A Histone Methyltransferase Inhibitor Can Reverse Epigenetically Acquired Drug Resistance in the Malaria Parasite Plasmodium falciparum

Malaria parasites invade and replicate within red blood cells (RBCs), extensively modifying their structure and gaining access to the extracellular environment by placing the plasmodial surface anion channel (PSAC) into the RBC membrane. Expression of members of the cytoadherence linked antigen gene 3 (clag3) family is required for PSAC activity, a process that is regulated epigenetically. PSAC is a well-established route of uptake for large, hydrophilic antimalarial compounds, and parasites can acquire resistance by silencing clag3 gene expression, thereby reducing drug uptake.

ABSTRACT

Malaria parasites invade and replicate within red blood cells (RBCs), extensively modifying their structure and gaining access to the extracellular environment by placing the plasmodial surface anion channel (PSAC) into the RBC membrane. Expression of members of the cytoadherence linked antigen gene 3 (clag3) family is required for PSAC activity, a process that is regulated epigenetically. PSAC is a well-established route of uptake for large, hydrophilic antimalarial compounds, and parasites can acquire resistance by silencing clag3 gene expression, thereby reducing drug uptake. We found that exposure to sub-IC₅₀ concentrations of the histone methyltransferase inhibitor chaetocin caused substantial changes in both clag3 gene expression and RBC permeability, and reversed acquired resistance to the antimalarial compound blasticidin S that is transported through PSACs. Chaetocin treatment also altered progression of parasites through their replicative cycle, presumably by changing their ability to modify chromatin appropriately to enable DNA replication. These results indicate that targeting histone modifiers could represent a novel tool for reversing epigenetically acquired drug resistance in P. falciparum.

INTRODUCTION

Malaria is a disease common in tropical and subtropical regions of the developing world, where it continues to cause significant morbidity and mortality, primarily among young children. The most virulent of the human malaria parasites, Plasmodium falciparum, is known for making extensive modifications to the membrane of the infected RBC (1). These include significant changes in the permeability of the RBC membrane to various solutes, in particular anions and small compounds (2). Targeting these modifications for novel intervention strategies is an attractive potential approach for fighting the disease. In particular, given that the increase in RBC membrane permeability provides access to the parasite, this trait could potentially be exploited in the development of new antimalarial strategies.

The changes in permeability to the RBC membrane resulting from infection by P. falciparum have been extensively studied. Experiments employing osmotic lysis or electrophysiology of infected cells have detected the induction by parasites of an anion pore or channel within the RBC membrane, alternatively referred to as the new permeability pathway (NPP) (3) or the plasmodial surface anion channel (PSAC) (4). A significant breakthrough in the understanding of the molecular nature of these channels came with the discovery that the parasite protein CLAG3, encoded by the cytoadherence linked antigen gene 3 (clag3), plays a pivotal role in RBC membrane permeability (5), and that it is likely an important component of the pore itself. Interestingly, this protein exists in two forms encoded by alternatively transcribed genes called clag3.1 and clag3.2, and certain PSAC properties are determined by which gene is expressed (5, 6).

Unlike the other members of the clag gene family, clag3.1 and clag3.2 display clonally variant expression and can thus exist in either a transcriptionally active or silent state, and this transcriptional state can be stably inherited through many cellular divisions. Clonally variant expression of these genes is controlled epigenetically through the incorporation of certain histone marks, specifically tri-methylation of the lysine in the 9th position of histone H3 (H3K9me3) into the chromatin surrounding the silent gene, while the opposing mark, acetylation at the same position (H3K9ac), is found at the transcriptionally active locus (7–9). Expression is also mutually exclusive, thus one gene displays the active H3K9ac mark while the silent modification H3K9me3 is enriched at the other allele (6, 10). Interestingly, it was demonstrated that cultured parasites can acquire resistance to large, hydrophilic drugs like blasticidin S or leupeptin by downregulating PSAC activity (11, 12) via simultaneous silencing of both clag3 alleles (6, 13). This unusual situation results from the incorporation of silent histone marks at both genes, thus downregulating pore activity and preventing drug uptake. Recent work by Mira-Martinez and colleagues further showed that antimalarial compounds known as bis-thiazolium salts require pore activity to enter infected cells, while compounds like doxycycline, azithromycin, and lumefantrine enter via an alternative route, thereby better defining the important role clag3 expression plays in the acquisition of resistance to a subset of compounds that kill parasites (14). The ability of parasites to control access of antimalarial compounds to the intracellular environment presents a potential new mechanism for the development of drug resistance in the field, which is a troubling possibility (15). However, with an understanding of the molecular basis of activation and silencing of clag3.1/3.2, it might be possible to manipulate clag3.1/3.2 gene activity and thereby reverse epigenetically acquired drug resistance and potentially increase accessibility of parasites to antimalarial compounds.

