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Microbiology Spectrum Mar 2023Toxoplasma gondii is an obligate intracellular parasite capable of infecting humans and animals. The organism has extraordinary metabolic resilience that allows it to...
Toxoplasma gondii is an obligate intracellular parasite capable of infecting humans and animals. The organism has extraordinary metabolic resilience that allows it to establish parasitism in varied nutritional milieus of diverse host cells. Our earlier work has shown that, despite flexibility in the usage of glucose and glutamine as the major carbon precursors, the production of pyruvate by glycolytic enzymes is central to the parasite's growth. Pyruvate is metabolized in a number of subcellular compartments, including the mitochondrion, apicoplast, and cytosol. With the objective of examining the mechanism and importance of the mitochondrial pool of pyruvate imported from the cytosol, we identified the conserved mitochondrial pyruvate carrier (MPC) complex, consisting of two subunits, MPC1 and MPC2, in T. gondii. The two parasite proteins could complement a yeast mutant deficient in growth on leucine and valine. Genetic ablation of either one or both subunits reduced the parasite's growth, mimicking the deletion of branched-chain ketoacid dehydrogenase (BCKDH), which has been reported to convert pyruvate into acetyl-coenzyme A (CoA) in the mitochondrion. Metabolic labeling of the MPC mutants by isotopic glucose revealed impaired synthesis of acetyl-CoA, correlating with a global decrease in carbon flux through glycolysis and the tricarboxylic acid (TCA) cycle. Disruption of MPC proteins exerted only a modest effect on the parasite's virulence in mice, further highlighting its metabolic flexibility. In brief, our work reveals the of pyruvate transport from the cytosol to the mitochondrion in the parasite, providing the missing link between glycolysis and the TCA cycle in T. gondii. T. gondii is a zoonotic parasite capable of infecting many warm-blooded organisms, including humans. Among others, a feature that allows it to parasitize multiple hosts is its exceptional metabolic plasticity. Although T. gondii can utilize different carbon sources, pyruvate homeostasis is critical for parasite growth. Pyruvate is produced primarily in the cytosol but metabolized in other organelles, such as the mitochondrion and apicoplast. The mechanism of import and physiological significance of pyruvate in these organelles remains unclear. Here, we identified the transporter of cytosol-derived pyruvate into the mitochondrion and studied its constituent subunits and their relevance. Our results show that cytosolic pyruvate is a major source of acetyl-CoA in the mitochondrion and that the mitochondrial pyruvate transporter is needed for optimal parasite growth. The mutants lacking the transporter are viable and virulent in a mouse model, underscoring the metabolic plasticity in the parasite's mitochondrion.
PubMed: 36920199
DOI: 10.1128/spectrum.05043-22 -
Parasitology International Jun 2023This work reports for the first time the presence and molecular characterization of Eimeria myoxi in the garden dormouse (Eliomys quercinus) from the Doñana Natural...
This work reports for the first time the presence and molecular characterization of Eimeria myoxi in the garden dormouse (Eliomys quercinus) from the Doñana Natural Area (Andalusia, SW Spain). Fresh faecal samples were collected from a total of 28 garden dormice, which were caught following current guidelines for the ethical use of animals in research, and processing by a standard flotation technique with saturated saline solution. Then, wet drops were examined microscopically, and the number of oocysts was semi-quantified. Eimeria oocysts were observed in 16 of the 28 (57.1%) faecal samples, showing most of them a very low number of oocysts (≤1 oocyst per microscopic field × 400). The unsporulated oocysts visualized in 16 faecal samples were subspherical and of length 19.2 ± 1.2 μm and width 17.4 ± 1.1 μm, being morphologically compatible with E. myoxi. This finding was supported by molecular analysis of the small subunit ribosomal RNA (SSU-rRNA) gene, identifying the same species in 22 of the 28 (78.6%) dormice, including 15 samples in which oocyst size was compatible with E. myoxi. Moreover, the subsequent analyses of the apicoplast open reading frame 470 (ORF470) and the mitochondrial cytochrome c oxidase subunit I (COI) genes confirmed the molecular identification of the isolates as E. myoxi. The phylogeny analyses were consistent with previous phylogenetic studies and support the existence of three lineages of rodent-infecting Eimeria species.
