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Contact (Thousand Oaks (Ventura County,... 2022Apicoplasts are critical for the growth of medically important parasites. It is now reported that they form contacts with the endoplasmic reticulum (ER) via two pore...
Apicoplasts are critical for the growth of medically important parasites. It is now reported that they form contacts with the endoplasmic reticulum (ER) via two pore channels thus enabling Ca trafficking. This highlights the dynamic physical association between organelles as a critical motif in Ca signaling.
PubMed: 37366509
DOI: 10.1177/25152564221095961 -
Microbiology Spectrum Jun 2023The human malaria parasite undergoes a noncanonical cell division, namely, endoreduplication, where several rounds of nuclear, mitochondrial, and apicoplast replication...
The human malaria parasite undergoes a noncanonical cell division, namely, endoreduplication, where several rounds of nuclear, mitochondrial, and apicoplast replication occur without cytoplasmic division. Despite its importance in biology, the topoisomerases essential for decatenation of replicated chromosome during endoreduplication remain elusive. We hypothesize that the topoisomerase VI complex, containing Plasmodium falciparum topiosomerase VIB (PfTopoVIB) and catalytic P. falciparum Spo11 (PfSpo11), might be involved in the segregation of the mitochondrial genome. Here, we demonstrate that the putative PfSpo11 is the functional ortholog of yeast Spo11 that can complement the sporulation defects of the yeast Δ strain, and the catalytic mutant Pfspo11Y65F cannot complement such defects. PfTopoVIB and PfSpo11 display a distinct expression pattern compared to the other type II topoisomerases of and are induced specifically at the late schizont stage of the parasite, when the mitochondrial genome segregation occurs. Furthermore, PfTopoVIB and PfSpo11 are physically associated with each other at the late schizont stage, and both subunits are localized in the mitochondria. Using PfTopoVIB- and PfSpo11-specific antibodies, we immunoprecipitated the chromatin of tightly synchronous early, mid-, and late schizont stage-specific parasites and found that both the subunits are associated with the mitochondrial genome during the late schizont stage of the parasite. Furthermore, PfTopoVIB inhibitor radicicol and atovaquone show synergistic interaction. Accordingly, atovaquone-mediated disruption of mitochondrial membrane potential reduces the import and recruitment of both subunits of PfTopoVI to mitochondrial DNA (mtDNA) in a dose-dependent manner. The structural differences between PfTopoVIB and human TopoVIB-like protein could be exploited for development of a novel antimalarial agent. This study demonstrates a likely role of topoisomerase VI in the mitochondrial genome segregation of Plasmodium falciparum during endoreduplication. We show that PfTopoVIB and PfSpo11 remain associated and form the functional holoenzyme within the parasite. The spatiotemporal expression of both subunits of PfTopoVI correlates well with their recruitment to the mitochondrial DNA at the late schizont stage of the parasite. Additionally, the synergistic interaction between PfTopoVI inhibitor and the disruptor of mitochondrial membrane potential, atovaquone, supports that topoisomerase VI is the mitochondrial topoisomerase of the malaria parasite. We propose that topoisomerase VI may act as a novel target against malaria.
Topics: Animals; Humans; Parasites; Atovaquone; Saccharomyces cerevisiae; Plasmodium falciparum; Malaria, Falciparum; Malaria; DNA, Mitochondrial; Protozoan Proteins; Endodeoxyribonucleases; Saccharomyces cerevisiae Proteins
PubMed: 37212694
DOI: 10.1128/spectrum.04980-22 -
Methods in Molecular Biology (Clifton,... 2023The ribosome is among the most complex and ancient cellular macromolecular assemblies that plays a central role in protein biosynthesis in all living cells. Its function...
