Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS) production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast) and mitoferrin-1 (human) that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1) showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes.
Leishmaniasis is a serious parasitic disease that affects 12 million people worldwide, with clinical manifestations ranging from self-healing cutaneous lesions to deadly visceralizing disease. A vaccine is not available, and new and less toxic drugs against this protozoan parasite are urgently needed. Following introduction into vertebrate hosts during a sand fly blood meal, Leishmania parasites undergo extensive changes in morphology and metabolism that are critical for adaptation to life inside host macrophages and replication as amastigotes. Earlier studies identified major events that occur during amastigote differentiation, but the signaling mechanism initiating this process remained poorly understood. Previously we demonstrated a novel role for the reactive oxygen species (ROS) H2O2 in initiating amastigote differentiation, a process proposed to be dependent on iron availability inside the parasite’s mitochondria. In this study we identify LMIT1, a Leishmania transmembrane protein that functions as a mitochondrial iron transporter and is conserved in other trypanosomatid protozoan parasites. Reduced LMIT1 expression impairs mitochondrial function in the infective amastigote stage, abolishing parasite virulence. Our findings identify LMIT1 as a promising new drug target, and support the conclusion that iron-dependent ROS signals generated in the mitochondria regulate differentiation of virulent Leishmania amastigotes.