The Suf Iron-Sulfur Cluster Synthesis Pathway Is Required for Apicoplast Maintenance in Malaria Parasites

The apicoplast organelle of the malaria parasite Plasmodium falciparum contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In P. falciparum, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome – a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis.     

Plasmodium chabaudi: effect of antimalarial drugs on gametocytogenesis.

The proportion of asexual blood-stage malaria parasites that develop into transmission stages (gametocytes) can increase in response to stress. We investigated whether stress imposed by a variety of antimalarial drugs administered before or during infection increased gametocyte production (gametocytogenesis) in vivo in the rodent malaria parasite, Plasmodium chabaudi. All methods of drug treatment greatly reduced the numbers of asexual parasites produced during an infection but resulted in either no reduction in numbers of gametocytes or a smaller reduction than that experienced by asexuals. We used a simple model to estimate temporal variation in gametocyte production. Temporal patterns of gametocytogenesis did not greatly differ between untreated and prophylaxis infections, with rates of gametocytogenesis always increasing as the infection progressed. In contrast, administration of drugs 5 days after infection stimulated increased rates of gametocytogenesis early in the infection, resulting in earlier peak gametocyte densities relative to untreated infections. Given the correlation between gametocyte densities and infectivity to mosquito vectors, and the high frequency of subcurative drug therapy and prophylaxis in human populations, these data suggest that antimalarial drugs may frequently have only a small effect on reducing malaria transmission and may help to explain the rapid spread of drug-resistant geno-types.     

Chemical rescue of malaria parasites lacking an apicoplast defines organelle function in blood-stage Plasmodium falciparum

Plasmodium spp parasites harbor an unusual plastid organelle called the apicoplast. Due to its prokaryotic origin and essential function, the apicoplast is a key target for development of new anti-malarials. Over 500 proteins are predicted to localize to this organelle and several prokaryotic biochemical pathways have been annotated, yet the essential role of the apicoplast during human infection remains a mystery. Previous work showed that treatment with fosmidomycin, an inhibitor of non-mevalonate isoprenoid precursor biosynthesis in the apicoplast, inhibits the growth of blood-stage P. falciparum. Herein, we demonstrate that fosmidomycin inhibition can be chemically rescued by supplementation with isopentenyl pyrophosphate (IPP), the pathway product. Surprisingly, IPP supplementation also completely reverses death following treatment with antibiotics that cause loss of the apicoplast. We show that antibiotic-treated parasites rescued with IPP over multiple cycles specifically lose their apicoplast genome and fail to process or localize organelle proteins, rendering them functionally apicoplast-minus. Despite the loss of this essential organelle, these apicoplast-minus auxotrophs can be grown indefinitely in asexual blood stage culture but are entirely dependent on exogenous IPP for survival. These findings indicate that isoprenoid precursor biosynthesis is the only essential function of the apicoplast during blood-stage growth. Moreover, apicoplast-minus P. falciparum strains will be a powerful tool for further investigation of apicoplast biology as well as drug and vaccine development.     

The novel putative transporter NPT1 plays a critical role in early stages of Plasmodium berghei sexual development

Transmission of Plasmodium species from a mammalian host to the mosquito vector requires the uptake, during an infected blood meal, of gametocytes, the precursor cells of the gametes. Relatively little is known about the molecular mechanisms involved in the developmental switch from asexual development to sexual differentiation or the maturation and survival of gametocytes. Here, we show that a gene coding for a novel putative transporter, NPT1, plays a crucial role in the development of Plasmodium berghei gametocytes. Parasites lacking NPT1 are severely compromised in the production of gametocytes and the rare gametocytes produced are unable to differentiate into fertile gametes. This is the earliest block in gametocytogenesis obtained by reverse genetics and the first to demonstrate the role of a protein with a putative transport function in sexual development. These results and the high degree of conservation of NPT1 in Plasmodium species suggest that this protein could be an attractive target for the development of novel drugs to block the spread of malaria.     

