Plasmodium sexual differentiation: how to make a female.

Sexual development is integral to the transmission of Plasmodium parasites between vertebrates and mosquitos. Recent years have seen great advances in understanding the gene expression that underlies commitment of asexual parasites to differentiate into sexual gametocyte stages, then how they mature and form gametes once inside a mosquito. Less well understood is how parasites differentially control development to become males or females. Plasmodium parasites are haploid at the time of sexual differentiation, but a clonal haploid line can produce both male and female gametocytes, so they presumably lack the sex-determining alleles present in some other eukaryotes. Though the molecular switch to initiate male or female development remains hidden, recent studies reveal regulatory proteins needed for the sex-specific maturation of male and female gametocytes. Yuda and collaborators report the characterization of a transcription factor necessary for female gametocyte maturation. With renewed attention on malaria elimination, sex has been an increasing focus because transmission-blocking strategies are likely to be an important component of elimination efforts.

A transcriptional switch underlies commitment to sexual development in malaria parasites.

The life cycles of many parasites involve transitions between disparate host species, requiring these parasites to go through multiple developmental stages adapted to each of these specialized niches. Transmission of malaria parasites (Plasmodium spp.) from humans to the mosquito vector requires differentiation from asexual stages replicating within red blood cells into non-dividing male and female gametocytes. Although gametocytes were first described in 1880, our understanding of the molecular mechanisms involved in commitment to gametocyte formation is extremely limited, and disrupting this critical developmental transition remains a long-standing goal. Here we show that expression levels of the DNA-binding protein PfAP2-G correlate strongly with levels of gametocyte formation. Using independent forward and reverse genetics approaches, we demonstrate that PfAP2-G function is essential for parasite sexual differentiation. By combining genome-wide PfAP2-G cognate motif occurrence with global transcriptional changes resulting from PfAP2-G ablation, we identify early gametocyte genes as probable targets of PfAP2-G and show that their regulation by PfAP2-G is critical for their wild-type level expression. In the asexual blood-stage parasites pfap2-g appears to be among a set of epigenetically silenced loci prone to spontaneous activation. Stochastic activation presents a simple mechanism for a low baseline of gametocyte production. Overall, these findings identify PfAP2-G as a master regulator of sexual-stage development in malaria parasites and mark the first discovery of a transcriptional switch controlling a differentiation decision in protozoan parasites.

Characterization of the accessible genome in the human malaria parasite Plasmodium falciparum.

Human malaria is a devastating disease and a major cause of poverty in resource-limited countries. To develop and adapt within hosts Plasmodium falciparum undergoes drastic switches in gene expression. To identify regulatory regions in the parasite genome, we performed genome-wide profiling of chromatin accessibility in two culture-adapted isogenic subclones at four developmental stages during the intraerythrocytic cycle by using the Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq). Tn5 transposase hypersensitivity sites (THSSs) localize preferentially at transcriptional start sites (TSSs). Chromatin accessibility by ATAC-seq is predictive of active transcription and of the levels of histone marks H3K9ac and H3K4me3. Our assay allows the identification of novel regulatory regions including TSS and enhancer-like elements. We show that the dynamics in the accessible chromatin profile matches temporal transcription during development. Motif analysis of stage-specific ATAC-seq sites predicts the in vivo binding sites and function of multiple ApiAP2 transcription factors. At last, the alternative expression states of some clonally variant genes (CVGs), including eba, phist, var and clag genes, associate with a differential ATAC-seq signal at their promoters. Altogether, this study identifies genome-wide regulatory regions likely to play an essential function in the developmental transitions and in CVG expression in P. falciparum.

Identification of Antimalarial Compounds That Require CLAG3 for Their Uptake by Plasmodium falciparum-Infected Erythrocytes.

