RESUMO
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.
Assuntos
Genes Essenciais , Glutamato-Cisteína Ligase/fisiologia , Leishmania infantum/enzimologia , Leishmania infantum/fisiologia , Oxidantes/toxicidade , Proteínas de Protozoários/fisiologia , Estresse Fisiológico , Animais , Antimônio/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Deleção de Genes , Teste de Complementação Genética , Glutamato-Cisteína Ligase/genética , Glutationa/análogos & derivados , Glutationa/biossíntese , Leishmania infantum/genética , Macrófagos/imunologia , Macrófagos/parasitologia , Proteínas de Protozoários/genética , Espermidina/análogos & derivados , Espermidina/biossínteseRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antimônio/farmacologia , Glutationa/análogos & derivados , Leishmania infantum/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Espermidina/análogos & derivados , Animais , Gluconato de Antimônio e Sódio/farmacologia , Linhagem Celular , Cisteína/metabolismo , DNA de Protozoário/biossíntese , DNA de Protozoário/genética , Resistência a Medicamentos , Glutationa/metabolismo , Leishmania infantum/crescimento & desenvolvimento , Microscopia Confocal , Monócitos/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Espermidina/metabolismo , TransfecçãoRESUMO
In the protozoan parasite Leishmania, drug resistance can be a complex phenomenon. Several metabolic pathways and membrane transporters are implicated in the resistance phenotype. To monitor the expression of these genes, we generated custom DNA microarrays with PCR fragments corresponding to 44 genes involved with drug resistance. Transcript profiling of arsenite and antimony resistant mutants with these arrays pinpointed a number of genes overexpressed in mutants, including the ABC transporter PGPA, the glutathione biosynthesis genes gamma-glutamylcysteine synthetase (GSH1) and the glutathione synthetase (GSH2). Competitive hybridisations with total RNA derived from sensitive and methotrexate resistant cells revealed the overexpression of genes coding for dihydrofolate reductase (DHFR-TS), pteridine reductase (PTR1) and S-adenosylmethionine synthase (MAT2) and a down regulation of one gene of the folate transporter (FT) family. By labelling the DNA of sensitive and resistant parasites we could also detect several gene amplification events using DNA microarrays including the amplification of the S-adenosyl homocysteine hydrolase gene (SAHH). Alteration in gene expression detected by microarrays was validated by northern blot analysis, while Southern blots indicated that most genes overexpressed were also amplified, although other mechanisms were also present. The microarrays were useful in the study of resistant parasites to pinpoint several genes linked to drug resistance.
