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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 -
Microbiology Spectrum Feb 2023Iron-sulfur [Fe-S] clusters are one of the most ancient and functionally versatile natural biosynthetic prosthetic groups required by various proteins involved in...
Iron-sulfur [Fe-S] clusters are one of the most ancient and functionally versatile natural biosynthetic prosthetic groups required by various proteins involved in important metabolic processes, including the oxidative phosphorylation of proteins, electron transfer, energy metabolism, DNA/RNA metabolism, and protein translation. Apicomplexan parasites harbor two possible [Fe-S] cluster assembly pathways: the iron-sulfur cluster (ISC) pathway in the mitochondria and the sulfur formation (SUF) pathway in the apicoplast. Glutaredoxin 5 (GRX5) is involved in the ISC pathway in many eukaryotes. However, the cellular roles of GRX5 in apicomplexan parasites remain to be explored. Here, we showed that Neospora caninum mitochondrial GRX5 (NcGRX5) deficiency resulted in aberrant mitochondrial ultrastructure and led to a significant reduction in parasite proliferation and virulence in mice, suggesting that NcGRX5 is important for parasite growth and . Comparative proteomics and energy metabolomics were used to investigate the effects of NcGRX5 on parasite growth and mitochondrial metabolism. The data showed that disruption of NcGRX5 downregulated the expression of mitochondrial electron transport chain (ETC) and tricarboxylic acid cycle (TCA) cycle proteins and reduced the corresponding metabolic fluxes. Subsequently, we identified 23 proteins that might be adjacent to or interact with NcGRX5 by proximity-based protein labeling techniques and proteomics. The interactions between NcGRX5 and two iron-sulfur cluster synthesis proteins (ISCS and ISCU1) were further confirmed by coimmunoprecipitation assays. In conclusion, NcGRX5 is important for parasite growth and may regulate mitochondrial energy metabolism by mediating the biosynthesis of iron-sulfur clusters. Iron-sulfur [Fe-S] clusters are among the oldest and most ubiquitous prosthetic groups, and they are required for a variety of proteins involved in important metabolic processes. The intracellular parasites in the phylum , including Plasmodium, Toxoplasma gondii, and Neospora caninum, harbor the ISC pathway involved in the biosynthesis of [Fe-S] clusters in mitochondria. These cofactors are required for a variety of important biological processes. However, little is known about the role of oxidoreductase glutaredoxins in these parasites. Our data indicate that NcGRX5 is an essential protein that plays multiple roles in several biological processes of N. caninum. NcGRX5 interacts with the mitochondrial iron-sulfur cluster synthesis proteins ISCS and ISCU1 and also regulates parasite energy metabolism. These data provide an insider's view of the metabolic regulation and iron-sulfur cluster assembly processes in the apicomplexan parasites.
Topics: Animals; Mice; Parasites; Glutaredoxins; Neospora; Mitochondria; Iron-Sulfur Proteins; Energy Metabolism; Iron; Sulfur
PubMed: 36541793
DOI: 10.1128/spectrum.03091-22 -
Frontiers in Chemistry 2022is the etiological agent of human malaria, one of the most widespread diseases in tropical and subtropical regions. Drug resistance is one of the biggest problems in...
is the etiological agent of human malaria, one of the most widespread diseases in tropical and subtropical regions. Drug resistance is one of the biggest problems in controlling the disease, which leads to the need to discover new antimalarial compounds. One of the most promissory drugs purposed is fosmidomycin, an inhibitor of the biosynthesis of isoprene units by the methylerythritol 4-phosphate (MEP) pathway, which in some cases failed in clinical studies. Once formed, isoprene units are condensed to form longer structures such as farnesyl and geranylgeranyl pyrophosphate, which are necessary for Heme O and A formation, ubiquinone, and dolichyl phosphate biosynthesis as well as for protein isoprenylation. Even though the natural substrates of polyprenyl transferases and synthases are polyprenyl pyrophosphates, it was already demonstrated that isoprenoid alcohols (polyprenols) such as farnesol (FOH) and geranylgeraniol (GGOH) can rescue parasites from fosmidomycin. This study better investigated how this rescue phenomenon occurs by performing drug-rescue assays. Similarly, to FOH and GGOH, it was observed that phytol (POH), a 20-carbon plant isoprenoid, as well as unsaponifiable lipid extracts from foods rescue parasites from the antimalarial effect of fosmidomycin. Contrarily, neither dolichols nor nonaprenol rescue parasites from fosmidomycin. Considering this, here we characterized the transport of FOH, GGOH, and POH. Once incorporated, it was observed that these substances are phosphorylated, condensed into longer isoprenoid alcohols, and incorporated into proteins and dolichyl phosphates. Through proteomic and radiolabelling approaches, it was found that prenylated proteins are naturally attached to several isoprenoids, derived from GGOH, dolichol, and POH if exogenously added. Furthermore, the results suggest the presence of at least two promiscuous protein prenyltransferases in the parasite: one enzyme which can use FPP among other unidentified substrates and another enzyme that can use GGPP, phytyl pyrophosphate (PPP), and dolichols, among other substrates not identified here. Thus, further evidence was obtained for dolichols and other isoprenoid products attached to proteins. This study helps to better understand the apicoplast-targeting antimalarial mechanism of action and a novel post-translational modification of proteins in .
