Dual targeting of aminoacyl-tRNA synthetases to the apicoplast and cytosol in Plasmodium falciparum

The causative agent of malaria, Plasmodium, possesses three translationally active compartments: the cytosol, the mitochondrion and a relic plastid called the apicoplast. Aminoacyl-tRNA synthetases to charge tRNA are thus required for all three compartments. However, the Plasmodiumfalciparum genome encodes too few tRNA synthetases to supply a unique enzyme for each amino acid in all three compartments. We have investigated the subcellular localisation of three tRNA synthetases (AlaRS, GlyRS and ThrRS), which occur only once in the nuclear genome, and we show that each of these enzymes is dually localised to the P. falciparum cytosol and the apicoplast. No mitochondrial fraction is apparent for these three enzymes, which suggests that the Plasmodium mitochondrion lacks at least these three tRNA synthetases. The unique Plasmodium ThrRS is the presumed target of the antimalarial compound borrelidin. Borrelidin kills P. falciparum parasites quickly without the delayed death effect typical of apicoplast translation inhibitors and without an observable effect on apicoplast morphology. By contrast, mupirocin, an inhibitor of the apicoplast IleRS, kills with a delayed death effect that inhibits apicoplast growth and division. Because inhibition of dual targeted tRNA synthetases should arrest translation in all compartments of the parasite, these enzymes deserve further investigation as potential targets for antimalarial drug development.     

Antibiotic inhibitors of organellar protein synthesis in Plasmodium falciparum.

Elongation factor Tu (EF-Tu) is encoded by the tuf gene of the plastid organelle of the malaria parasite Plasmodium falciparum. A range of structurally unrelated inhibitors of this GTP-dependent translation factor was shown to have antimalarial activity in blood cultures. The most active was the cyclic thiazolyl peptide amythiamicin A with an IC50 = 0.01 microM. Demonstrable complexes were formed in vitro between a recombinant version of P. falciparum EF-Tu(pl) and inhibitors that bind to different sites on EF-Tu; these included the antibiotics kirromycin, GE2270A and enacyloxin IIa.     

Reduced ribosomes of the apicoplast and mitochondrion of Plasmodium spp. and predicted interactions with antibiotics

Apicomplexan protists such as Plasmodium and Toxoplasma contain a mitochondrion and a relic plastid (apicoplast) that are sites of protein translation. Although there is emerging interest in the partitioning and function of translation factors that participate in apicoplast and mitochondrial peptide synthesis, the composition of organellar ribosomes remains to be elucidated. We carried out an analysis of the complement of core ribosomal protein subunits that are encoded by either the parasite organellar or nuclear genomes, accompanied by a survey of ribosome assembly factors for the apicoplast and mitochondrion. A cross-species comparison with other apicomplexan, algal and diatom species revealed compositional differences in apicomplexan organelle ribosomes and identified considerable reduction and divergence with ribosomes of bacteria or characterized organelle ribosomes from other organisms. We assembled structural models of sections of Plasmodium falciparum organellar ribosomes and predicted interactions with translation inhibitory antibiotics. Differences in predicted drug–ribosome interactions with some of the modelled structures suggested specificity of inhibition between the apicoplast and mitochondrion. Our results indicate that Plasmodium and Toxoplasma organellar ribosomes have a unique composition, resulting from the loss of several large and small subunit proteins accompanied by significant sequence and size divergences in parasite orthologues of ribosomal proteins.     

Interaction between sulphur mobilisation proteins SufB and SufC: evidence for an iron-sulphur cluster biogenesis pathway in the apicoplast of Plasmodium falciparum

The plastid of Plasmodium falciparum, the apicoplast, performs metabolic functions essential to the parasite. Various reactions in the plastid require the assembly of [Fe-S] prosthetic groups on participating proteins as well as the reductant activity of ferredoxin that is converted from its apo-form by the assembly of [Fe-S] clusters inside the apicoplast. The [Fe-S] assembly pathway involving sulphur mobilising Suf proteins has been predicted to function in the apicoplast with one component (PfSufB) encoded by the plastid genome itself. We demonstrate the ATPase activity of recombinant P. falciparum nuclear-encoded SufC and its localisation in the apicoplast. Further, an internal region of apicoplast SufB was used to detect PfSufB-PfSufC interaction in vitro; co-elution of SufB from parasite lysate with recombinant PfSufC on an affinity column also indicated an interaction of the two proteins. As a departure from bacterial SufB and similar to reported plant plastid SufB, apicoplast SufB exhibited ATPase activity, suggesting the evolution of specialised functions in the plastid counterparts. Our results provide experimental evidence for an active Suf pathway in the Plasmodium apicoplast.     

