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. Author manuscript; available in PMC: 2010 Feb 1.
Published in final edited form as: Environ Microbiol. 2009 Feb;11(2):483–493. doi: 10.1111/j.1462-2920.2008.01788.x

Recognition between symbiotic Vibrio fischeri and the hemocytes of Euprymna scolopes

Spencer V Nyholm 1,3,=, Jennifer J Stewart 1,4,=, Edward G Ruby 1,2, Margaret J McFall-Ngai 1,2,*
PMCID: PMC2652691  NIHMSID: NIHMS77454  PMID: 19196278

Summary

The light-organ crypts of the squid Euprymna scolopes permit colonization exclusively by the luminous bacterium Vibrio fischeri. Because the crypt interior remains in contact with seawater, the squid must not only foster the specific symbiosis but also continue to exclude other bacteria. Investigation of the role of the innate immune system in these processes revealed that macrophage-like hemocytes isolated from E. scolopes recognized and phagocytosed V. fischeri less than other closely related bacterial species common to the host’s environment. Interestingly, phagocytes isolated from hosts that had been cured of their symbionts bound five-times more V. fischeri cells than those from uncured hosts. No such change in the ability to bind other species of bacteria was observed, suggesting that the host adapts specifically to V. fischeri. Deletion of the gene encoding OmpU, the major outer membrane protein of V. fischeri, increased binding by hemocytes from uncured animals to the level observed for hemocytes from cured animals. Co-incubation with wild-type V. fischeri reduced this binding, suggesting they produce a factor that complements the mutant’s defect. Analyses of the phagocytosis of bound cells by fluorescence-activated cell sorting (FACS) indicated that, once binding to hemocytes had occurred, V. fischeri cells are phagocytosed as effectively as other bacteria. Thus, discrimination by this component of the squid immune system occurs at the level of hemocyte binding, and this response: (i) is modified by previous exposure to the symbiont and, (ii) relies on outer membrane and/or secreted components of the symbionts. These data suggest that regulation of host hemocyte binding by the symbiont may be one of many factors that contribute to specificity in this association.

Keywords: hemocyte, phagocytosis, bacteria, symbiosis, Euprymna scolopes, Vibrio fischeri

Introduction

A wide variety of both aquatic and terrestrial animals serve as the environment for life-long beneficial symbioses with a complement of extracellular bacteria that associate with their epithelial surfaces (McFall-Ngai, 2002; Dale and Moran, 2006). Most of these animal-microbe interactions are formed anew with each host generation by ambient bacteria that trade soil or seawater for a nutrient-rich environment of animal tissue. Because many of these associations are species specific, an effective communication must occur between the bacterium and its host, mediating both the harvesting of potential symbionts, and the establishment of a stable, long-term association. Increasing evidence suggests that, in both invertebrates and vertebrates, interactions between the bacteria and the host immune system play a critical role in this communication (Pruzzo et al., 2005; Burge et al., 2007; Pham et al., 2007). For example, in the healthy human gut, a system that has undergone intensive investigation, immune defense mechanisms are modulated in response to the normal microbiota (Hooper et al., 2001; Noverr and Huffnagle, 2004; Mazmanian et al., 2005; Cash et al., 2006; Liu et al., 2008). This effect is often referred to as the development of ‘tolerance’. However, recent studies suggest that the factors controlling beneficial bacteria-animal associations involve complex, active processes that promote dynamic interactions between the bacterium and its host, and are not a simple insensitivity to their presence (e.g., (Rakoff-Nahoum et al., 2004; Peterson et al., 2007; Salzman et al., 2007; Fujiwara et al., 2008).

The innate immune system appears to play a critical role in the conversation between beneficial microbes and their hosts (e.g., (Rakoff-Nahoum et al., 2004; Iwasaki, 2007; Vaishnava et al., 2008). Much of this interaction is mediated through signaling at the cell surfaces of the partners. In Gram-negative bacteria, microbe-associated molecular patterns (MAMPs), consisting of cell surface components such as capsule, lipopolysaccharide and outer membrane proteins (OMPs), play an important role in this mediation (Ofek and Doyle, 1994; Aeckersberg et al., 2001). In the host, recent studies of the role of innate immune responses in symbiosis have demonstrated that the underlying cellular and molecular mechanisms are well conserved throughout the animal kingdom, and the simplicity of invertebrate models has proven valuable in understanding the mechanisms of these responses (e.g., (Weis et al., 1996; Anselme et al., 2006; Silver et al., 2007; Ryu et al., 2008). During pathogenesis, the host’s cellular response aims to kill invading microorganisms principally through phagocytosis by blood cells, a process that is mediated in the vertebrates by polymorphonuclear leukocytes, monocytes and tissue macrophages, and in the invertebrates by macrophage-like hemocytes (Irving et al., 2005; Pruzzo et al., 2005). The stages of this process: recognition, binding, and internalization of microorganisms, are conserved in all systems studied (Canesi et al., 2002; Hoffmann et al., 2002; Lavine and Strand, 2002; Govind and Nehm, 2004; Magnadottir, 2006). The realization that significant numbers of beneficial bacteria occur in association with animals begs the question: At what point(s) do these bacteria avoid phagocytosis, and does this avoidance involve a reciprocal interplay between host and symbiont?

The binary association between the Hawaiian squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri offers an experimentally accessible model for defining the role of the host innate immune system in the dynamics between beneficial bacteria and animal tissue. The juvenile squid obtains its bacteria from the seawater each generation, yet the association formed is highly specific; i.e., only V. fischeri can colonize the tissues of a nascent light-emitting organ (McFall-Ngai and Ruby, 1991). The symbiont population, which is maintained throughout the life of the host, grows within polarized epithelium-lined crypt spaces of the highly vascularized, light organ (Fig. 1). Several aspects of the innate immune system have been implicated in the control of this association, but the role of the blood cells has remained unexplored.