Inhibitors of histone modifiers have been developed as potential therapeutic agents against numerous diseases, including malaria (16–18). The compounds typically work by altering the chromatin structure of the targeted cell sufficiently to disrupt gene expression patterns or prevent DNA replication, thus either killing the cell or preventing proliferation. Cell cycle progression in malaria parasites is unusual in that, unlike model eukaryotes that replicate by binary fission, malaria parasites undergo repeated rounds of unsynchronized genome replication and nuclear division in the absence of cell division (19, 20), a process called schizogony. What triggers each additional round of replication and how the cycle is regulated are largely unknown, although the Plasmodium-specific kinase CRK4 was shown to play a key role in this process (21). The heterochromatin mark H3K9me3 has been shown to affect preinitiation complex formation at origins (22) and rates of DNA replication (23) in higher eukaryotic cells, thus, inhibitors that affect H3K9me3 deposition would be predicted to have interesting effects on schizogony.

In addition to their development as therapeutic compounds, inhibitors that target various aspects of chromatin structure can also be used experimentally to probe the function of specific histone modifications and decipher mechanisms that regulate patterns of gene expression or cell replication. For example, we previously utilized sub-50% inhibitory concentration (IC₅₀) concentrations of the histone methyltransferase inhibitor chaetocin to investigate how changes in histone methylation efficiency affected patterns of var gene expression switching (24). This inhibitor targets histone methyltransferases of the SET3 family that deposit the histone mark H3K9me3 (25). In P. falciparum, this mark is devoted almost exclusively to regulating transcription of clonally variant gene families, which, in addition to var genes, also includes clag3.1/3.2 (8, 9), thus providing a potential tool for investigating mechanisms of acquired resistance to antimalarial compounds that enter infected cells through PSACs.

Here, we report that parasites grown in the presence of low doses of chaetocin display altered expression of clag3.1/3.2. In particular, exposure to chaetocin reestablishes PSAC activity and reverses drug resistance in parasites that have become drug-insensitive by downregulating clag3.1/3.2 expression. Thus, manipulation of clag3.1/3.2 expression could represent a new avenue for delivery of antimalarial compounds to parasitized red blood cells, as well as for reversing acquired drug resistance in P. falciparum. In addition, treatment with chaetocin slowed parasite progression through the multiple cycles of DNA replication that occur during schizogony, presumably by altering the ability of the parasites to make the appropriate chromatin modifications required for DNA replication. These observations provide insight into the poorly understood process by which parasites undergo repeated rounds of DNA replication in the absence of cell division.

RESULTS

Treatment with the histone methyltransferase inhibitor chaetocin reverses epigenetic silencing of clag3.1/3.2 expression.

Previous work showed that treatment with low levels of chaetocin induced significant changes in var gene expression (24), presumably by reducing incorporation of H3K9me3 into the surrounding chromatin. We were curious to see if treatment with chaetocin could similarly alter clag3.1/3.2 expression, and, more specifically, to determine if treatment with this compound could reverse the epigenetic silencing of both clag3 alleles observed in parasites that are resistant to blasticidin. To test this hypothesis, we utilized a line of FCB parasites that had previously been selected for the development of resistance to blasticidin and shown to have repressed clag3.1/3.2 expression (20) (a kind gift from the Desai lab); in tandem we used a blasticidin-sensitive FCB wild-type strain as control. We cultured blasticidin-resistant parasites in the presence of sub-IC₅₀ levels of chaetocin for 2 weeks, then determined steady-state levels of clag3.1 and clag3.2 transcripts using quantitative reverse transcriptase PCR (qRT-PCR).

