Complex Endosymbiosis II: The Nonphotosynthetic Plastid of Apicomplexa Parasites (The Apicoplast) and Its Integrated Metabolism.

Chloroplasts are essential organelles that are responsible for photosynthesis in a wide range of organisms that have colonized all biotopes on Earth such as plants and unicellular algae. Interestingly, a secondary endosymbiotic event of a red algal ancestor gave rise to a group of organisms that have adopted an obligate parasitic lifestyle named Apicomplexa parasites. Apicomplexa parasites are some of the most widespread and poorly controlled pathogens in the world. These infectious agents are responsible for major human diseases such as toxoplasmosis, caused by Toxoplasma gondii, and malaria, caused by Plasmodium spp. Most of these parasites harbor this relict plastid named the apicoplast, which is essential for parasite survival. The apicoplast has lost photosynthetic capacities but is metabolically similar to plant and algal chloroplasts. The apicoplast is considered a novel and important drug target against Apicomplexa parasites. This chapter focuses on the apicoplast of apicomplexa parasites, its maintenance, and its metabolic pathways.

An apicoplast-localized deubiquitinase contributes to the cell growth and apicoplast homeostasis of Toxoplasma gondii.

Toxoplasma gondii is among the most important parasites worldwide. The apicoplast is a unique organelle shared by all Apicomplexan protozoa. Increasing lines of evidence suggest that the apicoplast possesses its own ubiquitination system. Deubiquitination is a crucial step executed by deubiquitinase (DUB) during protein ubiquitination. While multiple components of ubiquitination have been identified in T. gondii, the deubiquitinases involved remain unknown. The aim of the current study was to delineate the localization of TgOTU7 and elucidate its functions. TgOTU7 was specifically localized at the apicoplast, and its expression was largely regulated during the cell cycle. Additionally, TgOTU7 efficiently breaks down ubiquitin chains, exhibits linkage-nonspecific deubiquitinating activity and is critical for the lytic cycle and apicoplast biogenesis, similar to the transcription of the apicoplast genome and the nuclear genes encoding apicoplast-targeted proteins. Taken together, the results indicate that the newly described deubiquitinase TgOTU7 specifically localizes to the apicoplast and affects the cell growth and apicoplast homeostasis of T. gondii.

F-actin and myosin F control apicoplast elongation dynamics which drive apicoplast-centrosome association in Toxoplasma gondii.

IMPORTANCE: Toxoplasma gondii and most other parasites in the phylum Apicomplexa contain an apicoplast, a non-photosynthetic plastid organelle required for fatty acid, isoprenoid, iron-sulfur cluster, and heme synthesis. Perturbation of apicoplast function results in parasite death. Thus, parasite survival critically depends on two cellular processes: apicoplast division to ensure every daughter parasite inherits a single apicoplast, and trafficking of nuclear encoded proteins to the apicoplast. Despite the importance of these processes, there are significant knowledge gaps in regards to the molecular mechanisms which control these processes; this is particularly true for trafficking of nuclear-encoded apicoplast proteins. This study provides crucial new insight into the timing of apicoplast protein synthesis and trafficking to the apicoplast. In addition, this study demonstrates how apicoplast-centrosome association, a key step in the apicoplast division cycle, is controlled by the actomyosin cytoskeleton.

Coordination of apicoplast transcription in a malaria parasite by internal and host cues.

The malaria parasite Plasmodium falciparum has a nonphotosynthetic plastid called the apicoplast, which contains its own genome. Regulatory mechanisms for apicoplast gene expression remain poorly understood, despite this organelle being crucial for the parasite life cycle. Here, we identify a nuclear-encoded apicoplast RNA polymerase σ subunit (sigma factor) which, along with the α subunit, appears to mediate apicoplast transcript accumulation. This has a periodicity reminiscent of parasite circadian or developmental control. Expression of the apicoplast subunit gene, apSig, together with apicoplast transcripts, increased in the presence of the blood circadian signaling hormone melatonin. Our data suggest that the host circadian rhythm is integrated with intrinsic parasite cues to coordinate apicoplast genome transcription. This evolutionarily conserved regulatory system might be a future target for malaria treatment.

