Vol. 174, No. 8
JOURNAL OF BACTERIOLOGY, Apr. 1992, p. 2620-2630 0021-9193/92/082620-11$02.00/0 Copyright © 1992, American Society for Microbiology
Purification and Determination of the Structure of Capsular Polysaccharide of Vibrio vulnificus M06-24 C.
P.
UNAIZA HAYAT,2 C. ABEYGUNAWARDANA,1 CHARLES FOX,' ANITA C. DAVID R. MANEVAL, JR.,2 C. ALLEN BUSH,1 AND J. GLENN MORRIS, JR.2*
REDDY,'
WRIGHT,2
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21228 and Center of Marine Biotechnology, Baltimore, Maryland 21202'; and Division of Geographic Medicine, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, and Department of Veterans Affairs Medical Center, Baltimore, Maryland 21218 Received 28 October 1991/Accepted 3 February 1992
Virulence of Vtbrio vulnificus has been strongly associated with encapsulation and an opaque colony morphology. Capsular polysaccharide was purified from a whole-cell, phosphate-buffered saline-extracted preparation of the opaque, virulent phase of V. vulnificus M06-24 (M06-24/0) by dialysis, centrifugation, enzymatic digestion, and phenol-chloroform extraction. Nuclear magnetic resonance spectroscopic analysis of the purified polysaccharide showed that the polymer was composed of a repeating structure with four sugar residues per repeating subunit: three residues of 2-acetamido-2,6-dideoxyhexopyranose in the a-gluco configuration (QuiNAc) and an additional residue of 2-acetamido hexouronate in the a-galactopyranose configuration (GalNAcA). The complete carbohydrate structure of the polysaccharide was determined by heteronuclear nuclear magnetic resonance spectroscopy and by high-performance anion-exchange chromatography. The 'H and "3C nuclear magnetic resonance spectra were completely assigned, and vicinal coupling relationships were used to establish the stereochemistry of each sugar residue, its anomeric configuration, and the positions of the glycosidic linkages. The complete structure is: [3)
QuipNAc at-(1->3)-GalPNAcA a-(1->3)-QuiPNAc QuipNAc o-(14)-A
c-(lJ
The polysaccharide was produced by a translucent phase variant of M06-24 (M06-24/T) but not by a translucent, acapsular transposon mutant (CVD752). Antibodies to the polysaccharide were demonstrable in serum from rabbits inoculated with M06-24/0.
Vibio vulnificus is a halophilic bacterium that can
implicated as possible virulence factors, including cytolysin (15), elastolytic protease (24), phospholipases (41), and collagenase (37); however, to date only the capsule and capsule-related factors have been clearly linked with pathogenicity (36, 43, 44, 45, 47). We previously reported the construction of translucent transposon mutants of V. vulnificus by using TnphoA; these mutants appear to be acapsular and do not revert to the opaque morphology (44). In the present paper we describe the purification and complete structure determination of the capsular polysaccharide (CPS) from the opaque, virulent strain V. vulnificus M06-24, and we report preliminary data on the analysis of polysaccharide preparations from a translucent phase variant and an acapsular transposon mutant of
cause
wound infections or a syndrome of primary septicemia that is frequently fatal (mortality, >50%) in persons with underlying liver disease or hemochromatosis or who are immunocompromised (7, 23, 29). The organism is common in the estuarine environment (20, 31). Wound infections are associated with seawater exposure, whereas primary septicemia apparently results from ingestion of V. vulnificus in raw oysters or other shellfish (29). In 1984, Kreger et al. (25) first described a major protective antigen of V. vulnificus that appeared, by electron microscopy, to be a ruthenium red-staining acidic polysaccharide layer or capsule located on the bacterial surface. The degree of encapsulation (estimated by electron microscopy) was subsequently correlated with colony morphology: encapsulated cells produced opaque colonies, whereas bacteria with a reduced ruthenium red-staining layer produced translucent colonies (36, 47). Opaque, encapsulated strains were found to shift to a translucent morphology at a rate of ca. 10-4 (36, 44, 47); shifts from translucent to opaque morphologies have been demonstrated at comparable frequencies (44). The opaque morphology has been correlated with increased virulence in mice, resistance to serum bactericidal activity, decreased hydrophobicity, and ability to utilize iron bound to saturated transferrin (36, 44). V. vulnificus produces a number of extracellular products that have been severe
*
this strain. (This work was presented in part at the Annual Meeting of the American Society for Microbiology, Dallas, Tex., on 6 through 9 May 1991.)
