Differential Expression of Vibrio vulnificus Capsular Polysaccharide

INFECTION AND IMMUNITY, May 1999, p. 2250–2257 0019-9567/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 67, No. 5

Differential Expression of Vibrio vulnificus Capsular Polysaccharide ANITA C. WRIGHT,1* JAN L. POWELL,2 MIKE K. TANNER,3 LYNNE A. ENSOR,2 ARTHUR B. KARPAS,4 J. GLENN MORRIS, JR.,2 AND MARCELO B. SZTEIN3 Center of Marine Biotechnology, University of Maryland Biotechnology Institute,1 and Veterans Affairs Medical Center and Department of Medicine2 and Center for Vaccine Development,3 University of Maryland School of Medicine, Baltimore, and National Institute of Child Health and Human Development, National Institutes of Health, Bethesda,4 Maryland Received 20 August 1998/Returned for modification 9 October 1998/Accepted 23 February 1999

Vibrio vulnificus is a human pathogen whose virulence has been associated with the expression of capsular polysaccharide (CPS). Multiple CPS types have been described; however, virulence does not appear to correlate with a particular CPS composition. Reversible-phase variation for opaque and translucent colony morphologies is characterized by changes in CPS expression, as suggested by electron microscopy of cells stained nonspecifically with ruthenium red. Isolates with opaque colony morphologies are virulent and appear to be more thickly encapsulated than naturally occurring translucent-phase variants, which have reduced, patchy, or absent CPS. Previously, we have shown that the virulence of translucent-phase variants was intermediate between opaque-phase variants and acapsular transposon mutants, suggesting a correlation between virulence and the amount of CPS expressed. In the present study, CPS expression of phase variants and genetically defined mutants of V. vulnificus M06-24/O was examined by using a CPS-specific monoclonal antibody with an enzyme-linked immunosorbent assay, flow cytometry, and immunoelectron microscopy. Semiquantitative analyses of CPS expression correlated well among these assays, confirming that the translucent-phase variant was intermediate in CPS expression and retained type I CPS-specific epitopes. Cell surface expression of CPS varied with the growth phase, increasing during logarithmic growth and declining in stationary culture. Significantly greater CPS expression (P 5 0.026) was observed for cells grown at 30°C than for those at 37°C. These studies confirm that phase variation and virulence in V. vulnificus correlate with the amount of CPS expressed and demonstrate the fluidity of bacterial polysaccharide expression in response to environmental conditions.

Vibrio vulnificus can be readily isolated from the water, sediment, fish, and shellfish of estuaries worldwide during summer months (8, 23, 24, 35, 37). Human disease produced by this organism is characterized by fulminating primary septicemia and is strongly associated with the consumption of raw oysters (3, 28). Persons with liver disease, hemochromatosis, or immune dysfunction are particularly susceptible, with mortalities that exceed 50% (3), and constitute the majority of fatal infections associated with seafood consumption in the United States (28). The virulence of V. vulnificus has been positively correlated with capsular polysaccharide (CPS) expression in a number of animal models (18, 32, 38, 40). Encapsulated isolates of V. vulnificus have opaque colony morphologies and exhibit a reversible-phase variation to translucent morphotypes with a reduced or patchy expression of surface polysaccharide, as observed by electron microscopy of cells stained nonspecifically with ruthenium red. The importance of CPS as a virulence determinant for V. vulnificus was confirmed by the loss of virulence phenotype in acapsular transposon mutants (38). The phenotype of partially encapsulated V. vulnificus translucentphase variants is intermediate between the fully encapsulated parent strains and acapsular transposon mutants, in terms of the virulence or sensitivity to phagocytosis and complementmediated cell lysis. These correlations suggest a positive rela-