As expected, nontreated FCB wild-type parasites that had not been selected for resistance to blasticidin displayed robust clag3 expression, with clag3.2 being the dominantly expressed allele (Fig. 1). When these parasites were selected for resistance to blasticidin, expression of clag3.2 was dramatically reduced, leading to much lower overall clag3 expression levels, although a low level of clag3.1 expression remained largely unchanged. This is consistent with previous reports showing that expression of clag3.2 is much more sensitive to blasticidin selection due to more efficient uptake of the drug when this allele is expressed (6). Removal of blasticidin pressure for 2 weeks led to a moderate increase in overall clag3 expression as the parasites began to revert to wild-type expression levels. The dominant allele however shifted to clag3.1, similar to what was previously reported for clag3 expression after removal of blasticidin selection (6). Interestingly, exposure to sub-IC₅₀ levels of chaetocin after removal of blasticidin selection resulted in a dramatic increase in clag3.1 expression (Fig. 1), indicating that inhibition of H3K9me3 deposition reversed the epigenetic silencing of clag3.1, thereby reactivating the gene. It is worth noting that while overall clag3 expression returned to levels similar to that observed in untreated parasites, expression was dominated by clag3.1, with clag3.2 expression levels remaining relatively low. This suggests that at this concentration of chaetocin, mutually exclusive expression remains intact and only one allele was reactivated.

FIG 1.

Steady-state mRNA levels expressed from clag3.1 and clag3.2 in cultured parasites. RNA was extracted from synchronized cultures of late-stage parasites from the P. falciparum FCB isolate. Levels of mRNA were determined using qRT-PCR from cDNA prepared from wild-type parasites (FCB-WT) or parasites that had been selected for resistance to blasticidin (FCB-BR). The resistant parasites were either cultured continuously in the presence of blasticidin (+blast), in the absence of blasticidin for 2 weeks (−blast 2 wks) or in the absence of blasticidin and in the presence of chaetocin for 2 weeks (−blast +chae 2 wks). Expression levels were normalized to the seryl tRNA synthetase housekeeping gene and are displayed as relative copy number ± SEM. Statistically significant differences were determined using t tests with P values as displayed above the compared values.

Chaetocin treatment can reverse drug resistance acquired through reduced anion channel activity.

In addition to blasticidin, solutes like sorbitol and alanine, as well as some large, hydrophilic antimalarial compounds, have been shown to enter the infected RBCs through the parasite-induced anion channels, thereby gaining access to the parasite (11–15). Resistance to these compounds can be acquired by either mutations in CLAG3 that alter properties of the channel (26) or, alternatively, by epigenetic repression of expression of both clag 3.1 and clag 3.2, as shown for the blasticidin-resistant FCB line (13). Given our observations that treating parasites with sub-IC₅₀ concentrations of chaetocin could derepress clag3.1/3.2 expression, we hypothesized that this could reverse resistance to drugs like blasticidin in parasites that had acquired resistance by downregulating channel activity.

To test this hypothesis, we again utilized the blasticidin-resistant line of FCB parasites that had previously been shown to have repressed clag3.1/3.2 expression (Fig. 1) (13). These parasites were grown in the presence of both chaetocin and blasticidin to determine the effect of chaetocin exposure on blasticidin resistance. The data in Fig. 1 show that the effect of chaetocin on clag3.1/3.2 expression was detectable after 2 weeks of exposure; however, the length of time required for changes in CLAG expression and altered pore activity were not known. To determine if chaeotocin exposure alters sensitivity to blasticidin over time, resistant FCB parasites were cultured in the presence of either blasticidin, chaetocin, or both compounds, and parasite growth was assayed daily by flow cytometry (Fig. 2). The blasticidin-resistant parasites displayed slightly slower growth in the presence of chaetocin (Fig. 2A, left panel), similar to the growth observed in wild-type FCB parasites grown in the presence of chaetocin (Fig. 2A, center panel). In the presence of both blasticidin and chaetocin, the FCB resistant parasites initially grew well; however, after approximately 6 days of exposure to both compounds, parasite growth was arrested and the parasites failed to continue to replicate (Fig. 2A, right panel).

FIG 2.

