Effects of natural compounds over the peroxidase activity of AhpCs and pathogenic bacteria survival

Abstract

Recent studies indicate that the resistance of bacteria to antibacterials may be directly related to oxidative stress, since antibacterials of different classes have the common ability to generate reactive oxygen species (ROSs), which cause damage to macromolecules, especially DNA, accentuating the mutation rates which can contribute to the appearance of super-resistant strains. ROSs are also produced by host immune cells to fight pathogens and studies suggest that inhibition of pathogen antioxidant enzymes is quite harmful for pathogens. Bacteria have several antioxidant enzymes capable of decomposing ROSs, with emphasis on the typical 2-Cys peroxiredoxins, called AhpC bacteria, which are extremely abundant and have high reactivity against hydroperoxides. The Prx 2-Cys are capable of decomposing the substrates using a cysteine residue (Cys peroxidase - CP) which, after decomposing the peroxide, forms a disulfide with a second cysteine residue (Cys of resolution - CR). The high reactivity of CP is achieved by interactions with the residues of Thr (in some cases Ser) and Arg, called catalytic triad (TC), absolutely conserved between the Prx 2-Cys. Recently, we demonstrated that the substitution of Thr for Ser on CT leads to functional and structural changes in these proteins, and that it exists mainly in bacteria. Eukaryotes also have typical Prx 2-Cys, however the environment of the active site is significantly different from AhpCs, because in eukaryotes the typical Prx 2-Cys have a C-terminal tail that protrudes into the active site. We believe that these two characteristics make AhpCs targets for chemotherapy capable of differentially inhibiting bacterial and host AhpCs. Although mammalian Prx 2-Cys inhibitors have been described, none have been characterized for the bacterial AhpC isoform. It is important to note that all the inhibitors described for Prx 2-Cys have in common an extensive hydrophobic skeleton, and that some of them effect the inhibition of Prx by binding to catalytic cysteines through an alpha, beta - unsaturated carbonyl system. Recently, our research group demonstrated that a natural compound (Adenantin-Adn) initially identified for human proteins is capable of efficiently inhibiting AhpC. Then, we looked for compounds similar to DNA from molecules isolated from the Brazilian flora and identified a sesquiterpene lactone (from CN3-LS) as an inhibitor of bacterial AhpCs containing Thr (IC50 = 0.553 ± 0.061mM) or Ser (IC50 = 0.460 ± 0.025 mM ) on CT. The compound showed cytotoxicity for Gram + bacteria (MIC50 ~ 100-350mM), but not for Gram -, however cytotoxicity was not evaluated in conjunction with immune cells. The objectives of this work are the expression and purification of AhpC from Pseudomonas aeruginosa (Gram bacteria - which contains Thr as part of the CT) and Staphylococcus epidermidis (Gram + bacteria with Ser on CT) and to evaluate the inhibitory capacity of natural compounds from the biota of the Brazil, which have characteristics similar to the compounds already described, aiming at the identification of bacterial AhpCs inhibitors. For this purpose, biochemical tests will be performed to determine the inhibition of activity and determination of IC50, computational analyzes, cytotoxicity tests of compounds identified on strains of isolated bacteria, compounds + antibacterials (kanamycin, ciprofloxacin or nonapeptide polymyxin) This project is already underway started and so far compounds of four different chemical classes have been tested, one of which, CN ABP1, was able to inhibit the AhpC of P. aerugisona. (AU)