Topics: Animals; Coccidiosis; Eimeria; Myoxidae; Oocysts; Phylogeny; Spain
PubMed: 36804597
DOI: 10.1016/j.parint.2023.102740 -
Veterinary Parasitology Mar 2023The apicoplast, which is the result of secondary endosymbiosis, is a distinctive subcellular organelle and a crucial therapeutic target for apicomplexan parasites. The...
The apicoplast, which is the result of secondary endosymbiosis, is a distinctive subcellular organelle and a crucial therapeutic target for apicomplexan parasites. The majority of apicoplast-resident proteins are encoded by the nuclear genome and target the apicoplast via bipartite targeting signals consisting of a signal peptide and a transit peptide. The properties and functions of these peptides are poorly understood, which hinders the identification of apicoplast proteins and the study for plastid evolution. Here, the targeting signals of the recently discovered apicoplast tRNA thiouridylase TgMnmA of Toxoplasma gondii were analyzed. Our data using a reporter (the enhanced green fluorescent protein) fused with individual fragments containing various numbers of its N-terminal amino acids unequivocally revealed that the first 28 amino acids of TgMnmA functioned as a signal peptide for cellular secretion. The N-terminal 150 amino acids were sufficient to direct the fusion protein to the apicoplast, whereas its deletion caused the fusion protein to be localized to the mitochondrion. Our data further demonstrated that the apicoplast, rhoptry, and mitochondrion shared similar targeting signals, indicating that the apicoplast localization peptide was trans-organellar in function. In addition, the apicoplast localization peptide was important for the healthy proliferation of tachyzoites. In conclusion, the targeting signals of the nucleus-encoded apicoplast-targeted protein TgMnmA have been mapped out and the importance of this localization peptide has been elucidated in the current study.
Topics: Animals; Toxoplasma; Apicoplasts; Protein Sorting Signals; Peptides; Protozoan Proteins; Amino Acids
PubMed: 36731210
DOI: 10.1016/j.vetpar.2023.109888 -
BioRxiv : the Preprint Server For... Jun 2023Toxoplasma gondii contains an essential plastid organelle called the apicoplast that is necessary for fatty acid, isoprenoid, and heme synthesis. Perturbations affecting...
Toxoplasma gondii contains an essential plastid organelle called the apicoplast that is necessary for fatty acid, isoprenoid, and heme synthesis. Perturbations affecting apicoplast function or inheritance lead to parasite death. The apicoplast is a single copy organelle and therefore must be divided so that each daughter parasite inherits an apicoplast during cell division. In this study we identify new roles for F-actin and an unconventional myosin motor, TgMyoF, in this process. First, loss of TgMyoF and actin lead to an accumulation of apicoplast vesicles in the cytosol indicating a role for this actomyosin system in apicoplast protein trafficking or morphological integrity of the organelle. Second, live cell imaging reveals that during division the apicoplast is highly dynamic, exhibiting branched, U-shaped and linear morphologies that are dependent on TgMyoF and actin. In parasites where movement was inhibited by the depletion of TgMyoF, the apicoplast fails to associate with the parasite centrosomes. Thus, this study provides crucial new insight into mechanisms controlling apicoplast-centrosome association, a vital step in the apicoplast division cycle, which ensures that each daughter inherits a single apicoplast.
PubMed: 36711828
DOI: 10.1101/2023.01.01.521342 -
Microbiology Resource Announcements Feb 2023We are reporting the nearly complete genome of Theileria equi (Piroplasmida, Apicomplexa), which contains four nuclear chromosomes, a mitochondrial genome, and an...
We are reporting the nearly complete genome of Theileria equi (Piroplasmida, Apicomplexa), which contains four nuclear chromosomes, a mitochondrial genome, and an apicoplast from the NVSL354 reference isolate. This report includes all six genetic molecules.
PubMed: 36688717
DOI: 10.1128/mra.00809-22 -
MBio Feb 2023Atg8 family proteins are highly conserved eukaryotic proteins with diverse autophagy and nonautophagic functions in eukaryotes. While the structural features required...
