Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii.
The goal of this work was to investigate the mechanism of drug resistance in Leishmania enriettii as a model system for drug resistance both in human leishmaniasis and on other parasitic diseases. Parasites were selected in increasing concentrations of vinblastine, an inhibitor of microtubule assembly, and resistant clones were isolated which grew in concentrations 5-30 times the IC50 (30 micrograms ml-1) of parental cells. The vinblastine-resistant parasites were also resistant to puromycin, an unrelated drug which inhibits protein synthesis. This cross-resistance to unrelated drugs had previously been observed in mammalian cells and recently in L. donovani. The proposed mechanism for this cross-resistance is drug efflux mediated by increased expression of a P-glycoprotein molecule encoded by a multidrug resistance (mdr) gene. Here we report the identification, cloning and sequencing of an mdr-like gene from L. enriettii, lemdr1, and demonstrate that this gene is amplified on an extrachromosomal circle of 35-40 kb in vinblastine-resistant L. enriettii. The longest open reading frame in the cloned gene is 1280 amino acids with a predicted protein of 140 kDa. The predicted protein has a structure similar to that for all other reported P-glycoproteins namely 12 transmembrane domains and 2 ATP binding sites, arranged in 2 similar half-molecules. Comparison of the primary amino acid sequence with other known mdr gene products demonstrates a significant homology with 37% amino acid identity with human mdr1 and 83% identity with the L. donovani ldmdr1 gene. The lemdr1 gene was cloned in the expression vector pALTNEO and transfected into wild-type L. enriettii and the resulting transfected cells were resistant to vinblastine but at lower levels than in the selected mutant cells.
Chow
L M
LM
Department of Tropical Public Health, Harvard School of Public Health, Boston, MA 02115.
Wong
A K
AK
Ullman
B
B
Wirth
D F
DF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D005111:Extrachromosomal Inheritance; D005784:Gene Amplification; D017125:Genes, Protozoan; D006801:Humans; D018313:Leishmania enriettii / Q000187:drug effects* / Q000235:genetics*; D007896:Leishmaniasis / Q000188:drug therapy; D008969:Molecular Sequence Data; D011691:Puromycin / Q000494:pharmacology; D015183:Restriction Mapping; D014747:Vinblastine / Q000494:pharmacology
D016054:DNA, Protozoan; D011691:Puromycin; D014747:Vinblastine