The antioxidative effect of de novo generated vitamin B₆ in Plasmodium falciparum validated by protein interference

The malaria parasite Plasmodium falciparum is able to synthesize de novo PLP (pyridoxal 5′-phosphate), the active form of vitamin B₆. In the present study, we have shown that the de novo synthesized PLP is used by the parasite to detoxify ¹O₂ (singlet molecular oxygen), a highly destructive reactive oxygen species arising from haemoglobin digestion. The formation of ¹O₂ and the response of the parasite were monitored by live-cell fluorescence microscopy, by transcription analysis and by determination of PLP levels in the parasite. Pull-down experiments of transgenic parasites overexpressing the vitamin B₆-biosynthetic enzymes PfPdx1 and PfPdx2 clearly demonstrated an interaction of the two proteins in vivo which results in an elevated PLP level from 12.5 μM in wild-type parasites to 36.6 μM in the PfPdx1/PfPdx2-overexpressing cells and thus to a higher tolerance towards ¹O₂. In contrast, by applying the dominant-negative effect on the cellular level using inactive mutants of PfPdx1 and PfPdx2, P. falciparum becomes susceptible to ¹O₂. Our results demonstrate clearly the crucial role of vitamin B₆ biosynthesis in the detoxification of ¹O₂ in P. falciparum. Besides the known role of PLP as a cofactor of many essential enzymes, this second important task of the vitamin B₆de novo synthesis as antioxidant emphasizes the high potential of this pathway as a target of new anti-malarial drugs.