Acyl-CoA binding protein is essential in bloodstream form Trypanosoma brucei. Mol Biochem Parasitol 112: 301– 304. Nare, B., Luba, J., Hardy, L.W., and Beverley, S.M. (1997a) New approaches to Leishmania chemotherapy: pteridine reductase 1 (PTR1) as a target and modulator of antifolate sensitivity. Parasitol 114: S101–S110. Nare, B., Hardy, L.W., and Beverley, S.M. (1997b) The roles of pteridine reductase 1 and dihydrofolate reductase- thymidylate synthase in pteridine metabolism in the protozoan parasite Leishmania major. J Biol Chem 272: 13883–13891. Nirmalan, N., Sims, P.F.G., and Hyde, J.E. (2004) Transla- tional up-regulation of antifolate drug targets in the human malaria parasite Plasmodium falciparum upon challenge with inhibitors. Mol Biochem Parasitol 136: 63–70. Ouellette, M., Drummelsmith, J., El Fadili, A., Kundig, C., Richard, D., and Roy, G. (2002) Pterin transport and metabolism in Leishmania and related trypanosomatid parasites. Int J Parasitol 32: 385–398. Rathod, P.K., and Reshmi, S. (1994) Susceptibility of Plas- modium falciparum to a combination of thymidine and ICI D1694, a quinazoline antifolate directed at thymidylate synthase. Antimicrob Agents Chemother 38: 476–480. Rathod, P.K., Khatri, A., Hubbert, T., and Milhous, W.K. (1989) Selective activity of 5-ﬂuoroorotic acid against Plasmodium falciparum in vitro. Antimicrob Agents Chemother 33: 1090–1094. Raz, B., Iten, M., Grether-Buhler, Y., Kaminsky, R., and Brun, R. (1997) 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. Roper, J.R., Guther, M.L.S., Milne, K.G., and Ferguson, M.A.J. (2002) Galactose metabolism is essential for the African sleeping sickness parasite Trypanosoma brucei. Proc Natl Acad Sci USA 99: 5884–5889. Senkovich, O., Bhatia, V., Garg, N., and Chattopadhyay, D. (2005) Lipophilic antifolate trimetrexate is a potent inhibitor of Trypanosoma cruzi: prospect for chemotherapy of Chagas’ disease. Antimicrob Agents Chemother 49: 3234– 3238. Tinkelenberg, B.A., Hansbury, M.J., and Ladner, R.D. (2002) dUTPase and uracil-DNA glycosylase are central modula- tors of antifolate toxicity in Saccharomyces cerevisiae. Cancer Res 62: 4909–4915. Titus, R.G., Gueiros-Filho, F.J., De Freitas, L.A.R., and Beverley, S.M. (1995) Development of a safe live Leish- mania vaccine line by gene replacement. Proc Natl Acad Sci USA 92: 10267–10271. Validation of DHFR–TS as a drug target 533 Tu, X., and Wang, C.C. (2005) Coupling of posterior cytosk- eletal morphogenesis to the G1/S transition in the Trypa- nosoma brucei cell cycle. Mol Biol Cell 16: 97–105. Van Triest, B., Pinedo, H.M., Giaccone, G., and Peters, G.J. (2000) Downstream molecular determinants of response to 5-ﬂuorouracil and antifolate thymidylate synthase inhibitors. Ann Oncology 11: 385–391. Veras, P.S.T., Brodskyn, C.I., Balestieri, F.M.P., De Freitas, L.A.R., Ramos, A.P.S., Queiroz, A.R.P., et al. (1999) A dhfr–ts –Leishmania major knockout mutant cross-protects against Leishmania amazonensis. Mem Inst Oswaldo Cruz 94: 491–496. Wang, Y.H., Zhao, R.B., and Goldman, I.D. (2003) Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line. Biochem Pharmacol 65: 1163–1170. Wilson, K., Berens, R.L., Sifri, C.D., and Ullman, B. (1994) Ampliﬁcation of the inosinate dehydrogenase gene in Try- panosoma brucei gambiense due to an increase in chro- mosome copy number. J Biol Chem 269: 28979–28987. van der Wilt, C.L., Backus, H.H.J., Smid, K., Comijn, L., Veerman, G., Wouters, D., et al. (2001) Modulation of both endogenous folates and thymidine enhance the therapeu- tic efficacy of thymidylate synthase inhibitors. Cancer Res 61: 3675–3681. Wirtz, E., Leal, S., Ochatt, C., and Cross, G.A.M. (1999) A tightly regulated inducible expression system for condi- tional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol Biochem Parasitol 99: 89–101. Wyllie, S., and Fairlamb, A.H. (2006) Differential toxicity of antimonial compounds and their effects on glutathione homeostasis in a human leukaemia monocyte cell line. Biochem Pharmacol 71: 257–267. Supplementary material This material is available as part of the online article from: http://www.blackwell-synergy.com/doi/abs/10.1111/ j.1365-2958.2008.06305.x (This link will take you to the article abstract). Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 69, 520–533