FIGURE 2.

OMVs carrying KatA hydrolyse/detoxify ROS and provide protection against ROS-mediated bacterial killing. (A) Comparison of catalase activity between OMVs derived from H. pylori 18943 wt and a bacterial lysate. An increased H₂O₂ hydrolysis on a weight basis was detected with OMVs compared to bacterial lysate. (B) Determination of KatA-dependent catalase activity in OMVs. Deletion of KatA in two different strains of H. pylori, 18943 and P12 resulted in abolished catalase activity in OMVs from H. pyloriΔkatA mutant strains compared to OMVs derived from the wild type counterparts. In (A) and (B), catalase activity was presented as nmol of H₂O₂ decomposed per mg of sample tested (mean ± SD; n = 3; ^∗p < 0.05; ^∗∗p < 0.01). (C) and (D), Helicobacter OMVs reduce the bactericidal activity of H₂O₂ and NaClO, and thus promoted bacterial survival. In (C), H. pylori P12ΔkatA devoid of catalase activity was challenged with 1 mM H₂O₂ preincubated with 40 μg/ml of OMV (equivalent to OMVs produced from 10⁹ CFU) (OMV^wt, OMV^ΔkatA or heat-inactivated OMVs; OMV^hia). In (D), H. pylori 18943ΔkatA devoid of KatA expression was exposed to 5% NaOCl that had been pre-treated with 0.1–10.0 μg of OMV^wt. Pure H₂O₂ and NaOCl in PBS that were fully bactericidal were included as a positive control. In (C), the viability of bacteria was assessed by plating and counting colony forming units (CFU). Only KatA-containing OMV^wt was able to neutralize H₂O₂ and promote survival of H. pylori P12ΔkatA from peroxidative killing. OMV lacking KatA (OMV^ΔkatA) or heat-inactived KatA (OMV^hia) did not show any protection of whole bacteria (mean ± SD; n = 3; ^∗p < 0.05; ^∗∗p < 0.01). In (D), the bactericidal activity of NaOCl was decreased by preincubation with increasing amounts of OMV^wt (equivalent to OMVs produced from 0.2 to 2 × 10⁸ CFU). The killing of H. pylori 18943ΔkatA by NaOCl was measured by the diameter of inhibition zone, which is the area without bacterial growth (mean ± SD; n = 3; ^∗∗p < 0.01; ^∗∗∗p < 0.001).