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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 -
Veterinary Research Jan 2024Toxoplasma gondii is among the most important parasites worldwide. The apicoplast is a unique organelle shared by all Apicomplexan protozoa. Increasing lines of evidence...
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.
Topics: Animals; Toxoplasma; Apicoplasts; Cell Cycle; Homeostasis; Deubiquitinating Enzymes; Protozoan Proteins
PubMed: 38233899
DOI: 10.1186/s13567-023-01261-y -
Parasitology International Aug 2021A key characteristic of eukaryotic cells is the presence of organelles with discrete boundaries and functions. Such subcellular compartmentalization into organelles... (Review)
Review
A key characteristic of eukaryotic cells is the presence of organelles with discrete boundaries and functions. Such subcellular compartmentalization into organelles necessitates platforms for communication and material exchange between each other which often involves vesicular trafficking and associated processes. Another way is via the close apposition between organellar membranes, called membrane contact sites (MCSs). Apart from lipid transfer, MCSs have been implicated to mediate in various cellular processes including ion transport, apoptosis, and organelle dynamics. In mammalian and yeast cells, contact sites have been reported between the membranes of the following: the endoplasmic reticulum (ER) and the plasma membrane (PM), ER and the Golgi apparatus, ER and endosomes (i.e., vacuoles, lysosomes), ER and lipid droplets (LD), the mitochondria and vacuoles, the nucleus and vacuoles, and the mitochondria and lipid droplets, whereas knowledge of MCSs in non-model organisms such as protozoan parasites is extremely limited. Growing evidence suggests that MCSs play more general and conserved roles in cell physiology. In this mini review, we summarize and discuss representative MCSs in divergent parasitic protozoa, and highlight the universality, diversity, and the contribution of MCSs to parasitism.
Topics: Cell Membrane; Entamoeba histolytica; Giardia lamblia; Organelles; Plasmodium; Signal Transduction; Toxoplasma; Trypanosoma brucei brucei
PubMed: 33933652
DOI: 10.1016/j.parint.2021.102372 -
Trends in Parasitology Oct 2019The discovery of a plastid in apicomplexan parasites was hoped to be a watershed moment in the treatment of parasitic diseases as it revealed drug targets that are... (Review)
Review
The discovery of a plastid in apicomplexan parasites was hoped to be a watershed moment in the treatment of parasitic diseases as it revealed drug targets that are implicitly divergent from host molecular processes. Indeed, this organelle, known as the apicoplast, has since been a productive therapeutic target for pharmaceutical interventions against infections by Plasmodium, Toxoplasma, Babesia, and Theileria. However, some inhibitors of the apicoplast are restricted in their treatment utility because of their slow-kill kinetics, and this characteristic is called the delayed death effect. Here we review the recent genetic and pharmacological experiments that interrogate the causes of delayed death and explore the foundation of this phenomenon in Plasmodium and Toxoplasma parasites.
Topics: Animals; Antiparasitic Agents; Apicoplasts; Humans; Parasitic Diseases; Plasmodium; Toxoplasma
PubMed: 31427248
DOI: 10.1016/j.pt.2019.07.010 -
Proceedings of the National Academy of... Aug 2023is responsible for toxoplasmosis, a disease that can be serious when contracted during pregnancy, but can also be a threat for immunocompromised individuals. Acute...
is responsible for toxoplasmosis, a disease that can be serious when contracted during pregnancy, but can also be a threat for immunocompromised individuals. Acute infection is associated with the tachyzoite form that spreads rapidly within the host. However, under stress conditions, some parasites can differentiate into cyst-forming bradyzoites, residing mainly in the central nervous system, retina and muscle. Because this latent form of the parasite is resistant to all currently available treatments, and is central to persistence and transmission of the parasite, specific therapeutic strategies targeting this developmental stage need to be found. contains a plastid of endosymbiotic origin called the apicoplast, which is an appealing drug target because it is essential for tachyzoite viability and contains several key metabolic pathways that are largely absent from the mammalian host. Its function in bradyzoites, however, is unknown. Our objective was thus to study the contribution of the apicoplast to the viability and persistence of bradyzoites during chronic toxoplasmosis. We have used complementary strategies based on stage-specific promoters to generate conditional bradyzoite mutants of essential apicoplast genes. Our results show that specifically targeting the apicoplast in both in vitro or in vivo-differentiated bradyzoites leads to a loss of long-term bradyzoite viability, highlighting the importance of this organelle for this developmental stage. This validates the apicoplast as a potential area to look for therapeutic targets in bradyzoites, with the aim to interfere with this currently incurable parasite stage.