Chaetocin treatment can reverse resistance to blasticidin. Blasticidin-resistant FCB parasites display reduced CLAG expression and are less sensitive to treatment with blasticidin. (A) Growth rates of resistant parasites grown in the presence of 450, 600, or 750 nM chaetocin, 2.5 μg/ml blasticidin, or both compounds over time. Sensitivity to blasticidin is observed after approximately 6 days of chaetocin treatment (red arrow, right panel). (B) Sybr-green based drug-sensitivity curves to determine sensitivity to blasticidin. The y axis displays Sybr green fluorescence as a percentage of that observed in parasites grown in the absence of blasticidin while the x axis displays the log of molar blasticidin concentration. Blasticidin-resistant parasites grown in the absence of chaetocin are shown in blue while parasites grown in the presence of 450, 600, or 750 nM chaetocin for 13 days are shown in red, green, or purple, respectively. (C) Blasticidin sensitivity curves for wild-type FCB parasites grown in the absence of chaetocin (blue) or in the presence of 450, 600, or 750 nM chaetocin (red, green, or purple, respectively). Values in (B) and (C) represent means ± SEM for triplicate wells on each plate (technical replicates) and in three independent plates (biological replicates). For statistical comparisons, mean EC₅₀ values for 0 nM chaetocin were compared to those treated with chaetocin by t tests (P < 0.05).

To obtain a more quantitative measurement of changes in sensitivity to blasticidin, we performed drug sensitivity assays to identify changes in IC₅₀ resulting from chaetocin treatment. Blasticidin-resistant FCB parasites were released from blasticidin pressure and were grown in the presence or absence of three different sub-IC₅₀ concentrations of chaetocin (450, 600, and 750 nM) for thirteen days, then blasticidin drug-sensitivity assays were performed over 72 h using a standard Sybr green growth assay (27). As previously reported, the FCB-BR line of parasites displayed a marked resistance to blasticidin; however, after exposure to all three concentrations of chaetocin these parasites became significantly more sensitive to blasticidin and were killed by concentrations known to kill most lines of P. falciparum (Fig. 2B). The shift in sensitivity to blasticidin resulting from chaetocin exposure was not simply the result of synergism between the two compounds, since chaetocin treatment had no effect on blasticidin sensitivity of wild-type parasites (Fig. 2C).

An alternative method for directly observing anion channel activity within the infected RBC membrane is via uptake of the fluorescent dye benzothiocarboxypurine, also known as PUR-1. This dye has been shown to enter infected RBCs through the parasite induced anion channel, where it forms a brightly fluorescent complex with parasite nucleic acids (28). Uninfected RBCs and cells infected with ring-stage parasites do not take up the dye. To investigate changes in channel activity resulting from treatment with chaetocin, we exposed synchronized cultures of chaetocin-treated and untreated parasites to PUR-1, then examined the degree of dye uptake using fluorescence microscopy. As expected, wild-type FCB parasites efficiently take up the dye and appear brightly fluorescent, while blasticidin-resistant parasites fail to take up the dye and fluoresce much more weakly (Fig. 3). In contrast, blasticidin-resistant parasites grown in the presence of chaetocin were brightly fluorescent, indicating they had reactivated the pore and were now readily taking up PUR1 (Fig. 3). These data provide additional evidence that treatment with sub-IC₅₀ levels of chaetocin can reactivate PSAC activity in parasites that have silenced CLAG expression.

FIG 3.

Uptake of PUR-1 in wild-type (top), blasticidin-resistant (middle), or blasticidin-resistant parasites grown in the presence of chaetocin (bottom). PUR-1 uptake assays were performed as described by Kelly et al. (28).

Chaetocin treatment alters DNA replication and transition through schizogony.

As a histone methyltransferase inhibitor, chaetocin acts by altering chromatin structure, in particular the formation of heterochromatin through the deposition of the histone mark H3K9me3. Given the established role of heterochromatin and H3K9me3 on DNA replication (29–31), we examined whether treatment with sub-IC₅₀ concentrations of chaetocin might affect progression through schizogony. Parasites of the 3D7 line, which had been cultured in the presence or absence of chaetocin, were tightly synchronized and allowed to progress through the entire replicative cycle. Levels of DNA and RNA were assayed by flow cytometry at 2-h intervals, thus enabling us to monitor cells as they entered and exited the asexual cycle. We observed that parasites treated with chaetocin had a reproducible delay at the onset of DNA replication, and this delay continued as the parasites progressed through trophozoites and toward schizonts (Fig. 4). However, by the end of the 48-h cycle, the chaetocin-treated parasites completed replication and reinvasion at rates similar to the untreated parasites, indicating that chaetocin treatment had affected the rate of DNA replication but not the ability of the parasites to complete schizogony.

FIG 4.

