchronization (59) was applied in order to remove remaining schizonts resulting in a highly synchronous ring stage parasite culture with a 4-h age window. Induction of gametocytogenesis was done as previously described (41,60). Briefly, GDV1-GFP-DD expression was achieved by the addition of 2 or 4 mM Shield-1 to the culture medium, and gametocyte cultures were treated with 50 mM N-acetyl-D-glucosamine (GlcNAc) for 5 days starting 72 h after Shield-1 addition to eliminate asexual parasites (61). Alternatively, asexual ring stage cultures with .10% parasitemia, cultured in the presence of choline, were synchronized with sorbitol (59) and washed twice in choline-free RPMI medium. Cells were kept in choline-free medium for the entirety of the assay. After one reinvasion cycle, cultures at trophozoite stage were treated with 50 mM GlcNAc (61) and kept on this for 5 days. Gametocytes were maintained in RPMI complete medium containing 0.25% Albumax and 0.25% sterile filtered human serum (Interstate Blood Bank, Inc., Memphis, TN, USA).
Growth assays in low-amino acid medium were performed using amino acid-restricted RPMI medium prepared as previously described (20). Briefly, complete medium was added in a 1/20 dilution to amino acid-free RPMI 1640 medium (catalog no. R9010-01; US Biological). This resulted in a 1:20 of the concentration of every amino acid compared to the standard complete RPMI-based medium.
For overexpression constructs, the full-length PfApiAT10 sequence was amplified from parasite gDNA and cloned into pARL-ama1 (46)-AIP-mCherry-yDHODH (62) using the XhoI and KpnI restriction  (82). P values displayed were determined with a two-tailed unpaired t test with Welch's correction. (F) For each condition, gametocytemia at day 10 postgametocyte induction was determined by counting between 702 and 7,693 (mean, 2,210) cells per condition in Giemsastained thin blood smears. The relative gametocytemia values (as a percentage) displayed were obtained by dividing the gametocytemia of glucosamine-treated cultures by the gametocytemia of the corresponding untreated cultures. Displayed are means 6 SD of independent growth experiments with the number of experiments (n) indicated. A two-tailed unpaired t test with Welch's and Benjamini-Hochberg correction was used to calculate multiplicity-adjusted P values for ApiAT2-GFP-glmS or ApiAT4-GFP-glmS versus 3D7-iGP parasites all cultured with 2.5 mM GLCN.
To perform loading controls and ensure equal loading of parasite material, rabbit antialdolase (65) antibodies were used. The corresponding immunoblots were incubated twice in stripping buffer (0.2 M glycine, 50 mM dithiothreitol, 0.05% Tween 20) at 55°C for 1 h and washed three times with Tris-buffered saline for 10 min before reprobing.
Transfection of P. falciparum. For transfection, Percoll-purified (58) parasites at late schizont stage were transfected with 50 mg plasmid DNA using Amaxa Nucleofector 2b (Lonza, Switzerland) as previously described (66). Transfectants were selected using either 4 nM WR99210 (Jacobus Pharmaceuticals), 0.9 mM DSM1 (67) (BEI Resources), or 2 mg/ml blasticidin S (Life Technologies, USA). In order to select for parasites carrying the genomic modification via the SLI system (38), G418 (ThermoFisher, USA) at a final concentration of 400 mg/ml was added to a culture with about 5% parasitemia. The selection process and integration test were performed as previously described (38).
Imaging. All fluorescence images were captured using a Zeiss Axioskop 2plus microscope with a Hamamatsu digital camera (model C4742-95) or a Leica D6B fluorescence microscope equipped with a Leica DFC9000 GT camera and a Leica Plan Apochromat 100Â/1.4 oil objective.
Microscopy of live-parasite-infected erythrocytes was performed as previously described (68). Briefly, parasites were incubated in standard culture medium with 1 mg/ml Hoechst-33342 (Invitrogen) for 15 min at 37°C prior to imaging. Infected erythrocytes (5.4 ml) were added on a glass slide and covered with a cover slip. Nuclei were stained with 1 mg/ml Hoechst-33342 (Invitrogen). Mitochondria were visualized by incubation of parasites with 20 nM MitoTracker Red 665 CMXRos (Invitrogen) for 15 min at 37°C prior to imaging. Contrast and intensities were linear adjusted for clarification and cropped images were assembled as panels using Fiji (69) and Adobe Photoshop CC 2021.
Growth assay. For growth assays of TGD cell lines, a flow cytometry assay, adapted from previously published assays (70,71), was performed to measure proliferation over 5 days. For growth under lowamino acid conditions, TGD and wild-type cell lines were cultured in parallel in standard and amino acid-depleted medium for 5 days. Each day parasite cultures were resuspended, and 20-ml samples were transferred to an Eppendorf tube. Eighty microliters of RPMI medium containing Hoechst-33342 and dihydroethidium (DHE) was added to obtain final concentrations of 5 mg/ml and 4.5 mg/ml, respectively. Samples were incubated for 20 min (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.
