The human malaria parasite Plasmodium falciparum can sense environmental changes and respond by antigenic switching.

The primary antigenic and virulence determinant of the human malaria parasite Plasmodium falciparum is a variant surface protein called PfEMP1. Different forms of PfEMP1 are encoded by a multicopy gene family called var, and switching between active genes enables the parasites to evade the antibody response of their human hosts. var gene switching is key for the maintenance of chronic infections; however, what controls switching is unknown, although it has been suggested to occur at a constant frequency with little or no environmental influence. var gene transcription is controlled epigenetically through the activity of histone methyltransferases (HMTs). Studies in model systems have shown that metabolism and epigenetic control of gene expression are linked through the availability of intracellular S-adenosylmethionine (SAM), the principal methyl donor in biological methylation modifications, which can fluctuate based on nutrient availability. To determine whether environmental conditions and changes in metabolism can influence var gene expression, P. falciparum was cultured in media with altered concentrations of nutrients involved in SAM metabolism. We found that conditions that influence lipid metabolism induce var gene switching, indicating that parasites can respond to changes in their environment by altering var gene expression patterns. Genetic modifications that directly modified expression of the enzymes that control SAM levels similarly led to profound changes in var gene expression, confirming that changes in SAM availability modulate var gene switching. These observations directly challenge the paradigm that antigenic variation in P. falciparum follows an intrinsic, programed switching rate, which operates independently of any external stimuli.

Single-cell RNA sequencing reveals a signature of sexual commitment in malaria parasites.

Pathogens have to balance transmission with persistence. For Plasmodium falciparum, the most widespread and virulent malaria parasite, persistence within its human host requires continuous asexual replication within red blood cells, while its mosquito-borne transmission depends on intra-erythrocytic differentiation into non-replicating sexual stages called gametocytes. Commitment to either fate is determined during the preceding cell cycle that begins with invasion by a single, asexually committed merozoite and ends, 48 hours later, with a schizont releasing newly formed merozoites, all committed to either continued asexual replication or differentiation into gametocytes. Sexual commitment requires the transcriptional activation of ap2-g (PF3D7_1222600), the master regulator of sexual development, from an epigenetically silenced state during asexual replication. AP2-G expression during this 'commitment cycle' prepares gene expression in nascent merozoites to initiate sexual development through a hitherto unknown mechanism. To maintain a persistent infection, the expression of ap2-g is limited to a sub-population of parasites (1-30%, depending on genetic background and growth conditions). As sexually committed schizonts comprise only a sub-population and are morphologically indistinguishable from their asexually committed counterparts, defining their characteristic gene expression has been difficult using traditional, bulk transcriptome profiling. Here we use highly parallel, single-cell RNA sequencing of malaria cultures undergoing sexual commitment to determine the transcriptional changes induced by AP2-G within this sub-population. By analysing more than 18,000 single parasite transcriptomes from a conditional AP2-G knockdown line and NF54 wild-type parasites at multiple stages of development, we show that sexually committed, AP2-G+ mature schizonts specifically upregulate additional regulators of gene expression, including other AP2 transcription factors, histone-modifying enzymes, and regulators of nucleosome positioning. These epigenetic regulators may act to facilitate the expression and/or repression of genes that are necessary for the initiation of gametocyte development in the subsequent cell cycle.

Activity of Epigenetic Inhibitors against Plasmodium falciparum Asexual and Sexual Blood Stages.

Earlier genetic and inhibitor studies showed that epigenetic regulation of gene expression is critical for malaria parasite survival in multiple life stages and a promising target for new antimalarials. We therefore evaluated the activity of 350 diverse epigenetic inhibitors against multiple stages of Plasmodium falciparum We observed ≥90% inhibition at 10 µM for 28% of compounds against asexual blood stages and early gametocytes, of which a third retained ≥90% inhibition at 1 µM.

A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium.

Commitment to and completion of sexual development are essential for malaria parasites (protists of the genus Plasmodium) to be transmitted through mosquitoes. The molecular mechanism(s) responsible for commitment have been hitherto unknown. Here we show that PbAP2-G, a conserved member of the apicomplexan AP2 (ApiAP2) family of DNA-binding proteins, is essential for the commitment of asexually replicating forms to sexual development in Plasmodium berghei, a malaria parasite of rodents. PbAP2-G was identified from mutations in its encoding gene, PBANKA_143750, which account for the loss of sexual development frequently observed in parasites transmitted artificially by blood passage. Systematic gene deletion of conserved ApiAP2 genes in Plasmodium confirmed the role of PbAP2-G and revealed a second ApiAP2 member (PBANKA_103430, here termed PbAP2-G2) that significantly modulates but does not abolish gametocytogenesis, indicating that a cascade of ApiAP2 proteins are involved in commitment to the production and maturation of gametocytes. The data suggest a mechanism of commitment to gametocytogenesis in Plasmodium consistent with a positive feedback loop involving PbAP2-G that could be exploited to prevent the transmission of this pernicious parasite.

