Lipid droplet dynamics are essential for the development of the malaria parasite Plasmodium falciparum.

Lipid droplets (LDs) are organelles central to lipid and energy homeostasis across all eukaryotes. In the malaria-causing parasite Plasmodium falciparum the roles of LDs in lipid acquisition from its host cells and their metabolism are poorly understood, despite the high demand for lipids in parasite membrane synthesis. We systematically characterised LD size, composition and dynamics across the disease-causing blood infection. Applying split fluorescence emission analysis and 3D Focused Ion Beam-Scanning Electron Microscopy, we observed a decrease in LD size in late schizont stages. LD contraction likely signifies a switch from lipid accumulation to lipid utilisation in preparation for parasite egress from host red blood cells. We demonstrate connections between LDs and several parasite organelles, pointing to potential functional interactions. Chemical inhibition of triacylglyerol (TAG) synthesis or break-down revealed essential LD functions for schizogony and in counteracting lipid toxicity. The dynamics of lipid synthesis, storage and utilisation in P. falciparum LDs might provide a target for new anti-malarial intervention strategies.

Harnessing cholesterol uptake of malaria parasites for therapeutic applications.

Parasites, such as the malaria parasite P. falciparum, are critically dependent on host nutrients. Interference with nutrient uptake can lead to parasite death and, therefore, serve as a successful treatment strategy. P. falciparum parasites cannot synthesise cholesterol, and instead source this lipid from the host. Here, we tested whether cholesterol uptake pathways could be 'hijacked' for optimal drug delivery to the intracellular parasite. We found that fluorescent cholesterol analogues were delivered from the extracellular environment to the intracellular parasite. We investigated the uptake and inhibitory effects of conjugate compounds, where proven antimalarial drugs (primaquine and artesunate) were attached to steroids that mimic the structure of cholesterol. These conjugated antimalarial drugs improved the inhibitory effects against multiple parasite lifecycle stages, multiple parasite species, and drug-resistant parasites, whilst also lowering the toxicity to human host cells. Steroids with introduced peroxides also displayed antimalarial activity. These results provide a proof-of-concept that cholesterol mimics can be developed as a drug delivery system against apicomplexan parasites with the potential to improve drug efficacy, increase therapeutic index, and defeat drug resistance.

Curiosities take the stage - role-play in parasitology teaching.

Careful observation of parasites, masters of camouflage, reveals an ingenious and fascinating world. However, students often perceive parasitology as impenetrable. What if a flamboyant flea circus director passionately introduced the multidimensional contexts of this discipline? Will role-play capture the imagination of students and guide them in their future learning?

Correction: A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites.

[This corrects the article DOI: 10.1371/journal.ppat.1011517.].

Analysis of Plasmodium falciparum Mitochondrial Electron Transport Chain Activity Using Seahorse XFe96 Extracellular Flux Assays.

The mitochondrial electron transport chain (ETC) is a multi-component pathway that mediates the transfer of electrons from metabolic reactions that occur in the mitochondrion to molecular oxygen (O2). The ETC contributes to numerous cellular processes, including the generation of cellular ATP through oxidative phosphorylation, serving as an electron sink for metabolic pathways such as de novo pyrimidine biosynthesis and for maintaining mitochondrial membrane potential. Proper functioning of the mitochondrial ETC is necessary for the growth and survival of apicomplexan parasites including Plasmodium falciparum, a causative agent of malaria. The mitochondrial ETC of P. falciparum is an attractive target for antimalarial drugs, due to its essentiality and its differences from the mammalian ETC. To identify novel P. falciparum ETC inhibitors, we have established a real-time assay to assess ETC function, which we describe here. This approach measures the O2 consumption rate (OCR) of permeabilized P. falciparum parasites using a Seahorse XFe96 flux analyzer and can be used to screen compound libraries for the identification of ETC inhibitors and, in part, to determine the targets of those inhibitors. Key features • With this protocol, the effects of candidate inhibitors on mitochondrial O2 consumption in permeabilized asexual P. falciparum parasites can be tested in real time. • Through the sequential injection of inhibitors and substrates into the assay, the molecular targets of candidate inhibitors in the ETC can, in part, be determined. • The assay is applicable for both drug discovery approaches and enquiries into a fundamental aspect of parasite mitochondrial biology.

A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites.

