The Human Malaria Parasite Plasmodium falciparum Is Not Dependent on Host Coenzyme A Biosynthesis

Pantothenate, a precursor of the fundamental enzyme cofactor coenzyme A (CoA), is essential for growth of the intraerythrocytic stage of human and avian malaria parasites. Avian malaria parasites have been reported to be incapable of de novo CoA synthesis and instead salvage CoA from the host erythrocyte; hence, pantothenate is required for CoA biosynthesis within the host cell and not the parasite itself. Whether the same is true of the intraerythrocytic stage of the human malaria parasite, Plasmodium falciparum, remained to be established. In this study we investigated the metabolic fate of [¹⁴C]pantothenate within uninfected and P. falciparum-infected human erythrocytes. We provide evidence consistent with normal human erythrocytes, unlike rat erythrocytes (which have been reported to possess an incomplete CoA biosynthesis pathway), being capable of CoA biosynthesis from pantothenate. We also show that CoA biosynthesis is substantially higher in P. falciparum-infected erythrocytes and that P. falciparum, unlike its avian counterpart, generates most of the CoA synthesized in the infected erythrocyte, presumably necessitated by insufficient CoA biosynthesis in the host erythrocyte. Our data raise the possibility that malaria parasites rationalize their biosynthetic activity depending on the capacity of their host cell to synthesize the metabolites they require.Pantothenate (vitamin B₅) is an essential nutrient for the virulent human malaria parasite Plasmodium falciparum, required to support the rapid growth and replication of the parasite during the intraerythrocytic stage of its life cycle (1–3). In bacteria, plants, and mammalian tissues, pantothenate serves as a precursor of coenzyme A (CoA),³ an essential enzyme cofactor involved in numerous metabolic reactions in the cell. Pantothenate is converted to CoA via five universal enzyme-mediated steps (see Fig. 1).Open in a separate windowFIGURE 1.The CoA biosynthesis pathway.Several decades ago, Trager (4) showed that pantothenate supported the survival of the avian malaria parasite Plasmodium lophurae during its development within duck erythrocytes in vitro. Trager (5, 6) later demonstrated, however, that CoA, and not pantothenate, stimulated exoerythrocytic growth of the intraerythrocytic stage of P. lophurae, and proposed that avian malaria parasites are incapable of metabolizing pantothenate to CoA, and instead rely on CoA synthesized by the host erythrocyte. In support of this proposal, CoA biosynthesis enzymes are readily detectable in duck erythrocytes, but appear to be absent from P. lophurae parasites isolated from their host erythrocyte (7, 8). Pantothenate is therefore required by the P. lophurae-infected duck erythrocyte for CoA biosynthesis within the host cell and not the parasite itself.By contrast with nucleated avian erythrocytes, mammalian erythrocytes are thought to be incapable of CoA biosynthesis. In the only study on the subject, Annous and Song (9) reported that although pantothenate is phosphorylated within rat erythrocytes (the first step in CoA biosynthesis), there is no evidence for the subsequent steps of the CoA biosynthesis pathway. Saliba et al. (10) confirmed that human erythrocytes similarly phosphorylate pantothenate, but did not investigate whether CoA synthesis also occurs in the cells. A lack of CoA biosynthesis in mammalian erythrocytes would seemingly place the burden of CoA synthesis squarely on malaria parasites that infect mammals (such as P. falciparum), contrary to the situation in birds. Although Saliba et al. (10) have shown that P. falciparum is capable of performing the first step in CoA biosynthesis, it remains to be established whether the parasite can metabolize the 4′-phosphopantothenate generated from pantothenate to CoA or, like P. lophurae, relies on CoA synthesized in the host erythrocyte for its normal growth and replication.In this study we followed the metabolism of pantothenate within uninfected human erythrocytes, P. falciparum-infected human erythrocytes, and isolated P. falciparum parasites. We provide evidence that both uninfected erythrocytes (which we show do take up pantothenate, albeit very slowly) and P. falciparum-infected erythrocytes synthesize CoA from pantothenate. CoA biosynthesis is, however, dramatically higher in the P. falciparum-infected cell. Furthermore, we show that P. falciparum parasites synthesize CoA in the absence of the host erythrocyte, and hence, by contrast with avian malaria parasites, the human malaria parasite does not rely on the host erythrocyte for CoA.     

