Structural characterization of redox active auto assembling large molecular AhpC complexes from Pseudomonas aeruginosa and Staphylococcus epidermidis using single-particle cryo-EM

• Grant number: 24/23466-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: January 05, 2026
• End date: July 04, 2026
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Marcos Antonio de Oliveira
• Grantee: Vitória Isabela Montanhero Cabrera
• Supervisor: Vitor Hugo Balasco Serrao
• Host Institution: Instituto de Biociências (IB-CLP). Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , SP, Brazil
• Institution abroad: University of California, Santa Cruz (UC Santa Cruz), United States
• Associated to the scholarship: 23/01263-7 - Molecular characterization of biological substrates and natural/synthetic compounds as inhibitors of typical 2-Cys peroxiredoxins (AhpC) from bacteria, BP.DR

Abstract

Multidrug-resistant (MDR) pathogenic bacteria have been a serious global public health concern, particularly since the beginning of this century. Bacterial infection stimulates the host's immune system to produce ROS and RNS through the respiratory burst mechanism. ROS are also produced through primary or secondary mechanisms of some antibiotics. To mitigate the oxidants produced by the immune system and antibiotics, bacteria defenses rely on several antioxidant enzymes, including AhpC, a peroxiredoxin (Prx) involved in the decomposition of a wide variety of hydroperoxides. AhpC decomposes hydroperoxides using a highly reactive cysteine so called peroxidatic (CP) that forms a disulfide with a second cysteine residue (resolution Cys - CR) during the catalytic cycle. The high reactivity of AhpC is related to a catalytic triad (CT), composed of two polar residues (Thr/Ser and Arg) that maintain the sulfur of CP in the thiolate form (S-). The substitution of Thr for Ser in some Prx CT was considered redundant for many years, but we have revealed that this substitution results in changes on the oligomeric structure and reactivity towards hydroperoxides. When the concentrations of hydroperoxides are very high, there is a loss of peroxidase activity due to the CP hyperoxidation into cysteine sulfinic acid (CP-SO2H), in an irreversible inactivation. The oligomeric state of these enzymes is closely related to their redox state: reduced forms are decamers, oxidized ones are dimers, and hyperoxidized species are decamers or self- assemble in high-molecular weight complexes (HMWC > 1GDa). In enzymes containing Ser, decamer dissociation does not occur after oxidation. In the pathogenic bacteria Pseudomonas aeruginosa and Staphylococcus epidermidis, AhpC (PaAhpC with Thr in the CT, and SeAhpC with Ser in the CT) are considered virulence factors. Nevertheless, no work has determined the structure of PaAhpC and SeAhpC in a hyperoxidized state. This project aims to determine the structure of distinct large molecular complexes of PaAhpC and SeAhpC to understand the biological roles that may be applied to combat infections caused by bacteria MDR strains. Therefore, we propose cooperation with Dr. Vitor Hugo Balasco Serrão (Biomolecular Cryo - Electron Microscopy Facility, University of California, - Santa Cruz, CA) to carry out experiments involving Cryogenic Electron Microscopy using single-particle analysis (SPA-cryo-EM), to obtain the protein structure in its reduced (decameric state) and hyperoxidized states (decamers HMW). Preliminary results have demonstrated project viability, which is here presented.