Plasmodium parasites mount an arrest response to dihydroartemisinin, as revealed by whole transcriptome shotgun sequencing (RNA-seq) and microarray study

Plasmodium parasite transcriptional responses to the anti-malarial drug DHA were studied. We
performed short treatments of drug at high concentration to mimic what the parasite
would have to respond to in a typical therapeutic intervention. The transcriptional
response to DHA for the P. falciparum K1 strain was first explored by microarray to permit comparison with other drug responses
at the same trophozoite stage in this strain. DHA exposure elicits a marked response
that is very similar among the three time-points tested (Fig. 1; Additional file 1). The majority of trophozoites are inviable after 1 h exposure to 500 nM DHA 10]. Therefore, the consistent transcriptomic pattern of DHA response in trophozoites
is a signature of loss of viability. Moreover, this pattern is consistent across independent
microarray studies measuring response to different artemisinin derivatives (Fig. 1a). This loss of viability pattern is obscured in the transcriptomic response to slower
acting anti-malarial drugs such as pyronaridine and chloroquine 15] and antifolates 3], perhaps because of confounding transcriptomic changes occurring through normal cell
cycle progression during the longer drug treatment period needed to observe a significant
transcriptional response.

Correlation analyses of ring and trophozoite DHA treatment profiles with the IDC show
that these parasite stages respond to the drug by global reprogramming of transcriptomes.
This is shown most markedly for trophozoites by the negative correlation between DHA
treatment profiles and mid-cycle IDC time-points corresponding to trophozoite maturation
(Figs. 1b and 2e). Apart from a brief early ring phase, this is the same period during the IDC when
parasites are most vulnerable to DHA 10], 12]. The transcriptome of rings is similarly reprogrammed in that there is negative correlation
of DHA-induced changes with ring and trophozoite time-points during the IDC (Fig. 2d). Moreover, there is extensive overlap of differentially expressed genes induced
by DHA between rings and trophozoites (Fig. 2g, h). The ring and trophozoite transcriptional response to DHA may thus be an arrest
mechanism to retard metabolic processes essential for maturation, as shown by down-regulation
of glycolysis and protein synthesis genes (Tables 1 and 2). In contrast, schizonts do not respond to DHA (Fig. 2f). The lack of transcriptional response in schizonts can be explained by their reduced
protein synthesis 16] and transcription 17]. Moreover, schizonts are also less able to down-regulate gene expression because
of an overall reduced rate of mRNA decay 18].

The maturation and metabolic status of the cell is critical in determining sensitivity
to ART across other cell types, since actively dividing mammalian cells are more susceptible
to ART-induced cell death than quiescent cells 19]. The arrest transcriptomic pattern in DHA-treated rings may be a trigger for a physiological
protective change, e.g. dormancy. We did not observe dormant parasites in our experiments
(Additional file 3) as we harvested parasites immediately after a short treatment period (dormant parasites
are observed after a 6–13 h lag 10], 12], 20], 21]). Strains with lower sensitivity to ART are better able to enter, or recover from
dormancy 22]–24]. A similar trait has been observed in parasite isolates from slow-clearing infections
1], 2] and a rodent malaria model 25], suggesting that in vitro tests of ART response correspond well to the situation in vivo. In trophozoites, the transcriptional response to DHA does not lead to a protective
physiological change, perhaps because parasites at this stage are committed to an
anaplerotic metabolic state in which glycolysis, TCA cycling and glutamiolysis occur
at high rates 26]. The commitment to anaplerosis may occur quite early in the IDC, since ART-resistant
strains are significantly more tolerant of 700 nM DHA than sensitive strains only
for early ring stages of development 2], 12]. Recently, it has been shown that ART-resistant clones from slow-clearing infection
isolates have elongated ring and shortened trophozoite development stages 21]. These adaptations enhance the parasite’s ability to become a dormant ring and minimize
the ART-vulnerable trophozoite stage.

