Toxoplasma gondii scavenges host-derived lipoic acid despite its de novo synthesis in the apicoplast.
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ABSTRACT:
SUBMITTER: Crawford MJ
PROVIDER: S-EPMC1500979 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Toxoplasma gondii is an obligate intracellular parasite that contains a relic plastid, called the apicoplast, deriving from a secondary endosymbiosis with an ancestral alga. Metabolic labelling experiments using [14C]acetate led to a substantial production of numerous glycero- and sphingo-lipid classes in extracellular tachyzoites. Syntheses of all these lipids were affected by the herbicide haloxyfop, demonstrating that their de novo syntheses necessarily required a functional apicoplast fatty acid synthase II. The complex metabolic profiles obtained and a census of glycerolipid metabolism gene candidates indicate that synthesis is probably scattered in the apicoplast membranes [possibly for PA (phosphatidic acid), DGDG (digalactosyldiacylglycerol) and PG (phosphatidylglycerol)], the endoplasmic reticulum (for major phospholipid classes and ceramides) and mitochondria (for PA, PG and cardiolipid). Based on a bioinformatic analysis, it is proposed that apicoplast produced acyl-ACP (where ACP is acyl-carrier protein) is transferred to glycerol-3-phosphate for apicoplast glycerolipid synthesis. Acyl-ACP is also probably transported outside the apicoplast stroma and irreversibly converted into acyl-CoA. In the endoplasmic reticulum, acyl-CoA may not be transferred to a three-carbon backbone by an enzyme similar to the cytosolic plant glycerol-3-phosphate acyltransferase, but rather by a dual glycerol-3-phosphate/dihydroxyacetone-3-phosphate acyltransferase like in animal and yeast cells. We further showed that intracellular parasites could also synthesize most of their lipids from scavenged host cell precursors. The observed appearance of glycerolipids specific to either the de novo pathway in extracellular parasites (unknown glycerolipid 1 and the plant like DGDG), or the intracellular stages (unknown glycerolipid 8), may explain the necessary coexistence of both de novo parasitic acyl-lipid synthesis and recycling of host cell compounds.
| S-EPMC1386017 | biostudies-literature
Project description:Toxoplasma gondii and related human parasites contain an essential plastid organelle called the apicoplast. Clinically used antibiotics and other inhibitors that disrupt apicoplast biogenesis cause a mysterious "delayed-death" phenotype in which parasite growth is unaffected during the first lytic cycle of inhibitor treatment but is severely inhibited in the second lytic cycle even after drug removal. Critical to understanding the complex downstream cellular effects of these drug classes are the timing of apicoplast loss during inhibitor treatment and how it relates to this peculiar growth phenotype. Here we show that, upon treatment with diverse classes of apicoplast inhibitors, newly replicated T. gondii parasites in the first lytic cycle initially form apicoplasts with defects in protein import or genome replication and eventually fail to inherit the apicoplast altogether. Despite the accumulation of parasites with defective or missing apicoplasts, growth is unaffected during the first lytic cycle, as previously observed. Strikingly, concomitant inhibition of host cell isoprenoid biosynthesis results in growth inhibition in the first lytic cycle and unmasks the apicoplast defects. These results suggest that defects in and even the complete loss of the apicoplast in T. gondii are partially rescued by scavenging of host cell metabolites, leading to death that is delayed. Our findings uncover host cell interactions that can alleviate apicoplast inhibition and highlight key differences in delayed-death inhibitors between T. gondii and Plasmodium falciparum.
| S-EPMC6355570 | biostudies-other
Project description:Apicomplexan parasites harbor a secondary plastid that has lost the ability to photosynthesize yet is essential for the parasite to multiply and cause disease. Bioinformatic analyses predict that 5-10% of all proteins encoded in the parasite genome function within this organelle. However, the mechanisms and molecules that mediate import of such large numbers of cargo proteins across the four membranes surrounding the plastid remain elusive. In this work, we identify a highly diverged member of the Tic20 protein family in Apicomplexa. We demonstrate that Tic20 of Toxoplasma gondii is an integral protein of the innermost plastid membrane. We engineer a conditional null-mutant and show that TgTic20 is essential for parasite growth. To characterize this mutant functionally, we develop several independent biochemical import assays to reveal that loss of TgTic20 leads to severe impairment of apicoplast protein import followed by organelle loss and parasite death. TgTic20 is the first experimentally validated protein import factor identified in apicoplasts. Our studies provide experimental evidence for a common evolutionary origin of import mechanisms across the innermost membranes of primary and secondary plastids.