Apicoplast biogenesis mediated by ATG8 requires the ATG12-ATG5-ATG16L and SNAP29 complexes in Toxoplasma gondii.

In apicomplexan parasites, the macroautophagy/autophagy machinery is repurposed to maintain the plastid-like organelle apicoplast. Previously, we showed that in Toxoplasma and Plasmodium, ATG12 interacts with ATG5 in a non-covalent manner, in contrast to the covalent interaction in most organisms. However, it remained unknown whether apicomplexan parasites have functional orthologs of ATG16L1, a protein that is essential for the function of the covalent ATG12-ATG5 complex in vivo in other organisms. Furthermore, the mechanism used by the autophagy machinery to maintain the apicoplast is unclear. We report that the ATG12-ATG5-ATG16L complex exists in Toxoplasma gondii (Tg). This complex is localized on isolated structures at the periphery of the apicoplast dependent on TgATG16L. Inducible depletion of TgATG12, TgATG5, or TgATG16L caused loss of the apicoplast and affected parasite growth. We found that a putative soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein, synaptosomal-associated protein 29 (TgSNAP29, Qbc SNARE), is required to maintain the apicoplast in T. gondii. TgSNAP29 depletion disrupted TgATG8 localization at the apicoplast. Additionally, we identified a putative ubiquitin-interacting motif-docking site (UDS) of TgATG8. Mutation of the UDS site abolished TgATG8 localization on the apicoplast but not lipidation. These findings suggest that the TgATG12-TgATG5-TgATG16L complex is required for biogenesis of the apicoplast, in which TgATG8 is translocated to the apicoplast via vesicles in a SNARE -dependent manner in T. gondii.Abbreviations: AID: auxin-inducible degron; CCD: coiled-coil domain; HFF: human foreskin fibroblast; IAA: indole-3-acetic acid; LAP: LC3-associated phagocytosis; NAA: 1-naphthaleneacetic acid; PtdIns3P: phosphatidylinositol-3-phosphate; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; UDS: ubiquitin-interacting motif-docking site; UIM: ubiquitin-interacting motif.

Ribonucleotide Misincorporation and Reverse Transcriptase Activities of Plasmodium falciparum Apicoplast DNA Polymerase.

Plasmodium falciparumis the most common and harmful causative agent of malaria worldwide. As a member of the phylum Apicomplexa, P. falciparum is characterized by the presence of a unique and essential organelle called the apicoplast. Reminiscent of an algal chloroplast, the apicoplast possesses its own genome, which is maintained by a single apicoplast DNA polymerase (apPol). Ribonucleotides misincorporated into the genome are among the most common lesions encountered by DNA polymerases, and the ability to replicate past these lesions varies widely among characterized enzymes. Here, we have investigated the ribonucleotide (rNTP) misincorporation frequency of apPol and determined its reverse transcriptase (RT) activity across templating ribonucleotides. Pre-steady-state kinetic experiments indicate that apPol does not have an unusually high discrimination between deoxy and ribonucleotides, with frequencies ranging between 104 and 106 depending on the identity of the ribonucleotide. Once incorporated into its template, apPol can replicate across ribonucleotides using its RT activity, but extension of a deoxynucleotide basepaired with the ribonucleotide is slow relative to a canonical basepair. Exonuclease assays indicate that apPol proofreads ribonucleotides an order of magnitude faster than extension, suggesting that most, but not all, misincorporated ribonucleotides will be excised. Although the components have not been identified, ribonucleotide excision repair or other tolerance mechanisms may exist in the P. falciparum apicoplast, and more targeted proteomic efforts will be needed to elucidate them.

Promising antimalarials targeting apicoplast DNA polymerase from Plasmodium falciparum.