Progression through schizogony in the presence of absence of chaetocin. Cultured parasites were grown in the absence (untreated) or presence (chaetocin treated) of the histone methyltransferase inhibitor chaetocin. Cultures were tightly synchronized and monitored by flow cytometry as they progressed through the replicative cycle. Proportions of rings, trophozoites, and schizonts were determined by both RNA content (thiazole orange fluorescence) and DNA content (Hoechst 33342 fluorescence) using a gating strategy described in the Materials and Methods section. The approximate time after initial red cell invasion is shown above each set of charts.

DISCUSSION

The development of resistance to antimalarial drugs remains an ongoing problem for the global effort to combat malaria. The spread of reduced sensitivity to the current most widely used antimalarial, artemisinin, has now been reported (32, 33), reinforcing the notion that the development of drug resistance is an ever-present problem. The acquisition of drug resistance by altering uptake pathways through downregulation of porins has been well documented in bacterial pathogens (34), highlighting the possibility that this type of drug resistance might arise for malaria parasites through the downregulation of PSACs.

Most examples of drug resistance result from genetic mutations that lead to amino acid changes in proteins that are either the direct target of the drug or that play a role in access of the drug to its target. Such genetic changes tend to be relatively stable, enabling rapid spread throughout a population, but also frequently inflict an associated fitness cost that can lead to slow reemergence of drug sensitivity when drug pressure is removed, as was observed for chloroquine resistance in some geographical regions (35). In contrast, the development of drug resistance through epigenetic changes, as described for the clag3.1/3.2 locus, is potentially a more easily reversible phenotype, thus avoiding some of the fitness costs associated with heritable genetic changes. The ability of parasites to rapidly switch between resistant and sensitive phenotypes could enable them to easily adapt to the presence or absence of a drug, thus further complicating malaria-containment strategies. Efficient changes in gene expression patterns through epigenetic switching have been well documented for the processes of antigenic variation and sexual differentiation (36), indicating that parasites are capable of such rapid changes in gene expression. The effect of chaetocin on clag3.1/3.2 expression, however, indicates that this type of epigenetic switching can itself be targeted, providing a possible method to address resistance that arises through an epigenetic mechanism. In this study, we have provided evidence that disruption of epigenetic silencing of gene expression of the mutually exclusively expressed genes clag3.1 and 3.2 has the ability to reverse resistance to drugs taken up through PSACs. Additionally, disrupting this recently discovered mechanism of resistance in P. falciparum during ongoing drug treatment enabled effective killing of the parasites with the same compound to which it had developed resistance. Compounds such as chaetocin, or derivatives thereof, may therefore provide an important tool when evaluating resistance dynamics during de novo drug development targeting PSACs. Several epigenetic modifying compounds are currently in clinical use, mainly to treat cancer (37, 38). These compounds may therefore hold the potential for use as part of future combination therapies with de novo-synthesized compounds targeting the malarial PSAC.

Stanojcic et al. described in detail the unusual dynamics of DNA replication displayed by malaria parasites over the course of a cycle of schizogony (39). Surprisingly, replication velocity slowed as schizogony progressed, the opposite of what is observed during S-phase progression in mammalian cells (40). These authors offered several possible explanations for the slowing of DNA replication rates, including reduced availability of nucleotides after several rounds of replication or changes in chromatin structure within various regions of the genome. Our data indicating that chaetocin treatment slows progression through the replicative cycle is consistent with the latter hypothesis. In model eukaryotes, reduction in H3K9me3 levels, a predicted result of chaetocin treatment, increases replication rates (22, 23). However, other studies have identified specific subsets of late-firing replication origins that are specifically associated with H3K9me3 (41, 42), indicating that this histone mark might play a role in recruiting the replication machinery to specific regions of the genome. Future work investigating the mechanisms regulating DNA replication during schizogony by malaria parasites is likely to identify additional characteristics quite different from what would be predicted from the study of model organisms. Such differences are both interesting for the insights they provide into the evolution of cellular replication in various eukaryotic lineages, as well as for the opportunities they provide for the development of novel strategies to combat malaria.

MATERIALS AND METHODS

Parasite culture.

All P. falciparum parasites were cultured according to standard procedures in media containing Albumax II (Gibco) without human serum. Parasites were incubated at 37°C in an atmosphere of 5% oxygen, 5% carbon dioxide, and 90% nitrogen. 3D7 and FCB parasites were obtained from MR4 (MRA-156, MR4, BEI-Resources) and blasticidin-resistant FCB (FCB-BR) parasites (13) were a kind gift from Sanjay Desai at the laboratory for Malaria and Vector Research, NIAID, NIH.

