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Communications Biology Jul 2023Transgenic luciferase-expressing Plasmodium falciparum parasites have been widely used for the evaluation of anti-malarial compounds. Here, to screen for anti-malarial...
Transgenic luciferase-expressing Plasmodium falciparum parasites have been widely used for the evaluation of anti-malarial compounds. Here, to screen for anti-malarial drugs effective against multiple stages of the parasite, we generate a P. falciparum reporter parasite that constitutively expresses NanoLuciferase (NanoLuc) throughout its whole life cycle. The NanoLuc-expressing P. falciparum reporter parasite shows a quantitative NanoLuc signal in the asexual blood, gametocyte, mosquito, and liver stages. We also establish assay systems to evaluate the anti-malarial activity of compounds at the asexual blood, gametocyte, and liver stages, and then determine the 50% inhibitory concentration (IC) value of several anti-malarial compounds. Through the development of this robust high-throughput screening system, we identify an anti-malarial compound that kills the asexual blood stage parasites. Our study highlights the utility of the NanoLuc reporter line, which may advance anti-malarial drug development through the improved screening of compounds targeting the human malarial parasite at multiple stages.
Topics: Humans; Animals; Antimalarials; Plasmodium falciparum; Animals, Genetically Modified; Biological Assay
PubMed: 37438491
DOI: 10.1038/s42003-023-05078-5 -
Nature Microbiology Dec 2023Malaria results in over 600,000 deaths annually, with the highest burden of deaths in young children living in sub-Saharan Africa. Molecular surveillance can provide...
Malaria results in over 600,000 deaths annually, with the highest burden of deaths in young children living in sub-Saharan Africa. Molecular surveillance can provide important information for malaria control policies, including detection of antimalarial drug resistance. However, genome sequencing capacity in malaria-endemic countries is limited. We designed and implemented an end-to-end workflow to detect Plasmodium falciparum antimalarial resistance markers and diversity in the vaccine target circumsporozoite protein (csp) using nanopore sequencing in Ghana. We analysed 196 clinical samples and showed that our method is rapid, robust, accurate and straightforward to implement. Importantly, our method could be applied to dried blood spot samples, which are readily collected in endemic settings. We report that P. falciparum parasites in Ghana are mostly susceptible to chloroquine, with persistent sulfadoxine-pyrimethamine resistance and no evidence of artemisinin resistance. Multiple single nucleotide polymorphisms were identified in csp, but their significance is uncertain. Our study demonstrates the feasibility of nanopore sequencing for malaria genomic surveillance in endemic countries.
Topics: Child; Humans; Child, Preschool; Plasmodium falciparum; Ghana; Nanopore Sequencing; Antimalarials; Malaria; Malaria, Falciparum; Drug Resistance
PubMed: 37996707
DOI: 10.1038/s41564-023-01516-6 -
Genome Medicine Nov 2023Malaria continues to be a major threat to global public health. Whole genome sequencing (WGS) of the underlying Plasmodium parasites has provided insights into the...
BACKGROUND
Malaria continues to be a major threat to global public health. Whole genome sequencing (WGS) of the underlying Plasmodium parasites has provided insights into the genomic epidemiology of malaria. Genome sequencing is rapidly gaining traction as a diagnostic and surveillance tool for clinical settings, where the profiling of co-infections, identification of imported malaria parasites, and detection of drug resistance are crucial for infection control and disease elimination. To support this informatically, we have developed the Malaria-Profiler tool, which rapidly (within minutes) predicts Plasmodium species, geographical source, and resistance to antimalarial drugs directly from WGS data.