The ribosome is among the most complex and ancient cellular macromolecular assemblies that plays a central role in protein biosynthesis in all living cells. Its function of translation of genetic information encoded in messenger RNA into protein molecules also extends to subcellular compartments in eukaryotic cells such as apicoplasts, chloroplasts, and mitochondria. The origin of mitochondria is primarily attributed to an early endosymbiotic event between an alpha-proteobacterium and a primitive (archaeal) eukaryotic cell. The timeline of mitochondrial acquisition, the nature of the host, and their diversification have been studied in great detail and are continually being revised as more genomic and structural data emerge. Recent advancements in high-resolution cryo-EM structure determination have provided architectural details of mitochondrial ribosomes (mitoribosomes) from various species, revealing unprecedented diversifications among them. These structures provide novel insights into the evolution of mitoribosomal structure and function. Here, we present a brief overview of the existing mitoribosomal structures in the context of the eukaryotic evolution tree showing their diversification from their last common ancestor.
Topics: Mitochondrial Ribosomes; Mitochondria; Ribosomes; Eukaryota; Eukaryotic Cells; Mitochondrial Proteins; Cryoelectron Microscopy; Ribosomal Proteins
PubMed: 37166629
DOI: 10.1007/978-1-0716-3171-3_2 -
ELife May 2023Iron-sulfur clusters (FeS) are ancient and ubiquitous protein cofactors that play fundamental roles in many aspects of cell biology. These cofactors cannot be scavenged...
Iron-sulfur clusters (FeS) are ancient and ubiquitous protein cofactors that play fundamental roles in many aspects of cell biology. These cofactors cannot be scavenged or trafficked within a cell and thus must be synthesized in any subcellular compartment where they are required. We examined the FeS synthesis proteins found in the relict plastid organelle, called the apicoplast, of the human malaria parasite . Using a chemical bypass method, we deleted four of the FeS pathway proteins involved in sulfur acquisition and cluster assembly and demonstrated that they are all essential for parasite survival. However, the effect that these deletions had on the apicoplast organelle differed. Deletion of the cysteine desulfurase SufS led to disruption of the apicoplast organelle and loss of the organellar genome, whereas the other deletions did not affect organelle maintenance. Ultimately, we discovered that the requirement of SufS for organelle maintenance is not driven by its role in FeS biosynthesis, but rather, by its function in generating sulfur for use by MnmA, a tRNA modifying enzyme that we localized to the apicoplast. Complementation of MnmA and SufS activity with a bacterial MnmA and its cognate cysteine desulfurase strongly suggests that the parasite SufS provides sulfur for both FeS biosynthesis and tRNA modification in the apicoplast. The dual role of parasite SufS is likely to be found in other plastid-containing organisms and highlights the central role of this enzyme in plastid biology.
Topics: Humans; Plasmodium falciparum; Apicoplasts; Sulfur; Iron; RNA, Transfer; Iron-Sulfur Proteins
PubMed: 37166116
DOI: 10.7554/eLife.84491 -
PeerJ 2023The spread of artemisinin (ART)-resistant threatens the control of malaria. Mutations in the propeller domains of Kelch13 () are strongly associated with ART...
BACKGROUND
The spread of artemisinin (ART)-resistant threatens the control of malaria. Mutations in the propeller domains of Kelch13 () are strongly associated with ART resistance. Ferredoxin (Fd), a component of the ferredoxin/NADP reductase (Fd/FNR) redox system, is essential for isoprenoid precursor synthesis in the plasmodial apicoplast, which is important for K13-dependent hemoglobin trafficking and ART activation. Therefore, Fd is an antimalarial drug target and mutations may modulate ART sensitivity. We hypothesized that loss of Fd/FNR function enhances the effect of mutation on ART resistance.
METHODS
In this study, methoxyamino chalcone (C3), an antimalarial compound that has been reported to inhibit the interaction of recombinant Fd and FNR proteins, was used as a chemical inhibitor of the Fd/FNR redox system. We investigated the inhibitory effects of dihydroartemisinin (DHA), C3, and iron chelators including deferiprone (DFP), 1-(-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) and deferiprone-resveratrol hybrid (DFP-RVT) against wild-type (WT), mutant, mutant, and double mutant parasites. Furthermore, we investigated the pharmacological interaction of C3 with DHA, in which the iron chelators were used as reference ART antagonists.
RESULTS
C3 showed antimalarial potency similar to that of the iron chelators. As expected, combining DHA with C3 or iron chelators exhibited a moderately antagonistic effect. No differences were observed among the mutant parasites with respect to their sensitivity to C3, iron chelators, or the interactions of these compounds with DHA.
