-
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 -
Translational Research : the Journal of... Aug 2018The proteasome plays a vital role throughout the life cycle as Plasmodium parasites quickly adapt to a new host and undergo a series of morphologic changes during... (Review)
Review
The proteasome plays a vital role throughout the life cycle as Plasmodium parasites quickly adapt to a new host and undergo a series of morphologic changes during asexual replication and sexual differentiation. Plasmodium carries 3 different types of protease complexes: typical eukaryotic proteasome (26S) that resides in the cytoplasm and the nucleus, a prokaryotic proteasome homolog ClpQ that resides in the mitochondria, and a caseinolytic protease complex ClpP that resides in the apicoplast. In silico prediction in conjunction with immunoprecipitation analysis of ubiquitin conjugates have suggested that over half of the Plasmodium falciparum proteome during asexual reproduction are potential targets for ubiquitination. The marked potency of multiple classes of proteasome inhibitors against all stages of the life cycle, synergy with the current frontline antimalarial, artemisinin, and recent advances identifying differences between Plasmodium and human proteasomes strongly support further drug development efforts.
Topics: Animals; Antimalarials; Humans; Life Cycle Stages; Malaria, Falciparum; Plasmodium falciparum; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Reproduction, Asexual
PubMed: 30009761
DOI: 10.1016/j.trsl.2018.04.007 -
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 -
International Journal of Molecular... Nov 2021Glutaredoxins (GRXs), important components of the intracellular thiol redox system, are involved in multiple cellular processes. In a previous study, we identified five...
Glutaredoxins (GRXs), important components of the intracellular thiol redox system, are involved in multiple cellular processes. In a previous study, we identified five GRXs in the apicomplexan parasite, . In the present study, we confirmed that the GRXs S14 and C5 are located in the apicoplast, which suggests unique functions for these proteins. Although single-gene deficiency did not affect the growth of parasites, a double knockout (Δ S14Δ C5) significantly reduced their reproductive capacity. However, there were no significant changes in redox indices (GSH/GSSG ratio, reactive oxygen species and hydroxyl radical levels) in double-knockout parasites, indicating that S14 and C5 are not essential for maintaining the redox balance in parasite cells. Key amino acid mutations confirmed that the Cys of S14 and Cys of C5 are important for parasite growth. Based on comparative proteomics, 79 proteins were significantly downregulated in double-knockout parasites, including proteins mainly involved in the electron transport chain, the tricarboxylic acid cycle and protein translation. Collectively, GRX S14 and GRX C5 coordinate the growth of parasites. However, considering their special localization, the unique functions of GRX S14 and GRX C5 need to be further studied.
Topics: Apicoplasts; Glutaredoxins; Glutathione; Neospora; Oxidation-Reduction; Protozoan Proteins; Reactive Oxygen Species
PubMed: 34769376
DOI: 10.3390/ijms222111946 -
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 -
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 -
Bioscience Reports Jun 2022The untangling or overwinding of genetic material is an inevitable part of DNA replication, repair, recombination, and transcription. Topoisomerases belong to a... (Review)
Review
The untangling or overwinding of genetic material is an inevitable part of DNA replication, repair, recombination, and transcription. Topoisomerases belong to a conserved enzyme family that amends DNA topology during various processes of DNA metabolism. To relax the genetic material, topoisomerases transiently break the phosphodiester bond on one or both DNA strands and remain associated with the cleavage site by forming a covalent enzyme-DNA intermediate. This releases torsional stress and allows the broken DNA to be re-ligated by the enzyme. The biological function of topoisomerases ranges from the separation of sister chromatids following DNA replication to the aiding of chromosome condensation and segregation during mitosis. Topoisomerases are also actively involved in meiotic recombination. The unicellular apicomplexan parasite, Plasmodium falciparum, harbors different topoisomerase subtypes, some of which have substantially different sequences and functions from their human counterparts. This review highlights the biological function of each identified Plasmodium topoisomerase along with a comparative analysis of their orthologs in human or other model organisms. There is also a focus on recent advancements towards the development of topoisomerase chemical inhibitors, underscoring the druggability of unique topoisomerase subunits that are absent in humans. Plasmodium harbors three distinct genomes in the nucleus, apicoplast, and mitochondria, respectively, and undergoes non-canonical cell division during the schizont stage of development. This review emphasizes the specific developmental stages of Plasmodium on which future topoisomerase research should focus.
