[Show abstract][Hide abstract] ABSTRACT: Heme metabolism is central to blood-stage infection by the malaria parasite, Plasmodium falciparum. Parasites retain a heme biosynthesis pathway but do not require its activity during infection of heme-rich erythrocytes, where they can scavenge host heme to meet metabolic needs. Nevertheless, heme biosynthesis in parasite-infected erythrocytes can be potently stimulated by exogenous 5-aminolevulinic acid (ALA), resulting in accumulation of the phototoxic intermediate, protoporphyrin IX (PPIX). Here we use photodynamic imaging, mass spectrometry, parasite gene disruption, and chemical probes to reveal that vestigial host enzymes in the cytoplasm of Plasmodium-infected erythrocytes contribute to ALA-stimulated heme biosynthesis and that ALA uptake depends on parasite-established permeability pathways. We show that PPIX accumulation in infected erythrocytes can be harnessed for antimalarial chemotherapy using luminol-based chemiluminescence and combinatorial stimulation by low-dose artemisinin to photoactivate PPIX to produce cytotoxic reactive oxygen. This photodynamic strategy has the advantage of exploiting host enzymes refractory to resistance-conferring mutations.
[Show abstract][Hide abstract] ABSTRACT: Phylum Apicomplexa comprises a large group of obligate intracellular parasites of high medical and veterinary importance. These organisms succeed intracellularly by effecting remarkable changes in a broad range of diverse host cells. The transformation of the host erythrocyte is particularly striking in the case of the malaria parasite Plasmodium falciparum. P. falciparum exports hundreds of proteins that mediate a complex cellular renovation marked by changes in the permeability, rigidity, and cytoadherence properties of the host erythrocyte. The past decade has seen enormous progress in understanding the identity and function of these exported effectors, as well as the mechanisms by which they are trafficked into the host cell. Here we review these advances, place them in the context of host manipulation by related apicomplexans, and propose key directions for future research. Expected final online publication date for the Annual Review of Biochemistry Volume 84 is June 02, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
[Show abstract][Hide abstract] ABSTRACT: Heme is an essential cofactor for aerobic organisms. Its redox chemistry is central to a variety of biological functions mediated by hemoproteins. In blood stages, malaria parasites consume most of the hemoglobin inside the infected erythrocytes, forming nontoxic hemozoin crystals from large quantities of heme released during digestion. At the same time, the parasites possess a heme de novo biosynthetic pathway. This pathway in the human malaria parasite Plasmodium falciparum has been considered essential and proposed as a potential drug target. However, we successfully disrupted the first and last genes of the pathway, individually and in combination. These knockout parasite lines, lacking δ-aminolevulinic acid synthase (ALAS) and/or ferrochelatase (FC), grew normally in blood-stage culture and exhibited no changes in sensitivity to heme-related antimalarial drugs. We developed a sensitive LC-MS/MS assay to monitor stable isotope incorporation into heme from its precursor 13C4-5-aminolevulinic acid (5-ALA), and this assay confirmed that de novo heme synthesis was ablated in FC knockout parasites. Disrupting the FC gene also caused no defects in gametocyte generation or maturation but resulted in a greater than 70% reduction in male gamete formation and completely prevented oocyst formation in female Anopheles stephensi mosquitoes. Our data demonstrate that the heme biosynthesis pathway is not essential for asexual blood-stage growth of P. falciparum parasites but is required for mosquito transmission. Drug inhibition of pathway activity is therefore unlikely to provide successful antimalarial therapy. These data also suggest the existence of a parasite mechanism for scavenging host heme to meet metabolic needs.
[Show abstract][Hide abstract] ABSTRACT: The universally conserved kinase-associated endopeptidase 1 (Kae1) protein family has established roles in N6-threonylcarbamoyl adenosine tRNA modification, transcriptional regulation, and telomere homeostasis. These functions are
performed in complex with a conserved core of protein binding partners. Herein we describe the localization, essentiality,
and protein-protein interactions for Kae1 in the human malaria parasite Plasmodium falciparum. We found that the parasite expresses one Kae1 protein in the cytoplasm and a second protein in the apicoplast, a chloroplast
remnant organelle involved in fatty acid, heme, and isoprenoid biosynthesis. To analyze the protein interaction networks for
both Kae1 pathways, we developed a new proteomic cross-validation approach. This strategy compares immunoprecipitation-MS
data sets across different cellular compartments to enrich for biologically relevant protein interactions. Our results show
that cytoplasmic Kae1 forms a complex with Bud32 and Cgi121 as in other organisms, whereas apicoplast Kae1 makes novel interactions
with multiple proteins in the apicoplast. Quantitative RT-PCR and immunoprecipitation studies indicate that apicoplast Kae1
and its partners interact specifically with the apicoplast ribosomes and with proteins involved in ribosome function. Together,
these data indicate an expanded, apicoplast-specific role for Kae1 in the parasite.