Fig. 1.

Fig. 1

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Vascularization of the E. scolopes light organ. A. Ventral dissection of an adult E. scolopes, revealing the bilobed light organ (circled in yellow). Eye (e) and tentacles (t) are also indicated. Scale bar = 1 cm. B. Higher magnification view of the light organ, dissected further to reveal the symbiont-containing central core (cc) tissue. Scale bar (for both B and C) = 2 mm. C. View of the same light organ, in which the circulatory system has been visualized by injecting the fluorochrome CellTracker Orange (Molecular Probes) through the cephalic artery (ca), located at the mantle/head interface. Fluorescent excitation reveals a high degree of vascularization of the central core (cc) tissue. Other highly vascularized organs in the vicinity include the brain (b) and the brachial hearts (bh).

Unlike other invertebrates, which may have several types of blood cells in their circulatory system, cephalopods (i.e., squid, octopus, and their relatives) have a single type of hemocyte. E. scolopes hemocytes traverse the epithelium into the crypt spaces where the symbionts reside and appear to ‘sample’ these spaces, not unlike the way that mammalian blood cells sample the enteric microbiota (Rescigno et al., 2001; Niess et al., 2005). Previous studies of cephalopod hemocytes have indicated that they behave like vertebrate macrophages, binding, engulfing and killing bacteria (Cowden and Curtis, 1981; Schipp et al., 1990). Within the crypts of newly colonized juvenile E. scolopes, hemocytes have been observed with internalized bacterial cells (Nyholm and McFall-Ngai, 1998). However, within the crypts of adult squid, hemocytes have never been observed with engulfed bacteria although host hemocytes are entirely surrounded by V. fischeri cells. These preliminary observations suggested that the squid’s hemocytes change or mature in response to the persistent presence of the symbionts, perhaps as part of the complex developmental program induced in the host by V. fischeri (Nyholm and McFall-Ngai, 2004). Such observations, along with the discovery that certain V. fischeri genes are required for normal colonization (Visick et al., 2000; Aeckersberg et al., 2001; Lupp and Ruby, 2005; Visick and Ruby, 2006; Whistler et al., 2007), have provided evidence that both the host and the symbiont participate in ensuring an exclusive, persistent partnership.

To gain insight into the nature of the interaction between squid hemocytes and bacteria during the establishment and maintenance of the light-organ association, we compared the responses of these host cells to V. fischeri other bacterial species common to the marine environment of the squid. We report evidence that the host hemocytes are ‘educated’ specifically to the presence of symbionts in the light organ, and that binding of V. fischeri prior to phagocytosis is inhibited both by host determinants and by a specific bacterial OMP.

Results

Binding of bacterial cells to E. scolopes hemocytes

In vitro binding assays demonstrated that host hemocytes that had been isolated from colonized adult animals recognized and bound the cells of four marine bacterial species (Table 1) to different degrees (Fig. 2). Specifically, host cells bound significantly more cells of V. harveyi and P. leiognathi than of V. fischeri and V. parahaemolyticus. Control experiments using unlabeled bacterial or host cells yielded identical results to those using GFP-expressing or stained cells (data not shown).

Table 1.

Bacterial strains and plasmids used in this study

Strain or plasmid Characteristicsa Reference
Strain
  Vibrio fischeri
   ES114 Wild-type isolate from E. scolopes light organ Boettcher and Ruby, 1990
   ESR1 Spontaneous Rrr ES114 derivative Aeckersberg et al., 2001
   OM3 ESR1 deletion mutant of ompU Aeckersberg et al., 2001
  Vibrio harveyi
   B392 Seawater isolate Reichelt and Baumann, 1973
 Vibrio parahaemolyticus
   KNH1 Seawater isolate Nyholm et al., 2000
Photobacterium leiognathi
   KNH6 Seawater isolate E. Stabb, pers. comm.
Plasmid
   pVO8 V. fischeri cloning vector; Cmr Visick and Ruby, 1998
   pFA9 pVO8 bearing intact ES114 ompU Aeckersberg et al., 2001
  pKV111 pVO8 bearing the gene encoding green fluorescent protein Nyholm et al., 2000
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a

Rrr, rifampicin resistance; Cmr, chloramphenicol resistance

Fig. 2.

Fig. 2

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Differential binding of bacteria by squid hemocytes. Above: representative confocal microscopy images of isolated squid hemocytes stained with CellTracker Orange. Differential interference contrast (DIC) views of the cells are superimposed to reveal the extended pseudopodia. The extent to which different species of bacteria (green) bound to the hemocytes’ external surfaces was easily visualized and enumerated. Scale bar = 10 μm. Below: mean number of bacteria bound to each hemocyte was determined for at least ten hemocytes per microscopic field (n = 5) in three replicate experiments. Error bars indicate standard errors of the mean (SEM); asterisks denote levels of binding that were significantly different (p < 0.05) from that of V. fischeri ES114, as determined by ANOVA pair-wise analysis.

To examine whether hemocyte behavior was affected by whether they were isolated from an animal that had V. fischeri in its light organ, the symbiont population was eliminated from a subset of animals by antibiotic treatment prior to the collection of the hemocytes. In control experiments, after 24 h of antibiotic exposure no V. fischeri colony-forming units (CFUs) were present in homogenates of the central-core tissue of treated light organs. A cohort of cured animals was maintained in the cured state for an additional four days, during which time bacteria-binding efficiency was determined for hemocytes isolated either from cured (naïve hemocytes) or symbiotic (normal hemocytes) animals (Fig. 3). No change was detected in the ability of the normal hemocytes to bind any of the three bacterial strains over the five-day experiment; however, by day 4 the ability of the naïve hemocytes to bind V. fischeri cells had become significantly greater, increasing to 5-fold by five days (Fig. 3). This increased activity toward V. fischeri cells was not the result of a general enhancement of binding by hemocytes of cured animals; binding to V. parahaemolyticus or V. harveyi, which are bound with low and high efficiency respectively, was unchanged over the course of the experiment.

