The Kelch13 compartment contains highly divergent vesicle trafficking proteins in malaria parasites.

Single amino acid changes in the parasite protein Kelch13 (K13) result in reduced susceptibility of P. falciparum parasites to artemisinin and its derivatives (ART). Recent work indicated that K13 and other proteins co-localising with K13 (K13 compartment proteins) are involved in the endocytic uptake of host cell cytosol (HCCU) and that a reduction in HCCU results in reduced susceptibility to ART. HCCU is critical for parasite survival but is poorly understood, with the K13 compartment proteins among the few proteins so far functionally linked to this process. Here we further defined the composition of the K13 compartment by analysing more hits from a previous BioID, showing that MyoF and MCA2 as well as Kelch13 interaction candidate (KIC) 11 and 12 are found at this site. Functional analyses, tests for ART susceptibility as well as comparisons of structural similarities using AlphaFold2 predictions of these and previously identified proteins showed that vesicle trafficking and endocytosis domains were frequent in proteins involved in resistance or endocytosis (or both), comprising one group of K13 compartment proteins. While this strengthened the link of the K13 compartment to endocytosis, many proteins of this group showed unusual domain combinations and large parasite-specific regions, indicating a high level of taxon-specific adaptation of this process. Another group of K13 compartment proteins did not influence endocytosis or ART susceptibility and lacked detectable vesicle trafficking domains. We here identified the first protein of this group that is important for asexual blood stage development and showed that it likely is involved in invasion. Overall, this work identified novel proteins functioning in endocytosis and at the K13 compartment. Together with comparisons of structural predictions it provides a repertoire of functional domains at the K13 compartment that indicate a high level of adaption of endocytosis in malaria parasites.

Critical interdependencies between Plasmodium nutrient flux and drugs.

Nutrient import and waste efflux are critical dependencies for intracellular Plasmodium falciparum parasites. Nutrient transport proteins are often lineage specific and can provide unique targets for antimalarial drug development. P. falciparum nutrient transport pathways can be a double-edged sword for the parasite, not only mediating the import of nutrients and excretion of waste products but also providing an access route for drugs. Here we briefly summarise the nutrient acquisition pathways of intracellular P. falciparum blood-stage parasites and then highlight how these pathways influence many aspects relevant to antimalarial drugs, resulting in complex and often underappreciated interdependencies.

The Plasmodium Lactate/H+ Transporter PfFNT Is Essential and Druggable In Vivo.

Malaria parasites in the blood stage express a single transmembrane transport protein for the release of the glycolytic end product l-lactate/H+ from the cell. This transporter is a member of the strictly microbial formate-nitrite transporter (FNT) family and a novel putative drug target. Small, drug-like FNT inhibitors potently block lactate transport and kill Plasmodium falciparum parasites in culture. The protein structure of Plasmodium falciparum FNT (PfFNT) in complex with the inhibitor has been resolved and confirms its previously predicted binding site and its mode of action as a substrate analog. Here, we investigated the mutational plasticity and essentiality of the PfFNT target on a genetic level, and established its in vivo druggability using mouse malaria models. We found that, besides a previously identified PfFNT G107S resistance mutation, selection of parasites at 3 × IC50 (50% inhibitory concentration) gave rise to two new point mutations affecting inhibitor binding: G21E and V196L. Conditional knockout and mutation of the PfFNT gene showed essentiality in the blood stage, whereas no phenotypic defects in sexual development were observed. PfFNT inhibitors mainly targeted the trophozoite stage and exhibited high potency in P. berghei- and P. falciparum-infected mice. Their in vivo activity profiles were comparable to that of artesunate, demonstrating strong potential for the further development of PfFNT inhibitors as novel antimalarials.

Gene-by-gene screen of the unknown proteins encoded on Plasmodium falciparum chromosome 3.

Taxon-specific proteins are key determinants defining the biology of all organisms and represent prime drug targets in pathogens. However, lacking comparability with proteins in other lineages makes them particularly difficult to study. In malaria parasites, this is exacerbated by technical limitations. Here, we analyzed the cellular location, essentiality, function, and, in selected cases, interactome of all unknown non-secretory proteins encoded on an entire P. falciparum chromosome. The nucleus was the most common localization, indicating that it is a hotspot of parasite-specific biology. More in-depth functional studies with four proteins revealed essential roles in DNA replication and mitosis. The mitosis proteins defined a possible orphan complex and a highly diverged complex needed for spindle-kinetochore connection. Structure-function comparisons indicated that the taxon-specific proteins evolved by different mechanisms. This work demonstrates the feasibility of gene-by-gene screens to elucidate the biology of malaria parasites and reveal critical parasite-specific processes of interest as drug targets.

