Lower Nitric Oxide Susceptibility of Trivalent Antimony-Resistant Amastigotes ofLeishmania infantum

Abstract

We previously documented the induction of Leishmania amastigote apoptosis by trivalent antimony (SbIII) and nitric oxide (NO). We demonstrate here that SbIII-resistant amastigotes were resistant to NO toxicity when delivered extracellularly by NO donors or intracellularly via macrophage activation. Shared biochemical targets for SbIII and NO resistance in Leishmania are discussed.Leishmania infantum or Leishmania chagasi is responsible for canine and human visceral leishmaniasis in both the Old and the New Worlds. Leishmania parasites develop as flagellated promastigotes in the insect vector and reside as intracellular nonflagellated amastigotes in the mammalian host, which are responsible for the clinical disease manifestations. Basic treatment of leishmaniasis consists of the administration of pentavalent antimony (SbV) in the form of sodium stibogluconate or meglumine antimoniate. The mode of action of SbV implicates its reduction by the host cell to the trivalent form (SbIII) (8,9,21,23). Besides this, successful chemotherapy in murine and canine models has been correlated with the efficiency of natural immunity (5, 26), and synergism between SbV and immunostimulant cytokines has been proven to be pertinent in the treatment of leishmaniasis (14,18). We recently demonstrated that both nitric oxide (NO) and SbIII lead to Leishmania amastigote cell death with some characteristics of apoptosis (11,22). Moreover, the activity of SbIII may be directly linked to the induction of reactive oxygen intermediates such as NO (24). In order to clarify more precisely the potency of NO and SbIII, we investigated the cross-resistance of SbIII-resistant parasites to NO.SbIII-resistant amastigotes previously described (20) were used in all experiments. The susceptibility to NO of wild-type (WT) amastigotes and amastigotes resistant to 120 g/ml potassium antimonyl tartrate (LiSbIIIR120) was ascertained using either acidified sodium nitrite (NaNO 2 ) or the NO donors SNAP (S-nitroso-N-acetylpenicillamine) and DETA-NONO-For NaNO 2 , 10 7 amastigotes per ml were incubated for 2 h at 37 Ϯ 1°C in the dark in 0.01 M phosphate-buffered saline (PBS), pH 4.5, in the presence of 0 to 10 mM NaNO 2 . After a wash in PBS, parasites were seeded in 96-well microplates at 2 ϫ 10 5 /well in 100 l of medium for axenic amastigotes (MAA) (13). To estimate parasite survival after NO treatment, amastigotes were cultured in the presence of 2.5 Ci of [ 3 H]thymidine for 24 h and then filtered through GF/C filters (Whatman International) for radioactivity determination. The relative inhibition of [ 3 H]thymidine incorporation by 50% (IC 50 ) was calculated. The NO donors SNAP and DETA-NONOate were added directly in the culture medium at concentrations ranging from 0 to 500 M, immediately after inoculation of 2 ϫ 10 5 amastigotes/100 l of MAA in 96-well microplates. After 72 h of incubation, the effective concentrations of NO donors inhibiting WT and LiSbIIIR120 amastigote growth by 50% (IC 50 ) were estimated by MTT test (21). IC 50 for WT and LiSbIIIR...

Resistance of Leishmania (Viannia) braziliensis to nitric oxide: correlation with antimony therapy and TNF-α production

“…Pentavalent antimony (Sb V ) must be reduced to Sb III to exhibit its leishmanicidal activity [18,19]. Sb III in turn induces the production of reactive oxygen and nitrogen radicals in macrophages infected with L. donovani [20].…”

Leishmania antimony resistance: what we know what we can learn from the field

“…Experimental evidence supports the notion that NO may be a crucial factor that can modify the antimony response of Leishmania populations. Axenic amastigotes of Leishmania that were selected in vitro for SbIII resistance expressed cross-resistance to NO (Holzmuller et al 2005). Although studies have not addressed the effect that NO resistance has on antimony cross-resistance, promastigotes of L. braziliensis isolated from antimony-unresponsive patients were generally less susceptible to NO (Souza et al 2010).…”

Mechanisms and consequences of persistence of intracellular pathogens: leishmaniasis as an example

“…In the case of Leishmania parasites partial resistance to ROI or reactive nitrogen intermediates (RNI) has been described for the extracellular (promastigote) and for the intracellular (amastigote) stage of certain Leishmania species (Miller et al. , 2000; Holzmuller et al. , 2005; 2006; Giudice et al.…”