Fig. 3.

Fig. 3

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Effect of curing squid of their symbiont population on hemocyte binding to different bacteria. Mean number of bacteria bound to hemocytes isolated from animals at different times after initiating antibiotic treatment to remove symbiotic V. fischeri. Adult squids were maintained in sterile seawater either in the normal symbiotic state or under antibiotic conditions that cured the light organ. Hemocytes were removed from either the symbiotic (normal) or cured (naïve) animals over 5 days for adherence assays (see Experimental procedures). The mean numbers of bacteria bound by symbiotic (white bars) or naïve (black bars) hemocytes were determined in three independent experiments. Error bars indicate SEM; asterisks denote levels of binding that were significantly different (p < 0.05) from those measured on day 1, as determined by ANOVA pair-wise analysis.

V. fischeri avoidance of binding to host hemocytes

To determine whether the avoidance of symbiotic hemocyte adherence by V. fischeri is mediated by bacterial surface components, we examined mutant strains of V. fischeri that were defective in the synthesis of flagella (Millikan and Ruby, 2004), type-4 pili (Stabb and Ruby, 2003) or a major OMP (Aeckersberg et al., 2001). Only strain OM3 (Table 1), which is unable to produce the outer membrane protein OmpU, showed a significant effect on hemocyte binding. When strain OM3 was exposed to adult hemocytes from symbiotic animals, its level of adherence was five-times higher than that of wild-type V. fischeri (Fig. 4). Complementing this mutation in trans with a wild-type copy of ompU that restores the OmpU protein (Aeckersberg et al., 2001) resulted in a specific decrease in hemocyte binding to the levels observed for wild-type V. fischeri. These data suggest that the ability of V. fischeri to avoid hemocyte recognition is due, at least in part, to the presence or activity of a bacterial OMP.

Fig. 4.

Fig. 4

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Effect of an ompU mutation on V. fischeri evasion of hemocyte recognition and binding. The mean numbers of wild-type (ES114) or ompU (OM3) V. fischeri cells that were bound by isolated hemocytes was determined in three independent experiments. Restoration of an intact copy of ompU on pFA9 eliminated the elevated level of hemocyte binding of strain OM3. Addition of the V. fischeri cloning vector pV08 had no significant effect on OM3 binding. Error bars indicate SEM; asterisks denote levels of binding that were significantly different (p < 0.05) from those of V. fischeri ES114, as determined by ANOVA pair-wise analysis.

One possible explanation for this result is that V. fischeri may produce a secreted signal that passes though the OmpU channel and contributes to bacterial evasion of the hemocyte defenses of the host. If this hypothesis were correct, one would predict that the presence of V. fischeri cells might influence how the hemocytes bind other bacterial species as well. To test this hypothesis, we conducted binding assays with GFP-labeled V. fischeri cells co-incubated with unlabeled cells of either V. parahaemolyticus, V. harveyi, or P. leiognathi. A combination of both fluorescence and differential-interference contrast (DIC) microscopy allowed the differentiation of the number of cells of each type of bacteria that were bound by the hemocytes. Incubation of V. fischeri with any of these other species resulted in a reproducible, but not statistically significant, increase in binding of V. fischeri cells by hemocytes (Fig. 5A). Conversely, the presence of wild-type V. fischeri cells did not cause any decrease in the binding of V. parahaemolyticus, V. harveyi or P. leiognathi to host hemocytes (Fig, 5B). In fact, the presence of V. fischeri resulted in a significant increase in binding of the V. parahaemolyticus and P. leiognathi to host hemocytes (Fig. 5B). However, co-incubation with wild-type V. fischeri cells reduced OM3 binding to the level of wild type, further supporting the notion that OmpU provides an activity or factor that complements the defect of the ompU mutant (Fig. 5C). This complementation required the presence of viable wild-type V. fischeri: neither heat-killed nor azide-killed cells protected the mutant from binding. These data suggest the involvement of a secreted signal whose activity provides a protective mechanism that is specific for V. fischeri.

Fig. 5.

Fig. 5

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Complementation of the V. fischeri ompU defect by viable, wild-type bacteria. A. Hemocyte binding of wild-type V. fischeri ES114 incubated either alone or in combination with cells of V. harveyi, V. parahaemolyticus, P. leiognathi, or V. fischeri OM3. B. Hemocyte binding of V. parahaemolyticus, P. leiognathi and V. harveyi in either the absence (white bars) or presence (black bars) of wild-type V. fischeri ES114. Error bars indicate SEM; asterisks denote levels of binding that were significantly different (p < 0.05) in the presence of V. fischeri ES114, as determined by ANOVA pair-wise analysis. C. Hemocyte binding of V. fischeri OM3 (ompU) either alone (white bar) or in the presence of wild-type V. fischeri ES114 cells (black bars). Values are the mean number of bacteria adhering to ten hemocytes in three independent experiments. Error bars indicate SEM; asterisk denotes levels of binding that were significantly different (p < 0.05) from those of V. fischeri ES114, as determined by ANOVA pair-wise analysis.