Clearing of hemozoin crystals in malaria parasites enables whole-cell STED microscopy.

Malaria is a devastating mosquito-borne parasitic disease that manifests when Plasmodium parasites replicate within red blood cells. During the development within the red blood cell, the parasite digests hemoglobin and crystalizes the otherwise toxic heme. The resulting hemozoin crystals limit imaging by STED nanoscopy owing to their high light-absorbing capacity, which leads to immediate cell destruction upon contact with the laser. Here, we establish CUBIC-P-based clearing of hemozoin crystals, enabling whole-cell STED nanoscopy of parasites within red blood cells. Hemozoin-cleared infected red blood cells could reliably be stained with antibodies, and hence proteins in the hemozoin-containing digestive vacuole membrane, as well as in secretory vesicles of gametocytes, could be imaged at high resolution. Thus, this process is a valuable tool to study and understand parasite biology and the potential molecular mechanisms mediating drug resistance. This article has an associated First Person interview with the first author of the paper.

PMRT1, a Plasmodium-Specific Parasite Plasma Membrane Transporter, Is Essential for Asexual and Sexual Blood Stage Development.

Membrane transport proteins perform crucial roles in cell physiology. The obligate intracellular parasite Plasmodium falciparum, an agent of human malaria, relies on membrane transport proteins for the uptake of nutrients from the host, disposal of metabolic waste, exchange of metabolites between organelles, and generation and maintenance of transmembrane electrochemical gradients for its growth and replication within human erythrocytes. Despite their importance for Plasmodium cellular physiology, the functional roles of a number of membrane transport proteins remain unclear, which is particularly true for orphan membrane transporters that have no or limited sequence homology to transporter proteins in other evolutionary lineages. Therefore, in the current study, we applied endogenous tagging, targeted gene disruption, conditional knockdown, and knockout approaches to investigate the subcellular localization and essentiality of six membrane transporters during intraerythrocytic development of P. falciparum parasites. They are localized at different subcellular structures-the food vacuole, the apicoplast, and the parasite plasma membrane-and four out of the six membrane transporters are essential during asexual development. Additionally, the plasma membrane resident transporter 1 (PMRT1; PF3D7_1135300), a unique Plasmodium-specific plasma membrane transporter, was shown to be essential for gametocytogenesis and functionally conserved within the genus Plasmodium. Overall, we reveal the importance of four orphan transporters to blood stage P. falciparum development, which have diverse intracellular localizations and putative functions. IMPORTANCE Plasmodium falciparum-infected erythrocytes possess multiple compartments with designated membranes. Transporter proteins embedded in these membranes not only facilitate movement of nutrients, metabolites, and other molecules between these compartments, but also are common therapeutic targets and can confer antimalarial drug resistance. Orphan membrane transporters in P. falciparum without sequence homology to transporters in other evolutionary lineages and divergent from host transporters may constitute attractive targets for novel intervention approaches. Here, we localized six of these putative transporters at different subcellular compartments and probed their importance during asexual parasite growth by using reverse genetic approaches. In total, only two candidates turned out to be dispensable for the parasite, highlighting four candidates as putative targets for therapeutic interventions. This study reveals the importance of several orphan transporters to blood stage P. falciparum development.

Proximity-dependent biotinylation approaches to study apicomplexan biology.

In the last 10 years, proximity-dependent biotinylation (PDB) techniques greatly expanded the ability to study protein environments in the living cell that range from specific protein complexes to entire compartments. This is achieved by using enzymes such as BirA* and APEX that are fused to proteins of interest and biotinylate proteins in their proximity. PDB techniques are now also increasingly used in apicomplexan parasites. In this review, we first give an overview of the main PDB approaches and how they compare with other techniques that address similar questions. PDB is particularly valuable to detect weak or transient protein associations under physiological conditions and to study cellular structures that are difficult to purify or have a poorly understood protein composition. We also highlight new developments such as novel smaller or faster-acting enzyme variants and conditional PDB approaches, providing improvements in both temporal and spatial resolution which may offer broader application possibilities useful in apicomplexan research. In the second part, we review work using PDB techniques in apicomplexan parasites and how this expanded our knowledge about these medically important parasites.

