Study on the roles of O-acetylserine (thiol) lyase and thiol dependent reductase 1 of Leishmania

Thiol dependent reductase 1 (TDR1) of Leishmania has been implicated in both the activation of pentavalent antimonial drugs and in the generation of drug-thiol conjugates facilitating drug-resistance. Reverse genetic studies were carried out on TDR1 to elucidate the role of the enzyme and to assess its potential value as a drug target against Leishmania. In a similar study, O¬acetylserine (thiol) lyase (OAS-TL), a key enzyme for the de novo synthesis of cysteine in Leishmania, was investigated as a potential target for new antileishmanial drugs. TDR1 of Leishmania is a 49.9 kDa protein of which the physiological role remains unclear. The protein has shown both thiol transferase activity and dehydroascorbate reductase activity. Due to its ability to reduce pentavalent antimonials to the active trivalent form in vitro, TDR1 has been suggested as playing a vital role in antimonial resistance in Leishmania. This part of the study was undertaken to clarify the role TDR1 plays in the parasite by investigating the effects of deleting the gene. Attempts were made to generate Δtdr1 null mutants in Leishmania donovani, but these were unsuccessful despite the fact that Δtdr1 null mutants exist for L. major and L. infantum. The mutants of these latter lines were studied to discover more on the roles of the proteins. L. major and L. infantum Δtdr1 null mutant promastigotes grow normally and do not display any change in total intracellular levels of cysteine, glutathione and trypanothione. The L. major Δtdr1 null mutants were able to survive and proliferate in parasitophorous vacuoles of peritoneal macrophages in vitro, with significantly higher numbers of parasites per infected macrophage compared to L. major wild-type. This suggests that the loss of TDR1 is beneficial to L. major when establishing an infection in macrophages. However the loss of TDR1 also causes hypersensitivity to the antimonial drug sodium stibogluconate, under the conditions tested. The data generated in this study indicate that the physiological function of TDR1 does not lie in the activation of pentavalent antimonials as has been previously suggested. The sulfur-containing amino acid cysteine plays a vital role in the synthesis of low molecular weight thiols, e.g. glutathione and trypanothione, as well as redox active thiol-containing proteins. In addition, cysteine is important for the stabilisation of tertiary and quaternary protein conformation due to its ability to form inter- and intra-chain disulfide bonds with other cysteine residues. In mammals, cysteine can either be taken up from the environment, or synthesised via the reverse trans-sulfuration pathway, involving the action of the enzymes cystathionine β-synthase and cystathionine γ-lyase, to generate cysteine from the essential amino acid methionine. In contrast, Leishmania parasites can synthesise cysteine in two ways but appear unable to salvage it effectively. They contain the reverse trans-sulfuration pathway, similar to mammals, and additionally, can generate cysteine through the sulfhydrylation pathway from serine, coenzyme A and sulfide, by utilising the enzymes serine acetyltransferase and O-acetylserine (thiol) lyase (OAS-TL). The aim of this study was to assess the suitability of OAS-TL as a potential drug target against Leishmania. In this study, Δoas-tl null mutants were generated in L. donovani, thus negating the sulfhydrylation pathway. The Δoas-tl null mutant promastigotes displayed a slight growth defect as well as a severe morphological alterations directly affecting cell body and flagellum length. In addition, the Δoas-tl null mutants were unable to survive in the parasitophorous vacuoles of peritoneal macrophages in vitro, suggesting that the exogenous supply of a source of cysteine (such as methionine) was not sufficiently high to support parasite proliferation. The finding that addition of high methionine concentrations to the medium facilitates parasite survival supports this idea. The data show that either differentiation of promastigotes into amastigotes or proliferation of amastigotes is detrimentally affected by the deletion of OAS-TL. Lines re-expressing OAS-TL were also generated in the Δoas-tl null mutants and were found to complement the phenotypes of the Δoas-tl null mutants identified in this study. The inability of Leishmania Δoas-tl null mutants to survive within macrophages, together with the absence of OAS-TL in the mammalian host, make it a suitable candidate for the identification of new drug targets in the search for novel chemotherapeutic agents against leishmaniasis.