[Show abstract][Hide abstract] ABSTRACT: To mediate its survival and virulence, the malaria parasite Plasmodium falciparum exports hundreds of proteins into the host erythrocyte. To enter the host cell, exported proteins must cross the parasitophorous vacuolar membrane (PVM) within which the parasite resides, but the mechanism remains unclear. A putative Plasmodium translocon of exported proteins (PTEX) has been suggested to be involved for at least one class of exported proteins; however, direct functional evidence for this has been elusive. Here we show that export across the PVM requires heat shock protein 101 (HSP101), a ClpB-like AAA+ ATPase component of PTEX. Using a chaperone auto-inhibition strategy, we achieved rapid, reversible ablation of HSP101 function, resulting in a nearly complete block in export with substrates accumulating in the vacuole in both asexual and sexual parasites. Surprisingly, this block extended to all classes of exported proteins, revealing HSP101-dependent translocation across the PVM as a convergent step in the multi-pathway export process. Under export-blocked conditions, association between HSP101 and other components of the PTEX complex was lost, indicating that the integrity of the complex is required for efficient protein export. Our results demonstrate an essential and universal role for HSP101 in protein export and provide strong evidence for PTEX function in protein translocation into the host cell.
[Show abstract][Hide abstract] ABSTRACT: For over a century, heme metabolism has been recognized to play a central role during intraerythrocytic infection by Plasmodium parasites, the causative agent of malaria. Parasites liberate vast quantities of potentially cytotoxic heme as a by-product of hemoglobin catabolism within the digestive vacuole, where heme is predominantly sequestered as inert crystalline hemozoin. Plasmodium spp. also utilize heme as a metabolic cofactor. Despite access to abundant host-derived heme, parasites paradoxically maintain a biosynthetic pathway. This pathway has been assumed to produce the heme incorporated into mitochondrial cytochromes that support electron transport. In this review, we assess our current understanding of the love-hate relationship between Plasmodium parasites and heme, we discuss recent studies that clarify several long-standing riddles about heme production and utilization by parasites, and we consider remaining challenges and opportunities for understanding and targeting heme metabolism within parasites. Expected final online publication date for the Annual Review of Microbiology Volume 68 is September 08, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
[Show abstract][Hide abstract] ABSTRACT: Given the threat of drug resistance, there is an acute need for new classes of antimalarial agents that act via a unique mechanism of action relative to currently used drugs. We have identified a set of druglike compounds within the Tres Cantos Anti-Malarial Set (TCAMS) which likely act via inhibition of a Plasmodium aspartic protease. Structure-activity relationship analysis and optimization of these aminohydantoins demonstrate that these compounds are potent nanomolar inhibitors of the Plasmodium aspartic proteases PM-II and PM-IV and likely one or more other Plasmodium aspartic proteases. Incorporation of a bulky group, such as a cyclohexyl group, on the aminohydantion N-3 position gives enhanced antimalarial potency while reducing inhibition of human aspartic proteases such as BACE. We have identified compound 8p (CWHM-117) as a promising lead for optimization as an antimalarial drug with a low molecular weight, modest lipophilicity, oral bioavailability, and in vivo antimalarial activity in mice.
[Show abstract][Hide abstract] ABSTRACT: eIF2α kinases are stress sensors that respond to external stimuli by phosphorylating the eukaryotic initiation factor eIF2α. This response downregulates cellular protein synthesis but induces the translation of select mRNAs to allow stress survival. In Plasmodium, there are three eIF2α kinases: PfeIK1 responds to amino acid deprivation, while the other two are involved in controlling parasite development. Of the latter kinases, PfPK4 is essential for intraerythrocytic survival, while PfeIK2 controls salivary gland sporozoite latency. The study of parasite eIF2α kinases is broadening the present view of the role of these kinases in cellular and organismal function.