Bacterial uptake by hemocytes

FACS analysis was used to determine whether a difference could be detected in the ability of the squid hemocytes to engulf cells of either V. fischeri or V. parahaemolyticus, two strains of bacteria isolated from the natural environment of the squid, that are bound at relatively low levels. Our results show that the hemocytes are capable of engulfing bound bacteria regardless of the strain that was used (Fig. 6). Specifically, hemocyte engulfment of V. fischeri and V. parahaemolyticus was compared. Engulfment was determined by whether or not bacterial cell fluorescence was susceptible to quenching. The fluorescence of cells not phagocytosed was quenched, whereas the fluorescence of engulfed cells was not affected. The cells were sorted into these two categories by FACS analysis. No measurable differences could be detected between the two bacterial species in the rates of phagocytic activity between 30 and 180 min after exposure (Fig. 6C). The fact that the hemocytes did not appear to discriminate between these two bacterial species during phagocytosis supports the idea that the critical event in evading removal by the hemocytes is associated with recognition and/or initial binding. No bacterial uptake was detected for any strain in the presence of the phagocytosis inhibitor cytochalasin D (data not shown).

Fig. 6.

Fig. 6

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Rates of phagocytosis of bacteria bound by hemocytes. A. Representative flow cytometry plot recorded 30 min after the start of the phagocytosis assay. The fluorescence of hemocytes exposed to TAMRA-labeled cells of V. fischeri ES114 was assessed by flow cytometry before (left panel) and after (right panel) the addition of trypan blue, which quenches the fluorescence of cells not phagocytosed. B. The fluorescence of hemocytes exposed to TAMRA-labeled cells of V. parahaemolyticus was assessed as described above. C. The extent of reduction in the mean fluorescence after the addition of the trypan blue (A and B) corresponded to the degree of internalization of the bound bacteria. This value was determined every 30 min after exposure of the bacteria to hemocytes, and was a measure of the relative rates of phagocytosis of V. fischeri (closed circle) and V. parahaemolyticus (closed triangle) cells.

Discussion

The major findings of this study offer insight into the role of innate immunity in the adaptation of bacteria to an environment whose importance is becoming increasingly recognized: the tissues of their specific animal host. In the squid-vibrio association both host and symbiont cells have characters that mediate this exclusive interplay. The ability to avoid adherence to hemocytes of E. scolopes varied among related bacterial species, and the hemocyte’s response was specifically altered by previous exposure of the host to V. fischeri. These data provide evidence that host hemocytes become conditioned to colonization or ‘educated’ by the presence of their coevolved bacterial symbiont. A coarse level of recognition has recently been shown in the innate immune response of Drosophila to challenge by bacterial pathogens (Pham et al., 2007); however, the ability of the innate immune system to distinguish between closely related members of one bacterial family, coupled with a mechanism(s) for immune education to a specific bacterial symbiont, are unprecedented among the invertebrates. In addition, we show that V. fischeri with mutations in the gene encoding the outer membrane protein OmpU are bound to host hemocytes at significantly higher levels than wild type. The data further showed that the critical step in hemocyte specificity in the squid-vibrio system is binding itself as, once bound, both symbiotic and non-symbiotic bacteria are phagocytosed by host hemocytes equally well.

Differences in hemocyte binding of bacteria

In all systems described, adherence of a microorganism or particle to a phagocyte is an essential step (Aderem and Underhill, 1999). Characterizations of the initial onset of the symbiosis have implicated hemocytes in the determination of specificity and the induction of development (Nyholm and McFall-Ngai, 1998; Koropatnick et al., 2004). Given that a subpopulation of E. scolopes hemocytes enters the microenvironment of the light-organ crypts (Nyholm and McFall-Ngai, 1998), the resistance of V. fischeri to adherence to host hemocytes may aid in the persistence of the symbiont in the light organ crypt spaces, and contribute to the long-term specificity of the association. The non-symbiont V. parahaemolyticus also did not adhere well to squid hemocytes. Since these hemocytes circulate throughout the host’s vasculature and serve as the only cellular component of the squid’s innate defenses, various environmental bacteria, including V. parahaemolyticus may interact with the hemocytes in contexts outside the light organ, i.e. during infections of other tissues. However, previous studies using this strain have demonstrated that V. parahaemolyticus is unable to migrate through the ducts and colonize the light organ of uncolonized hatchling squid (Nyholm et al., 2000). Analysis of the juvenile host has revealed multiple mechanisms to ensure specificity, both biomechanical (e.g., ciliary currents along the ducts leading to the crypt spaces) (Nyholm and McFall-Ngai, 2004) and biochemical (e.g., lectin-glycan interactions and oxidative stress) (Weis et al., 1996; Visick and Ruby, 1998; Nyholm et al., 2000). Thus, the behavior of the hemocytes is one of a set of specificity determinants in the system.

An apparent education of the hemocytes

The ability for the host hemocytes to react differently over time to V. fischeri, may be analogous to vertebrate immune ‘tolerance’ involving features of both partners. Curing of the light organ only affected the binding of hemocytes to V. fischeri (Fig 3), suggesting that some component of the host hemocytes changes after specific recognition and interaction with the symbiont. The mechanism, however, behind this conditioning remains unknown. Chronic infection by pathogenic bacteria can alter the response of the innate immune system over time. For example, tolerance of airway epithelia that are chronically exposed to Pseudomonas aeruginosa appears to be mediated by down-regulation of the NF-kappaB pathway (Wu et al., 2005).

The fact that the extent of V. fischeri binding increased with time after curing suggests that changes in the hemocytes, or turnover in the hemocyte population, takes several days. The development of hemocytes in cephalopods takes place in the specialized white body located adjacent to the optic lobe (Cowden and Curtis, 1981). However, the rate at which these cells develop and their duration in the circulatory system remain unknown. Because the light organ is highly vascularized, hemocytes may be exchanged between the circulatory system and the crypt spaces on a regular basis. A previous study has shown that exposure to different pathogens can alter the expression of MAMP receptors during the maturation of innate immune cells (Kokkinopoulos et al., 2005). In the squid-vibrio association, the host’s hemolymph may contain bacterial products from the symbionts that are carried to the white body and somehow alter the development of pattern recognition receptors on nascent hemocytes.

