Plasmodium berghei K13 mutations mediate in vivo artemisinin resistance that is reversed by proteasome inhibition

Simwela, N., Stokes, B. H., Aghabi, D., Bogyo, M., Fidock, D. A. and Waters, A. (2020) Plasmodium berghei K13 mutations mediate in vivo artemisinin resistance that is reversed by proteasome inhibition. mBio, 11, e02312-20. (doi: 10.1128/mBio.02312-20) (PMID:33173001)

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Abstract

The recent emergence of Plasmodium falciparum parasite resistance to the first line antimalarial drug artemisinin is of particular concern. Artemisinin resistance is primarily driven by mutations in the P. falciparum 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 P. falciparum K13 mutations into the K13 gene of an artemisinin-sensitive Plasmodium berghei rodent model of malaria. Introduction of the orthologous P. falciparum K13 F446I, M476I, Y493H, and R539T mutations into P. berghei K13 yielded gene-edited parasites with reduced susceptibility to dihydroartemisinin in the standard 24-h in vitro assay and increased survival in an adapted in vitro ring-stage survival assay. Mutant P. berghei 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 P. berghei, while the equivalents of the M476I and R539T mutations resulted in significant growth defects. Furthermore, a Plasmodium-selective proteasome inhibitor strongly synergized dihydroartemisinin action in these P. berghei K13 mutant lines, providing further evidence that the proteasome can be targeted to overcome artemisinin resistance. Taken together, our findings provide clear experimental evidence for the involvement of K13 polymorphisms in mediating susceptibility to artemisinins in vitro and, most importantly, under in vivo conditions.

Item Type:Articles
Additional Information:This work was supported in part by grants from the Wellcome Trust to A.P.W. (083811/Z/07/Z, 107046/Z/15/Z, and 104111/Z/14/Z). Partial funding for this work was provided by the NIH (R01 AI109023 to D.A.F. and R33 AI127581 to M.B. and D.A.F.), the Department of Defense (W81XWH-19-1-0086 to D.A.F.), and the Columbia University—University of Glasgow Research Exchange Program. N.V.S. is a Commonwealth Doctoral Scholar (MWCS-2017-789), funded by the UK government. B.H.S. gratefully acknowledges earlier support from the Columbia University Graduate Training Program in Microbiology and Immunology (T32 AI106711; Program Director, D. A. Fidock).
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Simwela, Nelson and Waters, Professor Andy and Aghabi, Ms Dana
Authors: Simwela, N., Stokes, B. H., Aghabi, D., Bogyo, M., Fidock, D. A., and Waters, A.
College/School:College of Medical Veterinary and Life Sciences
College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:mBio
Publisher:American Society for Microbiology
ISSN:2150-7511
ISSN (Online):2150-7511
Copyright Holders:Copyright © 2020 The Authors
First Published:First published in mBio 11:e02312-20
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
143385Conditional translational repression: a core regulatory mechanism of gene expression during development of the malaria parasite.Andrew WatersWellcome Trust (WELLCOTR)083811/Z/07/ZIII - Parasitology
172459Gene expression in Plasmodium parasites: the molecular mechanics of gametocytogenesis (and variant transcription of genes)Andrew WatersWellcome Trust (WELLCOTR)107046/Z/15/ZIII - Parasitology
170547The Wellcome Centre for Molecular Parasitology ( Core Support )Andrew WatersWellcome Trust (WELLCOTR)104111/Z/14/ZRIII - Parasitology