Cytochrome P450 Reductase functions in Trypanosoma cruzi

PORTAL PATRICIO; FLAWIÁ MIRTHA; PAVETO CRISTINA

Mar del Plata

Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2011

Sociedad Argentina de Protozoología

Plasma membrane microdomains are involved in many vital processes as phagocytosis, reproduction, entry process and intracellular replication of microbial pathogens. These heterogeneous sterol-enriched microdomains compartmentalize cellular processes. TcCPR-A, TcCPR-B and TcCPR-C are members of putative Cytochrome P450 reductases (CPR) family in T. cruzi. CYP51, a Cytochrome P450 enzyme (CYP) is a key enzyme in sterol synthesis present in Trypanosomatids. The recombinant proteins TcCPR-B and TcCPR-C expressed in E. coli were able to complement CYP activities in an in vitro reconstituted system. Moreover, ergosterol content was significantly increased in TcCPR-B and C overexpressing parasites. Ergosterol biosynthetic pathway is considered to be a selective drug target in fungi, Leishmania and Trypanosoma, since their mammalian hosts use cholesterol instead. We present here the results of complementation experiments showing that the introduction of the T. cruzi cpr-B gene into the cpr knock-out Saccharomyces cerevisiae strain (cpr-) WRΔ can restore the yeast normal growth. Interestingly, immunofluorescence studies demonstrate different subcellular localization for each enzyme, the ER and reservosome respectively. On the contrary, TcCPR-A overexpression was lethal, displaying aberrant cells, with abnormal morphology and ultrastructural alterations. Interestingly, it was recently reported that Tah18, a yeast orthologue for T. cruzi CPR-A, plays a “pro-death” role in response to oxidative stress, inducing cell death pathways. A possible participation of this CPR in stress induced apoptosis mechanisms in T. cruzi is being studied.