A male gametocyte osmiophilic body and microgamete surface protein of the rodent malaria parasite Plasmodium yoelii (PyMiGS) plays a critical role in male osmiophilic body formation and exflagellation

Anopheles mosquitoes transmit Plasmodium parasites of mammals, including the species that cause malaria in humans. Malaria pathology is caused by rapid multiplication of parasites in asexual intraerythrocytic cycles. Sexual stage parasites are also produced during the intraerythrocytic cycle and are ingested by the mosquito, initiating gametogenesis and subsequent sporogonic stage development. Here, we present a Plasmodium protein, termed microgamete surface protein (MiGS), which has an important role in male gametocyte osmiophilic body (MOB) formation and microgamete function. MiGS is expressed exclusively in male gametocytes and microgametes, in which MiGS localises to the MOB and microgamete surface. Targeted gene disruption of MiGS in a rodent malaria parasite Plasmodium yoelii 17XNL generated knockout parasites (ΔPyMiGS) that proliferate normally in erythrocytes and form male and female gametocytes. The number of MOB in male gametocyte cytoplasm is markedly reduced and the exflagellation of microgametes is impaired in ΔPyMiGS. In addition, anti‐PyMiGS antibody severely blocked the parasite development in the Anopheles stephensi mosquito. MiGS might thus be a potential novel transmission‐blocking vaccine target candidate.     

Dephospho‐CoA kinase,a nuclear‐encoded apicoplast protein,remains active and essential after Plasmodium falciparum apicoplast disruption

Malaria parasites contain an essential organelle called the apicoplast that houses metabolic pathways for fatty acid, heme, isoprenoid, and iron–sulfur cluster synthesis. Surprisingly, malaria parasites can survive without the apicoplast as long as the isoprenoid precursor isopentenyl pyrophosphate (IPP) is supplemented in the growth medium, making it appear that isoprenoid synthesis is the only essential function of the organelle in blood‐stage parasites. In the work described here, we localized an enzyme responsible for coenzyme A synthesis, DPCK, to the apicoplast, but we were unable to delete DPCK, even in the presence of IPP. However, once the endogenous DPCK was complemented with the E. coli DPCK (EcDPCK), we were successful in deleting it. We were then able to show that DPCK activity is required for parasite survival through knockdown of the complemented EcDPCK. Additionally, we showed that DPCK enzyme activity remains functional and essential within the vesicles present after apicoplast disruption. These results demonstrate that while the apicoplast of blood‐stage P. falciparum parasites can be disrupted, the resulting vesicles remain biochemically active and are capable of fulfilling essential functions.     

Early gametocytes of the malaria parasite Plasmodium falciparum specifically remodel the adhesive properties of infected erythrocyte surface

In Plasmodium falciparum infections the parasite transmission stages, the gametocytes, mature in 10 days sequestered in internal organs. Recent studies suggest that cell mechanical properties rather than adhesive interactions play a role in sequestration during gametocyte maturation. It remains instead obscure how sequestration is established, and how the earliest sexual stages, morphologically similar to asexual trophozoites, modify the infected erythrocytes and their cytoadhesive properties at the onset of gametocytogenesis. Here, purified P. falciparum early gametocytes were used to ultrastructurally and biochemically analyse parasite‐induced modifications on the red blood cell surface and to measure their functional consequences on adhesion to human endothelial cells. This work revealed that stage I gametocytes are able to deform the infected erythrocytes like asexual parasites, but do not modify its surface with adhesive ‘knob’ structures and associated proteins. Reduced levels of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins are exposed on the red blood cell surface bythese parasites, and the expression of the var gene family, which encodes 50–60 variants of PfEMP1, is dramatically downregulated in the transition from asexual development to gametocytogenesis. Cytoadhesion assays show that such gene expression changes and host cell surface modifications functionally result in the inability of stage I gametocytes to bind the host ligands used by the asexual parasite to bind endothelial cells. In conclusion, these results identify specific differences in molecular and cellular mechanisms of host cell remodelling and in adhesive properties, leading to clearly distinct host parasite interplays in the establishment of sequestration of stage I gametocytes and of asexual trophozoites.     

PfCCp proteins of Plasmodium falciparum: gametocyte-specific expression and role in complement-mediated inhibition of exflagellation

