Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development

Abstract

Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis; however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. IMPORTANCEDetermining the function(s) of sheathed flagella in bacteria has been challenging, because no known mutation results only in the loss of this outer membrane-derived casing. Nevertheless, the presence of a sheathed flagellum in such host-associated genera as Vibrio, Helicobacter, and Brucella has led to several proposed functions, including physical protection of the flagella and masking of their immunogenic flagellins. Using the squid-vibrio light organ symbiosis, we demonstrate another role, that of V. fischeri cells require rotating flagella to induce apoptotic cell death within surface epithelium, which is a normal step in the organ's development. Further, we present evidence that this rotation releases apoptosis-triggering lipopolysaccharide in the form of outer membrane vesicles. Such release may also occur by pathogens but with different outcomes for the host.

The Role of Hemocytes in the Hawaiian Bobtail Squid, Euprymna scolopes: A Model Organism for Studying Beneficial Host–Microbe Interactions

“…Morphogenesis of the light organ occurs after successful colonization, including apoptosis and regression of the ciliated appendages (McFall-Ngai and Ruby, 1991; Montgomery and McFall-Ngai, 1994; Foster and McFall-Ngai, 1998). These developmental events are triggered by MAMPs from V. fischeri including LPS and tracheal cytotoxin (TCT, a derivative of peptidoglycan), along with outer membrane vesicles (OMVs) (Foster et al, 2000; Koropatnick et al, 2004; Aschtgen et al, 2016). …”

“…Upon initiation of the symbiosis, as few as 3–5 V. fischeri cells induce hemocyte trafficking into the ciliated appendages of the nascent organ via signaling by TCT and OMVs (Koropatnick et al, 2007; Altura et al, 2013; Aschtgen et al, 2016). Hemocytes have also been shown to internalize OMVs (Aschtgen et al, 2016) but how these vesicles and other MAMPs are detected by hemocytes is unclear.…”

“…Upon initiation of the symbiosis, as few as 3–5 V. fischeri cells induce hemocyte trafficking into the ciliated appendages of the nascent organ via signaling by TCT and OMVs (Koropatnick et al, 2007; Altura et al, 2013; Aschtgen et al, 2016). Hemocytes have also been shown to internalize OMVs (Aschtgen et al, 2016) but how these vesicles and other MAMPs are detected by hemocytes is unclear. These cells do express a number of PRRs, including peptidoglycan recognition proteins (PGRPs), a Toll-like receptor (TLR), and galectins (see below; Collins et al, 2012a; Schleicher et al, 2014).…”

Impact and Influence of the Natural Vibrio-Squid Symbiosis in Understanding Bacterial–Animal Interactions

“…In addition to providing information directly about colonization, these tools provided insight into the role of LPS during colonization and for the evolution and the generation of torque at the flagellar motor (Post et al, 2012; Beeby et al, 2016). Furthermore, recent work suggests that the rotation of the flagellum-which is enclosed in an LPS sheath-stimulates outer membrane vesicle release and triggers the host immune response by promoting LPS release (Brennan et al, 2014; Aschtgen et al, 2016). …”

Lessons from simple marine models on the bacterial regulation of eukaryotic development

“…One of the best explored examples concerns the mutualistic interaction between the Hawaiian bobtail squid ( Euprymna scolopes ) and the bacterium Vibrio fischeri , in which colonization of the squid by environmental V. fischeri is required to induce the morphogenesis of the host’s light-organ (Table 1) [4]. The squid-Vibrio association is unusually amenable to laboratory manipulation, and as such, many of the relevant bacterial cues have been identified and their consequences for squid development explored in detail [6–9]. In contrast, few other marine bacterial-eukaryotic interactions are understood in molecular detail, in part because marine environments are host to dynamic and diverse bacterial communities.…”