An interplay between 2 signaling pathways: Melatonin-cAMP and IP3–Ca signaling pathways control intraerythrocytic development of the malaria parasite Plasmodium falciparum

Plasmodium falciparum spends most of its asexual life cycle within human erythrocytes, where proliferation and maturation occur. Development into the mature forms of P. falciparum causes severe symptoms due to its distinctive sequestration capability. However, the physiological roles and the molecular mechanisms of signaling pathways that govern development are poorly understood. Our previous study showed that P. falciparum exhibits stage-specific spontaneous Calcium (Ca²⁺) oscillations in ring and early trophozoites, and the latter was essential for parasite development. In this study, we show that luzindole (LZ), a selective melatonin receptor antagonist, inhibits parasite growth. Analyses of development and morphology of LZ-treated P. falciparum revealed that LZ severely disrupted intraerythrocytic maturation, resulting in parasite death. When LZ was added at ring stage, the parasite could not undergo further development, whereas LZ added at the trophozoite stage inhibited development from early into late schizonts. Live-cell Ca²⁺ imaging showed that LZ treatment completely abolished Ca²⁺ oscillation in the ring forms while having little effect on early trophozoites. Further, the melatonin-induced cAMP increase observed at ring and late trophozoite stage was attenuated by LZ treatment. These suggest that a complex interplay between IP₃–Ca²⁺ and cAMP signaling pathways is involved in intraerythrocytic development of P. falciparum.     

Cellular architecture of Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum is a protozoan parasite that is responsible for the most pathogenic form of human malaria. The particular virulence of this parasite derives from its ability to develop within the erythrocytes of its host and to subvert their function. The intraerythrocytic parasite devours haemoglobin, and remodels its host cell to cause adhesion to blood vessel walls. Ultrastructural studies of P. falciparum have played a major role in defining its cell architecture and in resolving cell biology controversies. Here we review some of the early studies and describe some recent developments in electron microscopy techniques that have revealed information about the organization of the parasite in the blood stage of development. We present images of P. falciparum at different stages of the life cycle and highlight some of the plasmodium-specific organelles, the haemoglobin digestive apparatus and the membrane structures that are elaborated in the host cell cytoplasm to traffic virulence proteins to the erythrocyte surface. We describe methods for whole cell ultrastructural imaging that can provide three-dimensional views of intraerythrocytic development.     

Plasmodium falciparum RuvB1 is an active DNA helicase and translocates in the 5′–3′ direction

RuvB family of protein contains two similar kinds of proteins i.e. RuvB1 and RuvB2 from yeast to human. These proteins belong to the AAA + class of proteins and are critical components of several multiprotein complexes involved in diverse cellular activities. There are two RuvB proteins annotated in the Plasmodium database but the identification of the third protein recently by our lab has raised the question why Plasmodium falciparum contains three RuvB proteins instead of two. Hence the biochemical characterizations of these proteins have become essential to understand the role of these proteins in the malaria parasite. Recently we have reported the characterization of the recombinant PfRuvB3, which contains ATPase activity but lacks DNA helicase activity. In the present study we report the phylogenetic analysis and detailed biochemical characterization of one of the other RuvB homologue RuvB1 from P. falciparum. PfRuvB1 shows considerable homology with human as well as yeast RuvB1 and contains Walker motif A and Walker motif B. The activity analysis of this protein revealed that PfRuvB1 is an ATPase and this activity increased significantly in the presence of ss-DNA. PfRuvB1 also contains DNA helicase activity and translocates preferentially in 5′ to 3′ direction. In vivo investigation of PfRuvB1 revealed that it is constitutively expressed during all the stages of intraerythrocytic cycle of P. falciparum and localizes mainly to the nucleus. These studies will make important contribution in understanding the role of RuvB protein in P. falciparum.     

