GFP‐targeting allows visualization of the apicoplast throughout the life cycle of live malaria parasites

Background information. The Plasmodium parasite, during its life cycle, undergoes three phases of asexual reproduction, these being repeated rounds of erythrocytic schizogony, sporogony within oocysts on the mosquito midgut wall and exo‐erythrocytic schizogony within the hepatocyte. During each phase of asexual reproduction, the parasite must ensure that every new daughter cell contains an apicoplast, as this organelle cannot be formed de novo and is essential for parasite survival. To date, studies visualizing the apicoplast in live Plasmodium parasites have been restricted to the blood stages of Plasmodium falciparum. Results. In the present study, we have generated Plasmodium berghei parasites in which GFP (green fluorescent protein) is targeted to the apicoplast using the specific targeting sequence of ACP (acyl carrier protein), which has allowed us to visualize this organelle in live Plasmodium parasites. During each phase of asexual reproduction, the apicoplast becomes highly branched, but remains as a single organelle until the completion of nuclear division, whereupon it divides and is rapidly segregated into newly forming daughter cells. We have shown that the antimicrobial agents azithromycin, clindamycin and doxycycline block development of the apicoplast during exo‐erythrocytic schizogony in vitro, leading to impaired parasite maturation. Conclusions. Using a range of powerful live microscopy techniques, we show for the first time the development of a Plasmodium organelle through the entire life cycle of the parasite. Evidence is provided that interference with the development of the Plasmodium apicoplast results in the failure to produce red‐blood‐cell‐infective merozoites.     

Mitochondrial lipoic acid scavenging is essential for Plasmodium berghei liver stage development

Lipoic acid is an essential cofactor for enzymes that participate in key metabolic pathways in most organisms. While in mammalian cells lipoylated proteins reside exclusively in the mitochondria, apicomplexan parasites of the genus Plasmodium harbour two independent lipoylation pathways in the mitochondrion and the apicoplast, a second organelle of endosymbiotic origin. Protein lipoylation in the apicoplast relies on de novo lipoic acid synthesis while lipoylation of proteins in the mitochondrion depends on scavenging of lipoic acid from the host cell. Here, we analyse the impact of lipoic acid scavenging on the development of Plasmodium berghei liver stage parasites. Treatment of P. berghei-infected HepG2 cells with the lipoic acid analogue 8-bromo-octanoic acid (8-BOA) abolished lipoylation of mitochondrial enzyme complexes in the parasite while lipoylation of apicoplast proteins was not affected. Parasite growth as well as the ability of the parasites to successfully complete liver stage development by merosome formation were severely impaired but not completely blocked by 8-BOA. Liver stage parasites were most sensitive to 8-BOA treatment during schizogony, the phase of development when the parasite grows and undergoes extensive nuclear division to form a multinucleated syncytium. Live cell imaging as well as immunofluorescence analysis and electronmicroscopy studies revealed a close association of both host cell and parasite mitochondria with the parasitophorous vacuole membrane suggesting that host cell mitochondria might be involved in lipoic acid uptake by the parasite from the host cell.     

Type II fatty acid synthesis is essential only for malaria parasite late liver stage development

Intracellular malaria parasites require lipids for growth and replication. They possess a prokaryotic type II fatty acid synthesis (FAS II) pathway that localizes to the apicoplast plastid organelle and is assumed to be necessary for pathogenic blood stage replication. However, the importance of FAS II throughout the complex parasite life cycle remains unknown. We show in a rodent malaria model that FAS II enzymes localize to the sporozoite and liver stage apicoplast. Targeted deletion of FabB/F , a critical enzyme in fatty acid synthesis, did not affect parasite blood stage replication, mosquito stage development and initial infection in the liver. This was confirmed by knockout of FabZ , another critical FAS II enzyme. However, FAS II-deficient Plasmodium yoelii liver stages failed to form exo-erythrocytic merozoites, the invasive stage that first initiates blood stage infection. Furthermore, deletion of FabI in the human malaria parasite Plasmodium falciparum did not show a reduction in asexual blood stage replication in vitro . Malaria parasites therefore depend on the intrinsic FAS II pathway only at one specific life cycle transition point, from liver to blood.     