Apicomplexans are widespread parasites of humans and other animals, and include the causative agents of malaria (Plasmodium species) and toxoplasmosis (Toxoplasma gondii). Existing anti-apicomplexan therapies are beset with issues around drug resistance and toxicity, and new treatment options are needed. The mitochondrial electron transport chain (ETC) is one of the few processes that has been validated as a drug target in apicomplexans. To identify new inhibitors of the apicomplexan ETC, we developed a Seahorse XFe96 flux analyzer approach to screen the 400 compounds contained within the Medicines for Malaria Venture 'Pathogen Box' for ETC inhibition. We identified six chemically diverse, on-target inhibitors of the ETC in T. gondii, at least four of which also target the ETC of Plasmodium falciparum. Two of the identified compounds (MMV024937 and MMV688853) represent novel ETC inhibitor chemotypes. MMV688853 belongs to a compound class, the aminopyrazole carboxamides, that were shown previously to target a kinase with a key role in parasite invasion of host cells. Our data therefore reveal that MMV688853 has dual targets in apicomplexans. We further developed our approach to pinpoint the molecular targets of these inhibitors, demonstrating that all target Complex III of the ETC, with MMV688853 targeting the ubiquinone reduction (Qi) site of the complex. Most of the compounds we identified remain effective inhibitors of parasites that are resistant to Complex III inhibitors that are in clinical use or development, indicating that they could be used in treating drug resistant parasites. In sum, we have developed a versatile, scalable approach to screen for compounds that target the ETC in apicomplexan parasites, and used this to identify and characterize novel inhibitors.

Plasmodium falciparum gametocytes display global chromatin remodelling during sexual differentiation.

BACKGROUND: The protozoan malaria parasite Plasmodium falciparum has a complex life cycle during which it needs to differentiate into multiple morphologically distinct life forms. A key process for transmission of the disease is the development of male and female gametocytes in the human blood, yet the mechanisms determining sexual dimorphism in these haploid, genetically identical sexual precursor cells remain largely unknown. To understand the epigenetic program underlying the differentiation of male and female gametocytes, we separated the two sexual forms by flow cytometry and performed RNAseq as well as comprehensive ChIPseq profiling of several histone variants and modifications. RESULTS: We show that in female gametocytes the chromatin landscape is globally remodelled with respect to genome-wide patterns and combinatorial usage of histone variants and histone modifications. We identified sex specific differences in heterochromatin distribution, implicating exported proteins and ncRNAs in sex determination. Specifically in female gametocytes, the histone variants H2A.Z/H2B.Z were highly enriched in H3K9me3-associated heterochromatin. H3K27ac occupancy correlated with stage-specific gene expression, but in contrast to asexual parasites this was unlinked to H3K4me3 co-occupancy at promoters in female gametocytes. CONCLUSIONS: Collectively, we defined novel combinatorial chromatin states differentially organising the genome in gametocytes and asexual parasites and unravelled fundamental, sex-specific differences in the epigenetic code. Our chromatin maps represent an important resource for future understanding of the mechanisms driving sexual differentiation in P. falciparum.

7-N-Substituted-3-oxadiazole Quinolones with Potent Antimalarial Activity Target the Cytochrome bc1 Complex.

The development of new antimalarials is required because of the threat of resistance to current antimalarial therapies. To discover new antimalarial chemotypes, we screened the Janssen Jumpstarter library against the P. falciparum asexual parasite and identified the 7-N-substituted-3-oxadiazole quinolone hit class. We established the structure-activity relationship and optimized the antimalarial potency. The optimized analog WJM228 (17) showed robust metabolic stability in vitro, although the aqueous solubility was limited. Forward genetic resistance studies uncovered that WJM228 targets the Qo site of cytochrome b (cyt b), an important component of the mitochondrial electron transport chain (ETC) that is essential for pyrimidine biosynthesis and an established antimalarial target. Profiling against drug-resistant parasites confirmed that WJM228 confers resistance to the Qo site but not Qi site mutations, and in a biosensor assay, it was shown to impact the ETC via inhibition of cyt b. Consistent with other cyt b targeted antimalarials, WJM228 prevented pre-erythrocytic parasite and male gamete development and reduced asexual parasitemia in a P. berghei mouse model of malaria. Correcting the limited aqueous solubility and the high susceptibility to cyt b Qo site resistant parasites found in the clinic will be major obstacles in the future development of the 3-oxadiazole quinolone antimalarial class.