tionship between the amount of expressed CPS and virulence and are consistent with observations in Escherichia coli in which enhanced virulence in mice correlated with growth conditions that significantly increased CPS expression (36). Bacteria that produce extracellular systemic infections frequently express polysaccharide capsules on their cell surfaces for the evasion of innate host defenses (13, 36). The amount of CPS expressed can vary with genetically determined phase variation (19, 25) or with environmental factors such as pH, nutrient levels, metal cation availability, and growth phase (21, 26, 31, 36). Differential expression suggests mechanisms by which bacteria respond to environmental signals to regulate biosynthesis and transport of CPS to the cell surface, thereby enhancing survival in the host and increasing virulence. Environmental conditions that facilitate CPS expression either in vivo or in vitro have not been described for V. vulnificus. However, both the reticuloendothelial system in mice (40) and the phagocytic hemocytes of oysters rapidly take up translucent isolates (15), whereas opaque encapsulated strains more readily avoid phagocytosis or may persist in oyster tissues. Therefore, CPS expression in V. vulnificus is a likely indicator of both virulence potential in mammals and the ability to colonize oysters. V. vulnificus also shows great diversity in its CPS structure (4, 16), and further studies are needed to relate both capsular expression and structure to biological function. Previous examination of CPS expression in V. vulnificus has relied on electron microscopy of cells stained with ruthenium red, which binds nonspecifically to negatively charged polysaccharides (18, 37).

* Corresponding author. Mailing address: Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt St., Baltimore, MD 21202. Phone: (410) 234-8827. Fax: (410) 234-8896. E-mail: [email protected]. 2250

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This dye does not provide a quantitative analysis or differentiate among CPS types or lipopolysaccharide (LPS) with long O-antigen side chains that may resemble CPS. Other methods for the evaluation of CPS expression can be hampered by a number of problems related to polysaccharide detection and quantification. Polysaccharide extraction efficiencies vary with composition or with the presence of other carbohydrates, and biochemical assays may detect only certain classes of sugars or require extensive hydrolysis (5). For example, hydrolysis of V. vulnificus M06-24/O CPS produces a disaccharide of uronic acid sugars that gives no reaction by standard phenol-sulfuric acid assays commonly used to detect neutral sugars (27). Capsular polysaccharides are notoriously poor immunogens and, when available, antibody-based analyses may not discriminate between total and cell surface-associated polysaccharide (36). In the present study, we produced V. vulnificus type I CPSspecific monoclonal antibodies by using purified CPS conjugated to tetanus toxoid for immunizations. Monoclonal antibodies which bound CPS and not LPS were used for semiquantitative analyses of CPS cell surface expression, as determined by flow cytometry (FC), enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy (IEM). The application of FC with LPS-specific antibodies (11, 12, 22) or CPS-specific lectins (31) has been used previously to evaluate surface expression of bacterial polysaccharides. However, the extensive use of FC analysis to quantify bacterial CPS has not been reported, possibly due to a lack of appropriate controls and antibodies. To our knowledge, this is the first report on the use of a monoclonal antibody for FC analysis of bacterial CPS. A variety of V. vulnificus strains, including phase variants, genetically defined mutants, and isolates with different capsular types, were examined throughout the growth phase at different temperatures. These studies demonstrated that CPS expression in V. vulnificus responds to environmental signals and confirmed the essential role of the capsule in virulence. MATERIALS AND METHODS Bacterial strains and growth conditions. Most of the V. vulnificus strains used in this study have been described elsewhere (38). M06-24/O is an encapsulated isolate with an opaque colony morphology and type I CPS (16) that was isolated from the blood of an infected individual, M06-24/T is a spontaneous translucentphase variant with reduced CPS expression, CVD752 is an acapsular transposon mutant of M06-24/O which is unable to synthesize CPS, and M06-24/31T is able to synthesize CPS but does not express a capsule on the surface as the result of a nonpolar insertion in the CPS transport gene wza (38a). Other V. vulnificus strains examined include V1015H (type 1 CPS) and B062316 (type 2 CPS) and eight other opaque clinical and environmental isolates whose CPS composition (types 2, 3, 4, 5, 8, 12, 14, and 15) had been previously determined (16). Strains were stored at 270°C in Luria broth (LB) (Difco, Detroit, Mich.) with 50% glycerol and streaked to LB agar for isolation and subsequent inoculation into LB with or without appropriate antibiotics. Production of V. vulnificus hyperimmune antiserum and CPS-specific monoclonal antibodies. For hyperimmune serum production, New Zealand White rabbits received multiple intravenous injections of live V. vulnificus cells at intervals of three to four times per week for 4 weeks with increasing doses ranging from 107 to 5 3 109 bacteria/injection. For monoclonal antibody production, V. vulnificus M06-24/O CPS was purified (27) and conjugated to tetanus toxoid as previously described (9). BALB/c mice (Charles River, Wilmington, Mass.) were immunized intraperitoneally with one of two CPS conjugates, VvPSTT-a or VvPSTT-b, prepared by conjugating tetanus toxoid to V. vulnificus CPS, with either carboxyl or hydroxyl activation of the polysaccharide, respectively. The antigen (ca. 5 mg) for inoculations was diluted 1:5 in sterile phosphatebuffered saline (PBS), pH 7, and mixed with an equal volume of Freund’s complete adjuvant. Animals were given a booster at about 4 weeks postinoculation with the antigen and Freund’s incomplete adjuvant and at 8 weeks with the antigen alone. Three to 5 days after the final booster, spleens were removed and processed by mechanical shearing. Splenocytes were mixed 10:1 with SP2/O cells in the presence of polyethylene glycol 4000. Plated cells were incubated at 37°C in 5% CO2 in the presence of hypoxanthine-aminopterin-thymidine until colonies had grown. Supernatants were collected and tested for reactivity against M06-24/O cells and purified CPS by ELISA (described below). Following limit-