Enzymes of type II fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella

Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter formation.     

Replication and partitioning of the apicoplast genome of Toxoplasma gondii is linked to the cell cycle and requires DNA polymerase and gyrase

Apicomplexans are the causative agents of numerous important infectious diseases including malaria and toxoplasmosis. Most of them harbour a chloroplast-like organelle called the apicoplast that is essential for the parasites’ metabolism and survival. While most apicoplast proteins are nuclear encoded, the organelle also maintains its own genome, a 35 kb circle. In this study we used Toxoplasma gondii to identify and characterise essential proteins involved in apicoplast genome replication and to understand how apicoplast genome segregation unfolds over time. We demonstrated that the DNA replication enzymes Prex, DNA gyrase and DNA single stranded binding protein localise to the apicoplast. We show in knockdown experiments that apicoplast DNA Gyrase A and B, and Prex are required for apicoplast genome replication and growth of the parasite. Analysis of apicoplast genome replication by structured illumination microscopy in T. gondii tachyzoites showed that apicoplast nucleoid division and segregation initiate at the beginning of S phase and conclude during mitosis. Thus, the replication and division of the apicoplast nucleoid is highly coordinated with nuclear genome replication and mitosis. Our observations highlight essential components of apicoplast genome maintenance and shed light on the timing of this process in the context of the overall parasite cell cycle.     

Targeting malaria parasite at the level of apicoplast: an update

In 1996, the discovery of a relic chloroplast in Plasmodium and Toxoplasma cells has strongly changed our vision of these parasites in the "Tree of Life", and has opened an unexpected new field of investigation in the search for antiparasitic treatments, including antimalarials. This review details our current understanding of the sophisticated evolution of the parasites of the Apicomplexa phylum and briefly covers a decade of research and development in drug discovery, trying to target the malaria parasite at the level of its plant-like organelle. Fifteen years after the discovery of the apicoplast and ten years after the publication of the genome of Plasmodium falciparum, it seems that we have completed a first phase of tests of available antibiotics and herbicides. In the human host, the liver phase is the only parasitic stage, for which biological functions harbored by the apicoplast, such as fatty acid biosynthesis, seem indispensable. During the erythrocytic phase, recent results have focused the attention on the processes controlling the biogenesis of the apicoplast, and one of the functions harbored by the apicoplast, i.e. the biosynthesis of isoprenoids, as major -promising targets for future treatments.     

Targeting and processing of nuclear-encoded apicoplast proteins in plastid segregation mutants of Toxoplasma gondii

The apicoplast is a distinctive organelle associated with apicomplexan parasites, including Plasmodium sp. (which cause malaria) and Toxoplasma gondii (the causative agent of toxoplasmosis). This unusual structure (acquired by the engulfment of an ancestral alga and retention of the algal plastid) is essential for long-term parasite survival. Similar to other endosymbiotic organelles (mitochondria, chloroplasts), the apicoplast contains proteins that are encoded in the nucleus and post-translationally imported. Translocation across the four membranes surrounding the apicoplast is mediated by an N-terminal bipartite targeting sequence. Previous studies have described a recombinant "poison" that blocks plastid segregation during mitosis, producing parasites that lack an apicoplast and siblings containing a gigantic, nonsegregating plastid. To learn more about this remarkable phenomenon, we examined the localization and processing of the protein produced by this construct. Taking advantage of the ability to isolate apicoplast segregation mutants, we also demonstrated that processing of the transit peptide of nuclear-encoded apicoplast proteins requires plastid-associated activity.     

<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.     

