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.

Advances in single-cell experimental design made possible by automated imaging platforms with feedback through segmentation.

Live optical microscopy has become an essential tool for studying the dynamical behaviors and variability of single cells, and cell-cell interactions. However, experiments and data analysis in this area are often extremely labor intensive, and it has often not been achievable or practical to perform properly standardized experiments on a statistically viable scale. We have addressed this challenge by developing automated live imaging platforms, to help standardize experiments, increasing throughput, and unlocking previously impossible ones. Our real-time cell tracking programs communicate in feedback with microscope and camera control software, and they are highly customizable, flexible, and efficient. As examples of our current research which utilize these automated platforms, we describe two quite different applications: egress-invasion interactions of malaria parasites and red blood cells, and imaging of immune cells which possess high motility and internal dynamics. The automated imaging platforms are able to track a large number of motile cells simultaneously, over hours or even days at a time, greatly increasing data throughput and opening up new experimental possibilities.

Effect of thioredoxin peroxidase-1 gene disruption on the liver stages of the rodent malaria parasite Plasmodium berghei.

Phenotypic observation of thioredoxin peroxidase-1 (TPx-1) gene-disrupted Plasmodium berghei (TPx-1 KO) in the liver-stage was performed with an in vitro infection system in order to investigate defective liver-stage development in a mouse infection model. Indirect immunofluorescence microscopy assay with anti-circumsporozoite protein antibody revealed that in the liver schizont stage, TPx-1 KO parasite cells were significantly smaller than cells of the wild-type parent strain (WT). Indirect immunofluorescence microscopy assay with anti-merozoite surface protein-1 antibody, which was used to evaluate late schizont-stage development, indicated that TPx-1 KO schizont development was similar to WT strain development towards the merozoite-forming stage (mature schizont). However, fewer merozoites were produced in the mature TPx-1 KO schizont than in the mature WT schizont. Taken together, the results suggest that TPx-1 may be involved in merozoite formation during liver schizont development.

Magnetic isolation of Plasmodium falciparum schizonts iRBCs to generate a high parasitaemia and synchronized in vitro culture.

BACKGROUND: The establishment of methods for an in vitro continuous culture of Plasmodium falciparum is essential for gaining knowledge into its biology and for the development of new treatments. Previously, several techniques have been used to synchronize, enrich and concentrate P. falciparum, although obtaining cultures with high parasitaemia continues being a challenging process. Current methods produce high parasitaemia levels of synchronized P. falciparum cultures by frequent changes of culture medium or reducing the haematocrit. However, these methods are time consuming and sometimes lead to the loss of synchrony. METHODS: A procedure that combines Percoll and sorbitol treatments, the use of magnetic columns, and the optimization of the in vitro culture conditions to reach high parasitaemia levels for synchronized Plasmodium falciparum cultures is described. RESULTS: A new procedure has been established using P. falciparum 3D7, combining previous reported methodologies to achieve in vitro parasite cultures that reach parasitaemia up to 40% at any intra-erythrocytic stage. High parasitaemia levels are obtained only one day after magnetic column purification without compromising the parasite viability and synchrony. CONCLUSIONS: The described procedure allows obtaining a large scale synchronized parasite culture at a high parasitaemia with less manipulations than other methods previously described.

Targeting Plasmodium PI(4)K to eliminate malaria.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

Plasmodium berghei MAPK1 displays differential and dynamic subcellular localizations during liver stage development.

Mitogen-activated protein kinases (MAPKs) regulate key signaling events in eukaryotic cells. In the genomes of protozoan Plasmodium parasites, the causative agents of malaria, two genes encoding kinases with significant homology to other eukaryotic MAPKs have been identified (mapk1, mapk2). In this work, we show that both genes are transcribed during Plasmodium berghei liver stage development, and analyze expression and subcellular localization of the PbMAPK1 protein in liver stage parasites. Live cell imaging of transgenic parasites expressing GFP-tagged PbMAPK1 revealed a nuclear localization of PbMAPK1 in the early schizont stage mediated by nuclear localization signals in the C-terminal domain. In contrast, a distinct localization of PbMAPK1 in comma/ring-shaped structures in proximity to the parasite's nuclei and the invaginating parasite membrane was observed during the cytomere stage of parasite development as well as in immature blood stage schizonts. The PbMAPK1 localization was found to be independent of integrity of a motif putatively involved in ATP binding, integrity of the putative activation motif and the presence of a predicted coiled-coil domain in the C-terminal domain. Although PbMAPK1 knock out parasites showed normal liver stage development, the kinase may still fulfill a dual function in both schizogony and merogony of liver stage parasites regulated by its dynamic and stage-dependent subcellular localization.

