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    • The life cycle of T cruzi requires both vertebrate

    2022-07-19

    The life cycle of T. cruzi requires both vertebrate and invertebrate hosts. In the insect (invertebrate) vector, the protozoan undergoes extracellular replication, whereas in the mammalian (vertebrate) host it replicates by obligate intracellular multiplication (Rodrigues et al., 2014). Hence, the parasite has to handle oxidative burst from the immune systems of its hosts, and from oxidative species inside mammalian cells. This oxidative burst generates superoxide anions (O2) and other reactive oxygen species (ROS), such as hydrogen peroxide (Gupta et al., 2009). Several molecules, including carbohydrates, proteins, lipids and nucleic acids can be oxidized by ROS, leading to deleterious effects in cells (Nakabeppu, 2014). ROS cause a variety of damage at the DNA level, from single- and double-strand breaks (SSBs and DSBs, respectively) to base oxidation. One of the most abundant and best-characterized oxidative lesions is 8-oxoguanine (8-oxoG). This modified base has a great biological importance, since it can mispair with vivo vu residues, increasing the frequency of spontaneous G:C to T:A mutations. It is estimated that there are approximately 10,000 8-oxoG residues per human cell nucleus (Nakabeppu, 2014, Ohno et al., 2006). Given the importance and the abundance of 8-oxoG in eukaryotic genomes, eukaryotes have evolved a multi-defense mechanism, the so-called GO system, which is able to counteract this type of lesion. Three enzymes comprise the GO system: MTH (MutT Homolog), which hydrolyses 8-oxodGTP to 8-oxoGMP in the nucleotide pool, preventing the incorporation of 8-oxoG into DNA; OGG1, a DNA glycosylase that removes 8-oxoG from 8-oxoG:C base pairs; and MYH (MutY Homolog), also a glycosylase, which excises adenine from 8-oxoG:A pairs (Tchou and Grollman, 1993, Michaels and Miller, 1992). Our research group has previously described TcMTH and TcOGG1 activities in T. cruzi oxidative response (Furtado et al., 2012, Aguiar et al., 2013). Overexpression of TcMTH led to improved growth of parasites in macrophage cultures when compared to wild type cells and overexpression of TcOGG1 increased DNA lesions of parasites treated with hydrogen peroxide (Furtado et al., 2012, Aguiar et al., 2013). OGG and MYH are involved in DNA Base Excision Repair (BER), a repair pathway that usually resolves oxidative DNA lesions, in which a set of enzymes act sequentially. First, a specific DNA glycosylase recognizes and removes the damaged base, resulting in an abasic site (AP site) that can be lethal if unrepaired. After that, an AP endonuclease cleaves the DNA backbone, leaving room for a phosphodiesterase to act. Finally, a DNA polymerase and a DNA ligase complete the DNA repair (Kim and Wilson, 2012). MutY and its eukaryotic counterpart MYH have been characterized in several organisms, including Escherichia coli, Mus musculus and Homo sapiens, among others (Nghiem et al., 1988, Ichinoe et al., 2004, McGoldrick et al., 1995). This enzyme is a monofunctional glycosylase, that is, it cleaves the base from DNA, but it does not possess lyase activity (Mazzei et al., 2013, de Oliveira et al., 2014). This protein can be localized both to the nucleus and mitochondria (Ohtsubo et al., 2000). Various MYH mutations have been linked to diseases, especially colorectal cancer (Al-Tassan et al., 2002, Nielsen et al., 2011, Mazzei et al., 2013). In silico analysis of the T. cruzi genome showed that this protozoan possesses one putative copy of MYH gene (El-Sayed et al., 2005a, El-Sayed et al., 2005b). Since MYH is an important protein involved in the reduction of the mutagenic events derived from oxidative stress, we decided to characterize this enzyme in T. cruzi, an organism that is subject to numerous and varied oxidative stress sources during its life cycle. Our results show that the T. cruzi enzyme is able to complement bacteria deficient in MutY gene, as well as recognize 8-oxoG paired with A. In addition, the overexpression of TcMYH in epimastigotes indicates that it could participate in responses to oxidative stress in the nuclear and mitochondrial genomes.