Vibrio cholerae CD81 (O1 classical ) was kindly provided by Dr. B. S. Srivastava (Microbiology Division, Central Drug Research Institute, Lucknow 226001, India); RC60, RC66, and RC69 (non-O1/O139) were kindly provided by Dr. A. Huq (Maryland Pathogen Research Institute, University of Maryland, College Park, MD 20742, USA). N16961 ATCC^® 39315^TM (O1 El Tor) was also used in some experiments. All cultures were grown in Luria-Bertani (LB) broth or agar under static conditions at 37°C. To radiolabel bacteria, strains were grown overnight in LB broth containing 10 μCi of [methyl-³H]thymidine (25 Ci/mmol) ml^-1, were then harvested by centrifugation (3,000 × g for 15 min at 4°C), washed three times with phosphate-buffered saline (PBS; 0.1 M KH₂PO₄, 0.1 M Na₂HPO₄, 0.15 M NaCl, pH 7.2–7.4), and resuspended in PBS at an A₆₅₀ of 1(2 × 10⁸ to 4 × 10⁸ bacteria ml^-1). The number of counts per minute (cpm) per milliliter and the number of bacteria per milliliter were evaluated in triplicate samples to calculate the efficiency of cell labeling (number of bacteria per cpm) that varied in different bacterial preparations from 180 to 350. Artificial sea water [ASW; 35‰ (wt/vol) salinity, pH 7.9], filtered onto 0.22 μm-pore-size Millipore filters (Bedford, MA, USA), was used throughout the experiments.

Mussels (M. galloprovincialis Lam.) were obtained from the depuration plant Casa del Pescatore (Cattolica, Italy). Animals were transferred to the laboratory, cleaned of epibiota and kept in an aquarium at 16°C in static tanks containing ASW (1 l/animal) for 1–3 days before use; sea water was changed daily. Hemolymph was extracted from the posterior adductor muscle of the mussels by using a sterile 1 ml syringe with a 18-gauge, 0.5-in. long needle. After the needle was removed, the hemolymph was filtered through sterile gauze and pooled. To prepare hemocyte monolayers, an approximately 0.3-ml portion of hemolymph (corresponding to about 2 × 10⁶ to 3 × 10⁶ cells) was seeded onto glass coverslips (20 by 22 mm) placed in plastic culture dishes. The coverslips were incubated at 18°C for 30 min. Non-adherent hemocytes were removed by gently washing the preparations three times with 3 ml of ASW (Zampini et al., 2003).

To obtain hemolymph serum (i.e., hemolymph free of cells), the whole hemolymph was centrifuged at 50 × g for 10 min; the supernatant was then passed through a filter (pore size, 0.22 μm).

Evaluation of bacterial adherence to hemocytes was performed as follow: aliquots (1.5 ml) of either ASW or hemolymph serum containing radiolabeled bacteria at a final concentration of 2 × 10⁷ to 3 × 10⁷ bacteria ml^-1 were added to monolayers, and the dishes were incubated with gentle shaking at either 4°C (to evaluate adhering bacteria) or 18°C (to evaluate associated bacteria = adhering + internalized). Triplicate preparations were made for each sample. After 60 min incubation, the cover slips were rinsed with cold ASW, and transferred to PICO-FLUOR^TM15 scintillation fluid (Packard Instruments Company Inc., Meriden, CT, USA). For each sample, the number of bacteria per monolayer was calculated using the efficiency of cell labeling. Background counts due to bacterial attachment to cover-slips were also evaluated (typically 50–250 cpm) and subtracted from the sample values. In experiments performed to define the chemical nature of the bacterial component(s) involved in interactions with hemocytes, bacteria were pretreated with pronase E and sodium meta-periodate. Pronase E was added to bacterial suspensions at a final concentration of 100 pgml^-1. The suspensions were then incubated for 1 h at 37°C in a shaking water bath and centrifuged. The pellets were resuspended in PBS to the original volume. Sodium meta-periodate pretreatment of bacteria was performed in PBS containing sodium meta-periodate at a final concentration of 1 mM. The suspensions were incubated at room temperature for 10 min, washed twice and resuspended in PBS.

To evaluate bacterial sensitivity to killing by hemocytes, V. cholerae suspensions (about 10⁷ bacteria ml^-1) were added to hemocyte monolayers at 18°C in the presence of hemolymph serum as described above. Triplicate preparations were made for each sampling time. Immediately after the inoculum (T = 0) and after 60 min of incubation at 18°C supernatants were collected from monolayers, and hemocytes were lysed by adding 5 ml of cold distilled water and by 10 min of agitation. The collected monolayer supernatants and hemocyte lysates were pooled and 10-fold serially diluted in PBS; aliquots (100 μl) of the diluted samples were plated onto LB agar, and after overnight incubation at 37°C the number of colony-forming units (CFU) per milliliter in the hemocyte monolayer (representing culturable bacteria survived to hemocyte bactericidal activity) was determined. Percentages of killing at 60 min were then determined relative to values obtained at T = 0. To evaluate the presence of endogenous bacteria in hemocytes, controls were performed with hemocyte monolayers without bacteria. The number of CFU in controls never exceeded 0.1% of those enumerated in experimental samples. To detect and correct for bacterial growth in hemolymph serum, separate samples were seeded with bacteria and 1.5 ml of sterile hemolymph serum. No appreciable bacterial growth was observed at the same time intervals used in killing experiments.

