Transgenic Trypanosoma cruzi: transformation with Crithidia fasciculata ODC gene. Expression and characterization of the foreign product

CARRILLO C; CEJAS S; GONZÁLEZ NS; ALGRANATI ID

Caxambú, Brazil

Congreso; XXVII Reunião Anual de Pesquisa em Doença de Chagas e XVI Reunião Anual de Protozoologia; 2000

Polyamines, putrescine, spermidine and spermine, are present in a vast majority of living organisms, playing important roles in macromolecules synthesis and in cellular proliferation and differentiation. The first step in their synthesis is controlled by the enzyme “ornithine decarboxylase” (ODC). Trypanosoma cruzi is different from other trypanosomatids in some physiological and metabolic aspects. In fact, T. cruzi is the only parasite where ODC activity is not detectable, neither in epimastigote nor in trypomastigote cultures. We have demonstrated that ODC activity absence is due to the absence of the ODC gene in T. cruzi. Then, epimastigote cultures were transfected with a recombinant vector carrying the complete code region of Crithidia fasciculata ODC gene. The transgenic trypanosome presented a high level of ODC gene expression transiently as well as stablely. Concomitantly with the expression, this parasite developed polyamine autothrophy and became sensible to the specific and irreversible ODC inhibitor, DFMO. The specific activity fluctuated continuously along the culture time, indicating a partial integration of the transfected gene into the T. cruzi genome. The foreign ODC activity presented kinetics parameters and molecular weight similar to the native ODC in C. fasciculata. However, the metabolic stability varied remarkably: since the ODC middle life in C. fasciculata was less than 30 minutes, it become larger than 4 hours in T. cruzi. This notable increase in the stability suggest a strong difference between both T. cruzi and C. fasciculata proteolytic machinery.