Inactivation of a Plasmodium apicoplast protein attenuates formation of liver merozoites

Malaria parasites undergo a population expansion inside the host liver before disease onset. Developmental arrest inside host hepatocytes elicits protective immune responses. Therefore, elucidation of the molecular mechanisms leading to mature hepatic merozoites, which initiate the pathogenic blood phase, also informs anti-malaria vaccine strategies. Using targeted gene deletion in the rodent model malaria parasite Plasmodium berghei, we show that a Plasmodium-specific Apicoplast protein plays an important role for Liver Merozoite formation (PALM). While the resulting knockout mutants develop normally for most of the life cycle, merozoite release into the blood stream and the ability to establish an infection are severely impaired. Presence of a signature blood-stage antigen, merozoite surface protein 1 and normal apicoplast morphology indicate that the inability to finalize merozoite segregation is a direct consequence of loss of PALM function. Experimental immunization of mice with as few as two doses of palm(-) sporozoites can elicit sterile protection up to 110 days after final immunization. Our data establish that a tailor-made arrest in the final steps of hepatic merozoite formation can induce strong protective immune responses and that malaria parasites employ a distinct apicoplast protein for efficient formation of pre-erythrocytic merozoites.     

Plasmodium salvages cholesterol internalized by LDL and synthesized de novo in the liver

Our previous morphological studies illustrated the association of sterols with Plasmodium infecting hepatocytes. Because malaria parasites cannot synthesize sterols, they must scavenge these lipids from the host. In this paper, we have examined the source/s of sterols for intrahepatic Plasmodium and evaluated the importance of sterols for liver stage development. We show that Plasmodium continuously diverts cholesterol from hepatocytes until release of merozoites. Removal of plasma lipoproteins from the medium results in a 70% reduction of cholesterol content in hepatic merozoites but these parasites remain infectious in animals. Plasmodium salvages cholesterol that has been internalized by low-density lipoprotein but reduced expression of host low-density lipoprotein receptors by 70% does not influence liver stage burden. Plasmodium is also able to intercept cholesterol synthesized by hepatocytes. Pharmacological blockade of host squalene synthase or downregulation of the expression of this enzyme by 80% decreases by twofold the cholesterol content of merozoites without further impacting parasite development. These data enlighten that, on one hand, malaria parasites have moderate need of sterols for optimal development in hepatocytes and, on the other hand, they can adapt to survive in cholesterol-restrictive conditions by exploitation of accessible sterols derived from alternative sources in hepatocytes to maintain proper infectivity.     

Plasmodium pyruvate dehydrogenase activity is only essential for the parasite's progression from liver infection to blood infection

Plasmodium parasites possess a single pyruvate dehydrogenase (PDH) enzyme complex that is localized to the plastid‐like organelle known as the apicoplast. Unlike most eukaryotes, Plasmodium parasites lack a mitochondrial PDH. The PDH complex catalyses the conversion of pyruvate to acetyl‐CoA, an important precursor for the tricarboxylic acid cycle and type II fatty acid synthesis (FAS II). In this study, using a rodent malaria model, we show that the PDH E1α and E3 subunits colocalize with the FAS II enzyme FabI in the apicoplast of liver stages but are not significantly expressed in blood stages. Deletion of the E1α or E3 subunit genes of Plasmodium yoelii PDH caused no defect in blood stage development, mosquito stage development or early liver stage development. However, the gene deletions completely blocked the ability of the e1α^‐ and e3^‐ parasites to form exo‐erythrocytic merozoites during late liver stage development, thus preventing the initiation of a blood stage infection. This phenotype is similar to that observed for deletions of genes involved in FAS II elongation. The data strongly support the hypothesis that the sole role of PDH is to provide acetyl‐CoA for FAS II.     

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.     