Non‐canonical metabolic pathways in the malaria parasite detected by isotope‐tracing metabolomics

The malaria parasite, Plasmodium falciparum, proliferates rapidly in human erythrocytes by actively scavenging multiple carbon sources and essential nutrients from its host cell. However, a global overview of the metabolic capacity of intraerythrocytic stages is missing. Using multiplex ¹³C‐labelling coupled with untargeted mass spectrometry and unsupervised isotopologue grouping, we have generated a draft metabolome of P. falciparum and its host erythrocyte consisting of 911 and 577 metabolites, respectively, corresponding to 41% of metabolites and over 70% of the metabolic reaction predicted from the parasite genome. An additional 89 metabolites and 92 reactions were identified that were not predicted from genomic reconstructions, with the largest group being associated with metabolite damage‐repair systems. Validation of the draft metabolome revealed four previously uncharacterised enzymes which impact isoprenoid biosynthesis, lipid homeostasis and mitochondrial metabolism and are necessary for parasite development and proliferation. This study defines the metabolic fate of multiple carbon sources in P. falciparum, and highlights the activity of metabolite repair pathways in these rapidly growing parasite stages, opening new avenues for drug discovery.     

Plasmodium falciparum DDX3X is a nucleocytoplasmic protein and requires N-terminal for DNA helicase activity

Malaria is a haemato-protozoan disease which causes thousands of deaths every year. Due to the alarming increase of drug resistant strains of P. falciparum, malaria is now becoming more deadly. Helicases are the most important components of the cellular machinery without which cells are unable to survive. The importance of helicases has been proven in variety of organisms. In this study we have reported detailed biochemical characterization of human homologue of DDX3X from Plasmodium falciparum (PfDDX3X). Our study revealed that PfDDX3X is ATP- dependent DNA helicase whereas in human host it is ATP-dependent RNA helicase. We show that N-terminal is essential for its activity and it is present in nucleus and cytoplasm in intraerythrocytic developmental stages of P. falciparum 3D7 strain. Also, it is highly expressed in the schizont stage of P. falciparum 3D7strain. The present study suggests that a protein can perform different functions in different systems. The present study will help to understand the basic biology of malaria parasite P. falciparum.     

Plasmodium falciparum RuvB2 translocates in 5′–3′ direction,relocalizes during schizont stage and its enzymatic activities are up regulated by RuvB3 of the same complex

Two similar proteins RuvB like1 (Rvb1/Pontin) and RuvB like2 (Rvb2/Reptin) of AAA + family of enzymes are present in yeast to human and are well known to be involved in diverse cellular activities. The human malaria parasite Plasmodium falciparum contains three different RuvB like proteins. Thus it has been of interest to explore why P. falciparum requires three RuvB like proteins and how these enzymes are biochemically regulated. In this study, we present the detailed biochemical characterization of PfRuvB2. The complex of PfRuvB3 was immunopurified and the presence of PfRuvB2 was confirmed. The in vitro interaction study shows that PfRuvB2 interacts only with PfRuvB3 but not with PfRuvB1. The recombinant as well as endogenous PfRuvB2 contains ATPase as well as weak DNA helicase activities. The presence of PfRuvB3 in the helicase reaction of PfRuvB2 increases the helicase activity significantly. Interestingly PfRuvB2/PfRuvB3 complex preferentially translocates and unwinds DNA in the 5′–3′ direction. In vivo studies showed that PfRuvB2 is expressed in all the asexual intraerythrocytic developmental stages and localizes mainly in the nucleus during merozoite, ring and trophozoite stages while during schizont stage it relocalizes partially in the nucleus and partially towards cytoplasm. As PfRuvB3 is specific to intraerythrocytic mitosis so we interpret that PfPuvB3 interacts with PfRuvB2 during schizont/intraerythrocytic mitosis and acts as its modulator mainly for the appreciable helicase activity.     

Deep Profiling of the Novel Intermediate-Size Noncoding RNAs in Intraerythrocytic Plasmodium falciparum

Intermediate-size noncoding RNAs (is-ncRNAs) have been shown to play important regulatory roles in the development of several eukaryotic organisms. However, they have not been thoroughly explored in Plasmodium falciparum, which is the most virulent malaria parasite infecting human being. By using Illumina/Solexa paired-end sequencing of an is-ncRNA-specific library, we performed a systematic identification of novel is-ncRNAs in intraerythrocytic P. falciparum, strain 3D7. A total of 1,198 novel is-ncRNA candidates, including antisense, intergenic, and intronic is-ncRNAs, were identified. Bioinformatics analyses showed that the intergenic is-ncRNAs were the least conserved among different Plasmodium species, and antisense is-ncRNAs were more conserved than their sense counterparts. Twenty-two novel snoRNAs were identified, and eight potential novel classes of P. falciparum is-ncRNAs were revealed by clustering analysis. The expression of randomly selected novel is-ncRNAs was confirmed by RT-PCR and northern blotting assays. An obvious different expressional profile of the novel is-ncRNA between the early and late intraerythrocytic developmental stages of the parasite was observed. The expression levels of the antisense RNAs correlated with those of their cis-encoded sense RNA counterparts, suggesting that these is-ncRNAs are involved in the regulation of gene expression of the parasite. In conclusion, we accomplished a deep profiling analysis of novel is-ncRNAs in P. falciparum, analysed the conservation and structural features of these novel is-ncRNAs, and revealed their differential expression patterns during the development of the parasite. These findings provide important information for further functional characterisation of novel is-ncRNAs during the development of P. falciparum.     