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.     

A Nondiscriminating Glutamyl-tRNA Synthetase in the Plasmodium Apicoplast: THE FIRST ENZYME IN AN INDIRECT AMINOACYLATION PATHWAY*

The malaria parasite Plasmodium falciparum and related organisms possess a relict plastid known as the apicoplast. Apicoplast protein synthesis is a validated drug target in malaria because antibiotics that inhibit translation in prokaryotes also inhibit apicoplast protein synthesis and are sometimes used for malaria prophylaxis or treatment. We identified components of an indirect aminoacylation pathway for Gln-tRNA^Gln biosynthesis in Plasmodium that we hypothesized would be essential for apicoplast protein synthesis. Here, we report our characterization of the first enzyme in this pathway, the apicoplast glutamyl-tRNA synthetase (GluRS). We expressed the recombinant P. falciparum enzyme in Escherichia coli, showed that it is nondiscriminating because it glutamylates both apicoplast tRNA^Glu and tRNA^Gln, determined its kinetic parameters, and demonstrated its inhibition by a known bacterial GluRS inhibitor. We also localized the Plasmodium berghei ortholog to the apicoplast in blood stage parasites but could not delete the PbGluRS gene. These data show that Gln-tRNA^Gln biosynthesis in the Plasmodium apicoplast proceeds via an essential indirect aminoacylation pathway that is reminiscent of bacteria and plastids.     

Targeting the malarial plastid via the parasitophorous vacuole

The malarial "apicoplast" derived from an algal plastid, has stimulated interest for its novel evolutionary biology and potential as a drug target. An endoplasmic reticulum-type signal sequence followed by a plastid targeting sequence are required to target proteins to the apicoplast but the pathway by which proteins are transported to the organelle is unknown. By stage regulating the expression of transgenes we show that early (0-12 h) in the parasite's development in red cells, newly synthesized green fluorescent protein that contains the plastid targeting sequence (plastid targeting sequence-green fluorescent protein (PTS-GFP)) is recruited into the parasite's secretory pathway. PTS-GFP in 0-12-h parasites is found released into the parasitophorous vacuole (PV) and in apposition with the Golgi. However, import into the apicoplast and processing to GFP does not occur until 18-36 h in development. In intermediate, 18-h parasites PTS-GFP resides in the PV. Quantitative exit of PTS-GFP from the PV and its conversion to GFP is seen at 36 h. The data suggest that: (i) import into the apicoplast is stage regulated and (ii) the PTS can signal endomembrane targeting from the PV to the apicoplast.     

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.     

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.     

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.     

Structure-based sequence alignment of elongation factors Tu and G with related GTPases involved in translation

The G domain and domain II in the crystal structure of Thermus thermophilus elongation factor G (EF-G) were compared with the homologous domains in Thermus aquaticus elongation factor Tu (EF-Tu). Sequence alignment derived from the structural superposition was used to define conserved sequence elements in domain II. These elements and previously known conserved sequence elements in the G domain were used to guide the alignment of the sequences of Sulfolobus acidocaldarius elongation factor 2, human elongation factor 2, and Escherichia coli initiation factor 2 and release factor 3 to the aligned sequences of EF-G and EF-Tu. This alignment, which deviates from previously published alignments, has evolutionary implications and leads to alternative interpretations of biochemical data concerning the interaction of elongation factors with the -sarcin/ricin region of the ribosome. A single conserved sequence motif in domain II was identified and used to further characterize the GTPase subfamily of translation factors and related proteins. It was shown that the motif is found in most if not all the members of the family. Apparently, the common characteristic of these GTPases is an extensive consensus structural unit that possibly accounts for a similar interaction with the ribosome and is composed of two domains homologous to the G domain and domain II in EF-Tu and EF-G.     

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.     

Elongation factor G with effector loop from elongation factor Tu is inactive in translocation

Elongation factors Tu and G (EF-Tu and EF-G) alternately interact with the ribosome during the elongation phase of protein biosynthesis. The function of both factors depends on GTP binding, and the factors are ascribed to a superfamily of G-proteins. All G-proteins contain the effector loop, a structural element that is important for the protein's interaction with its target molecule. In this study the effector loop of EF-G was replaced by the loop taken from EF-Tu. The EF-G with EF-Tu loop has markedly decreased GTPase activity and did not catalyze translocation. We conclude that these loops are not functionally interchangeable since the factors interact with different states of the ribosome.     

