Malaria remains a major global health problem. The appearance and spread of resistant Plasmodium falciparum, and also P. vivax, have resulted in the re-emergence of the disease. Contributing to the magnitude of the problem is the lack of a full understanding of the mechanisms responsible for the development of parasite drug resistance. It is now well recognized that drug resistance in malaria is multifactodal and that multiple genes/factors play a critical role. We have recently described, in the murine model P. berghei, an up-regulation in the expression of the gamma glutamyl cysteine (pbggcs) gene in lines resistant to chloroquine (RC) and mefloquine (N1100). We have also identified a multidrug resistant associated protein (pbmrp) orthologue gone in P. berghei. Functional studies are still needed, however, to assess the role of mrp and ggcs in malaria drug resistance. Our working hypothesis is that both ggcs and mrp, play a key role in the evolution of drug resistant phenotypes. The long-term goal is to elucidate mechanisms of Plasmodia quinoline resistance. Our objectives are: 1) to determine the functional role of pbggcs in malaria drug resistance, and initiate studies on the transcriptional regulation of the pbggcs gene, and 2) to characterize and determine the role of pbmrp gone in malaria drug resistance. To asses the role of the P. berghei ggcs gene in drug resistance, an over-expression transfection strategy will be used; the resulting pattern of drug resistance will be assayed. Expression of pbGGCS, glutathione levels and pbggcs-specific inhibition studies, in combination with drug sensitivity assays, will be done. The cis elements associated to the transcriptional regulation of the pbggcs gone will be identified and characterized; 5' and 3' UTRs will be analyzed. The pbggcs transcription start site will be determined and the activity of the pbggcs promoter will be analyzed. Expression of the pbmrp gene at the protein and the mRNA levels, as well as gone disruption studies, will be carried out. The proposed research should contribute to a better understanding of the mechanisms underlying drug resistance in Plasmodia and open new approaches to malaria chemotherapy. The high morbidity and mortality associated to this disease, as well as its resulting economic impact, makes such understanding not only needed, but warranted.
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