Helicobacter pylori Outer Membrane Vesicles Protect the Pathogen From Reactive Oxygen Species of the Respiratory Burst

Outer membrane vesicles (OMVs) play an important role in the persistence of Helicobacter pylori infection. Helicobacter OMVs carry a plethora of virulence factors, including catalase (KatA), an antioxidant enzyme that counteracts the host respiratory burst. We found KatA to be enriched and surface-associated in OMVs compared to bacterial cells. This conferred OMV-dependent KatA activity resulting in neutralization of H2O2 and NaClO, and rescue of surrounding bacteria from oxidative damage. The antioxidant activity of OMVs was abolished by deletion of KatA. In conclusion, enrichment of antioxidative KatA in OMVs is highly important for efficient immune evasion.


INTRODUCTION
Helicobacter pylori is a Gram-negative pathogen that commonly colonizes the gastric mucosa. Infection persists for a lifetime without antibiotic treatment although the pathogen constantly experiences hostile conditions including the acidic ventricle environment and host defense (Roesler et al., 2014). In order to survive against the highly acidic gastric juice (pH 1.0-3.0), H. pylori uses a series of acidic acclimation systems that neutralize the surrounding acid. Other virulence mechanisms include expression of abundant molecules at the surface for attachment and manipulation of host extracellular matrix proteins and serum resistance (Parker and Keenan, 2012;Richter et al., 2016). In addition, H. pylori is equipped with antioxidant molecules such as catalase (KatA), catalase-like protein (KatB), alkyl hydroperoxide reductase (AhpC), and superoxide dismutase (SOD) to detoxify reactive oxygen species (ROS) released from host immune cells during the respiratory burst (Wang et al., 2006). Furthermore, H. pylori constitutively releases outer membrane vesicles (OMVs) from its outer membrane (OM).
Outer membrane vesicles are cargos comprising an OM lipid bilayer enveloping several virulence factors. H. pylori OMVs have been extensively studied with respect to composition, proteome, and virulence functions (Mullaney et al., 2009;Olofsson et al., 2010), and play multiple roles in bacterial pathogenesis including biofilm formation, cancer development, and immune evasion (Parker and Keenan, 2012). Furthermore, OMVs display immunomudulatory effects by inducing IL-8 secretion from epithelial cells, activating phagoctyes, and suppressing immune cells of the adaptive immune system (Mullaney et al., 2009;Olofsson et al., 2010;Ko et al., 2016).
KatA, a 55 kDa catalase, is an essential virulence factor protecting H. pylori against the respiratory burst (Olofsson et al., 2010). In fact, KatA is upregulated during oxidative stress (Huang and Chiou, 2011). It is widely known that KatA detoxifies hydrogen peroxide (H 2 O 2 ) and hypochlorite (OCl − ) (Benoit and Maier, 2016). Additionally, we recently reported that KatA mediates vitronectin acquisition resulting in increased serum resistance (Richter et al., 2016). Interestingly, despite the lack of a signal peptide, Helicobacter KatA is ubiquitous with various topology including the bacterial surface, the cytosol and periplasmic space. KatA has also recently been identified in OMVs (Wang et al., 2006;Mullaney et al., 2009). However, little is known regarding the role of KatA in OMVs since previous studies have mainly focused on KatA in the cell-associated context.
We determined the importance of OMVs to eliminate extracellular ROS-mediated killing via KatA enrichment. Our data suggest a new mechanism of OMV-mediated H. pylori evasion from the attack of the innate immune system.