Topics: Animals; Female; Pregnancy; Humans; Toxoplasma; Apicoplasts; Central Nervous System; Cysts; Toxoplasmosis; Mammals
PubMed: 37590416
DOI: 10.1073/pnas.2309043120 -
Molecular Informatics May 2020Carbon (C), hydrogen (H), nitrogen (N), oxygen (O), and sulfur (S) atoms intrigue as they are the foundation for amino acid (AA) composition and the folding and...
Carbon (C), hydrogen (H), nitrogen (N), oxygen (O), and sulfur (S) atoms intrigue as they are the foundation for amino acid (AA) composition and the folding and functions of proteins and thus define and control the survival of a cell, the smallest unit of life. Here, we calculated the proteomic atom distribution in >1500 randomly selected species across the entire current phylogenetic tree and identified uracil-5-methyltransferase (U5MTase) of the protozoan parasite Plasmodium falciparum (Pf, strain Pf3D7), with a distinct atom and AA distribution pattern. We determined its apicoplast location and in silico 3D protein structure to refocus attention exclusively on U5MTase with tremendous potential for therapeutic intervention in malaria. Around 300 million clinical cases of malaria occur each year in tropical and subtropical regions of the world, resulting in over one million deaths annually, placing malaria among the most serious infectious diseases. Genomic and proteomic research of the clades of parasites containing Pf is progressing slowly and the functions of most of the ∼5300 genes are still unknown. We applied a 'bottom-up' comparative proteomic atomics analysis across the phylogenetic tree to visualize a protein molecule on its actual basis - i. e., its atomic level. We identified a protruding Pf3D7-specific U5MTase, determined its 3D protein structure, and identified potential inhibitory drug molecules through in silico drug screening that might serve as possible remedies for the treatment of malaria. Besides, this atomic-based proteome map provides a unique approach for the identification of parasite-specific proteins that could be considered as novel therapeutic targets.
Topics: Amino Acid Sequence; Amino Acids; Carbon; Computational Biology; Computer Simulation; Databases, Genetic; Humans; Hydrogen; Ligands; Malaria, Falciparum; Methyltransferases; Models, Chemical; Nitrogen; Oxygen; Phylogeny; Plasmodium falciparum; Protein Structure, Tertiary; Proteome; Proteomics; Protozoan Proteins; Sulfur; Uracil
PubMed: 31943843
DOI: 10.1002/minf.201900135 -
Biology of the Cell Jan 2021Like other apicomplexan parasites, Toxoplasma gondii harbours a four-membraned endosymbiotic organelle - the apicoplast. Apicoplast proteins are nuclear encoded and...
BACKGROUND INFORMATION
Like other apicomplexan parasites, Toxoplasma gondii harbours a four-membraned endosymbiotic organelle - the apicoplast. Apicoplast proteins are nuclear encoded and trafficked to the organelle through the endoplasmic reticulum (ER). From the ER to the apicoplast, two distinct protein trafficking pathways can be used. One such pathway is the cell's secretory pathway involving the Golgi, whereas the other is a unique Golgi-independent pathway. Using different experimental approaches, many apicoplast proteins have been shown to utilize the Golgi-independent pathway, whereas a handful of reports show that a few proteins use the Golgi-dependent pathway. This has led to an emphasis towards the unique Golgi-independent pathway when apicoplast protein trafficking is discussed in the literature. Additionally, the molecular features that drive proteins to each pathway are not known.
RESULTS
In this report, we systematically test eight apicoplast proteins, using a C-terminal HDEL sequence to assess the role of the Golgi in their transport. We demonstrate that dually localised proteins of the apicoplast and mitochondrion (TgSOD2, TgTPx1/2 and TgACN/IRP) are trafficked through the Golgi, whereas proteins localised exclusively to the apicoplast are trafficked independent of the Golgi. Mutants of the dually localised proteins that localised exclusively to the apicoplast also showed trafficking through the Golgi. Phylogenetic analysis of TgSOD2, TgTPx1/2 and TgACN/IRP suggested that the evolutionary origins of TgSOD2 and TgTPx1/2 lie in the mitochondrion, whereas TgACN/IRP appears to have originated from the apicoplast.