Topics: Biology; DNA Replication; DNA Topoisomerases, Type I; Humans; Mitosis; Plasmodium
PubMed: 35699968
DOI: 10.1042/BSR20212847 -
Essays in Biochemistry 2011Calcium is relevant for several vital functions in apicomplexan parasites, including host cell invasion, parasite motility and differentiation. The ER (endoplasmic... (Review)
Review
Calcium is relevant for several vital functions in apicomplexan parasites, including host cell invasion, parasite motility and differentiation. The ER (endoplasmic reticulum) and calcium-rich acidocalcisomes have been identified as major calcium stores. Other potential calcium-storage organelles include the Golgi, the mitochondrion, the apicoplast and the recently described plant-like vacuole in Toxoplasma gondii. Compared with most eukaryotic systems, apicomplexan parasites contain a reduced number of calcium-related genes, a vast majority of which remain uncharacterized. Several Ca²⁺-ATPases have been described in apicomplexans, several of which are annotated in the different genomes. There is experimental evidence for an IP3 (inositol 1,4,5-trisphosphate)-dependent calcium response in Plasmodium spp. and T. gondii, although no IP3 or ryanodine receptors have been identified. Genes encoding potential calcium channels are present in T. gondi, but not in Plasmodium spp. and Cryptosporidium spp. Effector calcium-binding proteins including calmodulins and CDPK (calcium-dependent protein kinase) genes mainly found in plants have also been described. The characterized CDPKs were found to play important roles in protein secretion, host cell invasion and parasite differentiation. Taken together, the available information on calcium storage and function in apicomplexans, although fragmented, suggest the existence of unique calcium-mediated pathways in these parasites. An in-depth functional characterization of the apicomplexan calcium-related genes could lead to the identification of novel therapeutic targets, and will improve our understanding of the role of calcium in parasite development and virulence.
Topics: Animals; Apicomplexa; Calcium; Calcium Channels; Calcium-Binding Proteins; Calcium-Transporting ATPases; Endoplasmic Reticulum; Inositol 1,4,5-Trisphosphate; Mitochondria; Protein Kinases; Protozoan Infections
PubMed: 22023444
DOI: 10.1042/bse0510097 -
Microbiology Resource Announcements Jun 2024We are reporting a genome containing four nuclear chromosomes, a mitochondrial genome, and an apicoplast from reference isolate NVSL348. This report includes a gapless...
We are reporting a genome containing four nuclear chromosomes, a mitochondrial genome, and an apicoplast from reference isolate NVSL348. This report includes a gapless assembly consisting of all six genetic molecules.
PubMed: 38651914
DOI: 10.1128/mra.00039-24 -
Proceedings of the National Academy of... Apr 2023Coenzyme A (CoA) biosynthesis is an excellent target for antimalarial intervention. While most studies have focused on the use of CoA to produce acetyl-CoA in the...
Coenzyme A (CoA) biosynthesis is an excellent target for antimalarial intervention. While most studies have focused on the use of CoA to produce acetyl-CoA in the apicoplast and the cytosol of malaria parasites, mitochondrial acetyl-CoA production is less well understood. In the current study, we performed metabolite-labeling experiments to measure endogenous metabolites in lines with genetic deletions affecting mitochondrial dehydrogenase activity. Our results show that the mitochondrion is required for cellular acetyl-CoA biosynthesis and identify a synthetic lethal relationship between the two main ketoacid dehydrogenase enzymes. The activity of these enzymes is dependent on the lipoate attachment enzyme LipL2, which is essential for parasite survival solely based on its role in supporting acetyl-CoA metabolism. We also find that acetyl-CoA produced in the mitochondrion is essential for the acetylation of histones and other proteins outside of the mitochondrion. Taken together, our results demonstrate that the mitochondrion is required for cellular acetyl-CoA metabolism and protein acetylation essential for parasite survival.
Topics: Plasmodium falciparum; Acetyl Coenzyme A; Acetylation; Mitochondria; Oxidoreductases
PubMed: 37068227
DOI: 10.1073/pnas.2210929120