Cysteine biosynthesis in Leishmania

Every year 2 million people are diagnosed with leishmaniasis and 350 million are at risk of becoming infected. Spread throughout 88 countries in the world, leishmaniasis is a group of diseases comprising visceral, mucocutaneous and cutaneous leishmaniasis as the main forms. Visceral leishmaniasis is the most severe form of the disease and is caused by Leishmania donovani. The parasite, Leishmania, is transmitted to humans by a sandfly vector. The absence of vector-control procedures and effective vaccines for humans makes chemotherapy the only weapon against leishmaniasis. Great efforts have been made to develop new drugs against these diseases, however toxic side effect and the constant cases of resistance call for new drug targets and drugs to be discovered and developed. Cysteine is a key building block of trypanothione, an antioxidant unique to trypanosomatids that plays a pivotal role for the survival of the parasites. Leishmania can obtain cysteine in two ways, using the sulphydrylation and trans-sulphuration pathways. Humans lack the sulphydrylation pathway, thus this, and especially cysteine synthase (CS), of Leishmania could be a potential drug target. In order to determine the relative importance of these pathways, the levels of thiols at different stages of promastigote growth of wild-type, mutants lacking CS (deltacs), and CS episomal re-expressor parasite lines were determined. It was found that during logarithmic phase the mutant parasites have significantly reduced levels of thiols, which is reversed in the CS re-expressing parasite line. The mRNA and protein levels of cystathionine β-synthase (CBS) were increased in deltacs L. donovani, while this decrease was reversed in the CS re-expessing line. These data suggest that the reverse trans-sulphuration pathway compensates for the loss of CS to some extent but that this is not sufficient to maintain thiol levels during logarithmic growth. It was further found that ornithine decarboxylase mRNA is up-regulated while the protein levels appear to be reduced in the deltacs parasite line. The latter was supported by the finding that the deltacs mutant parasites have low sensitivity to alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase. The studies on the recombinant L. major cysteine synthase (CS) have shown that it can be inhibited by small peptides based on the C-terminal of L. major serine acetyltransferase. The CPM assay showed promising results to be used in high throughput screening of CS inhibitors. The data overall suggests that the levels of thiols are dependent on an adequate supply of cysteine through the sulphydrylation pathway. How this affects polyamine biosynthesis is yet to be investigated.