H+-coupled pantothenate transport in the intracellular malaria parasite

Pantothenate, the precursor of coenzyme A, is an essential nutrient for the intraerythrocytic stage of the malaria parasite Plasmodium falciparum. Pantothenate enters the malaria-infected erythrocyte via new permeation pathways induced by the parasite in the host cell membrane (Saliba, K. J., Horner, H. A., and Kirk, K. (1998) J. Biol. Chem. 273, 10190-10195). We show here that pantothenate is taken up by the intracellular parasite via a novel H(+)-coupled transporter, quite different from the Na(+)-coupled transporters that mediate pantothenate uptake into mammalian cells. The plasmodial H(+):pantothenate transporter has a low affinity for pantothenate (K(m) approximately 23 mm) and a stoichiometry of 1 H(+):1 pantothenate. It is inhibited by low concentrations of the bioflavonoid phloretin and the thiol-modifying agent p-chloromercuribenzene sulfonate. On entering the parasite, pantothenate is phosphorylated (and thereby trapped) by an unusually high affinity pantothenate kinase (K(m) approximately 300 nm). The combination of H(+)-coupled transporter and kinase provides the parasite with an efficient, high affinity pantothenate uptake system, which is distinct from that of the host and is therefore an attractive target for antimalarial chemotherapy.     

Bongkrekic acid and the adenosinetriphosphate requirement of malaria parasites

The avian malaria parasite Plasmodium lophurae, when removed from its host erythrocytes into an appropriate medium, develops extracellularly in vitro. This development was inhibited by bongkrekic acid at concentrations down to 2 μg/ml. Adenosine triphosphate at high concentrations partly reversed the inhibition. Bongkrekic acid also inhibited intraerythrocytic development in vitro of the human malaria P. falciparum.     

Reconstructing the Qo Site of Plasmodium falciparum bc 1 Complex in the Yeast Enzyme

The bc ₁ complex of the mitochondrial respiratory chain is essential for Plasmodium falciparum proliferation, the causative agent of human malaria. Therefore, this enzyme is an attractive target for antimalarials. However, biochemical investigations of the parasite enzyme needed for the study of new drugs are challenging. In order to facilitate the study of new compounds targeting the enzyme, we are modifying the inhibitor binding sites of the yeast Saccharomyces cerevisiae to generate a complex that mimics the P. falciparum enzyme. In this study we focused on its Q_o pocket, the site of atovaquone binding which is a leading antimalarial drug used in treatment and causal prophylaxis. We constructed and studied a series of mutants with modified Q_o sites where yeast residues have been replaced by P. falciparum equivalents, or, for comparison, by human equivalents. Mitochondria were prepared from the yeast Plasmodium-like and human-like Q_o mutants. We measured the bc ₁ complex sensitivity to atovaquone, azoxystrobin, a Q_o site targeting fungicide active against P. falciparum and RCQ06, a quinolone-derivative inhibitor of P. falciparum bc ₁ complex.The data obtained highlighted variations in the Q_o site that could explain the differences in inhibitor sensitivity between yeast, plasmodial and human enzymes. We showed that the yeast Plasmodium-like Q_o mutants could be useful and easy-to-use tools for the study of that class of antimalarials.     