Atg8 family proteins are highly conserved eukaryotic proteins with diverse autophagy and nonautophagic functions in eukaryotes. While the structural features required for conserved autophagy functions of Atg8 are well established, little is known about the molecular changes that facilitated acquisition of divergent, nonautophagic functions of Atg8. The malaria parasite Plasmodium falciparum offers a unique opportunity to study nonautophagic functions of Atg8 family proteins because it encodes a single Atg8 homolog whose only essential function is in the inheritance of an unusual secondary plastid called the apicoplast. Here, we used functional complementation to investigate the structure-function relationship for this divergent Atg8 protein. We showed that the LC3-interacting region (LIR) docking site (LDS), the major interaction interface of the Atg8 protein family, is required for P. falciparum Atg8 (Atg8) apicoplast localization and function, likely via Atg8 lipidation. On the other hand, another region previously implicated in canonical Atg8 interactions, the N-terminal helix, is not required for apicoplast-specific Atg8 function. Finally, our investigations at the cellular level demonstrate that the unique apicomplexan-specific loop, previously implicated in interaction with membrane conjugation machinery in recombinant protein-based assays, is not required for membrane conjugation nor for the apicoplast-specific effector function of Atg8 in both P. falciparum and related Apicomplexa member Toxoplasma gondii. These results suggest that the effector function of apicomplexan Atg8 is mediated by structural features distinct from those previously identified for macroautophagy and selective autophagy functions. The most extensively studied role of Atg8 proteins is in autophagy. However, it is clear that they have other nonautophagic functions critical to cell function and disease pathogenesis that are so far understudied compared to their canonical role in autophagy. Mammalian cells contain multiple Atg8 paralogs that have diverse, specialized functions. Gaining molecular insight into their nonautophagic functions is difficult because of redundancy between the homologs and their role in both autophagy and nonautophagic pathways. Malaria parasites such as Plasmodium falciparum are a unique system to study a novel, nonautophagic function of Atg8 separate from its role in autophagy: they have only one Atg8 protein whose only essential function is in the inheritance of the apicoplast, a unique secondary plastid organelle. Insights into the molecular basis of Atg8's function in apicoplast biogenesis will have important implications for the evolution of diverse nonautophagic functions of the Atg8 protein family.
Topics: Animals; Apicoplasts; Autophagy-Related Protein 8 Family; Malaria; Mammals; Parasites; Protozoan Proteins; Structure-Activity Relationship
PubMed: 36625582
DOI: 10.1128/mbio.03642-21 -
Drug Research Mar 2023Malaria is one of the world's most devastating diseases, infecting well over 300 million people annually and killing between 2 and 3 million worldwide. Increasing...
Malaria is one of the world's most devastating diseases, infecting well over 300 million people annually and killing between 2 and 3 million worldwide. Increasing parasite resistance to many existing drugs is exacerbating disease. Resistance to commonly used malarial drugs is increasing the need to develop new drugs urgently. Due to the slow pace and substantial costs of new drug development, repurposing of old drugs which is recently increasingly becoming an attractive proposition of highly efficient and effective way of drug discovery led us to study the drug rifampicin for this purpose. The present paper aims to investigate the route of apicoplast-targeted proteins that putatively encode β subunits of RNA polymerase with an objective to develop an effective antimalarial drug. Homology searching for conserved binding site to the rifampicin drug and the functional analysis of B gene were done. Multiple Sequence alignment analysis of B was compared with that in - B and - B. Docking studies of - B complex was also done for finding binding affinity. The results of computational studies showed that rifampicin is a potential drug for malaria.
Topics: Humans; Rifampin; Molecular Docking Simulation; Drug Repositioning; Malaria, Falciparum; Mycobacterium tuberculosis; Malaria; Plasmodium falciparum; Antimalarials; Drug Design
PubMed: 36623818
DOI: 10.1055/a-1974-9028 -
Molecular Biology and Evolution Jan 2023Apicomplexans and related lineages comprise many obligate symbionts of animals; some of which cause notorious diseases such as malaria. They evolved from photosynthetic...
