Helicobacter pylori Outer Membrane Vesicles Engage TLR2 Through NapA to Promote Epithelial Association and Inflammatory Signalling

Abstract

Helicobacter pylori infection is characterized by chronic gastric inflammation driven by persistent activation of innate immune signalling pathways. Outer membrane vesicles (OMVs) released by H. pylori are important mediators of host–pathogen interactions. Although several mechanisms of OMV internalization have been described, how specific OMV-associated bacterial factors engage host innate immune receptors and couple vesicle association to inflammatory signalling remains poorly understood. Here, we investigated the contribution of OMV-associated virulence factors and Toll-like receptor 2 (TLR2) heterodimers to OMV–host cell interactions in gastric epithelial cells. Using engineered AGS and GES-1 reporter cells expressing TLR2/1 or TLR2/6, together with genetically defined OMVs, we found that H. pylori OMVs preferentially associated with TLR2-expressing cells and induced robust NF-κB activation. Comparative analysis of OMVs from wild-type and isogenic mutant strains identified Neutrophil-Activating Protein A (NapA) as a major determinant of TLR2-dependent OMV association and inflammatory signalling, whereas urease had a more limited contribution. Disruption of NapA or neutralization of TLR2 markedly reduced OMV association with gastric epithelial cells. Purified H. pylori lipopolysaccharide elicited a comparatively weak and TLR2/6-biased response, indicating that it does not account for the robust TLR2-dependent signalling induced by intact OMVs. Furthermore, OMV-associated NapA exhibited greater pro-inflammatory activity than soluble recombinant NapA, supporting a role for the vesicular context in enhancing TLR2-dependent signalling. Pharmacological inhibition with sulforaphane significantly attenuated OMV-induced TLR2/NF-κB activation. Collectively, these findings identify a previously unrecognized role for OMV-associated NapA in linking TLR2-dependent vesicle association with gastric epithelial cells to inflammatory signalling and support a model in which vesicular presentation