Parasite plastids: maintenance and functions

Malaria and related parasites retain a vestigial, but biosynthetically active, plastid organelle acquired far back in evolution from a red algal cell. The organelle appears to be essential for parasite transmission from cell to cell and carries the smallest known plastid genome. Why has this genome been retained? The genes it carries seem to be dedicated to the expression of just two "housekeeping" genes. We speculate that one of these, called ycf24 in plants and sufB in bacteria, is tied to an essential "dark" reaction of the organelle--fatty acid biosynthesis. "Ball-park" clues to the function of bacterial suf genes have emerged only recently and point to the areas of iron homeostasis, [Fe-S] cluster formation and oxidative stress. We present experimental evidence for a physical interaction between SufB and its putative partner SufC (ycf16). In both malaria and plants, SufC is encoded in the nucleus and specifies an ATPase that is imported into the plastid.     

Nucleus-encoded genes for plastid-targeted proteins in Helicosporidium: functional diversity of a cryptic plastid in a parasitic alga

Plastids are the organelles of plants and algae that house photosynthesis and many other biochemical pathways. Plastids contain a small genome, but most of their proteins are encoded in the nucleus and posttranslationally targeted to the organelle. When plants and algae lose photosynthesis, they virtually always retain a highly reduced "cryptic" plastid. Cryptic plastids are known to exist in many organisms, although their metabolic functions are seldom understood. The best-studied example of a cryptic plastid is from the intracellular malaria parasite, Plasmodium, which has retained a plastid for the biosynthesis of fatty acids, isoprenoids, and heme by the use of plastid-targeted enzymes. To study a completely independent transformation of a photosynthetic plastid to a cryptic plastid in another alga-turned-parasite, we conducted an expressed sequence tag (EST) survey of Helicosporidium. This parasite has recently been recognized as a highly derived green alga. Based on phylogenetic relationships to other plastid homologues and the presence of N-terminal transit peptides, we have identified 20 putatively plastid-targeted enzymes that are involved in a wide variety of metabolic pathways. Overall, the metabolic diversity of the Helicosporidium cryptic plastid exceeds that of the Plasmodium plastid, as it includes representatives of most of the pathways known to operate in the Plasmodium plastid as well as many others. In particular, several amino acid biosynthetic pathways have been retained, including the leucine biosynthesis pathway, which was only recently recognized in plant plastids. These two parasites represent different evolutionary trajectories in plastid metabolic adaptation.     

Impact of a plastid-bearing endocytobiont on apicomplexan genomes

Both the chromosomal and extrachromosomal components of the apicomplexan genome have been supplemented by genes from a plastid-bearing endocytobiont: probably an algal cell. The sequence of the apicomplexan plastid's vestigial genome indicates that a large number (>100) of genes of endocytobiotic origin must have transferred laterally to the host cell nucleus where they control maintenance of the plastid organelle and supply its functional components by means of post-translational protein trafficking. Should the nuclear genes prove to be less divergent phylogenetically than those left on the plastid genome, they might give better clues than we have at present to the origin of the plastid-bearing endocytobiont. Most of these nuclear genes still await discovery, but the on-going genome sequencing project will reveal the function of the organelle, as well as many "housekeeping" processes of interest on a wider front. The plastid's own protein synthetic machinery, being cyanobacterial in origin, offers conventional targets for antibiotic intervention, and this is discussed here using a structural model of elongation factor Tu. Uncovering the vital function(s) of the plastid organelle will provide new drug targets.     

Phylogenomic evidence supports past endosymbiosis, intracellular and horizontal gene transfer in Cryptosporidium parvum

Background

The apicomplexan parasite Cryptosporidium parvum is an emerging pathogen capable of causing illness in humans and other animals and death in immunocompromised individuals. No effective treatment is available and the genome sequence has recently been completed. This parasite differs from other apicomplexans in its lack of a plastid organelle, the apicoplast. Gene transfer, either intracellular from an endosymbiont/donor organelle or horizontal from another organism, can provide evidence of a previous endosymbiotic relationship and/or alter the genetic repertoire of the host organism. Given the importance of gene transfers in eukaryotic evolution and the potential implications for chemotherapy, it is important to identify the complement of transferred genes in Cryptosporidium.

