Understanding how heme is acquired and used by Trypansoma cruzi

MARCELO L MERLI; LUCÍA V FERRERO; JULIA A CRICCO

Newport, RI

Conferencia; Gordon Reasearch Conference, 2012 Tetrapyrroles, Chemistry & Biology of; 2012

Trypanosoma cruzi is the etiological agent of the Chagas? disease. This parasite presents a complex life cycle, altering between a mammalian hosts and an insect vector. It needs to adapt its metabolism to different environments and nutrient sources. T. cruzi, like other trypanosomatids, requires several essential cofactors where heme is included. Previous biochemical studies, have demonstrated the absence of a complete heme biosynthetic pathway that lately was corroborated when the genomic sequence of the parasite was solved, revealing the absence of the eight enzymes of the classical glycine pathway. This parasite has to import heme from the environment, distribute and insert it into different heme-proteins, some of them involved in essential metabolic routes. We are focusing in elucidating how heme is imported and distributed in the cell by T. cruzi, specially focused in its transport to the mitochondrion (trypanosomatids present only one mitochondria per cell, called mitochondrion). As a first approach to understand the heme uptake processes, we study the biochemical properties of the heme transport using a fluorescent heme analog in epimastigotes of T. cruzi. We observed that heme import is mediated by a membrane active transporter, depending on ATP. Recent results showed that heme uptake was impaired by the presence of ABC transport inhibitors causing a negative effect on parasite proliferation, reinforcing the essentialness of this cofactor in the T. cruzi life style (Cupello, et al., 2011, Acta Tropica 120(3): 211-218). Using the bioinformatics? tools and the genomic information available, we selected several putative transporter proteins, based on their protein sequence homology to active transporters, to conduct the biochemical assays that allow us to identify which of them are actually responsible for heme transport. Taking into account that heme is a highly toxic molecule, the mechanisms involves in the intracellular heme trafficking and the associated carriers or chaperons are not well understood in eukaryotic cells. Although the energetic metabolism of T. cruzi is not completely elucidated, different studies have demonstrated that this parasite depends on the mitochondrial respiratory chain activity during several stages of its life cycle. This way, heme has to be transported to mitochondria and inserted into the respiratory complexes. We will present studies on T. cruzi?s heme A biosynthesis, the essential cofactor of cytochrome c oxidase enzyme. We identified two ORFs in the T. cruzi genomic sequence, with homology to the S. cerevisiae Cox10 and Cox15 proteins. As a first approach to understand if T. cruzi is capable of producing heme A, we studied the products of these genes using S. cereviseae as a heterologous expression system. Our results demonstrated that these T. cruzi genes encode the enzymes (named TcCox10 and TcCox15, respectively) capable of synthesizing heme A when they were expressed in yeast (Buchensky, et al., 2010, FEMS Microbioloy Letters, 312(2), 133-141). We evaluated the level of mRNA of these genes by qRT-PCR, we observed a differential accumulation of these mRNA along the different T. cruzi life stages. Considering that heme A is a cofactor for the sole enzyme CcO, we can speculate about a relation between the mRNA level (amount of protein) and the energetic metabolism of this parasite. However, since mRNA level is not a direct measurement of protein accumulation and enzymatic activity, we need to estimate these parameters. Based on the predicted structure of TcCox10 (heme O synthase) and TcCox15 (heme A synthase) and previous studies on the yeast proteins, we designed different strategies to obtain specific antibodies against them and analyze their expression and subcellular location by Western blot and indirect immufluorescence analysis. Preliminary results shown that TcCox10 and TcCox15 are detected in the different life stages of T. cruzi. As an approach to evaluate the relevance of the heme A biosynthesis pathway and CcO activity in T. cruzi, we are designing strategies to study the effect of shutdown heme A synthesis on this parasite.