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  1. Drug resistance is an obstacle to global malaria control, as evidenced by the recent emergence and rapid spread of delayed artemisinin (ART) clearance by mutant forms of thePfKelch13 protein in Southeast Asia. Identifying genetic determinants of ART resistance in African-derived parasites is important for surveillance and for understanding the mechanism of resistance. In this study, we carried out long-term in vitro selection of two recently isolated West African parasites (from Pikine and Thiès, Senegal) with increasing concentrations of dihydroartemisinin (DHA), the biologically active form of ART, over a 4-y period. We isolated two parasite clones, one from each original isolate, that exhibited enhanced survival to DHA in the ring-stage survival assay. Whole-genome sequence analysis identified 10 mutations in seven different genes. We chose to focus on the gene encodingPfCoronin, a member of the WD40-propeller domain protein family, because mutations in this gene occurred in both independent selections, and the protein shares the β-propeller motif withPfKelch13 protein. For functional validation, whenpfcoroninmutations were introduced into the parental parasites by CRISPR/Cas9-mediated gene editing, these mutations were sufficient to reduce ART susceptibility in the parental lines. The discovery of a second gene for ART resistance may yield insights into the molecular mechanisms of resistance. It also suggests thatpfcoroninmutants could emerge as a nonkelch13type of resistance to ART in natural settings.

     
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  2. Abstract

    Ecologists have long studied the evolution of niche breadth, including how variability in environments can drive the evolution of specialism and generalism. This concept is of particular interest in viruses, where niche breadth evolution may explain viral disease emergence, or underlie the potential for therapeutic measures like phage therapy. Despite the significance and potential applications of virus–host interactions, the genetic determinants of niche breadth evolution remain underexplored in many bacteriophages. In this study, we present the results of an evolution experiment with a model bacteriophage system,Escherichia virus T4,in several host environments: exposure toEscherichia coliC, exposure toE. coliK‐12, and exposure to bothE. coliC andE. coliK‐12. This experimental framework allowed us to investigate the phenotypic and molecular manifestations of niche breadth evolution. First, we show that selection on different hosts led to measurable changes in phage productivity in all experimental populations. Second, whole—genome sequencing of experimental populations revealed signatures of selection. Finally, clear and consistent patterns emerged across the host environments, especially the presence of new mutations in phage structural genes—genes encoding proteins that provide morphological and biophysical integrity to a virus. A comparison of mutations found across functional gene categories revealed that structural genes acquired significantly more mutations than other categories. Our findings suggest that structural genes are central determinants in bacteriophage niche breadth.

     
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  3. Abstract

    While reverse genetics and functional genomics have long affirmed the role of individual mutations in determining protein function, there have been fewer studies addressing how large‐scale changes in protein sequences, such as in entire modular segments, influence protein function and evolution. Given how recombination can reassort protein sequences, these types of changes may play an underappreciated role in how novel protein functions evolve in nature. Such studies could aid our understanding of whether certain organismal phenotypes related to protein function—such as growth in the presence or absence of an antibiotic—are robust with respect to the identity of certain modular segments. In this study, we combine molecular genetics with biochemical and biophysical methods to gain a better understanding of protein modularity in dihydrofolate reductase (DHFR), an enzyme target of antibiotics also widely used as a model for protein evolution. We replace an integral α‐helical segment ofEscherichia coliDHFR with segments from a number of different organisms (many nonmicrobial) and examine how these chimeric enzymes affect organismal phenotypes (e.g., resistance to an antibiotic) as well as biophysical properties of the enzyme (e.g., thermostability). We find that organismal phenotypes and enzyme properties are highly sensitive to the identity of DHFR modules, and that this chimeric approach can create enzymes with diverse biophysical characteristics.

     
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