The parasitophorous vacuole nutrient channel is critical for drug access in malaria parasites and modulates the artemisinin resistance fitness cost.

Intraerythrocytic malaria parasites proliferate bounded by a parasitophorous vacuolar membrane (PVM). The PVM contains nutrient permeable channels (NPCs) conductive to small molecules, but their relevance for parasite growth for individual metabolites is largely untested. Here we show that growth-relevant levels of major carbon and energy sources pass through the NPCs. Moreover, we find that NPCs are a gate for several antimalarial drugs, highlighting their permeability properties as a critical factor for drug design. Looking into NPC-dependent amino acid transport, we find that amino acid shortage is a reason for the fitness cost in artemisinin-resistant (ARTR) parasites and provide evidence that NPC upregulation to increase amino acids acquisition is a mechanism of ARTR parasites in vitro and in human infections to compensate this fitness cost. Hence, the NPCs are important for nutrient and drug access and reveal amino acid deprivation as a critical constraint in ARTR parasites.

Characterization of Apicomplexan Amino Acid Transporters (ApiATs) in the Malaria Parasite Plasmodium falciparum.

During the symptomatic human blood phase, malaria parasites replicate within red blood cells. Parasite proliferation relies on the uptake of nutrients, such as amino acids, from the host cell and blood plasma, requiring transport across multiple membranes. Amino acids are delivered to the parasite through the parasite-surrounding vacuolar compartment by specialized nutrient-permeable channels of the erythrocyte membrane and the parasitophorous vacuole membrane (PVM). However, further transport of amino acids across the parasite plasma membrane (PPM) is currently not well characterized. In this study, we focused on a family of Apicomplexan amino acid transporters (ApiATs) that comprises five members in Plasmodium falciparum. First, we localized four of the P. falciparum ApiATs (PfApiATs) at the PPM using endogenous green fluorescent protein (GFP) tagging. Next, we applied reverse genetic approaches to probe into their essentiality during asexual replication and gametocytogenesis. Upon inducible knockdown and targeted gene disruption, a reduced asexual parasite proliferation was detected for PfApiAT2 and PfApiAT4. Functional inactivation of individual PfApiATs targeted in this study had no effect on gametocyte development. Our data suggest that individual PfApiATs are partially redundant during asexual in vitro proliferation and fully redundant during gametocytogenesis of P. falciparum parasites. IMPORTANCE Malaria parasites live and multiply inside cells. To facilitate their extremely fast intracellular proliferation, they hijack and transform their host cells. This also requires the active uptake of nutrients, such as amino acids, from the host cell and the surrounding environment through various membranes that are the consequence of the parasite's intracellular lifestyle. In this paper, we focus on a family of putative amino acid transporters termed ApiAT. We show expression and localization of four transporters in the parasite plasma membrane of Plasmodium falciparum-infected erythrocytes that represent one interface of the pathogen to its host cell. We probed into the impact of functional inactivation of individual transporters on parasite growth in asexual and sexual blood stages of P. falciparum and reveal that only two of them show a modest but significant reduction in parasite proliferation but no impact on gametocytogenesis, pointing toward dispensability within this transporter family.

The newly discovered role of endocytosis in artemisinin resistance.

Artemisinin and its derivatives (ART) are the cornerstone of malaria treatment as part of artemisinin combination therapy (ACT). However, reduced susceptibility to artemisinin as well as its partner drugs threatens the usefulness of ACTs. Single point mutations in the parasite protein Kelch13 (K13) are necessary and sufficient for the reduced sensitivity of malaria parasites to ART but several alternative mechanisms for this resistance have been proposed. Recent work found that K13 is involved in the endocytosis of host cell cytosol and indicated that this is the process responsible for resistance in parasites with mutated K13. These studies also identified a series of further proteins that act together with K13 in the same pathway, including previously suspected resistance proteins such as UBP1 and AP-2µ. Here, we give a brief overview of artemisinin resistance, present the recent evidence of the role of endocytosis in ART resistance and discuss previous hypotheses in light of this new evidence. We also give an outlook on how the new insights might affect future research.

