OhrR, a Redox-Regulated Transcription Factor: Elucidation of Biochemical, Structural, Genetic Mechanisms and Bacterial VirulenceCharacterization of the redox sensor OhrR and its regulatory function in Pseudomonas aeruginosa

• Grant number: 25/13376-6
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: November 01, 2025
• End date: June 30, 2029
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Luis Eduardo Soares Netto
• Grantee: Luca Paulino Otvos
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 13/07937-8 - Redoxome - Redox Processes in Biomedicine, AP.CEPID

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen classified by the WHO as a critical priority due to its high level of antibiotic resistance and its association with severe hospital-acquired infections. Its ability to adapt to hostile environments is closely linked to antimicrobial resistance and to its capacity to evade host defenses, such as oxidative stress generated by neutrophils, which includes the production of organic peroxides. P. aeruginosa employs specialized antioxidant systems, among which the Ohr-OhrR system stands out for its role in responding to organic peroxides. In this system, the enzyme Ohr detoxifies organic peroxides, while its expression is repressed by the redox-sensitive transcription factor OhrR, a member of the MarR family. Under oxidative conditions, OhrR undergoes modifications that lead to its dissociation from DNA, allowing ohr transcription to proceed. Notably, studies using Caenorhabditis elegans have demonstrated the involvement of P. aeruginosa OhrR (PaOhrR) in virulence.The specific redox properties of PaOhrR, the mechanisms underlying its regeneration, and the full extent of its regulon remain poorly understood. Preliminary data from our group indicate that PaOhrR is functionally active and highly reactive to organic peroxides. Our central hypothesis is that PaOhrR functions as a key redox sensor that coordinates the oxidative stress response and influences bacterial virulence. In this project, we propose to investigate the molecular mechanisms of PaOhrR-mediated transcriptional regulation. The objectives include: (i) biochemical and structural characterization of PaOhrR, focusing on its reactivity toward organic peroxides, DNA-binding properties, and the role of conserved residues; (ii) identification of the reducing systems involved in the regeneration of its functional form; (iii) generation of PaOhrR bacterial mutants via CRISPR-Cas9 to assess the impact of specific mutations on DNA-binding affinity and bacterial virulence; (iv) global mapping of the PaOhrR regulon through transcriptomic analyses; and (v) evaluation of the Ohr-OhrR system's role in oxidative lipid signaling during interaction with phagocytes, through lipidomic analyses in co-culture systems.This project is expected to provide a comprehensive understanding of the molecular mechanisms by which PaOhrR senses and responds to oxidative stress, contributing to the elucidation of the redox physiology and virulence determinants of P. aeruginosa. These insights may open new avenues for the development of therapeutic strategies targeting this multidrug-resistant pathogen.