How is Heme A synthesized in Trypanosoma cruzi?

MARCELO L. MERLI ; JULIA A. CRICCO

Mar del Plata

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

Sociedad Argentina de Protozoologia

Trypanosoma cruzi has requirements for different cofactors where heme is included. This cofactor serves as a prosthetic group for heme-proteins, most of them involved in several essential metabolic pathways. Because this organism is unable to synthesize heme, it must be acquired from the environment (the different hosts). Once heme is imported, it is distributed inside the cell and inserted into the target heme-proteins, available throughout different subcellular compartments. Heme-proteins form part of the respiratory chain complexes in the parasite mitochondrion, cytochromes involved in poliinsaturated fatty acid metabolism and sterol biosynthesis in the endoplasmic reticulum. As heme is a highly toxic molecule, it is well accepted that heme carriers or chaperons involved in its distribution exist. But, in eukaryotic cells, these type of proteins were not reported yet and the heme transport and distribution processes in trypanosomatids remain unknown. We are interested into elucidate how heme is imported and distributed in T. cruzi, specially we are focused in its traffic to the mitochondrion and its conversion into heme A, the essential cofactor only for cytochrome c oxidase complex (CcO). In our lab, we identified the codifying sequences for the T. cruzi enzymes involved in heme A biosynthesis, TcCox10 (heme O synthase) and TcCox15 (heme A synthase) and characterized their function in the yeast S. cerevisiae. The mRNA level of these genes (TcCOX10 and TcCOX15) were analyzed by qRT-PCR and we observed the presence of these mRNA along different parasite life stages. In order to have a direct protein measurement, we design strategies to determine the presence and amount of them by western blot analysis. We cloned an small fragment of TcCox10 or TcCox15 (extra-membrane peptides, both are integral membrane proteins) as GST-fusion proteins to be used to get specific antibodies. The obtained polyclonal antibodies were purified and used to evaluate TcCox10 and TcCox15 in T. cruzi. The presence of TcCox10 and TcCox15 was observed in epimastigotes by western blot analysis and their mitochondrial localization was confirmed by indirect immunofluorescence detection. We postulated that the observed changes in mRNA levels could be a form of regulation reflecting differences in respiratory requirements at different life stages. The presence of these protein were confirmed by western blot analysis but more studies need to be carry out to confirm the presence of them in the different life stages and evaluate their enzymatic activity.