The enemy within: lipid asymmetry in intracellular parasite-host interactions.

Eukaryotic pathogens with an intracellular parasitic lifestyle are shielded from extracellular threats during replication and growth. In addition to many nutrients, parasites scavenge host cell lipids to establish complex membrane structures inside their host cells. To counteract the disturbance of the host cell plasma membrane they have evolved strategies to regulate phospholipid asymmetry. In this review, the function and importance of lipid asymmetry in the interactions of intracellular protozoan parasites with the target and immune cells of the host are highlighted. The malaria parasite Plasmodium infects red blood cells and extensively refurbishes these terminally differentiated cells. Cholesterol depletion and an altered intracellular calcium ion homeostasis can lead to disruption in erythrocyte membrane asymmetry and increased exposure of phosphatidylserine (PS). Binding to the PS receptor on monocytes and macrophages results in phagocytosis and destruction of infected erythrocytes. Leishmania parasites display apoptotic mimicry by actively enhancing PS exposure on their surface to trigger increased infection of macrophages. In extracellular Toxoplasma gondii a P4-type ATPase/CDC50 co-chaperone pair functions as a flippase important for exocytosis of specialised secretory organelles. Identification and functional analysis of parasite lipid-translocating proteins, i.e. flippases, floppases, and scramblases, will be central for the recognition of the molecular mechanisms of parasite/host interactions. Ultimately, a better understanding of parasitic diseases, host immunity, and immune escape by parasites require more research on the dynamics of phospholipid bilayers of parasites and the infected host cell.

The role of cholesterol in invasion and growth of malaria parasites.

Malaria parasites are unicellular eukaryotic pathogens that develop through a complex lifecycle involving two hosts, an anopheline mosquito and a vertebrate host. Throughout this lifecycle, the parasite encounters widely differing conditions and survives in distinct ways, from an intracellular lifestyle in the vertebrate host to exclusively extracellular stages in the mosquito. Although the parasite relies on cholesterol for its growth, the parasite has an ambiguous relationship with cholesterol: cholesterol is required for invasion of host cells by the parasite, including hepatocytes and erythrocytes, and for the development of the parasites in those cells. However, the parasite is unable to produce cholesterol itself and appears to remove cholesterol actively from its own plasma membrane, thereby setting up a cholesterol gradient inside the infected host erythrocyte. Overall a picture emerges in which the parasite relies on host cholesterol and carefully controls its transport. Here, we describe the role of cholesterol at the different lifecycle stages of the parasites.

Pas-de-deux: African Plasmodium falciparum adaptations to sickle hemoglobin.

The molecular arms race between humans and Plasmodium falciparum in Africa resulted in selection of sickle-cell disease, which, on balance, protects heterozygote carriers against severe malaria. Band et al. discovered that parasites counter-adapt and can overcome disease resistance by identifying parasite genome signatures, termed P. falciparum sickle-associated (Pfsa) variants.

Analysis of sex-specific lipid metabolism of Plasmodium falciparum points to the importance of sphingomyelin for gametocytogenesis.

Male and female Plasmodium falciparum gametocytes are the parasite lifecycle stage responsible for transmission of malaria from the human host to the mosquito vector. Not only are gametocytes able to survive in radically different host environments, but they are also precursors for male and female gametes that reproduce sexually soon after ingestion by the mosquito. Here, we investigate the sex-specific lipid metabolism of gametocytes within their host red blood cell. Comparison of the male and female lipidome identifies cholesteryl esters and dihydrosphingomyelin enrichment in female gametocytes. Chemical inhibition of each of these lipid types in mature gametocytes suggests dihydrosphingomyelin synthesis but not cholesteryl ester synthesis is important for gametocyte viability. Genetic disruption of each of the two sphingomyelin synthase genes points towards sphingomyelin synthesis contributing to gametocytogenesis. This study shows that gametocytes are distinct from asexual stages, and that the lipid composition is also vastly different between male and female gametocytes, reflecting the different cellular roles these stages play. Taken together, our results highlight the sex-specific nature of gametocyte lipid metabolism, which has the potential to be targeted to block malaria transmission. This article has an associated First Person interview with the first author of the paper.

Role of the J Domain Protein Family in the Survival and Pathogenesis of Plasmodium falciparum.

