Evaluation of bioactive molecules as inhibitors for atioxidant system of typical 2-Cys peroxiredoxins (AhpC) in baceria

Author(s)

Melina Cardoso dos Santos

• Document type: Doctoral Thesis
• Press: São Vicente. 2021-04-07.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. São Vicente
• Defense date: 2021-03-03
• Advisor: Marcos Antonio de Oliveira

Abstract

The increase in resistance to antibiotics by microorganisms has grown dramatically and experts have warned of the rapid ineffectiveness of drugs against super-resistant bactéria strains. The bacterial resistance to antibiotics can be directly related to oxidative stress, since different antibiotics have the convergent capacity to produce reactive oxygen species, which cause damage to macromolecules, especially to DNA, increasing mutation rates, which can contribute to appearance of super-resistant strains. Reactive oxygen and nitrogen species are also produced by the host immune system to combat pathogens and studies indicate that inhibition of the expression of pathogen antioxidant enzymes is able to generate a significant intracellular increase in these species, which significantly interferes in their survival. It has recently been demonstrated that a natural compound named adenantin (Adn) is able to inhibit the peroxidase activity of typical 2-Cys peroxyredoxins (Prx) from humans (PrxI and PrxII), resulting in the mortality of tumor cells, but presenting low toxicity to normal cells . 2-Cys Prx are capable of decomposing hydroperoxides using a cysteine residue (CysP) and their high reactivity over different types of hydroperoxide is achieved by interactions with Thr/Ser and Arg residues, absolutely conserved between 2-Cys Prx. Together, CysP, Thr / Ser and Arg, are called catalytic triad (CT). Typical 2-Cys Prx are also present in bacteria and are named AhpC, being considered one of the fundamental oxidative defense fronts for the pathogen. In a previous project, we have evaluated the effects of the presence of the Thr or Ser residue on the TC of two yeast typical 2-Cys Prx on the inhibitory activity of Adn. We then demonstrated that Adn has higher affinity for enzymes containing Ser on TC and that the presence of Ser in this position exists essentially in prokaryotes, especially in bacteria. In the present study, continuing this work, we have found that AhpCs containing a Ser on TC are more resistant to overoxidation and would have higher holdase activity during thermal stress than AhpCs containing a Thr in that position. Since oxidative and thermal stress are barriers imposed to bacteria infection by the host organism, the inhibition of this defense system could be an important strategy in the fight against these pathogens. So, we have evaluated the inhibitory capacity of Adn over AhpCs and found that Adn has a higher affinity for AhpC of Escherichia coli (EcAhpC), regardless of the presence of Thr or Ser on TC (EcAhpCWT and EcAhpCT44S), than for human isoforms, presenting an IC50 value ≈ 5 and 50 times lower than for PrxI and PrxII, respectively. Additionally, we observed that Adn also inhibited E. coli Trx activity (EcTrx1) with higher efficiency than human Trx. We also found that Adn was toxic to Gram-positive bacteria, with IC50 values in the range of 110-140µM, and to Gram-negative (E. coli) when treated with polymyxin B nonapeptide (PMBN). Since PMBN also has antibiotic activity, its use in association with Adn may represent a potential new antimicrobial therapy. Additionally, we carry out a screening of natural compounds from the floristic biota of the State of São Paulo and identified a molecule capable of inhibiting the peroxidase activity of EcAhpC with similar efficiency to Adn, besides inhibiting EcTrx1 with less efficiency. This compound represents the first Prx inhibitor in the sesquiterpene lactone class, in addition to being the first inhibitor derived from Brazilian biodiversity. Finally, we have characterized a new thiol protein from Xylella fastidiosa and demonstrated that it is able to protect bacterial cells against cyanide and oxidative stress generated by H2O2. Collectively, the results obtained in this work contribute to the discovery of new approaches to combat pathogenic bacteria. (AU)

• FAPESP's process: 17/06263-4 - Evaluation of bioactive molecules as inhibitors for atioxidant system of typical 2-Cys peroxiredoxins (AhpC) in baceria
• Grantee: Melina Cardoso dos Santos
• Support Opportunities: Scholarships in Brazil - Doctorate