RESULTS
The online and command line versions of Malaria-Profiler detect ~ 250 markers from genome sequences covering Plasmodium speciation, likely geographical source, and resistance to chloroquine, sulfadoxine-pyrimethamine (SP), and other anti-malarial drugs for P. falciparum, but also providing mutations for orthologous resistance genes in other species. The predictive performance of the mutation library was assessed using 9321 clinical isolates with WGS and geographical data, with most being single-species infections (P. falciparum 7152/7462, P. vivax 1502/1661, P. knowlesi 143/151, P. malariae 18/18, P. ovale ssp. 5/5), but co-infections were identified (456/9321; 4.8%). The accuracy of the predicted geographical profiles was high to both continental (96.1%) and regional levels (94.6%). For P. falciparum, markers were identified for resistance to chloroquine (49.2%; regional range: 24.5% to 100%), sulfadoxine (83.3%; 35.4- 90.5%), pyrimethamine (85.4%; 80.0-100%) and combined SP (77.4%). Markers associated with the partial resistance of artemisinin were found in WGS from isolates sourced from Southeast Asia (30.6%).
CONCLUSIONS
Malaria-Profiler is a user-friendly tool that can rapidly and accurately predict the geographical regional source and anti-malarial drug resistance profiles across large numbers of samples with WGS data. The software is flexible with modifiable bioinformatic pipelines. For example, it is possible to select the sequencing platform, display specific variants, and customise the format of outputs. With the increasing application of next-generation sequencing platforms on Plasmodium DNA, Malaria-Profiler has the potential to be integrated into point-of-care and surveillance settings, thereby assisting malaria control. Malaria-Profiler is available online (bioinformatics.lshtm.ac.uk/malaria-profiler) and as standalone software ( https://github.com/jodyphelan/malaria-profiler ).
Topics: Humans; Animals; Antimalarials; Parasites; Coinfection; Malaria; Plasmodium; Malaria, Falciparum; Chloroquine; Malaria, Vivax; Drug Resistance; Plasmodium falciparum
PubMed: 37950308
DOI: 10.1186/s13073-023-01247-7 -
Nature Communications Jan 2024Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are...
Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism.
Topics: Animals; Humans; Plasmodium falciparum; Asparagine; Aspartate-tRNA Ligase; RNA, Transfer, Amino Acyl; Antimalarials; Mammals
PubMed: 38297033
DOI: 10.1038/s41467-024-45224-z -
Nature Communications Oct 2023Long-acting injectable medications, such as atovaquone, offer the prospect of a "chemical vaccine" for malaria, combining drug efficacy with vaccine durability. However,...
Long-acting injectable medications, such as atovaquone, offer the prospect of a "chemical vaccine" for malaria, combining drug efficacy with vaccine durability. However, selection and transmission of drug-resistant parasites is of concern. Laboratory studies have indicated that atovaquone resistance disadvantages parasites in mosquitoes, but lack of data on clinically relevant Plasmodium falciparum has hampered integration of these variable findings into drug development decisions. Here we generate atovaquone-resistant parasites that differ from wild type parent by only a Y268S mutation in cytochrome b, a modification associated with atovaquone treatment failure in humans. Relative to wild type, Y268S parasites evidence multiple defects, most marked in their development in mosquitoes, whether from Southeast Asia (Anopheles stephensi) or Africa (An. gambiae). Growth of asexual Y268S P. falciparum in human red cells is impaired, but parasite loss in the mosquito is progressive, from reduced gametocyte exflagellation, to smaller number and size of oocysts, and finally to absence of sporozoites. The Y268S mutant fails to transmit from mosquitoes to mice engrafted with human liver cells and erythrocytes. The severe-to-lethal fitness cost of clinically relevant atovaquone resistance to P. falciparum in the mosquito substantially lessens the likelihood of its transmission in the field.
Topics: Humans; Animals; Mice; Atovaquone; Parasites; Antimalarials; Malaria; Malaria, Falciparum; Plasmodium falciparum; Anopheles; Antiparasitic Agents; Vaccines
PubMed: 37828012
DOI: 10.1038/s41467-023-42030-x -
European Journal of Medicinal Chemistry Oct 2023A novel family of 4-aminoacridine derivatives was obtained by linking this heteroaromatic core to different trans-cinnamic acids. The 4-(N-cinnamoylbutyl)aminoacridines...