Serum was fractionated into low and high molecular mass fractions (LMM and HMM) by ultrafiltration using the Vivaflow 200 complete system (Vivascience AG, Feodor-Lynen-Strasse 21, 30625 Hannover, Germany) comprising a pump (240 V), tubing, 500 ml sample/diafiltration reservoir, and a membrane 5,000 MWCO PES for ultradiafiltration (Vivascience). The LMM 5000 cutoff was chosen since electrophoretic separation of mussel soluble serum proteins (in both 1D and also 2D gels used for proteomic studies in different conditions) generally yields protein bands with MM generally ≥10,000. A diafiltrate, i.e., a LMM containing all the compounds with molecular masses less than 5000 Da, and a retentate, i.e., a HMM containing all the compounds with molecular masses greater than 5000 Da were obtained and, after restoring the initial volume, were tested.

Experiments were repeated at least three times. Data shown in the Figures are the mean values ± standard deviation obtained in one representative experiment performed in triplicate. Data were analyzed for significance by the Mann–Whitney U test. Differences were considered significant at P < 0.05.

We studied interactions with hemocytes of V. cholerae strains that either do not carry the msha gene (RC60, RC66, and RC69 [non-O1/O139 serogroups]) or do not express it (CD81 [O1 serogroup, classical biotype]). Bacteria were added to hemocyte monolayers in both ASW and hemolymph serum at 18°C. At this temperature, the number of associated (adhering + internalized) bacteria was evaluated. Figure 1 shows that in hemolymph serum the association efficiency of the tested strains was 2.2–3.5-fold higher than in ASW (P ≤ 0.05).

To clarify to what extent the observed differences in association were due to different adhesion efficiencies, the same assays were performed at 4°C, a condition that almost completely inhibits the internalization process (Zampini et al., 2003; Figure 1). The presence of hemolymph serum increased the number of vibrios adhering to hemocytes by 2.1–2.6-fold in comparison to ASW (P ≤ 0.05; Figure 1). Moreover, as expected, at 4°C the number of bacteria interacting with hemocytes was lower than that at 18°C. Interestingly, the increase in both association with and adherence to mussel hemocytes of the tested strains was about twofold lower than that observed with the MSHA-positive V. cholerae N16961 strain, used for comparison.

Vibrio cholerae strains were then tested for their ability to resist to killing by hemocytes, both in the presence and in the absence of serum (Figure 2). It was found that, after 60 min incubation, the percentage of killed bacteria compared to that at T = 0 ranged from 9.3 to 14.7% in the experiments performed in serum and from 3.5 to 6.3% in experiments performed in ASW. Differences between the two experimental conditions were statistically significant (P ≤ 0.05). As for association and adhesion, the serum-mediated increase in killing efficiency of the tested strains by hemocytes was about twofold lower than that observed with V. cholerae N16961 strain carrying the MSHA.

As a whole, these results indicate that mussel serum, in addition to opsonins directed toward MSHA, contains factors that mediate binding between Vibrio cell wall component(s) and hemocyte surface receptor(s), and promote internalization and killing of these bacteria.

In a first attempt to define the chemical nature of bacterial surface components involved in V. cholerae interactions with hemolymph, adhesion to hemocytes of the tested strains in the presence of serum was evaluated after bacteria had been treated with either pronase E or sodium meta-periodate, which oxidizes polysaccharides. As shown in Figure 3, both treatments caused a decrease in serum-mediated attachment to hemocytes. In comparison to untreated controls, pronase E and sodium meta-periodate treatments reduced the interactions by 2.3–3.5 and 2.7–7.0-fold, respectively. Meta-periodate oxidation and pronase E digestion did not affect bacterial viability, as demonstrated by viable counts (data not shown).

To preliminarily assess the main chemical properties of serum factors capable to promote Vibrio strain interactions with hemocytes, hemolymph serum was first exposed (15 min) to different temperatures, ranging from 45 to 80°C, then used in experiments to evaluate efficiency of bacterial adhesion to hemocytes. Serum progressively lost its capability to promote V. cholerae attachment to hemocytes with increasing temperature from 45 to 80°C. In particular, the number of bacteria adhering to hemocytes in the presence of serum incubated at 80°C was 2.2–3.9-fold lower than that observed in the presence of untreated serum (Figure 3), and similar to those obtained in experiments performed in ASW (Figure 1).

Serum was then fractionated into LMM and HMM fractions by ultrafiltration (see Methods). LMM fraction contained all the compounds with molecular masses lower than 5000 Da, and HMM contained all the compounds with molecular masses greater than 5000 Da. Capability of both fractions to promote adhesion to and association with hemocytes of the tested bacteria was analyzed. As shown in Figure 4, whereas LMM fraction did not cause any statistically significant increase in bacterial interactions with hemocytes, the HMM fraction caused a 1.6–2.4-fold increase of both adhesion to and association with monolayers of the tested strains. When the experiments were performed using a mixture of both LMM and HMM fractions, results similar to those obtained with HMM alone were obtained (not shown).