MATERUILS AND METHODS Materials. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, sodium borohydride, and sodium acetate were purchased from Sigma Chemical Co. (St. Louis, Mo.). NaOH (50%, wt/wt) was purchased from Fisher Scientific Co. (Rockville, Md.). Sodium periodate and ethylene glycol were purchased from Aldrich Chemical Co. (Milwaukee, Wis.). The sample of Pseudomonas aeruginosa IID 1009
Corresponding author. 2620
VOL. 174, 1992
VIBRIO VULNIFICUS M06-24 CAPSULAR POLYSACCHARIDE
(ATCC 27585) was a generous gift from Shunji Kaya (Kokkaido University, Kokkaido, Japan). Bacterial strains. Opaque and translucent variants of clinical isolate V. vulnificus M06-24 were previously described (44). The opaque variant was designated M06-24/O, and the translucent phase variant was designated M06-24/T. A translucent, acapsular TnphoA mutant of M06-24/O was designated CVD752. CVD752 appears to be acapsular by electron microscopy, is not lethal to iron-loaded mice, is sensitive to serum bactericidal activity, and has increased hydrophobicity (44); the exact function of the mutated gene in this strain remains to be determined. Frozen stocks of all bacterial strains were maintained at -70°C in L broth containing 50% glycerol. CPS extraction and initial characterization. Isolates retrieved from frozen stocks were grown on L-agar plates overnight at 30°C. Bacteria from single colonies were suspended in L broth and incubated overnight at 30°C. One milliliter of broth culture was then spread on each liter of L agar in 28- by 48-cm pans and incubated overnight at 30°C. Cells from two pans were harvested and suspended in 80 ml of 50% Dulbecco phosphate-buffered saline. Bacteria were shaken at 200 rpm on a rotary shaker in 250-ml baffled polystyrene bottles for 30 min at room temperature. Cells and debris were removed by centrifugation (16,000 x g, 20 min, 4°C), and supernatants were dialyzed with multiple changes of distilled water and concentrated ca. twofold by ultrafiltration (10,000-nominal-molecular-weight stirred cell; Amicon, Beverly, Mass.). The retentates were then ultracentrifuged (154,000 x g, 16 h, 20°C), and the supernatants were removed and subjected to enzymatic digestion with RNase A (100 ,ug/ml), DNase I (50 ,ug/ml plus 1 mM MgCl2), and pronase (250 ,ug/ml) followed by sequential phenolchloroform extraction. The aqueous layer was dialyzed as described above, and the resultant sample was lyophilized. Purity was assessed by lack of detectable protein on silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (14), with a bicinochoninic acid protein assay (MicroBCA; Pierce Chemical Co., Rockford, Ill.) (38), and by wavelength scanning spectrophotometric analysis (Gilford spectrophotometer, Gilford Systems, Oberlin, Ohio). Lipopolysaccharide (LPS) concentrations were determined by a standard Limulus amoebocyte lysate assay (Sigma, St. Louis, Mo.) on serial 10-fold dilutions of CPS. The molecular size of the CPS was estimated by calculation of the Kd (partition coefficient) after gel filtration through Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, N.J.) (11, 32). A 20-mg sample of V. vulnificus M06-24/O polysaccharide was passed through a Dowex 50W-X8 (20- by 0.5-cm) column eluted with water, and the fractions were monitored by on-line UV A205. The UV-absorbing fractions were pooled and freeze-dried. The dried sample was again passed through a Bio-Gel P6 column (100 by 2 cm) with water, and the fractions eluted at void volume because of the polymer were collected and lyophilized (approximately 17 mg). This sample was used for all of the nuclear magnetic resonance (NMR) and high-performance liquid chromatography experiments.
Carbohydrate analysis. Before analysis, lyophilized polysaccharide samples (200 ,ug) were hydrolyzed in 4 N HCl (200 ,ul) for 2 h at 100°C. Acid was removed by evaporation with nitrogen gas, and the residue was dissolved in 200 ,ul of distilled water. The system used for high-performance anionexchange (HPAE) chromatography consisted of a Dionex BioLC gradient pump (Dionex Corp., Sunnyvale, Calif.)
2621
with a pulsed amperometric detector (PAD). A Carbopac PAl (4- by 250-mm) pellicular anion-exchange column (Dionex Corp.) equipped with a Carbopac guard column was used with a flow rate of 1 ml/min at room temperature. The Dionex eluant degas module was employed to sparge and pressurize the eluants with helium. In these experiments, eluant 1 was 15 mM NaOH and eluant 2 was 100 mM NaOH plus 150 mM sodium acetate, prepared by suitable dilution of a 50% NaOH solution with high-purity water. Sample injection (20 ,ul) was via a Dionex microinjection valve operated by a controlled helium source of 100 to 120 lb/in2. Detection was done with the PAD with a gold working electrode. The following pulse potentials and durations were used: El, 0.05 V (tl, 300 ms); E2, 0.65 V (t2, 180 msec); E3, -0.65 V (t3, 60 ms). An IBM PC computer interfaced with Dionex AI-450 software was used for data collection and handling. In these analyses, useful retention times for neutral monosaccharides and amino sugars were provided by eluant 1, and eluant 2 was effective in analysis for acidic monosaccharides. Carboxyl reduction. Carboxyl groups of uronic acids were reduced by the method of Taylor and Conrad (40). Solid 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (10 mg) was added to a 1-ml aqueous solution of polysaccharide (8 mg). The pH of the reaction mixture was maintained at 4.75 for 2 h by slowly adding 0.1 N HCl. After reduction with 2 M NaBH4 (400 ,ul, 30 min), the reaction mixture was lyophilized and codistilled three times with acidic methanol to remove excess borohydride. The crude reaction mixture was applied to a Bio-Gel P6 column (Bio-Rad, Richmond, Calif.) with water as the eluant, and the reduced polysaccharide was recovered in the void volume. Reduced polysaccharide (200 ,ug) was hydrolyzed and then analyzed for neutral monosaccharides by HPAE chromatography. NMR spectroscopy. The polysaccharide sample (15 mg) was exchanged in D20 and lyophilized for three cycles. NMR samples were prepared by dissolving dried samples in 450 ,ul of high-purity (99.96 atom% D) D20 (Merck Sharp and Dohme, St. Louis, Mo.). NMR spectra were recorded at 60°C on a General Electric GN-500 spectrometer. The observed 1H and 13C chemical shifts were measured relative to internal sodium 4,4-dimethyl-4-silapentane-1-sulfonate with acetone as the internal standard (2.225 and 31.07 ppm downfield from sodium 4,4-dimethyl-4-silapentane-1-sulfonate). Two-dimensional spectra were recorded without sample spinning. Data were acquired in the phase-sensitive mode by the method of States et al. (39). Double-quantumfiltered correlation spectroscopy (DQF-COSY) (33), homonuclear Hartman-Hann spectroscopy (HOHAHA) (4), and nuclear Overhauser spectroscopy (NOESY) (39) were recorded at 500 MHz with standard pulse sequences. The heteronuclear chemical shift correlation spectrum was recorded in proton detected mode with standard X-nucleus decoupling hardware in the GN-500 spectrometer with a 5-mm reverse polarization transfer probe. The pulse sequence used was that of Bax et al. (5), with WALTZ-16 (34) decoupling at the carbon frequency during the acquisition. Heteronuclear multiple-bond correlation (HMBC) spectra were recorded in the phase-sensitive mode (6). NMR data processing was carried out on VAX station 3200 with the FTNMR program of Dennis Hare (Hare Research Inc., Woodinville, Wash.). GN data were transferred via ethernet to a VAX station and converted to readable files by an in-house program (GENET). Experimental details and processing parameters are given in the figure legends. Immunogenicity of CPS. Antiserum to V vulnificus M06-