Identification of novel inner membrane complex and apical annuli proteins of the malaria parasite Plasmodium falciparum.

The inner membrane complex (IMC) is a defining feature of apicomplexan parasites, which confers stability and shape to the cell, functions as a scaffolding compartment during the formation of daughter cells and plays an important role in motility and invasion during different life cycle stages of these single-celled organisms. To explore the IMC proteome of the malaria parasite Plasmodium falciparum we applied a proximity-dependent biotin identification (BioID)-based proteomics approach, using the established IMC marker protein Photosensitized INA-Labelled protein 1 (PhIL1) as bait in asexual blood-stage parasites. Subsequent mass spectrometry-based peptide identification revealed enrichment of 12 known IMC proteins and several uncharacterized candidate proteins. We validated nine of these previously uncharacterized proteins by endogenous GFP-tagging. Six of these represent new IMC proteins, while three proteins have a distinct apical localization that most likely represents structures described as apical annuli in Toxoplasma gondii. Additionally, various Kelch13 interacting candidates were identified, suggesting an association of the Kelch13 compartment and the IMC in schizont and merozoite stages. This work extends the number of validated IMC proteins in the malaria parasite and reveals for the first time the existence of apical annuli proteins in P. falciparum. Additionally, it provides evidence for a spatial association between the Kelch13 compartment and the IMC in late blood-stage parasites.

Adipose tissue parasite sequestration drives leptin production in mice and correlates with human cerebral malaria.

Circulating levels of the adipokine leptin are linked to neuropathology in experimental cerebral malaria (ECM), but its source and regulation mechanism remain unknown. Here, we show that sequestration of infected red blood cells (iRBCs) in white adipose tissue (WAT) microvasculature increased local vascular permeability and leptin production. Mice infected with parasite strains that fail to sequester in WAT displayed reduced leptin production and protection from ECM. WAT sequestration and leptin induction were lost in CD36KO mice; however, ECM susceptibility revealed sexual dimorphism. Adipocyte leptin was regulated by the mechanistic target of rapamycin complex 1 (mTORC1) and blocked by rapamycin. In humans, although Plasmodium falciparum infection did not increase circulating leptin levels, iRBC sequestration, tissue leptin production, and mTORC1 activity were positively correlated with CM in pediatric postmortem WAT. These data identify WAT sequestration as a trigger for leptin production with potential implications for pathogenesis of malaria infection, prognosis, and treatment.

The ZIP Code of Vesicle Trafficking in Apicomplexa: SEC1/Munc18 and SNARE Proteins.

Apicomplexans are obligate intracellular parasites harboring three sets of unique secretory organelles termed micronemes, rhoptries, and dense granules that are dedicated to the establishment of infection in the host cell. Apicomplexans rely on the endolysosomal system to generate the secretory organelles and to ingest and digest host cell proteins. These parasites also possess a metabolically relevant secondary endosymbiotic organelle, the apicoplast, which relies on vesicular trafficking for correct incorporation of nuclear-encoded proteins into the organelle. Here, we demonstrate that the trafficking and destination of vesicles to the unique and specialized parasite compartments depend on SNARE proteins that interact with tethering factors. Specifically, all secreted proteins depend on the function of SLY1 at the Golgi. In addition to a critical role in trafficking of endocytosed host proteins, TgVps45 is implicated in the biogenesis of the inner membrane complex (alveoli) in both Toxoplasma gondii and Plasmodium falciparum, likely acting in a coordinated manner with Stx16 and Stx6. Finally, Stx12 localizes to the endosomal-like compartment and is involved in the trafficking of proteins to the apical secretory organelles rhoptries and micronemes as well as to the apicoplast.IMPORTANCE The phylum of Apicomplexa groups medically relevant parasites such as those responsible for malaria and toxoplasmosis. As members of the Alveolata superphylum, these protozoans possess specialized organelles in addition to those found in all members of the eukaryotic kingdom. Vesicular trafficking is the major route of communication between membranous organelles. Neither the molecular mechanism that allows communication between organelles nor the vesicular fusion events that underlie it are completely understood in Apicomplexa. Here, we assessed the function of SEC1/Munc18 and SNARE proteins to identify factors involved in the trafficking of vesicles between these various organelles. We show that SEC1/Munc18 in interaction with SNARE proteins allows targeting of vesicles to the inner membrane complex, prerhoptries, micronemes, apicoplast, and vacuolar compartment from the endoplasmic reticulum, Golgi apparatus, or endosomal-like compartment. These data provide an exciting look at the "ZIP code" of vesicular trafficking in apicomplexans, essential for precise organelle biogenesis, homeostasis, and inheritance.

