Table 1. Sensitivity of WT and DKO to DHFR–TS and PTR1 inhibitors. Inhibitor Methotrexate Aminopterin Raltitrexed Pemetrexed Pyrimethamine Trimetrexate 5-Fluoro-orotic acid O/129b Triamterene EC50 HMI9 (mM) 2.5 15.1 22.4 1.8 17.2 1.2 2.8 16.5 48.3 0.2 (3)a 2.3 (2) 2.1 (3) 0.1 (2) 2.7 (3) 0.1 (3) 0.3 (3) 1.7 (3) 5.1 (2) WT EC50 FDM (mM) 0.037 0.049 0.072 1.9 9.6 3.2 1.4 7.4 45.2 0.007 (2) 0.009 (3) 0.009 (3) 0.3 (2) 1.3 (2) 0.3 (3) 0.3 (3) 1.2 (2) 3.6 (2) Ratio EC50 68 308 311 0.9 1.8 0.4 1.6 2.2 1.1 EC50 HMI9 (mM) 17.9 6.5 (2) 78.5 5.9 (2) > 800 (2) > 800 (2) 35.6 4.5 (2) 4.1 0.6 (3) 4.2 0.7 (2) 17.9 2.1 (3) 15.9 1.1 (2) Validation of DHFR–TS as a drug target 525 DKO EC50 FDM (mM) 22.2 5.2 (2) 75.1 6.2 (2) > 800 (2) > 800 (2) 27.6 2.6 (2) 5.6 0.8 (2) 2.5 0.5 (3) 9.7 1.1 (3) 58.9 7.8 (2) Ratio EC50 0.8 1.0 - - 1.3 0.7 1.7 1.8 0.3 Ratio DKO/WT HMI9 7.2 5.2 > 36 > 444 2.1 1.9 1.9 0.9 0.3 FDM 484 1 533 > 11 111 > 421 2.9 1.8 1.7 1.3 1.3 a. n = number of independent experiments, where EC50 values are weighted means and standard errors of each independent determination. b. 2,4-Diamino-6,7-diisopropylpteridine. than WT to these same inhibitors in low-folate medium. Growth inhibition by the thymidylate synthase inhibitors, raltitrexed and pemetrexed, was completely abolished in DKO parasites, showing up to 11 000-fold resistance compared with WT. There was no marked change in sensitivity towards the lipophilic DHFR inhibitors pyrimethamine, trimetrexate or to 5-ﬂuoro-oroate when compared across media or cell type. Likewise, the known PTR1 inhibitors belonging to the 2,4-diaminopteridine class, O/129 and triamterene, were also unchanged. Can endogenous PTR1 compensate for lack of DHFR? PTR1 is known to reduce a broad spectrum of pterins and folates (Dawson et al., 2006), raising the intriguing possi- bility that it may compensate for changes in DHFR activity in SKO and DKO lines. PTR1 levels in each cell line were analysed for RNA and protein content by Northern and Western blotting relative to suitable controls (Fig. 6). Den- sitometry of RNA blots indicated that the absolute ratio of DHFR–TS in WT, SKO and DKO is 1:0.5:0 (Fig. 6A). Using INO1 [1-D-myo-inositol-3-phosphate synthase (Martin and Smith, 2005)] as a loading control (Fig. 6C), the corrected ratios are 1:0.6:0. PTR1 transcripts are only marginally increased in SKO and DKO cell lines (1:1.1:1.1 respectively; Fig. 6B). After correction with INO1, the corresponding values are 1:1.4:1.1. Protein blots were probed with polyclonal antisera raised against T. brucei PTR1 (Fig. 6D) and the endoplasmic reticulum chaperone BiP (Fig. 6E). PTR1 levels are equal for all three cell lines, with expression levels relative to BiP being 1:0.9:0.9 for WT, SKO and DKO respectively. Although PTR1 levels are unchanged in the knockout lines, it is possible that endogenous activity is nonethe- less sufficient to compensate for the loss of DHFR activity. To determine whether the thymidylate cycle could be sus- tained by endogenous PTR1, we constructed a recovery © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 69, 520–533 plasmid containing the thymidylate synthase gene from Escherichia coli. This plasmid (pLew82_EcTS) was able to complement E. coli thyA - cells grown on thymidine- deﬁcient medium indicating that TS was functional (Fig. 7A). This expression construct was transfected into DKO cells and lines selected for phleomycin resistance in HMI9 medium. Integration of the EcTS in the conditional DKO (cDKOTS) cells at the correct locus was conﬁrmed by Southern blot analysis using the EcTS as a probe (Fig. 7B). Growth was monitored in HMI9 (with and without thymidine and/or tetracycline) to determine whether the addition of TS alone could maintain the Fig. 6. Northern and Western blot analysis of WT, SKO and DKO cells. Trypanosomes were grown for 24 h in HMI9 medium in the absence (WT and SKO) or presence of thymidine (DKO). A–C. RNA blots probed with TbDHFR–TS, TbPTR1 and TbINO1 respectively. RNA size markers are indicated to the left of the panels. D and E. Protein blots of parasite lysates probed with antiserum to PTR1 and BiP respectively. Approximately 20 mg of protein was loaded per lane. Standard size markers are indicated to the right of the panels.