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.

Transcriptional plasticity of virulence genes provides malaria parasites with greater adaptive capacity for avoiding host immunity.

Chronic, asymptomatic malaria infections contribute substantially to disease transmission and likely represent the most significant impediment preventing malaria elimination and eradication. Plasmodium falciparum parasites evade antibody recognition through transcriptional switching between members of the var gene family, which encodes the major virulence factor and surface antigen on infected red blood cells. This process can extend infections for up to a year; however, infections have been documented to last for over a decade, constituting an unseen reservoir of parasites that undermine eradication and control efforts. How parasites remain immunologically "invisible" for such lengthy periods is entirely unknown. Here we show that in addition to the accepted paradigm of mono-allelic var gene expression, individual parasites can simultaneously express multiple var genes or enter a state in which little or no var gene expression is detectable. This unappreciated flexibility provides parasites with greater adaptive capacity than previously understood and challenges the dogma of mutually exclusive var gene expression. It also provides an explanation for the antigenically "invisible" parasites observed in chronic asymptomatic infections.

Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome.

The molecular chaperone heat shock protein 90 (Hsp90) has an essential but largely undefined role in maintaining proteostasis in Plasmodium falciparum, the most lethal malaria parasite. Herein, we identify BX-2819 and XL888 as potent P. falciparum (Pf)Hsp90 inhibitors. Derivatization of XL888's scaffold led to the development of Tropane 1, as a PfHsp90-selective binder with nanomolar affinity. Hsp90 inhibitors exhibit anti-Plasmodium activity against the liver, asexual blood, and early gametocyte life stages. Thermal proteome profiling was implemented to assess PfHsp90-dependent proteome stability, and the proteasome-the main site of cellular protein recycling-was enriched among proteins with perturbed stability upon PfHsp90 inhibition. Subsequent biochemical and cellular studies suggest that PfHsp90 directly promotes proteasome hydrolysis by chaperoning the active 26S complex. These findings expand our knowledge of the PfHsp90-dependent proteome and protein quality control mechanisms in these pathogenic parasites, as well as further characterize this chaperone as a potential antimalarial drug target.

A non-canonical sensing pathway mediates Plasmodium adaptation to amino acid deficiency.

Eukaryotes have canonical pathways for responding to amino acid (AA) availability. Under AA-limiting conditions, the TOR complex is repressed, whereas the sensor kinase GCN2 is activated. While these pathways have been highly conserved throughout evolution, malaria parasites are a rare exception. Despite auxotrophic for most AA, Plasmodium does not have either a TOR complex nor the GCN2-downstream transcription factors. While Ile starvation has been shown to trigger eIF2α phosphorylation and a hibernation-like response, the overall mechanisms mediating detection and response to AA fluctuation in the absence of such pathways has remained elusive. Here we show that Plasmodium parasites rely on an efficient sensing pathway to respond to AA fluctuations. A phenotypic screen of kinase knockout mutant parasites identified nek4, eIK1 and eIK2-the last two clustering with the eukaryotic eIF2α kinases-as critical for Plasmodium to sense and respond to distinct AA-limiting conditions. Such AA-sensing pathway is temporally regulated at distinct life cycle stages, allowing parasites to actively fine-tune replication and development in response to AA availability. Collectively, our data disclose a set of heterogeneous responses to AA depletion in malaria parasites, mediated by a complex mechanism that is critical for modulating parasite growth and survival.

Sexual differentiation in human malaria parasites is regulated by competition between phospholipid metabolism and histone methylation.

