Thiamine utilisation by the malaria parasite Plasmodium falciparum

Abstract

Thiamine (vitamin B1), is converted into an important cofactor for several enzymes and is an essential nutrient for the human malaria parasite Plasmodium falciparum. The thiamine biosynthesis capacity of the intraerythrocytic stage of P. falciparum is insufficient to meet the parasite's thiamine requirements and, therefore, the parasite needs to scavenge extracellular thiamine in order to support its rapid growth during the erythrocytic stage of its life cycle. Thiamine utilisation by the P. falciparum parasite might therefore serve as a potential antimalarial drug target. The mechanism of thiamine transport and its accumulation in the P. falciparum parasite was investigated in some detail. So too was the antiplasmodial activity of various thiamine analogues. Oxythiamine, a thiamine analogue, inhibited in vitro parasite proliferation via a thiamine-related pathway, as removal of thiamine from the culture medium increased the antiplasmodial activity of oxythiamine. The antiplasmodial mode of action of oxythiamine was investigated by targeted overexpression of key enzymes in thiamine metabolism and utilisation. Overexpression of thiamine pyrophosphokinase (PfTPK; the enzyme which converts thiamine into its active form, thiamine pyrophosphate, TPP) hypersensitised parasites to oxythiamine by up to 1700-fold, consistent with oxythiamine being a substrate for PfTPK and being converted into an antimetabolite. Parasites overexpressing the TPP-dependent enzymes, oxoglutarate dehydrogenase (PfOxoDH) and pyruvate dehydrogenase (PfPDH) are up to 15-fold more resistant to oxythiamine. HPLC analysis of strep-tagged PfOxoDH and PfPDH from pull-down assays of oxythiamine-treated parasites demonstrated the generation of oxythiamine pyrophosphate (OxPP) and its binding to these enzymes in situ. Taken together, the results are consistent with oxythiamine being converted into OxPP within the parasite and with this antimetabolite simultaneously inactivating at least two TPP-dependent enzymes located within distinct organelles. The ability of thiamine analogues, antimalarials and MDR pump inhibitors to inhibit thiamine transport was investigated. Surprisingly, thiamine analogues (including oxythiamine) did not inhibit thiamine transport, but quinine, quinidine and verapamil were effective inhibitors. Moreover, it was found that thiamine accumulation into K1 (chloroquine-resistant) parasites was 3-fold lower than that observed in 3D7 (chloroquine-sensitive) parasites. Due to the difference in thiamine accumulation between K1 and 3D7 parasites, and the inhibitory effect of quinine and verapamil (known inhibitors of PfCRT, the key mediator of chloroquine resistance) on thiamine accumulation, the possibility that PfCRT plays a role in thiamine accumulation by the parasite was investigated. Using previously-generated transgenic parasites with key mutations in PfCRT, data was generated consistent with a role for PfCRT in thiamine accumulation in P. falciparum parasites. Outcomes from this study have advanced our understanding of the utilisation of thiamine by P. falciparum and of the antiplasmodial mechanism of action of thiamine analogues, in particular, oxythiamine. Additionally, it has also resulted in the identification of a number of effective antimalarials in inhibiting thiamine transport by the parasites. The findings described in this thesis, therefore, validate thiamine-utilisation by P. falciparum as a potential target for future antimalarials.