During the intraerythrocytic asexual cycle malaria parasites acquire nutrients and other solutes through a broad selectivity channel localized at the membrane of the infected erythrocyte termed the plasmodial surface anion channel (PSAC). The protein product of the Plasmodium falciparum clonally variant clag3.1 and clag3.2 genes determines PSAC activity. Switches in the expression of clag3 genes, which are regulated by epigenetic mechanisms, are associated with changes in PSAC-dependent permeability that can result in resistance to compounds toxic for the parasite, such as blasticidin S. Here, we investigated whether other antimalarial drugs require CLAG3 to reach their intracellular target and consequently are prone to parasite resistance by epigenetic mechanisms. We found that the bis-thiazolium salts T3 (also known as albitiazolium) and T16 require the product of clag3 genes to enter infected erythrocytes. P. falciparum populations can develop resistance to these compounds via the selection of parasites with dramatically reduced expression of both genes. However, other compounds previously demonstrated or predicted to enter infected erythrocytes through transport pathways absent from noninfected erythrocytes, such as fosmidomycin, doxycycline, azithromycin, lumefantrine, or pentamidine, do not require expression of clag3 genes for their antimalarial activity. This suggests that they use alternative CLAG3-independent routes to access parasites. Our results demonstrate that P. falciparum can develop resistance to diverse antimalarial compounds by epigenetic changes in the expression of clag3 genes. This is of concern for drug development efforts because drug resistance by epigenetic mechanisms can arise quickly, even during the course of a single infection.

Expression of the Plasmodium falciparum Clonally Variant clag3 Genes in Human Infections.

Background: Many genes of the malaria parasite Plasmodium falciparum show clonally variant expression regulated at the epigenetic level. These genes participate in fundamental host-parasite interactions and contribute to adaptive processes. However, little is known about their expression patterns during human infections. A peculiar case of clonally variant genes are the 2 nearly identical clag3 genes, clag3.1 and clag3.2, which mediate nutrient uptake and are linked to resistance to some toxic compounds. Methods: We developed a procedure to characterize the expression of clag3 genes in naturally infected patients and in experimentally infected human volunteers. Results: We provide the first description of clag3 expression during human infections, which revealed mutually exclusive expression and identified the gene predominantly expressed. Adaptation to culture conditions or selection with a toxic compound resulted in isolate-dependent changes in clag3 expression. We also found that clag3 expression patterns were reset during transmission stages. Conclusions: Different environment conditions select for parasites with different clag3 expression patterns, implying functional differences between the proteins encoded. The epigenetic memory is likely erased before parasites start infection of a new human host. Altogether, our findings support the idea that clonally variant genes facilitate the adaptation of parasite populations to changing conditions through bet-hedging strategies.

Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes.

The product of the Plasmodium falciparum genes clag3.1 and clag3.2 plays a fundamental role in malaria parasite biology by determining solute transport into infected erythrocytes. Expression of the two clag3 genes is mutually exclusive, such that a single parasite expresses only one of the two genes at a time. Here we investigated the properties and mechanisms of clag3 mutual exclusion using transgenic parasite lines with extra copies of clag3 promoters located either in stable episomes or integrated in the parasite genome. We found that the additional clag3 promoters in these transgenic lines are silenced by default, but under strong selective pressure parasites with more than one clag3 promoter simultaneously active are observed, demonstrating that clag3 mutual exclusion is strongly favored but it is not strict. We show that silencing of clag3 genes is associated with the repressive histone mark H3K9me3 even in parasites with unusual clag3 expression patterns, and we provide direct evidence for heterochromatin spreading in P. falciparum. We also found that expression of a neighbor ncRNA correlates with clag3.1 expression. Altogether, our results reveal a scenario where fitness costs and non-deterministic molecular processes that favor mutual exclusion shape the expression patterns of this important gene family.

Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo.

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.

Transcriptional variation in the malaria parasite Plasmodium falciparum.

