Abstract
Antimony (SbIII) and miltefosine (MIL) are important drugs for the treatment of Leishmania parasite infections. The mitochondrion is likely to play a central role in SbIII and MIL induced cell death in this parasite. Enriched mitochondrial samples from Leishmania promastigotes selected step by step for in vitro resistance to SbIII and MIL were subjected to differential proteomic analysis. A shared decrease in both mutants in the levels of pyruvate dehydrogenase, dihydrolipoamide dehydrogenase, and isocitrate dehydrogenase was observed, as well as a differential abundance in two calcium-binding proteins and the unique dynamin-1-like protein of the parasite. Both mutants presented a shared increase in the succinyl-CoA:3-ketoacid-coenzyme A transferase and the abundance of numerous hypothetical proteins was also altered in both mutants. In general, the proteomic changes observed in the MIL mutant were less pronounced than in the SbIII mutant, probably due to the early appearance of a mutation in the miltefosine transporter abrogating the need for a strong mitochondrial adaptation. This study is the first analysis of the Leishmania mitochondrial proteome and offers powerful insights into the adaptations to this organelle during SbIII and MIL drug resistance.
Highlights
The Leishmania spp genus of protozoan parasites contains more than 20 species that are responsible for several maladies termed leishmaniasis
To verify that our protein samples were enriched for mitochondria we performed a western blot for HSP60 and to show that they did not contain nuclear contamination, we performed a western blot for histone 3 (Figure 1, insets)
When the same amount of protein (200 μg) derived from the 25%:28% fraction or from whole cell extracts was migrated on 2D gels, fewer spots were observed in the mitochondrial samples compared to whole cell extracts (Figure 1), indicating an enrichment of specific proteins in the mitochondrial samples
Summary
The Leishmania spp genus of protozoan parasites contains more than 20 species that are responsible for several maladies termed leishmaniasis. The parasites infect an estimated 12 million people in Asia, Europe, the Middle East, Africa, and South America [1]. Since their discovery in the 1940s, the toxic parenteral pentavalent antimony (SbV) compounds have been the mainstay of treatment for all types of leishmaniasis and are still the first-line treatment in most areas, clinical resistance is often observed. SbIII (antimony in its 3+ oxidation state) is sequestered in Leishmania conjugated to trypanothione (TSH) or glutathione (GSH) [3,4,5] through an ABC transporter termed MRPA [6], which correlates with an increase in the production of thiols in resistant isolates [4,7,8]. It has been demonstrated that SbIII induces apoptotic-like features including accumulation of reactive oxygen species (ROS), a drop in mitochondrial membrane potential, genomic
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