Our results demonstrate that CT has no affinity for LPS, which comprises the outer leaflet of the OM, and resides entirely within the lumen of the OMVs. We therefore hypothesize that during T2S, some CT remains in the periplasm, allowing it to become incorporated in the OMV lumen. Unlike soluble CT, OMV-associated CT does not bind to the GM1 receptor in the lipid rafts of the host cell membrane because of the toxin’s location within the OMV. We propose that CT secretion via OMVs and through the T2S system are therefore complementary mechanisms for CT delivery.