Malaria genetic variation has been extensively characterized, but the level of epigenetic plasticity remains largely unexplored. Here we provide a comprehensive characterization of transcriptional variation in the most lethal malaria parasite, Plasmodium falciparum, based on highly accurate transcriptional analysis of isogenic parasite lines grown under homogeneous conditions. This analysis revealed extensive transcriptional heterogeneity within genetically homogeneous clonal parasite populations. We show that clonally variant expression controlled at the epigenetic level is an intrinsic property of specific genes and gene families, the majority of which participate in host-parasite interactions. Intrinsic transcriptional variability is not restricted to genes involved in immune evasion, but also affects genes linked to lipid metabolism, protein folding, erythrocyte remodeling, or transcriptional regulation, among others, indicating that epigenetic variation results in both antigenic and functional variation. We observed a general association between heterochromatin marks and clonally variant expression, extending previous observations for specific genes to essentially all variantly expressed gene families. These results suggest that phenotypic variation of functionally unrelated P. falciparum gene families is mediated by a common mechanism based on reversible formation of H3K9me3-based heterochromatin. In changing environments, diversity confers fitness to a population. Our results support the idea that P. falciparum uses a bet-hedging strategy, as an alternative to directed transcriptional responses, to adapt to common fluctuations in its environment. Consistent with this idea, we found that transcriptionally different isogenic parasite lines markedly differed in their survival to heat-shock mimicking febrile episodes and adapted to periodic heat-shock with a pattern consistent with natural selection of pre-existing parasites.

Epigenetic switches in clag3 genes mediate blasticidin S resistance in malaria parasites.

Malaria parasites induce changes in the permeability of the infected erythrocyte membrane to numerous solutes, including toxic compounds. In Plasmodium falciparum, this is mainly mediated by PSAC, a broad-selectivity channel that requires the product of parasite clag3 genes for its activity. The two paralogous clag3 genes, clag3.1 and clag3.2, can be silenced by epigenetic mechanisms and show mutually exclusive expression. Here we show that resistance to the antibiotic blasticidin S (BSD) is associated with switches in the expression of these genes that result in altered solute uptake. Low concentrations of the drug selected parasites that switched from clag3.2 to clag3.1 expression, implying that expression of one or the other clag3 gene confers different transport efficiency to PSAC for some solutes. Selection with higher BSD concentrations resulted in simultaneous silencing of both clag3 genes, which severely compromises PSAC formation as demonstrated by blocked uptake of other PSAC substrates. Changes in the expression of clag3 genes were not accompanied by large genetic rearrangements or mutations at the clag3 loci or elsewhere in the genome. These results demonstrate that malaria parasites can become resistant to toxic compounds such as drugs by epigenetic switches in the expression of genes necessary for the formation of solute channels.

Expression of the MSPDBL2 antigen in a discrete subset of Plasmodium falciparum schizonts is regulated by GDV1 but may not be linked to sexual commitment.

The Plasmodium falciparum merozoite surface protein MSPDBL2 is a polymorphic antigen targeted by acquired immune responses, and normally expressed in only a minority of mature schizonts. The potential relationship of MSPDBL2 to sexual commitment is examined, as variable mspdbl2 transcript levels and proportions of MSPDBL2-positive mature schizonts in clinical isolates have previously correlated with levels of many sexual stage parasite gene transcripts, although not with the master regulator ap2-g. It is demonstrated that conditional overexpression of the gametocyte development protein GDV1, which promotes sexual commitment, also substantially increases the proportion of MSPDBL2-positive schizonts in culture. Conversely, truncation of the gdv1 gene is shown to prevent any expression of MSPDBL2. However, across diverse P. falciparum cultured lines, the variable proportions of MSPDBL2 positivity in schizonts do not correlate significantly with variable gametocyte conversion rates, indicating it is not involved in sexual commitment. Confirming this, examining a line with endogenous hemagglutinin-tagged AP2-G showed that the individual schizonts expressing MSPDBL2 are mostly different from those expressing AP2-G. Using a selection-linked integration system, modified P. falciparum lines were engineered to express an intact or disrupted version of MSPDBL2, showing the protein is not required for sexual commitment or early gametocyte development. Asexual parasite multiplication rates were also not affected by expression of either intact or disrupted MSPDBL2 in a majority of schizonts. Occurring alongside sexual commitment, the role of the discrete MSPDBL2-positive schizont subpopulation requires further investigation in natural infections where it is under immune selection. IMPORTANCE: Malaria parasites in the blood are remarkably variable, able to switch antigenic targets so they may survive within humans who have already developed specific immune responses. This is one of the challenges in developing vaccines against malaria. MSPDBL2 is a target of naturally acquired immunity expressed in minority proportions of schizonts, the end stages of each 2-day replication cycle in red blood cells which contain merozoites prepared to invade new red blood cells. Results show that the proportion of schizonts expressing MSPDBL2 is positively controlled by the expression of the regulatory gametocyte development protein GDV1. It was previously known that expression of GDV1 leads to increased expression of AP2-G which causes parasites to switch to sexual development, so a surprising finding here is that MSPDBL2-positive parasites are mostly distinct from those that express AP2-G. This discrete antigenic subpopulation of mostly asexual parasites is regulated alongside sexually committed parasites, potentially enabling survival under stress conditions.

