Profiling molecular factors associated with pyknosis and developmental arrest induced by an opioid receptor antagonist and dihydroartemisinin in Plasmodium falciparum

Malaria continues to be a devastating disease, largely caused by Plasmodium falciparum infection. We investigated the effects of opioid and cannabinoid receptor antagonists on the growth of intraerythrocytic P. falciparum. The delta opioid receptor antagonist 7-benzylidenenaltrexone (BNTX) and the cannabinoid receptor antagonists rimonaband and SR144528 caused growth arrest of the parasite. Notably BNTX and the established antimalarial drug dihydroartemisinin induced prominent pyknosis in parasite cells after a short period of incubation. We compared genome-wide transcriptome profiles in P. falciparum with different degrees of pyknosis in response to drug treatment, and identified 11 transcripts potentially associated with the evoking of pyknosis, of which three, including glutathione reductase (PfGR), triose phosphate transporter (PfoTPT), and a conserved Plasmodium membrane protein, showed markedly different gene expression levels in accordance with the degree of pyknosis. Furthermore, the use of specific inhibitors confirmed PfGR but not PfoTPT as a possible factor contributing to the development of pyknosis. A reduction in total glutathione levels was also detected in association with increased pyknosis. These results further our understanding of the mechanisms responsible for P. falciparum development and the antimalarial activity of dihydroartemisinin, and provide useful information for the development of novel antimalarial agents.


Introduction
Malaria is one of the world's most devastating diseases, particularly in the tropics, with an estimated global annual incidence of 212 million clinical cases and mortality of 429,000 in 2015 [1], largely due to Plasmodium falciparum infection.
The rapid emergence of drug-resistant Plasmodium strains has severely reduced the therapeutic efficacy of conventional antimalarial drugs and threatens the effectiveness of artemisinin (ART) combination therapy, which is currently used widely in the field [2][3][4][5]. In humans, the P. falciparum parasite lives mainly within red blood cells (RBCs) and develops through three distinct stages (ring, trophozoite, and schizont) during its cycle lasting approximately 48 h [6][7][8]. However, the mechanisms responsible for regulating the developmental cycle are poorly understood, and a more complete understanding of the functional molecules involved in developmental succession/arrest is needed [9][10][11]. Such information would facilitate the development of new classes of anti-malarial drugs targeting innovative metabolic pathways, with different mechanisms of action from currently available drugs, thus furthering the fight against malaria [12][13][14].
Miyata et al. [15][16] reported that several opioid receptor antagonists, including 7-benzylidenenaltrexone (BNTX), reversed chloroquine (CQ)-resistance in murine malaria caused by Plasmodium chabaudi, but had virtually no antimalarial effects themselves. These antagonists are interesting because opioids and opioid receptors are known to not only exert profound analgesic activity, but to also exert many pharmacological effects and activate second-messenger signaling cascades in various cells.
In the present study, we investigated the effects of opioid receptor antagonists, as well as cannabinoid receptor antagonists, which exert physiological functions similar to opioid receptor antagonists, on the intraerythrocytic development of P. falciparum. We also used genomewide transcriptome profiling to identify genes that were differentially expressed in parasites exposed to BNTX and an antimalarial drug dihydroarthemisinin (DHART) that caused prominent pyknosis, which preceded parasite death, to identify putative key molecules associated with the evoking of pyknosis in the parasite.
Cultures were synchronized at the ring stage by three successive exposures to 5% (w/v) Dsorbitol (Sigma-Aldrich) at 41-and 46-h intervals [19]. After the third sorbitol treatment, residual schizonts and cell debris were removed by isopycnic density centrifugation on 63% Percoll PLUS (GE Healthcare Bio-Sciences, Tokyo, Japan). Parasites synchronized at the ring stage were adjusted to 5.0% parasitemia, unless specified otherwise, and maintained for development experiments and for RNA preparation.

Assessment of parasite growth and evaluation of growth inhibition
Samples were taken at the indicated times after inoculation. Thin smears were made and stained with Giemsa. Parasitemia was determined by examining more than 10000 PfRBCs and/or uninfected RBCs. The growth rate was estimated by dividing the parasitemia of the test sample after the indicated incubation period by the initial parasitemia.
In all the experiments, culture wells were run in triplicate or quadruplicate. All experiments were repeated two to four times.

