The first engagement of partners in the Euprymna scolopes-Vibrio fischeri symbiosis is a two-step process initiated by a few environmental symbiont cells

A small number of host-associated V. fischeri are sufficient to initiate symbiosis

To characterize the initial events of host-symbiont interaction, we quantified, over time and with varying levels of inocula, fluorescently labeled environmental V. fischeri cells associating with the ciliated surface of the juvenile host's light organ. These experiments were performed in the background of natural seawater (~10⁶ naturally occurring, non-specific marine bacteria per milliliter). To study the timing of association, we exposed the animals to levels of V. fischeri that the host typically encounters in the environment (~5 × 10³ CFU/ml of seawater; (Jones et al., 2007)); such doses typically result in 100% colonization and initiation of TCT-induced hemocyte trafficking (Koropatnick et al., 2004). V. fischeri associated with the ciliated epithelium at low numbers beginning at 1 h post-exposure (Fig. 1); on average, one fluorescent bacterium per ciliated field was detected. The number of associated V. fischeri cells per field increased slightly, but significantly, over the first 3 h to ~4 fluorescent cells/ciliated field (Fig. 2A). Shortly after this time, migration into the pores began. With increasing inoculum size, we observed an increase in the number of bacteria associated with the ciliated field at 3 h post-exposure (Fig. 2B). When twice the bacterial dose was added (i.e., from 5,000 to 10,000 CFU/ml of seawater), about twice as many V. fischeri cells associated with host tissues, although the result was not statistically significant. With a 20-fold increase in dose, however, we observed a significant 200-fold increase in the number of associating bacteria, which suggests cooperativity above a certain inoculum size. Taken together, these data provide evidence that a very small population of V. fischeri interacts with host tissues under normal conditions.

We also examined the effects of increased inoculum size on hemocyte trafficking to the blood-sinus space underlying the ciliated epithelium of the light organ. Increased hemocyte trafficking was seen in response to the presence of the symbiont as was previously described (Koropatnick et al., 2007). Further, an increase in trafficking was also observed in response to increasing inoculum size (Fig. 2C).

V. fischeri cells intimately associate with cilia of the light organ's superficial epithelium

Because only a few associating symbiont cells are capable of inducing a robust host-cell phenotype, e.g., TCT-triggered hemocyte trafficking, we reasoned that the V. fischeri cells were not merely suspended above host tissues in host-secreted mucus as previously suggested by our work and the work of others (Nyholm et al., 2000; Yip et al., 2006), but are likely associated more closely with host cells. Examination of large aggregates of V. fischeri (i.e., those resulting from an exposure of the animal to 10⁶ CFU/ml) by scanning electron microscopy revealed multiple points of contact between V. fischeri and host cilia, as well as contact among the symbiont bacteria themselves (Fig. 3A).

Since the typical aggregates of a few symbiont cells were difficult to visualize by SEM and the fixation/dehydration steps for SEM may introduce artifacts, we also used confocal microscopy with live specimens to visualize the cilia-bacteria interactions. By staining the light organ of live animals with a fluorochrome that labels the abundant tubulin present in the cilia, we localized the fluorescent bacteria in close association with the cilia of the organ's surface. Although in the absence of the labeling of cilia, V. fischeri cells often appear to be at some distance from the surface (Nyholm et al., 2000), with the cilia labeled, all V. fischeri cells colocalized with the ciliary surfaces (Fig. 3B, C) whether the inoculum was at environmentally relevant (10³ CFU/ml) or higher (10⁵ CFU/ml) doses of bacteria. In areas that stained positively for mucus, but did not contain cilia, V. fischeri cells were not detected (Fig. 3C). These data suggest that V. fischeri cells at these initial stages are not suspended in a mucus matrix or biofilm of their making, but rather interact intimately with host-cell surfaces. As such, even a small number of symbionts have the potential to deliver signals, such as TCT, directly to the host cells with which they associate.

Bacterial features affect normal interaction with cilia

We then sought to explore a general mechanism underlying V. fischeri attachment to host cells. In these experiments, to examine whether non-viable V. fischeri associate with host cilia, the bacteria were either heat- or azide-killed to disrupt cellular function. Both treatments led to bacteria that associated with the ciliated surface of the organ at statistically significantly lower levels (Fig. 4). When compared to treatment with azide, however, only one-tenth as many heat-treated cells were able to interact (p = 0.056). These data suggest that while viability enables this host-bacterial association, a heat-labile cell-surface component likely also plays a role.

