The increasing incidence of infections resistant to the action of different classes of antibiotics together with the decline of the discovery of new efficient classes in the fight against pathogens poses a threat to global public health. In this context, there is a need to expand the understanding of microbial defense mechanisms in order to potentiate the inactivation and death of pathogens by these agents as well as to search for new classes that possess selective toxicity to the pathogens. Recent studies have shown that different classes of antibiotics generate high levels of reactive oxygen species (ROS) that contribute to bacterial cell death. This notion served to refine the aspects of the models proposed in many laboratories in the investigation of these molecules and the permanence of the infection caused by bacterial species. Alkylhydroperoxide reductase C (AhpCs) belong to a group of peroxidases termed typical 2-Cys peroxydreloxins (typical 2-Cys Prx), which have as characteristics the ability to decompose a wide variety of hydroperoxides using a cysteine residue called cysteine peroxidase (CP), which is part of the catalytic triad of these enzymes represented by an arginine (Arg) and a threonine (Thr) or a serine (Ser). The CP and Arg residues are strictly conserved among all typical Prx 2-Cys, however the Thr residue can be replaced by a Ser, something considered redundant until recently. Recently, we have demonstrated that Thr or Ser in the catalytic triad confers functional and structural differences between enzymes (Tairum et al., 2016). Bacterial Prx 2-Cys are the subject of a number of research studies to provide significant resistance to the host immune system, and initial results from our research group revealed that bacteria of the genus Staphylococcus, which possess Ser as part of the catalytic triad, cell line in the presence of the Prx 2-Cys Adenantin (Adn) inhibitor than the Eschericchia coli lines, which have Thr. This project aims to deepen the studies of inhibition of AhpCs of bacteria containing Thr or Ser in the catalytic triad using Salmonella typhimurium (Thr) and Staphylococcus aureus (Ser) by Adn, causing a wide range of serious infections in mammals. In order to do so the AhpC genes of S. typhimurium (StAhpC) and S. aureus (SaAhpC) will be cloned and the recombinant histidine tail containing proteins (6 × His) will be expressed in bacterial systems for purification by IMAC. To verify the influence of Thr Ser replacement, site-directed mutations will be performed to obtain enzymes carrying reciprocal substitutions of these amino acids (StAhpCT44S SaAhpCS46T). In order to evaluate the effects of Adn on the activity of the AhpCs will be carried out tests of peroxidase activity (oxidation of DTT and FOX). The effects of binding of this molecule on protein structure will be performed procedures involving molecular exclusion chromatography (SEC), circular dichroism (CD) spectroscopy and non-reducing SDS PAGE assays. The project is already underway and the SaAhpC expression and purification conditions have already been established and it is possible to obtain 10mg / L of cell culture, as well as to start the procedures to obtain SaAhpCT44S.
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