RNA preparation
Total parasite RNA was harvested using an RNase plus Mini Kit (Qiagen GmbH, Hilden, Germany) as described previously [11]. Briefly P. falciparum was isolated from infected RBCs at the end of the incubation period by lysing infected cells, and was preserved in RNAprotect Cell Reagent (Qiagen) to protect the nucleic acids from degradation. The concentration and purity of the harvested RNA were confirmed using an Agilent 2100 Bioanalyzer (Agilent Technologies Japan, Ltd., Tokyo, Japan) and NanoDrop ND-100 spectrophotometer (Thermo Fisher Scientific Inc., Tokyo, Japan).

Gene expression analysis and transcriptome profiling
Genome-wide transcriptome profiling of P. falciparum during the various developmental stages was performed by Cell Innovator Inc. (Fukuoka, Japan), using the Affymetrix GeneChip Plasmodium/Anopheles Genome Array (Affymetrix Inc., Cleveland, OH, USA) and GeneChip 3' IVT PLUS Expression kit (Affymetrix). The assay contains 5407 probe sets designed on the basis of the annotated genome and expressed sequence tags determined by the Malaria Genome-sequencing consortium. Each probe consists of 11-20 different oligonucleotide sequences (25-mers) designed to be exactly complementary to different parts of the coding region of the transcript.
The Plasmodium/Anopheles Genome Array was hybridized with 12.5 μg of amplified RNA, incubated for 16 h at 45˚C, and automatically washed and stained using a GeneChip Hybridization, Wash and Stain Kit (Affymetrix) on an Affymetrix GeneChip Fluidics station. The arrays were analyzed using a GeneChip Scanner 3000. All preparations were run on qualitycontrolled chips and had 3/5 signal ratios of <3. The expression values of the transcripts were computed using Affymetrix GeneChip Command Console Software, with the MAS5 algorithm. For analysis, the data were normalized using Gene Spring GX11.0 (Agilent Technologies) data-mining software, per-chip normalization to the 50th percentile of the measurements for the array, and per-gene normalization to the median measurement for the gene across all the arrays in the data set. In addition, the software was used to calculate fold changes between the experimental groups and controls for each gene. All experiments for transcriptome profiling were repeated twice using separate cultures.

Statistical analysis
The significance of differences between means was evaluated using multifactorial analysis of variance (ANOVA). All calculations were performed using GraphPad PRISM 5 (GraphPad Software, Inc., La Jolla, CA, USA). The P value for significance was 0.05, and all pairwise comparisons were made post hoc with Bonferroni's test. Error bars in the graphs indicate standard deviations. Statistically significant differences were also determined by unpaired t-tests for gene expression analysis. Differences in gene expression (transcript) levels with P < 0.05 and at least a 2.0-fold increase or decrease were considered significant.

Effect of opioid-and cannabinoid-receptor antagonists on growth of P. falciparum
We determined at first the effects of increasing concentrations of the opioid receptor antagonists BNTX δ1 opioid receptor antagonist), NTB (delta 2 opioid receptor antagonist), and NTX (mu opioid receptor antagonist) on the growth of asynchronous P. falciparum in PfRBCs. The addition of BNTX, but not NTB or NTX, caused cessation of growth in parasite cultures (Fig 1a).
We also examined the effects of different concentrations of the cannabinoid receptor antagonists (reverse agonists) RIMO (CB1 receptor antagonist) and SR144528 (CB2 receptor antagonist) on the growth of asynchronous P. falciparum and showed that both cannabinoid receptor antagonists caused cessation of parasite growth (Fig 1b).
P. falciparum parasites were cultured in the presence of graded concentrations of the delta opioid receptor agonist DPDPE [20][21] (0-40 μM) for 45 and 95 h, with or without BNTX. Parasites grown in the presence of DPDPE without BNTX resembled control parasites grown in complete medium at the 2 different time points, while BNTX-induced (4 μM) growth arrest was not recovered by the addition of DPDPE. This suggests that uncertain factor(s) other than delta opioid receptors may be involved in the effect of BNTX on parasite growth. It is also possible that the intracellular processing of DPDPE may be altered and it may thus fail to affect the parasites. Further studies are needed to clarify this issue.

