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Nature Communications May 2023In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite...
In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase δ. Mutation accumulation assays reveal a ~5-8 fold elevation in the mutation rate, with an increase of 13-28 fold in drug-pressured lines. Upon challenge with the spiroindolone PfATP4-inhibitor KAE609, high-level resistance is obtained more rapidly and at lower inocula than wild-type parasites. Selections also yield mutants with resistance to an "irresistible" compound, MMV665794 that failed to yield resistance with other strains. We validate mutations in a previously uncharacterised gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), as causal for resistance to MMV665794 and a panel of quinoxaline analogues. The increased genetic repertoire available to this "mutator" parasite can be leveraged to drive P. falciparum resistome discovery.
Topics: Animals; Plasmodium falciparum; Parasites; Malaria, Falciparum; Antimalarials; Mutation; Drug Resistance; Protozoan Proteins
PubMed: 37244916
DOI: 10.1038/s41467-023-38774-1 -
Trends in Parasitology May 2021Plasmodium falciparum pathogenesis is complex and intimately connected to vascular physiology. This is exemplified by cerebral malaria (CM), a neurovascular complication... (Review)
Review
Plasmodium falciparum pathogenesis is complex and intimately connected to vascular physiology. This is exemplified by cerebral malaria (CM), a neurovascular complication that accounts for most of the malaria deaths worldwide. P. falciparum sequestration in the brain microvasculature is a hallmark of CM and is not replicated in animal models. Numerous aspects of the disease are challenging to fully understand from clinical studies, such as parasite binding tropism or causal pathways in blood-brain barrier breakdown. Recent bioengineering approaches allow for the generation of 3D microvessels and organ-specific vasculature that provide precise control of vessel architecture and flow dynamics, and hold great promise for malaria research. Here, we discuss recent and future applications of bioengineered microvessels in malaria pathogenesis research.
Topics: Animals; Bioengineering; Brain; Humans; Microvessels; Parasitology; Plasmodium falciparum
PubMed: 33485788
DOI: 10.1016/j.pt.2020.12.008 -
Malaria Journal May 2021Loop-mediated isothermal amplification (LAMP) for malaria diagnosis at the point of care (POC) depends on the detection capacity of synthesized nucleic acids and the...
BACKGROUND
Loop-mediated isothermal amplification (LAMP) for malaria diagnosis at the point of care (POC) depends on the detection capacity of synthesized nucleic acids and the specificity of the amplification target. To improve malaria diagnosis, new colorimetric LAMP tests were developed using multicopy targets for Plasmodium vivax and Plasmodium falciparum detection.
METHODS
The cytochrome oxidase I (COX1) mitochondrial gene and the non-coding sequence Pvr47 for P. vivax, and the sub-telomeric sequence of erythrocyte membrane protein 1 (EMP1) and the non-coding sequence Pfr364 for P. falciparum were targeted to design new LAMP primers. The limit of detection (LOD) of each colorimetric LAMP was established and assessed with DNA extracted by mini spin column kit and the Boil & Spin method from 28 microscopy infections, 101 malaria submicroscopic infections detected by real-time PCR only, and 183 negatives infections by both microscopy and PCR.
RESULTS
The LODs for the colorimetric LAMPs were estimated between 2.4 to 3.7 parasites/µL of whole blood. For P. vivax detection, the colorimetric LAMP using the COX1 target showed a better performance than the Pvr47 target, whereas the Pfr364 target was the most specific for P. falciparum detection. All microscopic infections of P. vivax were detected by PvCOX1-LAMP using the mini spin column kit DNA extraction method and 81% (17/21) were detected using Boil & Spin sample preparation. Moreover, all microscopic infections of P. falciparum were detected by Pfr364-LAMP using both sample preparation methods. In total, PvCOX1-LAMP and Pfr364-LAMP detected 80.2% (81 samples) of the submicroscopic infections using the DNA extraction method by mini spin column kit, while 36.6% (37 samples) were detected using the Boil & Spin sample preparation method.
CONCLUSION
The colorimetric LAMPs with multicopy targets using the COX1 target for P. vivax and the Pfr364 for P. falciparum have a high potential to improve POC malaria diagnosis detecting a greater number of submicroscopic Plasmodium infections.
