Cation ATPase (ATP4) Orthologue Replacement in the Malaria Parasite Plasmodium knowlesi Reveals Species-Specific Responses to ATP4-Targeting Drugs.

Several unrelated classes of antimalarial compounds developed against Plasmodium falciparum target a parasite-specific P-type ATP-dependent Na pump, PfATP4. We have previously shown that other malaria parasite species infecting humans are less susceptible to these compounds. Here, we generated a series of transgenic Plasmodium knowlesi orthologue replacement (OR) lines in which the endogenous locus was replaced by a recodonized atp4 () coding region or the orthologous coding region from P.

Artemisinin susceptibility in the malaria parasite Plasmodium falciparum: propellers, adaptor proteins and the need for cellular healing.

Studies of the susceptibility of Plasmodium falciparum to the artemisinin family of antimalarial drugs provide a complex picture of partial resistance (tolerance) associated with increased parasite survival in vitro and in vivo. We present an overview of the genetic loci that, in mutant form, can independently elicit parasite tolerance. These encode Kelch propeller domain protein PfK13, ubiquitin hydrolase UBP-1, actin filament-organising protein Coronin, also carrying a propeller domain, and the trafficking adaptor subunit AP-2μ.

The Artemisinin Susceptibility-Associated AP-2 Adaptin μ Subunit is Clathrin Independent and Essential for Schizont Maturation.

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of to artemisinin, associated with mutations in Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in , we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication.

Modification of and Markedly Reduces Ring-Stage Susceptibility of Plasmodium falciparum to Artemisinin .

Management of uncomplicated malaria worldwide is threatened by the emergence in Asia of carrying variants of the locus and exhibiting reduced susceptibility to artemisinin. Mutations in two other genes, and , are associated with artemisinin resistance in rodent malaria and with clinical failure of combination therapy in African malaria patients. Transgenic clones, each carrying orthologues of mutations in and associated with artemisinin resistance in , were derived by Cas9 gene editing.

Rapid and iterative genome editing in the malaria parasite provides new tools for research.

Tackling relapsing and zoonotic infections is critical to reducing malaria incidence and mortality worldwide. Understanding the biology of these important and related parasites was previously constrained by the lack of robust molecular and genetic approaches. Here, we establish CRISPR-Cas9 genome editing in a culture-adapted strain and define parameters for optimal homology-driven repair.

Transient temperature fluctuations severely decrease P. falciparum susceptibility to artemisinin in vitro.

Clinical studies suggest that outcomes for hospitalised malaria patients can be improved by managed hypothermia during treatment. We examined the impact of short pulses of low temperature on ring-stage susceptibility of Plasmodium falciparum to artemisinin in vitro. The usually artemisinin-sensitive clone 3D7 exhibited substantially reduced ring-stage susceptibility to a 4-h pulse of 700 nM dihydro-artemisinin administered during a 5-h pulse of low temperature down to 17 °C.

The pPSU Plasmids for Generating DNA Molecular Weight Markers.

Visualizing nucleic acids by gel electrophoresis is one of the most common techniques in molecular biology, and reference molecular weight markers or ladders are commonly used for size estimation. We have created the pPSU1 & pPSU2 pair of molecular weight marker plasmids which produce both 100 bp and 1 kb DNA ladders when digested with two common restriction enzymes. The 100 bp ladder fragments have been optimized to migrate appropriately on both agarose and native polyacrylamide, unlike many currently available DNA ladders.

Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome.

The Polycomb group of epigenetic enzymes represses expression of developmentally regulated genes in many eukaryotes. This group includes the Polycomb repressive complex 1 (PRC1), which ubiquitylates nucleosomal histone H2A Lys 119 using its E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. However, the molecular mechanism of nucleosome substrate recognition by PRC1 or other chromatin enzymes is unclear.