Artemisinin resistance phenotypes and K13 inheritance in a
            <i>Plasmodium falciparum</i>
            cross and
            <i>Aotus</i>
            model

Sá, Juliana M.; Kaslow, Sarah R.; Krause, Michael; Melendez-Muniz, Viviana A.; Salzman, Rebecca E.; Kite, Whitney A.; Zhang, Min; Barros, Roberto R. Moraes; Mu, Jianbing; Han, Paul K.; Mershon, J. Patrick; Figan, Christine E.; Caleon, Ramoncito L.; Rahman, Rifat; Gibson, Tyler J.; Amaratunga, Chanaki; Nishiguchi, Erika P.; Breglio, Kimberly F.; Engels, Theresa; Velmurugan, Soundarapandian; Ricklefs, Stacy M.; Straimer, Judith; Gnädig, Nina F.; Deng, Bingbing; Liu, Anna; Diouf, Ababacar; Miura, Kazutoyo; Tullo, Gregory; Eastman, Richard T.; Chakravarty, Sumana; James, Eric R.; Udenze, Kenneth O.; Li, Suzanne; Sturdevant, Daniel E.; Gwadz, Robert W.; Porcella, Stephen F.; Long, Carole A.; Fidock, David A.; Thomas, Marvin L.; Fay, Michael P.; Sim, B. Kim Lee; Hoffman, Stephen L.; Adams, John; Fairhurst, Rick M.; Su, Xin; Wellems, Thomas E.

doi:10.1073/pnas.1813386115

Cited by 48 publications

(52 citation statements)

References 52 publications

(75 reference statements)

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“…Previous P. falciparum genetic crosses exhibited significant segregation distortion at several loci [13, 16, 17, 25]. We explored overlap between distorted regions in all the published P. falciparum crosses and our two new crosses and included a previously published bulk analysis of selection in uncloned progeny of the MKK2835xNHP1337 cross [33] (S2 Fig).…”

Section: Resultsmentioning

confidence: 99%

“…P. falciparum has the potential to be a particularly powerful genetic mapping system because of its unusually high recombination rate of 11-13.3 kb/cM [13, 16, 17], a haploid state for most of the life cycle, and the ability to clone every F 1 progeny in vitro , creating effectively immortal mapping populations in a single generation. Also, P. falciparum has a small genome (23 Mb) and a high-quality reference assembly [18] with frequent annotation updates [19, 20]; consequently, re-sequencing and comprehensive analysis of the genome of F 1 progeny is simple and cost effective [21].…”

Section: Introductionmentioning

confidence: 99%

“…This has resulted in only four genetic crosses being performed over a thirty-year period. F 1 mapping populations from all four previous P. falciparum genetic crosses have been small, containing 33, 35, 15 [21] and most recently 27 individual recombinant progeny [13]. When compared to the thousands of F 1 progeny possible in many plants and fruit flies [22], these numbers are small indeed.…”

Section: Introductionmentioning

confidence: 99%

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Surprising variation in the outcome of two malaria genetic crosses using humanized mice: implications for genetic mapping and malaria biology

