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Scientific Reports Aug 2023Malaria parasites carry out fatty acid synthesis (FAS) in their apicoplast organelle via a bacterially related (type II) enzymatic pathway. In the vertebrate host,...
Malaria parasites carry out fatty acid synthesis (FAS) in their apicoplast organelle via a bacterially related (type II) enzymatic pathway. In the vertebrate host, exoerythrocytic Plasmodium stages rely on FAS, whereas intraerythrocytic stages depend on scavenging FA from their environment. In the mosquito, P. falciparum oocysts express and rely on FAS enzymes for sporozoite formation, but P. yoelii oocysts do not express, nor depend on, FAS enzymes and thus rely on FA scavenging to support sporogony. In P. berghei, FAS enzymes are similarly expendable for sporogony, indicating it conforms to the P. yoelii scenario. We show here that P. berghei, unexpectedly, expresses FAS enzymes throughout oocyst development. These findings indicate that P. berghei can employ FAS alongside FA scavenging to maximise sporogony and transmission, and is more similar to P. falciparum than previously assumed with respect to FA acquisition by the oocyst. The ability of oocysts to switch between FAS and scavenging could be an important factor in the non-competitive relationship of resource exploitation between Plasmodium parasites and their mosquito vectors, which shapes parasite virulence both in the insect and vertebrate.
Topics: Animals; Oocysts; Plasmodium berghei; Mosquito Vectors; Malaria, Falciparum; Anopheles; Fatty Acids; Protozoan Proteins
PubMed: 37543672
DOI: 10.1038/s41598-023-39708-z -
Microbiology Spectrum Aug 2023The intracellular protozoan parasite Babesia gibsoni infects canine erythrocytes and causes babesiosis. The hazards to animal health have increased due to the rise of B....
The intracellular protozoan parasite Babesia gibsoni infects canine erythrocytes and causes babesiosis. The hazards to animal health have increased due to the rise of B. gibsoni infections and medication resistance. However, the lack of high-quality full-genome sequencing sets has expanded the obstacles to the development of pathogeneses, drugs, and vaccines. In this study, the whole genome of was sequenced, assembled, and annotated. The genomic size of was 7.94 Mbp in total. Four chromosomes with the size of 0.69 Mb, 2.10 Mb, 2.77 Mb, and 2.38 Mb, respectively, 1 apicoplast (28.4 Kb), and 1 mitochondrion (5.9 Kb) were confirmed. KEGG analysis revealed 2,641 putative proteins enriched on 316 pathways, and GO analysis showed 7,571 annotations of the nuclear genome in total. Synteny analysis showed a high correlation between and B. bovis. A new divergent point of occurred around 297.7 million years ago, which was earlier than that of , B. ovata, and B. bigemina. Orthology analysis revealed 22 and 32 unique genes compared to several spp. and apicomplexan species. The metabolic pathways of were characterized, pointing to a minimal size of the genome. A species-specific secretory protein SA1 and 19 homologous genes were identified. Selected specific proteins, including apetala 2 (AP2) factor, invasion-related proteins BgAMA-1 and BgRON2, and rhoptry function proteins BgWH_04g00700 were predicted, visualized, and modeled. Overall, whole-genome sequencing provided molecular-level support for the diagnosis, prevention, clinical treatment, and further research of . The whole genome of was first sequenced, annotated, and disclosed. The key part of genome composition, four chromosomes, was comparatively analyzed for the first time. A full-scale phylogeny evolution analysis based on the whole-genome-wide data of was performed, and a new divergent point on the evolutionary path was revealed. In previous reports, molecular studies were often limited by incomplete genomic data, especially in key areas like life cycle regulation, metabolism, and host-pathogen interaction. With the whole-genome sequencing of , we provide useful genetic data to encourage the exploration of new terrain and make it feasible to resolve the theoretical and practical problems of babesiosis.
Topics: Animals; Dogs; Babesia; Babesiosis; Whole Genome Sequencing; Genomics; Genome; Dog Diseases
PubMed: 37432130
DOI: 10.1128/spectrum.00721-23 -
Proceedings of the National Academy of... Jul 2023The malaria parasite has a nonphotosynthetic plastid called the apicoplast, which contains its own genome. Regulatory mechanisms for apicoplast gene expression remain...
