Gaétan Roy

Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models.

We have expressed the reporter firefly luciferase gene (LUC) in Leishmania donovani and Leishmania major either as part of episomal vectors or integrated into the parasite genome under the control of their respective ribosomal promoter regions. An excellent linear correlation between parasite number and luciferase activity was observed with all the transfectants. LUC-expressing recombinant parasites were useful to monitor Leishmania spp. infections in macrophages or in animal models. For prolonged growth in absence of drug selection, such as within animal models, quantitation of parasites is more reliable when the reporter gene LUC is stably integrated in the parasite genome. These recombinant strains should be useful tools to monitor Leishmania growth under a number of conditions.

DNA Transformation of Leishmania infantum Axenic Amastigotes and Their Use in Drug Screening

ABSTRACT Protocols for DNA electroporation in Leishmaniapromastigote cells are well established. More recently, in vitro culture of axenic Leishmania amastigotes became possible. We have established conditions for DNA transformation of axenically grown Leishmania infantum amastigotes. Parameters for DNA electroporation of Leishmania axenic amastigotes were systematically studied using luciferase-mediated transient transfection. Cell lines expressing stable luciferase activity were then selected, and their ability to be used in an in vitro drug screening procedure was determined. A model was established, using axenic amastigotes expressing luciferase activity, for rapidly determining the activity of drugs directly against both axenic and intracellular amastigotes. For intracellular amastigotes, the 50% effective concentrations of pentamidine, sodium stibogluconate (Pentostam), meglumine (Glucantime), and potassium antimonyl tartrate determined with the luciferase assay were 0.2 μM (0.12 μg/ml), 55 μg/ml, 95 μg/ml, and 0.12 μg/ml, respectively; these values are in agreement with values determined by more labor-intensive staining methods. We also showed the usefulness of luciferase-expressing parasites for analyzing drug resistance. The availability of luciferase-expressing amastigotes for use in high-throughput screening should facilitate the search for new antileishmanial drugs.

High level arsenite resistance in Leishmania tarentolae is mediated by an active extrusion system.

Leishmania tarentolae cells selected for resistance to the oxyanions pentavalent or trivalent antimonials or to trivalent arsenicals exhibited cross-resistance to the other oxyanions. The basis for resistance in these mutants was studied by transport experiments using radioactive arsenite. All mutants exhibiting high level resistance to arsenite showed a marked decrease in the steady-state accumulation of arsenite. Decreased accumulation was also observed in antimonials-resistant mutants cross-resistant to various concentrations of arsenite. Cells depleted of endogenous energy reserves with metabolic inhibitors were loaded with radioactive arsenite; following addition of glucose, rapid efflux of arsenite was observed from arsenite mutant cells. Mutants resistant to high levels of arsenicals exhibited amplification of the P-glycoprotein related gene ltpgpA or of a linear amplicon of unknown function. However, the efflux-mediated arsenite resistance did not correlate with the amplification of the ltpgpA gene or with the presence of the linear amplicon. The calcium channel blocker verapamil and arsenite act in synergy in cells exhibiting the efflux system. Overall the oxyanion efflux system in Leishmania shares several properties with other resistance efflux systems mediated by transporters.

Role of the ABC Transporter MRPA (PGPA) in Antimony Resistance in Leishmania infantum Axenic and Intracellular Amastigotes

ABSTRACT Antimonial compounds are the mainstay for the treatment of infections with the protozoan parasite Leishmania. We present our studies on Leishmania infantum amastigote parasites selected for resistance to potassium antimonyl tartrate [Sb(III)]. Inside macrophages, the Sb(III)-selected cells are cross-resistant to sodium stibogluconate (Pentostam), the main drug used against Leishmania. Putative alterations in the level of expression of more than 40 genes were compared between susceptible and resistant axenic amastigotes using customized DNA microarrays. The expression of three genes coding for the ABC transporter MRPA (PGPA), S-adenosylhomocysteine hydrolase, and folylpolyglutamate synthase was found to be consistently increased. The levels of cysteine were found to be increased in the mutant. Transfection of the MRPA gene was shown to confer sodium stibogluconate resistance in intracellular parasites. This MRPA-mediated resistance could be reverted by using the glutathione biosynthesis-specific inhibitor buthionine sulfoximine. These results highlight for the first time the role of MRPA in antimony resistance in the amastigote stage of the parasite and suggest a strategy for reversing resistance.

Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species

The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolaes gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage.

Reduced infectivity of a Leishmania donovani biopterin transporter genetic mutant and its use as an attenuated strain for vaccination.

ABSTRACT 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.

Pterin transport and metabolism in Leishmania and related trypanosomatid parasites.

The folate metabolic pathway has been exploited successfully for the development of antimicrobial and antineoplasic agents. Inhibitors of this pathway, however, are not useful against Leishmania and other trypanosomatids. Work on the mechanism of methotrexate resistance in Leishmania has dramatically increased our understanding of folate and pterin metabolism in this organism. The metabolic and cellular functions of the reduced form of folates and pterins are beginning to be established and this work has led to several unexpected findings. Moreover, the currently ongoing sequencing efforts on trypanosomatid genomes are suggesting the presence of several gene products that are likely to require folates and pterins. A number of the properties of folate and pterin metabolism are unique suggesting that these pathways are valid and worthwhile targets for drug development.

Autonomous replication of bacterial DNA plasmid oligomers in Leishmania

Extrachromosomal amplicons are frequently observed in drug-resistant Leishmania. A dominant selectable marker, the neomycin phosphotransferase gene, was introduced by gene targeting in a circular amplicon derived from the H locus of Leishmania in a mutant cell. This recombinant amplicon was isolated and transfected in a wild-type cell. The amplicon was kept in the wild-type cells, provided the selective pressure was maintained, suggesting that it was capable of autonomous replication. Novel Leishmania expression vectors suited for stable transfections were made to isolate, by a high transformation assay, the putative origin of replication in the amplicons. However, these plasmids, which did not contain a single Leishmania nucleotide, were found as extrachromosomal circular oligomers in Leishmania transfectants. Their relative stability, in addition to changes in their methylation pattern, indicated that these plasmids were most likely replicating. No specific sequences seem to be required for replication (and expression) in Leishmania, therefore precluding the isolation of origins of replication by genetic transformation.

The γ-glutamylcysteine synthetase gene of Leishmania is essential and involved in response to oxidants

Gamma‐glutamylcysteine synthetase, encoded by the GSH1 gene, is the rate‐limiting enzyme in the biosynthesis of glutathione and of trypanothione in Leishmania. The importance of GSH1 was assessed by generating GSH1 null mutants in Leishmania infantum. Removal of even a single wild‐type allelic copy of GSH1 invariably led to the generation of an extra copy of GSH1, maintaining two intact wild‐type alleles. However, by first supplementing the parasites with a rescue plasmid, we succeeded in obtaining both a single and null chromosomal GSH1 mutants. Parasites with one intact GSH1 chromosomal allele lost the rescuing plasmid but not the double knockout, when grown in the absence of antibiotic, indicating the essentiality of the GSH1 gene in Leishmania. Heterozygous mutants with one allele‐inactivated transcribed less GSH1 mRNA and synthesized less glutathione and trypanothione. These mutants were more susceptible to oxidative stresses in vitro as promastigotes and showed decreased survival inside activated macrophages producing reactive oxygen or nitrogen species. These mutants showed a significant decreased survival in the presence of antimony (SbV) compared with control cells. All phenotypes were reverted in the add‐back mutant, thus proving the importance of thiols in dealing with oxidants including the action of antimonials.

ABC transporters in Leishmania and their role in drug resistance.

ABC transporters have been found in several parasitic protozoa including Leishmania. At least two Leishmania ABC transporters are involved in drug resistance. One is PgpA, which is involved in resistance to arsenic and antimony-containing compounds. Antimonials are the drug of choice against Leishmania infections. Transfection and biochemical studies suggest that PgpA recognizes metals conjugated to thiols. The second ABC transporter is closely related to mammalian P-glycoproteins and confers resistance to anticancer drugs by a mechanism that remains to be elucidated. Additional ABC transporters are likely to be present in Leishmania and these are discussed in relation to the phenomenon of antimony resistance.