2622
J. BACTERIOL.
REDDY ET AL.
0-
(D B4
Cs
4.B35
DS AS D3_ 4.0 3.23 A42 4m
1 0
A3
00 DI
Alo
~~~~~~~0
0
0
0
48
4. 0
35:2
2.4
1.6
2
ppm FIG. 1. Normal 'H spectrum and phase-sensitive HMQC spectrum of the polysaccharide from V. vulnificus M06-24/O at 500 MHz. The data matrix was 2 x 256 x 1K complex data points. The sweep widths were + 1,201 Hz in the t2 dimension and 25,000 Hz in the t, dimension. Sine bell apodization with a 900 phase shift and gaussian line broadening of 3 Hz were used in the t, and t2 dimensions, respectively. Zero filling was used in the t, dimension to obtain a 1K x 1K real matrix.
24/0 was obtained after intravenous injection of live bacteria grown on L agar (42). Ouchterlony double immunodiffusion (13) was done on micro-Ouchterlony plates (Calbiochem, San Diego, Calif.) with undiluted antisera (5 ,ul) in center wells and doubling dilutions (starting with an initial concentration of 5 ,ug in a 5-,ul volume) of CPS in outer wells. Plates were kept humid and incubated overnight at room temperature, dried, stained with acid fuchsin (0.2% in 50% methanol with 10% acetic acid) for 1 h, and destained with the same solvents. Vibriocidal titers were determined as previously described (26). RESULTS Encapsulated strain M06-24/O. (i) Polysaccharide extraction. Approximately 8.6 mg of CPS was extracted from the ca. 14 g (wet weight) of cells (ca. 1.9 x 1012 CFU) harvested from each 1-liter pan of L agar. This material contained
undetectable levels of protein (no bands on silver-stained sodium dodecyl sulfate-polyacrylamide electrophoresis; 3)-GalPNAcA oa-(1l >3)-QuiPNAc a-(l->], QuipNAc cx-(1->4)-7 D
Unencapsulated variants: V. vulnificus M06-24/T and CVD 752. Identical extraction procedures were used to evaluate surface material from V. vulnificus M06-24/T (the translucent phase variant of M06-24/O) and CVD752 (an acapsular transposon mutant of M06-24/O). Both preparations contained undetectable levels of protein; spectrophotometric scans demonstrated no A260 or A280. LPS detected by the Limulus amoebocyte lysate assay was present at trace levels of 3)-QuiNH2; 4, GalNH2A.
1VIBRIO VOL. 174, 1992
17, 30). GalNAcA (with variable 0 acetylation at the C-3 position) is found as a linear homopolymer in the Vi CPSs of Salmonella typhi, Salmonella paratyphi C, and Citrobacter freundii (10, 16). Both QuiNAc and GalNAcA have been identified in 0-specific chains of LPS of P. aeruginosa strains (18, 19, 46). QuiNAc is also present in the 0specific chain of Vibrio cholerae 02 LPS (21). Based on the Limulus amoebocyte lysate assay, the capsular material that we obtained had trace quantities of LPS. However, V. vulnificus M06-24/0 LPS obtained by a hot phenol-water extraction method appears to have no sugars in common with the CPS (27), suggesting that the CPS is distinct from LPS. The structure and significance of material other than the CPS obtained in the polysaccharide preparations from M06-24/T and CVD752 remain to be determined. Our studies were conducted with a single V. vulnificus strain and its derivatives. Shimada and Sakazaki have identified seven 0 groups among V. vulnificus isolates (35), whereas Martin and Siebeling have characterized five 0 groups (with a possible sixth group reported) (28). These data indicate that there is diversity in the carbohydrate composition of V. vulnificus LPS; no data are currently available on the sugar composition of the 0 side chains associated with these LPS types. One report (presented in abstract form [22]) suggested that the surface carbohydrate composition of a clinical V. vulnificus strain differed from that of an environmental strain. However, no data were given on the colony morphology of the strains studied: it is possible that the observed differences reflected differences comparable to those seen between our opaque and translucent phase variants. Further studies are needed to determine whether there are differences in capsular composition among V. vulnificus strains and to evaluate possible correlations between capsular type and virulence. The immunogenicity of the V. vulnificus CPS was demonstrated in double immunodiffusion studies. M06-24/0 antiserum to live cells reacted strongly with homologous CPS. The antiserum also reacted with "capsular" preparations from M06-24/T, although bands of precipitation were only seen with relatively high concentrations of the surface extracted material. These findings are compatible with HPAE chromatographic data suggesting that M06-24/T expresses the CPS but that the relative proportion to total material extracted was lower than that with M06-24/0. Antiserum to M06-24/0 gave no reaction to material extracted from CVD752, confirming HPAE chromatographic analysis indicating that this acapsular mutant does not contain the CPS found in the encapsulated parent strain. Although normal (nonimmune) serum does not kill encapsulated V. vulnificus strains (36, 44), antiserum to M06-24/0 (with demonstrable anti-capsular antibodies) had high vibriocidal titers against the encapsulated form of the organism. This is compatible with the hypothesis (25) that anticapsular antibodies play an important role in complement-mediated bacterial killing and in protection against disease. Prior studies from our laboratory and from other investigators have shown that the capsule is a critical virulence factor for V. vulnificus (36, 45, 47). Our data on capsule composition and structure provide a necessary basis for analysis of CPS genetics and expression; these data, in turn, should help to improve our understanding of the pathogenesis of this highly virulent organism.
