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

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.

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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.

References

  1. 1

    WHO. World Malaria Report 2015 1–280 (WHO Press, 2015).

  2. 2

    Dondorp, A. M. Artemisinin resistance in Plasmodium falciparum malaria. N. Engl. J. Med. 361, 455–467 (2009).

    CAS  Article  Google Scholar 

  3. 3

    Ashley, E. A. et al. Spread of artemisinin resistance in Plasmodium falciparum malaria. N. Engl. J. Med. 371, 411–423 (2014).

    Article  Google Scholar 

  4. 4

    Nagle, A. et al. Imidazolopiperazines: lead optimization of the second-generation antimalarial agents. J. Med. Chem. 55, 4244–4273 (2012).

    CAS  Article  Google Scholar 

  5. 5

    Wu, T. et al. Imidazolopiperazines: hit to lead optimization of new antimalarial agents. J. Med. Chem. 54, 5116–5130 (2011).

    CAS  Article  Google Scholar 

  6. 6

    Leong, F. J. et al. A first-in-human randomized, double-blind, placebo-controlled, single- and multiple-ascending oral dose study of novel imidazolopiperazine KAF156 to assess its safety, tolerability, and pharmacokinetics in healthy adult volunteers. Antimicrob. Agents Chemother. 58, 6437–6443 (2014).

    Article  Google Scholar 

  7. 7

    Kuhen, K. L. et al. KAF156 is an antimalarial clinical candidate with potential for use in prophylaxis, treatment, and prevention of disease transmission. Antimicrob. Agents Chemother. 58, 5060–5067 (2014).

    Article  Google Scholar 

  8. 8

    Ding, X. C., Ubben, D. & Wells, T. N. A framework for assessing the risk of resistance for anti-malarials in development. Malaria J. 11, 292 (2012).

    Article  Google Scholar 

  9. 9

    Jimenez-Diaz, M. B. et al. (+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium. Proc. Natl Acad. Sci. USA 111, E5455–E5462 (2014).

    CAS  Article  Google Scholar 

  10. 10

    Baragana, B. et al. A novel multiple-stage antimalarial agent that inhibits protein synthesis. Nature 522, 315–320 (2015).

    CAS  Article  Google Scholar 

  11. 11

    Flannery, E. L., Fidock, D. A. & Winzeler, E. A. Using genetic methods to define the targets of compounds with antimalarial activity. J. Med. Chem. 56, 7761–7771 (2013).

    CAS  Article  Google Scholar 

  12. 12

    Meister, S. et al. Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery. Science 334, 1372–1377 (2011).

    CAS  Article  Google Scholar 

  13. 13

    LaMonte, G. et al. Mutations in the Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL) confer multidrug resistance. mBio 7, e00696-16 (2016).

    Article  Google Scholar 

  14. 14

    Martin, R. E., Henry, R. I., Abbey, J. L., Clements, J. D. & Kirk, K. The ‘permeome’ of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum. Genome Biol. 6, R26 (2005).

    Article  Google Scholar 

  15. 15

    Valderramos, S. G. & Fiddock, D. A. Transporters involved in resistance to antimalarial drugs. Trends Pharmacol. Sci. 27, 594–601 (2006).

    CAS  Article  Google Scholar 

  16. 16

    Ecker, A., Lehane, A. M., Clain, J. & Fidock, D. A. PfCRT and its role in antimalarial drug resistance. Trends Parasitol. 28, 504–514 (2012).

    CAS  Article  Google Scholar 

  17. 17

    Johnson, J. D. et al. Assessment and continued validation of the malaria SYBR green I-based fluorescence assay for use in malaria drug screening. Antimicrob. Agents Chemother. 51, 1926–1933 (2007).

    CAS  Article  Google Scholar 

  18. 18

    Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567–580 (2001).

    CAS  Article  Google Scholar 

  19. 19

    Yan, N. Structural biology of the major facilitator superfamily transporters. Annu. Rev. Biophys. 44, 257–283 (2015).

    CAS  Article  Google Scholar 

  20. 20

    Pedersen, B. P. et al. Crystal structure of a eukaryotic phosphate transporter. Nature 496, 533–536 (2013).

