Increased thiol levels in antimony-resistant Leishmania infantum isolated from treatment-refractory visceral leishmaniasis in Brazil

Abstract

BACKGROUND Treatment-refractory visceral leishmaniasis (VL) has become an important problem in many countries. OBJECTIVES We evaluated the antimony-resistance mechanisms of Leishmania infantum isolated from VL patients refractory or responsive to treatment with pentavalent antimony. METHODS Strains isolated from antimony-refractory patients (in vitro antimony-resistant isolates) and antimony-responsive patients (in vitro antimony-sensitive isolates) were examined. Morphological changes were evaluated by transmission electron microscopy after trivalent antimony exposure. P-glycoprotein (P-gp) efflux pump activity was evaluated using the pump-specific inhibitor verapamil hydrochloride, and the role of thiol in trivalent antimony resistance was investigated using the enzymatic inhibitor L-buthionine sulfoximine. FINDINGS Antimony treatment induced fewer alterations in the cellular structure of L. infantum resistant isolates than in that of sensitive isolates. P-gp efflux activity was not involved in antimony resistance in these isolates. Importantly, the resistant isolates contained higher levels of thiol compared to the sensitive isolates, and inhibition of thiol synthesis in the resistant isolates recovered their sensitivity to trivalent antimony treatment, and enhanced the production of reactive oxygen species in promastigotes exposed to the drug. MAIN CONCLUSIONS Our results demonstrate that isolates from patients with antimony-refractory VL exhibited higher thiol levels than antimony-sensitive isolates. This indicates that redox metabolism plays an important role in the antimony-resistance of New World VL isolates.

Fig. 1. ultrastructural images obtained from transmission electron microscopy (TEM) in different conditions. Late log phase promastigotes were left untreated (A-D) or treated with 615 µM of Sb^III (E-H) for 48 h. Images are representative of the major alterations observed in each isolate and condition. SbR: antimony-resistant isolate; SbS: antimony-sensitive isolate. Ed: electrodensity; Gh: “Ghost” cells; Va: cytoplasmic vacuoles; Sm: absence of subpellicular microtubules; Ms: mitochondria swelling; Md: mitochondria disorganization. In the untreated parasites: N: nucleus; K: kinetoplast; M: mitochondria; FP: flagellar pocket; F: flagellum. Bars: A, B, D, E, F, H: 0.5 µM; C, G: 1 µM.

Fig. 2. (A) rhodamine 123 uptake and accumulation mean fluorescence intensity (MFI) with or without verapamil blockade in Leishmania infantum promastigotes. Statistical analysis was performed by Kruskal-Wallis test. Results are the mean ± standard error of the mean (SEM) of three independent experiments. (B) Promastigote viability after exposure to Sb^III for 48 h with or without verapamil blockade. Each isolate was exposed to its own IC₅₀. Statistical analysis was performed by ANOVA with Tukey's post-test. Statistical significance was defined in p < 0.05. Data shown are from two independent experiments performed in quintuplicate. SbS: antimony sensitive isolate; SbR: antimony resistant isolate.

Fig. 3. (A) thiol levels were measured (MFI) in promastigotes using CellTracker™ fluorescent probe (1 µM). Data are the mean ± standard error of the mean (SEM) of five independent experiments. (B) Parasite viability of promastigotes untreated and treated with Sb^III (at the IC₅₀ of each isolate) in the presence or absence of L-buthionine sulfoximine (BSO). Data are the mean ± SEM of two independent experiments performed in quintuplicate. (C) ROS production after exposition to IC₅₀ Sb^III with or without BSO measured by 2′,7′-dichlorofluorescein fluorescence (MFI). Statistical analysis was performed by ANOVA followed by Tukey's post-test. Statistical significance was defined as p < 0.05. Data represent the mean ± SEM of two independent experiments performed in quintuplicate. SbS: antimony sensitive isolate; SbR: antimony resistant isolate.

Fig. 4. schematic model of the antimony-resistance mechanism in promastigotes of Leishmania infantum isolated from refractory patients from Brazil. In drug resistant isolates, higher thiol metabolism of thiol results in thiol-metal complex formation and drug inactivation. Additionally, the thiol component can buffer Sb^III-induced ROS. Thereby, the parasites can survive at high concentrations of Sb^III.