<i>Plasmodium</i> thiamine pyrophosphokinase is essential for sporozoite formation and activation of an antiplasmodial thiamine analogue.

Abstract

Oxythiamine, a thiamine analogue, inhibits <i>Plasmodium falciparum</i> proliferation by acting as an antimetabolite of vitamin B₁. To elucidate in more detail its underlying mechanism of action, in vitro drug pressure was employed to generate oxythiamine-resistant <i>P. falciparum</i> lines. Whole-genome sequencing revealed that resistance was conferred by a single-point mutation in the thiamine pyrophosphokinase (TPK) gene. The mutated TPK has reduced activity, thereby likely limiting the conversion of oxythiamine into its active toxic form. To investigate the functional role of TPK across the parasite life cycle, a <i>TPK</i>-knockout line was generated in <i>Plasmodium berghei</i>. <i>TPK</i>-knockout parasites displayed a minor fitness cost during intraerythrocytic proliferation that could be overcome by infecting reticulocytes, but their sensitivity to oxythiamine was reduced fivefold in vivo, consistent with the hypothesis that activation of oxythiamine via TPK is essential for its antiplasmodial activity. In the <i>Anopheles</i> vector, <i>TPK</i>-knockout parasites produced a similar number of oocysts as wild-type parasites, but oocyst maturation was impaired and sporozoite formation was completely inhibited. These findings underscore an essential role for TPK in mediating the antiplasmodial activity of oxythiamine and reveal its critical function in sporogony within the mosquito, supporting its potential as a transmission-blocking target for antimalarial intervention.