Plasmodium falciparum has dedicated an unusually large proportion of its genome to molecular chaperones (2% of all genes), with the heat shock protein 40 (Hsp40) family (now called J domain proteins, JDPs) exhibiting evolutionary radiation into 49 members. A large number of the P. falciparum JDPs (PfJDPs) are predicted to be exported, with certain members shown experimentally to be present in the erythrocyte cytosol (PFA0660w and PFE0055c) or erythrocyte membrane (ring-infected erythrocyte surface antigen, RESA). PFA0660w and PFE0055c are associated with an exported plasmodial Hsp70 (PfHsp70-x) within novel mobile structures called J-dots, which have been proposed to be dedicated to the trafficking of key membrane proteins such as erythrocyte membrane protein 1 (PfEMP1). Well over half of the PfJDPs appear to be essential, including the J-dot PfJDP, PFE0055c, while others have been found to be required for growth under febrile conditions (e.g. PFA0110w, the ring-infected erythrocyte surface antigen protein [RESA]) or involved in pathogenesis (e.g. PF10_0381 has been shown to be important for protrusions of the infected red blood cell membrane, the so-called knobs). Here we review what is known about those PfJDPs that have been well characterised, and may be directly or indirectly involved in the survival and pathogenesis of the malaria parasite.

Sex-specific Separation of Plasmodium falciparum Gametocyte Populations.

Plasmodium falciparum is a unicellular eukaryotic parasite that causes malaria in humans. The parasite is spread by Anopheles mosquitoes after ingestion of sexual stage parasites known as gametocytes. Malaria transmission depends on parasites switching from the disease-causing asexual blood forms to male and female gametocytes. The current protocol allows the simultaneous isolation of male and female parasites from the same population to study this critical lifecycle stage in a sex-specific manner. We have generated a transgenic P. falciparum cell line that expresses a GFP-tagged parasite protein in female, but not male, parasites. Gametocyte production is stress induced and, through a series of steps, sexual stage parasites are enriched relative to uninfected red blood cells or red blood cells infected with asexual stage parasites. Finally, male and female gametocytes are separated by fluorescence-activated cell sorting. This protocol allows for the separation of up to 12 million live male and female parasites from the same population, which are amenable to further analysis.

Of membranes and malaria: phospholipid asymmetry in Plasmodium falciparum-infected red blood cells.

Malaria is a vector-borne parasitic disease with a vast impact on human history, and according to the World Health Organisation, Plasmodium parasites still infect over 200 million people per year. Plasmodium falciparum, the deadliest parasite species, has a remarkable ability to undermine the host immune system and cause life-threatening disease during blood infection. The parasite's host cells, red blood cells (RBCs), generally maintain an asymmetric distribution of phospholipids in the two leaflets of the plasma membrane bilayer. Alterations to this asymmetry, particularly the exposure of phosphatidylserine (PS) in the outer leaflet, can be recognised by phagocytes. Because of the importance of innate immune defence numerous studies have investigated PS exposure in RBCs infected with P. falciparum, but have reached different conclusions. Here we review recent advancements in our understanding of the molecular mechanisms which regulate asymmetry in RBCs, and whether infection with the P. falciparum parasite results in changes to PS exposure. On the balance of evidence, it is likely that membrane asymmetry is disrupted in parasitised RBCs, though some methodological issues need addressing. We discuss the potential causes and consequences of altered asymmetry in parasitised RBCs, particularly for in vivo interactions with the immune system, and the role of host-parasite co-evolution. We also examine the potential asymmetric state of parasite membranes and summarise current knowledge on the parasite proteins, which could regulate asymmetry in these membranes. Finally, we highlight unresolved questions at this time and the need for interdisciplinary approaches to uncover the machinery which enables P. falciparum parasites to hide in mature erythrocytes.

Breakdown in membrane asymmetry regulation leads to monocyte recognition of P. falciparum-infected red blood cells.