The importance of OmpU

This study provides evidence that the V. fischeri outer membrane porin OmpU plays a role in preventing symbiont adhesion to host hemocytes. An opposite role for OMPs, i.e., the mediating of adherence to immune cells, has been documented in bacterial pathogenesis (Negm and Pistole, 1999; Soulas et al., 2000; Biswas et al., 2001). The finding that co-incubation of wild type with ompU mutants rescues the mutant phenotype suggests that ompU is acting as a porin, i.e., transporting something into the medium recognized V. fischeri cells. The finding that co-incubation with wild-type V. fischeri does not rescue non-symbiont strains suggests an exclusivity to the response. V. cholerae OMP’s have been shown to function in a channel capacity, facilitating the movement of proteins into and out of the cell (Chakrabarti et al., 1996; Provenzano et al., 2001).

Unlike the non-symbiont marine species (V. parahaemolyticus, P. leiognathi and V. harveyi) used for comparison in the previously described experiments, V. fischeri strain OM3 does enter the light organ crypts of E. scolopes. However, this mutant has been shown to be defective in interacting with the host E. scolopes during the onset of the symbiosis compared to wild-type V. fischeri (Aeckersberg et al., 2001). The strain OM3 was tested for its ability to resist adherence by hemocytes from both symbiotic and cured squid. Because this mutant is more easily recognized and bound by host hemocytes, this phenomenon may explain why this mutant is defective in interacting with the squid host. Also, the fact that the ompU mutant could resist adherence by hemocytes in vitro when co-incubated with wild-type V. fischeri may also explain the complementation of this mutant’s defect during animal colonization when co-infected with wild-type V. fischeri. How OmpU mediates resistance to binding host hemocytes, and the identity of other symbiont molecules involved in adhesion to these cells are important points that remain to be determined. Interestingly, the related pathogen, V. vulnificus has been shown to require the presence of its homologous OmpU protein to bind a fibronectin-coated surface (Goo et al., 2006); however, similar analyses comparing the V. fischeri ompU mutant and its parent indicated that both attached equally well to fibronectin (data not shown).

Conclusions

Innate immunity is often cited solely for its role as an effector system in dealing with potential pathogens, the molecular basis of which may involve broad recognition of MAMPs. However, the role of this ubiquitous metazoan function in mediating very specific and selective communications with mutualistic microorganisms is poorly understood. Here we show that squid host hemocytes are capable of altering their response to symbiotic and non-symbiotic bacteria. This study lays the groundwork for using the E. scolopes/V. fischeri association for studying the interactions between a host’s innate immune system and its beneficial bacterial symbionts.

Experimental procedures

General procedures

Adult E. scolopes were collected from the shallow reef flats of Oahu, Hawaii and maintained in running seawater tables as described previously (Nyholm and McFall-Ngai, 1998). All chemicals were obtained from Sigma-Aldrich, Inc., unless otherwise stated.

Isolation of host hemocytes

Animals were anesthetized by placing them in a 2% solution of ethanol in seawater. For experiments using isolated hemocytes, hemolymph was withdrawn from the cephalic artery, located between the eyes (Fig. 1), using a sterile 1-ml syringe with a 28-gauge needle. An average of ~5,000 hemocytes μl−1 of hemolymph were obtained using this method; animals served as donors multiple times, and a total of approximately 106 hemocytes were obtained from each individual. Freshly collected hemocytes were washed and resuspended in Squid Ringer’s solution (S-Ringers; 530 mM NaCl, 10 mM KCl, 25 mM MgCl2, 10 mM CaCl2 and 10 mM HEPES buffer, pH 7.5). Hemocyte concentrations were determined by hemocytometer, and approximately 10,000 cells were added to chamber slides, and allowed to adhere to the glass for 10 min at room temperature. At this density, the hemocytes form a uniform monolayer on the glass slide surface.

Curing squid of symbiotic bacteria

To cure adult E. scolopes of their population of V. fischeri symbionts, squid were maintained individually in 5-gal tanks containing Instant Ocean (IO) artificial seawater (Aquarium Systems). For one set of animals chloramphenicol (Cm) and gentamicin (Gn) were added to the seawater to a final concentration of 20 μg ml−1 of each. The concentration of antibiotics used in these experiments effectively eliminates V. fischeri symbionts from the light organ without compromising the health of the host in any detectable way. The animals were transferred daily into fresh IO, either with or without antibiotics, for 5 days, and samples of hemolymph were removed at noon each day as described above. The resulting two sets of hemocytes were designated either ‘normal’ (untreated/symbiotic) or ‘naïve’ (treated/cured).

Twenty-four hours after initiating the treatment, the effectiveness of the antibiotics was determined by sacrificing a subset of animals, dissecting out and homogenizing the central core of the light organ (which normally contains the symbionts), and plating an aliquot of the homogenate on SWT agar. Untreated adult light organs contain >108 V. fischeri CFUs (Ruby and Asato, 1993); the absence of CFUs in the treated light organs were considered evidence of curing.

Preparation of bacterial cultures

Bacterial strains (Table 1) were cultured in a seawater nutrient medium (SWT) as previously described (Nyholm et al., 2000). The growth kinetics and yields of the different bacteria were roughly the same in this medium. The optical density at 600 nm (OD600) was taken periodically and, at an OD600 of 0.7 (i.e., mid-exponential growth-phase), the culture was centrifuged at 8,000 g to pellet the bacterial cells. Bacterial cell number, as determined by optical density, was confirmed by the plating of serial dilutions of cultures and determination of colony-forming units. The pellet was washed by resuspension in S-Ringer’s solution and re-centrifugation a total of three times. The bacteria were finally resuspended to a concentration of 2.0 × 108 cells ml−1 of S-Ringer’s.

