Population genomics and transcriptomics of Plasmodium falciparum in Cambodia and Vietnam uncover key components of the artemisinin resistance genetic background.

The emergence of Plasmodium falciparum parasites resistant to artemisinins compromises the efficacy of Artemisinin Combination Therapies (ACTs), the global first-line malaria treatment. Artemisinin resistance is a complex genetic trait in which nonsynonymous SNPs in PfK13 cooperate with other genetic variations. Here, we present population genomic/transcriptomic analyses of P.

Identification of complex Plasmodium falciparum genetic backgrounds circulating in Africa: a multicountry genomic epidemiology analysis.

Background: The population structure of the malaria parasite Plasmodium falciparum can reveal underlying adaptive evolutionary processes. Selective pressures to maintain complex genetic backgrounds can encourage inbreeding, producing distinct parasite clusters identifiable by population structure analyses.

Methods: We analysed population structure in 3783 P falciparum genomes from 21 countries across Africa, provided by the MalariaGEN Pf7 dataset.

Peeling the onion: how complex is the artemisinin resistance genetic trait of malaria parasites?

The genetics of Plasmodium as an intracellular, mostly haploid, sexually reproducing, eukaryotic organism with a complex life cycle, presents unprecedented challenges in studying drug resistance. This article summarizes current knowledge on the genetic basis of artemisinin resistance (AR) - a main component of current drug therapies for falciparum malaria. Although centered on nonsynonymous single-nucleotide polymorphisms (nsSNPs), we describe multifaceted resistance mechanisms as part of a complex, cumulative genetic trait that involves regulation of expression by a wide array of polymorphisms in noncoding regions.

Inhibition of falcilysin from Plasmodium falciparum by interference with its closed-to-open dynamic transition.

In the absence of an efficacious vaccine, chemotherapy remains crucial to prevent and treat malaria. Given its key role in haemoglobin degradation, falcilysin constitutes an attractive target. Here, we reveal the mechanism of enzymatic inhibition of falcilysin by MK-4815, an investigational new drug with potent antimalarial activity.

The artemisinin-induced dormant stages of Plasmodium falciparum exhibit hallmarks of cellular quiescence/senescence and drug resilience.

Recrudescent infections with the human malaria parasite, Plasmodium falciparum, presented traditionally the major setback of artemisinin-based monotherapies. Although the introduction of artemisinin combination therapies (ACT) largely solved the problem, the ability of artemisinin to induce dormant parasites still poses an obstacle for current as well as future malaria chemotherapeutics. Here, we use a laboratory model for induction of dormant P.

Identification of an inhibitory pocket in falcilysin provides a new avenue for malaria drug development.

Identification of new druggable protein targets remains the key challenge in the current antimalarial development efforts. Here we used mass-spectrometry-based cellular thermal shift assay (MS-CETSA) to identify potential targets of several antimalarials and drug candidates. We found that falcilysin (FLN) is a common binding partner for several drug candidates such as MK-4815, MMV000848, and MMV665806 but also interacts with quinoline drugs such as chloroquine and mefloquine.

Sterile protection against malaria by repeated blood stage infection in the monkey model.

The malaria parasite remains a major global public health challenge, and no vaccine is approved for use in humans. Here, we assessed whether strain-transcendent immunity can be achieved by repeated infection in monkeys. Sterile immunity was achieved after two homologous infections, whereas subsequent heterologous challenge provided only partial protection.

Identification and structural validation of purine nucleoside phosphorylase from Plasmodium falciparum as a target of MMV000848.

About 247 million cases of malaria occurred in 2021 with Plasmodium falciparum accounting for the majority of 619,000 deaths. In the absence of a widely available vaccine, chemotherapy remains crucial to prevent, treat, and contain the disease. The efficacy of several drugs currently used in the clinic is likely to suffer from the emergence of resistant parasites.