Button-Simons

Kumar

Carmago

et al. 2019

Preprint

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29Genetic crosses are most powerful for linkage analysis when progeny numbers are high, 30when parental alleles segregate evenly and, for hermaphroditic organisms, when numbers of 31 inbred progeny are minimized. We previously developed a novel genetic crossing platform for 32 the human malaria parasite Plasmodium falciparum, an obligately sexual, hermaphroditic 33 protozoan, using mice carrying human hepatocytes (the human liver-chimeric FRG NOD huHep 34 mouse) as the vertebrate host. Here we examine the statistical power of two different genetic 35 crosses -(1) between a laboratory parasite (NF54) of African origin and a patient-derived Asian 36 parasite, and (2) between two sympatric patient-derived Asian parasites. We generated >140 37 unique recombinant clones over a 12-month period from the four parental genotypes, doubling 38 the number of unique recombinant progeny generated in the previous 30 years. Both crosses 39show bi-parental inheritance of plastid markers amongst recombinant progeny, in contrast to 40 previous crosses (conducted using chimpanzee hosts) which carried single dominant plastid 41 genotypes. Both crosses show distinctive segregation patterns. The allopatric African/Asian cross 42 has minimal levels of inbreeding (2% of clonal progeny are inbred) and extreme skews in marker 43 segregation, while in the sympatric Asian cross, inbred progeny predominate (66% of clonal 44 progeny are inbred) and parental alleles segregate evenly. Using simulations, we demonstrate 45 that these progeny arrays (particularly the sympatric Asian cross) have excellent power to map 46 3 large-effect mutations to a 31 kb interval and can capture complex, epistatic interactions that 47 were far beyond the capacity of previous malaria crosses to detect. The extreme segregation 48 distortion in the allopatric African/Asian cross erodes power to detect linkage in several genome 49 regions, but the repeatable distortions observed offer promising alternative approaches to 50 identifying genes underlying traits of interest. These crosses show surprising variation in marker 51 segregation, nevertheless, the increased progeny numbers improve our ability to rapidly map 52 biomedically important parasite traits. 53 54 Author Summary 55Understanding how genome mutations contribute to newly emerging drug resistance in 56 parasites like Plasmodium falciparum is important to monitor the spread of drug resistance. This 57 scenario has been playing out in Southeast Asia with the emergence and spread of artemisinin 58 resistance. Here we show that new P. falciparum genetic crosses, using mice carrying human 59 liver cells and infused with human red blood cells (the human liver-chimeric FRG NOD 60 huHep/huRBC mouse), provide an important new tool for understanding complex interactions 61 underlying drug resistance phenotypes. We report two new genetic maps with 84 and 60 unique 62 recombinant progeny, which doubles the number of progeny available from 4 previous P. 63 falciparum genetic crosses. Through extensive simulations we show...

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Surprising variation in the outcome of two malaria genetic crosses using humanized mice: implications for genetic mapping and malaria biology

Button-Simons

Kumar

Carmago

et al. 2019

Preprint

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“…Currently, the K13 C580Y polymorphism is the most prevalent and dominant ART-resistant mutation in SEA (6,30). A recent genetic cross of the K13 C580Y ART-resistant line with an Aotus monkey-infecting PF strain provided evidence, in this non-human primate model, that parasites carrying the C580Y mutation could display increased survival in in vitro RSAs with no accompanying in vivo ART resistance (31).…”

Section: Introductionmentioning

confidence: 97%

Plasmodium bergheiK13 Mutations MediateIn VivoArtemisinin Resistance That is Reversed by Proteasome Inhibition