The arrest response to ART in P. falciparum rings and trophozoites, perhaps acting as a trigger to prevent the parasite from
maturing, implies that key metabolic functions (not yet identified) may be disrupted
by ART. Plasmodium is exquisitely sensitive to ART, in part because of oxidative damage mediated by
interaction of ART with heme, a major source of molecular iron. Although hemoglobin-heme
is highly abundant in the parasite’s food vacuole, this may not be the major site
for the drug’s killing effect since the ingested content containing red cell cytosolic
proteins such as haemoglobin and catalase can help to detoxify artemisinin 27]. Another site of parasite heme and molecular iron is the mitochondrion, in which
these cofactors are essential for the functions of cytochromes and iron-sulfur proteins.
Although some genes with mitochondrial function are differentially expressed in response
to DHA, e.g. up-regulation of PF3D7_0915000 (ndh2) and PF3D7_1034400 (sdha) genes in trophozoites (Additional file 4), there is no concerted regulation of genes with mitochondrial function as shown
by the absence of related significant GO terms (Tables 1–3). The lack of a coordinated parasite response regarding mitochondrial function suggests
that late rings and trophozoites are committed to mitochondrial biogenesis, which
is perhaps linked to commitment to anaplerosis (see above). Maturing parasites committed
in this manner are vulnerable to ART because mitochondrial biogenesis exposes the
cell to the oxidative stress effects of ART acting in combination with heme and molecular
iron. The inability of committed cells to arrest mitochondrial biogenesis imposes
a futile energy expenditure, since mitochondrial function is ablated by ART (shown
by rapid loss of mitochondrial membrane potential following ART treatment 28], 29]). Merozoites are less vulnerable to ART 11], because they are equipped with poorly developed mitochondria and have low metabolic
requirements. Schizonts have low metabolic requirement and are less sensitive to ART
than trophozoites as they have fully developed mitochondria. We therefore postulate
that maturing parasites committed to mitochondrial biogenesis are fated to ART-induced
death, whereas young uncommitted cells can become dormant. Our data are thus consistent
with the proposal in 30] that Plasmodium maintains expression of most mitochondrial genes, which enables rapid recovery of
mitochondrial function when exiting from ART-induced dormancy.

Among genes showing significant down-regulation in response to DHA in Plasmodium rings and trophozoites, it is notable that cytosolic ribosome function is represented
among significant GO terms from both microarray and RNA-seq data (Tables 1, 2 and 4). This coordinated down-regulation is particularly striking for DHA-treated rings,
since expression of these genes is maximal during the first 20 h of normal development
31]. The coordinated down-regulation of Plasmodium ribosomal protein genes is not surprising since they possess a common regulatory
element in their promoters, the G-box, and putative positive and negative transcription
factors to control their expressions have been described 32]. It is interesting to note that concerted repression of yeast cytosolic ribosomal
protein genes also occurs under hydrogen peroxide stress, and this response is dependent
on thiol peroxidase enzymes 33]. Plasmodium lack catalase and GPx enzymes, and so they rely on thiol peroxidase enzymes and the
NADPH pathway for maintaining redox balance 34]. It is tempting to speculate that thiol peroxidase enzymes become oxidized under
ART stress similar to hydrogen peroxide stress. The accumulation of these oxidized
enzymes could trigger a peroxide-signalling event 35] that activates the observed gross transcriptional response in parasites. One potential
regulator of such a peroxide-signalling event is the nuclear thiol peroxidase PfnPrx,
which is essential and has intimate association with most of the parasite genome 36].

Of the significant GO terms among up-regulated trophozoite genes in response to DHA
(Table 3), apicoplast function was recently shown by qRT-PCR to be up-regulated in P. falciparum recovering from DHA treatment; the up-regulation of pyruvate metabolism in the apicoplast
is thought to compensate for reduced ATP levels caused by down-regulation of glycolysis
and TCA cycle 30]. Among the up-regulated genes representing significant protein phosphorylation GO
terms are 18 FIKK kinase genes (Additional file 4). FIKK kinases are exported to the host red cell and play important roles in pathogenesis
37], 38]. In accordance with the findings made by Natalang et al. for artesunate response
4], many other genes for exported proteins from different families such as hyp, PHIST,
Pfmc-2TM, PfEMP1, RESA, ETRAMP, SURFIN are significantly up-regulated in DHA-treated
P. falciparum trophozoites (Additional file 4). The expressions of Plasmodium translocon of exported proteins (PTEX) genes recently shown to be essential for protein
export 39], 40] are also significantly up-regulated in DHA-treated trophozoites (Additional file
4). Increased protein export may be the final act of dying parasites to restore the
energy deficit, since exported proteins are essential for uptake of nutrients 40].