Early engagement of V. fischeri cells is a two-step process: attachment to host cilia followed by bacterial-bacterial cell adhesion

We used V. fischeri mutants defective in exopolysaccharide production, which have phenotypes associated with early engagements with the host (Yip et al. 2006), as a tool by which to study initial partner-cell interactions. To assess the effect of symbiont exopolysaccharide production on bacterial association with the ciliated field, we exposed squid to either V. fischeri mutants unable to produce exopolysaccharide (sypG⁻ and rscS⁻), or a mutant that overexpresses exopolysaccharide (rscS⁺⁺) (Table 1). We analyzed the data by normalizing the counts to wild-type controls. The sypG⁻ and rscS⁻ mutants had consistently, but not always significantly, fewer bacterial cells associating with host cilia than wild type at all time points and inoculum levels assayed. This lower level of cell association was only statistically significant at high inocula of sypG⁻ and rscS⁻ (10⁵ CFU/ml) at 3 h post-exposure and with short exposures (1 h) of 10³ CFU/ml for sypG⁻ (Fig. 5A). Although the bacteria associated with host cells at numbers close to those of wild type, a greater distance between individual sypG⁻ mutant cells was detected, indicating the disruption of the aggregation phenotype (Fig. 5B), as had been previously described (Yip et al., 2006). In contrast, symbiont cells that overexpressed exopolysaccharide (rscS⁺⁺) had significantly higher numbers of V. fischeri cells associating with the light-organ surface compared to wild-type at all time points and inocula (Fig. 5A), and were as closely associated with one another (Fig. 5C). The results of these experiments suggest that the V. fischeri exopolysaccharide is not required for normal association with host cilia but is required for aggregation. As such, they provide evidence of a two-step process: attachment of V. fischeri to host cilia, followed by aggregation mediated by the exopolysaccharide on the surface of these symbionts.

To determine which of these two steps, i.e., adherence to the cilia or exopolysaccharide-mediated aggregation, induces the characteristic hemocyte trafficking into associated host tissues, we quantified hemocytes in the blood sinus underlying the superficial epithelium. Neither the under-expression of exopolysaccharide (sypG⁻) nor the over-expression of exopolysaccharide (rscS⁺⁺) significantly affected the number of hemocytes that trafficked to the blood sinus space of the light organ anterior appendage (Fig 5D).

We also examined four other mutants defective in outer envelope features (Table 1). The strains used included three mutants defective in early colonization: 1) a pilus mutant (ΔpilA2) (Stabb and Ruby, 2003); 2) an outer membrane protein mutant (ΔompU) (Aeckersberg et al., 2001); and, 3) a mutant defective in O-antigen production (waal∷Tnerm) (Post et al., 2012). We also examined a mutant in the V. fischeri mam7 gene. The Mam7 protein was an attractive candidate for involvement in initial attachment, as in V. cholerae, the product encoded by this gene is involved in the early binding of V. cholerae to host microvilli (Krachler et al., 2011), and so its homolog in V. fischeri may mediate binding to host membranes. Further, the V. fischeri homolog has the same in vitro activity as the V. cholerae protein (Fig. S1). None of these mutants had either an effect on attachment or an association defect under any condition (Fig. S2).

Association with host cilia is not specific to symbiotic V. fischeri

To examine the specificity of attachment to host cilia, we assessed the ability of a V. fischeri strain that is not symbiotic in the squid host to associate with light-organ cilia (Table 1). We exposed the animal to V. fischeri MJ11, the symbiont of the Japanese pinecone fish Monocentris japonica, which is typically unable to colonize the squid (Mandel et al., 2009). No significant difference was found in any of the six inoculum conditions tested (Fig. 6A). These data indicate that the defects in colonization of this strain are not likely due to defects in association of the cells with host cilia. MJ11 does not have the rscS gene (Mandel et al., 2009), which renders them unable to aggregate, but similar to rscS⁻ ES114, they show no defect in association with the cilia.

Vibrio parahaemolyticus KNH1, an isolate from a near-shore habitat in Hawaii where both the host squid and free-living, symbiosis-competent V. fischeri co-occur, was also analyzed. KNH1 forms aggregates outside the squid light organ in the absence of V. fischeri cells, but is unable to colonize the animal (Nyholm and McFall-Ngai, 2003); as the aggregate matures, V. fischeri outcompetes V. parahaemolyticus. Under the conditions of the experiments reported here, V. parahaemolyticus KNH1 cells were able to associate with the cilia at levels statistically indistinguishable from the symbiont at all time points and inocula (Fig. 6A).

V. parahaemolyticus is known to be out-competed in the aggregate by wild-type V. fischeri (Nyholm and McFall-Ngai, 2003). To determine whether association with the cilia played a role in dominance, we exposed the squid to V. parahaemolyticus and wild-type V. fischeri ES114, and counterstained the cilia to observe the extent of close association of these cells with host tissues. We detected no change in localization of V. parahaemolyticus. In addition, we observed that this non-symbiotic species continued to localize to the cilia, even in the presence of increasing levels of wild-type V. fischeri (Fig. 6B).