VULNIFICUS M06-24 CAPSULAR POLYSACCHARIDE
2629
ACKNOWLEDGMENTS We thank Shunji Kaya and Tord Holme for their generous donation of purified polysaccharide standards. Sarvamangala Devi provided data on the partition coefficient of the polysaccharide. Support for these studies was provided in part by a Designated Research Initiative Fund grant from the University of Maryland at Baltimore (to J.G.M.), by a grant from the Department of Veterans Affairs (to J.G.M.), and by Public Health Service grant DE-09445 from the National Institutes of Health (to C.A.B.). REFERENCES 1. Abeygunawardana, C., C. A. Bush, and J. 0. Cisar. 1990. Complete structure of the polysaccharide from Streptococcus sanguis J22. Biochemistry 29:234-248. 2. Abeygunawardana, C., C. A. Bush, and J. 0. Cisar. 1991. Complete structure of the cell surface polysaccharide of Streptococcus oralis ATCC 10557: a receptor for lectin-mediated interbacterial adherence. Biochemistry 30:6528-6540.
3. Abeygunawardana, C., C. A. Bush, and J. 0. Cisar. 1991. Complete structure of the cell surface polysaccharide of Streptococcus oralis C104: a 600-MHz NMR study. Biochemistry 30:8568-8577. 4. Bax, A., and D. G. Davis. 1985. MLEV-16 based two-dimensional homonuclear magnetization transfer spectroscopy. J.
Magn. Reson. 65:355-360. 5. Bax, A., R. H. Griffey, and B. L. Howkins. 1983. Correlation of proton and nitrogen-15 chemical shifts by multiple quantum
NMR. J. Magn. Reson. 55:301-315. 6. Bax, A., and M. F. Summers. 1986. 'H and 13C assignments from sensitivity-enhanced detection of heteronuclear multiple-bond connectivity by 2D multiple quantum NMR. J. Am. Chem. Soc. 108:2093-2094. 7. Blake, P. A., M. M. Merson, R. E. Weaver, D. G. Hollis, and P. C. Heublein. 1979. Disease caused by a marine vibrio: clinical characteristics and epidemiology. N. Engl. J. Med. 300:1-5. 8. Boulnois, G. J., and I. S. Roberts. 1990. Genetics of capsular polysaccharide production in bacteria. Curr. Top. Microbiol. Immunol. 150:1-18. 9. Bush, C. A. 1988. High resolution NMR in the determination of structure in complex carbohydrates. Bull. Magn. Reson. 78: 186-191. 10. Daniels, E. M., R. Schneerson, W. M. Eagen, S. C. Szu, and J. B. Robbins. 1989. Characterization of the Salmonella paratyphi C Vi polysaccharide. Infect. Immun. 57:3159-3164. 11. Devi, S. J. N., R. Schneerson, W. Egan, W. F. Vann, J. B. Robbins, and J. Shiloach. 1991. Identity between polysaccharide
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20. Kaysner, C. A., C. Abeyta, Jr., M. M. Wakell, A. DePaola, Jr., R. F. Scott, and J. M. Leitch. 1987. Virulent strains of Vibrio vulnificus isolated from estuaries of the United States west coast. Appl. Environ. Microbiol. 53:1349-1351. 21. Kenne, L., B. Lindberg, and E. Schweda. 1988. Structural studies of the 0-antigen from Vibrio cholerae 02. Carbohydr. Res. 180:285-294. 22. Kilgore, S. G., and B. J. Plotkin. 1987. Extracellular polysaccharide of Vibrio vulnificus. Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, abstr. no. D-171, p. 100. 23. Klontz, K. C., S. Lieb, M. Schreiber, H. T. Janowski, L. M.
