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Abstract
Leishmania infantum is a protozoan parasite that can infect macrophages from different mammalian species causing visceral leishmaniasis. One distinctive feature between Leishmania and their hosts is their thiol-based metabolism, which in the parasites largely depends on an unique dithiol conjugate known as trypanothione. The need to maintain redox homeostasis is a constant during the lifetime of any cell, although in the case of Leishmania this is perhaps even more relevant since they can be exposed to powerful macrophage-derived oxidants. Thus, antioxidant defense might be determinant for the successful establishment of L. infantum infection. In this matter, cytosolic antioxidant enzymes are probably very suitably located to deal with the oxidative attack derived from an exogenous origin. In this thesis we particularly focused on the cytosolic trypanothione-dependent system for hydroperoxide detoxification that includes the oxidoreductases tryparedoxin (TXN) and classical 2-cysteine peroxiredoxin (2-Cys PRX). We demonstrated that the cytosolic tryparedoxin (LiTXN1) is essential for the survival of L. infantum throughout their life cycle, i.e. both the promastigote and the disease-causing amastigote stages depend on LiTXN1 expression to survive. Furthermore, we showed that L. infantum can upregulate the expression of LiTXN1 from a single allele, suggesting that the parasites require more than 50% of wild-type content of the enzyme to sustain normal development. Besides the cytosolic LiTXN1, L. infantum possess a redox-active mitochondrial enzyme (LiTXN2) and also two other TXN-like sequences with the potential to encode functional proteins. Here we also described the characterization of these L. infantum TXN-like proteins (LiTXN3 and LiTXN5), evidencing that they cannot function as classical TXN enzymes. Moreover, we showed thatLiTXN3 is associated to the mitochondrial membrane and LiTXN5 to the endoplasmic reticulum membrane. Together our findings clearly show that in L. infantum no other molecule can functionally substitute for LiTXN1 and also that no other TXN-like protein is capable of substituting LiTXN2 in reduction of mitochondrial 2-Cys PRX.
In the trypanothione/TXN antioxidant pathway classical 2-Cys PRXs are the enzymes that directly react with hydroperoxides. In other organisms besides reducing hydrogen peroxide and small chain organic hydroperoxides, 2-Cys PRXs also display peroxynitrite reductase activity. This study supports that the same is true for both L. infantum cytosolic 2-Cys PRXs (LiTXNPx1 and LiTXNPx2). To investigate if increased infectivity is directly linked to the capacity of the parasites to cope with macrophagederived reactive oxygen and nitrogen species, we used L. infantum lines overexpressing one of the cytosolic 2-Cys PRX (LiTXNPx2) in ex vivo infection assays. Our results provide evidence that upregulation of LiTXNPx2 enhanced the parasites capacity to survive inside two different types of host cells, murine and human macrophages. This suggests that indeed the antioxidant defense of these parasites might be a significant factor in the infectious process.