Validation of DHFR–TS as a drug target 521 Fig. 1. Pathway of thymidylate synthesis and primary site of action of inhibitors. SHMT, serine hydroxymethyltransferase; DHFR, dihydrofolate reductase; TS, thymidylate synthase; O/129, 2,4-diamino-6,7-diispropylpteridine; PTR1, pteridine reductase 1; TK, thymidine kinase; H2F, dihydrofolate; H4F, tetrahydrofolate; CH2-CH4F, 5, 10-methylene-tetrahydrofolate; FdUMP, 5-ﬂuorodeoxyuridylate; GCS, glycine cleavage system. (Nare et al., 1997a; Linares et al., 2006). Although the reason for this is not at all clear for African trypanosomes, in Leishmania spp., pteridine reductase (PTR1; EC 1.5.1.33) may serve to modulate or by-pass inhibition of DHFR by classical inhibitors such as methotrexate (Nare et al., 1997a). Gene knockout studies in avirulent lines of Leishmania major have shown that mutants of DHFR–TS can be readily generated when supplemented with thymidine (Cruz and Beverley, 1990; Cruz et al., 1991). In contrast, virulent Leishmania show plasticity in chromosome number in order to maintain at least one copy of DHFR–TS (Cruz et al., 1993). Thus, DHFR–TS may have another unidentiﬁed function in virulent lines in addition to de novo synthesis of thymidine in avirulent lines. Whether this requires DHFR, TS or both proteins is not clear. Neither is it clear whether endogenous PTR1 activity is sufficient to replace DHFR in the thymidylate cycle. Very little is known about folate metabolism in African trypanosomes. Comparative genomics indicates that Trypanosoma brucei lacks a number of genes in folate- dependent pathways that are present in L. major indicat- ing that extrapolation from L. major to T. brucei may not be straightforward (Berriman et al., 2005). Moreover, from a drug discovery perspective, although T. brucei possess both DHFR–TS and PTR1 (Gamarro et al., 1995; Dawson et al., 2006), these enzymes show signiﬁcant differences in structure and/or sensitivity to inhibitors to their Leish- mania counterparts (Meek et al., 1985; Hardy et al., 1997; Luba et al., 1998; Chowdhury et al., 1999; Gourley et al., 1999; 2001). In this study we use genetic and chemical methods to examine the roles of DHFR–TS in T. brucei and assess its potential as a drug target. We also examine the sensitivity of T. brucei bloodstream forms to known DHFR–TS inhibitors in a novel culture medium containing physiological levels of folate to assess the robustness of the currently accepted standard method for whole cell phenotypic screening of antifols (Raz et al., 1997). Results Generation of knockout mutants To assess the essentiality of DHFR–TS, bloodstream- form trypanosomes were transfected with linear con- structs containing the drug resistance genes, puromycin N-acetyl transferase (PAC) or hygromycin phosphotrans- ferase (HYG) ﬂanked by the 3′- and 5′-untranslated regions (3′-UTR and 5′-UTR respectively) of DHFR–TS. Stable drug-resistant lines were obtained after selection with either puromycin or hygromycin in HMI9 medium. This culture medium contains 160 mM thymidine which serves as a nutritional by-pass for loss of DHFR–TS activity. The resulting single-knockout (SKO) line contain- ing PAC was then transfected with the HYG knockout construct and selected for resistance to puromycin and hygromycin to obtain a double-knockout (DKO) line. A Southern blot of a restriction enzyme digest of genomic DNA from wild type (WT) (DHFR–TS/DHFR–TS), SKO (DHFR–TS/dhfr–ts::PAC) and DKO (dhfr–ts::PAC/dhfr– ts::HYG) conﬁrmed that both drug selectable markers had correctly integrated into the S427 genome via homolo- gous recombination at the DHFR–TS locus (Fig. 2). Effects of folate or thymidine on growth Cell lines were continuously passaged in various media to determine growth phenotypes. In HMI9 medium (Fig. 3A), WT and SKO cells grew at similar rates with doubling times of 14.0 0.1 and 14.5 0.1 h respectively. The DKO cell line grew at a decreased rate in the presence of puromycin plus hygromycin with a generation time of © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 69, 520–533