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.     

The non-photosynthetic, pathogenic green alga Helicosporidium sp. has retained a modified, functional plastid genome

A fragment of the Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) plastid genome has been sequenced. The genome architecture was compared to that of both a non-photosynthetic relative (Prototheca wickerhamii) and a photosynthetic relative (Chlorella vulgaris). Comparative genomic analysis indicated that Helicosporidium and Prototheca are closely related genera. The analyses also revealed that the Helicosporidium sp. plastid genome has been rearranged. In particular, two ribosomal protein-encoding genes (rpl19 and rps23) appeared to have been transposed, or lost from the Helicosporidium sp. plastid genome. RT-PCR reactions demonstrated that the retained plastid genes were transcribed, suggesting that, despite rearrangement(s), the Helicosporidium sp. plastid genome has remained functional. The modified plastid genome architecture is a novel apomorphy that indicates that the Helicosporidia are highly derived green algae, more so than Prototheca spp. As such, they represent a promising model to study organellar genome reorganizations in parasitic protists.     

Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway

The plastid of Plasmodium falciparum (or 'apicoplast') is the evolutionary homolog of the plant chloroplast and represents a vestige of a photosynthetic past. Apicoplast indispensability indicates that it still provides essential functions to parasites. Similar to plant chloroplasts, the apicoplast is dependent on many nucleus-encoded genes to provide these functions. The apicoplast is surrounded by four membranes, two more than plant chloroplasts. Thus, protein targeting to the apicoplast must overcome additional membrane barriers. In P.falciparum we have analyzed apicoplast targeting using green fluorescent protein (GFP). We demonstrate that protein targeting is at least a two-step process mediated by bipartite N-terminal pre-sequences that consist of a signal peptide for entry into the secretory pathway and a plant-like transit peptide for subsequent import into the apicoplast. The P.falciparum transit peptide is exceptional compared with other known plastid transit peptides in not requiring serine or threonine residues. The pre-sequence components are removed stepwise during apicoplast targeting. Targeting GFP to the apicoplast has also provided the first opportunity to examine apicoplast morphology in live P. falciparum.     

Detection of Helicosporidium spp. in metagenomic DNA

Distinct isolates of the invertebrate pathogenic alga Helicosporidium sp., collected from different insect hosts and different geographic locations, were processed to sequence the 18S rDNA and β-tubulin genes. The sequences were analyzed to assess genetic variation within the genus Helicosporidium and to design Helicosporidium-specific 18S rDNA primers. The specificity of these primers was demonstrated by testing not only on the Helicosporidium sp. isolates, but also on two trebouxiophyte algae known to be close Helicosporidium relatives, Prototheca wickerhamii and Prototheca zopfii. The genus-specific primers were used to develop a culture-independent assay aimed at detecting the presence of Helicosporidium spp. in environmental waters. The assay was based on the PCR amplification of 18SrDNA gene fragments from metagenomic DNA preparations, and it resulted in the amplification of detectable products for all sampled sites. Phylogenetic analyses that included the environmental sequences demonstrated that all amplification products clustered in a strongly supported, monophyletic Helicosporidium clade, thereby validating the metagenomic approach and the taxonomic origin of the produced environmental sequences. In addition, the phylogenetic analyses established that Helicosporidium spp. isolated from coleopteran hosts are more closely related to each other than they are to the isolate collected from a dipteran host. Finally, the phylogenetic trees depicted intergeneric relationships that supported a Helicosporidium-Prototheca cluster but did not support a Helicosporidium-Coccomyxa grouping, suggesting that pathogenicity to invertebrates evolved at least twice independently within the trebouxiophyte green algae.     

Processing of an apicoplast leader sequence in Plasmodium falciparum and the identification of a putative leader cleavage enzyme

The plastid (apicoplast) of the malaria-causing parasite Plasmodium falciparum was derived via a secondary endosymbiotic process. As in other secondary endosymbionts, numerous genes for apicoplast proteins are located in the nucleus, and the encoded proteins are targeted to the organelle courtesy of a bipartite N-terminal extension. The first part of this leader sequence is a signal peptide that targets proteins to the secretory pathway. The second, so-called transit peptide region is required to direct proteins from the secretory pathway across the multiple membranes surrounding the apicoplast. In this paper we perform a pulse-chase experiment and N-terminal sequencing to show that the transit peptide of an apicoplast-targeted protein is cleaved, presumably upon import of the protein into the apicoplast. We identify a gene whose product likely performs this cleavage reaction, namely a stromal-processing peptidase (SPP) homologue. In plants SPP cleaves the transit peptides of plastid-targeted proteins. The P. falciparum SPP homologue contains a bipartite N-terminal apicoplast-targeting leader. Interestingly, it shares this leader sequence with a Delta-aminolevulinic acid dehydratase homologue via an alternative splicing event.     