Suspensions of non-viable V. fischeri were produced by preparing the cells as described above, and exposing them either to 42°C for 20 min, or to 0.01% NaN3 for 20 min. Treated bacteria were then washed three times with S-Ringer’s solution, and the loss of viability was confirmed both by microscopy (the bacteria were stained with propidium iodine, which is excluded from living cells) and by spreading the cell suspension on SWT agar to observe CFUs.

Strains harboring the GFP-encoding plasmid pKV111 (Table 1) were used to visualize and quantify bacterial binding to squid hemocytes using fluorescent microscopy (Nyholm et al., 2000). During initial culturing, 2.5 μg of Cm was added per ml SWT to maintain pKV111 in the bacteria; however, no Cm was added to the S-Ringer’s solution used during the hemocyte binding assays.

Hemocyte/bacteria binding assay

To determine the extent of hemocyte binding, bacteria were added either as individual strains, or as pairs of strains at a ratio of 1:1. In all cases a total of 50 bacteria were added per hemocyte; bacterial cell numbers were confirmed by plating and quantification of colony-forming units. The hemocyte/bacteria mixtures were incubated in S-Ringer’s solution at 25°C for 1 h, a time determined to yield the maximum level of binding (data not shown). The cytoplasm of the hemocytes was then stained with 0.005% CellTracker Orange (Molecular Probes) to visualize the cells. Bacteria associated with individual hemocytes were viewed by fluorescence and differential interference contrast (DIC) using a Zeiss LSM 510 laser-scanning confocal microscope, and enumerated over the entire surface of the animal cell.

Quantification of hemocytic phagocytosis of bacteria

To detect bacteria with a fluorescence-activated cell sorter (FACS), cells were labeled by incubation for 20 min in a solution of 1.0 mg tetramethylrhodamine carboxylic acid (TAMRA, Molecular Probes) per ml of S-Ringer’s solution. The cells were then washed three times, and resuspended in S-Ringer’s solution. To determine the efficiency of trypan-blue quenching, the fluorescence level of labeled cells of V. fischeri ES114 or V. parahaemolyticus KNH1 (unquenched) was determined by flow cytometry. Trypan blue was then added to the suspension and the fluorescence of the labeled bacteria (quenched) was measured again. For both strains the reduction in mean fluorescence levels indicated a quenching of 95% of the initial fluorescence (data not shown).

An aliquot of 2.0 × 105 TAMRA-labeled V. fischeri ES114 or V. parahaemolyticus KNH1 cells were added to 500 μl of Squid Ringer’s solution containing 10,000 hemocytes. The mixture was incubated for 30–60 min at 25°C to allow binding. Phagocytosis was then measured using an Altra FACS (Beckman-Coulter) equipped with an argon laser operating at excitation wavelengths of 488/630 nm. The results were analyzed using PC-compatible Expo32 MultiComp software (Beckman-Coulter), as well as Macintosh-compatible FlowJo software (Tree Star). Cells were gated using forward- and side-light scatter to discriminate between eukaryotic cells and bacteria. The mean fluorescence of at least 10,000 hemocytes was determined using excitation and emission filters appropriate for the TAMRA dye, and fluorescent emission was monitored using the FL2 channel. To quench the fluorescence of extracellular bacteria, 0.2% trypan blue (Merck & Co.) in Squid Ringer’s solution was added to each tube, and the fluorescence was immediately re-counted. The difference in mean fluorescence between the untreated sample and the sample treated with trypan blue was a measure of the level of phagocytosis; the fluorescence of intracellular, or engulfed, bacteria will not be quenched by the dye, whereas the fluorescence of cells not engulfed would be susceptible to quenching. Phagocytic ability was defined as the percentage of hemocyte cells with one or more ingested bacteria (TAMRA fluorescent cells) within the total cell population (10,000 cells). In control experiments, phagocytosis was blocked by pre-incubating the hemocytes with cytocholasin D (10 μg ml−1) for 30 min at 25°C before inoculation.

Acknowledgments

We thank K.E. Selph (University of Hawaii) for performing the FACS analysis. This work was supported in part by grants from the National Institutes of Health (RR-12294) to E.G. Ruby and M.J. McFall-Ngai, and (AI-50661) to M.J. M., and from the National Science Foundation (IBN-9904601) to M.J.M. and E.G.R.