For Plasmodium falciparum, the most widespread and virulent malaria parasite that infects humans, persistence depends on continuous asexual replication in red blood cells, while transmission to their mosquito vector requires asexual blood-stage parasites to differentiate into non-replicating gametocytes. This decision is controlled by stochastic derepression of a heterochromatin-silenced locus encoding AP2-G, the master transcription factor of sexual differentiation. The frequency of ap2-g derepression was shown to be responsive to extracellular phospholipid precursors but the mechanism linking these metabolites to epigenetic regulation of ap2-g was unknown. Through a combination of molecular genetics, metabolomics and chromatin profiling, we show that this response is mediated by metabolic competition for the methyl donor S-adenosylmethionine between histone methyltransferases and phosphoethanolamine methyltransferase, a critical enzyme in the parasite's pathway for de novo phosphatidylcholine synthesis. When phosphatidylcholine precursors are scarce, increased consumption of SAM for de novo phosphatidylcholine synthesis impairs maintenance of the histone methylation responsible for silencing ap2-g, increasing the frequency of derepression and sexual differentiation. This provides a key mechanistic link that explains how LysoPC and choline availability can alter the chromatin status of the ap2-g locus controlling sexual differentiation.

New Agilent platform DNA microarrays for transcriptome analysis of Plasmodium falciparum and Plasmodium berghei for the malaria research community.

BACKGROUND: DNA microarrays have been a valuable tool in malaria research for over a decade but remain in limited use in part due their relatively high cost, poor availability, and technical difficulty. With the aim of alleviating some of these factors next-generation DNA microarrays for genome-wide transcriptome analysis for both Plasmodium falciparum and Plasmodium berghei using the Agilent 8 x 15 K platform were designed. METHODS: Probe design was adapted from previously published methods and based on the most current transcript predictions available at the time for P. falciparum or P. berghei. Array performance and transcriptome analysis was determined using dye-coupled, aminoallyl-labelled cDNA and streamlined methods for hybridization, washing, and array analysis were developed. RESULTS: The new array design marks a notable improvement in the number of transcripts covered and average number of probes per transcript. Array performance was excellent across a wide range of transcript abundance, with low inter-array and inter-probe variability for relative abundance measurements and it recapitulated previously observed transcriptional patterns. Additionally, improvements in sensitivity permitted a 20-fold reduction in necessary starting RNA amounts, further reducing experimental costs and widening the range of application. CONCLUSIONS: DNA microarrays utilizing the Agilent 8 x 15 K platform for genome-wide transcript analysis in P. falciparum and P. berghei mark an improvement in coverage and sensitivity, increased availability to the research community, and simplification of the experimental methods.

Safe, Effective, and Inexpensive Clearance of Mycoplasma Contamination from Cultures of Apicomplexan Parasites with Sparfloxacin.

Most commercial products cannot be used for clearance of Mycoplasma contamination from cultures of apicomplexan parasites due to the parasites' dependence on the apicoplast, an essential organelle with DNA replication and translation machinery of cyanobacterial origin. The lone exception, mycoplasma removal agent (MRA), is relatively expensive, and some mycoplasma strains have shown resistance to clearance with MRA. Here, we report that the fluoroquinolone antibiotic sparfloxacin is a safe, effective, and inexpensive alternative for treatment of mycoplasma contamination in cultures of apicomplexan parasites. Sparfloxacin cleared both MRA-sensitive and MRA-resistant mycoplasma species from P. falciparum cultures at 1 and 4 µg/mL, respectively. We show that cultures of three different apicomplexan parasites can be maintained at concentrations of sparfloxacin required to clear mycoplasma without resulting in substantial deleterious effects on parasite growth. We also describe an alternative low-cost, in-house PCR assay for detecting mycoplasma. These findings will be useful to laboratories maintaining apicomplexan parasites in vitro, especially in low-resource environments, where the high cost of commercial products creates an economic barrier for detecting and eliminating mycoplasma from culture. IMPORTANCE These findings will be useful to laboratories maintaining apicomplexan parasites in vitro, especially in low-resource environments, where the high cost of commercial products creates an economic barrier for detecting and eliminating Mycoplasma from culture.

The transcriptional regulator HDP1 controls expansion of the inner membrane complex during early sexual differentiation of malaria parasites.

Transmission of Plasmodium falciparum and other malaria parasites requires their differentiation from asexual blood stages into gametocytes, the non-replicative sexual stage necessary to infect the mosquito vector. This transition involves changes in gene expression and chromatin reorganization that result in the activation and silencing of stage-specific genes. However, the genomes of malaria parasites have been noted for their limited number of transcriptional and chromatin regulators, and the molecular mediators of these changes remain largely unknown. We recently identified homeodomain protein 1 (HDP1) as a DNA-binding protein, first expressed in gametocytes, that enhances the expression of key genes critical for early sexual differentiation. The discovery of HDP1 marks a new class of transcriptional regulator in malaria parasites outside of the better-characterized ApiAP2 family. Here, using molecular biology, biochemistry and microscopy techniques, we show that HDP1 is essential for gametocyte maturation, facilitating the necessary upregulation of inner membrane complex components during early gametocytogenesis that gives P. falciparum gametocytes their characteristic shape.