Identification of plant-like galactolipids in Chromera velia, a photosynthetic relative of malaria parasites

Apicomplexa are protist parasites that include Plasmodium spp., the causative agents of malaria, and Toxoplasma gondii, responsible for toxoplasmosis. Most Apicomplexa possess a relict plastid, the apicoplast, which was acquired by secondary endosymbiosis of a red alga. Despite being nonphotosynthetic, the apicoplast is otherwise metabolically similar to algal and plant plastids and is essential for parasite survival. Previous studies of Toxoplasma gondii identified membrane lipids with some structural features of plastid galactolipids, the major plastid lipid class. However, direct evidence for the plant-like enzymes responsible for galactolipid synthesis in Apicomplexan parasites has not been obtained. Chromera velia is an Apicomplexan relative recently discovered in Australian corals. C. velia retains a photosynthetic plastid, providing a unique model to study the evolution of the apicoplast. Here, we report the unambiguous presence of plant-like monogalactosyldiacylglycerol and digalactosyldiacylglycerol in C. velia and localize digalactosyldiacylglycerol to the plastid. We also provide evidence for a plant-like biosynthesis pathway and identify candidate galactosyltranferases responsible for galactolipid synthesis. Our study provides new insights in the evolution of these important enzymes in plastid-containing eukaryotes and will help reconstruct the evolution of glycerolipid metabolism in important parasites such as Plasmodium and Toxoplasma.     

Nucleus-encoded,plastid-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) indicates a single origin for chromalveolate plastids

Plastids (the photosynthetic organelles of plants and algae) originated through endosymbiosis between a cyanobacterium and a eukaryote and subsequently spread to other eukaryotes by secondary endosymbioses between two eukaryotes. Mounting evidence favors a single origin for plastids of apicomplexans, cryptophytes, dinoflagellates, haptophytes, and heterokonts (together with their nonphotosynthetic relatives, termed chromalveolates), but so far, no single molecular marker has been described that supports this common origin. One piece of evidence comes from plastid-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which originated by a gene duplication of the cytosolic form. However, no plastid GAPDH has been characterized from haptophytes, leaving an important piece of the puzzle missing. We have sequenced genes encoding cytosolic, mitochondrion-targeted, and plastid-targeted GAPDH proteins from a number of haptophytes and heterokonts and found haptophyte homologs that branch within a strongly supported clade of chromalveolate plastid-targeted genes, being more closely related to an apicomplexan homolog than was expected. The evolution of plastid-targeted GAPDH supports red algal ancestry of apicomplexan plastids and raises a number of questions about the importance of plastid loss and the possibility of cryptic plastids in nonphotosynthetic lineages such as ciliates.     

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.     

Characterization of two malaria parasite organelle translation elongation factor G proteins: the likely targets of the anti-malarial fusidic acid

Malaria parasites harbour two organelles with bacteria-like metabolic processes that are the targets of many anti-bacterial drugs. One such drug is fusidic acid, which inhibits the translation component elongation factor G. The response of P. falciparum to fusidic acid was characterised using extended SYBR-Green based drug trials. This revealed that fusidic acid kills in vitro cultured P. falciparum parasites by immediately blocking parasite development. Two bacterial-type protein translation elongation factor G genes are identified as likely targets of fusidic acid. Sequence analysis suggests that these proteins function in the mitochondria and apicoplast and both should be sensitive to fusidic acid. Microscopic examination of protein-reporter fusions confirm the prediction that one elongation factor G is a component of parasite mitochondria whereas the second is a component of the relict plastid or apicoplast. The presence of two putative targets for a single inhibitory compound emphasizes the potential of elongation factor G as a drug target in malaria.     

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.     