Recently, mutations in the PF3D7_1343700 gene encoding K13 protein have been associated
with artemisinin resistance in Southeast Asia 41]. Interestingly, this gene is significantly up-regulated in response to DHA in trophozoites
(Additional files 1 and 4). K13 mutations in ART resistant parasites appear to reduce ubiquitinylation of proteins,
including PfP13K 42]. The expression of the PfPI3K encoding gene PF3D7_0515300 does not change in response
to DHA (Additional files 1 and 4). The up-regulation of K13 in response to DHA could reduce PfP13K protein through
the ubiquitin/proteasome pathway, and in turn lead to reduced phosphatidyl inositol-3-phosphate
(PI(3)P). Reduced PI(3)P could lead to interference of protein export from the endoplasmic
reticulum (ER), which is dependent on PI(3)P 43]. Interference of protein export would presumably lead to ER stress, for which P. falciparum is notably vulnerable 44]. Among the genes with altered expression during normal development in ART resistant
parasites, genes encoding proteins in the unfolded protein response (UPR) are notably
up-regulated 7]. The key genes in this pathway, i.e. PF3D7_1108600 (ERC), PF3D7_0917900 (HSP70-2
or BiP), PF3D7_0322000 (CYP19A), PF3D7_1357800 (TCP-1/cpn60), PF3D7_0306800 (TCP1-b)
and PF3D7_0718500 (prefoldin) are significantly down-regulated in P. falciparum ring and/or trophozoites exposed to DHA (Additional file 4). Furthermore, the P. berghei orthologues of CYP19A and ERC (PBANKA_121650 and PBANKA_093840, respectively) are
also significantly down-regulated in response to DHA (Additional file 5). From these observations, it appears that ART resistant parasites have adaptations
in the arrest transcriptional response to specifically counteract the ER stress induced
by ART, in particular reversal of expression changes that could actually sensitize
the parasite to drug. This trend is also apparent for genes with antioxidant function,
which are up-regulated in DHA-resistant parasites to counteract the oxidant stress
induced by the drug 9]. Among genes with known antioxidant function, the PF3D7_1438900 (thioredoxin peroxidase1),
PF3D7_1457200 (thioredoxin 1), and PF3D7_1121600 (EXP1, an exported glutathione transferase
which mediates sensitivity to artesunate 45]) genes are down-regulated in response to DHA in P. falciparum trophozoites (Additional file 4) and the orthologues in P. berghei (Additional file 5).

The in vivo DHA response in P. berghei was strikingly similar to the ring and trophozoite responses in P. falciparum, as shown by the high correspondence of differentially expressed orthologous genes
from RNA-seq data (Fig. 4) and with the same significant representation of cytosolic ribosome function by GO
analysis (Table 4). The transcriptional responses between species were similar despite the fact that
the P. berghei were not synchronous and the read depth was much lower for P. berghei owing to contaminating mouse mRNA, as shown by the high percentage of reads mapping
to Mus musculus (Additional file 6). This could mean that the transcriptional response in ring and trophozoite stages
to DHA (see above) is conserved among Plasmodium parasite species with similar sensitivity to the drug, although this would need to
be investigated further with synchronous parasites.

Although similar overall patterns of DHA-response in P. falciparum trophozoites were found among microarray and RNA-seq data, there was rather poor
agreement in terms of specific genes showing significant changes in expression (Fig. 3). The P. falciparum microarray data analyzed in this study were generated from two-channel platforms.
These data must be normalized using aggressive techniques such as locally weighted
linear regression to correct for intensity-dependent dye bias due to differences in
physical properties of the two cyanine fluorescent dyes. Data normalization in this
fashion can be inaccurate though when central assumptions are violated, for example
when a high proportion of genes are differentially expressed, and/or there is a skewed
direction of change in gene expression. In this situation, external RNA controls may
be required 46], 47]. This approach is rarely used though as it depends crucially on the quality of the
external controls, and appropriate probes for the controls designed on the array.
Normalization of RNA-seq data is simpler than microarray as it involves re-scaling
of global read counts. In some cases where mRNA content varies greatly among cell
types, external controls may be required for normalization, as proposed in microarray
experiments 48]. However, the standard Trimmed Mean of M-values (TMM) normalization method, as we
have used in our RNA-seq experiments, is robust to skewed changes provided mRNA contents
are similar 49]. For the DHA-responsive genes found by RNA-seq, we found a clear bias in that highly
expressed genes were skewed towards down-regulation, and the opposite skew for low-expressed
genes. In contrast, there was no marked bias from normalized microarray data (Fig. 3). We think that the microarray normalization procedure removes the skew, which leads
to inaccurate normalization, and consequently inaccuracy in identification of differentially
expressed genes. In contrast, RNA-seq data normalization is more accurate, as shown
by greater statistical support for differentially expressed genes and high reproducibility,
even among different species.