Features of autophagic cell death in Plasmodium liver-stage parasites.

Analyzing molecular determinants of Plasmodium parasite cell death is a promising approach for exploring new avenues in the fight against malaria. Three major forms of cell death (apoptosis, necrosis and autophagic cell death) have been described in multicellular organisms but which cell death processes exist in protozoa is still a matter of debate. Here we suggest that all three types of cell death occur in Plasmodium liver-stage parasites. Whereas typical molecular markers for apoptosis and necrosis have not been found in the genome of Plasmodium parasites, we identified genes coding for putative autophagy-marker proteins and thus concentrated on autophagic cell death. We characterized the Plasmodium berghei homolog of the prominent autophagy marker protein Atg8/LC3 and found that it localized to the apicoplast. A relocalization of PbAtg8 to autophagosome-like vesicles or vacuoles that appear in dying parasites was not, however, observed. This strongly suggests that the function of this protein in liver-stage parasites is restricted to apicoplast biology.

Bioluminescence imaging of P. berghei Schizont sequestration in rodents.

We describe a technology for imaging the sequestration of infected red blood cells (iRBC) of the rodent malaria parasite Plasmodium berghei both in the bodies of live mice and in dissected organs, using a transgenic parasite that expresses luciferase. Real-time imaging of sequestered iRBC is performed by measuring bioluminescence produced by the enzymatic reaction in parasites between the luciferase enzyme and its substrate luciferin injected into the mice several minutes prior to imaging. The bioluminescence signal is detected by a sensitive I-CCD photon-counting video camera. Using a reporter parasite that expresses luciferase under the control of a schizont-specific promoter (i.e., the ama-1 promoter), the schizont stage is made visible when detecting bioluminescence signals. Schizont sequestration is imaged during short-term infections with parasites that are synchronized in development or during ongoing infections. Real-time in vivo imaging of iRBC will provide increased insights into the dynamics of sequestration and its role in pathology, and can be used to evaluate strategies that prevent sequestration.

Novel RuvB nuclear ATPase is specific to intraerythrocytic mitosis during schizogony of Plasmodium falciparum.

RuvB protein belongs to AAA+ family of enzymes involved in diverse cellular activities. In addition to the annotated two RuvB proteins in Plasmodium falciparum database, we report that a third RuvB protein is also present. The amino acid sequence analysis has revealed that P. falciparum RuvB3 (PfRuvB3) possesses Walker motif A, Walker motif B, sensor I and sensor II conserved motifs similar to yeast and human RuvB like proteins. The phylogenetic analysis revealed that PfRuvB3 is closely related to yeast RuvB like proteins which are essential for the survival of yeast. The biochemical characterization of recombinant PfRuvB3 confirms its ssDNA dependent ATPase activity. Using the truncated derivatives we show that Walker motif A is essential for the enzymatic activity of PfRuvB3. Using the immunodepletion assays we further show that the ATPase activity is attributable to PfRuvB3 protein. The endogenous P. falciparum RuvB3 contains the characteristic ATPase and some DNA helicase activities. The confocal microscopy analysis showed that this protein is mainly expressed during intraerythrocytic schizont stages of the parasite and is localized to the nuclear region. Once merozoite comes out from schizont, PfRuvB3 protein distinctly relocalized to the subnuclear region. The co-localization studies with a nucleolar marker PfNop1 further suggest that in P. falciparum RuvB3 localizes into a discrete nuclear compartment. On the basis of these studies it can be speculated that P. falciparum RuvB3 is most likely required for intraerythrocytic schizogony.