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ing dilution cloning of selected cell lines, CPS-reactive monoclonal antibodies were isotyped (MabCheck; Sterogene Bioseparations, Inc., Arcadia, Calif.) and specificity was examined by ELISA or Western blot analyses as described below. Based on their high reactivity to purified CPS from V. vulnificus M06-24/O, hybridoma cell lines 7/G4-D2 (immunoglobulin A [IgA] derived from VvPSTTb), 1004-B7 (IgG3 derived form VvPSTT-a), and 1002-C4 (IgM derived from VvPSTT-a) were selected for subsequent studies. CPS expression in V. vulnificus strains as determined by ELISA. For wholecell ELISA, bacterial strains described above were grown in LB to mid-log phase (optical density at 600 nm [OD600] 5 0.7), washed once in PBS, and diluted to an initial concentration of 107 cells/well. Twofold serial dilutions of these cells were prepared in triplicate in 96-well microtiter plates (Immulon 1; Dynex Technologies, Chantilly, Va.) and incubated at 4°C overnight to allow cells to bind to the plates. Bacterial cells were also disrupted by sonication, and the membrane and soluble fractions were collected by differential centrifugation. Serial dilutions of antigen were prepared in triplicate in microtiter plates as described above. The excess antigen was removed by washing with 0.05% Tween in PBS, followed by blocking unbound sites with PBS containing 5% fetal bovine serum (FBS) (Gibco BRL, Gaithersburg, Md.) at 37°C for 1 h. Hybridoma supernatants were diluted 1:20 in PBS–1% FBS, 100 ml of the dilution was added to each well, and the plates were incubated for 1 h at 37°C. After the plates were washed, as described above, 100 ml of alkaline phosphatase-labeled goat antimouse (IgG-IgA-IgM) antibody (1 mg/ml) (Kirkegaard and Perry, Gaithersburg, Md.) diluted with PBS–1% FBS was added to all wells and incubated as before. The plates were washed a final time, a substrate (pNPP; Kirkegaard and Perry) was added to all wells, and the OD405 was determined after 30 min of incubation at 37°C. Negative controls for whole cells and cell fractions of each strain employed the above method without a primary antibody, and the mean OD405 of these samples was subtracted from the experimental value to determine the binding of CPS-specific antibody. A comparable specificity was seen with all CPS-specific monoclonal antibodies; however, higher titers were observed with 7/D4-G2. Western blot analysis. Purified V. vulnificus CPS and LPS preparations were analyzed by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described by Laemmli (20) and compared to molecular weight standards (Bio-Rad Laboratories, Hercules, Calif.). Gels were silver stained for LPS (17) or were transferred to a nitrocellulose membrane for Western blot analysis (14). Western blots were incubated with the primary antibody (either hyperimmune polyclonal serum to V. vulnificus M06-24/O whole cells or monoclonal antibody) and diluted in a blocking buffer consisting of 5% nonfat dry milk with 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), for 2 h at 4°C with shaking. The secondary antibody, alkaline phosphatase-labeled goat anti-rabbit or anti-mouse Ig (1 mg/ml) (Kirkegaard and Perry) diluted in the blocking buffer, was incubated with membranes for 1 h at room temperature with shaking, followed by development in a buffered substrate (Kirkegaard and Perry) of 5-bromo-4-chloro-3indolyl phosphate and nitroblue tetrazolium. FC analysis of CPS surface expression with a CPS-specific monoclonal antibody. One milliliter of overnight cultures grown to an OD600 of 1.1 was inoculated into 100 ml of LB in 250-ml baffled flasks prewarmed to 30° or 37°C and incubated with shaking at 200 rpm. Cultures were sampled, and the OD600 were recorded at 0, 1, 2, 4, 6, and 24 h postinoculation. Cells (100 ml) were adjusted to a standard OD600 of 0.6, pelleted by centrifugation at 3,000 3 g for 1 min, washed in 1 ml of PBS with 0.02% azide (PBSA), and resuspended in V. vulnificus CPS-specific hybridoma supernatants (described above) diluted 1:2 in PBSA. Following incubation on ice, cells were washed as above, incubated with goat IgA-specific fluorescein isothiocyanate (FITC) antibody conjugate (PharMingen, San Diego, Calif.) in PBSA (1 mg/ml), washed again in PBSA, and stored in 1% formaldehyde at 4°C. All incubations were for 30 min on ice. Triplicate samples were analyzed for all samples with V. vulnificus M06-24 variants and the 7/G4-D2 IgA monoclonal antibody. Analysis of V. vulnificus strains (V1015H and B062316) and other monoclonal antibodies represented single samples. Negative controls included cells incubated without either primary or secondary antibody, as well as incubation with an irrelevant mouse IgA antibody (Sigma, St. Louis, Mo.). Surface-associated CPS was detected by measuring FITC fluorescence with a Coulter Epics Elite flow cytometer-cell sorter system (Coulter Cytometry, Hialeah, Fla.) with excitation from a 488-nm argon laser, and emissions were collected through a 525-nm band-pass filter. Bacteria were gated on the basis of log forward scatter and log side scatter, so as to exclude instrumentation noise. Approximately 10,000 bacteria were collected for each histogram. The percentage of cells binding antibody and mean fluorescence channel (MFCh), as a measure of the fluorescence intensity, was recorded. Samples (n 5 3) were averaged, and standard deviations were calculated. F test analysis on the sum of squares of the percentage of cells binding antibody was calculated at each time point by using analysis of variance, and the significance of difference in CPS expression at different temperatures was determined by a standard t test (33). IEM of V. vulnificus strains to visualize CPS production. V. vulnificus strains were embedded, immunolabeled, and observed by transmission electron microscopy as previously described (41). Briefly, cells grown overnight at 30°C on LB agar were washed in 3.5% saline, pelleted by centrifugation (2,000 3 g for 15 min at 4°C), fixed, and embedded in LR white. Ultrathin sections were collected on nickel grids (Electron Microscopy Sciences, Fort Washington, Pa.) which were incubated specimen side down in 5% goat serum in PBS (GS-PBS) for 15 min