Topics: Colorimetry; Electron Transport Complex IV; Malaria, Falciparum; Malaria, Vivax; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Plasmodium falciparum; Plasmodium vivax; Protozoan Proteins
PubMed: 34011373
DOI: 10.1186/s12936-021-03753-8 -
Trends in Biochemical Sciences Feb 2022The difficulty of faithfully recapitulating malarial protein complexes in heterologous expression systems has long impeded structural study for much of the Plasmodium... (Review)
Review
The difficulty of faithfully recapitulating malarial protein complexes in heterologous expression systems has long impeded structural study for much of the Plasmodium falciparum proteome. However, recent advances in single-particle cryo electron microscopy (cryoEM) now enable structure determination at atomic resolution with significantly reduced requirements for both sample quantity and purity. Combined with recent developments in gene editing, these advances open the door to structure determination and structural proteomics of macromolecular complexes enriched directly from P. falciparum parasites. Furthermore, the combination of cryoEM with the rapidly emerging use of in situ cryo electron tomography (cryoET) to directly visualize ultrastructures and protein complexes in the native cellular context will yield exciting new insights into the molecular machinery underpinning malaria parasite biology and pathogenesis.
Topics: Animals; Cryoelectron Microscopy; Malaria; Malaria, Falciparum; Parasites; Plasmodium falciparum
PubMed: 34887149
DOI: 10.1016/j.tibs.2021.10.006 -
Trends in Parasitology Jun 2021Recent progress in genomics and molecular genetics has empowered novel approaches to study gene functions in disease-causing pathogens. In the human malaria parasite... (Review)
Review
Recent progress in genomics and molecular genetics has empowered novel approaches to study gene functions in disease-causing pathogens. In the human malaria parasite Plasmodium falciparum, the application of genome-based analyses, site-directed genome editing, and genetic systems that allow for temporal and quantitative regulation of gene and protein expression have been invaluable in defining the genetic basis of antimalarial resistance and elucidating candidate targets to accelerate drug discovery efforts. Using examples from recent studies, we review applications of some of these approaches in advancing our understanding of Plasmodium biology and illustrate their contributions and limitations in characterizing parasite genomic loci associated with antimalarial drug responses.
Topics: Antimalarials; Drug Resistance; Genome, Protozoan; Genomics; Humans; Malaria, Falciparum; Molecular Biology; Plasmodium falciparum
PubMed: 33715941
DOI: 10.1016/j.pt.2021.02.007 -
Nature Communications May 2024The human infectious reservoir of Plasmodium falciparum is governed by transmission efficiency during vector-human contact and mosquito biting preferences. Understanding...
The human infectious reservoir of Plasmodium falciparum is governed by transmission efficiency during vector-human contact and mosquito biting preferences. Understanding biting bias in a natural setting can help target interventions to interrupt transmission. In a 15-month cohort in western Kenya, we detected P. falciparum in indoor-resting Anopheles and human blood samples by qPCR and matched mosquito bloodmeals to cohort participants using short-tandem repeat genotyping. Using risk factor analyses and discrete choice models, we assessed mosquito biting behavior with respect to parasite transmission. Biting was highly unequal; 20% of people received 86% of bites. Biting rates were higher on males (biting rate ratio (BRR): 1.68; CI: 1.28-2.19), children 5-15 years (BRR: 1.49; CI: 1.13-1.98), and P. falciparum-infected individuals (BRR: 1.25; CI: 1.01-1.55). In aggregate, P. falciparum-infected school-age (5-15 years) boys accounted for 50% of bites potentially leading to onward transmission and had an entomological inoculation rate 6.4x higher than any other group. Additionally, infectious mosquitoes were nearly 3x more likely than non-infectious mosquitoes to bite P. falciparum-infected individuals (relative risk ratio 2.76, 95% CI 1.65-4.61). Thus, persistent P. falciparum transmission was characterized by disproportionate onward transmission from school-age boys and by the preference of infected mosquitoes to feed upon infected people.
Topics: Humans; Anopheles; Animals; Plasmodium falciparum; Malaria, Falciparum; Male; Adolescent; Child; Child, Preschool; Female; Kenya; Mosquito Vectors; Insect Bites and Stings; Adult; Feeding Behavior; Young Adult; Infant
PubMed: 38816383
DOI: 10.1038/s41467-024-49080-9 -
Molecular Microbiology May 2021Malaria is one of the most life-threatening infectious diseases worldwide, caused by infection of humans with parasites of the genus Plasmodium. The complex life cycle... (Review)
Review
Malaria is one of the most life-threatening infectious diseases worldwide, caused by infection of humans with parasites of the genus Plasmodium. The complex life cycle of Plasmodium parasites is shared between two hosts, with infection of multiple cell types, and the parasite needs to adapt for survival and transmission through significantly different metabolic environments. Within the blood-stage alone, parasites encounter changing levels of key nutrients, including sugars, amino acids, and lipids, due to differences in host dietary nutrition, cellular tropism, and pathogenesis. In this review, we consider the mechanisms that the most lethal of malaria parasites, Plasmodium falciparum, uses to sense nutrient levels and elicit changes in gene expression during blood-stage infections. These changes are brought about by several metabolic intermediates and their corresponding sensor proteins. Sensing of distinct nutritional signals can drive P. falciparum to alter the key blood-stage processes of proliferation, antigenic variation, and transmission.