References

  1. Aderem A, Underhill DM. Mechanisms of phagocytosis in macrophages. Annu Rev Immunol. 1999;17:593–623. doi: 10.1146/annurev.immunol.17.1.593. [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. Anselme C, Vallier A, Balmand S, Fauvarque MO, Heddi A. Host PGRP gene expression and bacterial release in endosymbiosis of the weevil Sitophilus zeamais. Appl Environ Microbiol. 2006;72:6766–6772. doi: 10.1128/AEM.00942-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Biswas I, Germon P, McDade K, Scott JR. Generation and surface localization of intact M protein in Streptococcus pyogenes are dependent on sagA. Infect Immun. 2001;69:7029–7038. doi: 10.1128/IAI.69.11.7029-7038.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burge EJ, Madigan DJ, Burnett LE, Burnett KG. Lysozyme gene expression by hemocytes of Pacific white shrimp, Litopenaeus vannamei, after injection with Vibrio. Fish Shellfish Immunol. 2007;22:327–339. doi: 10.1016/j.fsi.2006.06.004. [DOI] [PubMed] [Google Scholar]
  6. Canesi L, Gallo G, Gavioli M, Pruzzo C. Bacteria-hemocyte interactions and phagocytosis in marine bivalves. Microsc Res Tech. 2002;57:469–476. doi: 10.1002/jemt.10100. [DOI] [PubMed] [Google Scholar]
  7. Cash HL, Whitham CV, Behrendt CL, Hooper LV. Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science. 2006;313:1126–1130. doi: 10.1126/science.1127119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chakrabarti SR, Chaudhuri K, Sen K, Das J. Porins of Vibrio cholerae: purification and characterization of OmpU. J Bacteriol. 1996;178:524–530. doi: 10.1128/jb.178.2.524-530.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cowden RR, Curtis SK. Invertebrate Blood Cells. In: Ratcliffe NA, Rowley AF, editors. Cephalopods. New York: Academic Press; 1981. pp. 301–323. [Google Scholar]
  10. Dale C, Moran NA. Molecular interactions between bacterial symbionts and their hosts. Cell. 2006;126:453–465. doi: 10.1016/j.cell.2006.07.014. [DOI] [PubMed] [Google Scholar]
  11. Fujiwara D, Wei B, Presley LL, Brewer S, McPherson M, Lewinski MA, et al. Systemic control of plasmacytoid dendritic cells by CD8+ T cells and commensal microbiota. J Immunol. 2008;180:5843–5852. doi: 10.4049/jimmunol.180.9.5843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goo SY, Lee HJ, Kim WH, Han KL, Park DK, Lee HJ, et al. Identification of OmpU of Vibrio vulnificus as a fibronectin-binding protein and its role in bacterial pathogenesis. Infect Immun. 2006;74:5586–5594. doi: 10.1128/IAI.00171-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Govind S, Nehm RH. Innate immunity in fruit flies: a textbook example of genomic recycling. PLoS Biol. 2004;2:E276. doi: 10.1371/journal.pbio.0020276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hoffmann A, Levchenko A, Scott ML, Baltimore D. The IkappaB-NF-kappaB signaling module: temporal control and selective gene activation. Science. 2002;298:1241–1245. doi: 10.1126/science.1071914. [DOI] [PubMed] [Google Scholar]
  15. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science. 2001;291:881–884. doi: 10.1126/science.291.5505.881. [DOI] [PubMed] [Google Scholar]
  16. Irving P, Ubeda JM, Doucet D, Troxler L, Lagueux M, Zachary D, et al. New insights into Drosophila larval haemocyte functions through genome-wide analysis. Cell Microbiol. 2005;7:335–350. doi: 10.1111/j.1462-5822.2004.00462.x. [DOI] [PubMed] [Google Scholar]
  17. Iwasaki A. Mucosal dendritic cells. Annu Rev Immunol. 2007;25:381–418. doi: 10.1146/annurev.immunol.25.022106.141634. [DOI] [PubMed] [Google Scholar]
  18. Kokkinopoulos I, Jordan WJ, Ritter MA. Toll-like receptor mRNA expression patterns in human dendritic cells and monocytes. Mol Immunol. 2005;42:957–968. doi: 10.1016/j.molimm.2004.09.037. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Lavine MD, Strand MR. Insect hemocytes and their role in immunity. Insect Biochem Mol Biol. 2002;32:1295–1309. doi: 10.1016/s0965-1748(02)00092-9. [DOI] [PubMed] [Google Scholar]
  21. Liu CH, Lee SM, Vanlare JM, Kasper DL, Mazmanian SK. Regulation of surface architecture by symbiotic bacteria mediates host colonization. Proc Natl Acad Sci U S A. 2008;105:3951–3956. doi: 10.1073/pnas.0709266105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lupp C, Ruby EG. Vibrio fischeri uses two quorum-sensing systems for the regulation of early and late colonization factors. J Bacteriol. 2005;187:3620–3629. doi: 10.1128/JB.187.11.3620-3629.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Magnadottir B. Innate immunity of fish (overview) Fish Shellfish Immunol. 2006;20:137–151. doi: 10.1016/j.fsi.2004.09.006. [DOI] [PubMed] [Google Scholar]
  24. Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell. 2005;122:107–118. doi: 10.1016/j.cell.2005.05.007. [DOI] [PubMed] [Google Scholar]
  25. McFall-Ngai MJ. Unseen forces: the influence of bacteria on animal development. Dev Biol. 2002;242:1–14. doi: 10.1006/dbio.2001.0522. [DOI] [PubMed] [Google Scholar]
  26. McFall-Ngai MJ, Ruby EG. Symbiont recognition and subsequent morphogenesis as early events in an animal-bacterial mutualism. Science. 1991;254:1491–1494. doi: 10.1126/science.1962208. [DOI] [PubMed] [Google Scholar]
  27. Millikan DS, Ruby EG. Vibrio fischeri flagellin A Is essential for normal motility and for symbiotic competence during initial squid light organ colonization. J Bacteriol. 2004;186:4315–4325. doi: 10.1128/JB.186.13.4315-4325.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Negm RS, Pistole TG. The porin OmpC of Salmonella typhimurium mediates adherence to macrophages. Can J Microbiol. 1999;45:658–669. [PubMed] [Google Scholar]
