Investigation of oxidizing biological substrates of AhpC: fundamental aspects and applications to combat pathogenic bacteria.

Abstract

Multiple drug resistance (MDR) bacterial strains are a global health threat and the search for new biological targets is a global interest. Recent surveys indicate that annually about 1.3 million deaths are directly caused by bacteria resistant to multiple antibiotics worldwide. Among them, MDR strains of Escherichia coli account for almost one third of deaths. The immune system of hosts has several strategies to combat infection by pathogenic bacteria, such as oxidative and nitrosative explosion by immune cells, characterized by producing a wide variety of reactive oxygen species (ROS) and nitrogen (RNS), which intend to annihilate the pathogen preventing its establishment. Additionally, several antibiotics are also able to generate EROS, directly or indirectly, which contributes to the control of infections. On the other hand the pathogens, in the course of evolution, developed highly efficient enzymes to decompose these species neutralizing the oxidative defenses of the hosts and the effects of certain antibiotics. Among the species produced, hydroperoxides represent an extremely diverse group of compounds whose toxicity is mitigated by several enzymes called peroxidases, especially bacteria, for peroxiredoxin (Prx) AhpC. This Prx is capable of decompose hydroperoxides using a highly reactive cysteine called peroxidases (CP) which during the catalytic cycle forms a disulfide with a second cysteine residue called resolution (CR). In order for them to perform another catalytic cycle disulfide needs to be reduced, a task usually performed by the Trx or AhpF systems. When hydroperoxide concentrations are very high, peroxidase activity is lost due to hyperoxidation of CP in cysteine sulfinic acid (CP-SO2H). In bacteria there is no system capable of reducing this intermediate and the inactive enzyme is degraded. Recently, we have demonstrated that AhpC is much more sensitive to hyperoxidation by synthetic organic hydroperoxides than by hydrogen peroxide. In E. coli, AhpC is one of the ten most expressed proteins and, in general, in pathogenic bacteria, these enzymes are related to infection, establishment and survival in their hosts, subduing the oxidative explosion generated by the immune system and antibiotics. AhpCs were identified due to the fact that ”ahpc strains have high sensitivity to organic peroxides including long-chain fatty acid derivatives which are generated in large quantities as a consequence of oxidative explosion and treatment with certain antibiotics. Despite the importance of knowing more deeply the natural substrates of AhpCs, no systematic investigation has been carried out to date. The objectives of this project aim to investigate which organic peroxides derived from long-chain lipids may be relevant as biological substrates of E. coli AhpC, through methodologies involving computational analyses (construction of theoretical structural model and evaluation of substrate binding by molecular docking) and biochemical (activity assays by NADPH oxidation, determination of kinetic parameters, evaluation of hyperoxidation by kinetic assays and non-reducing SDS-PAGE, among others). We believe that the results generated in this project have importance in basic science assisting in a greater understanding of the decomposition of lipid peroxides in bacteria and also in the applied field, since the determination of biological substrates can pave the way for the identification of inhibitors of this group of proteins with implications in the treatment of infectious diseases caused by MDR bacteria.