PubMed: 36531309
DOI: 10.3389/fchem.2022.1035548 -
Frontiers in Cellular and Infection... 2022The spread of artemisinin resistant parasites is of global concern and highlights the need to identify new antimalarials for future treatments. Azithromycin, a...
INTRODUCTION
The spread of artemisinin resistant parasites is of global concern and highlights the need to identify new antimalarials for future treatments. Azithromycin, a macrolide antibiotic used clinically against malaria, kills parasites two mechanisms: 'delayed death' by inhibiting the bacterium-like ribosomes of the apicoplast, and 'quick-killing' that kills rapidly across the entire blood stage development.
METHODS
Here, 22 azithromycin analogues were explored for delayed death and quick-killing activities against (the most virulent human malaria) and (a monkey parasite that frequently infects humans).
RESULTS
Seventeen analogues showed improved quick-killing against both species, with up to 38 to 20-fold higher potency over azithromycin after less than 48 or 28 hours of treatment for and , respectively. Quick-killing analogues maintained activity throughout the blood stage lifecycle, including ring stages of parasites (<12 hrs treatment) and were >5-fold more selective against than human cells. Isopentenyl pyrophosphate supplemented parasites that lacked an apicoplast were equally sensitive to quick-killing analogues, confirming that the quick killing activity of these drugs was not directed at the apicoplast. Further, activity against the related apicoplast containing parasite and the gram-positive bacterium did not show improvement over azithromycin, highlighting the specific improvement in antimalarial quick-killing activity. Metabolomic profiling of parasites subjected to the most potent compound showed a build-up of non-haemoglobin derived peptides that was similar to chloroquine, while also exhibiting accumulation of haemoglobin-derived peptides that was absent for chloroquine treatment.
DISCUSSION
The azithromycin analogues characterised in this study expand the structural diversity over previously reported quick-killing compounds and provide new starting points to develop azithromycin analogues with quick-killing antimalarial activity.
Topics: Animals; Humans; Antimalarials; Azithromycin; Parasites; Plasmodium falciparum; Malaria, Falciparum; Chloroquine; Malaria
PubMed: 36530422
DOI: 10.3389/fcimb.2022.1063407 -
PLoS Pathogens Nov 2022Many apicomplexan parasites harbor a non-photosynthetic plastid called the apicoplast, which hosts important metabolic pathways like the methylerythritol 4-phosphate...
