The plastid DNA of the malaria parasite Plasmodium falciparum is replicated by two mechanisms

In common with other apicomplexan parasites, Plasmodium falciparum, a causative organism of human malaria, harbours a residual plastid derived from an ancient secondary endosymbiotic acquisition of an alga. The function of the 35 kb plastid genome is unknown, but its evolutionary origin and genetic content make it a likely target for chemotherapy. Pulsed field gel electrophoresis and ionizing radiation have shown that essentially all the plastid DNA comprises covalently closed circular monomers, together with a tiny minority of linear 35 kb molecules. Using two-dimensional gels and electron microscopy, two replication mechanisms have been revealed. One, sensitive to the topoisomerase inhibitor ciprofloxacin, appears to initiate at twin D-loops located in a large inverted repeat carrying duplicated rRNA and tRNA genes, whereas the second, less drug sensitive, probably involves rolling circles that initiate outside the inverted repeat.     

Evolution of Cryptosporidium parvum lactate dehydrogenase from malate dehydrogenase by a very recent event of gene duplication

We have expressed the L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (malDH) genes from the apicomplexan Cryptosporidium parvum (CpLDH1 and CpMalDH1) as maltose-binding protein (MBP) fusion proteins in Escherichia coli. The substrate specificities, enzymatic kinetics, and oligomeric states of these two parasite enzymes have been characterized. By taking advantage of recently completed and ongoing apicomplexan genome sequencing projects, we identified additional MalDH genes from Plasmodium spp., Toxoplasma gondii, and Eimeria tenella that were previously unavailable. All apicomplexan MalDHs appeared to be cytosolic and no organellar homologs were identified from the completely sequenced P. falciparum genome and other ongoing apicomplexan genome-sequencing projects. Using these expanded apicomplexan LDH and MalDH sequence databases, we reexamined their phylogenetic relationships and reconfirmed their relationship to alpha-proteobacterial MalDHs. All LDH and MalDH enzymes from apicomplexans were monophyletic within the LDH-like MalDH group (i.e., MalDH resembling LDH) as a sister to alpha-proteobacterial MalDHs. All apicomplexan LDHs, with the exception of CpLDH1, formed a separate clade from their MalDH counterparts, indicating that these LDHs were evolved from an ancestral apicomplexan MalDH by a gene duplication coupled with functional conversion before the expansion of apicomplexans. Finally, CpLDH1 was consistently placed together with CpMalDH1 within the apicomplexan MalDH cluster, confirming an early working hypothesis that CpLDH1 was probably evolved from the same ancestor of CpMalDH1 by a very recent gene duplication that occurred after C. parvum diverged from other apicomplexans.     

Protein synthesis in the plastid of Plasmodium falciparum.

The plastid organelle of malarial and other apicomplexan parasites contains ribosome-like particles as well as a genome dedicated largely to specifying components of a protein expression system. We have identified plastid ribosomes using hybridization studies and show that in erythrocytic stages of Plasmodium falciparum a subset of polysomes carries plastid-specified rRNAs and mRNA, supporting the idea that protein synthesis is active in the plastid.     

Phylogeny of the Large Extrachromosomal DNA of Organisms in the Phylum Apicomplexa

ABSTRACT. Organisms in the phylum Apicomplexa appear to have a large extrachromosomal DNA which is unrelated to the mitochondrial DNA. Based on the apparent gene content of the large (35 kb) extrachromosomal DNA of Plasmodium falciparum , it has been suggested that it is a plastid-like DNA, which may be related to the plastid DNA of rhodophytes. However, phylogenetic analyses have been inconclusive. It has been suggested that this is due to the unusually high A + T content of the Plasmodium falciparum large extrachromosomal DNA. To further investigate the evolution of the apicomplexan large extrachromosomal DNA, the DNA sequence of the organellar ribosomal RNA gene from Toxoplasma gondii , was determined. The Toxoplasma gondii rDNA sequence was most similar to the large extrachromosomal rDNA of Plasmodium falciparum , but was much less A + T rich. Phylogenetic analyses were carried out using the LogDet transformation to minimize the impact of nucleotide bias. These studies support the evolutionary relatedness of the Toxoplasma gondii rDNA with the large extrachromosomal rDNA of Plasmodium falciparum and with the organellar rDNA of another parasite in the phylum Apicomplexa, Babesia bovis. These analyses also suggest that the apicomplexan large extra-chromosomal DNA may be more closely related to the plastid DNA of euglenoids than to those of rhodophytes.     

