Daniel E. Goldberg

The interface between the malaria parasite Plasmodium spp. and its host erythrocyte critically mediates nutrient uptake, lipid exchange, and protein export required for parasite replication and thus pathogenesis. Approximately half of this interface consists of narrow membrane contact sites, a region defined by only 3 to 4 nm of aqueous space between the parasite plasma membrane and the parasitophorous vacuole membrane. Despite its extent, the region remains largely uncharacterized and inaccessible, not least due to its narrowness. The sole identified resident is the Plasmodium falciparum Niemann-Pick Type C1-Related protein, PfNCR1, an essential Patched/NPC1-like cholesterol transporter, suggesting this conserved transporter family has evolved an unconventional adaptation for localizing to membrane contact sites that can reveal a mechanism to access the region. Here, using live-cell microscopy and assessment of cholesterol homeostasis, we identify a helix–linker–helix (HLH) domain as the necessary modification for efficient PfNCR1 localization. We find that correct localization is required for the parasite to maintain low cholesterol levels, connecting targeting to function. Using a glycosylphosphatidylinositol-anchored fluorescent reporter as a minimal membrane-associated protein, we show that the HLH domain is sufficient to drive localization to the narrow membrane contact site region. Mechanistically, we demonstrate that targeting depends on the physicochemical character of the targeting domain rather than a specific primary sequence, identifying amphipathicity as a determining property of the HLH domain. Taken together, we propose physicochemical partitioning as a fundamental principle for protein targeting at the Plasmodium –erythrocyte interface, and for driving direct functional interactions of a Patched/NPC1-like protein with a second membrane.

Malaria blood-stage parasites digest ~80% of host cell hemoglobin within a degradative vacuole, releasing heme that is detoxified by sequestration into hemozoin crystals. Although essential for survival and a validated drug target, the mechanisms of heme biomineralization remain unclear. Here, we study the parasite’s Heme Detoxification Protein (HDP), previously proposed to mediate hemozoin formation, using genetic, microscopic, bioenergetic, and proteomic approaches. Endogenous tagging reveals that HDP localizes to the mitochondrion, not the digestive vacuole. HDP inactivation has no effect on heme biomineralization, but causes mitochondrial depolarization, proguanil hypersensitivity, and developmental arrest, which is rescued by bypassing respiratory-chain-dependent pyrimidine biosynthesis. HDP knockout abolishes mitochondrial electron flow due to loss of complexes III and IV, consistent with impaired mitochondrial protein synthesis. Integration of structural modelling with quantitative proteomics places HDP within the mitoribosomal large subunit. Here, we show that HDP is essential for mitochondrial function and does not contribute to hemozoin formation.

Plasmodium falciparum possesses one of the most AT-rich genomes in nature (80.6%). A consequence is an asparagine-rich proteome. A quarter of P. falciparum proteins possess poly-asparagine repeats that can extend more than 100 residues. The role of these repeats has remained a mystery in the biology of this parasite. Here, we report that the poly-asparagine repeat-containing Nterminus of the histone acetyltransferase PfGCN5 associates with the C-terminal catalytic domain after cleavage in the nucleus. Deletion of the repeat destabilizes the N-terminal polypeptide, leading to impaired parasite development and growth, particularly under stress conditions. Using high-resolution mass spectrometry and western blotting analysis, we uncovered a profound effect of the poly-asparagine repeat on acetylation of histones H3, H3.3, and H4. These findings suggest that the poly-asparagine repeat contributes to PfGCN5 acetyltransferase activity, a role previously attributed solely to its C-terminal domain. This report of a function for a poly-asparagine repeat in P. falciparum expands our understanding of a pervasive characteristic of its proteome.

