Translational up-regulation of antifolate drug targets in the human malaria parasite Plasmodium falciparum upon challenge with inhibitors

The thymidylate cycle in Plasmodium falciparum is essential for cell growth and replication, and dihydrofolate reductase (DHFR), a key enzyme in this cycle, is the target of important antimalarial drugs such as pyrimethamine and cycloguanil. Following previous work, where we found no evidence of upregulation of the dhfr-ts gene upon challenge with pyrimethamine, we investigated the expression at the protein level of the bifunctional gene product, which also carries thymidylate synthase (TS) activity. Challenge of parasite cultures with fluoro-substituted bases that are specific TS inhibitors at levels close to the IC50 resulted in five to seven-fold increases in enzyme level, as monitored by both DHFR and TS activities, while pyrimethamine and another DHFR-binding inhibitor, WR99210, induced smaller but still significant increases of ∼three-fold. However, when parasites were challenged with tetracycline, an antimalarial not directed at the folate pathway, although an increase was consistently seen above untreated controls, this was at a level of ∼1.8-fold. These increases reflect enhanced synthesis of the DHFR-TS enzyme, rather than liberation of a latent activity, as they were completely abolished if cultures were pre-incubated with cycloheximide to block de novo protein synthesis. Moreover, none of the above antimalarial drugs was found to significantly alter absolute levels of the dhfr-ts mRNA under the conditions of challenge used. We conclude that, in common with mammalian systems, where a similar phenomenon has been reported, malaria parasites are able to significantly relieve translational constraint when faced with antifolate drug challenge. The data indicate that there is a specific component in addition to a low-level non-specific increment, and that binding to the TS domain of the DHFR-TS protein appears to be better able to relieve this constraint than binding to the DHFR domain. © 2004 Elsevier B.V. All rights reserved.