Morphological arrest of development with BNTX
We also determined the effects of different concentrations of BNTX on the developmental progression of P. falciparum parasites synchronized at the ring stage and allowed to develop for 24 h, sufficient time for development to the early schizont. BNTX arrested parasite development during the ring-trophozoite progression. All stages of parasite development were observed at the concentrations of BNTX at 1.25 μM (0.74 x IC 50 ) and 5 μM (2.96 x IC 50 ) (Fig 2a), but morphological developmental arrest occurred at the high concentration at 20 μM (11.8 x IC 50 ), with the appearance of features such as cytoplasmic elimination, decreased size, and pyknosis (nuclear shrinkage) (Fig 2a and 2b). We referred to parasites in this state, which preceded parasite death, as pyknotic. Early development of pyknosis in P. falciparum following treatment with BNTX and DHART We examined the timing of pyknosis after incubation with the test drugs. P. falciparum synchronized at the ring stage were cultured in the presence of BNTX, RIMO, SR144528, and DHART and CQ (antimalarial drugs for a control), respectively, and in control medium. The distribution of the parasites among the different developmental stages was determined at 4, 12, and 24 h during the first cycle of growth. Pyknotic forms accumulated notably among P. falciparum cultured for 12 h and 24 h in the presence of BNTX or DHART, and to a lesser extent RIMO, but not in parasites cultured in SR144528 or CQ, and in control medium (Fig 3). The Resumption of parasite development after exposure to DHART but not BNTX Some parasites treated with ART derivatives are known to enter a state of quiescence, or dormancy [22][23]. To test for this possibility, we exposed parasites to BNTX (20 μM and 80 μM) or DHART (50 nM and 200 nM) for 24 or 72 h, followed by culture for 10-21 days, to detect any resumption of parasite growth. There was no growth recovery of parasites cultured with BNTX, even after 21 days. In contrast, parasites cultured with DHART showed initial growth arrest, but a few parasites (<0.001%) resumed growth as early as 3 days (50 nM) or 5 days (200 nM), even after 72 h exposure. These parasites grew and demonstrated a normal morphology. These imply that BNTX killed all the parasites at the tested doses and durations, while DHART killed most parasites, but a small percentage was still alive, suggesting that the tested doses and durations of DHART may have been inadequate for complete killing of the parasites, or induce dormant parasites.
Transcriptome profiling of development-arrested parasites BNTX, DHART, and RIMO arrested development of early intraerythrocytic stages of P. falciparum by inducing pyknosis. We aimed to identify the factors associated with the observed pyknosis and subsequent developmental arrest in P. falciparum by genome-wide transcriptome profiling of parasites cultured in the presence of BNTX (BNTX-12h), DHART (DHART-12h), or RIMO (RIMO-12h), respectively, to identify genes that were differentially expressed in line with the extent of pyknosis.
Genes related to transcripts that were up-or down-regulated in P. falciparum cultured in BNTX-12h, DHART-12h, or RIMO-12h in comparison with those kept for 12 h in complete medium (no antagonist/inhibitor, GFSRPMI-12h) were selected and predicted using genome data for P. falciparum (3D7 strain) in PlasmoDB (http://www.plasmodb.org) and GenBank (National Center for Biotechnology Information, NCBI; http://www.ncbi.nlm.nih.gov). In line with the morphological assessment of parasite development, the numbers of transcripts with profoundly changed levels in comparison with GFSRPMI-12h varied among the BNTX-12h, DHART-12h, and RIMO-12h groups (Fig 4). BNTX-12h (high incidence of pyknosis) induced transcripts of <0. 25 (Fig 4). The large number of unique transcripts detected suggested that the impacts on gene transcription varied markedly between the different drugs, and that parasite-growth arrest may be induced by different factors.

Effect of GR inhibitors on growth of P. falciparum
The possible involvement of PfGR in parasite pyknosis following exposure to BNTX and DHART was examined by adding the GR inhibitors, 2-AAPA and BCNU to the cultures. These inhibitors stopped the growth of parasites in asynchronous cultures (Fig 5a and 5b).

Change in redox state among development-arrested parasites
The fact that GR maintains intracellular concentrations of GSH suggests that pyknosis may be linked to the cellular redox state. We therefore quantified total GSH and GSSG levels under each experimental condition, and in GFSRPMI-12h (control) and normal RBCs. The levels of total GSH and GSSG were significantly decreased in all PfRBC samples compared with normal RBCs. The greatest significant reductions in total GSH levels occurred in the BNTX-12h (high pyknosis), followed by the DHART-12h (moderate pyknosis), and RIMO-12h (low pyknosis) groups (Fig 7). These results suggest that PfGR may be involved in pyknosis by affecting GSH levels (redox state)."

