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Journal of Cellular and Molecular... Oct 2023The widespread emergence of antimalarial drug resistance has created a major threat to public health. Malaria is a life-threatening infectious disease caused by...
The widespread emergence of antimalarial drug resistance has created a major threat to public health. Malaria is a life-threatening infectious disease caused by Plasmodium spp., which includes Apicoplast DNA polymerase and Plasmodium falciparum cysteine protease falcipain-2. These components play a critical role in their life cycle and metabolic pathway, and are involved in the breakdown of erythrocyte hemoglobin in the host, making them promising targets for anti-malarial drug design. Our current study has been designed to explore the potential inhibitors from haplopine derivatives against these two targets using an in silico approach. A total of nine haplopine derivatives were used to perform molecular docking, and the results revealed that Ligands 03 and 05 showed strong binding affinity compared to the control compound atovaquone. Furthermore, these ligand-protein complexes underwent molecular dynamics simulations, and the results demonstrated that the complexes maintained strong stability in terms of RMSD (root mean square deviation), RMSF (root mean square fluctuation), and Rg (radius of gyration) over a 100 ns simulation period. Additionally, PCA (principal component analysis) analysis and the dynamic cross-correlation matrix showed positive outcomes for the protein-ligand complexes. Moreover, the compounds exhibited no violations of the Lipinski rule, and ADMET (absorption, distribution, metabolism, excretion, and toxicity) predictions yielded positive results without indicating any toxicity. Finally, density functional theory (DFT) and molecular electrostatic potential calculations were conducted, revealing that the mentioned derivatives exhibited better stability and outstanding performance. Overall, this computational approach suggests that these haplopine derivatives could serve as a potential source for developing new, effective antimalarial drugs to combat malaria. However, further in vitro or in vivo studies might be conducted to determine their actual effectiveness.
PubMed: 37724615
DOI: 10.1111/jcmm.17940 -
BMC Infectious Diseases Sep 2023Malaria cases in non-endemic zero-indigenous case areas are most likely to have been imported whatever of the route of importation. In countries recently declared...
BACKGROUND
Malaria cases in non-endemic zero-indigenous case areas are most likely to have been imported whatever of the route of importation. In countries recently declared malaria-free and now without local transmission, imported cases remain a threat to re-introduction of the disease and a burden on the health system.
CASE PRESENTATION
Three days after returning from a long trip to malaria- endemic countries; Abyei-Sudan, Chad and Uganda, a 41-year-old male resident from Jericho, Palestine, suffered paroxysms of fever, general fatigue, myalgia, arthralgia, headache, and a strong desire to vomit. Thin and thick Giemsa-stained blood smears were prepared and examined microscopically using oil immersion. Immature trophozoites (ring forms) were seen to parasitize approximately 10% of the erythrocytes revealing hyperparasitemia equivalent to > 100,000 parasites/ µl indicating severe malaria [1, 2]. The double chromatin configuration (headphones) and accolé (applique) position are both indicative of Plasmodium falciparum infection. The 18S rRNA- PCR targeting the rPLU6-rPLU5 region was used to confirm the diagnosis. The next-generation sequencing (NGS) method was carried out according to the manufacturer's instructions (Illumina® DNA Prep, (M) Tagmentation kit (20060060), Illumina) to identify Plasmodium spp. Furthermore, NGS produced a whole-genome sequence of 22.8Mbp of the 14 chromosomes and 25Kbp of the apicoplast. A BLAST search of the apicoplast DNA and selected chromosomal DNA revealed that P. falciparum was the causative agent. The merozoite surface protein-1 (msp-1) was used to construct a phylogenetic tree of 26 P. falciparum, including the one isolated from the patient from Jericho, which clustered with the Sudanese isolate indicating genetic relatedness between the two.
CONCLUSION
The travel history together with signs and symptoms of malaria, followed by prompt diagnosis using conventional microscopic inspection of Giemsa-stained films together with molecular DNA tracking tools like msp-1 were key means in tracking the place of origin of infection in the case of travel to multiple destination.
Topics: Humans; Adult; Plasmodium falciparum; Merozoite Surface Protein 1; Phylogeny; Malaria; Malaria, Falciparum; Azure Stains; DNA, Ribosomal
PubMed: 37723449
DOI: 10.1186/s12879-023-08583-4 -
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 -
Proceedings of the National Academy of... Aug 2023Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissecting -secreted protein functions are limited....
Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissecting -secreted protein functions are limited. We report knockER, a unique DiCre-mediated knock-sideways approach to sequester secreted proteins in the ER by inducible fusion with a KDEL ER-retrieval sequence. We show conditional ER sequestration of diverse proteins is not generally toxic, enabling loss-of-function studies. We employed knockER in multiple species to interrogate the trafficking, topology, and function of an assortment of proteins that traverse the secretory pathway to diverse compartments including the apicoplast (ClpB1), rhoptries (RON6), dense granules, and parasitophorous vacuole (EXP2, PTEX150, HSP101). Taking advantage of the unique ability to redistribute secreted proteins from their terminal destination to the ER, we reveal that vacuolar levels of the PTEX translocon component HSP101 but not PTEX150 are maintained in excess of what is required to sustain effector protein export into the erythrocyte. Intriguingly, vacuole depletion of HSP101 hypersensitized parasites to a destabilization tag that inhibits HSP101-PTEX complex formation but not to translational knockdown of the entire HSP101 pool, illustrating how redistribution of a target protein by knockER can be used to query function in a compartment-specific manner. Collectively, our results establish knockER as a unique tool for dissecting secreted protein function with subcompartmental resolution that should be widely amenable to genetically tractable eukaryotes.
Topics: Plasmodium falciparum; Protozoan Proteins; Plasmodium; Protein Transport; Biological Transport; Erythrocytes
PubMed: 37552754
DOI: 10.1073/pnas.2308676120 -
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