Results

We have identified 31 genes of likely plastid/endosymbiont (n = 7) or prokaryotic (n = 24) origin using a phylogenomic approach. The findings support the hypothesis that Cryptosporidium evolved from a plastid-containing lineage and subsequently lost its apicoplast during evolution. Expression analyses of candidate genes of algal and eubacterial origin show that these genes are expressed and developmentally regulated during the life cycle of C. parvum.

Conclusions

Cryptosporidium is the recipient of a large number of transferred genes, many of which are not shared by other apicomplexan parasites. Genes transferred from distant phylogenetic sources, such as eubacteria, may be potential targets for therapeutic drugs owing to their phylogenetic distance or the lack of homologs in the host. The successful integration and expression of the transferred genes in this genome has changed the genetic and metabolic repertoire of the parasite.     

Biogenesis and maintenance of the apicoplast in model apicomplexan parasites

The apicoplast is a non-photosynthetic relict plastid of Apicomplexa that evolved from a secondary symbiotic system. During its evolution, most of the genes derived from its alga ancestor were lost. Only genes involved in several valuable metabolic pathways, such as the synthesis of isoprenoid precursors, heme, and fatty acids, have been transferred to the host genome and retained to help these parasites adapt to a complex life cycle and various living environments. The biological function of an apicoplast is essential for most apicomplexan parasites. Considering their potential as drug targets, the metabolic functions of this symbiotic organelle have been intensively investigated through computational and biological means. Moreover, we know that not only organellar metabolic functions are linked with other organelles, but also their biogenesis processes have developed and evolved to tailor their biological functions and proper inheritance. Several distinct features have been found in the biogenesis process of apicoplasts. For example, the apicoplast borrows a dynamin-related protein (DrpA) from its host to implement organelle division. The autophagy system has also been repurposed for linking the apicoplast and centrosome during replication and the division process. However, many vital questions remain to be answered about how these parasites maintain and properly inherit this symbiotic organelle. Here we review our current knowledge about its biogenesis process and discuss several critical questions remaining to be answered in this field.     

Plastid genes in a non-photosynthetic dinoflagellate

Dinoflagellates are a diverse group of protists, comprising photosynthetic and heterotrophic free-living species, as well as parasitic ones. About half of them are photosynthetic with peridinin-containing plastids being the most common. It is uncertain whether non-photosynthetic dinoflagellates are primitively so, or have lost photosynthesis. Studies of heterotrophic species from this lineage may increase our understanding of plastid evolution. We analyzed an EST project of the early-diverging heterotrophic dinoflagellate Crypthecodinium cohnii looking for evidence of past endosymbiosis. A large number of putative genes of cyanobacterial or algal origin were identified using BLAST, and later screened by metabolic function. Phylogenetic analyses suggest that several proteins could have been acquired from a photosynthetic endosymbiont, arguing for an earlier plastid acquisition in dinoflagellates. In addition, intact N-terminal plastid-targeting peptides were detected, indicating that C. cohnii may contain a reduced plastid and that some of these proteins are imported into this organelle. A number of metabolic pathways, such as heme and isoprenoid biosynthesis, seem to take place in the plastid. Overall, these data indicate that C. cohnii is derived from a photosynthetic ancestor and provide a model for loss of photosynthesis in dinoflagellates and their relatives. This represents the first extensive genomic analysis of a heterotrophic dinoflagellate.     

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.     