Protein Phosphorylation in Parasites, 10/2013: pages 123-130; , ISBN: 9783527332359
[Show abstract][Hide abstract] ABSTRACT: Malaria parasites multiply in human erythrocytes through schizogony, a process characterised by nuclear divisions in the absence of cytokinesis, leading to the formation of a multinucleated schizont from which individual daughter cells are subsequently generated. Here, we provide evidence that parasites lines lacking Pfcrk-5, an atypical cyclin-dependent kinase, display a reduced parasitemia growth rate linked to a decrease in the number of daughter nuclei produced by each schizont. We show that in vitro activity of recombinant Pfcrk-5 is indeed cyclin-dependent, and that the enzyme localises to the nuclear periphery. Thus, Pfcrk-5 is part of a regulatory pathway that mediates the proliferation rate of Plasmodium falciparum through the control of nuclear divisions during schizogony.
[Show abstract][Hide abstract] ABSTRACT: One-fourth of Plasmodium falciparum proteins have asparagine repeats that increase the propensity for aggregation, especially at elevated temperatures that occur routinely in malaria-infected patients. Here we report that a Plasmodium Asn repeat-containing protein (PFI1155w) formed aggregates in mammalian cells at febrile temperatures, as did a yeast Asn/Gln-rich protein (Sup35). Co-expression of the cytoplasmic P. falciparum heat shock protein 110 (PfHsp110c) prevented aggregation. Human or yeast orthologs were much less effective. All-Asn and all-Gln versions of Sup35 were protected from aggregation by PfHsp110c, suggesting that this chaperone is not limited to handling runs of asparagine. PfHsp110c gene-knockout parasites were not viable and conditional knockdown parasites died slowly in the absence of protein-stabilizing ligand. When exposed to brief heat shock, these knockdowns were unable to prevent aggregation of PFI1155w or Sup35 and died rapidly. We conclude that PfHsp110c protects the parasite from harmful effects of its asparagine repeat-rich proteome during febrile episodes.
[Show abstract][Hide abstract] ABSTRACT: Intraerythrocytic malaria parasites send hundreds of effector proteins into the host cell. Diverse modes of export have been proposed for different proteins. In this issue, Grüring et al. (2012) present findings that bring the models together.
[Show abstract][Hide abstract] ABSTRACT: The human malaria parasite Plasmodium falciparum is auxotrophic for most amino acids. Its amino acid needs are met largely through the degradation of host erythrocyte hemoglobin; however the parasite must acquire isoleucine exogenously, because this amino acid is not present in adult human hemoglobin. We report that when isoleucine is withdrawn from the culture medium of intraerythrocytic P. falciparum, the parasite slows its metabolism and progresses through its developmental cycle at a reduced rate. Isoleucine-starved parasites remain viable for 72 h and resume rapid growth upon resupplementation. Protein degradation during starvation is important for maintenance of this hibernatory state. Microarray analysis of starved parasites revealed a 60% decrease in the rate of progression through the normal transcriptional program but no other apparent stress response. Plasmodium parasites do not possess a TOR nutrient-sensing pathway and have only a rudimentary amino acid starvation-sensing eukaryotic initiation factor 2α (eIF2α) stress response. Isoleucine deprivation results in GCN2-mediated phosphorylation of eIF2α, but kinase-knockout clones still are able to hibernate and recover, indicating that this pathway does not directly promote survival during isoleucine starvation. We conclude that P. falciparum, in the absence of canonical eukaryotic nutrient stress-response pathways, can cope with an inconsistent bloodstream amino acid supply by hibernating and waiting for more nutrient to be provided.
Proceedings of the National Academy of Sciences 10/2012; 109(47). DOI:10.1073/pnas.1209823109 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Malaria parasites generate vast quantities of heme during blood-stage infection via hemoglobin digestion and limited de novo biosynthesis, but it remains unclear if parasites metabolize heme for utilization or disposal. Recent in vitro experiments with a heme oxygense (HO)-like protein from P. falciparum suggested that parasites may enzymatically degrade some heme to the canonical HO product, biliverdin (BV), or its downstream metabolite, bilirubin (BR). To directly test for BV and BR production by P. falciparum parasites, we DMSO-extracted equal numbers of infected and uninfected erythrocytes and developed a sensitive LC-MS/MS assay to quantify these tetrapyrroles. We found comparable low levels of BV and BR in both samples, suggesting the absence of HO activity in parasites. We further tested live parasites by targeted expression of a fluorescent BV-binding protein within the parasite cytosol, mitochondrion, and plant-like plastid. This probe could detect exogenously added BV but gave no signal indicative of endogenous BV production within parasites. Finally, we recombinantly expressed and tested the proposed heme-degrading activity of the HO-like protein, PfHO. Although PfHO bound heme and protoporphyrin IX with modest affinity, it did not catalyze heme degradation in vivo within bacteria or in vitro in UV absorbance and HPLC assays. These observations are consistent with the lack of a heme-coordinating His residue in PfHO and suggest an alternative function within parasites. We conclude that P. falciparum parasites lack a canonical HO pathway for heme degradation and thus rely fully on alternative mechanisms for heme detoxification and iron acquisition during blood-stage infection.