Genetic validation of FKBP35 as an antimalarial drug target.

accounts for the majority of over 600,000 malaria-associated deaths annually. Parasites resistant to nearly all antimalarials have emerged and the need for drugs with alternative modes of action is thus undoubted. The FK506-binding protein FKBP35 has gained attention as a promising drug target due to its high affinity to the macrolide compound FK506 (tacrolimus).

Eukaryotic Translation Initiation Factor 3 is Stabilized by Quinazoline-Quinoline Bisubstrate Inhibitors.

Malaria drug resistance is hampering the fight against the deadliest parasitic disease affecting over 200 million people worldwide. We recently developed quinoline-quinazoline-based inhibitors (as compound ) as promising new antimalarials. Here, we aimed to investigate their mode of action by using thermal proteome profiling (TPP).

Short tandem repeat polymorphism in the promoter region of cyclophilin 19B drives its transcriptional upregulation and contributes to drug resistance in the malaria parasite Plasmodium falciparum.

Resistance of the human malaria parasites, Plasmodium falciparum, to artemisinins is now fully established in Southeast Asia and is gradually emerging in Sub-Saharan Africa. Although nonsynonymous SNPs in the pfk13 Kelch-repeat propeller (KREP) domain are clearly associated with artemisinin resistance, their functional relevance requires cooperation with other genetic factors/alterations of the P. falciparum genome, collectively referred to as genetic background.

Human peroxiredoxin 6 is essential for malaria parasites and provides a host-based drug target.

The uptake and digestion of host hemoglobin by malaria parasites during blood-stage growth leads to significant oxidative damage of membrane lipids. Repair of lipid peroxidation damage is crucial for parasite survival. Here, we demonstrate that Plasmodium falciparum imports a host antioxidant enzyme, peroxiredoxin 6 (PRDX6), during hemoglobin uptake from the red blood cell cytosol.

Stochastic expression of invasion genes in Plasmodium falciparum schizonts.

Genetically identical cells are known to exhibit differential phenotypes in the same environmental conditions. These phenotypic variants are linked to transcriptional stochasticity and have been shown to contribute towards adaptive flexibility of a wide range of unicellular organisms. Here, we investigate transcriptional heterogeneity and stochastic gene expression in Plasmodium falciparum by performing the quasilinear multiple annealing and looping based amplification cycles (MALBAC) based amplification and single cell RNA sequencing of blood stage schizonts.

Reverse transcription PCR to detect low density malaria infections.

Targeted malaria elimination strategies require highly sensitive tests to detect low density malaria infections (LDMI). Commonly used methods for malaria diagnosis such as light microscopy and antigen-based rapid diagnostic tests (RDTs) are not sensitive enough for reliable identification of infections with parasitaemia below 200 parasites per milliliter of blood. While targeted malaria elimination efforts on the Thailand-Myanmar border have successfully used high sample volume ultrasensitive quantitative PCR (uPCR) to determine malaria prevalence, the necessity for venous collection and processing of large quantities of patient blood limits the widespread tractability of this method.

Mediator Complex of the Malaria Parasite Associates with Evolutionarily Novel Subunits.

The eukaryotic Mediator is a large and conserved multisubunit protein complex that directly contacts RNA polymerase II and impinges on multiple aspects of gene expression. The genome of the human malaria parasite has been predicted to encode several Mediator subunits. We provide physical evidence for the presence of a Mediator complex in by using coimmunoprecipitation and mass spectrometry to identify interaction partners of the highly conserved Mediator subunit PfMed31.

Artemisinin resistance in the malaria parasite, Plasmodium falciparum, originates from its initial transcriptional response.

The emergence and spread of artemisinin-resistant Plasmodium falciparum, first in the Greater Mekong Subregion (GMS), and now in East Africa, is a major threat to global malaria elimination ambitions. To investigate the artemisinin resistance mechanism, transcriptome analysis was conducted of 577 P. falciparum isolates collected in the GMS between 2016-2018.

The parasitophorous vacuole nutrient channel is critical for drug access in malaria parasites and modulates the artemisinin resistance fitness cost.