Baldy, and R. A. Gunn. 1988. Syndromes of Vibrio vulnificus
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infections: clinical and epidemiologic features in Florida cases, 1981-1987. Ann. Intern. Med. 109:318-323. Kothary, M. M., and A. S. Kreger. 1987. Purification and characterization of an elastolytic protease of Vibrio vulnificus. J. Gen. Microbiol. 133:1783-1791. Kreger, A. S., L. D. Gray, and J. Testa. 1984. Protection of mice against Vibno vulnificus disease by vaccination with surface antigen preparations and anti-surface antigen antisera. Infect. Immun. 45:537-543. Lefkowitz, A., G. S. Fout, G. Losonsky, S. S. Wasserman, E. Israel, and J. G. Morris, Jr. 1991. A serosurvey of pathogens associated with shellfish: prevalence of antibodies to Vibrio species and Norwalk agent in the Chesapeake bay region. Am. J. Epidemiol., in press. Maneval, D., and G. P. Reddy. Unpublished data. Martin, S. J., and R. J. Siebeling. 1991. Identification of Vibrio vulnificus 0 serovars with antilipopolysaccharide monoclonal antibody. J. Clin. Microbiol. 29:1684-1688. Morris, J. G., Jr., and R. E. Black. 1985. Cholera and other vibrioses in the United States. N. Engl. J. Med. 312:343-350. Moxon, E. R., and J. S. Kroll. 1990. The role of bacterial polysaccharide capsules as virulence factors. Curr. Top. Microbiol. Immunol. 150:65-85. Oliver, J. D., R. A. Warner, and D. R. Cleland. 1983. Distribution of Vibrio vulnificus and other lactose-fermenting vibrios in the marine environment. Appl. Environ. Microbiol. 45:985-998. Orskov, F., I. Orskov, A. Sutton, R. Schneerson, W. Lin, W. Egan, G. E. Hoff, and J. B. Robbins. 1979. Form variation in Escherichia coli Kl: determined by 0-acetylation of the capsular polysaccharide. J. Exp. Med. 149:669-685. Rance, M., 0. W. Sorensen, G. Bodenhausen, G. Wagner, E. R. Ernst, and K. Wuthrich. 1983. Improved spectral resolution in COSY 'H NMR spectra of proteins via double quantum filtering. Biochem. Biophys. Res. Commun. 117:479-485.
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34. Shaka, A. J., J. Keeler, T. Frenkiel, and R. Freeman. 1983. An improved sequence for broadband decoupling: WALTZ-16. J. Magn. Reson. 52:335-338. 35. Shimada, T., and R. Sakazaki. 1984. On the serology of Vibrio vulnificus. Jpn. J. Med. Sci. Biol. 37:241-246. 36. Simpson, L. M., V. K. White, S. F. Zane, and J. D. Oliver. 1987. Correlation between virulence and colony morphology in Vibno
vulnificus. Infect. Immun. 55:269-272. 37. Smith, G. G., and J. R. Merkel. 1982. Collagenolytic activity of
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39. 40.
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Infect. Immun. 35:1155-1156. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk. 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76-85. States, D. J., R. A. Hakerborn, and D. J. Ruben. 1982. A two-dimensional nuclear overhauser experiment with pure absorption phase in four quadrants. J. Magn. Reson. 48:286-292. Taylor, R. L., and H. E. Conrad. 1972. Stoichiometric depolymerization of polysaccharides and glycosaminoslyuronans to monosaccharides following reduction of their carbodiimideactivated carboxyl groups. Biochemistry 11:1383-1388. Testa, J., L. W. Daniel, and A. S. Kreger. 1984. Extracellular phospholipase A2 and lysophospholipase produced by Vibrio
vulnificus. Infect. Immun. 45:458-463.
42. Vial, P. A., R. Robins-Browne, H. Lior, V. Prado, J. B. Kaper, D. Maneval, A.-e.-d. Elsayed, and M. M. Levine. 1988. Characterization of enteroadherent-aggregative E. coli, a putative agent of diarrheal disease. J. Infect. Dis. 158:70-79. 43. Wright, A. C., and J. G. Morris, Jr. 1991. The extracellular cytolysin of Vibrio vulnificus: inactivation and relationship to virulence in mice. Infect. Immun. 59:192-197. 44. Wright, A. C., L. M. Simpson, J. D. Oliver, and J. G. Morris, Jr. 1990. Phenotypic evaluation of acapsular transposon mutants of
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45. Wright, A. C., L. M. Simpson, R. G. Russell, and J. G. Morris, Jr. 1991. Vibrio vulnificus protease. Abstr. Gen. Meet. Am. Soc. Microbiol. 1991, abstr. no. B-99, p. 42. 46. Yokota, S., S. Kaya, S. Sawada, T. Kawamura, Y. Araki, and E. Ito. 1987. Characterization of a polysaccharide component of lipopolysaccharide from Pseudomonas aeruginosa IID 1008 (ATCC 27584) as D-rhamnan. Eur. J. Biochem. 167:203-209. 47. Yoshida, S. I., M. Ogawa, and Y. Mizuguchi. 1985. Relation of capsular materials and colony opacity to Vibrio vulnificus. Infect. Immun. 47:446-451.
JOURNAL OF BACTERIOLOGY, Apr. 1992, p. 2620-2630 0021-9193/92/082620-11$02.00/0 Copyright © 1992, American Society for Microbiology
Purification and Determination of the Structure of Capsular Polysaccharide of Vibrio vulnificus M06-24 C.
P.
