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ACS Infectious Diseases Jan 2024Replication of the malarial parasite in human erythrocytes requires massive zinc fluxes, necessitating the action of zinc transporters across the parasite plasma and...
Replication of the malarial parasite in human erythrocytes requires massive zinc fluxes, necessitating the action of zinc transporters across the parasite plasma and organellar membranes. Although genetic knockout studies have been conducted on a few "orphan" zinc transporters in spp., none of them have been functionally characterized. We used the recombinant Zrt-/Irt-like protein (ZIP1) and specific antibodies generated against it to explore the subcellular localization, function, metal-ion selectivity, and response to cellular zinc levels. ZIP1 expression was enhanced upon the depletion of cytosolic Zn. The protein transitioned from the processed to unprocessed form through blood stages, localizing to the apicoplast in trophozoites and to the parasite plasma membrane in schizonts and gametocytes, indicating stage-specific functional role. The ZIP1 dimer mediated Zn influx in proteoliposomes. It exhibited preferential binding to Zn compared to Fe, with the selectivity for zinc being driven by a C-terminal histidine-rich region conserved only in primate-infecting species.
Topics: Animals; Humans; Plasmodium falciparum; Apicoplasts; Parasites; Cell Membrane; Erythrocytes
PubMed: 38163252
DOI: 10.1021/acsinfecdis.3c00426 -
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 -
BioRxiv : the Preprint Server For... Nov 2023Here we report the discovery of MED6-189, a new analogue of the kalihinol family of isocyanoterpene (ICT) natural products. MED6-189 is effective against drug-sensitive...
Here we report the discovery of MED6-189, a new analogue of the kalihinol family of isocyanoterpene (ICT) natural products. MED6-189 is effective against drug-sensitive and -resistant strains blocking both intraerythrocytic asexual replication and sexual differentiation. This compound was also effective against and . In vivo efficacy studies using a humanized mouse model of malaria confirms strong efficacy of the compound in animals with no apparent hemolytic activity or apparent toxicity. Complementary chemical biology, molecular biology, genomics and cell biological analyses revealed that MED6-189 primarily targets the parasite apicoplast and acts by inhibiting lipid biogenesis and cellular trafficking. Genetic analyses in revealed that a mutation in , which encodes a component of the parasite secretory machinery, reduced susceptibility to the drug. The high potency of MED6-189 and , its broad range of efficacy, excellent therapeutic profile, and unique mode of action make it an excellent addition to the antimalarial drug pipeline.
PubMed: 38045341
DOI: 10.1101/2023.11.21.568162 -
Scientific Reports Nov 2023In recent phylogenetic studies, bat Polychromophilus and ungulate Plasmodium, two relatively understudied haemosporidian parasites within the Apicomplexa phylum, have...
In recent phylogenetic studies, bat Polychromophilus and ungulate Plasmodium, two relatively understudied haemosporidian parasites within the Apicomplexa phylum, have often been overlooked. Instead, the focus has been primarily on haemosporidian parasites in primates, rodents, and birds. Several phylogenetic analyses of bat Polychromophilus have relied on limited datasets and short informative DNA sequences. As a result of these inherent limitations, the substantiation of their evolutionary stance has encountered a diminished degree of robust validation. This study successfully obtained complete mitochondrial genome sequences from 11 Polychromophilus parasites originating from Hipposideros gentilis and Myotis siligoensis bats for the first time. Additionally, the authors have sequenced the apicoplast caseinolytic protease C genes from Polychromophilus murinus and a potentially new Polychromophilus species. These mitochondrial genomes range in length from 5994 to 6001 bp and consist of three protein-coding genes (PCGs), seven small subunit ribosomal RNA genes (SSU rRNA), 12 large subunit ribosomal RNA genes (LSU rRNA), and seven miscellaneous RNA genes. Phylogenetic analyses using Bayesian Inference and Maximum Likelihood methods indicated robust support for the grouping of ungulate Plasmodium and bat Polychromophilus in a single clade separate from other Plasmodium spp., confirming previous reports, albeit with stronger evidence in this study. The divergence between Polychromophilus in bats and Plasmodium in ungulates occurred approximately 29.61 to 55.77 million years ago (Mya), with a node age estimated at 40.63 Mya. These findings highlight that the genus Plasmodium, which includes species found in ungulates, birds, reptiles, and other mammals, does not form a monophyletic group. By incorporating Polychromophilus in bats and Plasmodium in ungulates, this study contributes significantly to understanding the phylogenetic relationships within the Haemosporida order. It provides valuable insights into the evolutionary history and interconnections among these diverse parasites, thereby expanding knowledge in this field.