KatA Catalase Is Enriched in H. pylori-Derived OMVs
Helicobacter pylori KatA has been predicted as one of the periplasmic proteins that accounts for 7.4% of the total OMV proteome (Mullaney et al., 2009;Olofsson et al., 2010). Since most OM proteins are also located at the surface of vesicles (Bonnington and Kuehn, 2014), we wanted to investigate whether KatA localizes at the outer surface of H. pylori OMVs. As visualized by TEM, we found deposition of gold-labeled anti-KatA pAb at the surface of intact bacteria and OMVs of H. pylori wild type (wt) ( Figure 1A). However, no KatA was detected on any samples derived from the KatA-deficient H. pylori katA mutant. This suggested a similar surface exposure of KatA on OMVs as seen on intact bacteria. Further enumeration of antibody deposition revealed that more KatA was detected at the "blebbing areas" of wild type bacteria as compared to the "nonblebbing areas, " and this appearance was almost similar to the OMVs ( Figure 1B). This observation prompted our interest to compare the amount of KatA present in the OMVs and OM of H. pylori. Interestingly, we observed that OMVs contained sevenfold more KatA (18.37 ± 6.24 ng/µg sample) than bacterial OM (2.42 ± 0.24 ng/µg sample) ( Figure 1C and Supplementary Figure S1).

KatA Enriched OMVs Exhibit Catalase Activity
Helicobacter KatA of intact bacteria is known to actively hydrolyse H 2 O 2 and detoxify ClO − (Wang et al., 2006;Benoit and Maier, 2016). Interestingly, we found that the H 2 O 2 hydrolysis activity in OMVs was significantly (p < 0.05) higher than the H. pylori wt whole cell lysate (Figure 2A). We further investigated whether KatA could contribute to the antioxidant activity of Helicobacter OMV. As shown in Figure 2B, OMVs isolated from the strain H. pylori 18943 wt exhibited a strong . Arrows indicate OMVs that are vesiculating from the "blebbing area" at the bacterial surface (left panels) or purified OMVs (right panels). Visualization by Transmission Electron Microscopy (TEM) was performed on a Philips/FEICM 100 TWIN transmission electron microscope, and images were documented with a side-mounted Olympus Veleta camera having a resolution of 2048 × 2048 pixels (2k × 2K) and ITEM acquisitions software. (B) KatA is accumulated in OMVs at the bacterial surface, and in the released OMV fraction. The number of anti-KatA IgG pAb-gold particles per µm from 50 randomly selected TEM image profiles were calculated, and corresponded to at least 1000 different bacteria. (C) A significantly higher concentration of KatA is present in OMVs compared to the OM fraction. Estimation of KatA concentrations in OM and OMVs was done by western blotting as shown in Supplementary Figure S1. For (B) and (C), statistical differences were calculated by two-way ANOVA and two-tailed Student's t-test, respectively (mean ± SD; n = 3; * p < 0.05; * * * p < 0.001).
Frontiers in Microbiology | www.frontiersin.org , catalase activity was presented as nmol of H 2 O 2 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 H 2 O 2 and NaClO, and thus promoted bacterial survival. In (C), H. pylori P12 katA devoid of catalase activity was challenged with 1 mM H 2 O 2 preincubated with 40 µg/ml of OMV (equivalent to OMVs produced from 10 9 CFU) (OMV wt , OMV katA or heat-inactivated OMVs; OMV hia ). In (D), H. pylori 18943 katA devoid of KatA expression was exposed to 5% NaOCl that had been pre-treated with 0.1-10.0 µg of OMV wt . Pure H 2 O 2 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 H 2 O 2 and promote survival of H. pylori P12 katA from peroxidative killing. OMV lacking KatA (OMV katA ) 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 × 10 8 CFU). The killing of H. pylori 18943 katA 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).
catalase activity based upon hydrolysis of H 2 O 2 compared to OMVs of the H. pylori 18943 katA mutant that had an abolished KatA activity. In parallel, a similar H 2 O 2 hydrolysis activity was also found with OMVs isolated from another H. pylori strain (P12), whereas no activity was observed with the corresponding KatA-deficient H. pylori P12 mutant.

