While a few of the bacteria that live in and on the bodies of humans and other animals are harmful and can cause disease, most others can offer benefits to their hosts. Many bacteria—including some important human pathogens—have tails called flagella that rotate to move the bacteria inside its host. However, the immune system can detect parts of these flagella and eliminate the pathogen.

Bacterial flagella are made from filaments of proteins, and some flagella are also enclosed by a sheath that is similar to the outer membrane that encloses certain bacteria. The function of this sheath is unclear, although some researchers have suggested that it might prevent the immune system from detecting the proteins in the flagellum. Now, by studying the interactions between the Hawaiian bobtail squid and a marine bacterium, Brennan et al. show that the sheath can actually alert the host that the bacteria are around.

The Hawaiian bobtail squid collects bioluminescent bacteria within a so-called ‘light organ’. This organ undergoes a number of developmental changes to house the bacteria, and the squid then uses the light from the bacteria to mask its own shadow, which helps it to avoid being detected by predators. Brennan et al. compared how wild-type bacteria and mutant bacteria that either had no flagella, or had flagella that did not rotate, interacted with young squid. Only bacteria with working flagella were able to trigger the normal development of the squid’s light organ, which suggests that the rotating flagella are releasing the signal that tells the squid that the beneficial bacteria are present.

Brennan et al. demonstrated that the rotation of sheathed flagella led to the release of a molecule called lipopolysaccharide. This molecule is known to activate the immune system in animals, and it is one of the bacterial signals that the squid responds to. Moreover, when the flagella of other bacteria with sheaths—such as those that cause cholera—are rotating, there is also an increase in the release of lipopolysaccharide. However, rotation of the flagella of bacteria without sheaths has no such effect. The next challenge will be to test the importance of this release of lipopolysaccharide from rotating flagella on the outcome of bacterial diseases of humans and other animals.

Bacterial flagella are important virulence factors that facilitate tissue tropism in host–microbe interactions. Besides mediating such migration, flagella serve additional functions, such as adherence to or invasion of host cells (Young et al., 2000; Hayashi et al., 2001; Giron et al., 2002). However, in most vertebrate models of pathogenesis, it has been difficult to separate the distinct contribution of flagellar motility in bacterial migration from its other roles, such as immune-system activation (Feuillet et al., 2006; Andersen-Nissen et al., 2007). Bacterial flagella are highly conserved on a molecular level, but display diversity in morphology, number and subcellular localization between species. One such variation is the presence of an outer membrane-derived sheath (Fuerst and Perry, 1988; Geis et al., 1993; Ferooz and Letesson, 2010). While the flagella of most bacteria are unsheathed, such a structure surrounds the filament of several polarly flagellated bacteria, including several with a host-associated lifestyle. The biological function(s) of the sheath remains unknown, but it has been posited to allow these bacteria to evade innate immune recognition of the enclosed flagellins (Gewirtz et al., 2004; Yoon and Mekalanos, 2008). This hypothesized role for the flagellar sheath presents a conundrum: what is the effect of coating a rapidly spinning structure with lipopolysaccharide (LPS), another potent immunostimulatory molecule? As the tools with which to study the flagellar sheath are limited, we sought to use the model symbiosis between Vibrio fischeri and the Hawaiian bobtail squid, in which the events of initial colonization can be observed in real-time, to probe whether the sheathed flagellum plays a role in inducing a symbiont-specific host response.

V. fischeri is a gram-negative, bioluminescent bacterium that is found both in seawater and as a beneficial symbiont colonizing the light-emitting organs of certain fishes and squids. In the mutualism with the Hawaiian bobtail squid, Euprymna scolopes, symbiosis begins when a newly hatched squid specifically recruits V. fischeri cells from ambient seawater. The bacteria first aggregate on ciliated appendages of the light organ, and then migrate through pores and down ducts, ultimately reaching internal crypts (Figure 1A) where the symbiont population is housed (Nyholm and McFall-Ngai, 2004). Successful colonization of the crypts ultimately requires flagellar motility, as amotile mutants are unable to colonize the light-organ to luminous levels (Graf et al., 1994; Brennan et al., 2013). However, the symbionts are sensed by host cells even before they reach the crypts. Indeed, as few as 3–5 bacteria interacting with the ciliated cells of the surface appendages signal both organ-wide changes in host transcription and immune-cell (hemocyte) trafficking (Koropatnick et al., 2007; Kremer et al., 2013). Colonizing V. fischeri cells also induce an extensive morphogenesis of the light organ, beginning with the programmed cell death (apoptosis) of the ciliated appendages that begins within 6 hr of initial exposure to the symbiont, and continues until their complete regression (Montgomery and McFall-Ngai, 1994; Foster et al., 2000).