The factors that influence symbiont dominance are not well understood in the squid-vibrio system. To determine whether the exopolysaccharide-induced, bacteria-bacteria associations described above influence dominance by wild-type V. fischeri over V. parahaemolyticus, we competed V. parahaemolyticus against either the wild-type V. fischeri or the rscS⁻ mutant. The rscS⁻ mutation in V. fischeri did not compromise its ability to dominate V. parahaemolyticus in the aggregate. However, no significant difference in the relative levels of association with the host’s ciliated epithelium was detected at the inocula tested (Fig. 6C). These data provide evidence that dominance of V. fischeri occurs at a point downstream of both the attachment and aggregation steps

An exquisitely sensitive recognition system

The picture emerging from this and other studies indicates elaborate biomechanical and biochemical underpinnings that lead to the ultimate resolution of the exclusive partnership between Euprymna scolopes and Vibrio fischeri. While residing for hours on the light organ surface, 3–5 viable V. fischeri cells engage host cells first and then conspecific cells (Fig. 3A, Fig. 4), a finding that suggests an active process with molecular and/or biochemical dialog between and among partnering cells. Recent data have provided support for this possibility. Genetic studies of V. fischeri have demonstrated that, before entering host tissues, they must be capable of adapting to nitric oxide in host-shed mucus on the surface of the organ (Davidson et al., 2004; Wang et al., 2010; Altura et al., 2011). During these early hours, the host is also undergoing changes beyond the hemocyte trafficking described here. At first hatching, the light-organ crypt spaces, where the symbionts will eventually reside, are permissive, allowing all environmental particles < 5 microns to enter into these spaces; however, no particles or cells, including any V. fischeri cells that might enter, persist. Then, during the ~2–3 h period of V. fischeri aggregation, the host crypts experience a restrictive period when nothing enters (Nyholm et al., 2002). After this ‘restrictive’ period, the light-organ crypts exclude all cells other than those of V. fischeri. Taken together, these data provide several lines of evidence for an early period of adaptation by the partners, for which the host cilia-V. fischeri interactions described here may play a critical role.

The ability of the host to discriminate V. fischeri from most other environmental cells may result in the host’s detection of exported peptidoglycan monomers (TCT) produced by the attaching cells. Hemocyte trafficking is a response to symbiont TCT (Koropatnick et al., 2004) and the onset of hemocyte trafficking correlates with V. fischeri attachment to the cilia. While other Vibrio spp. can induce host hemocyte trafficking (Koropatnick et al., 2007), V. fischeri is the dominant Vibrio spp. in the host environment (Jones et al., 2007) and, when it is present, it outcompetes other Vibrio spp. These features suggest that the response is specific to V. fischeri under normal conditions. One other possible explanation for hemocyte trafficking during this time is that the host may merely be responding to the presence of the 5000 V. fischeri CFU/ml in the seawater. Extrapolating from data on the export of TCT by culture-grown V. fischeri cells (Koropatnick et al., 2004), about 5,000 CFU/ml in seawater would produce about 2 pM TCT, well below the detection limit of host cells (Koropatnick et al., 2004). This amount is likely an overestimate under the conditions of aggregation, as the cells are not growing in the seawater nor in the aggregates (Nyholm and McFall-Ngai, 2003). As such, peptidoglycan turnover and TCT export is likely to be attenuated. The finding that the host responds to 3–5 attaching V. fischeri cells provides evidence for very high specific activity of TCT as well as for requirement for direct contact with host cells for delivery of this MAMP.

The role of symbiont features in initial host-symbiont engagement

The finding that heat-killed V. fischeri cells were more perturbed than azide-killed cells in their association with the host raises the possibility of a heat-labile surface component that is involved in the formation of the interactions of these cells with host cilia. Mutants in surface features known to be important in bacterial pathogenesis, including those encoding a pilin protein, a porin, LPS O-antigen, and the Mam7 protein were normal in their ability both to associate with the host cilia and to aggregate (Fig. S1). Further analyses with these mutants in the squid-vibrio system will determine whether they mediate normal engagement and interactions with the microvillous surfaces of the crypt epithelia. While mutants defective in these molecular features of the cell envelope had no detectable defects in initial interactions with the host, because the partners are interacting at this time with their surfaces, screens directed at production of mutants in outer membrane molecules (Kaufman and Taylor, 1994) promise to be a fruitful area of future research on this system.