Gametocyte quantification assay. Giemsa-stained blood smears at day 10 postinduction of GDV1 expression were obtained, and at least 10 fields of view were recorded using a 63Â 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 was determined manually in .700 erythrocytes per sample.
glmS-based knockdown. The glmS-based knockdown assay was adapted from previously published assays (40,72). To induce knockdown, highly synchronous early ring stage parasites were split in two dishes, 2.5 mM glucosamine was added to one of them, and parasite growth was measured by fluorescence-activated cell sorting (FACS) after two and four parasite replication cycles. Parasite cultures were inspected daily by Giemsa smears and, if necessary, diluted to avoid growth bias caused by high parasitemia. As an additional control, the same amount of glucosamine was also added to 3D7 wild-type parasites. For all analyses, medium was changed daily, and fresh glucosamine was added every day.
Knockdown was quantified by fluorescence live-cell microscopy using schizonts about 40 h after glucosamine treatment. Parasites of similar sizes 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 (69), and background was subtracted in each image. The data were visualized with GraphPad Prism version 8 (GraphPad Software, USA).
For knockdown experiments in gametocytes, synchronized ring stage cultures were induced by the addition of Shield-1 as described above. At day 3 postinduction, the culture was spilt into two dishes, and one dish was cultured in the presence of 2.5 mM glucosamine for the remaining 10 days. Knockdown was quantified by fluorescence live-cell microscopy at day 7 and 10 postinduction as described above.
Drug assays. Drug assays were adapted from previously described assays (73)(74)(75). Briefly, 3D7-iGP and 3D7-iGP-ApiAT10-TGD parasites were synchronized to a 4-h time window resulting in 0-to 4-h ring stage parasites. At 24 hpi, parasitemia was determined by flow cytometry, and the drug susceptibility assays were set up in black 96-well microtiter plates (Thermo Scientific) with 0.1% starting parasitemia and 2% hematocrit in a final volume of 200 ml. In each plate, infected erythrocytes in the absence of drugs treated with dimethyl sulfoxide (DMSO) only served as positive controls, while uninfected red blood cells (RBCs) served as negative controls (for background subtraction). Parasites were incubated with various concentrations of dihydroartemisinin (DHA) (catalog no. AG-CN2-0468; Adipogen, Switzerland) (0 to 50 nM) and atovaquone (catalog no. 23802; Cayman) (0 to 16 NM).
After 96 h of incubation, parasite growth was determined by measuring the fluorescence of SYBR Gold (Invitrogen). Therefore, 100 ml/well supernatant was discarded without disturbing the RBC layer and 100 ml of lysis buffer (20 mM Tris, 0.008% saponin, 0.08% Triton X-100, 1Â SYBR Gold) was added to each well. Plates were incubated in the dark for 2 h at room temperature before measuring fluorescence using the EnVision Multimode plate reader (PerkinElmer) as described previously (75). In order to calculate 50% infective concentration (IC 50 ) values, the measured values were normalized to the uninfected erythrocytes and plotted in GraphPad Prism version 8 (GraphPad Software, USA) as a percentage of the value for the DMSO control. Dose-response curves were generated using nonlinear regression (curve fit . dose-response inhibition . (log) inhibitor versus normalized response-variable slope).
Quantitative real-time PCR. Parasites at different time points (8,16,32, and 44 hpi) were harvested for 3D7-ApiAT2-TGD, 3D7-ApiAT4-TGD, 3D7-ApiAT8-TGD, 3D7-ApiAT10-TGD, and 3D7-WT to obtain RNA samples for quantitative real-time PCR (qPCR). Highly synchronous ring stage parasite cultures were grown for another 40 h, and TRIzol samples were harvested in the following cycle. Volumes of prewarmed TRIzol used for infected erythrocyte lysis and storage of RNA samples depended on the parasite stage: ring stages were lysed in 5Â volumes, trophozoites in 10Â volumes, and schizonts in 20Â volumes of the settled cell pellet. RNA was purified and checked for the absence of genomic DNA. cDNA synthesis with random hexamers, and quantitative real-time PCR was performed exactly as previously described (76). Primers for each of the apiat genes, for genes to control for parasite stages (sbp1 [77], tom22, and ama1) and for housekeeping genes (arginyl-tRNA synthetase [76] and fructose-bisphosphate aldolase [78] genes) are listed in Table S1. Amplification efficiencies of the primer pairs were determined over 6 log 10 dilutions of gDNA (10 ng to 0.0001 ng) and were shown to have similar values between 1.915 and 2.001 (Table S1). Expression of apiat genes and controls were analyzed in relation to expression of the arginyl-tRNA synthetase gene (normalizer).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.