Topics: Animals; Gene Expression; Humans; Life Cycle Stages; Malaria, Falciparum; Nutrients; Plasmodium falciparum
PubMed: 33236377
DOI: 10.1111/mmi.14652 -
Proceedings of the National Academy of... Mar 20225-methylcytosine (mC) is an important epitranscriptomic modification involved in messenger RNA (mRNA) stability and translation efficiency in various biological...
5-methylcytosine (mC) is an important epitranscriptomic modification involved in messenger RNA (mRNA) stability and translation efficiency in various biological processes. However, it remains unclear if mC modification contributes to the dynamic regulation of the transcriptome during the developmental cycles of parasites. Here, we characterize the landscape of mC mRNA modifications at single nucleotide resolution in the asexual replication stages and gametocyte sexual stages of rodent () and human () malaria parasites. While different representations of mC-modified mRNAs are associated with the different stages, the abundance of the mC marker is strikingly enhanced in the transcriptomes of gametocytes. Our results show that mC modifications confer stability to the transcripts and that a ortholog of NSUN2 is a major mRNA mC methyltransferase in malaria parasites. Upon knockout of (), marked reductions of mC modification were observed in a panel of gametocytogenesis-associated transcripts. These reductions correlated with impaired gametocyte production in the knockout rodent malaria parasites. Restoration of the gene in the knockout parasites rescued the gametocyte production phenotype as well as mC modification of the gametocytogenesis-associated transcripts. Together with the mRNA mC profiles for two species of , our findings demonstrate a major role for NSUN2-mediated mC modifications in mRNA transcript stability and sexual differentiation in malaria parasites.
Topics: 5-Methylcytosine; Germ Cells; Plasmodium falciparum; Plasmodium yoelii; Protozoan Proteins; RNA, Messenger; Transcriptome
PubMed: 35210361
DOI: 10.1073/pnas.2110713119 -
Trends in Parasitology Aug 2021Strategies to counteract or prevent emerging drug resistance are crucial for the design of next-generation antimalarials. In the past, resistant parasites were generally... (Review)
Review
Strategies to counteract or prevent emerging drug resistance are crucial for the design of next-generation antimalarials. In the past, resistant parasites were generally identified following treatment failures in patients, and compounds would have to be abandoned late in development. An early understanding of how candidate therapeutics lose efficacy as parasites evolve resistance is important to facilitate drug design and improve resistance detection and monitoring up to the postregistration phase. We describe a new strategy to assess resistance to antimalarial compounds as early as possible in preclinical development by leveraging tools to define the Plasmodium falciparum resistome, predict potential resistance risks of clinical failure for candidate therapeutics, and inform decisions to guide antimalarial drug development.
Topics: Antimalarials; Drug Resistance; Malaria, Falciparum; Plasmodium falciparum; Risk Assessment
PubMed: 34001441
DOI: 10.1016/j.pt.2021.04.006 -
PloS One 2022Despite ongoing efforts to control malaria infection, progress in lowering the number of deaths and infections appears to have stalled. The continued high incidence of...
Despite ongoing efforts to control malaria infection, progress in lowering the number of deaths and infections appears to have stalled. The continued high incidence of malaria infection and mortality is in part due to emergence of parasites resistant to frontline antimalarials. This highlights the need for continued identification of novel protein drug targets. Mitochondrial functions in Plasmodium falciparum, the deadliest species of human malaria parasite, are targets of validated antimalarials including atovaquone and proguanil (Malarone). Thus, there has been great interest in identifying other essential mitochondrial proteins as candidates for novel drug targets. Garnering an increased understanding of the proteomic landscape inside the P. falciparum mitochondrion will also allow us to learn about the basic biology housed within this unique organelle. We employed a proximity biotinylation technique and mass spectrometry to identify novel P. falciparum proteins putatively targeted to the mitochondrion. We fused the leader sequence of a mitochondrially targeted chaperone, Hsp60, to the promiscuous biotin ligase TurboID. Through these experiments, we generated a list of 122 "putative mitochondrial" proteins. To verify whether these proteins were indeed mitochondrial, we chose five candidate proteins of interest for localization studies using ectopic expression and tagging of each full-length protein. This allowed us to localize four candidate proteins of unknown function to the mitochondrion, three of which have previously been assessed to be essential. We suggest that phenotypic characterization of these and other proteins from this list of 122 could be fruitful in understanding the basic mitochondrial biology of these parasites and aid antimalarial drug discovery efforts.
Topics: Antimalarials; Atovaquone; Biotinylation; Drug Combinations; Humans; Malaria; Malaria, Falciparum; Plasmodium falciparum; Proguanil; Proteomics
PubMed: 35984838
DOI: 10.1371/journal.pone.0273357