Trypanosoma cruzi as a model system to study the expression of exogenous genes coding for polyamine biosynthetic enzymes. Induction of DFMO resistance in transgenic parasites.
Trypanosoma cruzi, the etiologic agent of Chagas' disease, is a polyamine auxotroph organism because its genome contains neither ornithine decarboxylase (ODC) nor arginine decarboxylase (ADC) genes, presumably lost during evolution. After transformation with a recombinant plasmid bearing the complete coding region of Crithidia fasciculata ODC gene, the transgenic parasites were able to synthesize putrescine and simultaneously became susceptible to alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. We have studied the emergence of DFMO-resistant T. cruzi after one-step selection of ODC-transformed parasites cultivated in the presence of high levels of the drug (5 mM). Our results have indicated a duplication of the ODC gene copy number in the drug-resistant cell line. The ODC transcripts and the corresponding translation products showed very significant increases (about 7- and 25-fold, respectively) in DFMO-resistant parasites, while the ODC enzymatic activity was 5 times higher than in drug-sensitive T. cruzi. The unequal increases of ODC protein and enzymatic activity in DFMO-resistant protozoa strongly suggest that in addition to gene amplification and enhanced transcription and translation, the assembly of ODC polypeptide chains into dimeric active enzyme molecules might also contribute to regulate the development of DFMO resistance.
Carrillo
Carolina
C
Fundación Instituto Leloir, IIBBA-CONICET, and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1405BWE-Buenos Aires, Argentina.
González
Nélida S
NS
Algranati
Israel D
ID
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D015317:Biogenic Polyamines / Q000096:biosynthesis*; D015152:Blotting, Northern; D015139:Blotting, Southern; D017931:DNA Primers; D000518:Eflornithine / Q000494:pharmacology*; D015870:Gene Expression; D009955:Ornithine Decarboxylase / Q000235:genetics*; D065108:Ornithine Decarboxylase Inhibitors; D016133:Polymerase Chain Reaction; D014349:Trypanosoma cruzi / Q000201:enzymology / Q000235:genetics*
D015317:Biogenic Polyamines; D017931:DNA Primers; D065108:Ornithine Decarboxylase Inhibitors; D009955:Ornithine Decarboxylase; D000518:Eflornithine