Co-amplification 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 multifactorial. 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 one of these novel genes. It corresponds to gsh1, which encodes gamma-glutamylcysteine synthetase, an enzyme involved in the rate-limiting step 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 metals. However, when gsh1 was transfected in partial revertants, it conferred resistance. As pgpA is frequently co-amplified with gsh1, we co-transfected the two genes into both wild-type and partial revertants. Arsenite 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 previous suggestion that PgpA recognizes metals conjugated to thiols. Furthermore, amplification of gsh1 overcomes the rate-limiting step in the synthesis of trypanothione, contributing to resistance. In addition, the results suggest that at least one more factor acts synergistically with the gsh1 gene product.
Grondin
K
K
Centre de Recherche en Infectiologie du Centre de Recherche du CHUL and Département de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada.
Haimeur
A
A
Mukhopadhyay
R
R
Rosen
B P
BP
Ouellette
M
M
D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D018053:Arsenites / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics; D005784:Gene Amplification; D017125:Genes, Protozoan; D005822:Genetic Vectors; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D007891:Leishmania / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000032:analysis; D014162:Transfection
D018528:ATP-Binding Cassette Transporters; D018053:Arsenites; D008562:Membrane Glycoproteins; C106891:PgpA protein, Leishmania; D015800:Protozoan Proteins; D013438:Sulfhydryl Compounds; D005721:Glutamate-Cysteine Ligase; C015001:arsenite