Nucleus-encoded periplastid-targeted EFL in chlorarachniophytes

Chlorarachniophytes are cercozoan amoeboflagellates that acquired photosynthesis by enslaving a green alga, which has retained a highly reduced nucleus called a nucleomorph. The nucleomorph lacks many genes necessary for its own maintenance and expression, suggesting that some genes have been moved to the host nucleus and their products are now targeted back to the periplastid compartment (PPC), the reduced eukaryotic cytoplasm of the endosymbiont. Protein trafficking in chlorarachniophytes is therefore complex, including nucleus-encoded plastid-targeted proteins, nucleomorph-encoded plastid-targeted proteins, and nucleus-encoded periplastid-targeted proteins. A major gap in our understanding of this system is the PPC-targeted proteins because none have been described in any chlorarachniophytes. Here we describe the first such protein, the GTPase EFL. EFL was characterized from 7 chlorarachniophytes, and 2 distinct types were found. One is related to foraminiferan EFL and lacks an amino-terminal extension. The second, distantly related, type encodes an amino-terminal extension consisting of a signal peptide followed by sequence sharing many characteristics with transit peptides from nucleus-encoded plastid-targeted proteins and which we conclude is most likely PPC targeted. Western blotting with antibodies specific to putative host and PPC-targeted EFL from the chlorarachniophytes Bigelowiella natans and Gymnochlora stellata is consistent with posttranslational cleavage of the leaders from PPC-targeted proteins. Immunolocalization of both proteins in B. natans confirmed the cytosolic location of the leaderless EFL and a distinct localization pattern for the PPC-targeted protein but could not rule out a plastid location (albeit very unlikely). We sought other proteins with a similar leader and identified a eukaryotic translation initiation factor 1 encoding a bipartite extension with the same properties. Transit peptide sequences were characterized from all 3 classes of targeted protein by comparing all examples of each class from expressed sequence tag surveys of B. natans and G. stellata. No recognizable difference between plastid- and PPC-targeted proteins was observed, but nucleomorph-encoded transit peptides differ, likely reflecting high AT content of nucleomorph genomes. Taken together, the data suggest that the system that directs proteins to the PPC in chlorarachniophytes uses a bipartite targeting sequence, as does the PPC-targeting system that evolved independently in cryptomonads.     

On the evolutionary origins of apicoplasts: revisiting the rhodophyte vs. chlorophyte controversy

Apicomplexans are parasites of great medical and veterinary importance. They contain a vestigial plastid, the apicoplast, that originated through the secondary endosymbiosis of the photosynthetic unicellular alga. The nature of this alga remains controversial. Here, we revisit the available evidence and critically summarize the "green vs. red" debate.     

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.     

Evidence for Golgi-independent transport from the early secretory pathway to the plastid in malaria parasites

The malaria parasite Plasmodium falciparum harbours a relict plastid (termed the apicoplast) that has evolved by secondary endosymbiosis. The apicoplast is surrounded by four membranes, the outermost of which is believed to be part of the endomembrane system. Nuclear-encoded apicoplast proteins have a two-part N-terminal extension that is necessary and sufficient for translocation across these four membranes. The first domain of this N-terminal extension resembles a classical signal peptide and mediates translocation into the secretory pathway, whereas the second domain is homologous to plant chloroplast transit peptides and is required for the remaining steps of apicoplast targeting. We explored the initial, secretory pathway component of this targeting process using green fluorescent reporter protein constructs with modified leaders. We exchanged the apicoplast signal peptide with signal peptides from other secretory proteins and observed correct targeting, demonstrating that apicoplast targeting is initiated at the general secretory pathway of P. falciparum. Furthermore, we demonstrate by immunofluorescent labelling that the apicoplast resides on a small extension of the endoplasmic reticulum (ER) that is separate from the cis-Golgi. To define the position of the apicoplast in the endomembrane pathway in relation to the Golgi we tracked apicoplast protein targeting in the presence of the secretory inhibitor Brefeldin A (BFA), which blocks traffic between the ER and Golgi. We observe apicoplast targeting in the presence of BFA despite clear perturbation of ER to Golgi traffic by the inhibitor, which suggests that the apicoplast resides upstream of the cis-Golgi in the parasite's endomembrane system. The addition of an ER retrieval signal (SDEL) - a sequence recognized by the cis-Golgi protein ERD2 - to the C-terminus of an apicoplast-targeted protein did not markedly affect apicoplast targeting, further demonstrating that the apicoplast is upstream of the Golgi. Apicoplast transit peptides are thus dominant over an ER retention signal. However, when the transit peptide is rendered non-functional (by two point mutations or by complete deletion) SDEL-specific ER retrieval takes over, and the fusion protein is localized to the ER. We speculate either that the apicoplast in P. falciparum resides within the ER directly in the path of the general secretory pathway, or that vesicular trafficking to the apicoplast directly exits the ER.     

The apicoplast: a new member of the plastid family

Protozoan parasites of the phylum Apicomplexa include pathogens such as Plasmodium, Toxoplasma and Cryptosporidium. They have been shown to contain a vestigial nonphotosynthetic plastid, the apicoplast, which might have arisen by secondary endosymbiosis. Little is known about the function of the apicoplast but the parasites exhibit delayed cell death when their apicoplast is impaired. The discovery of the apicoplast opens an unexpected opportunity to link current fundamental research on plant and algal plastids to the physiology of apicomplexans. For example, the apicoplast might provide new targets for innovative drugs that act as herbicides and do not affect the mammalian host.