Simulated microgravity perturbs actin polymerization to promote nitric oxide-associated migration in human immortalized Eahy926 cells

Apicomplexan parasites obligatorily invade and multiply within eukaryotic cells. Phylogenetically, they are related to a group of algae which, during their evolution, have acquired a secondary endosymbiont. This organelle, which in the parasite is called the apicoplast, is highly reduced compared to the endosymbionts of algae, but still contains many plant-specific biosynthetic pathways. The malaria parasite Plasmodium falciparum infects mammalian erythrocytes which are devoid of intracellular compartments and which largely lack biosynthetic pathways. Despite the limited resources of nutrition, the parasite grows and generates up to 32 merozoites which are the infectious stages of the complex life cycle. A large part of the intra-erythrocytic development takes place in the so-called parasitophorous vacuole, a compartment which forms an interface between the parasite and the cytoplasm of the host cell. In the course of parasite growth, the host cell undergoes dramatic alterations which on one hand contribute directly to the symptoms of severe malaria and which, on the other hand, are also required for parasite survival. Some of these alterations facilitate the acquisition of nutrients from the extracellular environment which are not provided by the host cell. Here, we describe the cell biologically unique interactions between an intracellular eukaryotic pathogen and its metabolically highly reduced host cell. We further discuss current models to explain the appearance of pathogen-induced novel physiological properties in a host cell which has lost its genetic programme.     

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.     

Experimental Genetics of Plasmodium berghei NFU in the Apicoplast Iron-Sulfur Cluster Biogenesis Pathway

Eukaryotic pathogens of the phylum Apicomplexa contain a non-photosynthetic plastid, termed apicoplast. Within this organelle distinct iron-sulfur [Fe-S] cluster proteins are likely central to biosynthesis pathways, including generation of isoprenoids and lipoic acid. Here, we targeted a nuclear-encoded component of the apicoplast [Fe-S] cluster biosynthesis pathway by experimental genetics in the murine malaria parasite Plasmodium berghei. We show that ablation of the gene encoding a nitrogen fixation factor U (NifU)-like domain containing protein (NFUapi) resulted in parasites that were able to complete the entire life cycle indicating redundant or non-essential functions. nfu ^– parasites displayed reduced merosome formation in vitro, suggesting that apicoplast NFUapi plays an auxiliary role in establishing a blood stage infection. NFUapi fused to a combined fluorescent protein-epitope tag delineates the Plasmodium apicoplast and was tested to revisit inhibition of liver stage development by azithromycin and fosmidomycin. We show that the branched apicoplast signal is entirely abolished by azithromycin treatment, while fosmidomycin had no effect on apicoplast morphology. In conclusion, our experimental genetics analysis supports specialized and/or redundant role(s) for NFUapi in the [Fe-S] cluster biosynthesis pathway in the apicoplast of a malarial parasite.     

Selective inhibition of a two-step egress of malaria parasites from the host erythrocyte

Escape from the host erythrocyte by the invasive stage of the malaria parasite Plasmodium falciparum is a fundamental step in the pathogenesis of malaria of which little is known. Upon merozoite invasion of the host cell, the parasite becomes enclosed within a parasitophorous vacuole, the compartment in which the parasite undergoes growth followed by asexual division to produce 16-32 daughter merozoites. These daughter cells are released upon parasitophorous vacuole and erythrocyte membrane rupture. To examine the process of merozoite release, we used P. falciparum lines expressing green fluorescent protein-chimeric proteins targeted to the compartments from which merozoites must exit: the parasitophorous vacuole and the host erythrocyte cytosol. This allowed visualization of merozoite release in live parasites. Herein we provide the first evidence in live, untreated cells that merozoite release involves a primary rupture of the parasitophorous vacuole membrane followed by a secondary rupture of the erythrocyte plasma membrane. We have confirmed, with the use of immunoelectron microscopy, that parasitophorous vacuole membrane rupture occurs before erythrocyte plasma membrane rupture in untransfected wild-type parasites. We have also demonstrated selective inhibition of each step in this two-step process of exit using different protease inhibitors, implicating the involvement of distinct proteases in each of these steps. This will facilitate the identification of the parasite and host molecules involved in merozoite release.     