[Show abstract][Hide abstract] ABSTRACT: Although caused by vastly different pathogens, the world's three most serious infectious diseases, tuberculosis, malaria, and HIV-1 infection, share the common problem of drug resistance. The pace of drug development has been very slow for tuberculosis and malaria and rapid for HIV-1. But for each disease, resistance to most drugs has appeared quickly after the introduction of the drug. Learning how to manage and prevent resistance is a major medical challenge that requires an understanding of the evolutionary dynamics of each pathogen. This Review summarizes the similarities and differences in the evolution of drug resistance for these three pathogens.
[Show abstract][Hide abstract] ABSTRACT: Plasmepsins are the aspartic proteases of Plasmodium that play key roles in the survival of the parasite in its host. The plasmepsins of the digestive vacuole play an important role in hemoglobin degradation, providing the parasite with a vital source of nutrients. Recently, plasmepsin V has been shown to be an essential protease, processing hundreds of parasite proteins for export into the host erythrocyte. The functions of the remaining plasmepsins have yet to be discovered. Over the past decade, much effort has been placed towards developing plasmepsin inhibitors as antimalarial agents, particularly targeting the digestive vacuole. This review will highlight some of the recent work in this field with a particular focus on target druggability and strategies for identifying plasmepsins inhibitors as effective antimalarial drugs. Given recent advances in understanding the fundamental roles of the various plasmepsins, it is likely that the most effective antimalarial plasmepsin targets will be the non-digestive vacuole plasmepsins.
Current topics in medicinal chemistry 03/2012; 12(5):445-55. DOI:10.2174/156802612799362959 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The newly synthesized benzimidazole compounds were suggested to be inhibitors of Plasmodium falciparum plasmepsin II and human cathepsin D by virtual screening of an internal library of synthetic compounds. This was confirmed by enzyme inhibition studies that gave IC(50) values in the low micromolar range (2-48μM). Ligand docking studies with plasmepsin II predicted binding of benzimidazole compounds at the center of the extended substrate-binding cleft. According to the plausible mode of binding, the pyridine ring of benzimidazole compounds interacted with S1' subsite residues whereas the acetophenone moiety was in contact with S1-S3 subsites of plasmepsin II active center. The benzimidazole derivatives were evaluated for capacity to inhibit the growth of intraerythrocytic P. falciparum in culture. Four benzimidazole compounds inhibited parasite growth at ⩽3μM. The most active compound 10, 1-(4-phenylphenyl)-2[2-(pyridinyl-2-yl)-1,3-benzdiazol-1-yl]ethanone showed an IC(50) of 160nM. The substitution of a phenyl group and a chlorine atom at the para position of the acetophenone moiety were shown to be crucial for antiplasmodial activity.
[Show abstract][Hide abstract] ABSTRACT: Histidine-rich protein II (HRPII) is an abundant protein released into the bloodstream by Plasmodium falciparum, the parasite that causes the most severe form of human malaria. Here, we report that HRPII binds tightly and selectively to coagulation-active glycosaminoglycans (dermatan sulfate, heparan sulfate, and heparin) and inhibits antithrombin (AT). In purified systems, recombinant HRPII neutralized the heparin-catalyzed inhibition of factor Xa and thrombin by AT in a Zn(2+)-dependent manner. The observed 50% inhibitory concentration (IC(50)) for the HRPII neutralization of AT activity is approximately 30nM for factor Xa inhibition and 90nM for thrombin inhibition. Zn(2+) was required for these reactions with a distribution coefficient (K(d)) of approximately 7μM. Substituting Zn(2+) with Cu(2+), but not with Ca(2+), Mg(2+), or Fe(2+), maintained the HRPII effect. HRPII attenuated the prolongation in plasma clotting time induced by heparin, suggesting that HRPII inhibits AT activity by preventing its stimulation by heparin. In the microvasculature, where erythrocytes infected with P falciparum are sequestered, high levels of released HRPII may bind cellular glycosaminoglycans, prevent their interaction with AT, and thereby contribute to the procoagulant state associated with P falciparum infection.