Malaria is caused by the parasite Plasmodium falciparum, which contains an essential non-photosynthetic plastid called the apicoplast. A single DNA polymerase, apPOL, is targeted to the apicoplast, where it replicates and repairs the genome. apPOL has no direct orthologs in mammals and is considered a promising drug target for the treatment and/or prevention of malaria. We previously reported screening the Malaria Box to identify MMV666123 as an inhibitor of apPOL. Herein we extend our studies and report structure-activity relationships for MMV666123 and identify key structural motifs necessary for inhibition. Although attempts to crystallize apPOL with the inhibitor were not fruitful, kinetic analysis and crystal structure determinations of WT and mutant apo-enzymes, facilitated model building and provided insights into the putative inhibitor binding site. Our results validate apPOL as an antimalarial target and provide an avenue for the design of high potency, specific inhibitors of apPOL and other A-family DNA polymerases.

The first apicoplast tRNA thiouridylase plays a vital role in the growth of Toxoplasma gondii.

Toxoplasmosis caused by the protozoan Toxoplasma gondii is one of the most common parasitic diseases in humans and almost all warm-blooded animals. Lys, Glu, and Gln-specific tRNAs contain a super-modified 2-thiourea (s2U) derivatives at the position 34, which is essential for all living organisms by maintaining the structural stability and aminoacylation of tRNA, and the precision and efficiency of codon recognition during protein translation. However, the enzyme(s) involved in this modification in T. gondii remains elusive. In this report, three putative tRNA-specific 2-thiolation enzymes were identified, of which two were involved in the s2U34 modification of tRNALys, tRNAGlu, and tRNAGln. One was named TgMnmA, an apicoplast-located tRNA-specific 2-thiolation enzyme in T. gondii. Knockout of TgMnmA showed that this enzyme is important for the lytic cycle of tachyzoites. Loss of TgMnmA also led to abnormities in apicoplast biogenesis and severely disturbed apicoplast genomic transcription. Notably, mice survived from the infection with 10 TgMnmA-KO RH tachyzoites. These findings provide new insights into s2U34 tRNA modification in Apicomplexa, and suggest TgMnmA, the first apicoplast tRNA thiouridylase identified in all apicomplexans, as a potential drug target.

The ferredoxin redox system - an essential electron distributing hub in the apicoplast of Apicomplexa.

The apicoplast, a relict plastid found in most species of the phylum Apicomplexa, harbors the ferredoxin redox system which supplies electrons to enzymes of various metabolic pathways in this organelle. Recent reports in Toxoplasma gondii and Plasmodium falciparum have shown that the iron-sulfur cluster (FeS)-containing ferredoxin is essential in tachyzoite and blood-stage parasites, respectively. Here we review ferredoxin's crucial contribution to isoprenoid and lipoate biosynthesis as well as tRNA modification in the apicoplast, highlighting similarities and differences between the two species. We also discuss ferredoxin's potential role in the initial reductive steps required for FeS synthesis as well as recent evidence that offers an explanation for how NADPH required by the redox system might be generated in Plasmodium spp.

Disrupting the plastidic iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability.

Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harboring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are crucial for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway, deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on the parasites. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis, and suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets.

Discovery of small molecule inhibitors of Plasmodium falciparum apicoplast DNA polymerase.

Malaria is caused by infection with protozoan parasites of the Plasmodium genus, which is part of the phylum Apicomplexa. Most organisms in this phylum contain a relic plastid called the apicoplast. The apicoplast genome is replicated by a single DNA polymerase (apPOL), which is an attractive target for anti-malarial drugs. We screened small-molecule libraries (206,504 compounds) using a fluorescence-based high-throughput DNA polymerase assay. Dose/response analysis and counter-screening identified 186 specific apPOL inhibitors. Toxicity screening against human HepaRG human cells removed 84 compounds and the remaining were subjected to parasite killing assays using chloroquine resistant P. falciparum parasites. Nine compounds were potent inhibitors of parasite growth and may serve as lead compounds in efforts to discover novel malaria drugs.

Apicoplast Dynamics During Plasmodium Cell Cycle.