Topics: Animals; Chiroptera; Phylogeny; Genome, Mitochondrial; Bayes Theorem; Plasmodium; Mammals; Haemosporida; Parasites; Rodentia; Primates
PubMed: 37985797
DOI: 10.1038/s41598-023-45551-z -
Cellular and Molecular Life Sciences :... Nov 2023During macroautophagy, the Atg8 protein is conjugated to phosphatidylethanolamine (PE) in autophagic membranes. In Apicomplexan parasites, two cysteine proteases, Atg4...
During macroautophagy, the Atg8 protein is conjugated to phosphatidylethanolamine (PE) in autophagic membranes. In Apicomplexan parasites, two cysteine proteases, Atg4 and ovarian tumor unit (Otu), have been identified to delipidate Atg8 to release this protein from membranes. Here, we investigated the role of cysteine proteases in Atg8 conjugation and deconjugation and found that the Plasmodium parasite consists of both activities. We successfully disrupted the genes individually; however, simultaneously, they were refractory to deletion and essential for parasite survival. Mutants lacking Atg4 and Otu showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast; however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling. These findings highlight a role for the Atg8 deconjugation pathway in organelle biogenesis and maintenance of the homeostatic cellular balance.
Topics: Animals; Mice; Cysteine Proteases; Parasites; Plasmodium berghei; Autophagy-Related Protein 8 Family; Autophagy; Malaria; Protozoan Proteins
PubMed: 37910326
DOI: 10.1007/s00018-023-05004-2 -
Tropical Biomedicine Sep 2023Malaria, caused by the unicellular Apicomplexan protozoa of the genus Plasmodium, is an infectious disease transmitted via female Anopheles mosquitoes. The sexual stage...
Malaria, caused by the unicellular Apicomplexan protozoa of the genus Plasmodium, is an infectious disease transmitted via female Anopheles mosquitoes. The sexual stage (gametocytes) of malaria parasites is the key to the transmission of parasites from vertebrate hosts to mosquitoes, representing critical bottleneck of the parasite life cycle. This study has established a systematic computational pipeline to achieve the genome-wide in silico analysis and find 708 novels potentially indispensable genes for gametocyte development, consisting of 644 protein coding genes, 56 ncRNA genes and 8 pseudogenes, with a total of 191 genes in the transmembrane, 29 protein coding genes to be exported proteins, and 58 genes in apicoplast regions. Furthermore, Gene Ontology analysis showed that the largest cluster was cellular processes with nucleus and cytosol highest, followed by molecular function with binding and oxidoreductase activities abundant. Meanwhile, when a text searched, using PlasmoDB, there were 300 genes with annotations of "putative", and 196 genes with annotations of "unknown function". These data would be helpful to provide potential targets for effective malaria transmission-blocking strategies.
Topics: Animals; Female; Plasmodium falciparum; Malaria, Falciparum; Malaria; Plasmodium; Anopheles
PubMed: 37897169
DOI: 10.47665/tb.40.3.012 -
PLoS Pathogens Oct 2023Isoprenoid precursor synthesis is an ancient and fundamental function of plastid organelles and a critical metabolic activity of the apicoplast in Plasmodium malaria...
Isoprenoid precursor synthesis is an ancient and fundamental function of plastid organelles and a critical metabolic activity of the apicoplast in Plasmodium malaria parasites [1-3]. Over the past decade, our understanding of apicoplast properties and functions has increased enormously [4], due in large part to our ability to rescue blood-stage parasites from apicoplast-specific dysfunctions by supplementing cultures with isopentenyl pyrophosphate (IPP), a key output of this organelle [5,6]. In this Pearl, we explore the interdependence between isoprenoid metabolism and apicoplast biogenesis in P. falciparum and highlight critical future questions to answer.
Topics: Animals; Apicoplasts; Parasites; Plasmodium falciparum; Malaria, Falciparum; Protozoan Proteins
PubMed: 37883328
DOI: 10.1371/journal.ppat.1011713 -
Biochimica Et Biophysica Acta.... Jan 2024ATG8/LC3-mediated autophagosome formation is a key rate-limiting step in the process of autophagy. The parasitic protist Toxoplasma gondii possesses a single ATG8...
