Bacterial outer membrane vesicles (OMVs) are nano‐sized compartments consisting of a lipid bilayer that encapsulates periplasm‐derived, luminal content. OMVs, which pinch off of Gram‐negative bacteria, are now recognized as a generalized secretion pathway which provides a means to transfer cargo to other bacterial cells as well as eukaryotic cells. Compared with other secretion systems, OMVs can transfer a chemically extremely diverse range of cargo, including small molecules, nucleic acids, proteins, and lipids to proximal cells. Although it is well recognized that OMVs can enter and release cargo inside host cells during infection, the mechanisms of host association and uptake are not well understood. This review highlights existing studies focusing on OMV‐host cell interactions and entry mechanisms, and how these entry routes affect cargo processing within the host. It further compares the wide range of methods currently used to dissect uptake mechanisms, and discusses potential sources of discrepancy regarding the mechanism of OMV uptake across different studies.

The Pacific Northwest outbreak of cryptococcosis, caused by a near-clonal lineage of the fungal pathogen Cryptococcus gattii, represents the most significant cluster of life-threatening fungal infections in otherwise healthy human hosts currently known. The outbreak lineage has a remarkable ability to grow rapidly within human white blood cells, using a unique ‘division of labour’ mechanism within the pathogen population, where some cells adopt a dormant behaviour to support the growth of neighbouring cells. Here we demonstrate that pathogenic ‘division of labour’ can be triggered over large cellular distances and is mediated through the release of extracellular vesicles by the fungus. Isolated vesicles released by virulent strains are taken up by infected host macrophages and trafficked to the phagosome, where they trigger the rapid intracellular growth of non-outbreak fungal cells that would otherwise be eliminated by the host. Thus, long distance pathogen-to-pathogen communication via extracellular vesicles represents a novel mechanism to control complex virulence phenotypes in Cryptococcus gattii and, potentially, other infectious species.

Outer membrane vesicles are nano-sized microvesicles shed from the outer membrane of Gram-negative bacteria and play important roles in immune priming and disease pathogenesis. However, our current mechanistic understanding of vesicle-host cell interactions is limited by a lack of methods to study the rapid kinetics of vesicle entry and cargo delivery to host cells. Here, we describe a highly sensitive method to study the kinetics of vesicle entry into host cells in real-time using a genetically encoded, vesicle-targeted probe. We found that the route of vesicular uptake, and thus entry kinetics and efficiency, are shaped by bacterial cell wall composition. The presence of lipopolysaccharide O antigen enables vesicles to bypass clathrin-mediated endocytosis, which enhances both their entry rate and efficiency into host cells. Collectively, our findings highlight the composition of the bacterial cell wall as a major determinant of secretion-independent delivery of virulence factors during Gram-negative infections.

21Outer membrane vesicles (OMVs) are proteo-liposomes universally shed by Gram-negative bacteria. 22 Their secretion is significantly enhanced by the transition into the intra-host milieu and OMVs have been 23 shown to play critical roles during pathogenesis. Enterohemorrhagic Escherichia coli O157 (EHEC), 24 causes diarrheal disease in humans, and soluble toxins including Shiga-like toxins that contribute to 25 disease severity and clinical complications including hemolytic uremic syndrome, have been shown to be 26 OMV associated. In addition to Shiga-like toxins, EHEC produces a type III secretion system (T3SS), and 27 T3SS effectors are associated with colonization and disease severity in vivo. Here, we show that type III 28 secreted substrates including translocators and effectors are incorporated into OMVs independent of type 29 III secretion activity. EHEC strains with non-functional type III secretion systems shed more OMVs and 30 vesicles enter host cells with accelerated kinetics compared to vesicles shed from wild type EHEC. The 31 T3SS effector translocated intimin receptor (Tir) is trafficked from OMVs into host cells and localizes to 32 the membrane. However, its clustering on the host membrane and co-localization with bacterial pedestals 33 is intimin-dependent. We further show that OMV-delivered Tir can cross-complement an effector-34 deficient EHEC strain, demonstrating that OMV-associated effectors reach the host cell in a biologically 35 intact form. Finally, we observe that the non-LEE encoded E3 ubiquitin ligase effector NleL is also 36 trafficked to host cells via OMVs, where it ubiquitinylates its target kinase JNK. Together, these data 37 demonstrate that trafficking of OMV-associated effectors is a novel and T3SS-independent pathway for 38 the delivery of active effectors to host cells. 41Enterohemorrhagic Escherichia coli (EHEC) are a leading cause of food-borne diarrheal disease world-42 wide. In some cases, gastrointestinal symptoms can be complicated by the development of hemolytic 43 uremic syndrome (HUS), which is linked with increased morbidity and mortality (1). Secreted toxins, 44 chiefly Shiga-like toxins, lead to the development of HUS, and the accessory toxins cytolethal distending 45 toxin V (CdtV) and hemolysin (Hly) are thought to contribute to HUS pathology (2, 3). However, many 46 more virulence factors are associated with primary colonization of the gastrointestinal tract, in particular 47 the locus of enterocyte effacement (LEE), which encodes for a type III secretion system (T3SS) and 48 associated effectors (4, 5). The T3SS is a needle-like conduit that translocates effector proteins into the 49 host cell where they manipulate host cellular signaling machinery, induce cytoskeletal rearrangements and 50 modulate immunity to facilitate infection. The T3SS needle is formed by the structural protein EspA (6), 51 and secretion activity is driven by the ATPase EscN (7). EscN directly interacts with T3SS chaperones as 52 well as secreted effectors (8). The T3SS in...