Enzymes for heme biosynthesis are found in both the mitochondrion and plastid of the malaria parasite Plasmodium falciparum

All eight enzymes required for de novo heme biosynthesis have been predicted from the nuclear genome of the human malaria parasite Plasmodium falciparum. We have studied the subcellular localization of three of these using a GFP reporter in live transfected parasites. The first enzyme in the pathway d-aminolevulinic acid synthase (ALAS) is targeted to the mitochondrion, but the next two enzymes porphobilinogen synthase (PBGS) and hydroxymethylbilane synthase (HMBS) are targeted to the plastid. An enzymatically active recombinant version of PBGS from P. falciparum was over-expressed and its activity found to be stimulated by Mg²⁺(and enhanced by Mn²⁺) but not by Zn²⁺. A hypothetical scheme for the exchange of intermediates in heme biosynthesis between the mitochondrion and plastid organelle, as well as organelle attachment is discussed.     

The methylerythritol phosphate pathway for isoprenoid biosynthesis in coccidia: presence and sensitivity to fosmidomycin

The apicoplast is a recently discovered, plastid-like organelle present in most apicomplexa. The methylerythritol phosphate (MEP) pathway involved in isoprenoid biosynthesis is one of the metabolic pathways associated with the apicoplast, and is a new promising therapeutic target in Plasmodium falciparum. Here, we check the presence of isoprenoid genes in four coccidian parasites according to genome database searches. Cryptosporidium parvum and C. hominis, which have no plastid genome, lack the MEP pathway. In contrast, gene expression studies suggest that this metabolic pathway is present in several development stages of Eimeria tenella and in tachyzoites of Toxoplasma gondii. We studied the potential of fosmidomycin, an antimalarial drug blocking the MEP pathway, to inhibit E. tenella and T. gondii growth in vitro. The drug was poorly effective even at high concentrations. Thus, both fosmidomycin sensitivity and isoprenoid metabolism differs substantially between apicomplexan species.     

Tertiary endosymbiosis driven genome evolution in dinoflagellate algae

Dinoflagellates are important aquatic primary producers and cause "red tides." The most widespread plastid (photosynthetic organelle) in these algae contains the unique accessory pigment peridinin. This plastid putatively originated via a red algal secondary endosymbiosis and has some remarkable features, the most notable being a genome that is reduced to 1-3 gene minicircles with about 14 genes (out of an original 130-200) remaining in the organelle and a nuclear-encoded proteobacterial Form II Rubisco. The "missing" plastid genes are relocated to the nucleus via a massive transfer unequaled in other photosynthetic eukaryotes. The fate of these characters is unknown in a number of dinoflagellates that have replaced the peridinin plastid through tertiary endosymbiosis. We addressed this issue in the fucoxanthin dinoflagellates (e.g., Karenia brevis) that contain a captured haptophyte plastid. Our multiprotein phylogenetic analyses provide robust support for the haptophyte plastid replacement and are consistent with a red algal origin of the chromalveolate plastid. We then generated an expressed sequence tag (EST) database of 5,138 unique genes from K. brevis and searched for nuclear genes of plastid function. The EST data indicate the loss of the ancestral peridinin plastid characters in K. brevis including the transferred plastid genes and Form II Rubisco. These results underline the remarkable ability of dinoflagellates to remodel their genomes through endosymbiosis and the considerable impact of this process on cell evolution.     

The unusual mitochondrial compartment of Cryptosporidium parvum

Recent studies, including the Cryptosporidium parvum Genome Project, have provided evidence for a mitochondrial-derived compartment in this parasite. This organelle appears to lack a genome, and thus must be entirely dependent on nuclear-encoded proteins. Here, we review the evidence for such an organelle in C. parvum and its probable function. There is no adequate treatment for infection by this parasite and so the elucidation of the role of this organelle and the effective targeting of its functions by antimicrobial agents might provide new treatments for infection by C. parvum.     