UNAIZA HAYAT,2 C. ABEYGUNAWARDANA,1 CHARLES FOX,' ANITA C. DAVID R. MANEVAL, JR.,2 C. ALLEN BUSH,1 AND J. GLENN MORRIS, JR.2*
REDDY,'
WRIGHT,2
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21228 and Center of Marine Biotechnology, Baltimore, Maryland 21202'; and Division of Geographic Medicine, Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, and Department of Veterans Affairs Medical Center, Baltimore, Maryland 21218 Received 28 October 1991/Accepted 3 February 1992
Virulence of Vtbrio vulnificus has been strongly associated with encapsulation and an opaque colony morphology. Capsular polysaccharide was purified from a whole-cell, phosphate-buffered saline-extracted preparation of the opaque, virulent phase of V. vulnificus M06-24 (M06-24/0) by dialysis, centrifugation, enzymatic digestion, and phenol-chloroform extraction. Nuclear magnetic resonance spectroscopic analysis of the purified polysaccharide showed that the polymer was composed of a repeating structure with four sugar residues per repeating subunit: three residues of 2-acetamido-2,6-dideoxyhexopyranose in the a-gluco configuration (QuiNAc) and an additional residue of 2-acetamido hexouronate in the a-galactopyranose configuration (GalNAcA). The complete carbohydrate structure of the polysaccharide was determined by heteronuclear nuclear magnetic resonance spectroscopy and by high-performance anion-exchange chromatography. The 'H and "3C nuclear magnetic resonance spectra were completely assigned, and vicinal coupling relationships were used to establish the stereochemistry of each sugar residue, its anomeric configuration, and the positions of the glycosidic linkages. The complete structure is: [3)
QuipNAc at-(1->3)-GalPNAcA a-(1->3)-QuiPNAc QuipNAc o-(14)-A
c-(lJ
The polysaccharide was produced by a translucent phase variant of M06-24 (M06-24/T) but not by a translucent, acapsular transposon mutant (CVD752). Antibodies to the polysaccharide were demonstrable in serum from rabbits inoculated with M06-24/0.
Vibio vulnificus is a halophilic bacterium that can
implicated as possible virulence factors, including cytolysin (15), elastolytic protease (24), phospholipases (41), and collagenase (37); however, to date only the capsule and capsule-related factors have been clearly linked with pathogenicity (36, 43, 44, 45, 47). We previously reported the construction of translucent transposon mutants of V. vulnificus by using TnphoA; these mutants appear to be acapsular and do not revert to the opaque morphology (44). In the present paper we describe the purification and complete structure determination of the capsular polysaccharide (CPS) from the opaque, virulent strain V. vulnificus M06-24, and we report preliminary data on the analysis of polysaccharide preparations from a translucent phase variant and an acapsular transposon mutant of
cause
wound infections or a syndrome of primary septicemia that is frequently fatal (mortality, >50%) in persons with underlying liver disease or hemochromatosis or who are immunocompromised (7, 23, 29). The organism is common in the estuarine environment (20, 31). Wound infections are associated with seawater exposure, whereas primary septicemia apparently results from ingestion of V. vulnificus in raw oysters or other shellfish (29). In 1984, Kreger et al. (25) first described a major protective antigen of V. vulnificus that appeared, by electron microscopy, to be a ruthenium red-staining acidic polysaccharide layer or capsule located on the bacterial surface. The degree of encapsulation (estimated by electron microscopy) was subsequently correlated with colony morphology: encapsulated cells produced opaque colonies, whereas bacteria with a reduced ruthenium red-staining layer produced translucent colonies (36, 47). Opaque, encapsulated strains were found to shift to a translucent morphology at a rate of ca. 10-4 (36, 44, 47); shifts from translucent to opaque morphologies have been demonstrated at comparable frequencies (44). The opaque morphology has been correlated with increased virulence in mice, resistance to serum bactericidal activity, decreased hydrophobicity, and ability to utilize iron bound to saturated transferrin (36, 44). V. vulnificus produces a number of extracellular products that have been severe
*
this strain. (This work was presented in part at the Annual Meeting of the American Society for Microbiology, Dallas, Tex., on 6 through 9 May 1991.)
MATERUILS AND METHODS Materials. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, sodium borohydride, and sodium acetate were purchased from Sigma Chemical Co. (St. Louis, Mo.). NaOH (50%, wt/wt) was purchased from Fisher Scientific Co. (Rockville, Md.). Sodium periodate and ethylene glycol were purchased from Aldrich Chemical Co. (Milwaukee, Wis.). The sample of Pseudomonas aeruginosa IID 1009
Corresponding author. 2620
VOL. 174, 1992
VIBRIO VULNIFICUS M06-24 CAPSULAR POLYSACCHARIDE
(ATCC 27585) was a generous gift from Shunji Kaya (Kokkaido University, Kokkaido, Japan). Bacterial strains. Opaque and translucent variants of clinical isolate V. vulnificus M06-24 were previously described (44). The opaque variant was designated M06-24/O, and the translucent phase variant was designated M06-24/T. A translucent, acapsular TnphoA mutant of M06-24/O was designated CVD752. CVD752 appears to be acapsular by electron microscopy, is not lethal to iron-loaded mice, is sensitive to serum bactericidal activity, and has increased hydrophobicity (44); the exact function of the mutated gene in this strain remains to be determined. Frozen stocks of all bacterial strains were maintained at -70°C in L broth containing 50% glycerol. CPS extraction and initial characterization. Isolates retrieved from frozen stocks were grown on L-agar plates overnight at 30°C. Bacteria from single colonies were suspended in L broth and incubated overnight at 30°C. One milliliter of broth culture was then spread on each liter of L agar in 28- by 48-cm pans and incubated overnight at 30°C. Cells from two pans were harvested and suspended in 80 ml of 50% Dulbecco phosphate-buffered