Methionine transport in the malaria parasite Plasmodium falciparum

The intraerythrocytic malaria parasite, Plasmodium falciparum, derives amino acids from the digestion of host cell haemoglobin. However, it also takes up amino acids from the extracellular medium. Isoleucine is absent from adult human haemoglobin and an exogenous source of isoleucine is essential for parasite growth. An extracellular source of methionine is also important for the normal growth of at least some parasite strains. In this study we have characterised the uptake of methionine by P. falciparum-infected human erythrocytes, and by parasites functionally isolated from their host cells by saponin-permeabilization of the erythrocyte membrane. Infected erythrocytes take up methionine much faster than uninfected erythrocytes, with the increase attributable to the flux of this amino acid via the New Permeability Pathways induced by the parasite in the erythrocyte membrane. Having entered the infected cell, methionine is taken up by the intracellular parasite via a saturable, temperature-dependent process that is independent of ATP, Na⁺ and H⁺. Substrate competition studies, and comparison of the transport of methionine with that of isoleucine and leucine, yielded results consistent with the hypothesis that the parasite has at its surface one or more transporters which mediate the flux into and out of the parasite of a broad range of neutral amino acids. These transporters function most efficiently when exchanging one neutral amino acid for another, thus providing a mechanism whereby the parasite is able to import important exogenous amino acids in exchange for surplus neutral amino acids liberated from the digestion of host cell haemoglobin.     

Glycerol: An unexpected major metabolite of energy metabolism by the human malaria parasite

Background

Malaria is the third most prevalent cause of infectious disease in the world. Resistance of the parasite to classical drugs makes the discovery of new and effective drugs more urgent. The oxidized derivative of hydroxy- cis terpenone (OHCT) is a synthetic molecule that is not toxic to cultured human liver cells at concentrations as high as 60 μM and inhibits activity of cytochrome P450s that metabolize many drugs.

Methods

OHCT activity against chloroquine-sensitive and -resistant strains of Plasmodium falciparum, and a P. falciparum clone that is partially resistant to artemisinin was assayed in vitro.

Results

OHCT at nanomolar concentrations was effective against all intraerythrocytic stages of P. falciparum and exhibited activity in vitro against both chloroquine-sensitive and -resistant strains of P. falciparum as well as a P. falciparum clone that is partially resistant to artemisinin. Moreover, OHCT exhibited potent activity against gametocytes, the form that is transmitted by mosquitoes and essential for the spread of malaria.

Conclusion

OHCT displays strong growth inhibitory activity against all stages of P. falciparum and no evidence of toxicity to human cells in culture. It is easily synthesized and has the potential for inhibiting metabolism of drugs used in combination therapies.     

Isolation and characterization of Plasmodium falciparum UAP56 homolog: evidence for the coupling of RNA binding and splicing activity by site-directed mutations

UAP56 (U2AF65 associated protein) is a member of the DEAD-box helicase family. Helicases are essential enzymes generally involved in the metabolism of nucleic acids. The gene encoding a member of DEAD-box family was cloned and characterized from the human malaria parasite Plasmodium falciparum. PfU52 is homologous to UAP56 and contains the RNA-dependent ATPase, RNA helicase and RNA binding activities. Using the parasite extract we report that PfU52 is involved in splicing reaction. Site-directed mutagenesis studies indicate that the conserved residues glycine 181, isoleucine 182 and arginine 206 are involved in RNA binding and this activity is required for the enzymatic activities of PfU52. PfU52 is expressed in all the intraerythrocytic developmental stages of the parasite. In the present study we have reported the detailed characterization of PfU52 from P. falciparum and these results advance the knowledge regarding the function of UAP56 in general.     

From the Genome to the Phenome: Tools to Understand the Basic Biology of Plasmodium falciparum

Malaria plagues one out of every 30 humans and contributes to almost a million deaths, and the problem could worsen. Our current therapeutic options are compromised by emerging resistance by the parasite to our front line drugs. It is thus imperative to better understand the basic biology of the parasite and develop novel drugs to stem this disease. The most facile approach to analyse a gene's function is to remove it from the genome or inhibit its activity. Although genetic manipulation of the human malaria parasite Plasmodium falciparum is a relatively standard procedure, there is no optimal method to perturb genes essential to the intraerythrocytic development cycle—the part of the life cycle that produces the clinical manifestation of malaria. This is a severe impediment to progress because the phenotype we wish to study is exactly the one that is so elusive. In the absence of any utilitarian way to conditionally delete essential genes, we are prevented from investigating the parasite's most vulnerable points. This review aims to focus on the development of tools identifying essential genes of P. falciparum and our ability to elicit phenotypic mutation.     

Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO’s biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P. falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite.     

