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The FEBS Journal Oct 2022The cysteine desulfurase SufS is a pyridoxal-5'-phosphate-dependent enzyme and is essential for the SUF system, which participates in iron-sulfur cluster biosynthesis....
The cysteine desulfurase SufS is a pyridoxal-5'-phosphate-dependent enzyme and is essential for the SUF system, which participates in iron-sulfur cluster biosynthesis. Inhibition of SufS in the SUF system by D-cycloserine (DCS) in Plasmodium falciparum apicoplast has recently been reported, indicating that SufS could be a target for malaria therapeutics. However, the mechanistic details underlying the inhibition of SufS by DCS have not yet been clarified. Moreover, inhibition of SufS by the other enantiomer, L-cycloserine (LCS), has not been investigated. Herein, we investigated the structure-based inhibition mechanisms of SufS by DCS and LCS using Bacillus subtilis SufS, whose catalytic mechanism has been well characterized in comparison to that of the P. falciparum SufS. Surprisingly, DCS- and LCS-mediated inhibitions of SufS occur via distinct mechanisms resulting in pyridoxamine-5'-phosphate (PMP) in DCS-mediated inhibition and PMP-3-hydroxyisoxazole adduct (PMP-isoxazole) in LCS-mediated inhibition. Biochemical and structural evaluation of SufS variants identified conserved His and Arg residues at the active site as the key determinants of the distinct inhibition mechanisms. The importance of structural elements involved in DCS and LCS-mediated inhibitions of SufS provides valuable insights for the structure-based design of new drugs targeting SufS. DATABASE: Structural data are available in PDB database under the accession numbers 6KFY, 7CEO, 7CEP, 7CEQ, 7CER, 7CES, 7CET, 7CEU, 7E6A, 7E6B, 7E6C, 7E6D, 7E6E, and 7E6F.
Topics: Carbon-Sulfur Lyases; Cycloserine; Iron; Phosphates; Pyridoxal Phosphate; Pyridoxamine; Sulfur
PubMed: 35395703
DOI: 10.1111/febs.16455 -
The Journal of Eukaryotic Microbiology Nov 2022Toxoplasma gondii is a member of the apicomplexan phylum, a group of single-celled eukaryotic parasites that cause significant human morbidity and mortality around the... (Review)
Review
Toxoplasma gondii is a member of the apicomplexan phylum, a group of single-celled eukaryotic parasites that cause significant human morbidity and mortality around the world. T. gondii harbors two organelles of endosymbiotic origin: a non-photosynthetic plastid, known as the apicoplast, and a single mitochondrion derived from the ancient engulfment of an α-proteobacterium. Due to excitement surrounding the novelty of the apicoplast, the T. gondii mitochondrion was, to a certain extent, overlooked for about two decades. However, recent work has illustrated that the mitochondrion is an essential hub of apicomplexan-specific biology. Development of novel techniques, such as cryo-electron microscopy, complexome profiling, and next-generation sequencing have led to a renaissance in mitochondrial studies. This review will cover what is currently known about key features of the T. gondii mitochondrion, ranging from its genome to protein import machinery and biochemical pathways. Particular focus will be given to mitochondrial features that diverge significantly from the mammalian host, along with discussion of this important organelle as a drug target.
Topics: Animals; Humans; Toxoplasma; Parasites; Cryoelectron Microscopy; Apicoplasts; Mitochondria; Mammals
PubMed: 35315174
DOI: 10.1111/jeu.12906 -
PLoS Pathogens Mar 2022Apicomplexa are obligate intracellular parasites responsible for major human infectious diseases such as toxoplasmosis and malaria, which pose social and economic... (Review)
Review
Apicomplexa are obligate intracellular parasites responsible for major human infectious diseases such as toxoplasmosis and malaria, which pose social and economic burdens around the world. To survive and propagate, these parasites need to acquire a significant number of essential biomolecules from their hosts. Among these biomolecules, lipids are a key metabolite required for parasite membrane biogenesis, signaling events, and energy storage. Parasites can either scavenge lipids from their host or synthesize them de novo in a relict plastid, the apicoplast. During their complex life cycle (sexual/asexual/dormant), Apicomplexa infect a large variety of cells and their metabolic flexibility allows them to adapt to different host environments such as low/high fat content or low/high sugar levels. In this review, we discuss the role of lipids in Apicomplexa parasites and summarize recent findings on the metabolic mechanisms in host nutrient adaptation.
Topics: Animals; Apicomplexa; Apicoplasts; Humans; Lipid Metabolism; Lipids; Parasites
PubMed: 35298557
DOI: 10.1371/journal.ppat.1010313 -
ELife Mar 2022Isopentenyl pyrophosphate (IPP) is an essential metabolic output of the apicoplast organelle in malaria parasites and is required for prenylation-dependent vesicular...