Toxoplasma gondii protease TgSUB1 is required for cell surface processing of micronemal adhesive complexes and efficient adhesion of tachyzoites.

Host cell invasion by Toxoplasma gondii is critically dependent upon adhesive proteins secreted from the micronemes. Proteolytic trimming of microneme contents occurs rapidly after their secretion onto the parasite surface and is proposed to regulate adhesive complex activation to enhance binding to host cell receptors. However, the proteases responsible and their exact function are still unknown. In this report, we show that T. gondii tachyzoites lacking the microneme subtilisin protease TgSUB1 have a profound defect in surface processing of secreted microneme proteins. Notably parasites lack protease activity responsible for proteolytic trimming of MIC2, MIC4 and M2AP after release onto the parasite surface. Although complementation with full-length TgSUB1 restores processing, complementation of Δsub1 parasites with TgSUB1 lacking the GPI anchor (Δsub1::ΔGPISUB1) only partially restores microneme protein processing. Loss of TgSUB1 decreases cell attachment and in vitro gliding efficiency leading to lower initial rates of invasion. Δsub1 and Δsub1::ΔGPISUB1 parasites are also less virulent in mice. Thus TgSUB1 is involved in micronemal protein processing and regulation of adhesive properties of macromolecular adhesive complexes involved in host cell invasion.

There and back again: malaria parasite single-cell transcriptomics comes full circle.

The Malaria Cell Atlas (MCA) is an ambitious, ongoing project to profile the intensity and heterogeneity of gene expression throughout the entire malaria parasite life cycle with single-cell resolution. Real et al. now complete the cycle by adding the transmission stages of the most virulent malaria parasite, Plasmodium falciparum, to this easy-to-use resource.

Activity Comparison of Epigenetic Modulators against the Hemoprotozoan Parasites Babesia divergens and Plasmodium falciparum.

Babesiosis is a tick-borne parasitic disease of humans and livestock that has dramatically increased in frequency and geographical range over the past few decades. Infection of cattle often causes large economic losses, and human infection can be fatal in immunocompromised patients. Unlike for malaria, another disease caused by hemoprotozoan parasites, limited treatment options exist for Babesia infections. As epigenetic regulation is a promising target for new antiparasitic drugs, we screened 324 epigenetic inhibitors against Babesia divergens blood stages and identified 75 (23%) and 17 (5%) compounds that displayed ≥90% inhibition at 10 and 1 µM, respectively, including over a dozen compounds with activity in the low nanomolar range. We observed differential activity of some inhibitor classes against Babesia divergens and Plasmodium falciparum parasites and identified pairs of compounds with a high difference in activity despite a high similarity in chemical structure, highlighting new insights into the development of epigenetic inhibitors as antiparasitic drugs.

Telomere length dynamics in response to DNA damage in malaria parasites.

Malaria remains a major cause of morbidity and mortality in the developing world. Recent work has implicated chromosome end stability and the repair of DNA breaks through telomere healing as potent drivers of variant antigen diversification, thus associating basic mechanisms for maintaining genome integrity with aspects of host-parasite interactions. Here we applied long-read sequencing technology to precisely examine the dynamics of telomere addition and chromosome end stabilization in response to double-strand breaks within subtelomeric regions. We observed that the process of telomere healing induces the initial synthesis of telomere repeats well in excess of the minimal number required for end stability. However, once stabilized, these newly created telomeres appear to function normally, eventually returning to a length nearing that of intact chromosome ends. These results parallel recent observations in humans, suggesting an evolutionarily conserved mechanism for chromosome end repair.

Competition between two MHC binding registers in a single peptide processed from myelin basic protein influences tolerance and susceptibility to autoimmunity.