DISCUSSION
The data suggest that inhibitors of the Fd/FNR redox system should be avoided as ART partner drugs in ART combination therapy for treating malaria.
Topics: Humans; Antimalarials; Plasmodium falciparum; Ferredoxins; Chalcone; Deferiprone; Malaria, Falciparum; Ferredoxin-NADP Reductase; Iron Chelating Agents
PubMed: 37131988
DOI: 10.7717/peerj.15187 -
FASEB Journal : Official Publication of... Jun 2023Glutaredoxins (Grxs) are ubiquitous antioxidant proteins involved in many molecular processes to protect cells against oxidative damage. Here, we study the roles of Grxs...
Glutaredoxins (Grxs) are ubiquitous antioxidant proteins involved in many molecular processes to protect cells against oxidative damage. Here, we study the roles of Grxs in the pathogenicity of Toxoplasma gondii. We show that Grxs are localized in the mitochondria (Grx1), cytoplasm (Grx2), and apicoplast (Grx3, Grx4), while Grx5 had an undetectable level of expression. We generated Δgrx1-5 mutants of T. gondii type I RH and type II Pru strains using CRISPR-Cas9 system. No significant differences in the infectivity were detected between four Δgrx (grx2-grx5) strains and their respective wild-type (WT) strains in vitro or in vivo. Additionally, no differences were detected in the production of reactive oxygen species, total antioxidant capacity, superoxide dismutase activity, and sensitivity to external oxidative stimuli. Interestingly, RHΔgrx1 or PruΔgrx1 exhibited significant differences in all the investigated aspects compared to the other grx2-grx5 mutant and WT strains. Transcriptome analysis suggests that deletion of grx1 altered the expression of genes involved in transport and metabolic pathways, signal transduction, translation, and obsolete oxidation-reduction process. The data support the conclusion that grx1 supports T. gondii resistance to oxidative killing and is essential for the parasite growth in cultured cells and pathogenicity in mice and that the active site CGFS motif was necessary for Grx1 activity.
Topics: Animals; Mice; Antioxidants; Glutaredoxins; Toxoplasma; Amino Acid Sequence; Virulence; Oxidation-Reduction; Oxidative Stress
PubMed: 37115746
DOI: 10.1096/fj.202201275R -
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 -
PLoS Biology Apr 2023With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a...
With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.
Topics: Humans; Plasmodium falciparum; Actins; Profilins; Protozoan Proteins; Malaria, Falciparum; Erythrocytes; Antimalarials
PubMed: 37053271
DOI: 10.1371/journal.pbio.3002066 -
BioRxiv : the Preprint Server For... Oct 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 ~4.5x. 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 catalogued 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.
PubMed: 36993606
DOI: 10.1101/2023.03.22.533773 -
PLoS Pathogens Mar 2023Exocytosis is a key active process in cells by which proteins are released in bulk via the fusion of exocytic vesicles with the plasma membrane. Soluble...
Exocytosis is a key active process in cells by which proteins are released in bulk via the fusion of exocytic vesicles with the plasma membrane. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-mediated vesicle fusion with the plasma membrane is essential in most exocytotic pathways. In mammalian cells, the vesicular fusion step of exocytosis is normally mediated by Syntaxin-1 (Stx1) and SNAP25 family proteins (SNAP25 and SNAP23). However, in Toxoplasma gondii, a model organism of Apicomplexa, the only SNAP25 family protein, with a SNAP29-like molecular structure, is involved in vesicular fusion at the apicoplast. Here, we reveal that an unconventional SNARE complex comprising TgStx1, TgStx20, and TgStx21 mediates vesicular fusion at the plasma membrane. This complex is essential for the exocytosis of surface proteins and vesicular fusion at the apical annuli in T. gondii.
Topics: Animals; Toxoplasma; Vesicular Transport Proteins; Cell Membrane; Exocytosis; Membrane Fusion; Qa-SNARE Proteins; Mammals
PubMed: 36972314
DOI: 10.1371/journal.ppat.1011288