Resistance of Leishmania donovani to sodium stibogluconate is related to the expression of host and parasite gamma-glutamylcysteine synthetase

Abstract

Sequencing studies showed that the gamma-glutamylcysteine synthetase (gamma-GCS) heavy chain genes from sodium stibogluconate (SSG)-resistant (SSG-R) and SSG-susceptible (SSG-S) Leishmania donovani strains were identical, indicating that SSG resistance was related to quantitative differences in gamma-GCS expression rather than gene interstrain polymorphisms. In vitro infection of murine macrophages with the SSG-R strain, but not the SSG-S strain, down regulated expression of host gamma-GCS, which would result in a reduction in intramacrophage glutathione (GSH) levels and promote an oxidative intramacrophage environment. This would inhibit, or minimize, the reduction of SSG pentavalent antimony to its more toxic trivalent form. Macrophage studies showed that the SSG-R strain expressed higher levels of gamma-GCS compared to the SSG-S strain, which would result in higher GSH levels, giving increased protection against oxidative stress and facilitating SSG efflux. However a similar differential effect on host and parasite gamma-GCS expression was not obtained when using tissues from infected mice. In this case gamma-GCS expression was organ and strain dependent for both the host and the parasite, indicating that environmental conditions have a profound effect on gamma-GCS expression. Consistent with the proposed mechanism from in vitro studies, increasing tissue GSH levels in the presence of SSG by cotreatment of L. donovani-infected mice with SSG solution and GSH incorporated into nonionic surfactant vesicles was more effective in reducing liver, spleen, and bone marrow parasite burdens than monotherapy with SSG. Together, these results indicate that SSG resistance is associated with manipulation of both host and parasite GSH levels by L. donovani.

FIG. 1.

γ-GCS levels in uninfected macrophages and macrophages infected with the SSG-S or SSG-R strain of L. donovani. The amount of mouse γ-GCS (left) or Leishmania γ-GCS (right) present was quantified using RT-PCR. The data are representative of three separate experiments, which show the same differences between treatments indicated.

FIG. 2.

Comparison of mouse (spleen, top left; liver, top right) and Leishmania (spleen, bottom left; liver, bottom right) γ-GCS levels in the livers and spleens of L. donovani-infected mice. Spleen and liver samples were obtained from mice infected with the SSG-S or SSG-R strain of L. donovani on day 14 postinfection. Mice were treated with PBS alone (controls), free SSG (SSG final dose, 70 mg Sb^v/kg), BSO-NIV (final BSO dose, 34.7 mg/kg) mixed 1:1 (vol/vol) with water, or BSO-NIV (final BSO dose, 34.7 mg/kg) mixed 1:1 (vol/vol) with SSG solution (SSG final dose, 70 mg Sb^v/kg; BSO-NIV/SSG treatment). The mean relative expression of the mouse γ-GCS plus SE and the Leishmania γ-GCS plus SE was determined using the mean value for the SSG-S strain as the normalizer. The data are representative of two separate experiments which show the same significant differences between treatments indicated. *, P < 0.05 compared to the relevant control.

FIG. 3.

Effect of different treatments on the spleen, liver, and bone marrow parasite burdens of mice infected with the SSG-S (left) or SSG-R (right) strains of L. donovani on day 14 postinfection. Mice infected with the SSG-S or SSG-R strain of L. donovani were treated with PBS alone (control), free SSG (SSG final dose, 70 mg Sb^v/kg), GSH-NIV (final GSH dose, 89 mg/kg) mixed 1:1 (vol/vol) with water, or GSH-NIV (final GSH dose 89 mg/kg) mixed 1:1 (vol/vol) with SSG solution (SSG final dose, 70 mg Sb^v/kg; GSH-NIV/SSG treatment). ***, P < 0.001 compared to the relevant control. The data are representative of three separate experiments, which show the same significant differences between the treatments indicated.

FIG. 4.

Effect of GSH treatment on the efficacy of SSG treatment in macrophages infected with the SSG-S or SSG-R strains of L. donovani. Macrophages, infected with SSG-S or SSG-R strain of L. donovani promastigotes, were treated with medium alone (control), SSG alone (0.575 mM Sb^v), or SSG (0.575 mM Sb^v) and GSH (2.5 mM), and the mean percent infection was determined. **, P < 0.01 compared to relevant control The data are representative of three separate experiments, which show the same significant differences between the treatments indicated.