Plasmodium vivax spleen-dependent protein 1 and its role in extracellular vesicles-mediated intrasplenic infections.

Recent studies indicate that human spleen contains over 95% of the total parasite biomass during chronic asymptomatic infections caused by Plasmodium vivax. Previous studies have demonstrated that extracellular vesicles (EVs) secreted from infected reticulocytes facilitate binding to human spleen fibroblasts (hSFs) and identified parasite genes whose expression was dependent on an intact spleen. Here, we characterize the P. vivax spleen-dependent hypothetical gene (PVX_114580). Using CRISPR/Cas9, PVX_114580 was integrated into P. falciparum 3D7 genome and expressed during asexual stages. Immunofluorescence analysis demonstrated that the protein, which we named P. vivax Spleen-Dependent Protein 1 (PvSDP1), was located at the surface of infected red blood cells in the transgenic line and this localization was later confirmed in natural infections. Plasma-derived EVs from P. vivax-infected individuals (PvEVs) significantly increased cytoadherence of 3D7_PvSDP1 transgenic line to hSFs and this binding was inhibited by anti-PvSDP1 antibodies. Single-cell RNAseq of PvEVs-treated hSFs revealed increased expression of adhesion-related genes. These findings demonstrate the importance of parasite spleen-dependent genes and EVs from natural infections in the formation of intrasplenic niches in P. vivax, a major challenge for malaria elimination.

Selective inhibition of an apicoplastic aminoacyl-tRNA synthetase from Plasmodium falciparum.

The resistance of malaria parasites to available drugs continues to grow, and this makes the need for new antimalarial therapies pressing. Aminoacyl-tRNA synthetases (ARSs) are essential enzymes and well-established antibacterial targets and so constitute a promising set of targets for the development of new antimalarials. Despite their potential as drug targets, apicoplastic ARSs remain unexplored. We have characterized the lysylation system of Plasmodium falciparum, and designed, synthesized, and tested a set of inhibitors based on the structure of the natural substrate intermediate: lysyl-adenylate. Here we demonstrate that selective inhibition of apicoplastic ARSs is feasible and describe new compounds that that specifically inhibit Plasmodium apicoplastic lysyl-tRNA synthetase and show antimalarial activities in the micromolar range.

Patterns of Heterochromatin Transitions Linked to Changes in the Expression of Plasmodium falciparum Clonally Variant Genes.

The survival of malaria parasites in the changing human blood environment largely depends on their ability to alter gene expression by epigenetic mechanisms. The active state of Plasmodium falciparum clonally variant genes (CVGs) is associated with euchromatin characterized by the histone mark H3K9ac, whereas the silenced state is characterized by H3K9me3-based heterochromatin. Expression switches are linked to euchromatin-heterochromatin transitions, but these transitions have not been characterized for the majority of CVGs. To define the heterochromatin distribution patterns associated with the alternative transcriptional states of CVGs, we compared H3K9me3 occupancy at a genome-wide level among several parasite subclones of the same genetic background that differed in the transcriptional state of many CVGs. We found that de novo heterochromatin formation or the complete disruption of a heterochromatin domain is a relatively rare event, and for the majority of CVGs, expression switches can be explained by the expansion or retraction of heterochromatin domains. We identified different modalities of heterochromatin changes linked to transcriptional differences, but despite this complexity, heterochromatin distribution patterns generally enable the prediction of the transcriptional state of specific CVGs. We also found that in some subclones, several var genes were simultaneously in an active state. Furthermore, the heterochromatin levels in the putative regulatory region of the gdv1 antisense noncoding RNA, a regulator of sexual commitment, varied between parasite lines with different sexual conversion rates. IMPORTANCE The malaria parasite P. falciparum is responsible for more than half a million deaths every year. P. falciparum clonally variant genes (CVGs) mediate fundamental host-parasite interactions and play a key role in parasite adaptation to fluctuations in the conditions of the human host. The expression of CVGs is regulated at the epigenetic level by changes in the distribution of a type of chromatin called heterochromatin. Here, we describe at a genome-wide level the changes in the heterochromatin distribution associated with the different transcriptional states of CVGs. Our results also reveal a likely role for heterochromatin at a particular locus in determining the parasite investment in transmission to mosquitoes. Additionally, this data set will enable the prediction of the transcriptional state of CVGs from epigenomic data, which is important for the study of parasite adaptation to the conditions of the host in natural malaria infections.