Mutations in the chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) in the malaria parasite P. falciparum . Mutant PfCRT variants, defined by the K76T substitution, mediate efflux of CQ from the parasite digestive vacuole (DV), the site of drug action. However, these mutations impair the native function of PfCRT, which is export of hemoglobin (Hb)-derived peptides from the DV for downstream utilization as amino acids. Mutations in amino acid transporter 1 (PfAAT1) have also been associated with CQ resistance through population genomics and in vitro genetic crosses. The S258L mutation in Africa and the F313S mutation in Southeast Asia, often accompanied by additional substitutions, are found in near-complete linkage disequilibrium with pfcrt mutations, although the mechanistic basis linking these mutations to PfCRT dysfunction and CQ resistance has remained unclear. Here, we show that PfAAT1 is essential in parasites harboring mutant PfCRT, but largely dispensable in a wild-type PfCRT background. Replacing the mutant pfcrt allele with the wild-type version in otherwise isogenic parasite lines abolished the growth defect associated with pfaat1 knockdown, indicating functional overlap between PfCRT and PfAAT1. Consistent with this, PfAAT1 knockdown results in accumulation of Hb-derived peptides. Field-derived PfAAT1 variants (S258L and F313S) confer a significant fitness advantage over the wild-type allele in the mutant PfCRT background, consistent with a gain-of-function that enhances compensation for impaired PfCRT peptide transport activity. Comparative growth analyses reveal that the F313S mutation imparts a pronounced fitness advantage over the wild-type PfAAT1 under cysteine- and methionine-limiting conditions, providing a functional basis for this gain-of-function phenotype. We find that PfAAT1 mutations do not independently alter CQ sensitivity. Our data suggest that pfaat1 allele selection in the field is driven primarily by epistatic interaction with pfcrt .

The Niemann-Pick Type C1-Related protein of the malaria parasite Plasmodium falciparum , PfNCR1, is a promising anti-malarial drug target facilitating cholesterol homeostasis at the interface of the malaria parasite with its host-red blood cell. PfNCR1 is localized to otherwise functionally uncharacterized regions covering ∼half of the host-parasite interface. These regions are defined by exceptionally narrow membrane contact sites, leaving only ∼3-4 nm vertical aqueous space in between the membranes. Determining the origin and functional consequence of localization to the closely apposed membrane is central for our understanding of PfNCR1 as drug target but also offers a window into the mechanism of the group of homologous proteins, associated with congenital conditions and cancer. Here we define the mechanism of PfNCR1’s membrane contact site localization and its implication for cholesterol transport. We identified a 141 amino acid long (amphipathic) helix - linker - (amphipathic) helix domain (“HLH domain”) unique to Plasmodium spp. , that is necessary for efficient localization of PfNCR1 to the narrow membrane contact sites. Mechanistically, we show that this localization relies on the HLH domain’s physicochemical properties. GPI-anchoring the isolated HLH domain or a version of the HLH domain in which the helices are replaced by the amphipathic helix of human ATG3 are sufficient to target a fluorescent protein to regions of endogenous PfNCR1. Functionally, we demonstrate that the degree of PfNCR1 localization to narrow contact sites qualitatively correlates with its ability to maintain cholesterol homeostasis, linking PfNCR1’s membrane contact to its recognized transport function. Collectively, the results establish the HLH domain as key element for PfNCR1’s localization and effectiveness in cholesterol transport while also opening avenues to probe the narrow membrane contact site regions with engineered proteins.

Artemisinin-based combination therapies (ACTs) remain the mainstay of treatment for Plasmodium falciparum malaria, despite reports of ACT treatment failure. ACTs consist of an artemisinin and a longer-lived partner drug, which is often a quinoline. Given that heme is central to the mechanism of action of artemisinins and some quinolines, we hypothesized that these antimalarials would exhibit strong drug-drug interactions. Previous studies using standard 48 hr or 72 hr assays identified additive to mildly antagonistic interactions between artemisinins and quinolines. Here, we sought to re-evaluate these interactions using a pulsing assay that better mimics the short in vivo half-life of artemisinins. We found that chloroquine (CQ), piperaquine (PPQ), and amodiaquine substantially antagonize dihydroartemisinin (DHA), the active metabolite of artemisinins. CQ-DHA antagonism was notably exacerbated in CQ-resistant parasites, resulting in a superantagonistic phenotype in isobolograms. Further, we found that CQ co-treatment conferred artemisinin resistance to Kelch 13 wild-type parasites in the ring-stage survival assay. Using a small molecule probe (Ac-H-FluNox) to measure chemically reactive heme in live parasites, we determined that quinolines block artemisinin activation by rendering cytosolic heme inert. Finally, we probed beyond traditional ACTs, evaluating interactions of the proposed triple ACT, DHA-PPQ-mefloquine, as well as OZ439-quinoline combinations, which were all found to be antagonistic. Collectively, these in vitro data suggest that peroxide-quinolines may have liabilities as combination therapies.