Metabolic maps and functions of the Plasmodium mitochondrion

The mitochondrion of Plasmodium species is a validated drug target. However, very little is known about the functions of this organelle. In this review, we utilize data available from the Plasmodium falciparum genome sequencing project to piece together putative metabolic pathways that occur in the parasite, comparing this with the existing biochemical and cell biological knowledge. The Plasmodium mitochondrion contains both conserved and unusual features, including an active electron transport chain and many of the necessary enzymes for coenzyme Q and iron-sulphur cluster biosynthesis. It also plays an important role in pyrimidine metabolism. The mitochondrion participates in an unusual hybrid haem biosynthesis pathway, with enzymes localizing in both the mitochondrion and plastid organelles. The function of the tricarboxylic acid cycle in the mitochondrion is unclear. We discuss directions for future research into this fascinating, yet enigmatic, organelle.     

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.     

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.     

The plastid in Apicomplexa: what use is it?

An extrachromosomal genome of between 27 and 35 kb has been described in several apicomplexan parasites including Plasmodium falciparum and Toxoplasma gondii. Examination of sequence data proved the genomes to be a remnant plastid genome, from which all genes encoding photosynthetic functions had been lost. Localisation studies had shown that the genome was located within a multi-walled organelle, anterior to the nucleus. This organelle had been previously described in ultrastructural studies of several genera of apicomplexa, but no function had been attributed to it. This invited review describes the evolution of knowledge on the apicomplexan plastid, then discusses current research findings on the likely role of the plastid in the Apicomplexa. How the plastid may be used to effect better drug treatments for apicomplexan diseases, and its potential as a marker for investigating phylogenetic relationships among the Apicomplexa, are discussed.     

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.     

The search for the missing link: a relic plastid in Perkinsus?

Perkinsus marinus (Phylum Perkinsozoa) is a protozoan parasite that has devastated natural and farmed oyster populations in the USA, significantly affecting the shellfish industry and the estuarine environment. The other two genera in the phylum, Parvilucifera and Rastrimonas, are parasites of microeukaryotes. The Perkinsozoa occupies a key position at the base of the dinoflagellate branch, close to its divergence from the Apicomplexa, a clade that includes parasitic protista, many harbouring a relic plastid. Thus, as a taxon that has also evolved toward parasitism, the Perkinsozoa has attracted the attention of biologists interested in the evolution of this organelle, both in its ultrastructure and the conservation, loss or transfer of its genes. A review of the recent literature reveals mounting evidence in support of the presence of a relic plastid in P. marinus, including the presence of multimembrane structures, characteristic metabolic pathways and proteins with a bipartite N-terminal extension. Further, these findings raise intriguing questions regarding the potential functions and unique adaptation of the putative plastid and/or plastid genes in the Perkinsozoa. In this review we analyse the above-mentioned evidence and evaluate the potential future directions and expected benefits of addressing such questions. Given the rapidly expanding molecular/genetic resources and methodological toolbox for Perkinsus spp., these organisms should complement the currently established models for investigating plastid evolution within the Chromalveolata.     

The apicoplast: a review of the derived plastid of apicomplexan parasites

The apicoplast is a plastid organelle, homologous to chloroplasts of plants, that is found in apicomplexan parasites such as the causative agents of Malaria Plasmodium spp. It occurs throughout the Apicomplexa and is an ancient feature of this group acquired by the process of endosymbiosis. Like plant chloroplasts, apicoplasts are semi-autonomous with their own genome and expression machinery. In addition, apicoplasts import numerous proteins encoded by nuclear genes. These nuclear genes largely derive from the endosymbiont through a process of intracellular gene relocation. The exact role of a plastid in parasites is uncertain but early clues indicate synthesis of lipids, heme and isoprenoids as possibilities. The various metabolic processes of the apicoplast are potentially excellent targets for drug therapy.     

Reduction and compaction in the genome of the apicomplexan parasite Cryptosporidium parvum

The complete genome of the apicomplexan parasite Cryptosporidium parvum reveals many new insights into apicomplexan biology and evolution, as well as the general process of genome reduction in parasites. The genome is globally compacted, but gene loss seems to be focused, in particular in relation to organelles. Massive losses of mitochondrial genes have taken place and there is no evidence of any plastid-related genes, providing a useful tool for examining putative plastid proteins in Plasmodium and other apicomplexans.