CONCLUSIONS AND SIGNIFICANCE
Collectively, with these results, for the first time, we establish that the driver of the Golgi-dependent trafficking route to the apicoplast is the dual localisation of the protein to the apicoplast and the mitochondrion.
Topics: Apicoplasts; Golgi Apparatus; Mitochondria; Protein Transport; Protozoan Proteins; Toxoplasma
PubMed: 33112425
DOI: 10.1111/boc.202000050 -
ELife Dec 2023Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by...
Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample by ~4.5×. Here, we apply U-ExM to the human malaria parasite during the asexual blood stage of its lifecycle to understand how this parasite is organized in three dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have cataloged 13 different structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of during its intraerythrocytic development to date and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology.
Topics: Humans; Plasmodium falciparum; Microscopy; Ascomycota; Malaria, Falciparum; Apicoplasts; Plaque, Amyloid
PubMed: 38108809
DOI: 10.7554/eLife.88088 -
Microorganisms Jun 2023is an obligate intracellular parasite of the phylum Apicomplexa and causes toxoplasmosis infections, a disease that affects a quarter of the world's population and has...
is an obligate intracellular parasite of the phylum Apicomplexa and causes toxoplasmosis infections, a disease that affects a quarter of the world's population and has no effective cure. Epigenetic regulation is one of the mechanisms controlling gene expression and plays an essential role in all organisms. Lysine deacetylases (KDACs) act as epigenetic regulators affecting gene silencing in many eukaryotes. Here, we focus on TgKDAC4, an enzyme unique to apicomplexan parasites, and a class IV KDAC, the least-studied class of deacetylases so far. This enzyme shares only a portion of the specific KDAC domain with other organisms. Phylogenetic analysis from the TgKDAC4 domain shows a putative prokaryotic origin. Surprisingly, TgKDAC4 is located in the apicoplast, making it the only KDAC found in this organelle to date. Transmission electron microscopy assays confirmed the presence of TgKDAC4 in the periphery of the apicoplast. We identified possible targets or/and partners of TgKDAC4 by immunoprecipitation assays followed by mass spectrometry analysis, including TgCPN60 and TgGAPDH2, both located at the apicoplast and containing acetylation sites. Understanding how the protein works could provide new insights into the metabolism of the apicoplast, an essential organelle for parasite survival.
PubMed: 37375060
DOI: 10.3390/microorganisms11061558 -
Biochemistry Dec 2022is the most common and harmful causative agent of malaria worldwide. As a member of the phylum Apicomplexa, is characterized by the presence of a unique and essential...
is the most common and harmful causative agent of malaria worldwide. As a member of the phylum Apicomplexa, is characterized by the presence of a unique and essential organelle called the apicoplast. Reminiscent of an algal chloroplast, the apicoplast possesses its own genome, which is maintained by a single apicoplast DNA polymerase (apPol). Ribonucleotides misincorporated into the genome are among the most common lesions encountered by DNA polymerases, and the ability to replicate past these lesions varies widely among characterized enzymes. Here, we have investigated the ribonucleotide (rNTP) misincorporation frequency of apPol and determined its reverse transcriptase (RT) activity across templating ribonucleotides. Pre-steady-state kinetic experiments indicate that apPol does not have an unusually high discrimination between deoxy and ribonucleotides, with frequencies ranging between 10 and 10 depending on the identity of the ribonucleotide. Once incorporated into its template, apPol can replicate across ribonucleotides using its RT activity, but extension of a deoxynucleotide basepaired with the ribonucleotide is slow relative to a canonical basepair. Exonuclease assays indicate that apPol proofreads ribonucleotides an order of magnitude faster than extension, suggesting that most, but not all, misincorporated ribonucleotides will be excised. Although the components have not been identified, ribonucleotide excision repair or other tolerance mechanisms may exist in the apicoplast, and more targeted proteomic efforts will be needed to elucidate them.
Topics: Apicoplasts; Ribonucleotides; Plasmodium falciparum; Proteomics; DNA-Directed DNA Polymerase; DNA; RNA-Directed DNA Polymerase
PubMed: 36346714
DOI: 10.1021/acs.biochem.2c00450