The Plasmodium Apicoplast Genome: Conserved Structure and Close Relationship of P. ovale to Rodent Malaria Parasites

Apicoplast, a nonphotosynthetic plastid derived from secondary symbiotic origin, is essential for the survival of malaria parasites of the genus Plasmodium. Elucidation of the evolution of the apicoplast genome in Plasmodium species is important to better understand the functions of the organelle. However, the complete apicoplast genome is available for only the most virulent human malaria parasite, Plasmodium falciparum. Here, we obtained the near-complete apicoplast genome sequences from eight Plasmodium species that infect a wide variety of vertebrate hosts and performed structural and phylogenetic analyses. We found that gene repertoire, gene arrangement, and other structural attributes were highly conserved. Phylogenetic reconstruction using 30 protein-coding genes of the apicoplast genome inferred, for the first time, a close relationship between P. ovale and rodent parasites. This close relatedness was robustly supported using multiple evolutionary assumptions and models. The finding suggests that an ancestral host switch occurred between rodent and human Plasmodium parasites.     

A plastid segregation defect in the protozoan parasite Toxoplasma gondii

Apicomplexan parasites--including the causative agents of malaria (Plasmodium sp.) and toxoplasmosis (Toxoplasma gondii)--harbor a secondary endosymbiotic plastid, acquired by lateral genetic transfer from a eukaryotic alga. The apicoplast has attracted considerable attention, both as an evolutionary novelty and as a potential target for chemotherapy. We report a recombinant fusion (between a nuclear-encoded apicoplast protein, the green fluorescent protein and a rhoptry protein) that targets to the apicoplast but grossly alters its morphology, preventing organellar segregation during parasite division. Apicoplast-deficient parasites replicate normally in the first infectious cycle and can be isolated by fluorescence-activated cell sorting, but die in the subsequent host cell, confirming the 'delayed death' phenotype previously described pharmacologically, and validating the apicoplast as essential for parasite viability.     

Toxoplasma gondii myosin F,an essential motor for centrosomes positioning and apicoplast inheritance

Members of the Apicomplexa phylum possess an organelle surrounded by four membranes, originating from the secondary endosymbiosis of a red alga. This so‐called apicoplast hosts essential metabolic pathways. We report here that apicoplast inheritance is an actin‐based process. Concordantly, parasites depleted in either profilin or actin depolymerizing factor, or parasites overexpressing the FH2 domain of formin 2, result in loss of the apicoplast. The class XXII myosin F (MyoF) is conserved across the phylum and localizes in the vicinity of the Toxoplasma gondii apicoplast during division. Conditional knockdown of TgMyoF severely affects apicoplast turnover, leading to parasite death. This recapitulates the phenotype observed upon perturbation of actin dynamics that led to the accumulation of the apicoplast and secretory organelles in enlarged residual bodies. To further dissect the mode of action of this motor, we conditionally stabilized the tail of MyoF, which forms an inactive heterodimer with endogenous TgMyoF. This dominant negative mutant reveals a central role of this motor in the positioning of the two centrosomes prior to daughter cell formation and in apicoplast segregation.     

The plastid of Toxoplasma gondii is divided by association with the centrosomes

Apicomplexan parasites harbor a single nonphotosynthetic plastid, the apicoplast, which is essential for parasite survival. Exploiting Toxoplasma gondii as an accessible system for cell biological analysis and molecular genetic manipulation, we have studied how these parasites ensure that the plastid and its 35-kb circular genome are faithfully segregated during cell division. Parasite organelles were labeled by recombinant expression of fluorescent proteins targeted to the plastid and the nucleus, and time-lapse video microscopy was used to image labeled organelles throughout the cell cycle. Apicoplast division is tightly associated with nuclear and cell division and is characterized by an elongated, dumbbell-shaped intermediate. The plastid genome is divided early in this process, associating with the ends of the elongated organelle. A centrin-specific antibody demonstrates that the ends of dividing apicoplast are closely linked to the centrosomes. Treatment with dinitroaniline herbicides (which disrupt microtubule organization) leads to the formation of multiple spindles and large reticulate plastids studded with centrosomes. The mitotic spindle and the pellicle of the forming daughter cells appear to generate the force required for apicoplast division in Toxoplasma gondii. These observations are discussed in the context of autonomous and FtsZ-dependent division of plastids in plants and algae.