Intraerythrocytic malaria parasites proliferate bounded by a parasitophorous vacuolar membrane (PVM). The PVM contains nutrient permeable channels (NPCs) conductive to small molecules, but their relevance for parasite growth for individual metabolites is largely untested. Here we show that growth-relevant levels of major carbon and energy sources pass through the NPCs.

A heat-shock response regulated by the PfAP2-HS transcription factor protects human malaria parasites from febrile temperatures.

Periodic fever is a characteristic clinical feature of human malaria, but how parasites survive febrile episodes is not known. Although the genomes of Plasmodium species encode a full set of chaperones, they lack the conserved eukaryotic transcription factor HSF1, which activates the expression of chaperones following heat shock. Here, we show that PfAP2-HS, a transcription factor in the ApiAP2 family, regulates the protective heat-shock response in Plasmodium falciparum.

Artemisinin-resistant K13 mutations rewire Plasmodium falciparum's intra-erythrocytic metabolic program to enhance survival.

The emergence and spread of artemisinin resistance, driven by mutations in Plasmodium falciparum K13, has compromised antimalarial efficacy and threatens the global malaria elimination campaign. By applying systems-based quantitative transcriptomics, proteomics, and metabolomics to a panel of isogenic K13 mutant or wild-type P. falciparum lines, we provide evidence that K13 mutations alter multiple aspects of the parasite's intra-erythrocytic developmental program.

A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples.

Background: Sequencing technology advancements opened new opportunities to use transcriptomics for studying malaria pathology and epidemiology. Even though in recent years the study of whole parasite transcriptome proved to be essential in understanding parasite biology there is no compiled up-to-date reference protocol for the efficient generation of transcriptome data from growing number of samples. Here, a comprehensive methodology on how to preserve, extract, amplify, and sequence full-length mRNA transcripts from Plasmodium-infected blood samples is presented that can be fully streamlined for high-throughput studies.

Cellular thermal shift assay for the identification of drug-target interactions in the Plasmodium falciparum proteome.

Despite decades of research, little is known about the cellular targets and the mode of action of the vast majority of antimalarial drugs. We recently demonstrated that the cellular thermal shift assay (CETSA) protocol in its two variants: the melt curve and the isothermal dose-response, represents a comprehensive strategy for the identification of antimalarial drug targets. CETSA enables proteome-wide target screening for unmodified antimalarial compounds with undetermined mechanisms of action, providing quantitative evidence about direct drug-protein interactions.

Whole-Cell Phenotypic Screening of Medicines for Malaria Venture Pathogen Box Identifies Specific Inhibitors of Late-Stage Development and Egress.

We report a systematic, cellular phenotype-based antimalarial screening of the Medicines for Malaria Venture Pathogen Box collection, which facilitated the identification of specific blockers of late-stage intraerythrocytic development of First, from standard growth inhibition assays, we identified 173 molecules with antimalarial activity (50% effective concentration [EC] ≤ 10 μM), which included 62 additional molecules over previously known antimalarial candidates from the Pathogen Box. We identified 90 molecules with EC of ≤1 μM, which had significant effect on the ring-trophozoite transition, while 9 molecules inhibited the trophozoite-schizont transition and 21 molecules inhibited the schizont-ring transition (with ≥50% parasites failing to proceed to the next stage) at 1 μM. We therefore rescreened all 173 molecules and validated hits in microscopy to prioritize 12 hits as selective blockers of the schizont-ring transition.

Transcriptome profiling reveals functional variation in Plasmodium falciparum parasites from controlled human malaria infection studies.

Background: The transcriptome of Plasmodium falciparum clinical isolates varies according to strain, mosquito bites, disease severity and clinical history. Therefore, it remains a challenge to directly interpret the parasite's transcriptomic information into a more general biological signature in a natural human malaria infection. These confounding variations can be potentially overcome with parasites derived from controlled-human malaria infection (CHMI) studies.