Apicoplast acetyl Co-A carboxylase of the human malaria parasite is not targeted by cyclohexanedione herbicides

Malaria parasites retain a relict plastid (apicoplast) from a photosynthetic ancestor. The apicoplast is a useful drug target but the specificity of compounds believed to target apicoplast fatty acid biosynthesis has become uncertain, as this pathway is not essential in blood stages of the parasite. Herbicides that inhibit the plastid acetyl Coenzyme A (Co-A) carboxylase of plants also kill Plasmodium falciparum in vitro, but their mode of action remains undefined. We characterised the gene for acetyl Co-A carboxylase in P. falciparum. The P. falciparum acetyl-CoA carboxylase gene product is expressed in blood stage parasites and accumulates in the apicoplast. Ablation of the gene did not render parasites insensitive to herbicides, suggesting that these compounds are acting off-target in blood stages of P. falciparum.     

Biochemical characterization of the apicoplast-targeted AAA+ ATPase ClpB from Plasmodium falciparum

ClpB is a molecular chaperone from the AAA+ superfamily of ATPases, which reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB is essential for infectivity and in-host survival of a number of pathogenic microorganisms, but systematic studies on ClpB from pathogens have not been reported yet. We purified and characterized one of the two ClpB isoforms from the malaria parasite Plasmodium falciparum, PfClpB1. PfClpB1 is targeted to the apicoplast, an essential plastid organelle that is a promising anti-malaria drug target. PfClpB1 contains all characteristic AAA+ sequence motifs, but the middle domain of PfClpB1 includes a 52-residue long non-conserved insert. Like in most AAA+ ATPases, ATP induces self-association of PfClpB1 into hexamers. PfClpB1 catalyzes the hydrolysis of ATP and its ATPase activity is activated in the presence of casein and poly-lysine. Similar to Escherichia coli ClpB, PfClpB1 reactivates aggregated firefly luciferase, but the PfClpB1-mediated aggregate reactivation is inhibited in the presence of E. coli DnaK, DnaJ, and GrpE. The lack of effective cooperation between PfClpB1 and the bacterial DnaK system may arise from the Plasmodium-specific sequence of the ClpB middle domain. Our results indicate that the chaperone activity of PfClpB1 may support survival of Plasmodium falciparum by maintaining the folding status and activity of apicoplast proteins.     

Functional role of the sarcin-ricin loop of the 23S rRNA in the elongation cycle of protein synthesis

The sarcin-ricin loop (SRL) is one of the longest conserved sequences in the 23S ribosomal RNA. The SRL has been accepted as crucial for the activity of the ribosome because it is targeted by cytotoxins such as α-sarcin and ricin that completely abolish translation. Nevertheless, the precise functional role of the SRL in translation is not known. Recent biochemical and structural studies indicate that the SRL is critical for triggering GTP hydrolysis on elongation factor Tu (EF-Tu) and elongation factor G (EF-G). To determine the functional role of the SRL in the elongation stage of protein synthesis, we analyzed mutations in the SRL that are known to abolish protein synthesis and are lethal to cells. Here, we show that the SRL is not critical for GTP hydrolysis on EF-Tu and EF-G. The SRL also is not essential for peptide bond formation. Our results, instead, suggest that the SRL is crucial for anchoring EF-G on the ribosome during mRNA-tRNA translocation.     

A role for falcilysin in transit peptide degradation in the Plasmodium falciparum apicoplast

Falcilysin (FLN) is a zinc metalloprotease thought to degrade globin peptides in the acidic vacuole of the human malaria parasite Plasmodium falciparum. The enzyme has been found to have acidic or neutral pH optima on different peptides and to have additional distribution outside the food vacuole. These data suggested that FLN has an additional function in the parasite. To further probe the functions of FLN, we created a transgenic parasite clone expressing a chromosomally encoded FLN-GFP fusion. Unexpectedly, FLN was found in the apicoplast, an essential chloroplast-like organelle. Nuclear encoded apicoplast proteins are targeted to the organelle by a bipartite N-terminal sequence comprised of a signal sequence followed by a positively charged transit peptide domain. Free transit peptides are thought to be toxic to the plastid and need to be rapidly degraded after proteolytic release from proproteins. We hypothesized that FLN may participate in transit peptide degradation in the apicoplast based on its preference for basic residues at neutral pH and on phylogenetic comparison with other M16 family metalloproteases. In vitro cleavage by FLN of the transit peptide from the apicoplast-resident acyl carrier protein supports this idea. The importance of FLN for parasite development is suggested by our inability to truncate the chromosomal FLN open reading frame. Our work indicates that FLN is an attractive target for antimalarial development.