Gregarina niphandrodes may lack both a plastid genome and organelle

Gregarines are early diverging apicomplexans that appear to be closely related to Cryptosporidium. Most apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess both plastids and corresponding plastid genomes. Cryptosporidium lacks both the organelle and the genome. To investigate the evolutionary history of plastids in the Apicomplexa, we tried to determine whether gregarines possess a plastid and/or its genome. We used PCR and dot-blot hybridization to determine whether the gregarine Gregarina niphandrodes possesses a plastid genome. We used an inhibitor of plastid function for any reduction in gregarine infection, and transmission electron microscopy to search for plastid ultrastructure. Despite an extensive search, an organelle of the appropriate ultrastructure in transmission electron microscopy, was not observed. Triclosan, an inhibitor of the plastid-specific enoyl-acyl carrier reductase enzyme, did not reduce host infection by G. niphandrodes. Plastid-specific primers produced amplicons with the DNA of Babesia equi, Plasmodium falciparum, and Toxoplasma gondii as templates, but not with G. niphandrodes DNA. Plastid-specific DNA probes, which hybridized to Babesia equi, failed to hybridize to G. niphandrodes DNA. This evidence indicates that G. niphandrodes is not likely to possess either a plastid organelle or its genome. This raises the possibility that the plastid was lost in the Apicomplexan following the divergence of gregarines and Cryptosporidium.     

Cell cycle-regulated vesicular trafficking of Toxoplasma APT1, a protein localized to multiple apicoplast membranes

The apicoplast is a relict plastid essential for viability of the apicomplexan parasites Toxoplasma and Plasmodium. It is surrounded by multiple membranes that proteins, substrates and metabolites must traverse. Little is known about apicoplast membrane proteins, much less their sorting mechanisms. We have identified two sets of apicomplexan proteins that are homologous to plastid membrane proteins that transport phosphosugars or their derivatives. Members of the first set bear N-terminal extensions similar to those that target proteins to the apicoplast lumen. While Toxoplasma gondii lacks this type of translocator, the N-terminal extension from the Plasmodium falciparum sequence was shown to be functional in T. gondii. The second set of translocators lacks an N-terminal targeting sequence. This translocator, TgAPT1, when tagged with HA, localized to multiple apicoplast membranes in T. gondii. Contrasting with the constitutive targeting of luminal proteins, the localization of the translocator varied during the cell cycle. Early-stage parasites showed circumplastid distribution, but as the plastid elongated in preparation for division, vesicles bearing TgAPT1 appeared adjacent to the plastid. After plastid division, the protein resumes a circumplastid colocalization. These studies demonstrate for the first time that vesicular trafficking likely plays a role in the apicoplast biogenesis.     

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.     

Nuclear-encoded, plastid-targeted genes suggest a single common origin for apicomplexan and dinoflagellate plastids

The phylum Apicomplexa encompasses a large number of intracellular protozoan parasites, including the causative agents of malaria (Plasmodium), toxoplasmosis (Toxoplasma), and many other human and animal diseases. Apicomplexa have recently been found to contain a relic, nonphotosynthetic plastid that has attracted considerable interest as a possible target for therapeutics. This plastid is known to have been acquired by secondary endosymbiosis, but when this occurred and from which type of alga it was acquired remain uncertain. Based on the molecular phylogeny of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes, we provide evidence that the apicomplexan plastid is homologous to plastids found in dinoflagellates-close relatives of apicomplexa that contain secondary plastids of red algal origin. Surprisingly, apicomplexan and dinoflagellate plastid-targeted GAPDH sequences were also found to be closely related to the plastid-targeted GAPDH genes of heterokonts and cryptomonads, two other groups that contain secondary plastids of red algal origin. These results address several outstanding issues: (1) apicomplexan and dinoflagellate plastids appear to be the result of a single endosymbiotic event which occurred relatively early in eukaryotic evolution, also giving rise to the plastids of heterokonts and perhaps cryptomonads; (2) apicomplexan plastids are derived from a red algal ancestor; and (3) the ancestral state of apicomplexan parasites was photosynthetic.     

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.     

Mining the Plasmodium genome database to define organellar function: what does the apicoplast do?

Apicomplexan species constitute a diverse group of parasitic protozoa, which are responsible for a wide range of diseases in many organisms. Despite differences in the diseases they cause, these parasites share an underlying biology, from the genetic controls used to differentiate through the complex parasite life cycle, to the basic biochemical pathways employed for intracellular survival, to the distinctive cell biology necessary for host cell attachment and invasion. Different parasites lend themselves to the study of different aspects of parasite biology: Eimeria for biochemical studies, Toxoplasma for molecular genetic and cell biological investigation, etc. The Plasmodium falciparum Genome Project contributes the first large-scale genomic sequence for an apicomplexan parasite. The Plasmodium Genome Database (http://PlasmoDB.org) has been designed to permit individual investigators to ask their own questions, even prior to formal release of the reference P. falciparum genome sequence. As a case in point, PlasmoDB has been exploited to identify metabolic pathways associated with the apicomplexan plastid, or 'apicoplast' - an essential organelle derived by secondary endosymbiosis of an alga, and retention of the algal plastid.     

