528 N. Sienkiewicz, S. Jarosławski, S. Wyllie and A. H. Fairlamb in DKO cells and the fact that genetic rescue can be achieved by ectopic expression of TbDHFR–TS, but not with functionally active EcTS, suggests that PTR1 is unable to compensate for complete loss of DHFR activity. PTR1 from T. brucei is fourfold less efficient in reducing dihydrofolate than the L. major enzyme and is less sensi- tive to inhibition by methotrexate (Dawson et al., 2006). However, it is pertinent to note that DHFR mutants in E. coli are always associated with compensatory muta- tions in ThyA to maintain the delicate balance between formation and removal of dihydrofolate by TS and DHFR respectively (Ahrweiler and Frieden, 1988; Howell et al., 1988). Thus, the failure to restore viability with EcTS could be due to an imbalance between PTR1 and TS activity, which is 10-fold higher than in WT cells. Moreover, although DHFR in T. brucei may be a drug target per se without recourse to inhibiting PTR1, ampliﬁcation of PTR1 could represent a potential resistance mechanism to DHFR inhibitors, as observed in Leishmania spp. (Bello et al., 1994; Nare et al., 1997b; Ouellette et al., 2002). However, unlike Leishmania spp., gene ampliﬁcation in African trypanosomes is extremely rare. To our knowl- edge, ampliﬁcation of the inosine monophosphate dehy- drogenase gene in Trypanosoma gambiense in response to selection by mycophenolic acid is the only example reported in the literature (Wilson et al., 1994). The fact that T. brucei DHFR–TS mutants are non- viable in vivo is in contrast to L. major DHFR–TS mutants, which cause persistent avirulent infections in mice. Presumably, this is due to sufficient amounts of thymidine being available in the parasitophorous vacuole of the host macrophage (Titus et al., 1995). This interest- ing phenomenon of persistence has been exploited as a potential live vaccination strategy in leishmaniasis (Veras et al., 1999). In complete contrast, T. brucei DKO lines are totally incapable of establishing or maintaining an infec- tion in mice. Given the narrow window of thymidine con- centrations that can support growth of T. brucei in vitro, this is likely to be a consequence of the extremely low concentrations of thymidine in plasma: ~1 mM in mice or ~0.1 mM in humans (Clarke et al., 2000). The underlying mechanisms involved in ‘thymine-less death’ in prokaryotes and eukaryotes are not fully under- stood (Ahmad et al., 1998). The primary event following inhibition of TS activity is extreme depletion of the dTTP pool with a concomitant increase in dUMP and its subse- quent phosphorylation to form dUTP (Van Triest et al., 2000; Ackland et al., 2003; Gmeiner, 2005). If the capacity of dUTPase to hydrolyse dUTP is exceeded, then a futile cycle of misincorporation of uracil into DNA, followed by base excision by uracil-DNA glycosylase, takes place, ultimately causing irreparable chromosome damage and cell death (Ahmad et al., 1998; Van Triest et al., 2000; Tinkelenberg et al., 2002). However, we found no evidence for death by an apoptotic-like mechanism as frequently observed in higher eukaryotes (Van Triest et al., 2000). Rather, the morphological phenotype of an enlarged ﬂagellar pocket observed here resembles the ‘big-eye’ phenotype reported for clathrin knock-down by RNAi in T. brucei (Allen et al., 2003). It is not immediately apparent what the common mechanism might be, if any. Further studies on the biochemical processes leading to cell death due to thymidine depletion are required. The inhibitors used in this study have uncovered some interesting features of folate metabolism in T. brucei with potential implications for target validation and drug discovery. Phenotypic drug screening against these para- sites using standard HMI9 medium is not ideal for inhibi- tors targeting folate and thymidylate metabolism. For example, high folate levels markedly attenuate the cyto- toxic effect of antifolate inhibitors such as methotrexate and aminopterin (both DHFR inhibitors) and raltitrexed (a TS inhibitor) that are structurally similar to physiological folates. The precise mechanism of attenuation remains to be determined, but the high folate in HMI9 medium could competitively inhibit drug uptake into cells, interfere with cellular retention by competition for polyglutamylation by folylpolyglutamyl synthetase or compete for the active sites of target enzymes as reported for mammalian systems (Kane et al., 1986; Jansen et al., 1989; 1998; Barnes et al., 1999; Backus et al., 2000; van der Wilt et al., 2001). Interestingly, the trypanocidal potency of the TS inhibitor, pemetrexed (Wang et al., 2003), is not affected by high folate levels in the medium, despite a pronounced (440-fold) decrease in potency against the DKO cell line. This may be due to a different mode of uptake or reduced polyglutamylation compared with the other folate analogues. Further transport and metabolism studies are required to test these possibilities. Deletion of DHFR–TS causes a huge loss in inhibitor potency (>500-fold) in cells cultured in low-folate medium, indicating that these enzyme activities are the primary targets for methotrexate, aminopterin, raltitrexed and pemetrexed. However, this effect is not observed with lipophilic inhibitors such as pyrimethamine, a potent inhibitor of TbDHFR (Gamarro et al., 1995), or trime- trexate, a potent inhibitor of Trypanosoma cruzi DHFR (Kiapp6.6 nM) which has an EC50 of 26 nM against T. cruzi amastigotes (Senkovich et al., 2005). Neither is it observed for 5-ﬂuoro-orotate, a prodrug of 5-FdUMP. The reason for this is not known. However, the high concen- trations (>1 mM) required to inhibit growth point to factors such as poor cellular pharmacokinetics (absorption, dis- tribution, metabolism and excretion) or poor potency against the target with other ‘off-target effects’ predomi- nating in cell killing. The latter explanation is likely in the case of 5-ﬂuo-orotate, as ﬂuorinated pyrimidine ana- logues are known to have pleiotropic effects such as © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 69, 520–533