Deciphering apicoplast targeting signals--feature extraction from nuclear-encoded precursors of Plasmodium falciparum apicoplast proteins.

The malaria causing protozoan Plasmodium falciparum contains a vestigal, non-photosynthetic plastid, the apicoplast. Numerous proteins encoded by nuclear genes are targeted to the apicoplast courtesy of N-terminal extensions. With the impending sequence completion of an entire genome of the malaria parasite, it is important to have software tools in place for prediction of subcellular locations for all proteins. Apicoplast targeting signals are bipartite; containing a signal peptide and a transit peptide. Nuclear-encoded apicoplast protein precursors were analyzed for characteristic features by statistical methods, principal component analysis, self-organizing maps, and supervised neural networks. The transit peptide contains a net positive charge and is rich in asparagine, lysine, and isoleucine residues. A novel prediction system (PATS, predict apicoplast-targeted sequences) was developed based on various sequence features, yielding a Matthews correlation coefficient of 0.91 (97% correct predictions) in a 40-fold cross-validation study. This system predicted 22% apicoplast proteins of the 205 potential proteins on P. falciparum chromosome 2, and 21% of 243 chromosome 3 proteins. A combination of the PATS results with a signal peptide prediction yields 15% potentially nuclear-encoded apicoplast proteins on chromosomes 2 and 3. The prediction tool will advance P. falciparum genome analysis, and it might help to identify apicoplast proteins as drug targets for the development of novel anti-malaria agents.     

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

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

Deciphering apicoplast targeting signals – feature extraction from nuclear-encoded precursors of Plasmodium falciparum apicoplast proteins

The malaria causing protozoan Plasmodium falciparum contains a vestigal, non-photosynthetic plastid, the apicoplast. Numerous proteins encoded by nuclear genes are targeted to the apicoplast courtesy of N-terminal extensions. With the impending sequence completion of an entire genome of the malaria parasite, it is important to have software tools in place for prediction of subcellular locations for all proteins. Apicoplast targeting signals are bipartite; containing a signal peptide and a transit peptide. Nuclear-encoded apicoplast protein precursors were analyzed for characteristic features by statistical methods, principal component analysis, self-organizing maps, and supervised neural networks. The transit peptide contains a net positive charge and is rich in asparagine, lysine, and isoleucine residues. A novel prediction system (PATS, predict apicoplast-targeted sequences) was developed based on various sequence features, yielding a Matthews correlation coefficient of 0.91 (97% correct predictions) in a 40-fold cross-validation study. This system predicted 22% apicoplast proteins of the 205 potential proteins on P. falciparum chromosome 2, and 21% of 243 chromosome 3 proteins. A combination of the PATS results with a signal peptide prediction yields 15% potentially nuclear-encoded apicoplast proteins on chromosomes 2 and 3. The prediction tool will advance P. falciparum genome analysis, and it might help to identify apicoplast proteins as drug targets for the development of novel anti-malaria agents.     

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.     

Inactivation of a Plasmodium apicoplast protein attenuates formation of liver merozoites

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

Plasmodium falciparum OTU‐like cysteine protease (PfOTU) is essential for apicoplast homeostasis and associates with noncanonical role of Atg8

The metabolic pathways associated with the mitochondrion and the apicoplast in Plasmodium, 2 parasite organelles of prokaryotic origin, are considered as suitable drug targets. In the present study, we have identified functional role of a novel ovarian tumour unit (OTU) domain‐containing cysteine protease of Plasmodium falciparum (PfOTU). A C‐terminal regulatable fluorescent affinity tag on native protein was utilised for its localization and functional characterization. Detailed studies showed vesicular localization of PfOTU and its association with the apicoplast. Degradation‐tag mediated knockdown of PfOTU resulted in abnormal apicoplast development and blocked development of parasites beyond early‐schizont stages in subsequent cell cycle; downregulation of PfOTU hindered apicoplast protein import. Further, the isoprenoid precursor‐mediated parasite growth‐rescue experiments confirmed that PfOTU knockdown specifically effect development of functional apicoplast. We also provide evidence for a possible biological function of PfOTU in membrane deconjugation of Atg8, which may be linked with the apicoplast protein import. Overall, our results show that the PfOTU is involved in apicoplast homeostasis and associates with the noncanonical function of Atg8 in maintenance of parasite apicoplast.     