The deadly malaria parasite, Plasmodium falciparum, contains a unique subcellular organelle termed the apicoplast, which is a clinically-proven antimalarial drug target. The apicoplast is a plastid with essential metabolic functions that evolved via secondary endosymbiosis. As an ancient endosymbiont, the apicoplast retained its own genome and it must be inherited by daughter cells during cell division. During the asexual replication of P. falciparum inside human red blood cells, both the parasite, and the apicoplast inside it, undergo massive morphological changes, including DNA replication and division. The apicoplast is an integral part of the cell and thus its development is tightly synchronized with the cell cycle. At the same time, certain aspects of its dynamics are independent of nuclear division, representing a degree of autonomy in organelle biogenesis. Here, we review the different aspects of organelle dynamics during P. falciparum intraerythrocytic replication, summarize our current understanding of these processes, and describe the many open questions in this area of parasite basic cell biology.

Critical role for isoprenoids in apicoplast biogenesis by malaria parasites.

Isopentenyl pyrophosphate (IPP) is an essential metabolic output of the apicoplast organelle in Plasmodium falciparum malaria parasites and is required for prenylation-dependent vesicular trafficking and other cellular processes. We have elucidated a critical and previously uncharacterized role for IPP in apicoplast biogenesis. Inhibiting IPP synthesis blocks apicoplast elongation and inheritance by daughter merozoites, and apicoplast biogenesis is rescued by exogenous IPP and polyprenols. Knockout of the only known isoprenoid-dependent apicoplast pathway, tRNA prenylation by MiaA, has no effect on blood-stage parasites and thus cannot explain apicoplast reliance on IPP. However, we have localized an annotated polyprenyl synthase (PPS) to the apicoplast. PPS knockdown is lethal to parasites, rescued by IPP and long- (C50) but not short-chain (≤C20) prenyl alcohols, and blocks apicoplast biogenesis, thus explaining apicoplast dependence on isoprenoid synthesis. We hypothesize that PPS synthesizes long-chain polyprenols critical for apicoplast membrane fluidity and biogenesis. This work critically expands the paradigm for isoprenoid utilization in malaria parasites and identifies a novel essential branch of apicoplast metabolism suitable for therapeutic targeting.

Pfprex from Plasmodium falciparum can bypass oxidative stress-induced DNA lesions.

Apicomplexans such as the malaria parasite Plasmodium falciparum possess a unique organelle known as the apicoplast that has its own circular genome. The apicoplast genome is AT rich and is subjected to oxidative stress from the byproducts of the normal biochemical pathways that operate in the apicoplast. It is expected that oxidative stress will lead to the appearance of DNA lesions such as 2-hydroxydeoxyadenine, thymine glycol, and 8-oxodeoxyguanine in the apicoplast genome. The apicoplast genome is replicated by the DNA polymerase activity present in the Pfprex enzyme. We have named the polymerase module of Pfprex as PfpPol and the enzyme belongs to the A family of DNA polymerases. Similar to other members of this family, PfpPol also exhibits high fidelity of DNA synthesis. We show that this enzyme is also capable of carrying out translesion DNA synthesis past common DNA lesions that arise due to oxidative stress. The residues N505 and Y509 from the fingers sub-domain, which are unique to PfpPol, play an important role in the ability of PfpPol to bypass the three lesions. The observed lesion-bypass ability of the Pfprex enzyme will minimize the adverse effects of oxidative stress on the apicoplast genome of the malaria parasite. These findings also have implications regarding the evolution of the machinery responsible for replication of organellar genomes.

Toxoplasma TgAtg8-TgAtg3 Interaction Primarily Contributes to Apicoplast Inheritance and Parasite Growth in Tachyzoite.

