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High-throughput decoding of antitrypanosomal drug efficacy and resistance

Abstract

The concept of disease-specific chemotherapy was developed a century ago. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work1,2, and the drugs that emerged remain in use for treating human African trypanosomiasis (HAT)3. The importance of understanding the mechanisms underlying selective drug action and resistance for the development of improved HAT therapies has been recognized, but these mechanisms have remained largely unknown. Here we use all five current HAT drugs for genome-scale RNA interference target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate antitrypanosomal drug action. RIT-seq profiling identifies both known drug importers4,5 and the only known pro-drug activator6, and links more than fifty additional genes to drug action. A bloodstream stage-specific invariant surface glycoprotein (ISG75) family mediates suramin uptake, and the AP1 adaptin complex, lysosomal proteases and major lysosomal transmembrane protein, as well as spermidine and N-acetylglucosamine biosynthesis, all contribute to suramin action. Further screens link ubiquinone availability to nitro-drug action, plasma membrane P-type H+-ATPases to pentamidine action, and trypanothione and several putative kinases to melarsoprol action. We also demonstrate a major role for aquaglyceroporins in pentamidine and melarsoprol cross-resistance. These advances in our understanding of mechanisms of antitrypanosomal drug efficacy and resistance will aid the rational design of new therapies and help to combat drug resistance, and provide unprecedented molecular insight into the mode of action of antitrypanosomal drugs.

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Figure 1: Identification of drug efficacy determinants in T. brucei.
Figure 2: A network of proteins link ISG75, endocytosis and lysosomal functions to suramin action.
Figure 3: aqp2/aqp3 -null cells are melarsoprol, pentamidine cross-resistant.
Figure 4: Determinants of drug efficacy in African trypanosomes.

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Data deposits

Sequence data from this study have been submitted to the European Nucleotide Archive at http://www.ebi.ac.uk/ena under accession number ERA071064.

References

  1. Ehrlich, P. Address in pathology, on chemiotherapy: delivered before the seventeenth international congress of medicine. BMJ 2, 353–359 (1913)

    CAS  Article  Google Scholar 

  2. Williamson, J. in The African Trypanosomiases (ed. Mulligan, H. W.) (Allen and Unwin, 1970)

    Google Scholar 

  3. Fairlamb, A. H. Chemotherapy of human African trypanosomiasis: current and future prospects. Trends Parasitol. 19, 488–494 (2003)

    CAS  Article  Google Scholar 

  4. Mäser, P., Sutterlin, C., Kralli, A. & Kaminsky, R. A nucleoside transporter from Trypanosoma brucei involved in drug resistance. Science 285, 242–244 (1999)

    Article  Google Scholar 

  5. Vincent, I. M. et al. A molecular mechanism for eflornithine resistance in African trypanosomes. PLoS Pathog. 6, e1001204 (2010)

    Article  Google Scholar 

  6. Wilkinson, S. R., Taylor, M. C., Horn, D., Kelly, J. M. & Cheeseman, I. A mechanism for cross-resistance to nifurtimox and benznidazole in trypanosomes. Proc. Natl Acad. Sci. USA 105, 5022–5027 (2008)

    ADS  CAS  Article  Google Scholar 

  7. Fèvre, E. M., Wissmann, B. V., Welburn, S. C. & Lutumba, P. The burden of human African trypanosomiasis. PLoS Negl. Trop. Dis. 2, e333 (2008)

    Article  Google Scholar 

  8. Pépin, J. & Milord, F. The treatment of human African trypanosomiasis. Adv. Parasitol. 33, 1–47 (1994)

    Article  Google Scholar 

  9. de Koning, H. P. Ever-increasing complexities of diamidine and arsenical crossresistance in African trypanosomes. Trends Parasitol. 24, 345–349 (2008)

    CAS  Article  Google Scholar 

  10. Alsford, S. et al. High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res. 21, 915–924 (2011)

    CAS  Article  Google Scholar 

  11. Carter, N. S. & Fairlamb, A. H. Arsenical-resistant trypanosomes lack an unusual adenosine transporter. Nature 361, 173–176 (1993)

    ADS  CAS  Article  Google Scholar 

  12. Matovu, E. et al. Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei. Eukaryot. Cell 2, 1003–1008 (2003)

    CAS  Article  Google Scholar 

  13. Schumann Burkard, G., Jutzi, P. & Roditi, I. Genome-wide RNAi screens in bloodstream form trypanosomes identify drug transporters. Mol. Biochem. Parasitol. 175, 91–94 (2011)

    CAS  Article  Google Scholar 

  14. Baker, N., Alsford, S. & Horn, D. Genome-wide RNAi screens in African trypanosomes identify the nifurtimox activator NTR and the eflornithine transporter AAT6. Mol. Biochem. Parasitol. 176, 55–57 (2011)

