Bacterial outer membrane vesicles provide an alternative pathway for trafficking of Escherichia coli O157 type III secreted effectors to epithelial cells.

Outer membrane vesicles (OMVs) are proteoliposomes shed by Gram-negative bacteria. Their secretion is enhanced by the transition into the intra-host milieu, and OMVs play critical roles during pathogenesis. Enterohemorrhagic Escherichia coli O157 (EHEC) can cause diarrheal disease in humans, and soluble toxins including Shiga-like toxins that contribute to disease severity and clinical complications like 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 both in the presence and absence of a functional T3SS. EHEC strains with non-functional T3SS 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 show that OMV-delivered Tir can cross-complement an effector-deficient EHEC strain and promote pedestal formation. Together, these data demonstrate that OMVs provide an alternative pathway for the delivery of EHEC T3SS cargo and that vesicle associated effectors are released from OMVs inside host cells and can retain biological activity.IMPORTANCEBacteria can package protein cargo into nanosized membrane blebs that are shed from the bacterial membrane and released into the environment. Here, we report that a type of pathogenic bacteria called enterohemorrhagic Escherichia coli O157 (EHEC) uses their membrane blebs (outer membrane vesicles) to package components of their type 3 secretion system and send them into host cells, where they can manipulate host signaling pathways including those involved in infection response, such as immunity. Usually, EHEC use a needle-like apparatus to inject these components into host cells, but packaging them into membrane blebs that get taken up by host cells is another way of delivery that can bypass the need for a functioning injection system.