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

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), 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.

Keywords: 1-Cys peroxiredoxin; Ascorbate; H(2)O(2) response; LsfA; Pseudomonas aeruginosa, sulfenic acid.

Fig. 1

Kinetics of LsfA oxidation by various peroxides. A) HRP competition assay, using HRP (5 μM), peroxynitrite (1 μM), and increasing concentrations of LsfA (0–4 μM). B) Peroxynitrite decay assay performed with a fixed concentration of peroxynitrite (20 μM), while varying LsfA concentrations (0–15 μM). C) HRP competition assay with fixed concentrations of HRP (2 μM) and H₂O₂ (0.91 μM), while increasing concentrations of LsfA (0–2 μM). D) Redox-dependent changes on the intrinsic fluorescence of LsfA (1 μM) were followed upon H₂O₂ treatment (1 μM). The curves of the first phase were fitted with first-order exponential functions to obtain the observed rate constants k_obs. Inset: Plot of k_obs versus H₂O₂ concentration, where the slope represents the second-order rate constant. E) Peroxides competition assay, using fixed concentrations of peroxynitrite (20 μM) and LsfA (15 μM)and increasing H₂O₂ concentrations (0, 10 and 20 μM). Reactions were monitored by the absorbance decay of peroxynitrite. F) Determination of k_obs by fitting first-order exponential functions to the second phase of reactions containing LsfA (1 μM) and increasing H₂O₂ concentrations (100–1500 μM). An illustrative experiment is depicted in panel D. G) Peroxynitrite competition assay using a fixed concentration of peroxynitrite (20 μM) and LsfA (15 μM) and varying t-BOOH concentration. H) Redox-dependent changes in the intrinsic fluorescence of LsfA (1 μM) followed upon t-BOOH treatment (100 μM). The initial phase was fitted with a first-order exponential to obtain kobs. Inset: Plot of kobs versus t-BOOH concentration, where the slope represents the second-order rate constant. I) k_obs were obtained by fitting first-order exponential functions to the second phase of reactions containing LsfA (1 μM) and increasing t-BOOH concentrations (100–1500 μM).

Fig. 2

Structural characterization of LsfA. A) Crystallographic structure of reduced LsfA (PDB ID: 6P0W), showing the characteristic β -strand dimerization interface. One subunit of the dimer is colored in copper and the other is shown in gray. The active site, containing the C_P, is indicated by the black arrows. B) Secondary-structure topology of LsfA, highlighting the arrangement of α-helices and β-strands, with the active site located at the N-terminal region of α-helix 2. C) Close-up view of the active site of reduced LsfA (PDB ID: 6P0W), with C_P shown within 2Fo-Fc electron density map. D) Close-up view of the active site of LsfA in the sulfonic acid state (PDB ID: 7KUU), also displayed with its corresponding 2Fo-Fc electron density map. Both electron density maps were contoured at 1.5 σ units in PyMOL. For additional details regarding electron density maps, please refer to Fig. S6.

Fig. 3

Structural comparison among Prx6 family members. A) Electrostatic surface representation of one subunit from each Prx6 structure. Blue color indicates positively charged areas, while red regions represent negatively charged areas. The black circle highlights the positively charged active site. B) and C) Superposition of the active sites from different Prx6-type of enzymes, emphasizing the highly mobile His residue, the conserved arginine and C_P in either reduced or (hyper)oxidized, respectively. The PDB codes are as follow: P. aeruginosa LsfA (7KUU and 6P0W), Homo sapiens Prdx6 (5B6M and 5B6N), Saccharomyces cerevisiae Prx1 (5YKJ), Arenicola marina Prx6 (2V2G), Aquifex aeolicus AhpC2 (5OVQ), Plasmodium yoelli 1-Cys (3TB2), Sulfolobus islandicus 1-Cys Prx (6Q5V), Thermococcus kodakaraensis Prx (6IU0), Aeropyrum pernix K1 Prx (2E2M and 3A5W) and Pyrococcus horikoshii Prx (3W6G).

Fig. 4

Roles of LsfA in the response of P. aeruginosa to oxidative insults. A) Wild type (WT), ΔlsfA or ΔlsfA complemented with LsfA (ΔlsfA + LsfA) strains were treated with 3 mM SIN-1 or 2.5 mM paraquat, for 30 min at 37 °C. Prior to treatment, cells were pre-incubated with 5 mM ATZ for 10 min, to inhibit catalase activity. Survival was determined by counting colony-forming units (CFUs) after 16 h at 37 °C, where data are expressed as the percentage of CFU relative to untreated controls (n = 3, ∗p < 0.05 by unpaired t-test). Untreated cells presented around 50 to 100 colonies after a one million times dilution. B) Halo inhibition assay. A filter disk containing either 2 % H₂O₂ or 1 % t-BOOH was placed on solid culture plates seeded with WT, ΔlsfA or ΔlsfA + LsfA strains. After 16 h of incubation at 37 °C, the diameter of the halo was measured. Data are expressed as the percentage relative to treated WT strain (n = 3, ∗∗p < 0.0020, ∗∗∗p = 0.0006 by unpaired t-test). The halo distances for WT and ΔlsfA treated with H₂O₂ were around 1 cm for the WT and 1.2 cm for the ΔlsfA. C and D) Oxidation ratio of Hyper7 probe expressed in WT (C) or in ΔlsfA (D) in OD = 7 in PBS at 37 °C, at increasing H₂O₂ concentrations as depicted in the legend. Results are expressed by a representative experiment of a biological replicate (n = 3) and as means of technical duplicates (n = 2).

Fig. 5

Capacity of P. aeruginosa to use ascorbate for supporting H₂O₂ removal in a LsfA dependent manner. Oxidation ratio of Hyper7 probe expressed in WT (A and C) or ΔlsfA (B and D) at an OD = 7 in PBS at 37 °C. The first dotted line indicates the addition of 4 mM of H₂O₂, while the second dotted line, 1 min later, marks the addition of either PBS (blue line) or ascorbate (5 mM in green or 8 mM in red line). Results are presented as a representative experiment of biological replicates (n = 3) and as the mean of technical duplicates. Mann-Whitney test showed a highly significant difference between PBS and ascorbate in WT strains ∗∗∗∗p < 0.0001, while no significant difference were observed in ΔlsfA strains.