Upregulation of Cysteine Synthase and Cystathionine β-Synthase Contributes to Leishmania braziliensis Survival under Oxidative Stress

Abstract

Cysteine metabolism is considered essential for the crucial maintenance of a reducing environment in trypanosomatids due to its importance as a precursor of trypanothione biosynthesis. Expression, activity, functional rescue, and overexpression of cysteine synthase (CS) and cystathionine ␤-synthase (C␤S) were evaluated in Leishmania braziliensis promastigotes and intracellular amastigotes under in vitro stress conditions induced by hydrogen peroxide (H 2 O 2 ), S-nitroso-N-acetylpenicillamine, or antimonial compounds. Our results demonstrate a stage-specific increase in the levels of protein expression and activity of L. braziliensis CS (LbrCS) and L. braziliensis C␤S (LbrC␤S), resulting in an increment of total thiol levels in response to both oxidative and nitrosative stress. The rescue of the CS activity in Trypanosoma rangeli, a trypanosome that does not perform cysteine biosynthesis de novo, resulted in increased rates of survival of epimastigotes expressing the LbrCS under stress conditions compared to those of wild-type parasites. We also found that the ability of L. braziliensis promastigotes and amastigotes overexpressing LbrCS and LbrC␤S to resist oxidative stress was significantly enhanced compared to that of nontransfected cells, resulting in a phenotype far more resistant to treatment with the pentavalent form of Sb in vitro. In conclusion, the upregulation of protein expression and increment of the levels of LbrCS and LbrC␤S activity alter parasite resistance to antimonials and may influence the efficacy of antimony treatment of New World leishmaniasis.T he intracellular protozoan parasite Leishmania causes a neglected infectious disease commonly referred to as leishmaniasis. Depending on the infecting species and the immune status of the host, leishmaniasis can result in a variety of clinical manifestations with cutaneous, mucocutaneous, or visceral involvement (1-3). Leishmania (Viannia) braziliensis, the causative agent of cutaneous leishmaniasis and mucocutaneous leishmaniasis, is the most prevalent species infecting humans in the Americas (4, 5).Leishmania spp. have a digenetic life cycle, alternating between flagellated promastigote forms in the midgut of the sand fly and obligatory intracellular amastigotes within macrophages of the mammalian host (6, 7). During this complex life cycle, these parasites are exposed to variable oxidative or nitrosative stresses induced by reactive oxygen species (ROS) or reactive nitrogen species (RNS), respectively, generated by the host immune system to avoid infection (8, 9). Antimonial compounds, such as sodium stibogluconate or Sb (Pentostan) or meglumine antimoniate (Glucantime), are still the first-choice drugs for human leishmaniasis treatment (10). Among these, the Sb pentavalent form (Sb V ) has been reported to indirectly induce oxidative and nitrosative stress on the parasite by stimulating infected macrophages (M) to generate ROS and nitric oxide (NO) via activation of phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), and mitogenactivated ...

The Uptake and Metabolism of Amino Acids, and Their Unique Role in the Biology of Pathogenic Trypanosomatids

“…In L. braziliensis amastigotes, CS levels and activity were increased together with CBS and CGL, whereas CBS is more active in promastigotes. The overexpression of either CBS or CS in L. braziliensis promastigotes increased the resistance of these parasites to oxidative imbalance, in agreement with the role of Cys as an anti-oxidant and its participation as a precursor of trypanothione biosynthesis [ 214 ]. CGL was also characterized in L. major and L. mexicana , where it was shown to have a cytosolic localization in promastigotes [ 215 ].…”

Deciphering the interplay between cysteine synthase and thiol cascade proteins in modulating Amphotericin B resistance and survival of Leishmania donovani under oxidative stress

“…Till date only a single study in L. braziliensis has been carried out to emphasize the crucial role of CS in oxidative stress tolerance [60]. To elucidate the role of CS in thiol mediated drug resistance in L .…”

Drug transporter and oxidative stress gene expression in human macrophages infected with benznidazole-sensitive and naturally benznidazole-resistant Trypanosoma cruzi parasites treated with benznidazole

“…The human monocytic cell line THP-1 (ATCC TIB202), derived from an acute monocytic leukaemia, was cultured and differentiated into macrophages as previously described [19]. THP-1-derived macrophages (THP-1 Mϕ) were used to evaluate the gene expression profile in THP-1 Mϕs infected with T. cruzi benznidazole-sensitive and naturally benznidazole-resistant strains and then treated with BZ.…”