ROS regulate differentiation of visceralizingLeishmaniaspecies into the virulent amastigote form

Abstract

AbstractLeishmaniavirulence and disease development critically depends on the ability ofLeishmaniapromastigotes to infect, differentiate into amastigote forms and replicate inside mammalian host macrophages. Understanding changes associated with amastigote differentiation in axenic culture conditions is a key to identifying virulence factors. Here we compared efficiency of the conventional pH–temperature-dependent shift method to induce amastigote differentiation with the recently identified trigger for differentiation mediated by mitochondrial reactive oxygen species (ROS). Using two different visceral leishmaniasis species,L. infantumand.L. donovani, we show that ROS-generating methods such as iron deprivation or exposure to sub-lethal concentrations of hydrogen peroxide (H2O2) or menadione are significantly more effective in promoting promastigote–amastigote differentiation than the low pH–high temperature shift, leading to higher survival rates, morphological changes and gene expression patterns characteristic of the amastigote stage. Notably, both H2O2and menadione-mediated differentiation did not require upregulation of the mitochondrial electron transport chain-associated protein p27, suggesting that treatment with oxidants bypasses the necessity to upregulate mitochondrial activity, a precondition for mROS generation. Our findings confirm that ROS-induced differentiation occurs in multipleLeishmaniaspecies, including the medically important visceralizing species, and provide mechanistic rationale for earlier reports demonstrating markedly increased virulence ofL. infantumpromastigotes pre-treated with oxidative reagents.