Novel Intracellular SbV Reducing Activity Correlates with Antimony Susceptibility in Leishmania donovani

Abstract

The standard treatment of human visceral leishmaniasis involves the use of pentavalent antimony (Sb V ). Its mechanism of action is unknown because of the limited information available about intracellular antimony metabolism and about the genes that regulate these processes. Herein, flow injection-inductively coupled plasma mass spectrometry (ICP-MS), flow injection hydride generation ICP-MS, and ion chromatography ICP-MS were used to measure antimony accumulation and intracellular metabolism in the human protozoan parasite Leishmania donovani. V reduction has yet to be identified, and it may or may not be enzymatic. This is the first description of intracellular Sb V reducing activity in Leishmania as well as in any prokaryotic or eukaryotic cell.Leishmania donovani is the major causative agent of visceral leishmaniasis. In nature, the parasite exists either as an extracellular promastigote found in the alimentary tract of sandflies or as an obligatory intracellular amastigote found in phagolysosomes of mammalian macrophages(1-3). During the last several years, a number of laboratories have utilized axenic culture of L. donovani amastigotes for the direct evaluation of cell biological and biochemical processes in the amastigote, devoid of the host macrophage (4 -7,8,9,10). This technique has also been used to investigate the mechanism of drug action and resistance as well as for screening of potential new drugs(11-13). The treatment of choice of human visceral leishmaniasis is the administration of pentavalent antimony (Sb V )-containing drugs such as sodium stibogluconate (Pentostam, Wellcome, Beckenham, UK) or meglumine antimoniate (Glucantime, Rhone-Poulenc, Paris, France). Despite extensive use of these compounds over the last decades, the mechanism of action remains unclear.It has been hypothesized that Sb V is not toxic to Leishmania, but rather that it is enzymatically reduced, probably by the host macrophage, to Sb III , the form of antimony that is highly Leishmania-toxic (14 -18). In contrast, it has been shown that Sb V is directly toxic to axenic amastigotes (12,13,19), thus negating the necessity of the macrophage for Sb V reduction. Furthermore, these data imply that either the parasite reduces Sb V to Sb III intracellularly, and thus Sb III is directly toxic to amastigotes, or that both oxidation states of antimony are active against Leishmania amastigotes. The modes of action of the two oxidation states of antimony (Sb III and Sb V ) on Leishmania are, as yet, not fully understood.Several groups have shown obligatory cross-resistance between Sb V , Sb III , and arsenite (As III ) in Leishmania tarentolae, Leishmania major, Leishmania mexicana, L. donovani and Leishmania infantum (11,[20][21][22]. In contrast, it has been suggested that, at least in L. donovani, such cross-resistance does not necessarily occur (12). Furthermore, it has been demonstrated that, when bound to organic compounds, structural similarities exist between Sb III and Sb V (23). For example, the trivalent antimony comp...

LeishmaniaTDR1 structure, a unique trimeric glutathione transferase capable of deglutathionylation and antimonial prodrug activation

“…However, TDR1, in the presence of glutathione, catalyzes the reduction of Sb(V) in vitro and hence can mediate activation of the antimonial prodrugs (1). This finding correlates with the observation that in Leishmania major the enzyme is significantly more abundant in the form of the parasite (the amastigote) that infects the mammalian host than in the promastigote or insect form and that the amastigotes are strikingly more sensitive to Sb(V) than promastigotes (1,6).…”

Dual Action of Antimonial Drugs on Thiol Redox Metabolism in the Human Pathogen Leishmania donovani

“…Although the mechanism of reduction of Sb V is not understood, the selective toxicity of Sb V for LdBOB axenic amastigotes (Fig. 5) supports the contention that only the amastigote stage is capable of reducing Sb V to Sb III (5). Trypanothione has been reported to nonenzymatically reduce Sb V to Sb III , particularly under acidic conditions (36).…”

An in vitro system for developmental and genetic studies of Leishmania donovani phosphoglycans

“…The primary anti-leishmanial agents used clinically are pentavalent antimonial compounds [33], and the LdB amastigotes were 30-fold more susceptible to sodium stibogluconate than promastigotes (166 g/ml versus >5000 g/ml; Table 2). This differential stems from the ability of amastigotes to reduce SbV to toxic SbIII derivatives [34], and accordingly the susceptibility of amastigotes and promastigotes were less pronounced with SbIII (3.5 g/ml versus 14 g/ml; Table 2). This was also seen when promastigotes were grown in amastigote media at 26 • C, suggesting that the effect arises from parasite stage, and not the acid media pH or other factors.…”

“…Another example of this concerns our studies of LPG biosynthetic genes, discussed below. Other investigators have emphasized the importance of studying drug susceptibility and metabolism in the amastigote form of the parasite, and have shown that in many cases in vitro amastigotes offer good models for this [13][14][15]34]. In this respect LdB seems to offer similar advantages; for example, LdB amastigotes are much more sensitive to the pentavalent antimionials than promastigotes (Table 2), as seen in amastigotes grown in macrophages.…”