| S-EPMC2533231 | biostudies-literature
Project description: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.
| S-EPMC4356647 | biostudies-literature
Project description: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.
| S-EPMC10795397 | biostudies-literature
Project description:BackgroundDNA 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.ResultsWe 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.ConclusionThe 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.
| S-EPMC4297366 | biostudies-literature
Project description:Members of the Apicomplexa phylum possess an organelle surrounded by four membranes, originating from the secondary endosymbiosis of a red alga. This so-called apicoplast hosts essential metabolic pathways. We report here that apicoplast inheritance is an actin-based process. Concordantly, parasites depleted in either profilin or actin depolymerizing factor, or parasites overexpressing the FH2 domain of formin 2, result in loss of the apicoplast. The class XXII myosin F (MyoF) is conserved across the phylum and localizes in the vicinity of the Toxoplasma gondii apicoplast during division. Conditional knockdown of TgMyoF severely affects apicoplast turnover, leading to parasite death. This recapitulates the phenotype observed upon perturbation of actin dynamics that led to the accumulation of the apicoplast and secretory organelles in enlarged residual bodies. To further dissect the mode of action of this motor, we conditionally stabilized the tail of MyoF, which forms an inactive heterodimer with endogenous TgMyoF. This dominant negative mutant reveals a central role of this motor in the positioning of the two centrosomes prior to daughter cell formation and in apicoplast segregation.
| S-EPMC3680736 | biostudies-literature
Project description:ImportanceToxoplasma 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.
| S-EPMC10653800 | biostudies-literature
Project description:Toxoplasma gondii, like most apicomplexan parasites, possesses an essential relict chloroplast, the apicoplast. Several apicoplast membrane proteins lack the bipartite targeting sequences of luminal proteins. Vesicles bearing these membrane proteins are detected during apicoplast enlargement, but the means of cargo selection remains obscure. We used a combination of deletion mutagenesis, point mutations and protein chimeras to identify a short motif prior to the first transmembrane domain of the T. gondii apicoplast phosphate transporter 1 (APT1) that is necessary for apicoplast trafficking. Tyrosine 16 was essential for proper localization; any substitution resulted in misdirection of APT1 to the Golgi body. Glycine 17 was also important, with significant Golgi body accumulation in the alanine mutant. Separation of at least eight amino acids from the transmembrane domain was required for full motif function. Similarly placed YG motifs are present in apicomplexan APT1 orthologs and the corresponding N-terminal domain from Plasmodium vivax was able to route T. gondii APT1 to the apicoplast. Differential permeabilization showed that both the N- and C-termini of APT1 are exposed to the cytosol. We propose that this YG motif facilitates APT1 trafficking via interactions that occur on the cytosolic face of nascent vesicles destined for the apicoplast.
| S-EPMC3324616 | biostudies-literature
Project description:Apicomplexan parasites are responsible for high impact human diseases such as malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular pathogens are dependent on both de novo lipid biosynthesis as well as the uptake of host lipids for biogenesis of parasite membranes. Genome annotations and biochemical studies indicate that apicomplexan parasites can synthesize fatty acids via a number of different biosynthetic pathways that are differentially compartmentalized. However, the relative contribution of each of these biosynthetic pathways to total fatty acid composition of intracellular parasite stages remains poorly defined. Here, we use a combination of genetic, biochemical, and metabolomic approaches to delineate the contribution of fatty acid biosynthetic pathways in Toxoplasma gondii. Metabolic labeling studies with [(13)C]glucose showed that intracellular tachyzoites synthesized a range of long and very long chain fatty acids (C14:0-26:1). Genetic disruption of the apicoplast-localized type II fatty-acid synthase resulted in greatly reduced synthesis of saturated fatty acids up to 18 carbons long. Ablation of type II fatty-acid synthase activity resulted in reduced intracellular growth that was partially restored by addition of long chain fatty acids. In contrast, synthesis of very long chain fatty acids was primarily dependent on a fatty acid elongation system comprising three elongases, two reductases, and a dehydratase that were localized to the endoplasmic reticulum. The function of these enzymes was confirmed by heterologous expression in yeast. This elongase pathway appears to have a unique role in generating very long unsaturated fatty acids (C26:1) that cannot be salvaged from the host.
| S-EPMC3281623 | biostudies-literature