The human malaria parasite Plasmodium falciparum relies on lipids to survive; this makes its lipid metabolism an attractive drug target. The lipid phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell membrane (RBC) bilayer; however, some studies suggest that infection with the intracellular parasite results in the presence of this lipid in the RBC membrane outer leaflet, where it could act as a recognition signal to phagocytes. Here, we used fluorescent lipid analogues and probes to investigate the enzymatic reactions responsible for maintaining asymmetry between membrane leaflets, and found that in parasitised RBCs the maintenance of membrane asymmetry was partly disrupted, and PS was increased in the outer leaflet. We examined the underlying causes for the differences between uninfected and infected RBCs using fluorescent dyes and probes, and found that calcium levels increased in the infected RBC cytoplasm, whereas membrane cholesterol was depleted from the erythrocyte plasma membrane. We explored the resulting effect of PS exposure on enhanced phagocytosis by monocytes, and show that infected RBCs must expend energy to limit phagocyte recognition, and provide experimental evidence that PS exposure contributes to phagocytic recognition of P. falciparum-infected RBCs. Together, these findings underscore the pivotal role for PS exposure on the surface of Plasmodium falciparum-infected erythrocytes for in vivo interactions with the host immune system, and provide a rationale for targeted antimalarial drug design.

An apicoplast-resident folate transporter is essential for sporogony of malaria parasites.

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co-factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2-deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single-membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2-deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.

Novel Method for the Separation of Male and Female Gametocytes of the Malaria Parasite Plasmodium falciparum That Enables Biological and Drug Discovery.

We developed a flow-cytometry-based method to separate and collect cocultured male and female Plasmodium falciparum gametocytes responsible for malaria transmission. The purity of the collected cells was estimated at >97% using flow cytometry, and sorted cells were observed by Giemsa-stained thin-smear and live-cell fluorescence microscopy. The expression of validated sex-specific markers corroborated the sorting strategy. Collected male and female gametocytes were used to confirm three novel sex-specific markers by quantitative real-time PCR that were more enriched in sorted male and female gametocyte populations than existing sex-specific markers. We also applied the method as a proof-of-principle drug screen that allows the identification of drugs that kill gametocytes in a sex-specific manner. Since the developed method allowed for the separation of male and female parasites from the same culture, we observed for the first time a difference in development time between the sexes: females developed faster than males. Hence, the ability to separate male and female gametocytes opens the door to a new field of sex-specific P. falciparum gametocyte biology to further our understanding of malaria transmission.IMPORTANCE The protozoan Plasmodium falciparum causes the most severe form of human malaria. The development of sexual forms (so-called gametocytes) is crucial for disease transmission. However, knowledge of these forms is severely hampered by the paucity of sex-specific markers and the inability to extract single sex gametocytes in high purity. Moreover, the identification of compounds that specifically affect one sex is difficult due to the female bias of the gametocytes. We have developed a system that allows for the separation of male and female gametocytes from the same population. Applying our system, we show that male and female parasites mature at different rates, which might have implications for transmission. We also identified new sex-specific genes that can be used as sex markers or to unravel sex-specific functions. Our system will not only aid in the discovery of much needed gametocidal compounds, but it also represents a valuable tool for exploring malaria transmission biology.

Distinct adaptations of a gametocyte ABC transporter to murine and human Plasmodium parasites and its incompatibility in cross-species complementation.

Parasites of the genus Plasmodium infect a wide range of mammalian hosts including humans, primates, bats and arboreal rodents. A hallmark of Plasmodium spp. is the very narrow host range, indicative of matching parasite-host coevolution. Accordingly, their respective genomes harbour many unique genes and gene families that typically encode proteins involved in host cell recognition and remodelling. Whether and to what extent conserved proteins that are shared across Plasmodium spp. also exert distinct species-specific roles remains largely untested. Here, we present detailed functional profiling of the female gametocyte-specific ATP-binding cassette transporter gABCG2 in the murine parasite Plasmodium berghei and compare our findings with data from the orthologous gene in the human parasite Plasmodium falciparum. We show that P. berghei gABCG2 is female-specific and continues to be expressed in zygotes and ookinetes. In contrast to a distinct localization to Iipid-rich gametocyte-specific spots as observed in P. falciparum, the murine malaria parasite homolog is found at the parasite plasma membrane. Plasmodium berghei lacking gABCG2 displays fast asexual blood-stage replication and increased proportions of female gametocytes, consistent with the corresponding P. falciparum knock-out phenotype. Strikingly, cross-species replacement of gABCG2 in either the murine or the human parasite did not restore normal growth rates. The lack of successful complementation despite high conservation across Plasmodium spp. is an indicator of distinct adaptations and tight parasite-host coevolution. Hence, incompatibility of conserved genes in closely related Plasmodium spp. might be more common than previously anticipated.