Two long non-coding RNAs generated from subtelomeric regions accumulate in a novel perinuclear compartment in Plasmodium falciparum.

Chromosome ends have been implicated in the default silencing of clonally variant gene families in the human malaria parasite Plasmodium falciparum. These chromosome regions are organized into heterochromatin, as defined by the presence of a repressive histone H3 lysine 9 trimethylated marker and heterochromatin protein 1. Here, we show that the non-coding subtelomeric region adjacent to virulence genes forms facultative heterochromatin in a cell cycle-dependent manner. We demonstrate that telomere-associated repeat elements (TAREs) and telomeres are transcribed as long non-coding RNAs (lncRNAs) during schizogony. Northern blot assays revealed two classes of lncRNAs: a ~4-kb transcript composed of telomere sequences and a TARE-3 element, and a >6-kb transcript composed of 21-bp repeats from TARE-6. These lncRNAs are transcribed by RNA polymerase II as single-stranded molecules. RNA-FISH analysis showed that these lncRNAs form several nuclear foci during the schizont stage, whereas in the ring stage, they are located in a single perinuclear compartment that does not co-localize with any known nuclear subcompartment. Furthermore, the TARE-6 lncRNA is predicted to form a stable and repetitive hairpin structure that is able to bind histones. Consequently, the characterization of the molecular interactions of these lncRNAs with nuclear proteins may reveal novel modes of gene regulation and nuclear function in P. falciparum.

A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes.

Clinical malaria is associated with the proliferation of Plasmodium parasites in human erythrocytes. The coordinated processes of parasite egress from and invasion into erythrocytes are rapid and tightly regulated. We have found that the plant-like calcium-dependent protein kinase PfCDPK5, which is expressed in invasive merozoite forms of Plasmodium falciparum, was critical for egress. Parasites deficient in PfCDPK5 arrested as mature schizonts with intact membranes, despite normal maturation of egress proteases and invasion ligands. Merozoites physically released from stalled schizonts were capable of invading new erythrocytes, separating the pathways of egress and invasion. The arrest was downstream of cyclic guanosine monophosphate-dependent protein kinase (PfPKG) function and independent of protease processing. Thus, PfCDPK5 plays an essential role during the blood stage of malaria replication.

Novel putative glycosylphosphatidylinositol-anchored micronemal antigen of Plasmodium falciparum that binds to erythrocytes.

We have identified a new Plasmodium falciparum erythrocyte binding protein that appears to be located in the micronemes of the merozoite stage of the parasite and membrane linked through a glycosylphosphatidylinositol (GPI) anchor. The protein is designated GPI-anchored micronemal antigen (GAMA) and was identified by applying a set of selection criteria to identify previously uncharacterized merozoite proteins that may have a role in cell invasion. The protein is also present in the proteomes of the sporozoite and ookinete micronemes and is conserved throughout the genus. GAMA contains a novel domain that may be constrained by disulfide bonds and a predicted C-terminal hydrophobic sequence that is presumably replaced by the GPI. The protein is synthesized late during schizogony, processed into two fragments that are linked by a disulfide bond, and translocated to an apical location, which is probably the micronemes. In a proportion of free merozoites GAMA can also be detected on the parasite surface. Following erythrocyte invasion the bulk of the protein is shed in a soluble form, although a short C-terminal fragment may be carried into the newly invaded red blood cell. The protein was shown to bind reversibly to erythrocytes and therefore represents a new example of a host cell binding protein.

Clinically uncomplicated Plasmodium falciparum malaria with high schizontaemia: a case report.

