Parasitic protozoa belonging to the order Kinetoplastida contain trypanothione as their major thiol. Trypanothione reductase (TR), the enzyme responsible for maintaining trypanothione in its reduced form, is thought to be central to the redox defence systems of trypanosomatids. To investigate further the physiological role of TR in Leishmania, we attempted to create TR‐knockout mutants by gene disruption in L.donovani and L.major strains using the selectable markers neomycin and hygromycin phosphotransferases. TR is likely to be an important gene for parasite survival since all our attempts to obtain a TR null mutant in L.donovani failed. Instead, we obtained mutants with a partial trisomy for the TR locus where, despite the successful disruption of two TR alleles by gene targeting, a third TR copy was generated as a result of genomic rearrangements involving the translocation of a TR‐containing region to a larger chromosome. Mutants of L.donovani and L.major possessing only one wild‐type TR allele express less TR mRNA and have lower TR activity compared with wild‐type cells carrying two copies of the TR gene. Significantly, these mutants show attenuated infectivity with a markedly decreased capacity to survive intracellularly within macrophages, provided that the latter are producing reactive oxygen intermediates.

No abstract

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...

We have previously shown that the Leishmania genome possess two widespread families of extinct retroposons termed Short Interspersed DEgenerated Retroposons (SIDER1/2) that play a role in post-transcriptional regulation. Moreover, we have demonstrated that SIDER2 retroposons promote mRNA degradation. Here we provide new insights into the mechanism by which unstable Leishmania mRNAs harboring a SIDER2 retroposon in their 3′-untranslated region are degraded. We show that, unlike most eukaryotic transcripts, SIDER2-bearing mRNAs do not undergo poly(A) tail shortening prior to rapid turnover, but instead, they are targeted for degradation by a site-specific endonucleolytic cleavage. The main cleavage site was mapped in two randomly selected SIDER2-containing mRNAs in vivo between an AU dinucleotide at the 5′-end of the second 79-nt signature (signature II), which represents the most conserved sequence amongst SIDER2 retroposons. Deletion of signature II abolished endonucleolytic cleavage and deadenylation-independent decay and increased mRNA stability. Interestingly, we show that overexpression of SIDER2 anti-sense RNA can increase sense transcript abundance and stability, and that complementarity to the cleavage region is required for protecting SIDER2-containing transcripts from degradation. These results establish a new paradigm for how unstable mRNAs are degraded in Leishmania and could serve as the basis for a better understanding of mRNA decay pathways in general.

Pterins are essential for the growth of Leishmania species, and recent work has led to the isolation of the biopterin transporter BT1. In this study, we inactivated the Leishmania donovani biopterin transporter BT1 by gene disruption mediated by homologous recombination. No transport of biopterin was detected in this mutant. The L. donovani BT1 null mutant showed a much lesser capacity for inducing infection in mice than wild-type parasites and could elicit protective immunity in mice susceptible to infection against a L. donovani challenge. Splenocytes isolated from mice immunized with the BT1 null mutant parasites produced significant amounts of interferon gamma following stimulation with L. donovani promastigotes as measured by enzyme-linked immunosorbent assay and enzyme-linked immunospot assays. Overall, these results show that by genetically manipulating the pterin transport in L. donovani, it is possible to generate an attenuated organism that could be part of a vaccination strategy.Leishmania is a protozoan parasite that is distributed worldwide, being endemic in 88 countries. Each year, 1.5 million new cases of cutaneous leishmaniasis and 500,000 new cases of visceral leishmaniasis are estimated (15). The latter, caused mainly by Leishmania donovani, can be fatal if left untreated. The treatment relies mainly on chemotherapy, and the mainstay consists in different formulations of pentavalent antimony, although alternatives, such as liposomal amphotericin B, are also effective (3, 15). Nonetheless, in some parts of the world, notably in the state of Bihar, India, more than 50% of the patients are unresponsive or relapse after conventional chemotherapy (42). Several approaches (killed parasites with or without BCG [a naturally attenuated form of Mycobacterium bovis]; subunit vaccines; DNA vaccines; attenuated organisms) have been used as vaccination strategies against Leishmania, but none has yet translated into an effective product (see references 14 and 31 for recent reviews).With the advent of gene transfection technology with Leishmania, a number of innovative approaches were used in attempts to generate vaccines. These include the inactivation of genes encoding enzymes thought to be important for parasite intracellular survival, including the dihydrofolate reductase gene (43), the cysteine proteinase genes (2), the HSP100 heat shock protein (16), and the trypanothione reductase (10; also unpublished data). Other approaches relying on the expression of antisense RNA (45) or of cytotoxic genes (26) were also used. Most of this work was done with Leishmania species giving rise to cutaneous lesions, and with the exception of antisense RNA, no recombinant attenuated L. donovani organisms produced by gene inactivation have been reported. In order to generate attenuated L. donovani organisms for vaccination purposes, we have targeted genes thought to be important for intracellular survival. Our first attempt was to inactivate trypanothione reductase (10