Isopentenyl pyrophosphate (IPP) is an essential metabolic output of the apicoplast organelle in 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- (C) but not short-chain (≤C) 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.
Topics: Animals; Apicoplasts; Malaria, Falciparum; Parasites; Plasmodium falciparum; Polyprenols; Protozoan Proteins; Terpenes
PubMed: 35257658
DOI: 10.7554/eLife.73208 -
The FEBS Journal Sep 2022Apicomplexans such as the malaria parasite Plasmodium falciparum possess a unique organelle known as the apicoplast that has its own circular genome. The apicoplast...
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.
Topics: Apicoplasts; DNA; Humans; Malaria; Oxidative Stress; Plasmodium falciparum; Protozoan Proteins
PubMed: 35220686
DOI: 10.1111/febs.16414 -
Microbiology Spectrum Feb 2022The apicoplast, which harbors key pathways involved in biosynthesis of vital metabolites, is a unique and essential nonphotosynthetic plastid organelle in apicomplexan...
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 and parasites. The Atg8-Atg3 interaction plays a key role in Atg8 lipidation and localization, and our previously work in has suggested that the core Atg8-family interacting motif (AIM) in TgAtg3, FADI, and the R27 residue of TgAtg8 contribute to TgAtg8-TgAtg3 interaction . However, little is known about the function of this interaction or its importance in tachyzoite growth in . Here, we generated two complemented cell lines, TgAtg3 and TgAtg8, 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 TgAtg3 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 tachyzoites. is 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 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.
Topics: Amino Acid Motifs; Apicoplasts; Humans; Mutation; Protein Binding; Protein Transport; Protozoan Proteins; Toxoplasma; Toxoplasmosis
PubMed: 35196797
DOI: 10.1128/spectrum.01495-21 -
The Journal of Biological Chemistry Mar 2022
PubMed: 35168039
DOI: 10.1016/j.jbc.2022.101705 -
MBio Feb 2021Ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) form a redox system that is hypothesized to play a central role in the maintenance and function of the apicoplast...
Ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) form a redox system that is hypothesized to play a central role in the maintenance and function of the apicoplast organelle of malaria parasites. The Fd/FNR system provides reducing power to various iron-sulfur cluster (FeS)-dependent proteins in the apicoplast and is believed to help to maintain redox balance in the organelle. While the Fd/FNR system has been pursued as a target for antimalarial drug discovery, Fd, FNR, and the FeS proteins presumably reliant on their reducing power play an unknown role in parasite survival and apicoplast maintenance. To address these questions, we generated genetic deletions of these proteins in a parasite line containing an apicoplast bypass system. Through these deletions, we discovered that Fd, FNR, and certain FeS proteins are essential for parasite survival but found that none are required for apicoplast maintenance. Additionally, we addressed the question of how Fd and its downstream FeS proteins obtain FeS cofactors by deleting the FeS transfer proteins SufA and NfuApi. While individual deletions of these proteins revealed their dispensability, double deletion resulted in synthetic lethality, demonstrating a redundant role in providing FeS clusters to Fd and other essential FeS proteins. Our data support a model in which the reducing power from the Fd/FNR system to certain downstream FeS proteins is essential for the survival of blood-stage malaria parasites but not for organelle maintenance, while other FeS proteins are dispensable for this stage of parasite development. Ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) form one of the few known redox systems in the apicoplast of malaria parasites and provide reducing power to iron-sulfur (FeS) cluster proteins within the organelle. While the Fd/FNR system has been explored as a drug target, the essentiality and roles of this system and the identity of its downstream FeS proteins have not been determined. To answer these questions, we generated deletions of these proteins in an apicoplast metabolic bypass line (PfMev) and determined the minimal set of proteins required for parasite survival. Moving upstream of this pathway, we also generated individual and dual deletions of the two FeS transfer proteins that deliver FeS clusters to Fd and downstream FeS proteins. We found that both transfer proteins are dispensable, but double deletion displayed a synthetic lethal phenotype, demonstrating their functional redundancy. These findings provide important insights into apicoplast biochemistry and drug development.
Topics: Animals; Ferredoxins; Parasites; Plasmodium falciparum; Apicoplasts; NADP; Proteins; Ferredoxin-NADP Reductase
PubMed: 35164549
DOI: 10.1128/mbio.03023-21 -
Frontiers in Cellular and Infection... 2021Boromycin is a boron-containing macrolide antibiotic produced by with potent activity against certain viruses, Gram-positive bacteria and protozoan parasites. Most...