Exopolysaccharde (EPS) production by V. fischeri is a well-studied essential feature of early interactions in the squid-vibrio system (Visick, 2009). Mutations in symbiont genes encoding molecules that synthesize the EPS or regulate its production are defective in colonization of the host animal. Examination of mutants defective in EPS production in the present study suggests that the formation of bacterial-bacterial interactions is a distinct process from association with the host cilia, and that aggregate formation in part is EPS-mediated. A two-step process during initial host engagement may also occur in Vibrio cholerae, where attachment of V. cholerae cells to chitin and mucin is mediated by the colonization factor GbpA (Wong et al., 2012), and bacterial-bacterial interaction is controlled by the toxin-coregulated pilus (Kirn et al., 2000). Whereas EPS has a profound effect on the ability of the bacteria to aggregate, it does not appear to play a role in either dominance within the aggregate by symbiosis competent V. fischeri over other related bacteria or in the induction of host hemocyte trafficking (Fig. 5D and Fig. 6C). The fact that EPS is nonessential to these processes indicates that bacterial aggregation is most critical for the effect it has on communication among V. fischeri cells participating in the aggregate. Additionally, the data indicate that formation of host-bacterial, rather than bacterial-bacterial interactions, are likely to play a role in the hemocyte-trafficking and dominance phenotypes.

We also found that V. fischeri cells are not capable of outcompeting nonsymbiotic Vibrio spp., specifically V. fischeri MJ11 and V. parahaemolyticus KNH1, for attachment sites on the cilia or for early aggregation. This capacity of nonspecific Gram-negative cells may be related to the large number of sites available for attachment on the host’s ciliated epithelial fields. These data also provide evidence that the dominance of V. fischeri observed in the mature aggregate (Nyholm and McFall-Ngai, 2003) is due to other factors, the production or presentation of which may be induced as downstream responses to earlier interactions of V. fischeri cells with host cilia.

A new model of early host-symbiont interaction in the squid-vibrio symbiosis

Earlier concepts of the squid-vibrio system postulated that host-shed mucus provides a sort of platform for aggregating symbiont bacteria. The data presented here on ciliary attachment, as well as earlier findings of abundant antimicrobials in the mucus matrix (Davidson et al., 2004, Troll et al., 2010), suggest that the mucus may be instead involved in the winnowing process, one in which V. fischeri is increasingly able to withstand biochemical challenges presented by the host. These data suggest a new model in which initial interactions between the host animal and V. fischeri cells involve a multi-step process (Fig. 7). Shortly after hatching, the host releases mucus stores from the light-organ ciliated epithelia in response to environmental peptidoglycan (Nyholm et al., 2002). Then, environmental V. fischeri cells associate with host cilia, a behavior that facilitates the delivery of TCT by a few cells to induce hemocyte trafficking into the blood sinus underlying the host ciliated epithelia (this paper, Koropatnick et al., 2007). Aggregation of V. fischeri occurs subsequently. This process, which is at least in part mediated by exopolysaccharide (Visick, 2009), must involve migration of symbiont cells along the ciliated surface to affect cell-cell contact. During late aggregation, V. fischeri cells become competitively dominant in the aggregate and the cell populations localize near pores on the light-organ surface, into which the cells migrate (Nyholm and McFall-Ngai, 2003). V. fischeri cells then disengage from the ciliated surface and migrate to these pores. There the cells sense chitobiose produced by the host, which they use for chemotaxis into host tissues (Mandel et al., 2012).

All horizontally transmitted symbioses undergo a habitat transition. The squid-vibrio system provides evidence of how intricate such a process can be. The findings presented here add pieces to the complex puzzle, but many questions remain unanswered. These questions include: What are the molecular mechanisms underlying attachment of V. fischeri cells to host cilia? Does attachment to the cilia induce changes in host gene expression and/or in the biochemistry of the environment around aggregating symbionts? How do the attached V. fischeri cells subsequently detach and move along the ciliated surface to form aggregates, eventually migrating to colonize host tissues? How is specificity in the mature aggregate of V. fischeri cells achieved? Although it is clear a good deal more research must be done on the system before an accurate view of this process is achieved, the continued application of available methods, as well as the development of new technologies, such as single-cell transcriptomics and proteomics, promises to provide exciting avenues for future investigations of this system.

General methods

Adult E. scolopes were captured off the coast of Oahu and bred in aquaria as previously described (Montgomery and McFall-Ngai, 1993). Juveniles obtained from the breeding colony were collected within 15–20 min of hatching and placed in unfiltered seawater (i.e., with ~10⁶ environmental bacteria), which has no detectable V. fischeri, under the following conditions: 1) no additions, to generate the aposymbiotic (APO) condition; or, 2) addition of various strains of symbiotic and non-symbiotic bacteria at varying doses and time periods indicated in the individual experiment. For inoculation, each strain was grown to mid-log phase and then diluted into seawater containing newly hatched animals grouped by treatment.

All V. fischeri and V. parahaemolyticus strains (Table 1) were grown shaking at 28°C in LBS (Luria-Bertani salt medium; Dunlap, 1989) or SWT (seawater-based tryptone media; Boettcher and Ruby, 1990) prior to inoculation of the animal. The numbers of cells added per mL of seawater was confirmed by determination of colony-forming units per mL (CFU/mL) on LBS-based agar plates. For antibiotic selection, chloramphenicol (Cam), tetracycline (Tet), erythromycin (Erm), and kanamycin (Kan) were used at concentrations of 5, 5, 5, and 50 µg/ml, respectively.

