LsfA Peroxiredoxin and Ascorbate interact in the response of Pseudomonas aeruginosa to H2O2

Abstract 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) involved in P. aeruginosa virulence. LsfA efficiently reduces various peroxides (106 - 107 M-1.s-1), while exhibited hyperoxidation resistance. Despite its substrate oxidizing promiscuity, LsfA displayed specific reduction by ascorbate. Moreover, the elucidation of the LsfA’s crystallographic structures in the reduced and sulfonic acid states at 2.4 and 2.0 Ǻ resolutions, respectively, unveiled possible ascorbate-binding residues. Small-angle X ray scattering (SAXS) and size-exclusion chromatography (SEC) confirmed LsfA is 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.