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Abstract: Parasitic protozoa are unable to synthesise purines de novo and thus depend on the uptake of nucleosides and nucleobases across their plasma membrane through specific transporters. A number of nucleoside and nucleobase transporters from Trypanosoma brucei brucei and Leishmania major have recently been characterised and shown to belong to the equilibrative nucleoside transporter (ENT) family. A number of studies have demonstrated the functional importance of particular transmembrane segments (TMS) in nucleoside-specific ENT proteins. TbNBT1, one of only three bona fide nucleobase-selective members of the ENT family, has previously been shown to be a high-affinity transporter for purine nucleobases and guanosine. In this study, we use the Saccharomyces cerevisiae expression system to build a biochemical model of how TbNBT1 recognises nucleobases. We next performed random in vitro and site-directed mutagenesis to identify residues critical for TbNBT1 function. The identification of residues likely to contribute to permeant binding, when combined with a structural model of TbNBT1 obtained by homology threading, yield a tentative three-dimensional model of the transporter binding site that is consistent with the binding model emerging from the biochemical data. The model strongly suggests the involvement of TMS5, TMS7 and TMS8 in TbNBT1 function. This situation is very similar to that concerning transporters of the major facilitator superfamily (MFS), one of which was used as a template for the threading. This point raises the possibility that ENT and MFS carriers, despite being considered evolutionarily distinct, might in fact share similar topologies and substrate translocations pathways. [Copyright &y& Elsevier]

Abstract: As in the countries edging the Mediterranean basin, Leishmania infantum zymodeme MON-1 is the main causative agent of visceral leishmaniasis in Morocco, where visceral leishmaniasis is most active in the North-Eastern slopes of the Rif mountains. The dog was confirmed to be the main reservoir of L. infantum MON-1, while the reservoir of L. infantum MON-24 causative agent of both infantile visceral leishmaniasis and cutaneous leishmaniasis has not yet been identified. Here we report the first detection of this last zymodeme in a dog in Morocco. The isolated strain was first identified by the use of genotyping markers and confirmed by isoenzyme analysis. Phylogenetic analysis with the use of concatenated sequences from 26 Leishmania donovani complex strains revealed strong geographical correlation with the MON-24 strain from Morocco clustering with other East African strains whereas two other MON-24 strains clustered with L. infantum strains. Interestingly, the two distinct populations of MON-24 identified with the use of genotyping markers cannot be distinguished by multilocus enzyme electrophoresis. [Copyright &y& Elsevier]