AhpC from Escherichia coli: Organic Substrates, Inhibitors, and Crystallographic Structure Determination for Applications in Combating Pathogenic Bacteria

• Grant number: 23/18481-7
• Support Opportunities: Scholarships in Brazil - Master
• Start date: June 01, 2024
• End date: May 31, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Marcos Antonio de Oliveira
• Grantee: Sabrina Vargas Batista
• Host Institution: Instituto de Biociências (IB-CLP). Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , SP, Brazil
• Associated research grant: 13/07937-8 - Redoxome - Redox Processes in Biomedicine, AP.CEPID

Abstract

The global increase in bacterial strains resistant to multiple drugs (MDR) poses a serious threat to public health due to the high costs of treatment and associated high lethality. Recent studies highlight those different classes of antibiotics, despite having distinct biological targets, share a common mechanism for eradicating pathogens, involving the generation of reactive oxygen species (ROS). Higher eukaryotic defense cells also produce ROS and reactive nitrogen species (RNS) during oxidative/nitrosative bursts, a crucial defense against pathogenic organisms. Among the species formed, peroxides are particularly relevant due to their ability to traverse biological membranes and generate toxic species like the hydroxyl radical. Antioxidant enzymes, especially peroxidases, play a vital role in the survival of pathogens, making them significant targets in the search for inhibitors. 2-Cys peroxiredoxins (2-Cys Prx) are crucial homodimeric proteins capable of decomposing peroxides using a reactive cysteine (CP). In bacteria, the AhpC isoform is widely distributed and abundant, playing an essential role in virulence. Recent studies indicate that the deletion of the ahpc gene reduces or eliminates virulence in various species of pathogenic bacteria. Recently, two natural compounds from plants were identified as inhibitors of bacterial isoforms, suggesting the capability of these enzymes to metabolize hydrophobic organic oxidant substrates. Hydroperoxides derived from long-chain lipids were identified as substrates with high affinity for E. coli AhpC, promoting inactivation through hyperoxidation of the catalytic residue even in small quantities. The project's objectives include determining the rates of oxidation and hyperoxidation of EcAhpC through rapid kinetics experiments and in vivo assays evaluating the effects of exposure to hydroperoxides and their combination with antibiotics in wild-type and mutant E. coli strains. Additionally, the project aims to advance the understanding of the molecular biology of EcAhpC through biophysical characterization approaches and crystallizations of the enzyme in different REDOX states and with ligands. The project also explores semi-synthetic compounds and natural endoperoxides as potential irreversible inhibitors of EcAhpC. The expected results will contribute to understanding the relationship between AhpC, antibiotic resistance, biological organic substrates, and inhibitors, offering insights for combating bacterial infections. The research aims to produce manuscripts for publication in international journals and/or intellectual property applications.