Microscopy

To prepare specimens for scanning electron microscopy (SEM) analyses, juvenile animals were exposed to V. fischeri cells at 10⁶ CFU/ml of seawater for 2–3 h. To confirm aggregate formation within an experiment, a subset of live animals was examined by confocal microscopy. The remainder of the animals was dropped into 1% osmium tetroxide in marine phosphate buffered saline (50 mM sodium phosphate, 0.45 M sodium chloride, pH 7.4) and incubated for 30 min at room temperature on a rotator. This unusual fixation procedure arrests ciliary beat on the ciliated surfaces of molluscan epithelia (Reed and Satir, 1986). Fixed animals were dehydrated in an ethanol series from 30–100%. Specimens were dried in a Tousimis Samdri 780 critical point dryer and mounted with colloidal silver onto stubs dorsal side down. The tissue covering the light organs was removed and the specimens were sputter coated with gold in a SeeVac Auto Conductavac IV. The samples were examined on a Hitachi S-570 LaB6 scanning electron microscope.

To visualize the cilia on the surface of the organ by confocal microscopy, samples were exposed at 45 min prior to the conclusion of a time course to 250 nM TubulinTracker in the seawater containing the juvenile animal. Mucus shed from these host tissues was visualized by incubating the animal at 30 min prior to conclusion of a time course with 10 µg/ml Alexa633 wheat germ agglutinin (WGA), a fluorochrome that labels the sialic acid and N-acetylglucosaminyl residues of the mucus. At the end of the incubation period, the animals were anesthetized in 2% ethanol in seawater, and the tissues were dissected to reveal the light organ surface. Confocal experiments were performed on a Zeiss 510 laser scanning confocal microscope; fluorochromes were obtained from Invitrogen Life Technologies (Carlsbad, CA).

To enumerate associating bacteria, we counted the number of individual RFP-expressing bacteria per ciliated field on one ciliated field per animal for 5 individual animals. When an aggregate was too large and dense to visualize individual cells, we estimated the number of cells present based on the volume of the aggregate. When various strains were characterized for their ability to associate with host tissues, the data were normalized to wild-type V. fischeri ES114.

For wild-type V. fischeri and V. parahaemolyticus co-infection assays, animals were exposed to both phylotypes simultaneously and coincubated throughout the duration of the experiment. In experiments where we visualized V. parahaemolyticus in association with the cilia, we used unlabeled V. fischeri and RFP-expressing V. parahaemolyticus and counterstained the cilia with TubulinTracker. In experiments where both phylotypes were visualized, we used an RFP-expressing V. fischeri strain with a GFP-expressing V. parahaemolyticus and the mucus was counterstained with Alexa633 WGA as described above. In the competition experiments comparing relative numbers of V. parahaemolyticus in the presence of either wild-type or rscS⁻ V. fischeri, the squid were exposed to equal inocula of each strain.

Assays with heat-or azide-killed V. fischeri

V. fischeri cells were grown as described above and then subjected to the following treatment for heat- or metabolic-inactivation. One hundred µL of V. fischeri culture at mid-log phase were transferred to an Eppendorf tube and incubated in a 50°C heat block for 1 h to heat inactivate the cells. To metabolically inactivate cells, they were handled similarly, but exposed to 0.1% sodium azide for 1 h at room temperature. Cells were checked for viability using propidium iodide staining as previously described (Nyholm and McFall-Ngai, 2003). For each of these conditions, we compared untreated to treated cultured bacteria by fluorescence microscopy to confirm that RFP fluorescence was not affected by the treatment.

Hemocyte trafficking assays

To determine numbers of hemocytes migrating into the blood sinus underlying the ciliated epithelium, animals were incubated with bacteria for 3 h and then fixed overnight in 4% paraformaldehyde in marine phosphate buffered saline (mPBS) (50 mM sodium phosphate, 0.45M sodium chloride, pH 7.4) at 4°C. Light organs were removed from the squid and permeabolized for 24 h in 1% Triton-X-100 in mPBS. To stain globular actin, which is highly abundant in hemocytes and thus used to localize hemocytes, the samples were incubated overnight in FITC-DNAse I. The samples were counterstained with rhodamine phalloidin to localize the actin cytoskeleton and with TOTO-3, to localize nuclei. Samples were mounted on glass slides as previously described (Altura et al., 2011) and hemocytes were visualized and their abundance determined by confocal microscopy.

Statistics

All statistical analyses were performed in Prism 5 Software as described in the figure legends.