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To address whether the sheathed flagellum mediates symbiont recognition, we first compared the appearance of apoptosis within the host’s ciliated epithelium (Figure 1A) in response to inoculation with either wild-type V. fischeri, or one of two amotile mutants: motB1 (Brennan et al., 2013), which bears normal flagella that do not rotate (Figure 1—figure supplements 1 and 2); and flrA, which is both amotile and aflagellate (Millikan and Ruby, 2003; Brennan et al., 2013; Figure 1—figure supplements 1 and 3). Such amotile mutants do not successfully colonize the light-organ deep crypts (Graf et al., 1994). However, as this apoptosis proceeds over the first few days of symbiotic colonization, we assessed this phenotype an early time-point (10 hr post-inoculation) to optimize apoptopic induction by V. fischeri while selecting conditions under which amotile mutants would not yet exhibit a colonization defect. As previously reported (Montgomery and McFall-Ngai, 1994), squid that were not exposed to V. fischeri (i.e., aposymbiotic, or ‘apo’) exhibited much lower levels of apoptosis (Figure 1B,F) than squid exposed to wild-type V. fischeri (Figure 1C,F). In contrast, exposure to either the motB1 or the flrA strain did not induce early-stage apoptosis above the background levels (Figure 1D–F). Because the actual location at which amotile mutants are arrested in symbiotic initiation has not been defined, we confirmed that both the location and number (Figure 1G,H) of bacteria at this early stage were indistinguishable in squid exposed to wild-type, flrA or motB1 cells. These data show that flagellar motility plays a role in inducing a specific host immune response that is independent of its later role in migration into the light-organ crypts (Brennan et al., 2013), suggesting that biosynthesis of the flagellum, and its subsequent functions, may mediate multiple aspects of symbiotic initiation.

Reports of flagellar-mediated activation of immunity have thus far been limited to responses to the filament protein, flagellin (Hayashi et al., 2001; Franchi et al., 2006). Despite the flagellar sheath, flagellin monomers are a prevalent component of the secretome of both V. cholerae (Xicohtencatl-Cortes et al., 2006) and V. fischeri (unpublished data). However, the failure of the fully flagellated motB1 mutant to induce normal apoptosis demonstrated that the expression of immunogenic flagellin is insufficient to activate apoptosis in the light organ and, instead, suggests that apoptosis is driven by flagellar rotation itself. Previous work (Foster et al., 2000) has shown that apoptosis of the light-organ ciliated epithelium is induced specifically by the lipid-A component of LPS (Figure 1—figure supplements 4 and 5); indeed, LPS is a common activator of innate-immune defenses across the animal kingdom (Choi et al., 1998; Manna and Aggarwal, 1999). However, no effect of flagellar motility on LPS presentation has been reported previously. We posited that, as sheath production and flagellar biogenesis may not be tightly coordinated (Richardson et al., 1990; Josenhans et al., 1995; Ferooz and Letesson, 2010), a sheathed flagellum might shed LPS due to a rotation-weakened association of the sheath with the spinning flagellar filament. This hypothesis is supported by negative-stain transmission electron microscopy (TEM) of sheathed flagella, which has revealed vesicle-like structures (Figure 2A), often at the distal tip of the flagellum, and ∼10–80 nm in diameter (Geis et al., 1993; Millikan and Ruby, 2004; Ferooz and Letesson, 2010). Such images are indicative of weaknesses between the flagellar sheath and shaft, which may be analogous to the foci of disassociation between the outer membrane and peptidoglycan required for the formation of cell-derived outer-membrane vesicles (OMVs) (McBroom and Kuehn, 2007). Thus, rotation of the flagellum would further destabilize these interactions, likely releasing LPS and activating an LPS-dependent immune response, rather than simply shielding against a host response.