Proteolipid of vacuolar H-ATPase of Plasmodium falciparum: cDNA cloning, gene organization and complementation of a yeast null mutant

Vacuolar H⁺-ATPase (V-ATPase), an electrogenic proton pump, is highly expressed in Plasmodium falciparum, the human malaria parasite. Although V-ATPase-driven proton transport is involved in various physiological processes in the parasite, the overall features of the V-ATPase of P. falciparum, including the gene organization and biogenesis, are far less known. Here, we report cDNA cloning of proteolipid subunit c of P. falciparum, the smallest and most highly hydrophobic subunit of V-ATPase. RT-PCR analysis as well as Northern blotting indicated expression of the proteolipid gene in the parasite cells. cDNA, which encodes a complete reading frame comprising 165 amino acids, was obtained, and its deduced amino acid sequence exhibits 52 and 57% similarity to the yeast and human counterparts, respectively. Southern blot analysis suggested the presence of a single copy of the proteolipid gene, with 5 exons and 4 introns. Upon transfection of the cDNA into a yeast null mutant, the cells became able to grow at neutral pH, accompanied by vesicular accumulation of quinacrine. In contrast, a mutated proteolipid with replacement of glutamate residue 138 with glutamine did not lead to recovery of the growth ability or vesicular accumulation of quinacrine. These results indicated that the cDNA actually encodes the proteolipid of P. falciparum and that the proteolipid is functional in yeast.     

Biochemical studies of membrane bound Plasmodium falciparum mitochondrial L-malate:quinone oxidoreductase,a potential drug target

Plasmodium falciparum is an apicomplexan parasite that causes the most severe malaria in humans. Due to a lack of effective vaccines and emerging of drug resistance parasites, development of drugs with novel mechanisms of action and few side effects are imperative. To this end, ideal drug targets are those essential to parasite viability as well as absent in their mammalian hosts. The mitochondrial electron transport chain (ETC) of P. falciparum is one source of such potential targets because enzymes, such as L-malate:quinone oxidoreductase (PfMQO), in this pathway are absent humans. PfMQO catalyzes the oxidation of L-malate to oxaloacetate and the simultaneous reduction of ubiquinone to ubiquinol. It is a membrane protein, involved in three pathways (ETC, the tricarboxylic acid cycle and the fumarate cycle) and has been shown to be essential for parasite survival, at least, in the intra-erythrocytic asexual stage. These findings indicate that PfMQO would be a valuable drug target for development of antimalarial with novel mechanism of action. Up to this point in time, difficulty in producing active recombinant mitochondrial MQO has hampered biochemical characterization and targeted drug discovery with MQO. Here we report for the first time recombinant PfMQO overexpressed in bacterial membrane and the first biochemical study. Furthermore, about 113 compounds, consisting of ubiquinone binding site inhibitors and antiparasitic agents, were screened resulting in the discovery of ferulenol as a potent PfMQO inhibitor. Finally, ferulenol was shown to inhibit parasite growth and showed strong synergism in combination with atovaquone, a well-described anti-malarial and bc₁ complex inhibitor.     

Malaria Parasite-Synthesized Heme Is Essential in the Mosquito and Liver Stages and Complements Host Heme in the Blood Stages of Infection

Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-¹⁴C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.     

Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) interacts with the band 3 receptor to promote erythrocyte invasion by malaria parasites

Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) is an erythrocyte binding protein known to be involved in malarial parasite invasion. Although anti-GAMA antibodies have been shown to block GAMA attachment to the erythrocyte surface and subsequently inhibit parasite invasion, little is known about the molecular mechanisms by which GAMA promotes the invasion process. In this study, LC-MS analysis was performed on the erythrocyte membrane to identify the specific receptor that interacts with GAMA. We found that ankyrin 1 and the band 3 membrane protein showed affinity for GAMA, and characterization of their binding specificity indicated that both Plasmodium falciparum and Plasmodium vivax GAMA bound to the same extracellular loop of band 3 (loop 5). In addition, we show the interaction between GAMA and band 3 was sensitive to chymotrypsin. Furthermore, antibodies against band 3 loop 5 were able to reduce the binding activity of GAMA to erythrocytes and inhibit the invasion of P. falciparum merozoites into human erythrocytes, whereas antibodies against P. falciparum GAMA (PfGAMA)-Tr3 only slightly reduced P. falciparum invasion. The identification and characterization of the erythrocyte GAMA receptor is a novel finding that identifies an essential mechanism of parasite invasion of host erythrocytes.