The malaria parasite has a nonphotosynthetic plastid called the apicoplast, which contains its own genome. Regulatory mechanisms for apicoplast gene expression remain poorly understood, despite this organelle being crucial for the parasite life cycle. Here, we identify a nuclear-encoded apicoplast RNA polymerase σ subunit (sigma factor) which, along with the α subunit, appears to mediate apicoplast transcript accumulation. This has a periodicity reminiscent of parasite circadian or developmental control. Expression of the apicoplast subunit gene, , together with apicoplast transcripts, increased in the presence of the blood circadian signaling hormone melatonin. Our data suggest that the host circadian rhythm is integrated with intrinsic parasite cues to coordinate apicoplast genome transcription. This evolutionarily conserved regulatory system might be a future target for malaria treatment.
Topics: Animals; Apicoplasts; Parasites; Cues; Plasmodium falciparum; Malaria; Protozoan Proteins
PubMed: 37406097
DOI: 10.1073/pnas.2214765120 -
BioRxiv : the Preprint Server For... Jun 2023is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of parasites. The diversity of parasites, coupled with the lack of potent...
UNLABELLED
is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of parasites. The diversity of parasites, coupled with the lack of potent inhibitors necessitates the discovery of novel conserved druggable targets for the generation of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of novel and conserved targets. CCG relies on parallel evolution of resistance in independent populations of evolutionarily-related spp. ( and ). We identified a potent antibabesial inhibitor from the Malaria Box, MMV019266. We were able to select for resistance to this compound in two species of achieving 10-fold or greater resistance after ten weeks of intermittent selection. After sequencing of multiple independently derived lines in the two species, we identified mutations in a single conserved gene in both species: a membrane-bound metallodependent phosphatase (putatively named PhoD). In both species, the mutations were found in the phoD-like phosphatase domain, proximal to the predicted ligand binding site. Using reverse genetics, we validated that mutations in PhoD confer resistance to MMV019266. We have also demonstrated that PhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of PhoD alter the sensitivity to MMV019266 in the parasite: overexpression of PhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting PhoD is a resistance mechanism. Together, we have generated a robust pipeline for identification of resistance loci, and identified PhoD as a novel determinant of resistance in species.
HIGHLIGHTS
Use of two species for evolution identifies a high confidence locus associated with resistance Resistance mutation in phoD was validated using reverse genetics in Perturbation of phoD using function genetics results in changes in the level of resistance to MMV019266Epitope tagging reveals localization to the ER/apicoplast, a conserved localization with a similar protein in diatoms Together, phoD is a novel resistance determinant in multiple .
PubMed: 37398106
DOI: 10.1101/2023.06.13.544849 -
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 -
PLoS Biology Jun 2023Lipoic acid is an essential biomolecule found in all domains of life and is involved in central carbon metabolism and dissimilatory sulfur oxidation. The machineries for...
Lipoic acid is an essential biomolecule found in all domains of life and is involved in central carbon metabolism and dissimilatory sulfur oxidation. The machineries for lipoate assembly in mitochondria and chloroplasts of higher eukaryotes, as well as in the apicoplasts of some protozoa, are all of prokaryotic origin. Here, we provide experimental evidence for a novel lipoate assembly pathway in bacteria based on a sLpl(AB) lipoate:protein ligase, which attaches octanoate or lipoate to apo-proteins, and 2 radical SAM proteins, LipS1 and LipS2, which work together as lipoyl synthase and insert 2 sulfur atoms. Extensive homology searches combined with genomic context analyses allowed us to precisely distinguish between the new and established pathways and map them on the tree of life. This not only revealed a much wider distribution of lipoate biogenesis systems than expected, in particular, the novel sLpl(AB)-LipS1/S2 pathway, and indicated a highly modular nature of the enzymes involved, with unforeseen combinations, but also provided a new framework for the evolution of lipoate assembly. Our results show that dedicated machineries for both de novo lipoate biogenesis and scavenging from the environment were implemented early in evolution and that their distribution in the 2 prokaryotic domains was shaped by a complex network of horizontal gene transfers, acquisition of additional genes, fusions, and losses. Our large-scale phylogenetic analyses identify the bipartite archaeal LplAB ligase as the ancestor of the bacterial sLpl(AB) proteins, which were obtained by horizontal gene transfer. LipS1/S2 have a more complex evolutionary history with multiple of such events but probably also originated in the domain archaea.
Topics: Thioctic Acid; Peptide Synthases; Phylogeny; Bacterial Proteins; Sulfur
PubMed: 37368881
DOI: 10.1371/journal.pbio.3002177 -
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 -
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