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We determined whether the motB1 and flrA mutants released less of this signal molecule than wild-type V. fischeri using the Limulus amoebocyte lysate (LAL) assay to quantify the amount of reactogenic LPS in cell-free supernatants prepared from exponentially growing cultures (Figure 2B). While all strains had similar growth characteristics (Figure 2—figure supplement 1), we observed a significant reduction in shed LPS by the motB1 mutant, whose flagella do not rotate, and an even greater reduction by the aflagellate flrA mutant. To confirm that these strains actually released less (and not just a less reactive) LPS, we directly measured the total LPS shed from these strains by purifying the lipid fraction from the cell-free culture supernatants, and visualizing the LPS by quantitative SDS-PAGE (Figure 2—figure supplement 2). This analysis revealed a substantial reduction in total LPS released by both the motB1 and flrA strains (Figure 2C), consistent with the data from the LAL assay (Figure 2B).

To further test our hypothesis, we next examined whether (i) genetic complementation of the motB1 mutant would restore normal levels of LPS shedding; (ii) disruption of flagellar rotation by a motB1-independent method would similarly lessen the LPS observed in culture supernatant; and, (iii) reduced LPS shedding would be observed when flagellar rotation is disrupted in other sheathed bacteria as well. As anticipated, expression of motB1 in trans restored both soft-agar motility and reactogenic LPS levels in cell-free culture supernatants (Figure 3, Figure 3—figure supplement 1). In addition, cell-free culture supernatants from a V. fischeri motX mutant, which lacks a different component of the flagellar motor than motB1, yet is also flagellated but amotile (Figure 4—figure supplements 1 and 2), had similarly reduced levels of reactogenic LPS (Figure 4). Taken together, these data support the notion that flagellar rotation promotes LPS release by V. fischeri cells.

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Finally, we asked whether flagellar rotation-mediated LPS release is characteristic of other bacteria with sheathed flagella by comparing the amount of LPS shed by wild-type Vibrio cholerae O395N1 and its amotile motAB-deletion derivative. As in V. fischeri, disruption of flagellar rotation in V. cholerae significantly reduced LPS release (Figure 5A), supporting the conclusion that this mechanism of LPS release is not unique to V. fischeri, but is conserved among bacteria with sheathed flagella. In contrast, a similar analysis of an Escherichia coli mutant unable to rotate its (unsheathed) flagella revealed no alteration in levels of released LPS (Figure 5B), further indicating that this behavior may be specific to bacteria that sheath their flagella. Nevertheless, it remains possible that rotation-mediated LPS release still occurs in other non-sheathed bacteria, or, perhaps, under other growth conditions.

Figure 5

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Using the model squid-vibrio symbiosis as a tool to probe the effect of the enigmatic flagellar sheath on host recognition, we uncover a previously unrecognized role for the rotation of sheathed flagella in mediating the release of the immunogenic molecule LPS in both V. fischeri and its pathogenic congener V. cholerae. The flagellar sheath has proved problematic to study; indeed, to our knowledge, there are no mutations that result in the loss of a sheath in otherwise sheathed flagella. Intriguingly, a few Vibrio species, including the human pathogen Vibrio parahaemolyticus, encode two independent flagellar structures: one set producing sheathed, the other unsheathed flagella (McCarter, 1999). However, pleiotropic effects of mutations in these distinct pathways as well as other technical limitations make a direct comparison of unsheathed and sheathed flagella within this species difficult to interpret (L McCarter, personal communication). Thus, the functional role of the sheath, even as it relates to bacterial motility, remains poorly characterized, and observation of this new phenomenon for the flagellar sheath furthers our understanding of this enigmatic structure and its contribution to bacterial virulence. As this behavior is difficult to probe experimentally, novel approaches, such as the modeling of the biophysical properties underlying lipid-membrane behavior at a sub-micron scale, are needed to inform our understanding of the interactions between the sheath and filament along the length of the flagellum, as well as the physical forces at play between a rotating flagellar sheath and the contiguous, but static, cellular outer membrane. While vesicle-like structures have only been reported at the flagellar tip of V. fischeri (Millikan and Ruby, 2004), another possible region from which LPS may be shed during rotation is the base of the flagellum where the basal body perturbs the outer membrane. Interestingly, cryotomographic imaging has revealed unusual features in the Vibrio flagellar basal body that may help stabilize this interaction (Chen et al., 2011).