Mosaic origin of the heme biosynthesis pathway in photosynthetic eukaryotes

Heme biosynthesis represents one of the most essential metabolic pathways in living organisms, providing the precursors for cytochrome prosthetic groups, photosynthetic pigments, and vitamin B(12). Using genomic data, we have compared the heme pathway in the diatom Thalassiosira pseudonana and the red alga Cyanidioschyzon merolae to those of green algae and higher plants, as well as to those of heterotrophic eukaryotes (fungi, apicomplexans, and animals). Phylogenetic analyses showed the mosaic character of this pathway in photosynthetic eukaryotes. Although most of the algal and plant enzymes showed the expected plastid (cyanobacterial) origin, at least one of them (porphobilinogen deaminase) appears to have a mitochondrial (alpha-proteobacterial) origin. Another enzyme, glutamyl-tRNA synthase, obviously originated in the eukaryotic nucleus. Because all the plastid-targeted sequences consistently form a well-supported cluster, this suggests that genes were either transferred from the primary endosymbiont (cyanobacteria) to the primary host nucleus shortly after the primary endosymbiotic event or replaced with genes from other sources at an equally early time, i.e., before the formation of three primary plastid lineages. The one striking exception to this pattern is ferrochelatase, the enzyme catalyzing the first committed step to heme and bilin pigments. In this case, two red algal sequences do not cluster either with the other plastid sequences or with cyanobacterial sequences and appear to have a proteobacterial origin like that of the apicomplexan parasites Plasmodium and Toxoplasma. Although the heterokonts also acquired their plastid via secondary endosymbiosis from a red alga, the diatom has a typical plastid-cyanobacterial ferrochelatase. We have not found any remnants of the plastidlike heme pathway in the nonphotosynthetic heterokonts Phytophthora ramorum and Phytophthora sojae.     

A subset of conserved tRNA genes in plastid DNA of nongreen plants.

The plastid genome of the nonphotosynthetic parasitic plant Epifagus virginiana contains only 17 of the 30 tRNA genes normally found in angiosperm plastid DNA. Although this is insufficient for translation, the genome is functional, so import of cytosolic tRNAs into plastids has been suggested. This raises the question of whether the tRNA genes that remain in E. virginiana plastid DNA are active or have just fortuitously escaped deletion. We report the sequences of 20 plastid tRNA loci from Orobanche minor, which shares a nonphotosynthetic ancestor with E. virginiana. The two species have 9 intact tRNA genes in common, the others being defunct in one or both species. The intron-containing trnLUAA gene is absent from E. virginiana, but it is intact, transcribed, and spliced in O. minor. The shared intact genes are better conserved than intergenic sequences, which indicates that these genes are being maintained by natural selection and, therefore, must be functional. For the most part, the tRNA species conserved in nonphotosynthetic plastids are also those that have never been found to be imported in plant mitochondria, which suggests that the same rules may govern tRNA import in the two organelles. A small photosynthesis gene, psbI, is still intact in O. minor, and computer simulations show that some small nonessential genes have an appreciable chance of escaping deletion.     

Unusual properties of Plasmodium falciparum actin: new insights into microfilament dynamics of apicomplexan parasites

Plasmodium falciparum, the etiologic agent of malaria, is a facultative intracellular parasite of the phylum Apicomplexa. A limited turnover of microfilaments takes place beneath the parasite plasma membrane, but the cytoplasm of apicomplexans is virtually devoid of F-actin. We produced Plasmodium actin in yeast. Purified recombinant Plasmodium actin polymerized inefficiently unless both gelsolin and phalloidin were added. The resulting actin polymers appeared fragmented in the fluorescence microscope. Plasmodium actin bound DNaseI about 200 times weaker than bovine non-muscle actin. Our findings suggest that the unique properties of Plasmodium actin can explain some of the unusual features of apicomplexan parasite microfilaments.     

The plastid in Plasmodium falciparum asexual blood stages: a three-dimensional ultrastructural analysis

The plastid in Plasmodium falciparum asexual stages is a tubular structure measuring about 0.5 micron x 0.15 micron in the merozoite, and 1.6 x 0.35 microns in trophozoites. Each parasite contains a single plastid until this organelle replicates in late schizonts. The plastid always adheres to the (single) mitochondrion, along its whole length in merozoites and early rings, but only at one end in later stages. Regions of the plastid are also closely related to the pigment vacuole, nuclear membrane and endoplasmic reticulum. In merozoites the plastid is anchored to a band of 2-3 subpellicular microtubules. Reconstructions show the plastid wall is characteristically three membranes thick, with regions of additional, complex membranes. These include inner and outer membrane complexes. The inner complex in the interior lumen is probably a rolled invagination of the plastid's inner membrane. The outer complex lies between the outer and middle wall membranes. The interior matrix contains ribosome-like granules and a network of fine branched filaments. Merozoites of P. berghei and P. knowlesi possess plastids similar in structure to those of P. falciparum. A model is proposed for the transfer of membrane lipid from the plastid to other organelles in the parasite.     