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INFECT. IMMUN. TABLE 1. Comparison of CPS expression by V. vulnificus strains

Straina

M06-24/O M06-24/T CVD752 M06-24/31T

ELISA (OD405)b

FCc

Whole cell

Membrane

Soluble

% Positive

MFCh

IEMd % labeled (no. of total cells)

0.63 6 0.016 0.33 6 0.023 0.03 6 0.007 0.23 6 0.087

0.95 6 0.051 0.13 6 0.006 0.01 6 0.008 0.58 6 0.010

0.66 6 0.083 0.07 6 0.011 0.00 0.03 6 0.008

74.7 6 3.06 43.7 6 3.06 0.6 6 0.15 2.9 6 0.35

37.3 6 5.12 19.7 6 2.27 3.5 6 0.20 4.2 6 0.20

93.0 (100) 31.1 (122) 0.6 (161) 3.2 (124)

a V. vulnificus strains include opaque (M06-24/O) and translucent (M06-24/T) phase variants, an acapsular transposon mutant (CVD752), and a CPS transport mutant (M06-24/31T). b ELISA values represent binding of the CPS-specific monoclonal antibody to whole cells or fractions of sonicates as described in Materials and Methods. c FC analysis shows the proportion of cells staining positive (% positive) or MFCh as an indication of the fluorescence intensity of cells staining positive with the M06-24/O CPS-specific monoclonal antibody as described in the text. Means (n 5 3) and standard deviations are shown for samples at 2 h postinoculation. d Percentage of cells with surface immunogold label and number of total cells counted.

and immunolabeled for 1 h with an anti-CPS monoclonal antibody from hybridoma supernatants diluted 1:1 in GS-PBS. Grids were washed in GS-PBS with a subsequent 15-min incubation in a secondary antibody conjugate (goat antimouse IgA labeled with 10-nm colloidal gold) diluted 1:50 in GS-PBS, followed by multiple washes in distilled water. Cells were negatively stained with 2% uranyl acetate for 5 min, followed by 0.2% lead citrate staining for 1 min. Observations were performed on a JEM-100CX II transmission electron microscope (80 kV) (JEOL Ltd., Tokyo, Japan).