KatA-Enriched OMVs Promote H. pylori Survival Against the ROS of the Oxidative Burst
We also wanted to determine whether OMV loaded with KatA could protect bacteria from the bactericidal activity of ROS (H 2 O 2 and ClO − ) (Wang et al., 2006;Benoit and Maier, 2016). First, H. pylori katA lacking the catalase activity was exposed to H 2 O 2 that had been pre-incubated with OMVs derived from H. pylori wt (OMV wt ) or the KatA-deficient mutant (OMV katA ).
Interestingly, H. pylori katA survived when OMV wt -pre-treated H 2 O 2 was added, but was completely killed in both H 2 O 2 or H 2 O 2 preincubated with OMV katA (Figure 2C). Since KatA activity is heat sensitive, OMV wt was also heat-inactivated at 60 • C to generate OMV wt−hia . We found that H 2 O 2 preincubated with OMV wt−hia remained bactericidal against the mutant H. pylori katA.
We subsequently performed a disk diffusion assay to examine the capacity of OMVs in protecting H. pylori from the toxicity of NaClO. As shown in Figure 2D, the inhibition zone of H. pylori katA growth caused by NaClO was gradually reduced in response to increasing amounts (0.1-10 µg) of OMV wt used for preincubation with NaClO. Taken together, our data indicated that H. pylori OMVs exhibiting KatA-dependent catalase activity successfully neutralized both H 2 O 2 and NaClO, and thus promoting bacterial survival when exposed to the bactericidal activity of ROS.

DISCUSSION
Helicobacter pylori has evolved several virulence mechanisms for persistent colonization and infection in the gastric mucosa and this includes release of OMVs (Parker and Keenan, 2012). Here, we deciphered a novel role of OMVs in the pathogenesis of H. pylori; OMVs act as antioxidative particles via enrichment of KatA at the surface of vesicles. To the best of our knowledge, the current study is the first report regarding enrichment of KatA in H. pylori OMVs. Intriguingly, the H. pylori virulence factors OipA and HtrA have also been reported to be enriched in OMVs (Olofsson et al., 2010). Production of toxic ROS, i.e., superoxide (O 2 •− ), nitrogen oxide (NO), H 2 O 2 , and OCl − by human polymorphonuclear cells (PMNs) during the respiratory burst is an important component of the innate defense to eradicate phagocytosed pathogens (Yang et al., 2013). Despite H. pylori infection induces massive influx of neutrophils into the gastric mucosa and production of ROS, the pathogen expresses KatA to survive at the surface of phagocytes, H. pylori is thus antiphagocytic and resistant against the respiratory burst-dependent killing (Ramarao et al., 2000;Wang et al., 2006). In contrast to bacterial cell-associated KatA, little is known regarding the KatA-dependent ROS resistance of OMVs.
We speculated that the accumulation of KatA in H. pylori OMVs, and thus higher catalase activity compared to bacterial cells (Figure 2A), may confer OMVs as an antioxidant cargo to protect bacteria from extracellular ROS of the respiratory burst. Bacterial interactions with PMNs result in an increase of extracellular H 2 O 2 and ClO − release by neutrophils that is ineffective, however, to efficiently eradicate non-phagocytosed H. pylori (Ramarao et al., 2000;Allen et al., 2005). In addition to KatA, other antioxidant proteins such as KatB and AhpC are also present in the OMV proteome (Mullaney et al., 2009;Olofsson et al., 2010). However, we found that the catalase activity of OMVs is solely attributed to KatA accumulation since the ability to hydrolyse H 2 O 2 was diminished in OMVs lacking KatA (Figure 2B). This could be due to the relatively low amount of KatB and AhpC compared to KatA in the H. pylori OMVs (Mullaney et al., 2009;Olofsson et al., 2010). Importantly, the KatA-dependent catalase activity of H. pylori OMVs is highly conserved among different strains ( Figure 2B), further suggesting OMVs as important antioxidant particles.
In this study, we employed the direct H 2 O 2 and NaClO bactericidal assay as an in vitro extracellular ROS respiratory burst model. Of note, H. pylori KatA counteracts the oxidative damage of H 2 O 2 and OCl − via different mechanisms, which are through its catalase hydrolysis activity and oxidation of KatA methionine residues, respectively (Wang et al., 2006;Benoit and Maier, 2016). Our results demonstrated that H. pylori OMVs effectively neutralized ROS and rescued bacteria from lethal oxidative damage (Figures 2C,D).
The strategy to promote bacterial infection by virulence factor enrichment in OMVs has also been reported with other pathogens. Bacteroides spp. escapes from antibiotics by decorating their OMV surface with cephalosporinases (Stentz et al., 2015). Aggregatibacter actinomycetemcomitans utilizes OMVs enriched with leukotoxin to induce immune cell apoptosis (Bonnington and Kuehn, 2014). Our finding pioneered the idea of virulence factor enrichment in OMVs as a novel virulence mechanism of H. pylori. This is exemplified by KatA in OMVs that, in turn, contributes to the novel antioxidative role of H. pylori OMVs, and thus enhanced bacterial defense against host innate immune attacks. We speculate that, during infection in gastric mucosa, H. pylori releases OMVs enriched with KatA to decrease or depelete the surrounding extracellular ROS released from the oxidative burst of influxed PMNs. This will allow H. pylori to escape towards nearby infection sites with lower ROS, thereby facilitating bacterial survival and colonization.
In conclusion, we have presented expanded insights on a novel potential virulence mechanism of H. pylori that provide additional knowledge regarding bacterial survival in a hostile PMN-rich environment.