saline. Bacteria were shaken at 200 rpm on a rotary shaker in 250-ml baffled polystyrene bottles for 30 min at room temperature. Cells and debris were removed by centrifugation (16,000 x g, 20 min, 4°C), and supernatants were dialyzed with multiple changes of distilled water and concentrated ca. twofold by ultrafiltration (10,000-nominal-molecular-weight stirred cell; Amicon, Beverly, Mass.). The retentates were then ultracentrifuged (154,000 x g, 16 h, 20°C), and the supernatants were removed and subjected to enzymatic digestion with RNase A (100 ,ug/ml), DNase I (50 ,ug/ml plus 1 mM MgCl2), and pronase (250 ,ug/ml) followed by sequential phenolchloroform extraction. The aqueous layer was dialyzed as described above, and the resultant sample was lyophilized. Purity was assessed by lack of detectable protein on silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (14), with a bicinochoninic acid protein assay (MicroBCA; Pierce Chemical Co., Rockford, Ill.) (38), and by wavelength scanning spectrophotometric analysis (Gilford spectrophotometer, Gilford Systems, Oberlin, Ohio). Lipopolysaccharide (LPS) concentrations were determined by a standard Limulus amoebocyte lysate assay (Sigma, St. Louis, Mo.) on serial 10-fold dilutions of CPS. The molecular size of the CPS was estimated by calculation of the Kd (partition coefficient) after gel filtration through Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, N.J.) (11, 32). A 20-mg sample of V. vulnificus M06-24/O polysaccharide was passed through a Dowex 50W-X8 (20- by 0.5-cm) column eluted with water, and the fractions were monitored by on-line UV A205. The UV-absorbing fractions were pooled and freeze-dried. The dried sample was again passed through a Bio-Gel P6 column (100 by 2 cm) with water, and the fractions eluted at void volume because of the polymer were collected and lyophilized (approximately 17 mg). This sample was used for all of the nuclear magnetic resonance (NMR) and high-performance liquid chromatography experiments.
Carbohydrate analysis. Before analysis, lyophilized polysaccharide samples (200 ,ug) were hydrolyzed in 4 N HCl (200 ,ul) for 2 h at 100°C. Acid was removed by evaporation with nitrogen gas, and the residue was dissolved in 200 ,ul of distilled water. The system used for high-performance anionexchange (HPAE) chromatography consisted of a Dionex BioLC gradient pump (Dionex Corp., Sunnyvale, Calif.)
2621
with a pulsed amperometric detector (PAD). A Carbopac PAl (4- by 250-mm) pellicular anion-exchange column (Dionex Corp.) equipped with a Carbopac guard column was used with a flow rate of 1 ml/min at room temperature. The Dionex eluant degas module was employed to sparge and pressurize the eluants with helium. In these experiments, eluant 1 was 15 mM NaOH and eluant 2 was 100 mM NaOH plus 150 mM sodium acetate, prepared by suitable dilution of a 50% NaOH solution with high-purity water. Sample injection (20 ,ul) was via a Dionex microinjection valve operated by a controlled helium source of 100 to 120 lb/in2. Detection was done with the PAD with a gold working electrode. The following pulse potentials and durations were used: El, 0.05 V (tl, 300 ms); E2, 0.65 V (t2, 180 msec); E3, -0.65 V (t3, 60 ms). An IBM PC computer interfaced with Dionex AI-450 software was used for data collection and handling. In these analyses, useful retention times for neutral monosaccharides and amino sugars were provided by eluant 1, and eluant 2 was effective in analysis for acidic monosaccharides. Carboxyl reduction. Carboxyl groups of uronic acids were reduced by the method of Taylor and Conrad (40). Solid 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (10 mg) was added to a 1-ml aqueous solution of polysaccharide (8 mg). The pH of the reaction mixture was maintained at 4.75 for 2 h by slowly adding 0.1 N HCl. After reduction with 2 M NaBH4 (400 ,ul, 30 min), the reaction mixture was lyophilized and codistilled three times with acidic methanol to remove excess borohydride. The crude reaction mixture was applied to a Bio-Gel P6 column (Bio-Rad, Richmond, Calif.) with water as the eluant, and the reduced polysaccharide was recovered in the void volume. Reduced polysaccharide (200 ,ug) was hydrolyzed and then analyzed for neutral monosaccharides by HPAE chromatography. NMR spectroscopy. The polysaccharide sample (15 mg) was exchanged in D20 and lyophilized for three cycles. NMR samples were prepared by dissolving dried samples in 450 ,ul of high-purity (99.96 atom% D) D20 (Merck Sharp and Dohme, St. Louis, Mo.). NMR spectra were recorded at 60°C on a General Electric GN-500 spectrometer. The observed 1H and 13C chemical shifts were measured relative to internal sodium 4,4-dimethyl-4-silapentane-1-sulfonate with acetone as the internal standard (2.225 and 31.07 ppm downfield from sodium 4,4-dimethyl-4-silapentane-1-sulfonate). Two-dimensional spectra were recorded without sample spinning. Data were acquired in the phase-sensitive mode by the method of States et al. (39). Double-quantumfiltered correlation spectroscopy (DQF-COSY) (33), homonuclear Hartman-Hann spectroscopy (HOHAHA) (4), and nuclear Overhauser spectroscopy (NOESY) (39) were recorded at 500 MHz with standard pulse sequences. The heteronuclear chemical shift correlation spectrum was recorded in proton detected mode with standard X-nucleus decoupling hardware in the GN-500 spectrometer with a 5-mm reverse polarization transfer probe. The pulse sequence used was that of Bax et al. (5), with WALTZ-16 (34) decoupling at the carbon frequency during the acquisition. Heteronuclear multiple-bond correlation (HMBC) spectra were recorded in the phase-sensitive mode (6). NMR data processing was carried out on VAX station 3200 with the FTNMR program of Dennis Hare (Hare Research Inc., Woodinville, Wash.). GN data were transferred via ethernet to a VAX station and converted to readable files by an in-house program (GENET). Experimental details and processing parameters are given in the figure legends. Immunogenicity of CPS. Antiserum to V vulnificus M06-
2622
J. BACTERIOL.
REDDY ET AL.