RESULTS Specificity of monoclonal antibodies for type I CPS. Monoclonal antibody 7/G4-D2 was specific for type 1 CPS, as demonstrated by whole-cell ELISA. Both V. vulnificus M06-24/O and M06-24/T phase variants bound the antibody, whereas the acapsular mutant CVD752 and CPS transport mutant M06-24/ 31T bound little or no antibody (Table 1). V. vulnificus V1015H with type 1 CPS was also positive, but V. vulnificus strains representing eight other CPS types with different carbohydrate compositions did not bind the 7/G4-D2 monoclonal antibody. A similar specificity was obtained with the 1004-B7 and 1002-C4 monoclonal antibodies described above (data not shown). Monoclonal antibody 7/G4-D2 exhibited the highest reactivity and was used exclusively for subsequent experiments. The specificity of the 7/G4-D2 (not shown) and 1004-B7 (Fig. 1A) monoclonal antibody for CPS was also confirmed by Western blot analysis, which demonstrated that the monoclonal antibody bound only purified V. vulnificus CPS and not LPS, while both were detected by a polyclonal antibody to whole cells (Fig. 1B). Silver staining of polysaccharides stains LPS but not CPS and was used to assure the purity of CPS preparations, which were negative by silver staining (data not shown). Comparison of semiquantitative analyses of CPS expression in V. vulnificus strains as determined by ELISA, FC, and IEM. As shown in Table 1, results from FC analyses of surface CPS expression for different V. vulnificus strains and variants were consistent with results obtained by either ELISA or IEM. In these experiments, cells of the virulent encapsulated strain V. vulnificus M06-24/O exhibited the greatest values for ELISA, IEM, or FC (for both fluorescence intensity and percentage of cells binding antibody), whereas the acapsular transposon mutant CVD752 was negative by these assays. Values for surface CPS expression of transport mutant M06-24/31T were also negligible by FC and IEM, although binding of antibody was observed by whole-cell ELISA. Translucent-phase variant M06-24/T exhibited intermediate values for all assays. Typical FC histograms (Fig. 2) show values for gated cells within the rectangle as to exclude background sheath noise. No detectable signal from gated cells was observed from any of the negative controls. Both acapsular CVD752 and CPS transport mutant M06-24/31T exhibited profiles that were similar to those of negative controls. CPS type I strain V1015H exhibited

FC histograms similar to those of M06-24/O, while the signal was not detected for type 2 strain B062316. The differential expression of CPS by V. vulnificus strains was confirmed by IEM. Electron micrographs (Fig. 3) demonstrated abundant surface CPS for M06-24/O, reduced expression for M06-24/T, and little or no surface CPS for mutant strains CVD752 and M06-24/31T. Differential expression of V. vulnificus CPS as determined by FC analysis. V. vulnificus M06-24/O, M06-24/T, M06-24/31T, and CVD752 were cultured in LB at 37°C, and cell densities were determined over time (data not shown). Encapsulated V. vulnificus M06-24/O cells were also grown at 30°C to determine the effects of growth temperature on CPS expression. Similar growth rates were observed for all strains during logarithmic growth (0 to 4 h postinoculation), and cultures reached stationary phase at 6 h. As shown in Fig. 4A, the percentage of encapsulated M06-24/O cells binding the CPS-specific monoclonal antibody 7/G4-D2 was consistent over time, although the percentage of positive M06-24/T cells declined in stationary phase. Capsule mutants CVD752 and M06-24/31T did not bind the anti-CPS monoclonal antibody. Significant differences (P 5 0.001) in the percentage of positive cells were observed among V. vulnificus M06-24/O, M06-24/T, and CVD752 for all time points. Fluorescence intensity (MChF) as an indicator of

FIG. 1. Specificity of anti-V. vulnificus CPS monoclonal antibody. (A) Western blots with CPS-specific monoclonal antibody (7/G4-D2) and V. vulnificus M06-24/O LPS (lane 2) or CPS (lane 3). (B) Western blots with polyclonal antibody to whole cells and purified V. vulnificus M06-24/O LPS (lane 2) or CPS (lane 3). Lanes 1, molecular mass standards (in kilodaltons).