Many apicomplexan parasites harbor a non-photosynthetic plastid called the apicoplast, which hosts important metabolic pathways like the methylerythritol 4-phosphate (MEP) pathway that synthesizes isoprenoid precursors. Yet many details in apicoplast metabolism are not well understood. In this study, we examined the physiological roles of four glycolytic enzymes in the apicoplast of Toxoplasma gondii. Many glycolytic enzymes in T. gondii have two or more isoforms. Endogenous tagging each of these enzymes found that four of them were localized to the apicoplast, including pyruvate kinase2 (PYK2), phosphoglycerate kinase 2 (PGK2), triosephosphate isomerase 2 (TPI2) and phosphoglyceraldehyde dehydrogenase 2 (GAPDH2). The ATP generating enzymes PYK2 and PGK2 were thought to be the main energy source of the apicoplast. Surprisingly, deleting PYK2 and PGK2 individually or simultaneously did not cause major defects on parasite growth or virulence. In contrast, TPI2 and GAPDH2 are critical for tachyzoite proliferation. Conditional depletion of TPI2 caused significant reduction in the levels of MEP pathway intermediates and led to parasite growth arrest. Reconstitution of another isoprenoid precursor synthesis pathway called the mevalonate pathway in the TPI2 depletion mutant partially rescued its growth defects. Similarly, knocking down the GAPDH2 enzyme that produces NADPH also reduced isoprenoid precursor synthesis through the MEP pathway and inhibited parasite proliferation. In addition, it reduced de novo fatty acid synthesis in the apicoplast. Together, these data suggest a model that the apicoplast dwelling TPI2 provides carbon source for the synthesis of isoprenoid precursor, whereas GAPDH2 supplies reducing power for pathways like MEP, fatty acid synthesis and ferredoxin redox system in T. gondii. As such, both enzymes are critical for parasite growth and serve as potential targets for anti-toxoplasmic intervention designs. On the other hand, the dispensability of PYK2 and PGK2 suggest additional sources for energy in the apicoplast, which deserves further investigation.
Topics: Animals; Toxoplasma; Apicoplasts; Metabolic Networks and Pathways; Parasites; Pyruvic Acid; Fatty Acids; Protozoan Proteins
PubMed: 36449552
DOI: 10.1371/journal.ppat.1011009 -
Pharmaceutics Nov 2022The methyl erythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential for malaria parasites and also for several human pathogenic bacteria, thus...
The methyl erythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential for malaria parasites and also for several human pathogenic bacteria, thus representing an interesting target for future antimalarials and antibiotics and for diagnostic strategies. We have developed a DNA aptamer (D10) against 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), the second enzyme of this metabolic route. D10 binds in vitro to recombinant DXR from and , showing at 10 µM a ca. 50% inhibition of the bacterial enzyme. In silico docking analysis indicates that D10 associates with DXR in solvent-exposed regions outside the active center pocket. According to fluorescence confocal microscopy data, this aptamer specifically targets in in vitro cultures the apicoplast organelle where the MEP pathway is localized and is, therefore, a highly specific marker of red blood cells parasitized by vs. naïve erythrocytes. D10 is also selective for the detection of MEP+ bacteria (e.g., and ) vs. those lacking DXR (e.g., ). Based on these results, we discuss the potential of DNA aptamers in the development of ligands that can outcompete the performance of the well-established antibody technology for future therapeutic and diagnostic approaches.
PubMed: 36432706
DOI: 10.3390/pharmaceutics14112515 -
Scientific Reports Nov 2022Naturally occurring human infections by zoonotic Plasmodium species have been documented for P. knowlesi, P. cynomolgi, P. simium, P. simiovale, P. inui, P. inui-like,...
Naturally occurring human infections by zoonotic Plasmodium species have been documented for P. knowlesi, P. cynomolgi, P. simium, P. simiovale, P. inui, P. inui-like, P. coatneyi, and P. brasilianum. Accurate detection of each species is complicated by their morphological similarities with other Plasmodium species. PCR-based assays offer a solution but require prior knowledge of adequate genomic targets that can distinguish the species. While whole genomes have been published for P. knowlesi, P. cynomolgi, P. simium, and P. inui, no complete genome for P. brasilianum has been available. Previously, we reported a draft genome for P. brasilianum, and here we report the completed genome for P. brasilianum. The genome is 31.4 Mb in size and comprises 14 chromosomes, the mitochondrial genome, the apicoplast genome, and 29 unplaced contigs. The chromosomes consist of 98.4% nucleotide sites that are identical to the P. malariae genome, the closest evolutionarily related species hypothesized to be the same species as P. brasilianum, with 41,125 non-synonymous SNPs (0.0722% of genome) identified between the two genomes. Furthermore, P. brasilianum had 4864 (82.1%) genes that share 80% or higher sequence similarity with 4970 (75.5%) P. malariae genes. This was demonstrated by the nearly identical genomic organization and multiple sequence alignments for the merozoite surface proteins msp3 and msp7. We observed a distinction in the repeat lengths of the circumsporozoite protein (CSP) gene sequences between P. brasilianum and P. malariae. Our results demonstrate a 97.3% pairwise identity between the P. brasilianum and the P. malariae genomes. These findings highlight the phylogenetic proximity of these two species, suggesting that P. malariae and P. brasilianum are strains of the same species, but this could not be fully evaluated with only a single genomic sequence for each species.