Supp Fig S1-S2

1. Adam EC, Mitchell BS, Schumacher DU, Grant G, Schumacher U. Pseudomonas aeruginosa II lectin stops human ciliary beating: therapeutic implications of fucose. Am J Respir Crit Care Med. 1997;155:2102–2104. doi: 10.1164/ajrccm.155.6.9196121. [DOI] [PubMed] [Google Scholar]
2. Aeckersberg F, Lupp C, Feliciano B, Ruby EG. Vibrio fischeri outer membrane protein OmpU plays a role in normal symbiotic colonization. J Bacteriol. 2001;183:6590–6597. doi: 10.1128/JB.183.22.6590-6597.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
3. Altura MA, Stabb E, Goldman W, Apicella M, McFall-Ngai MJ. Attenuation of host NO production by MAMPs potentiates development of the host in the squid-vibrio symbiosis. Cell Microbiol. 2011;13:527–537. doi: 10.1111/j.1462-5822.2010.01552.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
4. Anderton TL, Maskell DJ, Preston A. Ciliostasis is a key early event during colonization of canine tracheal tissue by Bordetella bronchiseptica. Microbiology. 2004;150:2843–2855. doi: 10.1099/mic.0.27283-0. [DOI] [PubMed] [Google Scholar]
5. Bajolet-Laudinat O, Girod-de Bentzmann S, Tournier JM, Madoulet C, Plotkowski MC, Chippaux C, Puchelle E. Cytotoxicity of Pseudomonas aeruginosa internal lectin PA-I to respiratory epithelial cells in primary culture. Infect Immun. 1994;62:4481–4487. doi: 10.1128/iai.62.10.4481-4487.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Balder R, Krunkosky TM, Nguyen CQ, Feezel L, Lafontaine ER. Hag mediates adherence of Moraxella catarrhalis to ciliated human airway cells. Infect Immun. 2009;77:4597–4608. doi: 10.1128/IAI.00212-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Boettcher KJ, Ruby EG. Depressed light emission by symbiotic Vibrio fischeri of the sepiolid squid Euprymna scolopes. J Bacteriol. 1990;172:3701–3706. doi: 10.1128/jb.172.7.3701-3706.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
8. Bright M, Bulgheresi S. A complex journey: transmission of microbial symbionts. Nat Rev Micriobiol. 2010;8:218–230. doi: 10.1038/nrmicro2262. [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Davidson SK, Koropatnick TA, Kossmehl R, Sycuro L, McFall-Ngai MJ. NO means ‘yes’ in the squid-vibrio symbiosis: nitric oxide (NO) during the initial stages of a beneficial association. Cell Microbiol. 2004;6:1139–1151. doi: 10.1111/j.1462-5822.2004.00429.x. [DOI] [PubMed] [Google Scholar]
10. Dunlap PV. Regulation of luminescence by cyclic AMP in cya-like and crp-like mutants of Vibrio fischeri. J Bacteriol. 1989;171:1199–1202. doi: 10.1128/jb.171.2.1199-1202.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Dunn AK, Millikan DS, Adin DM, Bose JL, Stabb EV. New rfp- and pES213-derived tools for analyzing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ. Appl Environ Microbiol. 2006;72:802–810. doi: 10.1128/AEM.72.1.802-810.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
12. Edwards JA, Groathouse NA, Boitano S. Bordetella bronchiseptica adherence to cilia is mediated by multiple adhesin factors and blocked by surfactant protein A. Infect Immun. 2005;73:3618–3626. doi: 10.1128/IAI.73.6.3618-3626.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
13. Emlet RB. Functional constraints on the evolution of larval forms of marine invertebrates: Experimental and comparative evidence. Integ Comp Bio. 1991;31:707–725. [Google Scholar]
14. Fadaee-Shohada MJ, Hirst RA, Rutman A, Roberts IS, O’Callaghan C, Andrew PW. The behavior of both Listeria monocytogenes and rat ciliated ependymal cells is altered during their co-culture. PLoS One. 2010;5:e10450. doi: 10.1371/journal.pone.0010450. [DOI] [PMC free article] [PubMed] [Google Scholar]
15. Freeman BC, Chen C, Beattie GA. Identification of the trehalose biosynthetic loci of Pseudomonas syringae and their contribution to fitness in the phyllosphere. Environ Microbiol. 2010;12:1486–1497. doi: 10.1111/j.1462-2920.2010.02171.x. [DOI] [PubMed] [Google Scholar]
16. Groathouse NA, Heinzen RA, Boitano S. Functional BvgAS virulence control system in Bordetella bronchiseptica is necessary for induction of Ca2+ transients in ciliated tracheal epithelial cells. Infect Immun. 2003;71:7208–7210. doi: 10.1128/IAI.71.12.7208-7210.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
