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

Abstract

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...

The Ubiquitination System within Bacterial Host–Pathogen Interactions

“…Enterohemorrhagic E. coli produces E3 ubiquitin ligase effector NleL that is transported to the host cell through outer membrane vesicles (OMVs), where it can ubiquitinylate c-Jun N-terminal kinase (JNK) and thus could have a suppressing effect on the proinflammatory signaling pathway [ 64 ].…”

Nuclear trafficking of bacterial effector proteins

“…The OMVs from several bacterial species were shown to deliver CDTs (Kieselbach, Zijnge, Granström, & Oscarsson, 2015; Rompikuntal et al, 2012) and T3SS effectors (Sirisaengtaksin, O'Donoghue, Jabbari, Roe, & Krachler, 2018) into eukaryotic cells. Similarly, Helicobacter pylori OMVs were found to contain putative nucleus‐targeting proteins for example , urease subunit A, peptidoglycan‐associated lipoprotein (also known as Omp18) (Lee, Jun, Kim, Baik, & Lee, 2015; Suganuma et al, 2008; L. Turner et al, 2015).…”

“…Some nuclear‐targeting proteins, such as CDTs, are released from bacterial cells within OMVs. Moreover, OMVs have been reported to traffic to the nucleus (Rompikuntal et al, 2012; Rosenberger et al, 2000; Rüter et al, 2010; Sirisaengtaksin et al, 2018). Together, these observations raise the question as to whether OMVs may serve as an important mechanism whereby nuclear‐targeting proteins and other bacterial components are delivered to the nucleus and subvert host cell functions during bacterial infection.…”