Topics: Animals; Humans; Parasites; Phylogeny; Plasmodium; Malaria; Sequence Analysis, DNA
PubMed: 36396703
DOI: 10.1038/s41598-022-20706-6 -
PLoS Pathogens Nov 2022Phosphoinositides are important second messengers that regulate key cellular processes in eukaryotes. While it is known that a single phosphoinositol-3 kinase (PI3K)...
Phosphoinositides are important second messengers that regulate key cellular processes in eukaryotes. While it is known that a single phosphoinositol-3 kinase (PI3K) catalyses the formation of 3'-phosphorylated phosphoinositides (PIPs) in apicomplexan parasites like Plasmodium and Toxoplasma, how its activity and PI3P formation is regulated has remained unknown. Present studies involving a unique Vps15 like protein (TgVPS15) in Toxoplasma gondii provides insight into the regulation of phosphatidyl-3-phosphate (PI3P) generation and unravels a novel pathway that regulates parasite development. Detailed investigations suggested that TgVPS15 regulates PI3P formation in Toxoplasma gondii, which is important for the inheritance of the apicoplast-a plastid like organelle present in most apicomplexans and parasite replication. Interestingly, TgVPS15 also regulates autophagy in T. gondii under nutrient-limiting conditions as it promotes autophagosome formation. For both these processes, TgVPS15 uses PI3P-binding protein TgATG18 and regulates trafficking and conjugation of TgATG8 to the apicoplast and autophagosomes, which is important for biogenesis of these organelles. TgVPS15 has a protein kinase domain but lacks several key residues conserved in conventional protein kinases. Interestingly, two critical residues in its active site are important for PI3P formation and parasitic functions of this kinase. Collectively, these studies unravel a signalling cascade involving TgVPS15, a novel effector of PI3-kinase in T. gondii and possibly other Apicomplexa, that regulate critical processes like apicoplast biogenesis and autophagy.
Topics: Animals; Apicoplasts; Toxoplasma; Autophagy; Autophagosomes; Parasites; Phosphatidylinositols; Protozoan Proteins
PubMed: 36318587
DOI: 10.1371/journal.ppat.1010922 -
Malaria Journal Oct 2022The resistance of Plasmodium falciparum to artemisinin-based (ART) drugs, the front-line drug family used in artemisinin-based combination therapy (ACT) for treatment of...
BACKGROUND
The resistance of Plasmodium falciparum to artemisinin-based (ART) drugs, the front-line drug family used in artemisinin-based combination therapy (ACT) for treatment of malaria, is of great concern. Mutations in the kelch13 (k13) gene (for example, those resulting in the Cys580Tyr [C580Y] variant) were identified as genetic markers for ART-resistant parasites, which suggests they are associated with resistance mechanisms. However, not all resistant parasites contain a k13 mutation, and clearly greater understanding of resistance mechanisms is required. A genome-wide association study (GWAS) found single nucleotide polymorphisms associated with ART-resistance in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2), and crt (chloroquine resistance transporter), in addition to k13 gene mutations, suggesting that these alleles contribute to the resistance phenotype. The importance of the FD and ARPS10 variants in ART resistance was then studied since both proteins likely function in the apicoplast, which is a location distinct from that of K13.
METHODS
The reported mutations were introduced, together with a mutation to produce the k13-C580Y variant into the ART-sensitive 3D7 parasite line and the effect on ART-susceptibility using the 0-3 h ring survival assay (RSA) was investigated.
RESULTS AND CONCLUSION
Introducing both fd-D193Y and arps10-V127M into a k13-C580Y-containing parasite, but not a wild-type k13 parasite, increased survival of the parasite in the RSA. The results suggest epistasis of arps10 and k13, with arps10-V127M a modifier of ART susceptibility in different k13 allele backgrounds.
Topics: Humans; Plasmodium falciparum; Antimalarials; Malaria, Falciparum; Apicoplasts; Genome-Wide Association Study; Drug Resistance; Protozoan Proteins; Artemisinins; Mutation
PubMed: 36303209
DOI: 10.1186/s12936-022-04330-3