Abstract
Leishmania virulence and disease development critically depends on the ability of Leishmania promastigotes to infect, differentiate into amastigote forms and replicate inside mammalian host macrophages. Understanding changes associated with amastigote differentiation in axenic culture conditions is 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. infantum and. L. donovani, we show that ROS-generating methods such as iron deprivation or exposure to sub-lethal concentrations of H2O2 or menadione are significantly more effective in promoting promastigoteamastigote 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 H2O2 and menadione-mediated differentiation did not require up-regulation of the mitochondrial electron transport chain (ETC)-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 multiple Leishmania species, including the medically important visceralizing species, and provide mechanistic rationale for earlier reports demonstrating markedly increased virulence of L. infantum promastigotes pre-treated with oxidative reagents.
Highlights
Leishmania is an eukaryotic intracellular protozoan parasite that causes a broad spectrum of human diseases referred to as leishmaniasis, which currently affect about 12 million people and threaten more than 350 million globally (WHO, 2010)
We examined if H2O2-mediated signalling is involved in triggering amastigote differentiation of other Leishmania species, in the medically important visceralizing species
We tried several conditions for differentiating L. infantum amastigotes in axenic culture, all involving shifting of promastigotes in culture to low pH/high temperature to mimic intra-macrophage conditions that are conducive for amastigote growth and replication (Sereno and Lemesre, 1997; Somanna et al, 2002)
Summary
Leishmania is an eukaryotic intracellular protozoan parasite that causes a broad spectrum of human diseases referred to as leishmaniasis, which currently affect about 12 million people and threaten more than 350 million globally (WHO, 2010). The ability to successfully induce promastigote to amastigote differentiation in axenic culture by mimicking some of the intra-phagolysosomal conditions inside host macrophages (namely, low pH and elevated temperature) has greatly aided the understanding of cellular changes that accompany parasite differentiation (Bates et al, 1992; Zilberstein and Shapira, 1994; Barak et al, 2005). These temperature-/pH-dependent protocols for axenic differentiation and culture of amastigote forms have been difficult to adapt for many Leishmania species, including isolates of the clinically important L. infantum and L. donovani visceralizing species. Sudden changes in pH and temperature have been reported to result in loss of Leishmania viability as a result of a high reactive oxygen species (ROS) surge (Alzate et al, 2006, 2007)
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