17. Hussa EA, O’Shea TM, Darnell CL, Ruby EG, Visick KL. Two-component response regulators of Vibrio fischeri: identification, mutagenesis, and characterization. J Bacteriol. 2007;189:5825–5838. doi: 10.1128/JB.00242-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
18. Jones BW, Maruyama A, Ouverney CC, Nishiguchi MK. Spatial and temporal distribution of the Vibrionaceae in coastal waters of Hawaii, Australia, and France. Microb. Ecol. 2007;54:314–323. doi: 10.1007/s00248-006-9204-z. [DOI] [PubMed] [Google Scholar]
19. Kaufman MR, Taylor RK. Identification of bacterial cell-surface virulence determinants with TnphoA. Methods Enzymol. 1994;235:426–428. doi: 10.1016/0076-6879(94)35159-7. [DOI] [PubMed] [Google Scholar]
20. Kirn TJ, Lafferty MJ, Sandoe CM, Taylor RK. Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio cholerae . Mol Microbiol. 2000;35:896–910. doi: 10.1046/j.1365-2958.2000.01764.x. [DOI] [PubMed] [Google Scholar]
21. Koropatnick TA, Engle JT, Apicella MA, Stabb EV, Goldman WE, McFall-Ngai MJ. Microbial factor-mediated development in a host-bacterial mutualism. Science. 2004;306:1186–1188. doi: 10.1126/science.1102218. [DOI] [PubMed] [Google Scholar]
22. Koropatnick TA, Kimbell JR, McFall-Ngai MJ. Responses of host hemocytes during the initiation of the squid-vibrio symbiosis. Biol Bull. 2007;212:29–39. doi: 10.2307/25066578. [DOI] [PubMed] [Google Scholar]
23. Krachler AM, Ham H, Orth K. Outer membrane adhesion factor multivalent adhesion molecule 7 initiates host cell binding during infection by Gram-negative pathogens. Proc Natl Acad Sci USA. 2011;108:11614–11619. doi: 10.1073/pnas.1102360108. [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Lee L. Mechanisms of mammalian ciliary motility: Insights from primary ciliary dyskinesia genetics. Gene. 2011;473:57–66. doi: 10.1016/j.gene.2010.11.006. [DOI] [PubMed] [Google Scholar]
25. Mandel MJ, Stabb EV, Ruby EG. Comparative genomics-based investigation of resequencing targets in Vibrio fischeri: focus on point miscalls and artefactual expansions. BMC Genomics. 2008;9:138. doi: 10.1186/1471-2164-9-138. [DOI] [PMC free article] [PubMed] [Google Scholar]
26. Mandel MJ, Wollenberg MS, Stabb EV, Visick KL, Ruby EG. A single regulatory gene is sufficient to alter bacterial host range. Nature. 2009;458:215–218. doi: 10.1038/nature07660. [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Mandel MJ, Schaefer AL, Brennan CA, Heath-Heckman EAC, DeLoney-Marino CR, McFall-Ngai MJ, Ruby EG. Squid-derived chitin oligosaccharides are a chemotactic signal for colonizing Vibrio fischeri . Appl Environ Microbiol. 2012;78:4620–4626. doi: 10.1128/AEM.00377-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
28. McFall-Ngai M. Host-microbe symbiosis: the squid-vibrio association--a naturally occurring, experimental model of animal/bacterial partnerships. Adv Exp Med Biol. 2008;635:102–112. doi: 10.1007/978-0-387-09550-9_9. [DOI] [PubMed] [Google Scholar]
29. Mewe M, Tielker D, Schonberg R, Schachner M, Jaeger KE, Schumacher U. Pseudomonas aeruginosa lectins I and II and their interaction with human airway cilia. J Laryngol Otol. 2005;119:595–599. doi: 10.1258/0022215054516313. [DOI] [PubMed] [Google Scholar]
30. Montgomery M, McFall-Ngai M. Embryonic development of the light organ of the sepiolid squid Euprymna scolopes Berry. Biol Bull. 1993;184:296–308. doi: 10.2307/1542448. [DOI] [PubMed] [Google Scholar]
31. Montgomery MK, McFall-Ngai M. Bacterial symbionts induce host organ morphogenesis during early postembryonic development of the squid Euprymna scolopes . Development. 1994;120:1719–1729. doi: 10.1242/dev.120.7.1719. [DOI] [PubMed] [Google Scholar]
32. Nelson EJ, Harris JB, Morris JG, Jr, Calderwood SB, Camilli A. Cholera transmission: the host, pathogen and bacteriophage dynamic. Nat Rev Microbiol. 2009;7:693–702. doi: 10.1038/nrmicro2204. [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Nyholm SV, Stabb EV, Ruby EG, McFall-Ngai MJ. Establishment of an animal-bacterial association: recruiting symbiotic vibrios from the environment. Proc Natl Acad Sci U S A. 2000;97:10231–10235. doi: 10.1073/pnas.97.18.10231. [DOI] [PMC free article] [PubMed] [Google Scholar]
