MONOALLELIC DELETION OF HMGR GENE IN Trypanosoma cruzi ASSOCIATES WITH DEFECTS IN EPIMASTIGOTE GROWTH AND DIFFERENTIATION

FERNANDO SÁNCHEZ-VALDÉZ, CECILIA PEREZ BRANDAN, PAULA FARAL-TELLO, CARLOS ROBELLO, MIGUEL ANGEL BASOMBRÍO

Mar del Plata

Congreso; X Congreso de Protozoologia y Enfermedades Parasitarias; 2014

For more than 40 years, Chagas disease treatment was based on the use of relatively toxic drugs that produce serious side effects and have restricted indications in adult patients and pregnant women. Currently, one of the promissing target for the development of novel anti-T. cruzi drugs is the ergosterol byoshyntesis pathway. Ergosterol is a central component of the parasite plasma membrane, essential for parasite viability and proliferation during its entire life cycle. 3-hydroxy-3-methylglutaryl coenzyme a reductase A (HMGR) is a single copy gene that encodes a mitochondrial key enzyme of the ergosterol pathway. This enzyme is specifically inhibited by mevinolin, a potent statin widely used in humans as a hypocholesterolemic agent. The present work aims to delete HMGR gene in the Tulahuén T. cruzi strain generating mutant clones with reduced HMGR expression and highly sensitivity to mevinolin. The mevinolin hyper-susceptibility constitutes an interesting approach to control the possible persistence of genetically-attenuated parasites to be used as experimental vaccines in murine models. Using the Gateway cloning technology, we generated vectors carrying the neomycin or hygromicin phosphotransferase gene flanked by HMGR 5´ and 3´ UTRs. The recombination fragment was PCR-amplified from the vector and transfected into Tulahuén epimastigotes. G418 resistant parasites could be selected after transfection. PCR and Southern blot analysis of a cloned recombinant population confirmed the deletion of one allele of the HMGR gene. No double knock-out parasites could be obtained after several transfections. Furthermore, we generate HMGR overexpressing parasites by transfecting the pTrex-HMGR plasmid to Tulahuén parasites. Mutant epimastigotes presented a significant decrease in growth and differentiation rate in axenic culture media. We are characterizing at present the sensitivity to mevinolin and the infectivity and immunoprotective behavior of mutants in murine models.