ATG8/LC3-mediated autophagosome formation is a key rate-limiting step in the process of autophagy. The parasitic protist Toxoplasma gondii possesses a single ATG8 homolog (TgATG8), which can localize to either cytosolic autophagosome involved in delivery of autophagic material in bradyzoites, or the outermost membrane of apicoplast, a nonphotosynthetic plastid-like organelle, responsible for maintaining homeostasis in tachyzoites. However, mechanisms that regulate TgATG8 remain insufficiently understood. Here, a TgATG7 conditional knockdown line that we have generated is severely impaired in parasite's growth and exhibits significant defects in the organelle level, strikingly with a fragmentation of the mitochondrial network and a loss of the apicoplast. Specific TgATG7 point mutant complemented line showed that these defects were associated with its E1-type enzyme activity. Both depletion of TgATG7 and mutation of its catalytic cysteine 1133 hindered TgATG8 lipidation and apicoplast localization. Unexpectedly, we also found that depletion of TgATG7 reduced the unlipidated TgATG8 protein level. Subsequently, we determined that TgATG7 was able to interact with TgATG8 directly via its C-terminal domain and multi-monoubiquitination stimulated proteasome-dependent degradation of TgATG8, while TgATG7 could inhibit the degradation through stabilization of TgATG8. Additionally, we identified a putative TgATG8 interacting fragment of TgATG7, 1281-1290aa. Depletion of the fragment impaired the parasite growth and apicoplast inheritance. To our knowledge, our study is the first to elucidate the role of TgATG7 and the ubiquitin-proteasome system in synergistically regulating the non-lipidated pool of TgATG8, suggesting a potential homeostatic mechanism responsible for balancing autophagic activity in T. gondii.
Topics: Animals; Toxoplasma; Autophagy-Related Proteins; Apicoplasts; Proteasome Endopeptidase Complex; Parasites
PubMed: 37739091
DOI: 10.1016/j.bbadis.2023.166891 -
MBio Oct 2023and most other parasites in the phylum Apicomplexa contain an apicoplast, a non-photosynthetic plastid organelle required for fatty acid, isoprenoid, iron-sulfur...
and most other parasites in the phylum Apicomplexa contain an apicoplast, a non-photosynthetic plastid organelle required for fatty acid, isoprenoid, iron-sulfur cluster, and heme synthesis. Perturbation of apicoplast function results in parasite death. Thus, parasite survival critically depends on two cellular processes: apicoplast division to ensure every daughter parasite inherits a single apicoplast, and trafficking of nuclear encoded proteins to the apicoplast. Despite the importance of these processes, there are significant knowledge gaps in regards to the molecular mechanisms which control these processes; this is particularly true for trafficking of nuclear-encoded apicoplast proteins. This study provides crucial new insight into the timing of apicoplast protein synthesis and trafficking to the apicoplast. In addition, this study demonstrates how apicoplast-centrosome association, a key step in the apicoplast division cycle, is controlled by the actomyosin cytoskeleton.
Topics: Apicoplasts; Toxoplasma; Actins; Centrosome; Nuclear Proteins; Myosins; Protozoan Proteins
PubMed: 37732764
DOI: 10.1128/mbio.01640-23 -
Parasitology Research Dec 2023The protozoan Toxoplasma gondii (T. gondii) is a zoonotic disease agent causing systemic infection in warm-blooded intermediate hosts including humans. During the acute...
The protozoan Toxoplasma gondii (T. gondii) is a zoonotic disease agent causing systemic infection in warm-blooded intermediate hosts including humans. During the acute infection, the parasite infects host cells and multiplies intracellularly in the asexual tachyzoite stage. In this stage of the life cycle, invasion, multiplication, and egress are the most critical events in parasite replication. T. gondii features diverse cell organelles to support these processes, including the apicoplast, an endosymbiont-derived vestigial plastid originating from an alga ancestor. Previous studies have highlighted that phytohormones can modify the calcium-mediated secretion, e.g., of adhesins involved in parasite movement and cell invasion processes. The present study aimed to elucidate the influence of different plant hormones on the replication of asexual tachyzoites in a human foreskin fibroblast (HFF) host cell culture. T. gondii replication was measured by the determination of T. gondii DNA copies via qPCR. Three selected phytohormones, namely abscisic acid (ABA), gibberellic acid (GIBB), and kinetin (KIN) as representatives of different plant hormone groups were tested. Moreover, the influence of typical cell culture media components on the phytohormone effects was assessed. Our results indicate that ABA is able to induce a significant increase of T. gondii DNA copies in a typical supplemented cell culture medium when applied in concentrations of 20 ng/μl or 2 ng/μl, respectively. In contrast, depending on the culture medium composition, GIBB may potentially serve as T. gondii growth inhibitor and may be further investigated as a potential treatment for toxoplasmosis.
Topics: Humans; Toxoplasma; Plant Growth Regulators; Toxoplasmosis; Abscisic Acid; DNA
PubMed: 37725257
DOI: 10.1007/s00436-023-07968-3