A novel family of 4-aminoacridine derivatives was obtained by linking this heteroaromatic core to different trans-cinnamic acids. The 4-(N-cinnamoylbutyl)aminoacridines obtained exhibited in vitro activity in the low- or sub-micromolar range against (i) hepatic stages of Plasmodium berghei, (ii) erythrocytic forms of Plasmodium falciparum, and (iii) early and mature gametocytes of Plasmodium falciparum. The most active compound, having a meta-fluorocinnamoyl group linked to the acridine core, was 20- and 120-fold more potent, respectively, against the hepatic and gametocyte stages of Plasmodium infection than the reference drug, primaquine. Moreover, no cytotoxicity towards mammalian and red blood cells at the concentrations tested was observed for any of the compounds under investigation. These novel conjugates represent promising leads for the development of new multi-target antiplasmodials.
Topics: Animals; Aminacrine; Aminoacridines; Antimalarials; Mammals; Plasmodium berghei; Plasmodium falciparum; Primaquine
PubMed: 37390511
DOI: 10.1016/j.ejmech.2023.115575 -
Journal of Vector Borne Diseases 2023Malaria is the most common parasitic infection in children and the most common cause of mortality by vector-borne disease in tropical countries. In these endemic... (Observational Study)
Observational Study
BACKGROUND & OBJECTIVES
Malaria is the most common parasitic infection in children and the most common cause of mortality by vector-borne disease in tropical countries. In these endemic countries there is limited published literature on the clinical profile and severity of Plasmodium vivax malaria in children. We highlight the clinical presentations and severity of malaria in children belonging to Uttar Pradesh, North India Methods: This observational study was conducted in a tertiary care hospital, in Moradabad, western Uttar Pradesh, India. Children (aged 6 months-18 years) hospitalized from June 2019 to May 2020 with a clinical picture consistent with malaria along with a positive rapid malaria antigen test (RMAT) and/or positive peripheral blood smear for malaria were enrolled. All data about the clinical profile and laboratory features were recorded. Results were analyzed for significance using appropriate statistical tests for continuous and categorical data.
RESULTS
One hundred children were enrolled in this study; 59 cases had Plasmodium vivax (PV) malaria, 33 cases had Plasmodium falciparum (PF) malaria, and 8 cases had mixed infections (both PV and PF). Among 59 children with P V malaria, 44 (74%) had severe malaria and 15 (26%) had uncomplicated malaria. Severe malarial anaemia (43.2%), followed by jaundice (36.4%), impaired renal function (21.6%), significant bleeding (18.9%), shock (18.9%), and prostration (16.2%), were the main severity parameters of malaria among these hospitalised children. Impaired renal function (P-value = 0.01) and unconsciousness (P-value = 0.02) were more frequent in Plasmodium falciparum cases. Other severity parameters were not significant between the vivax and falciparum species.
INTERPRETATION & CONCLUSION
We conclude that a significant proportion of severe malaria was caused by Plasmodium vivax in this region, where both species coexist. Plasmodium vivax malaria is no longer the benign entity it was around ten years ago in hospitalised children. Severe malarial anaemia was the most common severity parameter found in both Plasmodium vivax and P. falciparum species. The clinical presentation and a change in the severity parameters in vivax malaria indicate a recent shift in the disease severity from benign to fatal.
Topics: Child; Humans; Plasmodium vivax; Malaria, Vivax; Child, Hospitalized; Malaria, Falciparum; Malaria; Plasmodium falciparum; Anemia; India
PubMed: 37843235
DOI: 10.4103/0972-9062.331406 -
Blood Dec 2023The malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an...
The malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an important host factor for P falciparum infection through a forward genetic screen, but little is known about its regulation or function in these cells, nor how it may be used by the parasite. We found that CD44 can be efficiently deleted from primary human hematopoietic stem cells using CRISPR/Cas9 genome editing, and that the efficiency of ex vivo erythropoiesis to enucleated cultured red blood cells (cRBCs) is not affected by lack of CD44. However, the rate of P falciparum invasion was reduced in CD44-null cRBCs relative to isogenic wild-type control cells, validating CD44 as an important host factor for this parasite. We identified 2 P falciparum invasion ligands as binding partners for CD44, erythrocyte binding antigen 175 (EBA-175) and EBA-140 and demonstrated that their ability to bind to human erythrocytes relies primarily on their canonical receptors, glycophorin A and glycophorin C, respectively. We further show that EBA-175 induces phosphorylation of erythrocyte cytoskeletal proteins in a CD44-dependent manner. Our findings support a model in which P falciparum exploits CD44 as a coreceptor during invasion of human erythrocytes, stimulating CD44-dependent phosphorylation of host cytoskeletal proteins that alter host cell deformability and facilitate parasite entry.
Topics: Humans; Antigens, Protozoan; Cytoskeletal Proteins; Erythrocytes; Hyaluronan Receptors; Malaria, Falciparum; Plasmodium falciparum; Protein Binding; Protozoan Proteins
PubMed: 37832027
DOI: 10.1182/blood.2023020831 -
Journal of Cell Science Oct 2023The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including...
The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including transmembrane proteins, across multiple membranes to the RBC. However, the process by which these exported membrane proteins are extracted from the parasite plasma membrane for export remains unknown. To address this question, we fused the exported membrane protein, skeleton binding protein 1 (SBP1), with TurboID, a rapid, efficient and promiscuous biotin ligase (SBP1TbID). Using time-resolved proximity biotinylation and label-free quantitative proteomics, we identified two groups of SBP1TbID interactors - early interactors (pre-export) and late interactors (post-export). Notably, two promising membrane-associated proteins were identified as pre-export interactors, one of which possesses a predicted translocon domain, that could facilitate the export of membrane proteins. Further investigation using conditional mutants of these candidate proteins showed that these proteins were essential for asexual growth and localize to the host-parasite interface during early stages of the intraerythrocytic cycle. These data suggest that they might play a role in ushering membrane proteins from the parasite plasma membrane for export to the host RBC.
Topics: Animals; Humans; Biotinylation; Erythrocytes; Malaria; Plasmodium falciparum; Porins; Protein Transport; Protozoan Proteins
PubMed: 37772444
DOI: 10.1242/jcs.260506 -
Innate Immunity Jul 2023Malaria is often characterized by a complicated disease course due to multifaceted intrinsic genetic factors of the host and the parasite. This study aimed to...
Malaria is often characterized by a complicated disease course due to multifaceted intrinsic genetic factors of the host and the parasite. This study aimed to investigate the role of interleukin-27 () gene polymorphisms in malaria infection in a Saudi Arabian cohort. This case-control study obtained blood samples from 250 malaria patients with and 200 randomly identified healthy control subjects from the Malaria Center in the Jazan area. Malaria patients were grouped into three cohorts as follow: low (<500 parasites/µl of blood), moderate (500-1000 parasites/µl of blood), and high (>1000 parasites/µl of blood) parasitemia. The results show that the IL-27 variant rs181209 was significantly associated with malaria patients ( = 0.026). Similarly, the homozygous GG genotype of rs26528 was also associated with risk of developing malaria ( = 0.032). The minor allele C of variant rs181206 exhibited an association with low to moderate parasitemia ( = 0.046). Furthermore, the rs181209 AA genotype was statistically significant in age group 1-5 years ( = 0.049). In conclusion, this study suggests that variant rs181209 and rs26528 could be associated with the risk of malaria infection by in the population studied.
Topics: Humans; Infant; Child, Preschool; Interleukin-27; Plasmodium falciparum; Parasitemia; Case-Control Studies; Saudi Arabia; Malaria, Falciparum; Malaria; Polymorphism, Genetic
PubMed: 37306242
DOI: 10.1177/17534259231178594