  29. Niess JH, Brand S, Gu X, Landsman L, Jung S, McCormick BA, et al. CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science. 2005;307:254–258. doi: 10.1126/science.1102901. [DOI] [PubMed] [Google Scholar]
  30. Noverr MC, Huffnagle GB. Does the microbiota regulate immune responses outside the gut? Trends Microbiol. 2004;12:562–568. doi: 10.1016/j.tim.2004.10.008. [DOI] [PubMed] [Google Scholar]
  31. Nyholm SV, McFall-Ngai MJ. Sampling the light-organ microenvironment of Euprymna scolopes: description of a population of host cells in association with the bacterial symbiont Vibrio fischer. Biol Bull. 1998;195:89–97. doi: 10.2307/1542815. [DOI] [PubMed] [Google Scholar]
  32. Nyholm SV, McFall-Ngai MJ. The winnowing: establishing the squid-Vibrio symbiosis. Nat Rev Microbiol. 2004;2:632–642. doi: 10.1038/nrmicro957. [DOI] [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 USA. 2000;97:10231–10235. doi: 10.1073/pnas.97.18.10231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ofek I, Doyle RJ. Bacterial adhesion to cells and tissues. New York: Chapman & Hall; 1994. [Google Scholar]
  35. Peterson DA, McNulty NP, Guruge JL, Gordon JI. IgA response to symbiotic bacteria as a mediator of gut homeostasis. Cell Host Microbe. 2007;2:328–339. doi: 10.1016/j.chom.2007.09.013. [DOI] [PubMed] [Google Scholar]
  36. Pham LN, Dionne MS, Shirasu-Hiza M, Schneider DS. A specific primed immune response in Drosophila is dependent on phagocytes. PLoS Pathog. 2007;3:e26. doi: 10.1371/journal.ppat.0030026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Provenzano D, Lauriano CM, Klose KE. Characterization of the role of the ToxR-modulated outer membrane porins OmpU and OmpT in Vibrio cholerae virulence. J Bacteriol. 2001;183:3652–3662. doi: 10.1128/JB.183.12.3652-3662.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pruzzo C, Gallo G, Canesi L. Persistence of vibrios in marine bivalves: the role of interactions with haemolymph components. Environ Microbiol. 2005;7:761–772. doi: 10.1111/j.1462-2920.2005.00792.x. [DOI] [PubMed] [Google Scholar]
  39. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell. 2004;118:229–241. doi: 10.1016/j.cell.2004.07.002. [DOI] [PubMed] [Google Scholar]
  40. Reichelt JL, Baumann P. Taxonomy of the marine, luminous bacteria. Arch Mikrobiol. 1973;94:283–330. [Google Scholar]
  41. Rescigno M, Urbano M, Valzasina B, Francolini M, Rotta G, Bonasio R, et al. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol. 2001;2:361–367. doi: 10.1038/86373. [DOI] [PubMed] [Google Scholar]
  42. Ruby EG, Asato LM. Growth and flagellation of Vibrio fischeri during initiation of the sepiolid squid light organ symbiosis. Arch Microbiol. 1993;159:160–167. doi: 10.1007/BF00250277. [DOI] [PubMed] [Google Scholar]
  43. Ryu JH, Kim SH, Lee HY, Bai JY, Nam YD, Bae JW, et al. Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila. Science. 2008;319:777–782. doi: 10.1126/science.1149357. [DOI] [PubMed] [Google Scholar]
  44. Salzman NH, Underwood MA, Bevins CL. Paneth cells, defensins, and the commensal microbiota: a hypothesis on intimate interplay at the intestinal mucosa. Semin Immunol. 2007;19:70–83. doi: 10.1016/j.smim.2007.04.002. [DOI] [PubMed] [Google Scholar]
  45. Schipp R, Chung YS, Arnold JM. Symbiotic bacteria in the coelom of Nautilus. J Ceph Biol. 1990;1:59–74. [Google Scholar]
  46. Silver AC, Kikuchi Y, Fadl AA, Sha J, Chopra AK, Graf J. Interaction between innate immune cells and a bacterial type III secretion system in mutualistic and pathogenic associations. Proc Natl Acad Sci U S A. 2007;104:9481–9486. doi: 10.1073/pnas.0700286104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Soulas C, Baussant T, Aubry JP, Delneste Y, Barillat N, Caron G, et al. Outer membrane protein A (OmpA) binds to and activates human macrophages. J Immunol. 2000;165:2335–2340. doi: 10.4049/jimmunol.165.5.2335. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Vaishnava S, Behrendt CL, Hooper LV. Innate immune responses to commensal bacteria in the gut epithelium. J Pediatr Gastroenterol Nutr. 2008;46(Suppl 1):E10–11. doi: 10.1097/01.mpg.0000313823.93841.65. [DOI] [PubMed] [Google Scholar]
  50. Visick KL, Ruby EG. The periplasmic, group III catalase of Vibrio fischeri is required for normal symbiotic competence and is induced both by oxidative stress and approach to stationary phase. J Bacteriol. 1998;180:2087–2092. doi: 10.1128/jb.180.8.2087-2092.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Visick KL, Ruby EG. Vibrio fischeri and its host: it takes two to tango. Curr Opin Microbiol. 2006;9:632–638. doi: 10.1016/j.mib.2006.10.001. [DOI] [PubMed] [Google Scholar]
  52. Visick KL, Foster J, Doino J, McFall-Ngai M, Ruby EG. Vibrio fischeri lux genes play an important role in colonization and development of the host light organ. J Bacteriol. 2000;182:4578–4586. doi: 10.1128/jb.182.16.4578-4586.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Weis VM, Small AL, McFall-Ngai MJ. A peroxidase related to the mammalian antimicrobial protein myeloperoxidase in the Euprymna-Vibrio mutualism. Proc Natl Acad Sci USA. 1996;93:13683–13688. doi: 10.1073/pnas.93.24.13683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Whistler CA, Koropatnick TA, Pollack A, McFall-Ngai MJ, Ruby EG. The GacA global regulator of Vibrio fischeri is required for normal host tissue responses that limit subsequent bacterial colonization. Cell Microbiol. 2007;9:766–778. doi: 10.1111/j.1462-5822.2006.00826.x. [DOI] [PubMed] [Google Scholar]
  55. Wu Q, Lu Z, Verghese MW, Randell SH. Airway epithelial cell tolerance to Pseudomonas aeruginosa. Respir Res. 2005;6:26. doi: 10.1186/1465-9921-6-26. [DOI] [PMC free article] [PubMed] [Google Scholar]

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