COAMPLIFICATION OF THE GAMMA-GLUTAMYLCYSTEINE SYNTHETASE GENE GSH1 AND OF THE ABC TRANSPORTER GENE PGPA IN ARSENITE-RESISTANT LEISHMANIA-TARENTOLAE

Resistance to the oxyanion arsenite in the parasite Leishmania is mult ifactorial, We have described previously the frequent amplification of the ABC transporter gene pgpA, the presence of a non-PgpA thiol-metal efflux pump and increased levels of glutathione and trypanothione in resistant cells, Other loci are also amplified, although their role in resistance is unknown, By gene transfection, we have characterized on e of these novel genes, It corresponds to gsh1, which encodes gamma-gl utamylcysteine synthetase, an enzyme involved in the rate-limiting ste p of glutathione biosynthesis, Transfection of gsh1 in wild-type cells increased the levels of glutathione and trypanothione to levels found in resistant mutants, These transfectants were not resistant to metal s, However, when gsh1 was transfected in partial revertants, it confer red resistance, As pgpA is frequently co-amplified with gsh1, we co-tr ansfected the two genes into both wild-type and partial revertants. Ar senite resistance levels in wild-type cells could be accounted for by the contribution of PgpA alone, In the partial revertant, the gsh1 and pgpA gene product acted synergistically. These results support our pr evious suggestion that PgpA recognizes metals conjugated to thiols, Fu rthermore, amplification of gsh1 overcomes the rate-limiting step in t he synthesis of trypanothione, contributing to resistance, In addition , the results suggest that at least one more factor acts synergistical ly with the gsh1 gene product.