    CAS  Article  Google Scholar 

  15. Steverding, D. The development of drugs for treatment of sleeping sickness: a historical review. Parasit. Vectors 3, 15 (2010)

    Article  Google Scholar 

  16. Overath, P., Chaudhri, M., Steverding, D. & Ziegelbauer, K. Invariant surface proteins in bloodstream forms of Trypanosoma brucei. Parasitol. Today 10, 53–58 (1994)

    CAS  Article  Google Scholar 

  17. Peck, R. F. et al. The LAMP-like protein p67 plays an essential role in the lysosome of African trypanosomes. Mol. Microbiol. 68, 933–946 (2008)

    CAS  Article  Google Scholar 

  18. Leung, K. F., Riley, F. S., Carrington, M. & Field, M. C. Ubiquitylation and developmental regulation of invariant surface protein expression in trypanosomes. Eukaryot. Cell 10, 916–931 (2011)

    CAS  Article  Google Scholar 

  19. Koumandou, V. L. et al. Evolutionary reconstruction of the retromer complex and its function in Trypanosoma brucei. J. Cell Sci. 124, 1496–1509 (2011)

    CAS  Article  Google Scholar 

  20. Caffrey, C. R. et al. Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense. Mol. Biochem. Parasitol. 118, 61–73 (2001)

    CAS  Article  Google Scholar 

  21. Fairlamb, A. H. & Bowman, I. B. Uptake of the trypanocidal drug suramin by bloodstream forms of Trypanosoma brucei and its effect on respiration and growth rate in vivo. Mol. Biochem. Parasitol. 1, 315–333 (1980)

    CAS  Article  Google Scholar 

  22. Vansterkenburg, E. L. et al. The uptake of the trypanocidal drug suramin in combination with low-density lipoproteins by Trypanosoma brucei and its possible mode of action. Acta Trop. 54, 237–250 (1993)

    CAS  Article  Google Scholar 

  23. Scott, A. G., Tait, A. & Turner, C. M. Characterisation of cloned lines of Trypanosoma brucei expressing stable resistance to MelCy and suramin. Acta Trop. 60, 251–262 (1996)

    CAS  Article  Google Scholar 

  24. Natesan, S. K., Peacock, L., Matthews, K., Gibson, W. & Field, M. C. Activation of endocytosis as an adaptation to the mammalian host by trypanosomes. Eukaryot. Cell 6, 2029–2037 (2007)

    CAS  Article  Google Scholar 

  25. Uzcategui, N. L. et al. Cloning, heterologous expression, and characterization of three aquaglyceroporins from Trypanosoma brucei. J. Biol. Chem. 279, 42669–42676 (2004)

    CAS  Article  Google Scholar 

  26. Lanteri, C. A., Tidwell, R. R. & Meshnick, S. R. The mitochondrion is a site of trypanocidal action of the aromatic diamidine DB75 in bloodstream forms of Trypanosoma brucei. Antimicrob. Agents Chemother. 52, 875–882 (2008)

    CAS  Article  Google Scholar 

  27. Luo, S., Fang, J. & Docampo, R. Molecular characterization of Trypanosoma brucei P-type H+-ATPases. J. Biol. Chem. 281, 21963–21973 (2006)

    CAS  Article  Google Scholar 

  28. Fairlamb, A. H., Henderson, G. B. & Cerami, A. Trypanothione is the primary target for arsenical drugs against African trypanosomes. Proc. Natl Acad. Sci. USA 86, 2607–2611 (1989)

    ADS  CAS  Article  Google Scholar 

  29. Morris, J. C., Wang, Z., Drew, M. E. & Englund, P. T. Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi library. EMBO J. 21, 4429–4438 (2002)

    CAS  Article  Google Scholar 

  30. Alsford, S., Kawahara, T., Glover, L. & Horn, D. Tagging a T. brucei RRNA locus improves stable transfection efficiency and circumvents inducible expression position effects. Mol. Biochem. Parasitol. 144, 142–148 (2005)

    CAS  Article  Google Scholar 

  31. Berriman, M. et al. The genome of the African trypanosome Trypanosoma brucei. Science 309, 416–422 (2005)

    ADS  CAS  Article  Google Scholar 

  32. Ning, Z., Cox, A. J. & Mullikin, J. C. SSAHA: a fast search method for large DNA databases. Genome Res. 11, 1725–1729 (2001)

    CAS  Article  Google Scholar 

  33. Carver, T. et al. Artemis and ACT: viewing, annotating and comparing sequences stored in a relational database. Bioinformatics 24, 2672–2676 (2008)

