cruzi proteins were subjected to, which might include cleavage of the C-terminal region (which includes the his tag sequence); and (4) the yeast cells might accumulate extra amounts of ScCox10 and/or ScCox15 proteins, but might not do so for the T. cruzi ones, which may be MAPK Inhibitor Library more exposed to protease attack and degraded faster. The results obtained here did not differentiate between these hypotheses,
but they allowed us to postulate that the amount of T. cruzi proteins detected was sufficient to restore the respiratory capability of yeast mutants to WT levels, recovering the biosynthesis of heme A. Type aa3 cytochrome c oxidase was identified as the main terminal oxidase in epimastigotes, and the heme A signal was detected in epimastigotes using differential absorption spectroscopy (Stoppani et al., 1980; Affranchino et al., 1986). In addition, we showed that TcCOX10 and TcCOX15 sequences encode for functional HOS and HAS proteins in the yeast model. Selleckchem Opaganib In order to find out whether the TcCOX10 and TcCOX15 genes are being differentially transcribed during the life cycle, their mRNA levels were quantified by qRT-PCR at different life stages. We observed that both genes were transcribed, and the data obtained showed that TcCOX10 mRNA (Fig. 4a) varied more than TcCOX15 mRNA (Fig. 4b) during the life cycle. However, in amastigotes, we observed
that the amount of mRNA for both genes was significantly lower compared with the other stages (P<0.05 for all comparisons between amastigotes and every other stage for both genes, see Supporting Information, Appendix S1). Little is known about the metabolic changes that occur when the parasite invades
host cells. BCKDHB A recent study showed that when the parasite differentiates into an amastigote in the host cell cytoplasm, a metabolic switch occurs (Silber et al., 2009). It is possible that the differences observed in TcCOX10 and TcCOX15 gene expression could be related to the metabolic adaptation afforded by the parasite, reflecting alterations in respiratory requirements in the different life stages. Although mRNA quantification is not a direct measure of protein level or function, it is capable of reflecting a direct relationship. In a recent study, Wang et al. (2009) described the relationship between S. cerevisiae COX10 and COX15, proposing that these enzymes might play another unidentified role besides heme A biosynthesis. These results confirmed the expression of the genes encoding TcCox10 and TcCox15 enzymes from T. cruzi at different life stages. Notwithstanding, complementary studies are necessary to discern whether Cox10 and Cox15 could have another physiological function in T. cruzi. In conclusion, T. cruzi metabolism must adapt to different environments during its life cycle in which the parasite is under different nutritional pressures. It presents auxotrophies for various cofactors, including heme.