Localisation of Plasmodium falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic pathway in the apicoplast and characterisation of its catalytic properties

Uroporphyrinogen decarboxylase (UROD) is a key enzyme in the heme-biosynthetic pathway and in Plasmodium falciparum it occupies a strategic position in the proposed hybrid pathway for heme biosynthesis involving shuttling of intermediates between different subcellular compartments in the parasite. In the present study, we demonstrate that an N-terminally truncated recombinant P. falciparum UROD (r(Δ)PfUROD) over-expressed and purified from Escherichia coli cells, as well as the native enzyme from the parasite were catalytically less efficient compared with the host enzyme, although they were similar in other enzyme parameters. Molecular modeling of PfUROD based on the known crystal structure of the human enzyme indicated that the protein manifests a distorted triose phosphate isomerase (TIM) barrel fold which is conserved in all the known structures of UROD. The parasite enzyme shares all the conserved or invariant amino acid residues at the active and substrate binding sites, but is rich in lysine residues compared with the host enzyme. Mutation of specific lysine residues corresponding to residues at the dimer interface in human UROD enhanced the catalytic efficiency of the enzyme and dimer stability indicating that the lysine rich nature and weak dimer interface of the wild-type PfUROD could be responsible for its low catalytic efficiency. PfUROD was localised to the apicoplast, indicating the requirement of additional mechanisms for transport of the product coproporphyrinogen to other subcellular sites for its further conversion and ultimate heme formation.     

Thiolactomycin analogues as potential anti-Toxoplasma gondii agents

The discovery of new compounds active against Toxoplasma gondii is extremely important due to the severe disease caused by this pathogen in immunocompromised hosts and to congenital infection. Type II fatty acid biosynthesis has shown to be a promising target for drug intervention in toxoplasmosis. Here we describe the inhibitory effect of 8 thiolactomycin (TLM) analogues against tachyzoite-infected LLC-MK₂ cells. The TLM analogues demonstrated anti-T. gondii activity, arresting tachyzoite proliferation with IC₅₀ values in the micromolar level after 24 h and 48 h of treatment. Metabolic labelling of extracellular parasites treated with TLM analogues using [³H]acetate demonstrated that these drugs affected acylglycerol synthesis. The rapid reduction of parasite load suggests that these compounds have selective cytotoxic effects against T. gondii. Transmission electron microscopy demonstrated that TLM analogues interfered with membrane-bounded organelles and parasite division and this in turn affected parasite development and survival.     

The search for the missing link: a relic plastid in Perkinsus?

Perkinsus marinus (Phylum Perkinsozoa) is a protozoan parasite that has devastated natural and farmed oyster populations in the USA, significantly affecting the shellfish industry and the estuarine environment. The other two genera in the phylum, Parvilucifera and Rastrimonas, are parasites of microeukaryotes. The Perkinsozoa occupies a key position at the base of the dinoflagellate branch, close to its divergence from the Apicomplexa, a clade that includes parasitic protista, many harbouring a relic plastid. Thus, as a taxon that has also evolved toward parasitism, the Perkinsozoa has attracted the attention of biologists interested in the evolution of this organelle, both in its ultrastructure and the conservation, loss or transfer of its genes. A review of the recent literature reveals mounting evidence in support of the presence of a relic plastid in P. marinus, including the presence of multimembrane structures, characteristic metabolic pathways and proteins with a bipartite N-terminal extension. Further, these findings raise intriguing questions regarding the potential functions and unique adaptation of the putative plastid and/or plastid genes in the Perkinsozoa. In this review we analyse the above-mentioned evidence and evaluate the potential future directions and expected benefits of addressing such questions. Given the rapidly expanding molecular/genetic resources and methodological toolbox for Perkinsus spp., these organisms should complement the currently established models for investigating plastid evolution within the Chromalveolata.     

Large amounts of apicoplast nucleoid DNA and its segregation inToxoplasma gondii

Summary Apicoplasts (apicomplexan plastids) are nonphotosynthetic, secondary endosymbiotic plastids that are found in most apicomplexans. Although these organelles are essential for parasite survival, their functions, activities, and structures are not well understood. We examined the apicoplast nucleoid ofToxoplasma gondii from a morphological aspect by high-resolution epifluorescence microscopy and electron microscopy. We found unexpectedly large amounts of DNA in the nucleoid and the presence of several division-related structures. Initially, we identified the organellar nucleoids by staining with the DNA-specific dye 4,6-diamidino-2-phenylindole. A single nucleoid was observed per apicoplast, and the fluorescent spot representing the nucleoid was bright and spherical in contrast to the weak and filamentous spot representing the mitochondrial nucleoid. We also measured the DNA content of each apicoplast nucleoid by a video-intensified microscope photon-counting system and determined that the genomic copy number was at least 25, a figure over four times greater than that reported previously. Moreover, several groups of apicoplasts had significantly higher genomic copy numbers. The DNA molecules were accurately divided into two daughter apicoplasts just before nuclear division. In addition, we examined nucleoid segregation and the division apparatus using electron microscopy. However, we failed to observe nucleoid structures, suggesting that the apicoplasts are predominantly composed of nucleoid material. In addition, we observed cap structures at the termini of dividing apicoplasts, a possible plastid-dividing ring, and a microbody-like granule around the constriction. These structures may be involved in apicoplast division.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system - PD ring plastid-dividing ring     

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.     

The carbon and energy sources of the non-photosynthetic plastid in the malaria parasite

The malaria parasite harbours an indispensable plastid known as the ‘apicoplast’. The apicoplast’s exact role remains uncertain, but it houses components involved in fatty acid, isoprenoid and haem biosyntheses. These pathways offer opportunities to develop anti-malarials. In the absence of photosynthesis, how apicoplast anabolism is fuelled is unclear. Here we investigated plant-like transporters of the apicoplast and measured their substrate preferences using a novel cell-free assay system to explore the carbon and energy sources of the apicoplast. The transporters exchange triose phosphate and phosphoenolpyruvate for inorganic phosphate, demonstrating that the apicoplast taps into host-derived glucose to fuel its metabolism.     

Plasmodium falciparum LipB mutants display altered redox and carbon metabolism in asexual stages and cannot complete sporogony in Anopheles mosquitoes

Malaria is still one of the most important global infectious diseases. Emergence of drug resistance and a shortage of new efficient antimalarials continue to hamper a malaria eradication agenda. Malaria parasites are highly sensitive to changes in the redox environment. Understanding the mechanisms regulating parasite redox could contribute to the design of new drugs. Malaria parasites have a complex network of redox regulatory systems housed in their cytosol, in their mitochondrion and in their plastid (apicoplast). While the roles of enzymes of the thioredoxin and glutathione pathways in parasite survival have been explored, the antioxidant role of α-lipoic acid (LA) produced in the apicoplast has not been tested. To take a first step in teasing a putative role of LA in redox regulation, we analysed a mutant Plasmodium falciparum (3D7 strain) lacking the apicoplast lipoic acid protein ligase B (lipB) known to be depleted of LA. Our results showed a change in expression of redox regulators in the apicoplast and the cytosol. We further detected a change in parasite central carbon metabolism, with lipB deletion resulting in changes to glycolysis and tricarboxylic acid cycle activity. Further, in another Plasmodium cell line (NF54), deletion of lipB impacted development in the mosquito, preventing the detection of infectious sporozoite stages. While it is not clear at this point if the observed phenotypes are linked, these findings flag LA biosynthesis as an important subject for further study in the context of redox regulation in asexual stages, and point to LipB as a potential target for the development of new transmission drugs.     

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.