Additionally, our results show that flagellar rotation in V. fischeri promotes release of the immunogenic molecule LPS that, during initiation of symbiosis, is required for normal induction of apoptotic tissue development (Foster et al., 2000). Further, by activating the squid’s developmental program in response to symbiont exposure, the flagellar sheath may serve to promote recognition by the host immune system, suggesting the context-dependent nature (mutualism or pathogenesis) of this behavior. Perhaps masking one immunostimulatory molecule (e.g., flagellin) with another (e.g., LPS) can serve to modulate, rather than simply avoid (Gewirtz et al., 2004; Yoon and Mekalanos, 2008), detection by the host immune system.

Surprisingly, although both the flrA and motB1 mutants still release LPS in culture, albeit at a reduced level, we observed complete attenuation of apoptotic induction, suggesting that (i) the threshold of LPS sensed by host cells is exquisitely calibrated to the amount of LPS shed by V. fischeri; (ii) because V. fischeri cells are not dividing during symbiotic initiation, flagellar-mediated LPS release is the predominant source of LPS to which host cells are exposed; and/or (iii) the LPS released by wild-type V. fischeri is somehow presented in a more immunogenically active form than that released by either the flrA or motB1 mutant. This last point emphasizes the microheterogeneous chemical structures of LPS made by bacteria like V. fischeri (Phillips et al., 2011). This heterogeneity, as well as LPS’s insolubility in aqueous solution and the toxicity of spent medium to juvenile squid, makes directly comparing the chemical concentration or specific activity of LPS between culture and symbiotic settings problematic. In this symbiosis, the LPS is produced by a few non-growing bacterial cells at a tissue interface, in high flow-field conditions that further confound quantification.

Finally, if LPS is shed from the flagellar tip as vesicles, other molecules such as flagellin might be enclosed, and these components may act synergistically to induce host responses (although flagellin has not yet been characterized as a morphogen in this association). Alternatively, LPS released from either the tip or base of the flagellum might form vesicles with a smaller diameter than the cell-derived OMVs, thereby enabling tighter interaction with the host-cell surfaces. Further work will build upon these discoveries and clarify the mechanisms underlying signaling during initiation of the squid-vibrio symbiosis; such work includes characterizing the composition of LPS released by V. fischeri during flagellar rotation, identifying the squid receptors that recognize V. fischeri LPS (Goodson et al., 2005; Krasity et al., 2011), and determining any role flagellin may have in activating host responses.

While thus far demonstrated only in V. fischeri and V. cholerae, the phenomenon of flagellar-mediated LPS release may be conserved across other animal-associated bacteria with sheathed flagella. The human pathogens Brucella melitensis and Helicobacter pylori, whose sheathed flagellum and/or flagellar motility serve as important virulence factors (Ottemann and Lowenthal, 2002; Petersen et al., 2011), may similarly release the immunogenic molecule LPS during flagellar rotation and, thereby, regulate immune recognition during infection. However, the complexity of mammalian models of host–microbe interactions can limit efforts to separate the various contributions of flagellar motility, and highlights the role of invertebrate model systems in revealing the underlying principles that govern shared mechanisms of host–microbe interactions.

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Author details

1. Caitlin A Brennan

   Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, United States

   Contribution

   CAB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Jason R Hunt

   1. Department of Microbiology, The Carver College of Medicine, University of Iowa, Iowa City, United States
   2. Department of Internal Medicine, The Carver College of Medicine, University of Iowa, Iowa City, United States

   Contribution

   JRH, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Natacha Kremer

   Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, United States

   Contribution

   NK, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Benjamin C Krasity

   Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, United States

   Contribution

   BCK, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Michael A Apicella

   1. Department of Microbiology, The Carver College of Medicine, University of Iowa, Iowa City, United States
   2. Department of Internal Medicine, The Carver College of Medicine, University of Iowa, Iowa City, United States

   Contribution

   MAA, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Margaret J McFall-Ngai

   Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, United States

   Contribution

   MJM-N, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

7. Edward G Ruby

   Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, United States

   Contribution

   EGR, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   egruby@wisc.edu

   Competing interests

   The authors declare that no competing interests exist.

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