Bacterial Strains and Growth Conditions
Bacterial strains and growth conditions are listed in Table 1.

Transmission Electron Microscopy (TEM)
The localization of KatA at the surface of intact bacteria and OMVs was determined by purified rabbit anti-KatA polyclonal antibodies (pAb) labeled with 5 nm colloidal thiocyanate gold followed by TEM using negative staining (Olofsson et al., 2010).

Estimation of KatA Concentrations and Catalase Enzymatic Assays
Recombinant KatA (rKatA), OM, or OMVs sample were separated by SDS-PAGE followed by immunoblotting using anti-KatA pAb (Supplementary Figure S1) (Richter et al., 2016). Signal intensities generated from known amounts of rKatA were included as a standard curve for KatA estimation. Analysis was done by Image Lab software (Bio-Rad, Copenhagen, Denmark).
Bacterial lysates or OMVs were incubated with 1 mM H 2 O 2 in catalase buffer (50 mM Tris pH 7.4, 0.1% TritonX-100) for 30 min at room temperature. The reaction was terminated with 50 mM sodium azide, and residual H 2 O 2 was detected by OxiRed TM (Biovision, Milpitas, CA, United States) and horseradish peroxidase (Thermoscientific, Waltham, MA, United States) mixture. Plates were read at 570 nm on a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany).

H 2 O 2 Bactericidal Assay
H 2 O 2 (1 mM) was preincubated with 40 µg/ml of OMV for 1 h at 37 • C. Bacteria were resuspended in Brucella broth to an OD 600 of 0.1, and added to the OMV-treated H 2 O 2 . Mixtures were incubated for 3 h at 37 • C, and plated on chocolate agar for 5 days at 37 • C to enumerate the bacterial survival based on colony forming units (CFU). Control experiments were performed as described above by using only H 2 O 2 without OMVs.

Hypochlorous Acid-Based Disk Diffusion Sensitivity Assay
A sterilized filter paper (5.4 mm in diameter) was saturated with 20 µl of 5% NaClO that had been pre-incubated for 3 h with OMVs. Bacterial colonies were resuspended in PBS and evenly spread on chocolate agar. Filter papers were placed on top of the agar, and plates were incubated at 37 • C for 3 days. The diameter of inhibition zones was measured.

Statistical Analysis
Graph-Pad Prism R 7.0 (La Jolla, CA, United States) was used, and differences between groups or samples were considered statistically significant at p < 0.05.