Co-amplification of the γ-glutamylcysteine synthetase gene gsh1 and of the ABC transporter gene pgpA in arsenite-resistant Leishmania tarentolae also for the related metal arsenic . Katherine Grondin, Anass Haimeur,Gene amplification is observed frequently in arseniteRita Mukhopadhyay 1 , Barry P.Rosen 1 and resistant Leishmania cells (Detke et al., 1989; Marc Ouellette 2 Grondin et al., 1993;Singh et al., 1994) (Borst and Ouellette, 1995). PgpA is more similar to e-mail: marc.ouellette@crchul.ulaval.ca the human multidrug-associated protein MRP than to P-glycoproteins (Cole et al., 1992;Légaré et al., 1994). Resistance to the oxyanion arsenite in the parasiteTransfection experiments implicated pgpA in both arsenite Leishmania is multifactorial. We have described preand antimonite resistance, but resistance levels differed viously the frequent amplification of the ABC transdepending on which pgpA allele was used and in which porter gene pgpA, the presence of a non-PgpA thiolLeishmania species the genes were transfected (Callahan metal efflux pump and increased levels of glutathione and Beverley, 1991;Papadopoulou et al., 1994a). and trypanothione in resistant cells. Other loci are alsoWe have also observed, in L.tarentolae mutants selected amplified, although their role in resistance is unknown.for resistance to arsenite or antimonite, the presence of a By gene transfection, we have characterized one of non-pgpA efflux system (Dey et al., 1994; Papadopoulou these novel genes. It corresponds to gsh1, which encodes et al., 1996) that recognizes metals conjugated to various γ-glutamylcysteine synthetase, an enzyme involved in thiols including glutathione (GSH) and the rate-limiting step of glutathione biosynthesis.trypanothione (TSH) (Mukhopadhyay et al., 1996). GSH Transfection of gsh1 in wild-type cells increased the is the tripeptide γ-Glu-Cys-Gly and plays an important levels of glutathione and trypanothione to levels found role in the cellular defense against oxidative stress and in in resistant mutants. These transfectants were not the detoxification of several drugs and xenobiotics (Meister resistant to metals. However, when gsh1 was transfected and Anderson, 1983). TSH (Fairlamb et al., 1985), which in partial revertants, it conferred resistance. As pgpA consists of a spermidine moiety linked to two GSH is frequently co-amplified with gsh1, we co-transfected molecules, is the major reduced thiol in Leishmania the two genes into both wild-type and partial revertants. (Fairlamb and Cerami, 1992). Recently, we found a large Arsenite resistance levels in wild-type cells could be increase in TSH and smaller increases in cysteine and accounted for by the contribution of PgpA alone. In GSH in arsenite-resistant cell lines (Mukhopadhyay the partial revertant, the gsh1 and pgpA gene product et al., 1996). acted synergistically. These results support our previous suggestion that PgpA recognizes metals conjugResistance to oxyanions in Leishmania is, therefore, ated to thiols. Furthermore, amplification of gsh1 c...

No abstract

The protozoan parasite Leishmania often responds to drug pressure by amplifying part of its genome. At least two loci derived from the same 800 kb chromosome were amplified either as extrachromosomal circles or linear fragments after sodium arsenite selection. A 50 kb linear amplicon was detected in six independent arsenite mutants and revertants grown in absence of arsenite rapidly lost the amplicon and part of their resistance. The circular extrachromosomal amplicons, all derived from the H locus of Leishmania, were characterized more extensively. In all cases, direct repeated sequences appeared to be involved in the formation of circular amplicons. Most amplicons were generated after homologous recombination between two linked P-glycoprotein genes. This recombination event was, in two cases, associated with the loss of one allele of the chromosomal copy. A novel rearrangement point was found in a mutant where the amplicon was created by recombination between two 541 bp direct repeats surrounding the P-glycoprotein gene present at the H locus. It is also at one of these repeats that an H circle with large inverted duplications was formed. We propose that the presence of repeated sequences in the H locus facilitates the amplification of the drug resistance genes concentrated in this locus.