Regulation of foreign ODC and ADC genes expression in Trypanosoma cruzi

ALGRANATI ID; SERRA MP; CARRILLO C; GONZÁLEZ NS

Kazusa Akademia Center; Kisarazu-city, Chiba, Japón.

Conferencia; 2004 International Conference on Polyamines, Function and Clinical Application; 2004

Kazusa Akademia Center

REGULA TION OF FOREIGN ODC AND ADC GENES EXPRESSION IN TRYPANOSOMA CRUZI I.D. Algranati, M.P. Serra, C. Carrillo and N.S. González Fundación Instituto Leloir, CONICET and University of Buenos Aires, Patricias Argentinas 435, Buenos Aires (1405), Argentina Metabolic studies carried out in severallaboratories have shown that Trypanosoma cruzi epimastigotes are auxotrophic for polyamines because they are unable to synthesize putrescine. This deficiency, unique among all protozoa investigated so far, is due to the absence of ODC and ADC enzymatic activities which catalyse both possible pathways of putrescine biosynthesis. The described feature cannot be attributed to T. cruzi deficient uptake of ornithine or arginine nor to the presence of putative inhibitors of the enzymes in the parasite intracellular medium. Instead, genetic studies have demonstrated that the absence of putrescine-synthesizing enzymes is due to the lack ofthe corresponding genes probably lost from the T .cruzi genome during evolution. We have recentIy shown that Trypanosoma cruzi epimastigotes were able to express heterologous ODC and ADC enzymatic activities after transfection of wild-type parasites with recombinant plasmids bearing the complete coding regions of Crithidia fasciculata ODC and oat ADC genes. These results ruled out the possibility that the internal medium of T. cruzi epimastigotes might interfere with the expression ofhypothetical own ODC and ADC genes. Transgenic parasite containing Crithidia ODC coding region became autotrophic for polyamines and sensitive to DFMO. The continuous presence ofthis specific inhibitor in cultures of ODC-transformed T. cruzi evoked the resistance to the drug with a concomitant increase of                                                                      ,..                                       ""-                  ~ ODC ORF copy number per genome in T. cruzi DNA. Transformation with the oat ADC coding sequence allowed the appearance of ADC enzymatic activity but did not confer polyamine autotrophy to the transfected parasites. We have been able to follow the fate of the transforming recombinant plasmids by assays of restriction analysis and PCR amplification of DNA from transformed protozoa. Keywords: Trypanosoma cruzi, Polyamine auxotrophy, Foreign genes expression. References 1. Carrillo, C., Cejas, S., González, N. S., and Algranati, I.D. (1999) FEBS Lett.454, 192-196.                                                             . 2. Carrillo, C., Cejas, S., Huber, A., González, N.S., and AIgranati, I.D. (2003)J. Eukaryot.     Microbio!. 50,312-316.