Studies on a thiol-dependent reductase and ascorbate metabolism of leishmania

The intracellular protozoan parasite Leishmania causes leishmaniasis, a disease which is most prevalent in tropical and sub-tropical countries where it infects some two million people every year and kills around 60,000 of them. For decades pentavalent antimonial compounds have been the standard first-line drugs used to treat the disease and this remains the case despite increasing reports of drug-resistance. The mode of action of these drugs is not entirely understood, although it is generally accepted that in vivo reduction of the compounds from the pentavalent to a trivalent form is required for antileishmanial activity. The site of antimonial conversion and whether the reaction is catalysed by an enzyme remain controversial points. However, it was recently reported that L. donovani amastigotes were capable of reducing pentavalent antimonials to the trivalent form and that drug-resistant parasites were deficient in this activity, suggesting that a parasite enzyme did mediate drug toxicity. The identity of such an enzyme was investigated in this study. Arsenical and antimonial compounds are similar and several classes of proteins that exhibit arsenate reductase activity have been previously identified in other organisms. Whether Leishmania possessed an enzyme akin to one of these was assessed by attempting to purify enzymes from parasite lysates and by searching the L. major genome database for similar sequences to the arsenate reductases. The latter approach was successful and a gene fragment was identified that shared similarity with omega glutathione S-transferases (oGSTs), a class of glutaredoxin-like GSTs which are capable of reducing pentavalent methylated arsenicals in vitro. The sequence of the complete L. major gene was elucidated by 5' RACE, and was found to encode a protein twice the expected size with similar 3' and 5' halves. The protein was named thiol-dependent reductase, or TDRl. Active recombinant protein was successfully produced and its biochemical activities were found to coincide with oGSTs: TDRl was capable of reducing pentavalent arsenical and antimonial compounds to trivalent species, and possessed thioltransferase and dehydroascorbate reductase activities usually associated with glutaredoxins. TDRl, which was shown to probably reside in the parasite cytosol but may also be secreted, was found to be more abundant in amastigote than promastigote forms, which correlates with the antileishmanial stage-specificity of pentavalent antimonials. L. major TDRl knockout mutants were generated, and the protein was also over-expressed in parasites. Both these genetic manipulations resulted in mutants with enhanced infectivity. TDRl knockout parasites were more susceptible than wild type parasites to paraquat, which induces the production of intracellular superoxide. As its glutaredoxin-like in vitro activities suggest, this implies TDRl has a role in protecting the parasites from oxidative stress, although re-expression of TDRl did not reinstate resistance. (Abstract shortened by ProQuest.)