Apicomplexans and related lineages comprise many obligate symbionts of animals; some of which cause notorious diseases such as malaria. They evolved from photosynthetic ancestors and transitioned into a symbiotic lifestyle several times, giving rise to species with diverse non-photosynthetic plastids. Here, we sought to reconstruct the evolution of the cryptic plastids in the apicomplexans, chrompodellids, and squirmids (ACS clade) by generating five new single-cell transcriptomes from understudied gregarine lineages, constructing a robust phylogenomic tree incorporating all ACS clade sequencing datasets available, and using these to examine in detail, the evolutionary distribution of all 162 proteins recently shown to be in the apicoplast by spatial proteomics in Toxoplasma. This expanded homology-based reconstruction of plastid proteins found in the ACS clade confirms earlier work showing convergence in the overall metabolic pathways retained once photosynthesis is lost, but also reveals differences in the degrees of plastid reduction in specific lineages. We show that the loss of the plastid genome is common and unexpectedly find many lineage- and species-specific plastid proteins, suggesting the presence of evolutionary innovations and neofunctionalizations that may confer new functional and metabolic capabilities that are yet to be discovered in these enigmatic organelles.
Topics: Animals; Proteome; Plastids; Phylogeny; Photosynthesis; Metabolic Networks and Pathways
PubMed: 36610734
DOI: 10.1093/molbev/msad002 -
Current Opinion in Microbiology Feb 2023The apicoplast of Plasmodium falciparum is the only source of essential isoprenoid precursors and Coenzyme A (CoA) in the parasite. Isoprenoid precursor synthesis relies... (Review)
Review
The apicoplast of Plasmodium falciparum is the only source of essential isoprenoid precursors and Coenzyme A (CoA) in the parasite. Isoprenoid precursor synthesis relies on the iron-sulfur cluster (FeS) cofactors produced within the apicoplast, rendering FeS synthesis an essential function of this organelle. Recent reports provide important insights into the roles of FeS cofactors and the use of isoprenoid precursors and CoA both inside and outside the apicoplast. Here, we review the recent insights into the roles of these metabolites in blood-stage malaria parasites and discuss new questions that have been raised in light of these discoveries.
Topics: Animals; Humans; Apicoplasts; Parasites; Malaria; Plasmodium falciparum; Terpenes; Protozoan Proteins
PubMed: 36563485
DOI: 10.1016/j.mib.2022.102255 -
Parasitology Mar 2023The apicomplexan parasite causes seasonal foodborne outbreaks of the gastrointestinal illness cyclosporiasis. Prior to the coronavirus disease-2019 pandemic, annually...
The apicomplexan parasite causes seasonal foodborne outbreaks of the gastrointestinal illness cyclosporiasis. Prior to the coronavirus disease-2019 pandemic, annually reported cases were increasing in the USA, leading the US Centers for Disease Control and Prevention to develop a genotyping tool to complement cyclosporiasis outbreak investigations. Thousands of US isolates and 1 from China (strain CHN_HEN01) were genotyped by Illumina amplicon sequencing, revealing 2 lineages (A and B). The allelic composition of isolates was examined at each locus. Two nuclear loci (CDS3 and 360i2) distinguished lineages A and B. CDS3 had 2 major alleles: 1 almost exclusive to lineage A and the other to lineage B. Six 360i2 alleles were observed – 2 exclusive to lineage A (alleles A1 and A2), 2 to lineage B (B1 and B2) and 1 (B4) was exclusive to CHN_HEN01 which shared allele B3 with lineage B. Examination of heterozygous genotypes revealed that mixtures of A- and B-type 360i2 alleles occurred rarely, suggesting a lack of gene flow between lineages. Phylogenetic analysis of loci from whole-genome shotgun sequences, mitochondrial and apicoplast genomes, revealed that CHN_HEN01 represents a distinct lineage (C). Retrospective examination of epidemiologic data revealed associations between lineage and the geographical distribution of US infections plus strong temporal associations. Given the multiple lines of evidence for speciation within human-infecting , we provide an updated taxonomic description of , and describe 2 novel species as aetiological agents of human cyclosporiasis: sp. nov. and sp. nov. (Apicomplexa: Eimeriidae).
Topics: Humans; Cyclosporiasis; Cyclospora; Phylogeny; Retrospective Studies; COVID-19; Feces
PubMed: 36560856
DOI: 10.1017/S003118202200172X