Increased levels of thiols protect antimony unresponsive Leishmania donovani field isolates against reactive oxygen species generated by trivalent antimony

Abstract

The current trend of antimony (Sb) unresponsiveness in the Indian subcontinent is a major impediment to effective chemotherapy of visceral leishmaniasis (VL). Although contributory mechanisms studied in laboratory-raised Sb-R parasites include an up-regulation of drug efflux pumps and increased thiols, their role in clinical isolates is not yet substantiated. Accordingly, our objectives were to study the contributory role of thiols in the generation of Sb unresponsiveness in clinical isolates. Promastigotes were isolated from VL patients who were either Sb responsive (n=2) or unresponsive (n=3). Levels of thiols as measured by HPLC and flow cytometry showed higher basal levels of thiols and a faster rate of thiol regeneration in Sb unresponsive strains as compared with sensitive strains. The effects of antimony on generation of reactive oxygen species (ROS) in normal and thiol-depleted conditions as also their H2O2 scavenging activity indicated that in unresponsive parasites, Sb-mediated ROS generation was curtailed, which could be reversed by depletion of thiols and was accompanied by a higher H2O2 scavenging activity. Higher levels of thiols in Sb-unresponsive field isolates from patients with VL protect parasites from Sb-mediated oxidative stress, thereby contributing to the antimony resistance phenotype.

Figure 1A. Flow cytometric measurement of thiols using Mercury orange (MO)

Log phase promastigotes (unstained, a) of S1 (b) and R3 (c) were exposed to MO (500 μM) in acetone, incubated on ice for 5 minutes, washed and analysed for fluorescence in FL3 channel as described in Materials and Methods.

Figure 1B. Flow cytometric detection of basal intracellular thiols in Sb-S and Sb-R strains

Log phase promastigotes both from Sb-S (S1 and S2) and Sb-R (R1, R2 and R3) strains were labelled with Mercury Orange (MO) before (open bars,□) and after treatment with buthionine sulphoximine (3 mM, 48 h, 24°C) (filled bars,■) and fluorescence analyzed as described in Materials and Methods. Data are expressed as MFC ± SEM of at least three independent experiments in duplicates.

Figure 2A. H₂O₂ mediated increase of H₂DCFDA mediated fluorescence

Log phase promastigotes of S1 (unstained, a) were exposed to increasing concentrations of H₂O₂, 100 μM (b) and 1000 μM (c), probed with dichlorodihydrofluorescein diacetate (H₂DCFDA) and analysed for dichlorofluorescein (DCF) fluorescence in FL1 channel as described in Materials and Methods.

Figure 2B. Generation of ROS by Sb^III and Sb^V in Sb-S and Sb-R strains

Log phase promastigotes (open bars,□) both from Sb-S (S1 and S2) and Sb-R (R1, R2 and R3) strains were exposed to either Sb^III (filled bars,■) or Sb^V (hatched bars,) for 3 h at 37°C, labelled with dichlorodihydrofluorescein diacetate (H₂DCFDA) and dichlorofluorescein (DCF) fluorescence was analysed as described in Materials and Methods. Data are expressed as MFC ± SEM of at least three independent experiments in duplicates.

Figure 3. Kinetics of thiol regeneration in Sb-S and Sb-R strains

Log phase promastigotes from Sb-S strains, S1 (closed squares, -■-) and S2 (open squares, -□-), as also Sb-R strains R1 (inverted closed triangles, -▼-), R2 (open circles, -○-) and R3 (upright closed triangles, -▲-) were depleted of thiols using buthionine sulphoximine (3 mM, 48 h, 24°C); promastigotes were then washed and resuspended in Medium A, harvested at different time points, labelled with Mercury Orange (MO) and fluorescence analysed as described in Materials and methods. Data are expressed as mean ± SEM of % increment of MFC from 0 minute of at least three independent experiments in duplicates.

Figure 4. Effect of thiol depletion on Sb^III mediated ROS generation in Sb-S and Sb-R strains

Log phase promastigotes (open bars,□) from Sb-S (S1 and S2) and Sb-R (R1, R2 and R3) strains following removal of thiols (filled bars,■) were exposed to Sb^III (300 μg/ml) for 3 h at 37°C. Cells were labelled with dichlorodihydrofluorescein diacetate (H₂DCFDA) and dichlorofluorescein (DCF) fluorescence analysed as described in Materials and Methods. Data were expressed as MFC ± SEM of at least three independent experiments in duplicates.

Figure 5. Effect of Sb^III on thiol levels in Sb-S and Sb-R strains

Log phase promastigotes from Sb-S (S1 and S2) and Sb-R (R1, R2 and R3) were exposed to Sb^III (300 μg/ml) at 37°C for 3 h; they were then harvested, labelled with Mercury Orange (MO) and fluorescence analysed as described in Materials and Methods. Data were expressed as mean ± SEM of % retention of MFC from baseline of at least three independent experiments in duplicates.

Figure 6. Flow cytometric detection of ROS scavenging activity in Sb-S and Sb-R strains

Log phase promastigotes from Sb-S strains S1 (closed squares, -■-), S2 (open squares, -□-) and Sb-R strains R1 (inverted closed triangles, -▼-), R2 (upright open triangles, -Δ-) and R3 (open circles, -○-) were exposed to increasing concentration of H₂O₂ (0 - 1000 μM) at 37°C for 1 h, probed with dichlorodihydrofluorescein diacetate (H₂DCFDA) at 37°C for 45 for minutes and dichlorofluorescein (DCF) fluorescence was analysed as described in Materials and Methods. Data were expressed as MFC ± SEM of at least three independent experiments in duplicates.