Unique features of apicoplast DNA gyrases from Toxoplasma gondii and Plasmodium falciparum

Background

DNA gyrase, an enzyme once thought to be unique to bacteria, is also found in some eukaryotic plastids including the apicoplast of Apicomplexa such as Plasmodium falciparum and Toxoplasma gondii which are important disease-causing organisms. DNA gyrase is an excellent target for antibacterial drugs, yet such antibacterials seem ineffective against Apicomplexa. Characterisation of the apicoplast gyrases would be a useful step towards understanding why this should be so. While purification of active apicoplast gyrase has proved impossible to date, in silico analyses have allowed us to discover differences in the apicoplast proteins. The resulting predicted structural and functional differences will be a first step towards development of apicoplast-gyrase specific inhibitors.

Results

We have carried out sequence analysis and structural predictions of the enzymes from the two species and find that P. falciparum gyrase lacks a GyrA box, but T. gondii may retain one. All proteins contained signal/transport peptides for localization to the apicoplast but T. gondii Gyrase B protein lacks the expected hydrophobic region. The most significant difference is in the GyrA C-terminal domain: While the cores of the proteins, including DNA binding and cleavage regions are essentially unchanged, both apicoplast gyrase A proteins have C-terminal domains that are significantly larger than bacterial counterparts and are predicted to have different structures.

Conclusion

The apicoplast gyrases differ significantly from bacterial gyrases while retaining similar core domains. T. gondii Gyrase B may have an unusual or inefficient mechanism of localisation to the apicoplast. P.falciparum gyrase, lacks a GyrA box and is therefore likely to be inefficient in DNA supercoiling. The C-terminal domains of both apicoplast Gyrase A proteins diverge significantly from the bacterial proteins. We predict that an additional structural element is present in the C-terminal domain of both apicoplast Gyrase A proteins, including the possibility of a β-pinwheel with a non-canonical number of blades. These differences undoubtedly will affect the DNA supercoiling mechanism and have perhaps evolved to compensate for the lack of Topoisomerase IV in the apicoplast. These data will be useful first step towards further characterisation and development of inhibitors for apicoplast gyrases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0416-9) contains supplementary material, which is available to authorized users.     

A systematic screen to discover and analyze apicoplast proteins identifies a conserved and essential protein import factor

Parasites of the phylum Apicomplexa cause diseases that impact global health and economy. These unicellular eukaryotes possess a relict plastid, the apicoplast, which is an essential organelle and a validated drug target. However, much of its biology remains poorly understood, in particular its elaborate compartmentalization: four membranes defining four different spaces. Only a small number of organellar proteins have been identified in particular few proteins are known for non-luminal apicoplast compartments. We hypothesized that enlarging the catalogue of apicoplast proteins will contribute toward identifying new organellar functions and expand the realm of targets beyond a limited set of characterized pathways. We developed a bioinformatic screen based on mRNA abundance over the cell cycle and on phyletic distribution. We experimentally assessed 57 genes, and of 30 successful epitope tagged candidates eleven novel apicoplast proteins were identified. Of those, seven appear to target to the lumen of the organelle, and four localize to peripheral compartments. To address their function we then developed a robust system for the construction of conditional mutants via a promoter replacement strategy. We confirm the feasibility of this system by establishing conditional mutants for two selected genes--a luminal and a peripheral apicoplast protein. The latter is particularly intriguing as it encodes a hypothetical protein that is conserved in and unique to Apicomplexan parasites and other related organisms that maintain a red algal endosymbiont. Our studies suggest that this peripheral plastid protein, PPP1, is likely localized to the periplastid compartment. Conditional disruption of PPP1 demonstrated that it is essential for parasite survival. Phenotypic analysis of this mutant is consistent with a role of the PPP1 protein in apicoplast biogenesis, specifically in import of nuclear-encoded proteins into the organelle.     

The apicoplast

Parasites like malaria and Toxoplasma possess a vestigial plastid homologous to the chloroplasts of plants. The plastid (known as the apicoplast) is non-photosynthetic but retains many hallmarks of its ancestry including a circular genome that it synthesises proteins from and a suite of biosynthetic pathways of cyanobacterial origin. In this review, the discovery of the apicoplast and its integration, function and purpose are explored. New insights into the apicoplast fatty acid biosynthesis pathway and some novel roles of the apicoplast in vaccine development are reviewed.     

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.     

Nuclear and nucleoid localization are independently conserved functions in bacteriophage terminal proteins

Bacteriophage terminal proteins (TPs) prime DNA replication and become covalently linked to the DNA 5′‐ends. In addition, they are DNA‐binding proteins that direct early organization of phage DNA replication at the bacterial nucleoid and, unexpectedly, contain nuclear localization signals (NLSs), which localize them to the nucleus when expressed in mammalian cells. In spite of the lack of sequence homology among the phage TPs, these three properties share some common features, suggesting a possible evolutionary common origin of TPs. We show here that NLSs of three different phage TPs, Φ29, PRD1 and Cp‐1, are mapped within the protein region required for nucleoid targeting in bacteria, in agreement with a previously proposed common origin of DNA‐binding domains and NLSs. Furthermore, previously reported point mutants of Φ29 TP with no nuclear localization still can target the bacterial nucleoid, and Cp‐1 TP contains two independent NLSs, only one of them required for nucleoid localization. Altogether, our results show that nucleoid and nucleus localization sequence requirements partially overlap, but they can be uncoupled, suggesting that conservation of both features could have a common origin but, at the same time, they have been independently conserved during evolution.     