Heterochromatin formation in bistable chromatin domains controls the epigenetic repression of clonally variant Plasmodium falciparum genes linked to erythrocyte invasion.

Clonally variant gene expression is a common survival strategy used by many pathogens, including the malaria parasite Plasmodium falciparum. Among the genes that show variant expression in this parasite are several members of small gene families linked to erythrocyte invasion, including the clag and eba families. The active or repressed state of these genes is clonally transmitted by epigenetic mechanisms. Here we characterized the promoters of clag3.1, clag3.2 and eba-140, and compared nuclease accessibility and post-translational histone modifications between their active and repressed states. Activity of these promoters in an episomal context is similar between parasite subclones characterized by different patterns of expression of the endogenous genes. Variant expression is controlled by the euchromatic or heterochromatic state of bistable chromatin domains. Repression is mediated by H3K9me3-based heterochromatin, whereas the active state is characterized by H3K9ac. These marks are maintained throughout the asexual blood cycle to transmit the epigenetic memory. Furthermore, eba-140 is organized in two distinct chromatin domains, probably separated by a barrier insulator located within its ORF. The 5' chromatin domain controls expression of the gene, whereas the 3' domain shares the chromatin conformation with the upstream region of the neighbouring phista family gene, which also shows variant expression.

Malaria parasites do respond to heat.

The capacity of malaria parasites to respond to changes in their environment at the transcriptional level has been the subject of debate, but recent evidence has unambiguously demonstrated that Plasmodium spp. can produce adaptive transcriptional responses when exposed to some specific types of stress. These include metabolic conditions and febrile temperature. The Plasmodium falciparum protective response to thermal stress is similar to the response in other organisms, but it is regulated by a transcription factor evolutionarily unrelated to the conserved transcription factor that drives the heat shock (HS) response in most eukaryotes. Of the many genes that change expression during HS, only a subset constitutes an authentic response that contributes to parasite survival.

Plasmodium falciparum sexual conversion rates can be affected by artemisinin-based treatment in naturally infected malaria patients.

BACKGROUND: Artemisinins (ART) are the key component of the frontline antimalarial treatment, but their impact on Plasmodium falciparum sexual conversion rates in natural malaria infections remains unknown. This is an important knowledge gap because sexual conversion rates determine the relative parasite investment between maintaining infection in the same human host and transmission to mosquitoes. METHODS: The primary outcome of this study was to assess the impact of ART-based treatment on sexual conversion rates by comparing the relative transcript levels of pfap2-g and other sexual ring biomarkers (SRBs) before and after treatment. We analysed samples from previously existing cohorts in Vietnam, Burkina Faso and Mozambique (in total, n=109) collected before treatment and at 12 h intervals after treatment. As a secondary objective, we investigated factors that may influence the effect of treatment on sexual conversion rates. FINDINGS: In the majority of infections from the African cohorts, but not from Vietnam, we observed increased expression of pfap2-g and other SRBs after treatment. Estimated parasite age at the time of treatment was negatively correlated with the increase in pfap2-g transcript levels, suggesting that younger parasites are less susceptible to stimulation of sexual conversion. INTERPRETATION: We observed enhanced expression of SRBs after ART-based treatment in many patients, which suggests that in natural malaria infections sexual conversion rates can be altered by treatment. ART-based treatment reduces the potential of a treated individual to transmit the disease, but we hypothesise that under some circumstances this reduction may be attenuated by ART-enhanced sexual conversion. FUNDING: Spanish Agencia Estatal de Investigación (AEI), European Regional Development Fund (ERDF, European Union), Belgium Development Cooperation (DGD), Canadian University Health Network (UHN), TransGlobalHealth-Erasmus Mundus (European Union).