The phylogeny of colpodellids (Alveolata) using small subunit rRNA gene sequences suggests they are the free-living sister group to apicomplexans

In an attempt to reconstruct early alveolate evolution, we have examined the phylogenetic position of colpodellids by analyzing small subunit rDNA sequences from Colpodella pontica Myl'nikov 2000 and Colpodella sp. (American Type Culture Collection 50594). All phylogenetic analyses grouped the colpodellid sequences together with strong support and placed them strongly within the Alveolata. Most analyses showed colpodellids as the sister group to an apicomplexan clade, albeit with weak support. Sequences from two perkinsids, Perkinsus and Parvilucifera, clustered together and consistently branched as the sister group to dinoflagellates as shown previously. These data demonstrate that colpodellids and perkinsids are plesiomorphically similar in morphology and help provide a phylogenetic framework for inferring the combination of character states present in the last common ancestor of dinoflagellates and apicomplexans. We can infer that this ancestor was probably a myzocytotic predator with two heterodynamic flagella, micropores, trichocysts, rhoptries, micronemes, a polar ring, and a coiled open-sided conoid. This ancestor also very likely contained a plastid, but it is presently not certain whether it was photosynthetic, and it is not clear whether extant perkinsids or colpodellids have retained the organelle.     

Plant hormone cytokinins control cell cycle progression and plastid replication in apicomplexan parasites

Cytokinins are plant hormones that are involved in regulation of cell proliferation, cell cycle progression, and cell and plastid development. Here, we show that the apicomplexan parasites Toxoplasma gondii and Plasmodium berghei, an opportunistic human pathogen and a rodent malaria agent, respectively, produce cytokinins via a biosynthetic pathway similar to that in plants. Cytokinins regulate the growth and cell cycle progression of T. gondii by mediating expression of the cyclin gene TgCYC4. A natural form of cytokinin, trans-zeatin (t-zeatin), upregulated expression of this cyclin, while a synthetic cytokinin, thidiazuron, downregulated its expression. Immunofluorescence microscopy and quantitative PCR analysis showed that t-zeatin increased the genome-copy number of apicoplast, which are non-photosynthetic plastid, in the parasite, while thidiazuron led to their disappearance. Thidiazuron inhibited growth of T. gondii and Plasmodium falciparum, a human malaria parasite, suggesting that thidiazuron has therapeutic potential as an inhibitor of apicomplexan parasites.     

Alveolata histone-like proteins have different evolutionary origins

Prokaryotic histone-like proteins (Hlps) are abundant proteins found in bacterial and plastid nucleoids. Hlps are also found in the eukaryotic dinoflagellates and the apicomplexans, two major lineages of the Alveolata. It may be expected that Hlps of both groups were derived from the same ancestral Alveolates. However, our phylogenetic analyses suggest different origins for the dinoflagellate and the apicomplexan Hlps. The apicomplexan Hlps are affiliated with the cyanobacteria and probably originated from Hlps of the plastid genome. The dinoflagellate Hlps and the proteobacterial long Hlps form a clade that branch off from the node with the proteobacterial short Hlps.     

Extrachromosomal DNA in the Apicomplexa.

Malaria and related apicomplexan parasites have two highly conserved organellar genomes: one is of plastid (pl) origin, and the other is mitochondrial (mt). The organization of both organellar DNA molecules from the human malaria parasite Plasmodium falciparum has been determined, and they have been shown to be tightly packed with genes. The 35-kb circular DNA is the smallest known vestigial plastid genome and is presumed to be functional. All but two of its recognized genes are involved with genetic expression: one of the two encodes a member of the clp family of molecular chaperones, and the other encodes a conserved protein of unknown function found both in algal plastids and in eubacterial genomes. The possible evolutionary source and intracellular location of the plDNA are discussed. The 6-kb tandemly repeated mt genome is the smallest known and codes for only three proteins (cytochrome b and two subunits of cytochrome oxidase) as well as two bizarrely fragmented rRNAs. The organization of the mt genome differs somewhat among genera. The mtDNA sequence provides information not otherwise available about the structure of apicomplexan cytochrome b as well as the unusually fragmented rRNAs. The malarial mtDNA has a phage-like replication mechanism and undergoes extensive recombination like the mtDNA of some other lower eukaryotes.