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ABSTRACTMalaria elimination efforts in Southeast Asia have been hindered by multidrug-resistant Plasmodium falciparum. High-grade resistance to piperaquine (PPQ, used in combination with dihydroartemisinin) is associated with PfCRT mutations that arose in strains expressing the PfCRT Dd2 isoform, which mediates resistance to the related 4-aminoquinoline chloroquine (CQ). The PPQ-resistant PfCRT haplotype Dd2 + F145I mediates the highest level resistance but causes a significant growth defect in intra-erythrocytic parasites. Recently, three separate mutations (F131C, I347T and C258W) have been observed on Dd2 + F145I PfCRT either during extended parasite culture or in Southeast Asian isolates no longer subject to PPQ pressure. Competitive growth assays with pfcrt-edited parasites reveal that these compensatory mutations reduce the fitness defect caused by F145I. PPQ survival assays on edited lines show a loss of PPQ resistance in two of the three variants, including the field mutant (C258W). The latter restores CQ resistance. None of these variants alter parasite susceptibility to the first-line partner drug, mefloquine. Utilizing drug transport assays with purified PfCRT isoforms reconstituted into proteoliposomes, we identify differences in mutant PfCRT-mediated transport of PPQ and CQ. Molecular dynamics energy minimization calculations predict that these same mutations cause small but significant conformational changes in PfCRT regions implicated in drug interactions. Metabolomic analyses of isogenic parasite lines reveal differences in hemoglobin-derived peptide accumulation as a hallmark of PfCRT variation. These studies highlight the transient nature of PPQ resistance upon removal of drug pressure and suggest a strategy for employing this drug as part of multiple first-line therapies.IMPORTANCEOur study leverages gene editing techniques in Plasmodium falciparum asexual blood stage parasites to profile novel mutations in mutant PfCRT, an important mediator of piperaquine resistance, which developed in Southeast Asian field isolates or in parasites cultured for long periods of time. We provide evidence that increased parasite fitness of these lines is the primary driver for the emergence of these PfCRT variants. These mutations differentially impact parasite susceptibility to piperaquine and chloroquine, highlighting the multifaceted effects of single point mutations in this transporter. Molecular features of drug resistance and parasite physiology were examined in depth using proteoliposome-based drug uptake studies and peptidomics, respectively. Energy minimization calculations, showing how these novel mutations might impact the PfCRT structure, suggested a small but significant effect on drug interactions. This study reveals the subtle interplay between antimalarial resistance, parasite fitness, PfCRT structure, and intracellular peptide availability in PfCRT-mediated parasite responses to changing drug selective pressures.

Abstract 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.

Drug resistance to current antimalarials is rising and new drugs and targets are urgently needed. Here the authors identify Plasmodium falciparum acyl-CoA synthetase 10 as a new target whose inhibition leads to a decrease in triacylglycerols.

The skeletal system derives from multiple embryonic sources whose derivatives must develop in coordination to produce an integrated whole. In particular, interactions across the lateral somitic frontier, where derivatives of the somites and lateral plate mesoderm come into contact, are important for proper development. Many questions remain about genetic control of this coordination, and embryological information is incomplete for some structures that incorporate the frontier, including the sternum. Hox genes act in both tissues as regulators of skeletal pattern. Here, we used conditional deletion to characterize the tissue-specific contributions of Hoxa5 to skeletal patterning. We found that most aspects of the Hoxa5 skeletal phenotype are attributable to its activity in one or the other tissue, indicating largely additive roles. However, multiple roles are identified at the junction of the T1 ribs and the anterior portion of the sternum, or presternum. The embryology of the presternum has not been well described in mouse. We present a model for presternum development, and show that it arises from multiple, paired LPM-derived primordia. We show evidence that HOXA5 expression marks the embryonic precursor of a recently identified lateral presternum structure that is variably present in therians.

Brown adipose tissue (BAT) plays critical thermogenic, metabolic and endocrine roles in mammals, and aberrant BAT function is associated with metabolic disorders including obesity and diabetes. The major BAT depots are clustered at the neck and forelimb levels, and arise largely within the dermomyotome of somites, from a common progenitor with skeletal muscle. However, many aspects of BAT embryonic development are not well understood. Hoxa5 patterns other tissues at the cervical and brachial levels, including skeletal, neural and respiratory structures. Here, we show that Hoxa5 also positively regulates BAT development, while negatively regulating formation of epaxial skeletal muscle. HOXA5 protein is expressed in embryonic preadipocytes and adipocytes as early as embryonic day 12.5. Hoxa5 null mutant embryos and rare, surviving adults show subtly reduced iBAT and sBAT formation, as well as aberrant marker expression, lower adipocyte density and altered lipid droplet morphology. Conversely, the epaxial muscles that arise from a common dermomyotome progenitor are expanded in Hoxa5 mutants. Conditional deletion of Hoxa5 with Myf5/Cre can reproduce both BAT and epaxial muscle phenotypes, indicating that HOXA5 is necessary within Myf5-positive cells for proper BAT and epaxial muscle development. However, recombinase-based lineage tracing shows that Hoxa5 does not act cell-autonomously to repress skeletal muscle fate. Interestingly, Hoxa5-dependent regulation of adipose-associated transcripts is conserved in lung and diaphragm, suggesting a shared molecular role for Hoxa5 in multiple tissues. Together, these findings establish a role for Hoxa5 in embryonic BAT development.

The emergence of mutant K13-mediated artemisinin (ART) resistance in Plasmodium falciparum malaria parasites has led to widespread treatment failures across Southeast Asia. In Africa, K13-propeller genotyping confirms the emergence of the R561H mutation in Rwanda and highlights the continuing dominance of wild-type K13 elsewhere. Using gene editing, we show that R561H, along with C580Y and M579I, confer elevated in vitro ART resistance in some African strains, contrasting with minimal changes in ART susceptibility in others. C580Y and M579I cause substantial fitness costs, which may slow their dissemination in high-transmission settings, in contrast with R561H that in African 3D7 parasites is fitness neutral. In Cambodia, K13 genotyping highlights the increasing spatio-temporal dominance of C580Y. Editing multiple K13 mutations into a panel of Southeast Asian strains reveals that only the R561H variant yields ART resistance comparable to C580Y. In Asian Dd2 parasites C580Y shows no fitness cost, in contrast with most other K13 mutations tested, including R561H. Editing of point mutations in ferredoxin or mdr2, earlier associated with resistance, has no impact on ART susceptibility or parasite fitness. These data underline the complex interplay between K13 mutations, parasite survival, growth and genetic background in contributing to the spread of ART resistance.