Transcriptional Analysis of Tightly Synchronized Plasmodium falciparum Intraerythrocytic Stages by RT-qPCR.

In Plasmodium falciparum, the parasite responsible for the most severe forms of human malaria, many fundamental processes are controlled at the transcriptional level. Studies on diverse aspects of basic parasite biology as well as molecular epidemiology studies often rely on the ability to accurately measure transcript levels, but this is complicated by the cyclic expression patterns of the majority of malaria parasite genes. Here, we provide a complete workflow to measure transcript levels in P. falciparum intraerythrocytic blood stages, overcoming the confounding factors that are commonly encountered. The method described covers all the steps from synchronization of parasite cultures to reverse transcriptase quantitative PCR (RT-qPCR) analysis.

Expression Patterns of Plasmodium falciparum Clonally Variant Genes at the Onset of a Blood Infection in Malaria-Naive Humans.

Clonally variant genes (CVGs) play fundamental roles in the adaptation of Plasmodium falciparum to fluctuating conditions of the human host. However, their expression patterns under the natural conditions of the blood circulation have been characterized in detail for only a few specific gene families. Here, we provide a detailed characterization of the complete P. falciparum transcriptome across the full intraerythrocytic development cycle (IDC) at the onset of a blood infection in malaria-naive human volunteers. We found that the vast majority of transcriptional differences between parasites obtained from the volunteers and the parental parasite line maintained in culture occurred in CVGs. In particular, we observed a major increase in the transcript levels of most genes of the pfmc-2tm and gbp families and of specific genes of other families, such as phist, hyp10, rif, or stevor, in addition to previously reported changes in var and clag3 gene expression. Increased transcript levels of individual pfmc-2tm, rif, and stevor genes involved activation in small subsets of parasites. Large transcriptional differences correlated with changes in the distribution of heterochromatin, confirming their epigenetic nature. Furthermore, the similar expression of several CVGs between parasites collected at different time points along the blood infection suggests that the epigenetic memory for multiple CVG families is lost during transmission stages, resulting in a reset of their transcriptional state. Finally, the CVG expression patterns observed in a volunteer likely infected by a single sporozoite suggest that new epigenetic patterns are established during liver stages. IMPORTANCE The ability of malaria parasites to adapt to changes in the human blood environment, where they produce long-term infection associated with clinical symptoms, is fundamental for their survival. CVGs, regulated at the epigenetic level, play a major role in this adaptive process, as changes in the expression of these genes result in alterations in the antigenic and functional properties of the parasites. However, how these genes are expressed under the natural conditions of the human circulation and how their expression is affected by passage through transmission stages are not well understood. Here, we provide a comprehensive characterization of the expression patterns of these genes at the onset of human blood infections, which reveals major differences with in vitro-cultured parasites. We also show that, during transmission stages, the previous expression patterns for many CVG families are lost, and new patterns are established.

A heat-shock response regulated by the PfAP2-HS transcription factor protects human malaria parasites from febrile temperatures.

Periodic fever is a characteristic clinical feature of human malaria, but how parasites survive febrile episodes is not known. Although the genomes of Plasmodium species encode a full set of chaperones, they lack the conserved eukaryotic transcription factor HSF1, which activates the expression of chaperones following heat shock. Here, we show that PfAP2-HS, a transcription factor in the ApiAP2 family, regulates the protective heat-shock response in Plasmodium falciparum. PfAP2-HS activates the transcription of hsp70-1 and hsp90 at elevated temperatures. The main binding site of PfAP2-HS in the entire genome coincides with a tandem G-box DNA motif in the hsp70-1 promoter. Engineered parasites lacking PfAP2-HS have reduced heat-shock survival and severe growth defects at 37 °C but not at 35 °C. Parasites lacking PfAP2-HS also have increased sensitivity to imbalances in protein homeostasis (proteostasis) produced by artemisinin, the frontline antimalarial drug, or the proteasome inhibitor epoxomicin. We propose that PfAP2-HS contributes to the maintenance of proteostasis under basal conditions and upregulates specific chaperone-encoding genes at febrile temperatures to protect the parasite against protein damage.