Introduction of Scaffold Nitrogen Atoms Renders Inhibitors of the Malarial l-Lactate Transporter, PfFNT, Effective against the Gly107Ser Resistance Mutation.

The spreading of malaria parasites, Plasmodium falciparum, with resistance to all known drugs calls for novel classes of inhibitors with new modes of action. Recently, we discovered and validated the plasmodial l-lactate transporter, PfFNT, as a novel antimalarial drug target. However, treatment of parasites with a screening hit from the malaria box compound collection, MMV007839, gave rise to a PfFNT Gly107Ser resistance mutation decreasing inhibitor affinity by 2 orders of magnitude. Here, we show that newly introduced nitrogen atoms into the inhibitor scaffold can act as hydrogen bond acceptor sites to the serine hydroxyl. The gain in affinity led to almost equal inhibition of wildtype PfFNT and the Gly107Ser mutation. The most potent inhibitor of this work, BH267.meta, killed cultured P. falciparum parasites with nanomolar efficacy and did not give rise to new resistance formation in vitro. Its deduced pharmacokinetic properties appear suitable for further drug development.

Endocytosis in Plasmodium and Toxoplasma Parasites.

Endocytosis is critical for many functions in eukaryotic cells. Uptake of host cell cytosol, an indispensable endocytic process in malaria blood-stage parasites, has been known for a long time. However, it is only recently that the proteins involved in this process have started to emerge. Unexpectedly, some of these proteins revealed a critical role for endocytosis in artemisinin resistance. More recently, endocytosis was discovered in both intracellular and extracellular Toxoplasma gondii parasites. Here, we review these findings, compare the endolysosomal systems of Toxoplasma and Plasmodium parasites, and present current knowledge about endocytic mechanisms in apicomplexans.

A Kelch13-defined endocytosis pathway mediates artemisinin resistance in malaria parasites.

Artemisinin and its derivatives (ARTs) are the frontline drugs against malaria, but resistance is jeopardizing their effectiveness. ART resistance is mediated by mutations in the parasite's Kelch13 protein, but Kelch13 function and its role in resistance remain unclear. In this study, we identified proteins located at a Kelch13-defined compartment. Inactivation of eight of these proteins, including Kelch13, rendered parasites resistant to ART, revealing a pathway critical for resistance. Functional analysis showed that these proteins are required for endocytosis of hemoglobin from the host cell. Parasites with inactivated Kelch13 or a resistance-conferring Kelch13 mutation displayed reduced hemoglobin endocytosis. ARTs are activated by degradation products of hemoglobin. Hence, reduced activity of Kelch13 and its interactors diminishes hemoglobin endocytosis and thereby ART activation, resulting in parasite resistance.

Epigenetic reader complexes of the human malaria parasite, Plasmodium falciparum.

Epigenetic regulatory mechanisms are central to the development and survival of all eukaryotic organisms. These mechanisms critically depend on the marking of chromatin domains with distinctive histone tail modifications (PTMs) and their recognition by effector protein complexes. Here we used quantitative proteomic approaches to unveil interactions between PTMs and associated reader protein complexes of Plasmodium falciparum, a unicellular parasite causing malaria. Histone peptide pull-downs with the most prominent and/or parasite-specific PTMs revealed the binding preference for 14 putative and novel reader proteins. Amongst others, they highlighted the acetylation-level-dependent recruitment of the BDP1/BDP2 complex and identified an PhD-finger protein (PHD 1, PF3D7_1008100) that could mediate a cross-talk between H3K4me2/3 and H3K9ac marks. Tagging and interaction proteomics of 12 identified proteins unveiled the composition of 5 major epigenetic complexes, including the elusive TBP-associated-factor complex as well as two distinct GCN5/ADA2 complexes. Furthermore, it has highlighted a remarkable degree of interaction between these five (sub)complexes. Collectively, this study provides an extensive inventory of PTM-reader interactions and composition of epigenetic complexes. It will not only fuel further explorations of gene regulation amongst ancient eukaryotes, but also provides a stepping stone for exploration of PTM-reader interactions for antimalarial drug development.

EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites.

Intracellular malaria parasites grow in a vacuole delimited by the parasitophorous vacuolar membrane (PVM). This membrane fulfils critical roles for survival of the parasite in its intracellular niche such as in protein export and nutrient acquisition. Using a conditional knockout (KO), we here demonstrate that the abundant integral PVM protein exported protein 1 (EXP1) is essential for parasite survival but that this is independent of its previously postulated function as a glutathione S-transferase (GST). Patch-clamp experiments indicated that EXP1 is critical for the nutrient-permeable channel activity at the PVM. Loss of EXP1 abolished the correct localisation of EXP2, a pore-forming protein required for the nutrient-permeable channel activity and protein export at the PVM. Unexpectedly, loss of EXP1 affected only the nutrient-permeable channel activity of the PVM but not protein export. Parasites with low levels of EXP1 became hypersensitive to low nutrient conditions, indicating that EXP1 indeed is needed for nutrient uptake and experimentally confirming the long-standing hypothesis that the channel activity measured at the PVM is required for parasite nutrient acquisition. Hence, EXP1 is specifically required for the functional expression of EXP2 as the nutrient-permeable channel and is critical for the metabolite supply of malaria parasites.

Delayed death in the malaria parasite Plasmodium falciparum is caused by disruption of prenylation-dependent intracellular trafficking.

Apicomplexan parasites possess a plastid organelle called the apicoplast. Inhibitors that selectively target apicoplast housekeeping functions, including DNA replication and protein translation, are lethal for the parasite, and several (doxycycline, clindamycin, and azithromycin) are in clinical use as antimalarials. A major limitation of such drugs is that treated parasites only arrest one intraerythrocytic development cycle (approximately 48 hours) after treatment commences, a phenotype known as the 'delayed death' effect. The molecular basis of delayed death is a long-standing mystery in parasitology, and establishing the mechanism would aid rational clinical implementation of apicoplast-targeted drugs. Parasites undergoing delayed death transmit defective apicoplasts to their daughter cells and cannot produce the sole, blood-stage essential metabolic product of the apicoplast: the isoprenoid precursor isopentenyl-pyrophosphate. How the isoprenoid precursor depletion kills the parasite remains unknown. We investigated the requirements for the range of isoprenoids in the human malaria parasite Plasmodium falciparum and characterised the molecular and morphological phenotype of parasites experiencing delayed death. Metabolomic profiling reveals disruption of digestive vacuole function in the absence of apicoplast derived isoprenoids. Three-dimensional electron microscopy reveals digestive vacuole fragmentation and the accumulation of cytostomal invaginations, characteristics common in digestive vacuole disruption. We show that digestive vacuole disruption results from a defect in the trafficking of vesicles to the digestive vacuole. The loss of prenylation of vesicular trafficking proteins abrogates their membrane attachment and function and prevents the parasite from feeding. Our data show that the proximate cause of delayed death is an interruption of protein prenylation and consequent cellular trafficking defects.

PfVPS45 Is Required for Host Cell Cytosol Uptake by Malaria Blood Stage Parasites.

During development in human erythrocytes, the malaria parasite Plasmodium falciparum internalizes a large part of the cellular content of the host cell. The internalized cytosol, consisting largely of hemoglobin, is transported to the parasite's food vacuole where it is degraded, providing nutrients and space for growth. This host cell cytosol uptake (HCCU) is crucial for parasite survival but the parasite proteins mediating this process remain obscure. Here, we identify P. falciparum VPS45 as an essential factor in HCCU. Conditional inactivation of PfVPS45 led to an accumulation of host cell cytosol-filled vesicles within the parasite and inhibited the delivery of hemoglobin to the parasite's digestive vacuole, resulting in arrested parasite growth. A proportion of these HCCU vesicle intermediates was positive for phosphatidylinositol 3-phosphate, suggesting endosomal characteristics. Thus PfVPS45 provides insight into the elusive machinery of the ingestion pathway in a parasite that contains an endolysosomal system heavily repurposed for protein secretion.