Interaction between 1-Cys peroxiredoxin and ascorbate in the response to H₂O₂ exposure in Pseudomonas aeruginosa

Pseudomonas aeruginosa, a leading cause of hospital-acquired infections, triggers host defenses, including oxidant release by phagocytes. Targeting bacterial antioxidants could reduce pathogen infectivity. This study investigates LsfA, a 1-Cys peroxiredoxin (Prx), member of the Prx6 subfamily, involved in P. aeruginosa virulence. LsfA efficiently reduced various peroxides (10⁶-10⁷ M^-1s^-1), while exhibiting hyperoxidation resistance (k_{hyperoxidation} ∼10² M^-1s^-1). Despite its substrate oxidizing promiscuity, LsfA displayed specific reduction by ascorbate (2.2 × 10³ M^-1s^-1). Moreover, elucidating the LsfA's crystallographic structures in the reduced and sulfinic/sulfonic acid states at 2.4 and 2.0 Å resolutions unveiled possible residues related to ascorbate binding. Small-angle X-ray scattering (SAXS) and size-exclusion chromatography (SEC) confirmed LsfA as a dimer regardless of its oxidative state. Microbiological assays, including a real-time analysis employing Hyper7, a genetically encoded probe, showed that ascorbate enhanced H₂O₂ removal in a LsfA-dependent manner. Hence, our integrated structural, biochemical, and microbiological analyses underscored the significance of the ascorbate-LsfA pathway in P. aeruginosa response to H₂O₂.