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UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes

Nature Microbiology volume 1, Article number: 16166 (2016) Cite this article

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Abstract

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.

Malaria is a global health menace, with 3.2 billion people at risk of infection and approximately 438,000 deaths in 2015 (ref. 1). In recent years, mounting evidence of resistance to the artemisinin component of artemisinin-based combination therapies (ACTs), the current standard of care for Plasmodium falciparum malaria, has been observed2,3. As the emergence of artemisinin resistance threatens recent success in malaria control, it is critical that alternative drugs are developed.

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Figure 1: Selection of imidazolopiperazine-resistant mutants.
Figure 2: Characterization of resistance mutations.
Figure 3: CRISPR/Cas9 mutation validation and localization of PfUGT and PfACT.
Figure 4: GNF179-resistant mutants display poor fitness.
Figure 5: Cross resistance against a panel of imidazolopiperazine analogues and unrelated antimalarial compounds.

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Acknowledgements

M.Y.L. is supported by the Economic Development Board—Industrial Postgraduate Programme (EDB-IPP) scholarship. G.L. is supported by an A.P. Giannini Post-Doctoral Fellowship. Work at UCSD was supported by grants from the National Institutes of Health (NIH; R01 AI090141 and R01 AI103058) to E.A.W. D.A.F. acknowledges support from the Medicines for Malaria Venture. B.M. and L.R. are supported by the Singapore Immunology Network under the Agency for Science, Technology and Research (A*STAR, Singapore). R.W. is a Research Associate at the National Fund for Scientific Research FNRS-FRS (Belgium). The authors thank C. Jensen (Leiden University Medical Center, Netherlands) for the donation of the P. berghei ANKA strain.

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Author notes

  1. Michelle Yi-Xiu Lim and Gregory LaMonte: These authors contributed equally to this work.

Authors and Affiliations

  1. Novartis Institute for Tropical Diseases, 138670, Singapore

    Michelle Yi-Xiu Lim, Bee Huat Tan, Bianca F. Tjahjadi, Adeline Chua, Peter Gedeck, Ghislain M. C. Bonamy, Bryan K. S. Yeung, Liting Lim, Thierry T. Diagana & Pablo Bifani

  2. Department of Pharmacy, Faculty of Science, National University of Singapore, 119077, Singapore

    Michelle Yi-Xiu Lim & Paul Chi-Lui Ho

  3. Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, 92093, California, USA

    Gregory LaMonte, Christin Reimer, Victoria Corey, Marie Nachon & Elizabeth A. Winzeler

  4. Department of Microbiology & Immunology, Columbia University Medical Center, New York, 10032, New York, USA

    Marcus C. S. Lee & David A. Fidock

  5. Malaria Programme, Wellcome Trust Sanger Institute, Wellcome Genome Campus, CB10 1SA, Cambridge, UK

    Marcus C. S. Lee

  6. Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 117545, Singapore

    Bianca F. Tjahjadi, Benoit Malleret, Thierry T. Diagana & Pablo Bifani

  7. Laboratory of Biopolymers and Supramolecular Nanomaterials, Faculty of Pharmacy, Université Libre de Bruxelles, Brussels, Belgium

    René Wintjens

  8. Singapore Immunology Network (SIgN), A*Star, 138648, Singapore

    Benoit Malleret & Laurent Renia

  9. Department of Biochemistry and Biophysics, Center for Advanced Technology, University of California, San Francisco, 94143, California, USA

    Eric D. Chow

  10. Division of Infectious Diseases, Department of Medicine, Columbia University Medical Center, New York, 10032, New York, USA

    David A. Fidock

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  1. Michelle Yi-Xiu Lim
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Contributions

M.Y.-X.L., G.L., M.C.S.L., E.A.W. and P.B. designed the experiments. M.Y.-X.L., G.L., M.C.S.L., C.R., B.H.T., V.C., B.F.T., A.C., M.N., B.M., E.D.C. and L.L. performed the experiments. Modelling work was performed by R.W. M.Y.-X.L., G.L., M.C.S.L., C.R., V.C., M.N., E.D.C. and P.G. analysed the data. G.M.C.B., P.C.-L.H., L.R., D.A.F. and T.T.D. contributed support. M.Y.-X.L., G.L., M.C.S.L., B.K.S.Y., D.A.F., E.A.W. and P.B. wrote and proofread the manuscript. E.A.W. and P.B. gave technical support and conceptual advice. The manuscript was edited by all authors.

Corresponding authors

Correspondence to Elizabeth A. Winzeler or Pablo Bifani.

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Supplementary Tables 1–3, Supplementary Figures 1–4 (PDF 1035 kb)

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Lim, MX., LaMonte, G., Lee, M. et al. UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes. Nat Microbiol 1, 16166 (2016). https://doi.org/10.1038/nmicrobiol.2016.166

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