A new source of reactive oxygen species in Leishmania amazonensis: Characterization of a heme-activated NADPH oxidase-like

Leishmania amazonensis is one of leishmaniasis’ causative agent, a disease that has no cure and leads the appearance of cutaneous lesions. Recently, our group showed that heme activates a Na + /K + ATPase in these parasites through a signaling cascade involving hydrogen peroxide (H 2 O 2 ) generation. Heme has a pro-oxidant activity and signaling capacity, but the mechanism by which this molecule increases H 2 O 2 levels in L. amazonensis has not been elucidated yet. In the present work, we investigated the source of H 2 O 2 stimulated by heme, ruling out the participation of mitochondria and raising the possibility of a NADPH oxidase (Nox) instead. Although the absence of a Nox sequence in trypanosomatids’ genome, it was described a ferric iron reductase (LFR1) in L. amazonensis, an enzyme with a very similar Nox structure which reduces iron instead of oxygen. Based on our experiments, we believe that LFR1 is a bifunctional enzyme, reducing oxygen or iron, and both activities are modulated by heme, whose extracellular concentration varies according to the parasite life cycle. Comparing L. amazonensis WT H 2 O 2 production to LFR1 overexpressing (OE) mutant we verified that OE cells produce more H 2 O 2 than WT cells. In addition, heterologous expression of LFR1 in mammalian HEK293 cells showed higher H 2 O 2 production when compared to cells transfected only with plasmid containing GFP. This is the first time that a Nox activity is described in trypanosomatids. This finding could help to better understand the parasite life cycle and may also clarify the action mechanism of some antileishmanial drugs that increase the level of ROS. Besides, LFR1 is the only protein, so far, that has the ability to reduce iron and to generate H 2 O 2 , suggesting a common ancestor of NADPH oxidase and iron reductase families.