Effect of TPT inhibitor on growth of P. falciparum
To examine the possible effects of PfoTPT activity on the development of P. falciparum, we cultured PfRBCs in the presence of graded concentrations of the selective TPT inhibitor DIDS. DIDS prevented parasite growth in asynchronous cultures at relatively high concentrations (Fig 8a). The effects of different concentrations of DIDS on the development of parasites synchronized at the ring stage were also tested. DIDS did not arrest parasite development during the ring-trophozoite-schizont-stage transitions, and the levels of pyknosis were similar to those in control cultures, even at high concentrations of DIDS (Fig 8b).

Discussion
The delta 1 opioid receptor antagonist, BNTX, and cannabinoid receptor antagonists, RIMO and SR144528, caused growth arrest of intraerythrocytic P. falciparum. BNTX, as well as the antimalarial drug DHART, notably induced pyknosis in the parasite cells after a short period of incubation. We compared genome-wide transcriptome responses in P. falciparum following exposure to BNTX, DHART, and RIMO, which induced different pyknotic responses, and selected 11 transcripts potentially associated with pyknosis. Three of these, PfGR, PfoTPT, and conserved Plasmodium membrane protein, showed markedly different gene expression levels among parasites exposed to the three different agents, reflecting the degree of pyknosis. The genes identified in this study may encode key factor(s) triggering pyknosis, or factors concomitant with developmental arrest. We further examined the roles of PfGR and PfoTPT using specific inhibitors. The GR inhibitors 2-AAPA and BCNU [24][25][26], but not the TPT inhibitor DIDS [27][28], caused marked cessation of P. falciparum growth associated with a high degree of pyknosis, similar to the levels caused by BNTX and DHART. These results suggest that PfGR may play a major role in the development of pyknosis in P. falciparum. However, the additional involvement of other proteins associated with pyknosis cannot be completely excluded.
The use of opioid analgesics has a long history in clinical settings, though the comprehensive action of opioids is still not fully understood [29][30]. Opioids, including endogenous and exogenous opioids, act at opioid receptors (namely, kappa, mu, delta, and nociceptin/orphanin opioid receptors) [30][31]. In addition to pain modulation and addiction, opioid receptors are widely involved in various physiological and pathophysiological activities, including the regulation of membrane-ion homeostasis, cell proliferation, and neurodegenerative disorders [29]. Opioid receptors are typical G protein-coupled receptors and activate canonical second-messenger signaling cascades to influence diverse cellular functions [29][30]. In the current study, BNTX, but not NTB and NTX, effectively inhibited the successive ring-trophozoite-schizont progression of P. falciparum during early developmental stages, associated with the development of pyknosis in ring forms. BNTX demonstrated a resistance-reversing effect in CQresistant murine malaria caused by P. chabaudi infection in vivo, but virtually no antimalarial effects [15][16]. In contrast, BNTX exhibited a direct antimalarial effect on P. falciparum in vitro in the current study. These apparently conflicting results may be produced by in vivo versus in vitro experiments. Among the opioid antagonists tested, only the delta 1 antagonist BNTX exerted a profound growth-arresting effect on P. falciparum. BNTX has a Michael acceptor structure, which differs from NTX which otherwise has a similar formula to BNTX [15][16], suggesting that the Michael acceptor moiety may be involved in the induction of developmental arrest by BNTX. However, no opioid-receptor gene with clear homology to human opioid receptors has yet been annotated in P. falciparum (PlasmoDB). The molecule(s) involved in the interaction between BNTX and P. falciparum, and the mechanisms responsible for growth arrest and gene-expression regulation in the parasite remain to be determined.
Cannabinoids are known to exert a variety of central and peripheral physiological functions similar to opioids. Endogenous and exogenous cannabinoids act at cannabinoid receptors, which comprise two pharmacologically distinct receptors, CB1 and CB2, both of which regulate a variety of central and peripheral physiological functions in humans and other animals. At the cellular level, these receptors are critically involved in proliferation, motility, adhesion, and apoptosis of various cell types [32][33][34]. Cannabinoid receptors are typical G protein-coupled receptors, and their activation leads to inhibition of adenylyl cyclase and consequent reductions in cyclic AMP accumulation. In addition, both CB1 and CB2 receptors regulate the phosphorylation and activation of different members of the mitogen-activated protein kinase family, and mitogen-activated protein kinase cascades are thus involved in cannabinoid regulation of cell survival/death, glucose metabolism, and ionic current control in various cells [32]. Notably, although the selective CB1 antagonist RIMO and the selective CB2 antagonist SR144528 induced few populations of pyknotic parasites, their IC 50 values were almost comparable to that of BNTX. Additional studies are currently underway to elucidate the roles of cannabinoid antagonists in P. falciparum growth.