The sexual phase of the malaria parasite Plasmodium falciparum is essential for transmission of the disease and is accompanied by the co-ordinated expression of sexual stage proteins. Six of these proteins belong to a highly conserved apicomplexan family of multi-domain adhesion proteins, termed PfCCps. PfCCp1, PfCCp2 and PfCCp3 are co-dependently expressed in the parasitophorous vacuole associated with the gametocyte plasma membrane. PfCCp2 and PfCCp3 also play an essential role for parasite development in the mosquito. We show that the six PfCCp proteins are expressed in stages II-V of gametocytogenesis as well as during early gamete formation. The proteins are expressed in association with the surface of both male and female gametocytes and macrogametes, but are not present in exflagellating microgametes. Further, the newly described protein PfCCp4 co-localizes with the transmission blocking candidate Pfs230, with which it forms a protein complex. In contrast to the phenotypes that are observed following targeted gene disruption of PfCCp2, PfCCp3 or Pfs230, the lack of PfCCp4 expression does not inhibit parasite development in the mosquito vector. This indicates a non-essential role for this protein during parasite transmission. Exflagellation assays revealed that antibodies directed against distinct domains of PfCCp1 through PfCCp4 and PfFNPA support a complement-mediated decrease in gametocyte emergence. We conclude that the six PfCCp proteins are specifically expressed during gametocytogenesis and gamete formation, and that select members may represent prospective candidates for transmission blocking vaccines.     

Targeting and sequestration of truncated Pfs230 in an intraerythrocytic compartment during Plasmodium falciparum gametocytogenesis

For malaria to be transmitted, the Plasmodium falciparum parasite must invade an erythrocyte and undergo gametocytogenesis. When mature intraerythrocytic gametocytes are taken up in a blood meal by a mosquito they emerge as gametes and, once fertilized, continue to differentiate into infectious sporozoites. One of the major proteins associated with the surface of the parasite during gamete differentiation is Pfs230, a 360 kDa member of a family of P. falciparum proteins that contains a repeated cysteine motif domain. To characterize the role of different regions of Pfs230, the gene was disrupted by targeted integration and clones isolated that expressed distinct sections of Pfs230. Independent clones D1.356 a and b express the first 452 amino acids (aa) of Pfs230 and do not contain a cysteine motif domain, whereas clones D2.850 a and b express the first 950 aa, including the first cysteine motif domain. Although both sets of clones undergo gametogenesis and produce morphologically normal gametes, neither truncated Pfs230 is located on the surface of the gamete. In clones D1.356 a and b, the 452 aa Pfs230 is secreted into the parasitophorous vacuole and released as a soluble protein when the parasite emerges from the erythrocyte as a gamete. In marked contrast, the 950 aa form of Pfs230 expressed by clones D2.850 a and b is sequestered in a novel tubular compartment in the erythrocyte cytoplasm. This sexual-stage tubular intraerythrocytic compartment (STIC) is not recognized by antibodies specific for proteins associated with the parasitophorous vacuole membrane (Pfs16 or Exp-1) or Maurer's clefts (Pfsbp 1 or mAb LWL1) or intraerythrocytic asexual parasite proteins (PfEMP2 or HRP II).     

Gametocytes: insights gained during a decade of molecular monitoring

In vertebrate hosts, malaria parasites produce specialized male and female sexual stages (gametocytes). Soon after being taken up by a mosquito, gametocytes rapidly produce gametes and, once mated, they infect their vector and can be transmitted to new hosts. Despite being the parasite stages that were first identified (over a century ago), gametocytes have remained elusive, and basic questions remain concerning their biology. However, the postgenomic era has substantiated information on the specialized molecular machinery of gametocytogenesis and expedited the development of molecular tools to detect and quantify gametocytes. The application of such highly sensitive and specific tools has opened up novel approaches and provided new insights into gametocyte biology. Here, we review the discoveries made during the past decade, highlight unanswered questions and suggest new directions.     

Functional characterization of both MAP kinases of the human malaria parasite Plasmodium falciparum by reverse genetics

The kinome of the human malaria parasite Plasmodium falciparum includes two genes encoding mitogen-activated protein kinase (MAPK) homologues, pfmap-1 and pfmap-2, but no clear orthologue of the MAPK kinase (MAPKK) family, raising the question of the mode of activation and function of the plasmodial MAPKs. Functional studies in the rodent malaria model Plasmodium berghei recently showed the map-2 gene to be dispensable for asexual growth and gametocytogenesis, but essential for male gametogenesis in the mosquito vector. Here, we demonstrate by using a reverse genetics approach that the map-2 gene is essential for completion of the asexual cycle of P. falciparum, an unexpected result in view of the non-essentiality of the orthologous gene for P. berghei erythrocytic schizogony. This validates Pfmap-2 as a potential target for chemotherapeutic intervention. In contrast, the other P. falciparum MAPK, Pfmap-1, is required neither for in vitro schizogony and gametocytogenesis in erythrocytes, nor for gametogenesis and sporogony in the mosquito vector. However, Pfmap-2 protein levels are elevated in pfmap-1(-) parasites, suggesting that Pfmap-1 fulfils an important function in asexual parasites that necessitates compensatory adaptation in parasites lacking this enzyme.     