BACKGROUND: The treatment options for acute Plasmodium falciparum malaria are based on the clinician classifying the patient as uncomplicated or severe according to the clinical and parasitological findings. This process is not always straightforward. CASE PRESENTATION: An adult male presented to a clinic on the western border of Thailand with a physical examination and P. falciparum trophozoite count (1.2% of infected red blood cells, IRBC) from malaria blood smear, consistent with a diagnosis of uncomplicated P. falciparum infection. However, the physician on duty treated the patient for severe malaria based on the reported P. falciparum schizont count, which was very high (0.3% IRBC), noticeably in relation to the trophozoite count and schizont:trophozoite ratio 0.25:1. On intravenous artesunate, the patient deteriorated clinically in the first 24 hours. The trophozoite count increased from 1.2% IRBC at baseline to 20.5% IRBC 18 hours following the start of treatment. By day three, the patient recovered and was discharged on day seven having completed a seven-day treatment with artesunate and mefloquine. CONCLUSION: The malaria blood smear provides only a guide to the overall parasite biomass in the body, due to the ability of P. falciparum to sequester in the microvasculature. In severe malaria, high schizont counts are associated with worse prognosis. In low transmission areas or in non-immune travelers the presence of schizonts in the peripheral circulation is an indication for close patient supervision. In this case, an unusually high schizont count in a clinically uncomplicated patient was indicative of potential deterioration. Prompt treatment with intravenous artesunate is likely to have been responsible for the good clinical outcome in this case.

Enhanced detection of gametocytes by magnetic deposition microscopy predicts higher potential for Plasmodium falciparum transmission.

BACKGROUND: Aggregated haemozoin crystals within malaria-infected erythrocytes confer susceptibility of parasitized cells to a magnetic field. Here the utility of this method for diagnosis of human malaria is evaluated in a malaria-endemic region of Papua New Guinea (PNG). METHODS AND FINDINGS: Individuals with Plasmodium falciparum malaria symptoms (n = 55) provided samples for conventional blood smear (CBS) and magnetic deposition microscopy (MDM) diagnosis. Standard Giemsa staining and light microscopy was performed to evaluate all preparations. Plasmodium falciparum parasitaemia observed on MDM slides was consistently higher than parasitaemia observed by (CBS) for ring (CBS = 2.6 vs. MDM = 3.4%; t-test P-value = 0.13), trophozoite (CBS = 0.5 vs. MDM = 1.6%; t-test P-value = 0.01), schizont (CBS = 0.003 vs. MDM = 0.1%; t-test P-value = 0.08) and gametocyte (CBS = 0.001 vs. MDM = 0.4%; t-test P-value = 0.0002) parasitaemias. Gametocyte prevalence determined by CBS compared to MDM increased from 7.3% to 45%, respectively. CONCLUSION: MDM increased detection sensitivity of P. falciparum-infected, haemozoin-containing erythrocytes from infected humans while maintaining detection of ring-stage parasites. Gametocyte prevalence five-fold higher than observed by CBS suggests higher malaria transmission potential in PNG endemic sites compared to previous estimates.

Influence of oxygen on asexual blood cycle and susceptibility of Plasmodium falciparum to chloroquine: requirement of a standardized in vitro assay.

OBJECTIVE: The main objective of this study was to assess the influence of gas mixtures on in vitro Plasmodium falciparum growth and 50% inhibitory concentration (IC50) for chloroquine. METHODS: The study was performed between February 2004 and December 2005. 136 Plasmodium falciparum isolates were used to evaluate gas mixtures effect on IC50 for chloroquine by isotopic microtest. The oxygen effect on asexual blood cycle of 3D7 and W2 clones was determined by thin blood smears examination and tritiated hypoxanthine uptake. RESULTS: From 5% O2 to 21% O2 conditions, no parasiticide effect of O2 concentration was observed in vitro on the clones 3D7 and W2. A parasitostatic effect was observed during the exposure of mature trophozoïtes and schizonts at 21% O2 with an increase in the length of schizogony. The chloroquine IC50 at 10% O2 were significantly higher than those at 21% O2, means of 173.5 nM and 121.5 nM respectively (p < 0.0001). In particular of interest, among the 63 isolates that were in vitro resistant to chloroquine (IC50 > 100 nM) at 10% O2, 17 were sensitive to chloroquine (IC50 < 100 nM) at 21% O2. CONCLUSION: Based on these results, laboratories should use the same gas mixture to realize isotopic microtest. Further studies on comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol to survey malaria drug resistance.