Analysis of clag3.1 and clag3.2 steady-state RNA.

RNA was extracted from synchronized late-stage parasites, including late trophozoites and schizonts, 48 h after isolation of schizonts using magnetic separation (43). RNA extraction was performed using TRIzol LD reagent (Invitrogen) as described (44). RNA was purified using the PureLink RNA minikit (Ambion) according to the manufacturer’s protocol and afterward treated with DNase I (Invitrogen). cDNA synthesis was performed with Superscript II RNase H reverse transcriptase (Invitrogen) with random primers (Invitrogen) as described by the manufacturer. Total RNA (800 ng) was used for each cDNA synthesis reaction and a control reaction without reverse transcriptase was performed in parallel. Quantitative reverse transcriptase PCR (qRT-PCR) analysis was done using the relative standard curve method. All qRT-PCRs were performed in triplicate with an ABI Prism 7900HT (Applied Biosystems) using iTaq SYBR green Supermix (Bio-Rad) and previously described primers specific for either clag3.1 or clag3.2 (6). Expression levels were normalized to the seryl tRNA synthetase housekeeping gene.

Uptake of PUR-1.

PUR-1 uptake assays were performed as described by Kelly et al. (28). Benzothiocarboypurine (PUR-1) was obtained from Sigma-Aldrich and dissolved in ethanol at a concentration of 10 mM and diluted to 5 μM in complete medium. Equal volumes of PUR-1 solution and aliquots of synchronized P. falciparum cultures were mixed and allowed to incubate for 10 min. Cells were then washed twice with culture medium and observed on a Leica DMI 6000b fluorescence microscope (excitation wavelength 476 nm, emission detection between 500 and 550 nm) using a Leica DFC 360FX camera.

Drug-sensitivity assays.

Drug-sensitivity assays were performed as described by Smilkstein et al. (27). Aliquots of 100 μl of parasite culture were distributed into clear, 96-well plates at a starting parasitemia of 0.2 to 0.5% and 2% haematocrit. Blasticidin S was obtain from Sigma-Aldrich and diluted appropriately in complete medium to achieve final concentrations in a 96-well culture plate ranging from 0.01 μM to 72.9 μM for blasticidin-sensitive parasites lines and 0.48 μM to 3.2 mM for blasticidin-resistant parasite lines. After addition of parasites and RBCs, each well of the 96-well plate had a total volume of 200 μl per well. Plates were incubated at 37°C in an airtight chamber containing 5% oxygen, 5% carbon dioxide, and 90% nitrogen. Cultures were allowed to incubate for 72 h. To assay for growth, the cultures were resuspended and 150 μl from each well was transferred to a 96-well black plate and placed at –80°C overnight. Plates were then thawed and 100 μl of SYBR green solution (0.2 μl SYBR green/ml lysis buffer) was added to each well. Plates were incubated in the dark at room temperature for 1 h with shaking. SYBR green incorporation was measured with a SpectraMax Gemini using an excitation wavelength of 490 nm and 530 nm detection. All assays were performed with triplicate wells on each plate (technical replicates) and in three independent plates (biological replicates). Data were analyzed using GraphPad Prism software by plotting counts against the log of the drug concentration, normalized, and curve fitted by nonlinear regression (sigmoidal dose-response/variable slope equation) to yield IC₅₀ values.

Flow cytometry and assays for cell cycle progression.

Progression through schizony was determined by flow cytometric analysis of parasite RNA and DNA content as previously described (45). Briefly, parasites of the 3D7 line that had been exposed to chaetocin for 2 weeks, were stained at 37°C with 16 μM Hoechst 33342 and 0.1 μg/ml thiazole orange for 30 min at 1% hematocrit in incomplete medium followed by a single wash in phosphate-buffered saline (PBS). Cells were then diluted to 0.1% hematocrit in PBS and analyzed using a Cytek DxP11 flow cytometer for Hoechst 33342 DNA-staining (375 nm laser, 450/50 emission filter) and thiazole orange RNA-staining (488 nm laser, 550/30 emission filter). For each sample, 50,000 infected RBCs (DNA⁺) were gated into ring (DNA^low/RNA^low), trophozoite (DNA^low/RNA^high), and schizont (DNA^high/RNA^high) stages.