The antimalarial activity of ART derivatives is known to result from alterations in the parasite's redox balance caused by their endoperoxides. ART derivatives interact with hemin to produce reactive oxygen species, resulting cellular damage [35][36]. In the current study, PfGR gene expression was markedly down-regulated, and it was thus predicted to be the main molecular factor associated with the development of pyknosis or dormancy in P. falciparum following treatment with either DHART or BNTX. A reduction in intracellular levels of GSH, as detected in the current study, may also increase reactive oxygen species-induced damage. GR is a homodimeric flavoenzyme that maintains high intracellular concentrations of GSH by catalyzing the reduction of glutathione disulfide (GSSG). GR and GSH thus perform central functions in cellular redox metabolism in various cells and in P. falciparum [37][38]. Although the functions of GSH and GSH-related enzymes, including GR, in relation to the antimalarial activity of DHART in P. falciparum remain poorly understood, intracellular GSH levels and protein S-glutathionylation have been proposed to be involved in the mechanism of ARTderivative action in Plasmodium spp. [39][40]. Kelch propeller domain (K13-propeller) proteins have also been associated with ART resistance in P. falciparum [41][42]. The human kelch domain-containing protein KEAP1, which showed maximum homology with the P. falciparum K13 propeller protein, induced the expression of antioxidant enzymes [43], and it is possible that the P. falciparum K13-propeller protein may perform a similar function, inducing the expression of antioxidant enzymes. Consistent with this possible mechanism, DHART reduced the expression of PfGR in association with growth arrest of P. falciparum in the current study. In addition to inducing endoperoxides, the prominent antimalarial effect of ART derivatives may involve the redox machinery via PfGR. Further studies are needed to determine the role of PfGR in the efficacy of DHART in terms of arresting parasite development.
During the erythrocytic stage of their life cycle, malaria parasites are exposed to oxidative stress produced by toxic heme and other hemoglobin-decomposition products [44][45]. The GSH system is believed to play a key role in the defense of malaria parasites against oxidative stress. This is particularly important in the development of CQ-resistance, and CQ-resistant parasites have been reported to exhibit increased GSH content. Despite the lack of conclusive evidence for a link between GSH levels and CQ response, currently available information suggests that P. falciparum could enhance its resistance to CQ by bolstering GSH levels [38,[44][45][46][47]. However, it should be noted that CQ only caused limited pyknosis in the current study, implying that the antimalarial effect of CQ may differ from that of DHART. Further research is needed to identify a link between GSH, PfGR, CQ, and the development of pyknosis.
Two sugar phosphate transporters are present in apicoplast membranes of P. falciparum: PfoTPT (PF3D7_0508300), which is located in the outermost apicoplast membrane, and a phosphoenolpyruvate/phosphate translocator (PfiTPT, PF3D7_0530200), located in the innermost apicoplast membrane [48][49]. PfoTPT and PfiTPT transport substrates, including dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, and phosphoenolpyruvate. TPT gene knockout was lethal for Plasmodium berghei in a murine model [50], while transcripts corresponding to PfoTPT were markedly reduced in P. falciparum in cultures with added BNTX or DHART. However, DIDS, which selectively binds TPTs and blocks their transporting ability [51][52], had no visible effect on the development of pyknosis, in contrast to parasites cultured in the presence of BNTX, 2-AAPA, and BCNU, despite causing general growth arrest. This implies that decreased PfoTPT transcription may not be responsible for pyknosis.
In conclusion, we identified molecular factors potentially associated with the development of pyknosis in P. falciparum by genome-wide transcriptome profiling of parasites exposed to BNTX and DHART, and by using specific inhibitors. The results suggest that PfGR may play a critical role in the development of pyknosis in P. falciparum, and provide important information for the development of novel, effective antimalarial strategies. Our understanding of PfGR functions remains incomplete, and genetic evidence is required to confirm the mechanisms underlying the role of PfGR in parasite pyknosis. Further studies are also needed to clarify the target molecules of BNTX, DHART, and cannabinoid antagonists in P. falciparum.