Artemisinin-based combination therapies (ACTs) remain the mainstay of treatment for Plasmodium falciparum malaria, despite reports of ACT treatment failure. ACTs consist of an artemisinin and a longer-lived partner drug, which is often a quinoline. Given that heme is central to the mechanism of action of artemisinins and some quinolines, we hypothesized that these antimalarials would exhibit strong drug-drug interactions. Previous studies using standard 48 h or 72 h assays identified additive to mildly antagonistic interactions between artemisinins and quinolines. Here, we sought to re-evaluate these interactions using a pulsing assay that better mimics the short in vivo half-life of artemisinins. We found that chloroquine (CQ), piperaquine (PPQ), and amodiaquine substantially antagonize dihydroartemisinin (DHA), the active metabolite of artemisinins. CQ-DHA antagonism was notably exacerbated in CQ-resistant parasites, resulting in a superantagonistic phenotype in isobolograms. Further, we found that CQ co-treatment conferred artemisinin resistance to Kelch 13 wild type parasites in the ring stage survival assay. Using a small molecule probe to measure chemically reactive heme in live parasites, we determined that quinolines block artemisinin activation by rendering cytosolic heme inert. Finally, we probed beyond traditional ACTs, evaluating interactions of the proposed triple ACT, DHA-PPQ-Mefloquine, as well as OZ439-quinoline combinations, which were all found to be antagonistic. Collectively, these in vitro data suggest that peroxide-quinolines may have liabilities as combination therapies.

Throughout their intraerythrocytic development, malaria parasites digest up to 80% of the host cell’s hemoglobin within a specialized degradative compartment known as the digestive vacuole. This process releases heme, which is detoxified by sequestration into bioinert hemozoin crystals. Although heme biomineralization is essential for blood-stage survival and a validated drug target, its underlying mechanisms remain unclear. Initially identified as a potent inducer of β-hematin crystallization in vitro , the parasite’s H eme D etoxification P rotein (HDP) has been proposed to execute a similar role in the formation of hemozoin crystals in cellulo . Here, we investigate the function of HDP in live Plasmodium falciparum parasites, integrating experimental genetic approaches with quantitative microscopy, cellular bioenergetics and whole-proteome profiling. Endogenous tagging revealed that HDP localizes to the mitochondrion rather than the digestive vacuole. Conditional inactivation of HDP resulted in a gradual loss of mitochondrial membrane potential, preceding developmental arrest. Bypassing the essential role of the respiratory chain in pyrimidine biosynthesis – either through exogenous electron acceptors or expression of a ubiquinone-independent dihydroorotate dehydrogenase – rescued HDP-deficient parasites, indicating a role in maintaining respiratory chain activity. Consistent with this, electron flow through complex IV was abolished in rescued HDP-null parasites, rendering them hypersensitive to proguanil, an antimalarial that synergizes with respiratory chain inhibitors. We found that loss of HDP leads to a marked reduction of complexes III and IV, whose integrity depends on mitochondrial protein biosynthesis. Integration of quantitative proteomic data with structure-guided homology modelling supports a role for HDP as part of the large mitoribosomal subunit at the inter-subunit contact site. By contrast, HDP loss did not affect the quantity of hemozoin or other heme species, crystal morphology, or sensitivity to the hemozoin-targeting drug chloroquine. Together, these findings challenge previous models linking HDP to hemozoin formation and instead reveal an essential role for HDP in mitochondrial protein biosynthesis.

Plasmodium falciparum possesses one of the most AT-rich genomes in nature (80.6%). A consequence is an asparagine-rich proteome. A quarter of P. falciparum proteins possess poly-asparagine repeats that can extend more than 100 residues. The role of these repeats has remained a mystery in the biology of this parasite. Here, we find that the poly-asparagine repeat-containing N terminus of the histone acetyltransferase PfGCN5 associates with the C-terminal catalytic domain after cleavage in the nucleus. Deletion of the repeat destabilizes the N-terminal polypeptide, leading to impaired parasite development and growth, particularly under stress conditions. Using high-resolution mass spectrometry and western blotting analysis, we uncovered a profound effect of the poly-asparagine repeat on acetylation of histones H3, H3.3, and H4. These findings suggest that the poly-asparagine repeat contributes to PfGCN5 acetyltransferase activity, a role previously attributed solely to its C-terminal domain. This report of a function for a poly-asparagine repeat in P. falciparum expands our understanding of a pervasive characteristic of its proteome.