RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite virulence.

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Abstract.Chagas disease (CD) is caused by the protozoan parasite Trypanosoma cruzi that infects a broad range of triatomines and mammalian species, including man. It afflicts 8 million people in Latin America, and its incidence is increasing in nonendemic countries owing to rising international immigration and nonvectorial transmission routes such as blood donation. Since the 1960s, the only drugs available for the clinical treatment of this infection have been benznidazole (BZ) and nifurtimox (NFX). Treatment with these trypanocidal drugs is recommended in both the acute and chronic phases of CD. These drugs have low cure rates mainly during the chronic phase, in addition both drugs present side effects that may result in the interruption of the treatment. Thus, more efficient and better-tolerated new drugs or pharmaceutical formulations containing BZ or NFX are urgently needed. Here, we review the drugs currently used for CD chemotherapy, ongoing clinical assays, and most-promising new experimental drugs. In addition, the mechanism of action of the commercially available drugs, NFX and BZ, the biodistribution of the latter, and the potential for novel formulations of BZ based on nanotechnology are discussed. Taken together, the literature emphasizes the urgent need for new therapies for acute and chronic CD.

Summary10-Formyl tetrahydrofolate (10-CHO-THF) is a key metabolite in C1 carbon metabolism, arising through the action of formate-tetrahydrofolate ligase (FTL) and/or 5,10-methenyltetrahydrofolate cyclohydrolase/ 5,10-methylene tetrahydrofolate dehydrogenase (DHCH). Leishmania major possesses single DHCH1 and FTL genes encoding exclusively cytosolic proteins, unlike other organisms where isoforms occur in the mitochondrion as well. Recombinant DHCH1 showed typical NADP + -dependent methylene tetrahydrofolate DH and 5,10-methenyltetrahydrofolate CH activities, and the DH activity was potently inhibited by a substrate analogue 5,10-CO-THF (Ki 105 nM), as was Leishmania growth (EC50 1.1 mM). Previous studies showed null ftl -mutants were normal, raising the possibility that loss of the purine synthetic pathway had rendered 10-CHO-THF dispensable in evolution. We were unable to generate dhch1 -null mutants by gene replacement, despite using a wide spectrum of nutritional supplements expected to bypass DHCH function. We applied an improved method for testing essential genes in Leishmania, based on segregational loss of episomal complementing genes rather than transfection; analysis of 1400 events without successful loss of DHCH1 again established its requirement. Lastly, we employed 'genetic metabolite complementation' using ectopically expressed FTL as an alternative source of 10-CHO-THF; now dhch1 -null parasites were readily obtained. These data establish a requirement for 10-CHO-THF metabolism in L. major, and provide genetic and pharmacological validation of DHCH as a target for chemotherapy, in this and potentially other protozoan parasites.