The apicoplast, which harbors key pathways involved in biosynthesis of vital metabolites, is a unique and essential nonphotosynthetic plastid organelle in apicomplexan parasites. Intriguingly, autophagy-related protein 8 (Atg8), a highly conserved eukaryotic protein, can localize to the outermost membrane of the apicoplast and modulate its inheritance in both Toxoplasma and Plasmodium parasites. The Atg8-Atg3 interaction plays a key role in Atg8 lipidation and localization, and our previously work in Toxoplasma has suggested that the core Atg8-family interacting motif (AIM) in TgAtg3, 239FADI242, and the R27 residue of TgAtg8 contribute to TgAtg8-TgAtg3 interaction in vitro. However, little is known about the function of this interaction or its importance in tachyzoite growth in Toxoplasma gondii. Here, we generated two complemented cell lines, TgAtg3F239A/I242A and TgAtg8R27E, based on the TgAtg3 and TgAtg8 conditional knockdown cell lines, respectively. We found that both mutant complemented cell lines were severely affected in terms of tachyzoite growth and displayed delayed death upon conditional knockdown of endogenous TgAtg3 or TgAtg8. Intriguingly, both complemented lines appeared to be defective in TgAtg8 lipidation and apicoplast inheritance. Moreover, we showed that the interaction of TgAtg8 and TgAtg3 is critical for TgAtg8 apicoplast localization. In addition, we found that the TgAtg3F239A/I242A complemented line exhibits an integral mitochondrial network upon ablation of endogenous TgAtg3, which is distinct from TgAtg3-depleted parasites with a fragmented mitochondrial network. Taken together, this work solidifies the contribution of the TgAtg8-TgAtg3 interaction to apicoplast inheritance and the growth of T. gondii tachyzoites. IMPORTANCEToxoplasma gondiiis a widespread intracellular parasite infecting a variety of warm-blooded animals, including humans. Current frontline treatment of toxoplasmosis suffers many drawbacks, including toxicity, drug resistance, and failure to eradicate tissue cysts, underscoring the need to identify novel drug targets for suppression or treatment of toxoplasmosis. TgAtg8 is thought to serve multiple functions in lipidation and is considered essential to the growth and development of both tachyzoites and bradyzoites. Here, we show that Toxoplasma gondii has adapted a conserved Atg8-Atg3 interaction, required for canonical autophagy in other eukaryotes, to function specifically in apicoplast inheritance. Our finding not only highlights the importance of TgAtg8-TgAtg3 interaction in tachyzoite growth but also suggests that this interaction is a promising drug target for the therapy of toxoplasmosis.

Toxoplasma gondii apicoplast-resident ferredoxin is an essential electron transfer protein for the MEP isoprenoid-biosynthetic pathway.

Apicomplexan parasites, such as Toxoplasma gondii, are unusual in that each cell contains a single apicoplast, a plastid-like organelle that compartmentalizes enzymes involved in the essential 2C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis. The last two enzymatic steps in this organellar pathway require electrons from a redox carrier. However, the small iron-sulfur cluster-containing protein ferredoxin, a likely candidate for this function, has not been investigated in this context. We show here that inducible knockdown of T. gondii ferredoxin results in progressive inhibition of growth and eventual parasite death. Surprisingly, this phenotype is not accompanied by ultrastructural changes in the apicoplast or overall cell morphology. The knockdown of ferredoxin was instead associated with a dramatic decrease in cellular levels of the last two metabolites in isoprenoid biosynthesis, 1-hydroxy-2-methyl-2-(E)- butenyl-4-pyrophosphate, and isomeric dimethylallyl pyrophosphate/isopentenyl pyrophosphate. Ferredoxin depletion was also observed to impair gliding motility, consistent with isoprenoid metabolites being important for dolichol biosynthesis, protein prenylation, and modification of other proteins involved in motility. Significantly, pharmacological inhibition of isoprenoid synthesis of the host cell exacerbated the impact of ferredoxin depletion on parasite replication, suggesting that the slow onset of parasite death after ferredoxin depletion is because of isoprenoid scavenging from the host cell and leading to partial compensation of the depleted parasite metabolites upon ferredoxin knockdown. Overall, these findings show that ferredoxin has an essential physiological function as an electron donor for the 2C-methyl-D-erythritol 4-phosphate pathway and is a potential drug target for apicomplexan parasites.

The metabolic pathways and transporters of the plastid organelle in Apicomplexa.

The apicoplast is the relict of a plastid organelle found in several disease-causing apicomplexan parasites such as Plasmodium spp. and Toxoplasma gondii. In these organisms, the organelle has lost its photosynthetic capability but harbours several fitness-conferring or essential metabolic pathways. Although maintaining the apicoplast and fuelling the metabolic pathways within requires the challenging constant import and export of numerous metabolites across its four membranes, only few apicoplast transporters have been identified to date, most of which are orphan transporters. Here we review the roles of metabolic pathways within the apicoplast and what is currently known about the few identified apicoplast metabolite transporters. We discuss what metabolites must get in and out of the apicoplast, the many transporters that are yet to be discovered, and what role these might play in parasite metabolism and as putative drug targets.

