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Developmental regulation of mRNA levels in trypanosomatid protozoa is determined post-transcriptionally and often involves sequences located in the 3-untranslated regions (3-UTR) of the mRNAs. We have previously identified a developmentally regulated gene family in Leishmania encoding the amastin surface proteins and showed that stage-specific accumulation of the amastin mRNA is mediated by sequences within the 3-UTR. Here we identified a 450-nt region within the amastin 3-UTR that can confer amastigote-specific gene expression by a novel mechanism that increases mRNA translation without an increase in mRNA stability. Remarkably, this 450-nt 3-UTR element is highly conserved among a large number of Leishmania mRNAs in several Leishmania species. Here we show that several of these mRNAs are differentially expressed in the intracellular amastigote stage of the parasite and that the 450-nt conserved element in their 3-UTRs is responsible for stage-specific gene regulation. We propose that the 450-nt conserved element, which is unlike any other regulatory element identified thus far, is part of a common mechanism of stage-regulated gene expression in Leishmania that regulates mRNA translation in response to intracellular stresses.Parasites of the genus Leishmania cause cutaneous, mucocutaneous, and visceral infections affecting ϳ400,000 people each year of the 397 million that are at risk worldwide (1). During its digenetic life cycle, Leishmania alternates between the alimentary tract of the sand fly vector as an extracellular promastigote and the acidic phagolysosomes of macrophages as an intracellular amastigote. Differentiation of the parasite into the amastigote form is a prerequisite for its intracellular survival. Several environmental factors including acidic pH, elevated temperature, and the harmful phagolysosomal milieu trigger cytodifferentiation accompanied by the differential expression of a variety of genes (2-6). Such stage-specific gene expression is crucial for adaptation because Leishmania differentiates from an extracellular to an intracellular parasite. Gene regulation in Leishmania and related trypanosomatids shares unique features that include polycistronic transcription of large RNA units by an ␣-amanitin-sensitive RNA polymerase II, probably in the absence of promoter elements, and pre-mRNA processing into monocistronic mRNAs through a post-transcriptional control mediated by trans-splicing and polyadenylation (7-9). trans-Splicing and polyadenylation are mechanistically coupled in trypanosomatids and recognize regulatory signals that consist of polypyrimidine-rich sequences (10,11).Numerous examples in Leishmania species support the notion that developmental regulation of mRNA levels is determined post-transcriptionally by sequences located in the 3Ј-untranslated regions (3Ј-UTR) 1 that usually control mRNA stability (12-17). More recently, a novel mechanism of stagespecific regulation affecting pre-mRNA processing has been reported in Leishmania mexicana (18). The role of 3Ј-UTRs and/or int...

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Monitoring Trypanosoma spread using real-time imaging in vivo provides a fast method to evaluate parasite distribution especially in immunoprivileged locations. Here, we generated monomorphic and pleomorphic recombinant Trypanosoma brucei expressing the Renilla luciferase. In vitro luciferase activity measurements confirmed the uptake of the coelenterazine substrate by live parasites and light emission. We further validated the use of Renilla luciferase-tagged trypanosomes for real-time bioluminescent in vivo analysis. Interestingly, a preferential testis tropism was observed with both the monomorphic and pleomorphic recombinants. This is of importance when considering trypanocidal drug development, since parasites might be protected from many drugs by the blood-testis barrier. This hypothesis was supported by our final study of the efficacy of treatment with trypanocidal drugs in T. brucei-infected mice. We showed that parasites located in the testis, as compared to those located in the abdominal cavity, were not readily cleared by the drugs.

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