Experimental allergic encephalomyelitis (EAE) is an animal model for multiple sclerosis induced by stimulating myelin basic protein (MBP)-specific T cells. The MBP-specific repertoire in B10.PL mice is shaped by tolerance mechanisms that eliminate MBP121-150-specific T cells. In contrast, MBPAc1-11-specific T cells escape tolerance and constitute the encephalitogenic repertoire. To determine if this differential tolerance is caused by differences in the abundance of MBP epitopes generated by processing, MBP peptides were eluted from I-Au complexes and analyzed by mass spectrometry. Peptides were identified from both the NH2-terminal and MBP121-150 regions. Unexpectedly, MBPAc1-18 and Ac1-17, which contain the MBPAc1-11 epitope, were much more abundant than MBP121-150 peptides. The results demonstrate that competition between two I-Au binding registers, a low affinity register defined by MBPAc1-11 and a high affinity register defined by MBP5-16, prevents most of the NH2-terminal naturally processed peptides from binding in the MBPAc1-11 register. The small fraction of MBPAc1-18 bound in the MBPAc1-11 register is not sufficient to induce tolerance but provides a ligand for MBPAc1-11-specific T cells during disease. These results provide a basis for both the lack of tolerance to MBPAc1-11 and the ability of this epitope to become a target during autoimmunity.

Disruption of the Plasmodium falciparum Life Cycle through Transcriptional Reprogramming by Inhibitors of Jumonji Demethylases.

Little is known about the role of the three Jumonji C (JmjC) enzymes in Plasmodium falciparum (Pf). Here, we show that JIB-04 and other established inhibitors of mammalian JmjC histone demethylases kill asexual blood stage parasites and are even more potent at blocking gametocyte development and gamete formation. In late stage parasites, JIB-04 increased levels of trimethylated lysine residues on histones, suggesting the inhibition of P. falciparum Jumonji demethylase activity. These epigenetic defects coincide with deregulation of invasion, cell motor, and sexual development gene programs, including gene targets coregulated by the PfAP2-I transcription factor and chromatin-binding factor, PfBDP1. Mechanistically, we demonstrate that PfJmj3 converts 2-oxoglutarate to succinate in an iron-dependent manner consistent with mammalian Jumonji enzymes, and this catalytic activity is inhibited by JIB-04 and other Jumonji inhibitors. Our pharmacological studies of Jumonji activity in the malaria parasite provide evidence that inhibition of these enzymatic activities is detrimental to the parasite.

Revisiting the initial steps of sexual development in the malaria parasite Plasmodium falciparum.

Human to vector transmission of malaria requires that some blood-stage parasites abandon asexual growth and convert into non-replicating sexual forms called gametocytes. The initial steps of gametocytogenesis remain largely uncharacterized. Here, we study this part of the malaria life cycle in Plasmodium falciparum using PfAP2-G, the master regulator of sexual conversion, as a marker of commitment. We demonstrate the existence of PfAP2-G-positive sexually committed parasite stages that precede the previously known committed schizont stage. We also found that sexual conversion can occur by two different routes: the previously described route in which PfAP2-G-expressing parasites complete a replicative cycle as committed forms before converting into gametocytes upon re-invasion, or a direct route with conversion within the same cycle as initial PfAP2-G expression. The latter route is linked to early PfAP2-G expression in ring stages. Reanalysis of published single-cell RNA-sequencing (RNA-seq) data confirmed the presence of both routes. Consistent with these results, using plaque assays we observed that, in contrast to the prevailing model, many schizonts produced mixed plaques containing both asexual parasites and gametocytes. Altogether, our results reveal unexpected features of the initial steps of sexual development and extend the current view of this part of the malaria life cycle.

Generation of Transmission-Competent Human Malaria Parasites with Chromosomally-Integrated Fluorescent Reporters.

Malaria parasites have a complex life cycle that includes specialized stages for transmission between their mosquito and human hosts. These stages are an understudied part of the lifecycle yet targeting them is an essential component of the effort to shrink the malaria map. The human parasite Plasmodium falciparum is responsible for the majority of deaths due to malaria. Our goal was to generate transgenic P. falciparum lines that could complete the lifecycle and produce fluorescent transmission stages for more in-depth and high-throughput studies. Using zinc-finger nuclease technology to engineer an integration site, we generated three transgenic P. falciparum lines in which tdtomato or gfp were stably integrated into the genome. Expression was driven by either stage-specific peg4 and csp promoters or the constitutive ef1a promoter. Phenotypic characterization of these lines demonstrates that they complete the life cycle with high infection rates and give rise to fluorescent mosquito stages. The transmission stages are sufficiently bright for intra-vital imaging, flow cytometry and scalable screening of chemical inhibitors and inhibitory antibodies.