Bacterial outer membrane vesicles provide an alternative pathway for trafficking of type III secreted effectors into epithelial cells

ABSTRACT Outer membrane vesicles (OMVs) are proteo-liposomes universally shed by Gram-negative bacteria. Their secretion is significantly enhanced by the transition into the intra-host milieu and OMVs have been shown to play critical roles during pathogenesis. Enterohemorrhagic Escherichia coli O157 (EHEC), causes diarrheal disease in humans, and soluble toxins including Shiga-like toxins that contribute to disease severity and clinical complications including hemolytic uremic syndrome, have been shown to be OMV associated. In addition to Shiga-like toxins, EHEC produces a type III secretion system (T3SS), and T3SS effectors are associated with colonization and disease severity in vivo. Here, we show that type III secreted substrates including translocators and effectors are incorporated into OMVs independent of type III secretion activity. EHEC strains with non-functional type III secretion systems shed more OMVs and vesicles enter host cells with accelerated kinetics compared to vesicles shed from wild type EHEC. The T3SS effector translocated intimin receptor (Tir) is trafficked from OMVs into host cells and localizes to the membrane. However, its clustering on the host membrane and co-localization with bacterial pedestals is intimin-dependent. We further show that OMV-delivered Tir can cross-complement an effector-deficient EHEC strain, demonstrating that OMV-associated effectors reach the host cell in a biologically intact form. Finally, we observe that the non-LEE encoded E3 ubiquitin ligase effector NleL is also trafficked to host cells via OMVs, where it ubiquitinylates its target kinase JNK. Together, these data demonstrate that trafficking of OMV-associated effectors is a novel and T3SS-independent pathway for the delivery of active effectors to host cells.