34. Nyholm SV, Deplancke B, Gaskins HR, Apicella MA, McFall-Ngai MJ. Roles of Vibrio fischeri and nonsymbiotic bacteria in the dynamics of mucus secretion during symbiont colonization of the Euprymna scolopes light organ. Appl Environ Microbiol. 2002;68:5113–5122. doi: 10.1128/AEM.68.10.5113-5122.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Nyholm SV, McFall-Ngai MJ. Dominance of Vibrio fischeri in secreted mucus outside the light organ of Euprymna scolopes: the first site of symbiont specificity. Appl Environ Microbiol. 2003;69:3932–3937. doi: 10.1128/AEM.69.7.3932-3937.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Post DM, Yu L, Krasity BC, Choudhury B, Mandel MJ, Brennan CA, Ruby EG, McFall-Ngai MJ, Gibson BW, Apicella MA. The O-antigen and core carbohydrate of Vibrio fischeri lipopolysaccharide: Composition and analysis of their role in Euprymna scolopes light organ colonization. J Biol Chem. 2012;287:8515–30. doi: 10.1074/jbc.M111.324012. [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Riisgard HU, Larsen PS. Minireview: Ciliary filter feeding and bio-fluid mechanics - present understanding and unsolved problems. Limnol Oceanogr. 2001;46:882–891. [Google Scholar]
38. Ruby EG, Lee KH. The Vibrio fischeri-Euprymna scolopes light organ association: Current ecological paradigms. Appl Environ Microbiol. 1998;64:805–812. doi: 10.1128/aem.64.3.805-812.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Shah AS, Ben-Shahar Y, Moninger TO, Kline JN, Welsh MJ. Motile cilia of human airway epithelia are chemosensory. Science. 2009;325:1131–1134. doi: 10.1126/science.1173869. [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Shibata S, Visick KL. Sensor kinase RscS induces the production of antigenically distinct outer membrane vesicles that depend on the symbiosis polysaccharide locus in Vibrio fischeri . J Bacteriol. 2012;194:185–194. doi: 10.1128/JB.05926-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Soane MC, Jackson A, Maskell D, Allen A, Keig P, Dewar A, et al. Interaction of Bordetella pertussis with human respiratory mucosa in vitro. Respir Med. 2000;94:791–799. doi: 10.1053/rmed.2000.0823. [DOI] [PubMed] [Google Scholar]
42. Stabb EV, Ruby EG. Contribution of pilA to competitive colonization of the squid Euprymna scolopes by Vibrio fischeri . Appl Environ Microbiol. 2003;69:820–826. doi: 10.1128/AEM.69.2.820-826.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Taylor MW, Schupp PJ, de Nys R, Kjelleberg S, Steinberg PD. Biogeography of bacteria associated with the marine sponge Cymbastela concentrica . Environ Microbiol. 2005;7:419–433. doi: 10.1111/j.1462-2920.2004.00711.x. [DOI] [PubMed] [Google Scholar]
44. Troll JV, Bent EH, Pacquette N, Wier AM, Goldman WE, Silverman N, McFall-Ngai MJ. Taming the symbiont for coexistence: a host PGRP neutralizes a bacterial symbiont toxin. Environ Microbiol. 2010;12:2190–2203. doi: 10.1111/j.1462-2920.2009.02121.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
45. Visick KL. An intricate network of regulators controls biofilm formation and colonization by Vibrio fischeri . Mol Microbiol. 2009;74:782–789. doi: 10.1111/j.1365-2958.2009.06899.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
46. Wang D, Yu S, Chen W, Zhang D, Shi X. Enumeration of Vibrio parahaemolyticus in oyster tissues following artificial contamination and depuration. Lett Appl Microbiol. 2010;51:104–108. doi: 10.1111/j.1472-765X.2010.02865.x. [DOI] [PubMed] [Google Scholar]
47. Wang Y, Dunn AK, Wilneff J, McFall-Ngai MJ, Spiro S, Ruby EG. Vibrio fischeri flavohaemoglobin protects against nitric oxide during initiation of the squid-vibrio symbiosis. Mol Microbiol. 2010;78:903–915. doi: 10.1111/j.1365-2958.2010.07376.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
48. Wong E, Vaaje-Kolstad G, Ghosh A, Hurtado-Guerrero R, Konarev PV, Ibrahim AF, et al. The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces. PLoS Pathog. 2012;8:e1002373. doi: 10.1371/journal.ppat.1002373. [DOI] [PMC free article] [PubMed] [Google Scholar]
49. Yip ES, Geszvain K, DeLoney-Marino CR, Visick KL. The symbiosis regulator RscS controls the syp gene locus, biofilm formation and symbiotic aggregation by Vibrio fischeri . Mol Microbiol. 2006;62:1586–1600. doi: 10.1111/j.1365-2958.2006.05475.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Supplementary Materials