    CAS  Article  Google Scholar 

  21. 21

    Quistgaard, E. M., Low, C., Guettou, F. & Nordlund, P. Understanding transport by the major facilitator superfamily (MFS): structures pave the way. Nat. Rev. Mol. Cell Biol. 17, 123–132 (2016).

    CAS  Article  Google Scholar 

  22. 22

    Lee, M. C. & Fidock, D. A. CRISPR-mediated genome editing of Plasmodium falciparum malaria parasites. Genome Med. 6, 63 (2014).

    Article  Google Scholar 

  23. 23

    Ginsburg, H. Malaria Parasite Metabolic Pathways (2015), http://mpmp.huji.ac.il/maps/ERGolgiVacuole.html

  24. 24

    Lee, M. C., Moura, P. A., Miller, E. A. & Fidock, D. A. Plasmodium falciparum Sec24 marks transitional ER that exports a model cargo via a diacidic motif. Mol. Microbiol. 68, 1535–1546 (2008).

    CAS  Article  Google Scholar 

  25. 25

    Hayakawa, Y. et al. Structure of tyroscherin, an antitumor antibiotic against IGF-1-dependent cells from Pseudallescheria sp. J. Antibiot. (Tokyo) 57, 634–638 (2004).

    CAS  Article  Google Scholar 

  26. 26

    Hediger, M. A. et al. The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteins. Pflugers Arch. 447, 465–468 (2004).

    CAS  Article  Google Scholar 

  27. 27

    Prasad, R. & Rawal, M. K. Efflux pump proteins in antifungal resistance. Front. Pharmacol. 5, 202 (2014).

    Article  Google Scholar 

  28. 28

    Kumar, S. et al. Bacterial multidrug efflux pumps of the major facilitator superfamily as targets for modulation. Infect. Disord. Drug Targets 16, 28–43 (2016).

    CAS  Article  Google Scholar 

  29. 29

    Dos Santos, S. C., Teixeira, M. C., Dias, P. J. & Sa-Correia, I. MFS transporters required for multidrug/multixenobiotic (MD/MX) resistance in the model yeast understanding their physiological function through post-genomic approaches. Front. Physiol. 5, 180 (2014).

    Article  Google Scholar 

  30. 30

    Aurrecoechea, C. et al. PlasmoDB: a functional genomic database for malaria parasites. Nucleic Acids Res. 37, D539–D543 (2009).

    CAS  Article  Google Scholar 

  31. 31

    Perlin, M. H., Andrews, J. & Toh, S. S. Essential letters in the fungal alphabet: ABC and MFS transporters and their roles in survival and pathogenicity. Adv. Genet. 85, 201–253 (2014).

    CAS  Article  Google Scholar 

  32. 32

    Jack, D. L., Yang, N. M. & Saier, M. H. Jr. The drug/metabolite transporter superfamily. Eur. J. Biochem. 268, 3620–3639 (2001).

    CAS  Article  Google Scholar 

  33. 33

    Tran, C. V. & Saier, M. H. Jr. The principal chloroquine resistance protein of Plasmodium falciparum is a member of the drug/metabolite transporter superfamily. Microbiology 150, 1–3 (2004).

    CAS  Article  Google Scholar 

  34. 34

    Martin, R. E. & Kirk, K. The malaria parasite's chloroquine resistance transporter is a member of the drug/metabolite transporter superfamily. Mol. Biol. Evol. 21, 1938–1949 (2004).

    CAS  Article  Google Scholar 

  35. 35

    Ng, B. G. et al. Mosaicism of the UDP-galactose transporter SLC35A2 causes a congenital disorder of glycosylation. Am. J. Hum. Gent. 92, 632–636 (2013).

    CAS  Article  Google Scholar 

  36. 36

    Sprong, H. et al. Association of the Golgi UDP-galactose transporter with UDP-galactose:ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum. Mol. Biol. Cell 14, 3482–3493 (2003).

    CAS  Article  Google Scholar 

  37. 37

    Dorre, K. et al. A new case of UDP-galactose transporter deficiency (SLC35A2-CDG): molecular basis, clinical phenotype, and therapeutic approach. J. Inherit. Metab. Dis. 38, 931–940 (2015).