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FIG. 2. Fluorescence histograms showing differential binding of CPS-specific monoclonal antibody to V. vulnificus strains with variable surface CPS expression. V. vulnificus strains were stained with the anti-CPS 7/G4-D2 monoclonal antibody and analyzed by FC as described in Materials and Methods. Shown are representative histograms corresponding to V. vulnificus M06-24/O (A), M0624/T (B), M06-24/31T (C), and CVD752 (D). The percentages of positive cells and MFCh are shown for each histogram.

the amount of CPS expression by the encapsulated strains was maximal during logarithmic growth at 2 h of incubation for both M06-24/O and M06-24/T (Fig. 4B). Surface CPS expression declined as cells approached stationary phase at 4 h postinoculation. M06-24/O cells grown at 30°C for 2 h exhibited significantly (P 5 0.026) greater fluorescence intensity than those that were incubated at 37°C for 2 h. DISCUSSION Despite the important role of bacterial polysaccharides in the evasion of innate host defenses, relatively little is known about the physical parameters or mechanisms that influence CPS expression. The differential expression of CPS may enhance bacterial survival by alternately exposing or masking more hydrophobic surface structures needed for adhesion dur-

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ing colonization or for evasion of macrophages and complement during systemic infection (7, 30, 34). For example, decreased CPS expression in group B streptococci correlates with adhesion to mucous membranes during initial infection, whereas during systemic phases of disease the expression increases when the need for the antiphagocytic capsule is greater (13, 39). CPS expression appears to be required for the virulence of V. vulnificus, and animal models have suggested that the amount of CPS expressed may be a factor in the observed differences among opaque- and translucent-phase variants. However, tremendous diversity in CPS carbohydrate composition has been reported (4, 16), and previous studies, including our own, examined CPS expression by using nonspecific analyses that may not discriminate different polysaccharide types or distinguish CPS from other hydrophilic or negatively charged surface structures. Therefore, we produced monoclonal antibody specific for V. vulnificus type I CPS for the evaluation of CPS expression. CPS expression for naturally occurring phase variants of V. vulnificus, as well as for genetically defined mutants, was evaluated by ELISA, IEM, or FC analyses with a CPS-specific monoclonal antibody. Difficulties in obtaining monoclonal or polyclonal antibodies to purified capsule preparations have been reported (29), but increased antibody responses can be achieved by the use of protein carriers (6). Previously described tetanus toxoid conjugates to purified V. vulnificus CPS elicit the greatest polyclonal response in mice (10) and were used to produce monoclonal antibodies in mice. Various isotypes were characterized, and the specificity was confirmed by selective reactivity to strains that expressed the type I capsule. The translucent-phase variant M06-24/T also bound all type 1-specific monoclonal antibodies, indicating the conservation of the CPS epitope(s) and supporting the hypothesis that the differences between CPS phase variants of V. vulnificus are quantitative and not qualitative. All assays indicated that the opaque encapsulated strain bound the greatest amount of antibody, while the translucent phenotype was intermediate and V. vulnificus mutants that were acapsular or did not express surface CPS did not bind the CPS-specific antibody. FC analysis demonstrated significant, consistent, and reproducible differences among strains for both the percentage and fluorescence intensity of cells binding the antibody and confirmed that CPS is differentially expressed in V. vulnificus phase variants. FC analysis can elucidate both qualitative and quantitative changes in bacterial polysaccharide expression. For example, FC analysis based on the affinity of wheat germ agglutinin lectin to N-acetylglucosamine discriminated phase variations of CPS within mixed populations of streptococci (31), and the examination of heterogeneous populations via fluorescenceassisted cell sorting documented reversible changes in LPS structure in E. coli (11, 12). Interestingly, about 30% of the cells from the V. vulnificus encapsulated strain bound little or no monoclonal antibody as determined by FC. Although a loss of surface antigen may result as an artifact of sample preparation, these cells could represent a down-regulation of CPS expression or an increased phase variation within the population and warrant further investigation. An apparent advantage of FC analysis was the absence of detectable surface CPS from the CPS transport mutant, as surface antigen was detected by whole-cell ELISA, presumably due to the release of intracellular CPS from dead or dying cells. A more precise FC quantification of surface proteins in staphylococci has been obtained by using calibration beads sized to approximate bacteria (2-mm diameter) and coated with a known amount of monoclonal antibody for the determination of a flow cytometric standard curve (1). Although the polymeric nature of capsules