Simwela

Stokes

Aghabi

et al. 2020

Preprint

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The recent emergence of Plasmodium falciparum (PF) parasite resistance to the first line antimalarial drug artemisinin is of particular concern. Artemisinin resistance is primarily driven by mutations in the PF K13 protein, which enhance survival of early ring stage parasites treated with the artemisinin active metabolite dihydroartemisinin in vitro and associate with delayed parasite clearance in vivo. However, association of K13 mutations with in vivo artemisinin resistance has been problematic due to the absence of a tractable model. Herein, we have employed CRISPR/Cas9 genome editing to engineer selected orthologous PF K13 mutations into the K13 gene of an artemisinin-sensitive, P. berghei (PB) rodent model of malaria. Introduction of the orthologous PF K13 F446I, M476I, Y493H and R539T mutations into PB K13 produced gene-edited parasites with reduced susceptibility to dihydroartemisinin in the standard 24-hour in vitro assay and increased survival in an adapted in vitro ring-stage survival assay. Mutant PB K13 parasites also displayed delayed clearance in vivo upon treatment with artesunate and achieved faster recrudescence upon treatment with artemisinin. Orthologous C580Y and I543T mutations could not be introduced into PB while the equivalent of the M476I and R539T mutations resulted in significant growth defects. Furthermore, a Plasmodium-selective proteasome inhibitor strongly synergized dihydroartemisinin action in these PB K13 mutant lines, providing further evidence that the proteasome can be targeted to overcome ART resistance. Taken together, our work provides clear experimental evidence for the involvement of K13 polymorphisms in mediating susceptibility to artemisinins in vitro, and most importantly under in vivo conditions.IMPORTANCERecent successes in malaria control have been seriously threatened by the emergence of Plasmodium falciparum parasite resistance to the frontline artemisinin drugs in Southeast Asia. P. falciparum artemisinin resistance is associated with mutations in the parasite K13 protein, which associates with a delay in the time required to clear the parasites upon treatment with the drug. Gene editing technologies have been used to validate the role of several candidate K13 mutations in mediating P. falciparum artemisinin resistance in vitro under laboratory conditions. Nonetheless, the causal role of these mutations under in vivo conditions has been a matter of debate. Here, we have used CRISPR/Cas9 gene editing to introduce K13 mutations associated with artemisinin resistance into the related rodent-infecting parasite, P. berghei. Phenotyping of these P. berghei K13 mutant parasites provides evidence of their role in mediating artemisinin resistance in vivo, which supports in vitro artemisinin resistance observations. However, we were unable to introduce some of the P. falciparum K13 mutations (C580Y, I543T) into the corresponding amino acid residues, while other introduced mutations (M476I, R539T equivalents) carried a pronounced fitness cost. Our study provides evidence of a clear causal role of K13 mutations in modulating susceptibility to artemisinins in vitro and in vivo using the well-characterized P. berghei model. We also show that inhibition of the P. berghei proteasome offsets parasite resistance to artemisinins in these mutant lines.

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“…3D7xHB3 (43), HB3xDD2 (44), 7G8xGB4 (14), and 803xGB4 (33). In addition to the 3D7 canonical reference genome (12), we generated high-quality draft reference assemblies for the remaining five parental genomes (HB3, DD2, 7G8, GB4, and 803) using PacBio RSII sequencing (Supplemental Table 3).…”

Section: Core Accessory and Repetitive Region Dnm Detection In P Fmentioning

confidence: 99%

Detection of simple and complexde novomutations without, with, or with multiple reference sequences

Garimella

Iqbal

Krause

et al. 2019

Preprint

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The characterization of de novo mutations in regions of high sequence and structural diversity from whole genome sequencing data remains highly challenging. Complex structural variants tend to arise in regions of high repetitiveness and low complexity, challenging both de novo assembly, where short-reads do not capture the long-range context required for resolution, and mapping approaches, where improper alignment of reads to a reference genome that is highly diverged from that of the sample can lead to false or partial calls. Long-read technologies can potentially solve such problems but are currently unfeasible to use at scale. Here we present Corticall, a graph-based method that combines the advantages of multiple technologies and prior data sources to detect arbitrary classes of genetic variant. We construct multi-sample, coloured de Bruijn graphs from shortread data for all samples, align long-read-derived haplotypes and multiple reference data sources to restore graph connectivity information, and call variants using graph path-finding algorithms and a model for simultaneous alignment and recombination. We validate and evaluate the approach using extensive simulations and use it to characterize the rate and spectrum of de novo mutation events in 119 progeny from four Plasmodium falciparum experimental crosses, using long-read data on the parents to inform reconstructions of the progeny and to detect several known and novel non-allelic homologous recombination events.

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Artemisinin resistance phenotypes and K13 inheritance in a Plasmodium falciparum cross and Aotus model

Cited by 48 publications

References 52 publications

Surprising variation in the outcome of two malaria genetic crosses using humanized mice: implications for genetic mapping and malaria biology

Surprising variation in the outcome of two malaria genetic crosses using humanized mice: implications for genetic mapping and malaria biology

Plasmodium bergheiK13 Mutations MediateIn VivoArtemisinin Resistance That is Reversed by Proteasome Inhibition

Detection of simple and complexde novomutations without, with, or with multiple reference sequences

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