Organelle Dynamics in Apicomplexan Parasites.

Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium falciparum, are the cause of many important human and animal diseases. While T. gondii tachyzoites replicate through endodyogeny, during which two daughter cells are formed within the parental cell, P. falciparum replicates through schizogony, where up to 32 parasites are formed in a single infected red blood cell and even thousands of daughter cells during mosquito- or liver-stage development. These processes require a tightly orchestrated division and distribution over the daughter parasites of one-per-cell organelles such as the mitochondrion and apicoplast. Although proper organelle segregation is highly essential, the molecular mechanism and the key proteins involved remain largely unknown. In this review, we describe organelle dynamics during cell division in T. gondii and P. falciparum, summarize the current understanding of the molecular mechanisms underlying organelle fission in these parasites, and introduce candidate fission proteins.

Properties of Plasmodium falciparum with a Deleted Apicoplast DNA Gyrase.

Malaria parasites have three genomes: a nuclear genome, a mitochondrial genome, and an apicoplast genome. Since the apicoplast is a plastid organelle of prokaryotic origin and has no counterpart in the human host, it can be a source of novel targets for antimalarials. Plasmodium falciparum DNA gyrase (PfGyr) A and B subunits both have apicoplast-targeting signals. First, to test the predicted localization of this enzyme in the apicoplast and the breadth of its function at the subcellular level, nuclear-encoded PfGyrA was disrupted using CRISPR/Cas9 gene editing. Isopentenyl pyrophosphate (IPP) is known to rescue parasites from apicoplast inhibitors. Indeed, successful growth and characterization of PfΔGyrA was possible in the presence of IPP. PfGyrA disruption was accompanied by loss of plastid acyl-carrier protein (ACP) immunofluorescence and the plastid genome. Second, ciprofloxacin, an antibacterial gyrase inhibitor, has been used for malaria prophylaxis, but there is a need for a more detailed description of the mode of action of ciprofloxacin in malaria parasites. As predicted, PfΔGyrA clone supplemented with IPP was less sensitive to ciprofloxacin but not to the nuclear topoisomerase inhibitor etoposide. At high concentrations, however, ciprofloxacin continued to inhibit IPP-rescued PfΔGyrA, possibly suggesting that ciprofloxacin may have an additional nonapicoplast target in P. falciparum. Overall, we confirm that PfGyrA is an apicoplast enzyme in the malaria parasite, essential for blood-stage parasites, and a possible target of ciprofloxacin but perhaps not the only target.

The apicoplast of Haemoproteus columbae: A comparative study of this organelle genome in Haemosporida.

Apicomplexa is a phylum of parasitic protozoa; among them are the order Haemosporida, vector-borne parasites that include those that cause malaria (genus Plasmodium). Most Apicomplexa species have a non-photosynthetic plastid or apicoplast. Given its unique metabolic pathways, this organelle is considered a target for malaria therapeutics. Regardless of its importance, there is a paucity of complete apicoplast genome data hindering comparative studies. Here, the Haemoproteus (Haemoproteus) columbae apicoplast genome (lineage HAECOL1) was obtained using next-generation sequencing. This genome was included in a comparative analysis with other plastids. This 29.8 kb circular genome shares the same structure found in Plasmodium parasites. It is A + T rich (87.7%), comparable but at the higher end of A + T content observed in Plasmodium species (85.5-87.2%). As expected, considering its high A + T content, the synonymous codon usage (RSCU) and the effective number of codons (ENc) showed a moderate codon bias. Several apicoplast genes have a phylogenetic signal. However, unlike mitochondrial genes, single-gene phylogenies have low support in haemosporidian clades that diverged recently. The H. columbae apicoplast genome suggests that the apicoplast function may be conserved across Haemosporida. This parasite could be a model to study this organelle in a non-mammalian system.