Boromycin is a boron-containing macrolide antibiotic produced by with potent activity against certain viruses, Gram-positive bacteria and protozoan parasites. Most antimalarial antibiotics affect plasmodial organelles of prokaryotic origin and have a relatively slow onset of action. They are used for malaria prophylaxis and for the treatment of malaria when combined to a fast-acting drug. Despite the success of artemisinin combination therapies, the current gold standard treatment, new alternatives are constantly needed due to the ability of malaria parasites to become resistant to almost all drugs that are in heavy clinical use. antiplasmodial activity screens of tetracyclines (omadacycline, sarecycline, methacycline, demeclocycline, lymecycline, meclocycline), macrolides (oleandomycin, boromycin, josamycin, troleandomycin), and control drugs (chloroquine, clindamycin, doxycycline, minocycline, eravacycline) revealed boromycin as highly potent against and the zoonotic . In contrast to tetracyclines, boromycin rapidly killed asexual stages of both species already at low concentrations (~ 1 nM) including multidrug resistant strains (Dd2, K1, 7G8). In addition, boromycin was active against stage V gametocytes at a low nanomolar range (IC: 8.5 ± 3.6 nM). Assessment of the mode of action excluded the apicoplast as the main target. Although there was an ionophoric activity on potassium channels, the effect was too low to explain the drug´s antiplasmodial activity. Boromycin is a promising antimalarial candidate with activity against multiple life cycle stages of the parasite.
Topics: Animals; Anti-Bacterial Agents; Antimalarials; Borates; Malaria, Falciparum; Plasmodium falciparum
PubMed: 35096650
DOI: 10.3389/fcimb.2021.802294 -
Parasites & Vectors Jan 2022Data on the genus Sarcocystis in insectivores are limited. The Asian gray shrew Crocidura attenuata is one of the most common species of the insectivore family Soricidae...
BACKGROUND
Data on the genus Sarcocystis in insectivores are limited. The Asian gray shrew Crocidura attenuata is one of the most common species of the insectivore family Soricidae in South Asia and Southeast Asia. To our knowledge, species of Sarcocystis have never been recorded previously in this host.
METHODS
Tissues were obtained from 42 Asian gray shrews caught in 2017 and 2018 in China. Sarcocysts were observed using light microscopy (LM) and transmission electron microscopy (TEM). To describe the parasite life cycle, muscle tissues of the host infected with sarcocysts were force-fed to two beauty rat snakes Elaphe taeniura. Individual sarcocysts from different Asian gray shrews, and oocysts/sporocysts isolated from the small intestines and feces of the experimental snakes, were selected for DNA extraction, and seven genetic markers, namely, two nuclear loci [18S ribosomal DNA (18S rDNA) and internal transcribed spacer region 1 (ITS1)], three mitochondrial genes [cytochrome oxidase subunit 1 (cox1), cox3 and cytochrome b], and two apicoplast genes (RNA polymerase beta subunit and caseinolytic protease C), were amplified, sequenced and analyzed.
RESULTS
Sarcocysts were found in 17 of the 42 (40.5%) Asian gray shrews. Under LM, the microscopic sarcocysts showed saw- or tooth-like protrusions measuring 3.3-4.5 μm. Ultrastructurally, the sarcocyst wall contained numerous lancet- or leaf-like villous protrusions, similar to those described for type 9h of the common cyst wall classification. The experimental beauty rat snakes shed oocysts/sporocysts measuring 11.9-16.7 × 9.2-10.6 μm with a prepatent period of 10-11 days. Comparison of the newly obtained sequences with those previously deposited in GenBank revealed that those of 18S rDNA and cox1 were most similar to those of Sarcocystis scandentiborneensis recorded in the tree shrews Tupaia minor and Tupaia tana (i.e., 97.6-98.3% and 100% identity, respectively). Phylogenetic analysis based on 18S rDNA or ITS1 sequences placed this parasite close to Sarcocystis spp. that utilize small animals as intermediate hosts and snakes as the known or presumed definitive host. On the basis of morphological and molecular characteristics and host specificity, the parasite was proposed as a new species, named Sarcocystis attenuati.
CONCLUSIONS
Sarcocysts were recorded in Asian gray shrews, to our knowledge for the first time. Based on morphological and molecular characterization, a new species of parasite is proposed: Sarcocystis attenuati. According to the LM and TEM results, S. attenuati sarcocysts are distinct from those of Sarcocystis spp. in other insectivores and those of S. scandentiborneensis in tree shrews. The 18S rDNA or cox1 sequences of Sarcocystis attenuati shared high similarity with those of Sarcocystis scandentiborneensis, Sarcocystis zuoi, Sarcocystis cf. zuoi in the Malayan field rat, and Sarcocystis sp. in the greater white-toothed shrew. Therefore, we suggest that more research on the relationships of these closely related taxa should be undertaken in the future.
Topics: Animals; China; Cyclooxygenase 1; DNA, Protozoan; DNA, Ribosomal; Phylogeny; Polymerase Chain Reaction; RNA, Ribosomal, 18S; Sarcocystis; Sarcocystosis; Shrews
PubMed: 35012619
DOI: 10.1186/s13071-021-05136-z