Pbcrk-1, the Plasmodium berghei orthologue of P. falciparum cdc-2 related kinase-1 (Pfcrk-1), is essential for completion of the intraerythrocytic asexual cycle

The molecular mechanisms underlying gametocytogenesis in malaria parasites are not understood. Plasmodium falciparum cdc2-related kinase 1 (pfcrk-1), a gene that is expressed predominantly in gametocytes, bears homology to the PITSLRE subfamily of cyclin-dependent kinases and has been hypothesized to function as a negative regulator of the cell cycle. We attempted to knock-out pbcrk-1, the P. berghei orthologue of pfcrk-1, but were unable to recover P. berghei parasites with a disrupted pbcrk-1 locus. In contrast, an integration event at this locus that did not result in a loss-of-function of the pbcrk-1 gene was readily observed. This strongly suggests that a functional pbcrk-1 gene product is essential to intraerythrocytic asexual multiplication.     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> apicoplast and its transcriptional regulation through calcium signaling

Malaria has been present since ancient time and remains a major global health problem in developing countries. Plasmodium falciparum belongs to the phylum Apicomplexan, largely contain disease-causing parasites and characterized by the presence of apicoplast. It is a very essential organelle of P. falciparum responsible for the synthesis of key molecules required for the growth of the parasite. Indispensable nature of apicoplast makes it a potential drug target. Calcium signaling is important in the establishment of malaria parasite inside the host. It has been involved in invasion and egress of merozoites during the asexual life cycle of the parasite. Calcium signaling also regulates apicoplast metabolism. Therefore, in this review, we will focus on the role of apicoplast in malaria biology and its metabolic regulation through Ca⁺⁺ signaling.     

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.     

Falciparum malaria in naturally infected human patients: I. Ultrastructural differences between malaria pigments in intraerythrocytic asexual and sexual forms

Venous blood samples were taken from patients naturally infected with the human malaria parasite Plasmodium falciparum. Two types of malaria pigment (MP) particles have been demonstrated in intraerythrocytic asexual forms (trophozoites and schizonts), while a single type was detected in gametocytes. Type I MP particles, found in both asexual and sexual forms, are electron-dense. It is suggested that these are proteinaceous and may be intermediate, utilizable metabolic products that serve as a food reserve during development of the parasite in the human host and also during the growth cycle of the sexual form in the mosquito. In asexual forms, type I particles occur within food vacuoles (FV) containing semidigested hemoglobin (Hg), while they are unenveloped in the cytoplasm of the sexual forms. Type II MP particles, found in electron-lucent residual bodies, are crystalloid and of low electron density. It is suggested that these are the final, waste product of Hg digestion in the asexual forms. © 1993 Wiley-Liss, Inc.     

Plasmodium falciparum LipB mutants display altered redox and carbon metabolism in asexual stages and cannot complete sporogony in Anopheles mosquitoes

Malaria is still one of the most important global infectious diseases. Emergence of drug resistance and a shortage of new efficient antimalarials continue to hamper a malaria eradication agenda. Malaria parasites are highly sensitive to changes in the redox environment. Understanding the mechanisms regulating parasite redox could contribute to the design of new drugs. Malaria parasites have a complex network of redox regulatory systems housed in their cytosol, in their mitochondrion and in their plastid (apicoplast). While the roles of enzymes of the thioredoxin and glutathione pathways in parasite survival have been explored, the antioxidant role of α-lipoic acid (LA) produced in the apicoplast has not been tested. To take a first step in teasing a putative role of LA in redox regulation, we analysed a mutant Plasmodium falciparum (3D7 strain) lacking the apicoplast lipoic acid protein ligase B (lipB) known to be depleted of LA. Our results showed a change in expression of redox regulators in the apicoplast and the cytosol. We further detected a change in parasite central carbon metabolism, with lipB deletion resulting in changes to glycolysis and tricarboxylic acid cycle activity. Further, in another Plasmodium cell line (NF54), deletion of lipB impacted development in the mosquito, preventing the detection of infectious sporozoite stages. While it is not clear at this point if the observed phenotypes are linked, these findings flag LA biosynthesis as an important subject for further study in the context of redox regulation in asexual stages, and point to LipB as a potential target for the development of new transmission drugs.