Dephospho‐CoA kinase,a nuclear‐encoded apicoplast protein,remains active and essential after Plasmodium falciparum apicoplast disruption

Malaria parasites contain an essential organelle called the apicoplast that houses metabolic pathways for fatty acid, heme, isoprenoid, and iron–sulfur cluster synthesis. Surprisingly, malaria parasites can survive without the apicoplast as long as the isoprenoid precursor isopentenyl pyrophosphate (IPP) is supplemented in the growth medium, making it appear that isoprenoid synthesis is the only essential function of the organelle in blood‐stage parasites. In the work described here, we localized an enzyme responsible for coenzyme A synthesis, DPCK, to the apicoplast, but we were unable to delete DPCK, even in the presence of IPP. However, once the endogenous DPCK was complemented with the E. coli DPCK (EcDPCK), we were successful in deleting it. We were then able to show that DPCK activity is required for parasite survival through knockdown of the complemented EcDPCK. Additionally, we showed that DPCK enzyme activity remains functional and essential within the vesicles present after apicoplast disruption. These results demonstrate that while the apicoplast of blood‐stage P. falciparum parasites can be disrupted, the resulting vesicles remain biochemically active and are capable of fulfilling essential functions.     

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.     

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.     

Protein targeting to the malaria parasite plastid

The relict plastid, or apicoplast, of the malaria parasite Plasmodium falciparum is an essential organelle and a promising drug target. Most apicoplast proteins are nuclear encoded and post-translationally targeted into the organelle using a bipartite N-terminal extension, consisting of a typical endomembrane signal peptide and a plant-like transit peptide. Apicoplast protein targeting commences through the parasite's secretory pathway. We review recent experimental evidence suggesting that the apicoplast resides in the mainstream endomembrane system proximal to the Golgi. Further, we explore possible mechanisms for translocation of nuclear-encoded apicoplast proteins across the four bounding membranes. Recent insights into the composition of the transit peptide and how it is cleaved and degraded after use are also examined. Characterization of apicoplast targeting has not only shed light on how this group of parasites mediate intracellular protein trafficking events but also it has helped identify new targets for therapeutics. The distinctive leader sequences of apicoplast proteins make them readily identifiable, allowing assembly of a virtual organelle metabolome from the genome. Such analysis has lead to the identification of several biochemical pathways that are absent from the human host and thus represent novel therapeutic targets for parasitic infection.     

Lipid kinases are essential for apicoplast homeostasis in Toxoplasma gondii

Phosphoinositides regulate numerous cellular processes by recruiting cytosolic effector proteins and acting as membrane signalling entities. The cellular metabolism and localization of phosphoinositides are tightly regulated by distinct lipid kinases and phosphatases. Here, we identify and characterize a unique phosphatidylinositol 3 kinase (PI3K) in Toxoplasma gondii, a protozoan parasite belonging to the phylum Apicomplexa. Conditional depletion of this enzyme and subsequently of its product, PI(3)P, drastically alters the morphology and inheritance of the apicoplast, an endosymbiotic organelle of algal origin that is a unique feature of many Apicomplexa. We searched the T. gondii genome for PI(3)P‐binding proteins and identified in total six PX and FYVE domain‐containing proteins including a PIKfyve lipid kinase, which phosphorylates PI(3)P into PI(3,5)P₂. Although depletion of putative PI(3)P‐binding proteins shows that they are not essential for parasite growth and apicoplast biology, conditional disruption of PIKfyve induces enlarged apicoplasts, as observed upon loss of PI(3)P. A similar defect of apicoplast homeostasis was also observed by knocking down the PIKfyve regulatory protein ArPIKfyve, suggesting that in T. gondii, PI(3)P‐related function for the apicoplast might mainly be to serve as a precursor for the synthesis of PI(3,5)P₂. Accordingly, PI3K is conserved in all apicomplexan parasites whereas PIKfyve and ArPIKfyve are absent in Cryptosporidium species that lack an apicoplast, supporting a direct role of PI(3,5)P₂ in apicoplast homeostasis. This study enriches the already diverse functions attributed to PI(3,5)P₂ in eukaryotic cells and highlights these parasite lipid kinases as potential drug targets.     

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.