    CAS  Article  Google Scholar 

  38. 38

    Kanamori, A. et al. Expression cloning and characterization of a cDNA encoding a novel membrane protein required for the formation of O-acetylated ganglioside: a putative acetyl-CoA transporter. Proc. Natl Acad. Sci. USA 94, 2897–2902 (1997).

    CAS  Article  Google Scholar 

  39. 39

    Varki, A. & Diaz, S. The transport and utilization of acetyl coenzyme A by rat liver Golgi vesicles. O-acetylated sialic acids are a major product. J. Biol. Chem. 260, 6600–6608 (1985).

    CAS  PubMed  Google Scholar 

  40. 40

    Jonas, M. C., Pehar, M. & Puglielli, L. AT-1 is the ER membrane acetyl-CoA transporter and is essential for cell viability. J. Cell Sci. 123, 3378–3388 (2010).

    CAS  Article  Google Scholar 

  41. 41

    Hirabayashi, Y., Nomura, K. H. & Nomura, K. The acetyl-CoA transporter family SLC33. Mol. Aspects Med. 34, 586–589 (2013).

    CAS  Article  Google Scholar 

  42. 42

    McNamara, C. W. et al. Targeting Plasmodium PI(4)K to eliminate malaria. Nature 504, 248–253 (2013).

    CAS  Article  Google Scholar 

  43. 43

    Walch-Solimena, C. & Novick, P. The yeast phosphatidylinositol-4-OH kinase pik1 regulates secretion at the Golgi. Nat. Cell Biol. 1, 523–525 (1999).

    CAS  Article  Google Scholar 

  44. 44

    Kruger, T., Sanchez, C. P. & Lanzer, M. Complementation of Saccharomyces cerevisiae Pik1ts by a phosphatidylinositol 4-kinase from Plasmodium falciparum. Mol. Biochem. Parasitol. 172, 149–151 (2010).

    Article  Google Scholar 

  45. 45

    Roemer, T. et al. Confronting the challenges of natural product-based antifungal discovery. Chem. Biol. 18, 148–164 (2011).

    CAS  Article  Google Scholar 

  46. 46

    Goodman, C. D. et al. Parasites resistant to the antimalarial atovaquone fail to transmit by mosquitoes. Science 352, 349–353 (2016).

    CAS  Article  Google Scholar 

  47. 47

    Trager, W. & Jensen, J. B. Human malaria parasites in continuous culture. Science 193, 673–675 (1979).

    Article  Google Scholar 

  48. 48

    Fidock, D. A., Rosenthal, P. J., Croft, S. L., Brun, R. & Nwaka, S. Antimalarial drug discovery: efficacy models for compound screening. Nat. Rev. Drug Discov. 3, 509–520 (2004).

    CAS  Article  Google Scholar 

  49. 49

    Kariuki, M. M. et al. Plasmodium falciparum: purification of the various gametocyte developmental stages from in vitro-cultivated parasites. Am. J. Trop. Med. Hyg. 59, 505–508 (1998).

    CAS  Article  Google Scholar 

  50. 50

    Manary, M. J. et al. Identification of pathogen genomic variants through an integrated pipeline. BMC Bioinformatics 15, 63 (2014).

    Article  Google Scholar 

  51. 51

    Ng, C. L. et al. CRISPR-Cas9-modified pfmdr1 protects Plasmodium falciparum asexual blood stages and gametocytes against a class of piperazine-containing compounds but potentiates artemisinin-based combination therapy partner drugs. Mol. Microbiol. 101, 381–393 (2016).

    CAS  Article  Google Scholar 

  52. 52

    Malleret, B. et al. A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development. Sci. Rep. 1, 118 (2011).

    Article  Google Scholar 

  53. 53

    Nkrumah, L. J. et al. Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase. Nat. Methods 3, 615–621 (2006).

    CAS  Article  Google Scholar 

  54. 54

    Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

    CAS  Article  Google Scholar 

  55. 55

    Fiser, A. & Sali, A. Modeller: generation and refinement of homology-based protein structure models. Methods Enzymol. 374, 461–491 (2003).

    CAS  Article  Google Scholar 

  56. 56

    Kraulis, P. J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  57. 57

    Merritt, E. A. & Bacon, D. J. Raster3D Photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997).

    CAS  Article  Google Scholar 

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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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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.

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Correspondence to Elizabeth A. Winzeler or Pablo Bifani.

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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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