    CAS  Article  Google Scholar 

  34. Buschini, A. et al. Genotoxicity revaluation of three commercial nitroheterocyclic drugs: nifurtimox, benznidazole, and metronidazole. J. Parasitol. Res. 2009, 463575 (2009)

    Article  Google Scholar 

  35. Redmond, S., Vadivelu, J. & Field, M. C. RNAit: an automated web-based tool for the selection of RNAi targets in Trypanosoma brucei. Mol. Biochem. Parasitol. 128, 115–118 (2003)

    CAS  Article  Google Scholar 

  36. Alsford, S. & Horn, D. Single-locus targeting constructs for reliable regulated RNAi and transgene expression in Trypanosoma brucei. Mol. Biochem. Parasitol. 161, 76–79 (2008)

    CAS  Article  Google Scholar 

  37. Mackey, Z. B., O'Brien, T. C., Greenbaum, D. C., Blank, R. B. & McKerrow, J. H. A cathepsin B-like protease is required for host protein degradation in Trypanosoma brucei. J. Biol. Chem. 279, 48426–48433 (2004)

    CAS  Article  Google Scholar 

  38. Singh, P. K., Tack, B. F., McCray, P. B., Jr & Welsh, M. J. Synergistic and additive killing by antimicrobial factors found in human airway surface liquid. Am. J. Physiol. Lung Cell. Mol. Physiol. 279, L799–L805 (2000)

    CAS  Article  Google Scholar 

  39. Räz, B., Iten, M., Grether-Buhler, Y., Kaminsky, R. & Brun, R. The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T. b. rhodesiense and T. b. gambiense) in vitro.. Acta Trop. 68, 139–147 (1997)

    Article  Google Scholar 

  40. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K., eds. Current Protocols in Molecular Biology (John Wiley and Sons, 1998).

  41. Leung, K. F., Dacks, J. B. & Field, M. C. Evolution of the multivesicular body ESCRT machinery; retention across the eukaryotic lineage. Traffic 9, 1698–1716 (2008)

    CAS  Article  Google Scholar 

  42. Ziegelbauer, K. & Overath, P. Organization of two invariant surface glycoproteins in the surface coat of Trypanosoma brucei. Infect. Immun. 61, 4540–4545 (1993)

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Kelley, R. J., Brickman, M. J. & Balber, A. E. Processing and transport of a lysosomal membrane glycoprotein is developmentally regulated in African trypanosomes. Mol. Biochem. Parasitol. 74, 167–178 (1995)

    CAS  Article  Google Scholar 

  44. Lingnau, A., Zufferey, R., Lingnau, M. & Russell, D. G. Characterization of tGLP-1, a Golgi and lysosome-associated, transmembrane glycoprotein of African trypanosomes. J. Cell Sci. 112, 3061–3070 (1999)

    CAS  PubMed  Google Scholar 

  45. Allen, C. L., Liao, D., Chung, W. L. & Field, M. C. Dileucine signal-dependent and AP-1-independent targeting of a lysosomal glycoprotein in Trypanosoma brucei. Mol. Biochem. Parasitol. 156, 175–190 (2007)

    CAS  Article  Google Scholar 

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Acknowledgements

We thank J. Morris, Z. Wang, M. Drew and P. Englund for the RNAi plasmid library, V. Yardley for antitrypanosomal drugs, J. Bangs for anti-p67 and CatL sera, D. Russell for anti-GLP1 sera, A. Varghese for assistance with preliminary Sanger sequencing and J. Kelly, M. Taylor and B. Wren for comments on the draft manuscript. The work was funded by grants from The Wellcome Trust (093010/Z/10/Z at the London School of Hygiene & Tropical Medicine, 085775/Z/08/Z at The Wellcome Trust Sanger Institute and 090007/Z/09/Z at The University of Cambridge). N.B. was supported by a Bloomsbury colleges PhD studentship.

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Contributions

S.A., N.B., L.G. and K.F.L. carried out the T. brucei manipulation and analyses, S.E., A.S.-F. and D.J.T. carried out the Illumina sequencing and mapping, D.H. coordinated the study and S.A., M.C.F., M.B. and D.H. wrote the paper.

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Correspondence to David Horn.

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Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-6 with legends, Supplementary Table 1 and additional references. Growth curves, sequence read-density signatures, EC50 data and comparative genomic information and HAT drug information are included. (PDF 497 kb)

Supplementary Data 1

This file shows two spreadsheets. The first (a) shows all 'primary' and 'secondary' hits from the HAT drug resistance screens with comments and links to databases. The second (b) shows all genes associated with >9 sequence reads in the HAT drug resistance screens. (XLS 362 kb)

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Alsford, S., Eckert, S., Baker, N. et al. High-throughput decoding of antitrypanosomal drug efficacy and resistance. Nature 482, 232–236 (2012). https://doi.org/10.1038/nature10771

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