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FIG. 3. Immunoelectron micrographs of V. vulnificus strains showing differential expression of CPS. Bacterial thin sections were immunolabeled with a type I-specific anti-CPS monoclonal antibody (7/G4-D2) and visualized with a gold-labeled secondary goat anti-mouse IgA conjugate (arrowheads) as described in the text. Strains included the following V. vulnificus phase variants and mutants: M06-24/O (A), M06-24/T (B), M06-24/31T (C), and CVD752 (D). Bars, 500 nm.

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FIG. 4. Percentages of cells binding V. vulnificus CPS-specific monoclonal antibody and fluorescence intensity over time. Surface expression of CPS as determined by FC analysis for V. vulnificus M06-24/O (M06/O), M06-24/T (M06/T), M06-24/31T (M06/31T), and CVD752 over time is shown as either a percentage of cells binding the CPS-specific monoclonal antibody (A) or MFCh as a measure of relative fluorescence intensity (B). M06-24/O cells were grown at either 37° or 30°C. Error bars represent standard deviations. Significant differences in the distribution of results from the F test were detected among M06-24/O, M06-24/T, and CVD752 at each time point (P 5 0.001). M06-24/O cells grown at 30°C exhibited significantly greater (P 5 0.026) fluorescence intensity than those grown at 37°C for 2 h postinoculation.

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and variation in chain length may complicate the determination of the number of exposed epitopes, this methodology may be applicable to a more rigorous quantification of bacterial polysaccharides. Our data demonstrated that FC offers a semiquantitative method for the rapid examination of the differential expression of CPS in V. vulnificus. This report is the first description of variable CPS expression for V. vulnificus in response to growth conditions. Expression peaked during logarithmic growth and declined as cells reached stationary phase. These results are in agreement with similar descriptions of CPS kinetics in E. coli (36) and group B Streptococcus (26). LPS expression in E. coli, on the other hand, differs from that of CPS and is maximal in stationary phase (12). Differences in CPS expression observed during the transition to stationary phase may relate to the availability of nutrients or the depletion of autoinducers present in logarithmic growth (2). CPS expression also varied with incubation temperature and was greater at 30°C than at 37°C. Therefore, these data indicate that the expression of CPS in V. vulnificus is sensitive to both genetically determined phase variation and changing environmental conditions. The polysaccharide capsule of V. vulnificus may contribute to the evasion of innate immune defenses in both vertebrate and invertebrate hosts. The genetic basis for phase variation, as well as environmental signals that regulate in vivo expression of CPS in V. vulnificus, has not been determined. However, the ubiquity of V. vulnificus in coastal waters and oysters supports the assumption that the estuarine environment is the primary habitat of this species, and regulatory response elements are more likely to have evolved as adaptations to environmental conditions rather than to the physiological milieu of vertebrate hosts. For example, increased CPS expression was observed at 30°C, closely corresponding to mean water temperatures reported during the summer months in the Gulf of Mexico when densities and disease incidence are greatest (35). Other parameters that correlate with the disease prevalence and environmental distribution may coincidentally prime this organism for enhanced virulence in humans and increased survival in oysters by increasing CPS expression. Thus, understanding the regulation of CPS expression in V. vulnificus may help define the virulence potential and elucidate strategies for the management of environmental reservoirs.

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6.

7.

8. 9.

10.

11.

12. 13. 14. 15.

16. 17. 18. 19. 20. 21.

ACKNOWLEDGMENTS Antigen preparations of V. vulnificus CPS conjugated to tetanus toxoid were kindly provided by S. Devi, technical assistance was provided by Kathy A. Strauss, and statistical analyses were performed by Anne Sill. Funding was provided in part by a Merit Review grant from the Department of Veterans Affairs and by a grant from the SaltonstallKennedy Grant Program, National Oceanic and Atmospheric Administration.

22. 23. 24. 25.

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