0-
(D B4
Cs
4.B35
DS AS D3_ 4.0 3.23 A42 4m
1 0
A3
00 DI
Alo
~~~~~~~0
0
0
0
48
4. 0
35:2
2.4
1.6
2
ppm FIG. 1. Normal 'H spectrum and phase-sensitive HMQC spectrum of the polysaccharide from V. vulnificus M06-24/O at 500 MHz. The data matrix was 2 x 256 x 1K complex data points. The sweep widths were + 1,201 Hz in the t2 dimension and 25,000 Hz in the t, dimension. Sine bell apodization with a 900 phase shift and gaussian line broadening of 3 Hz were used in the t, and t2 dimensions, respectively. Zero filling was used in the t, dimension to obtain a 1K x 1K real matrix.
24/0 was obtained after intravenous injection of live bacteria grown on L agar (42). Ouchterlony double immunodiffusion (13) was done on micro-Ouchterlony plates (Calbiochem, San Diego, Calif.) with undiluted antisera (5 ,ul) in center wells and doubling dilutions (starting with an initial concentration of 5 ,ug in a 5-,ul volume) of CPS in outer wells. Plates were kept humid and incubated overnight at room temperature, dried, stained with acid fuchsin (0.2% in 50% methanol with 10% acetic acid) for 1 h, and destained with the same solvents. Vibriocidal titers were determined as previously described (26). RESULTS Encapsulated strain M06-24/O. (i) Polysaccharide extraction. Approximately 8.6 mg of CPS was extracted from the ca. 14 g (wet weight) of cells (ca. 1.9 x 1012 CFU) harvested from each 1-liter pan of L agar. This material contained
undetectable levels of protein (no bands on silver-stained sodium dodecyl sulfate-polyacrylamide electrophoresis; 3)-GalPNAcA oa-(1l >3)-QuiPNAc a-(l->], QuipNAc cx-(1->4)-7 D
Unencapsulated variants: V. vulnificus M06-24/T and CVD 752. Identical extraction procedures were used to evaluate surface material from V. vulnificus M06-24/T (the translucent phase variant of M06-24/O) and CVD752 (an acapsular transposon mutant of M06-24/O). Both preparations contained undetectable levels of protein; spectrophotometric scans demonstrated no A260 or A280. LPS detected by the Limulus amoebocyte lysate assay was present at trace levels of 3)-QuiNH2; 4, GalNH2A.
1VIBRIO VOL. 174, 1992
17, 30). GalNAcA (with variable 0 acetylation at the C-3 position) is found as a linear homopolymer in the Vi CPSs of Salmonella typhi, Salmonella paratyphi C, and Citrobacter freundii (10, 16). Both QuiNAc and GalNAcA have been identified in 0-specific chains of LPS of P. aeruginosa strains (18, 19, 46). QuiNAc is also present in the 0specific chain of Vibrio cholerae 02 LPS (21). Based on the Limulus amoebocyte lysate assay, the capsular material that we obtained had trace quantities of LPS. However, V. vulnificus M06-24/0 LPS obtained by a hot phenol-water extraction method appears to have no sugars in common with the CPS (27), suggesting that the CPS is distinct from LPS. The structure and significance of material other than the CPS obtained in the polysaccharide preparations from M06-24/T and CVD752 remain to be determined. Our studies were conducted with a single V. vulnificus strain and its derivatives. Shimada and Sakazaki have identified seven 0 groups among V. vulnificus isolates (35), whereas Martin and Siebeling have characterized five 0 groups (with a possible sixth group reported) (28). These data indicate that there is diversity in the carbohydrate composition of V. vulnificus LPS; no data are currently available on the sugar composition of the 0 side chains associated with these LPS types. One report (presented in abstract form [22]) suggested that the surface carbohydrate composition of a clinical V. vulnificus strain differed from that of an environmental strain. However, no data were given on the colony morphology of the strains studied: it is possible that the observed differences reflected differences comparable to those seen between our opaque and translucent phase variants. Further studies are needed to determine whether there are differences in capsular composition among V. vulnificus strains and to evaluate possible correlations between capsular type and virulence. The immunogenicity of the V. vulnificus CPS was demonstrated in double immunodiffusion studies. M06-24/0 antiserum to live cells reacted strongly with homologous CPS. The antiserum also reacted with "capsular" preparations from M06-24/T, although bands of precipitation were only seen with relatively high concentrations of the surface extracted material. These findings are compatible with HPAE chromatographic data suggesting that M06-24/T expresses the CPS but that the relative proportion to total material extracted was lower than that with M06-24/0. Antiserum to M06-24/0 gave no reaction to material extracted from CVD752, confirming HPAE chromatographic analysis indicating that this acapsular mutant does not contain the CPS found in the encapsulated parent strain. Although normal (nonimmune) serum does not kill encapsulated V. vulnificus strains (36, 44), antiserum to M06-24/0 (with demonstrable anti-capsular antibodies) had high vibriocidal titers against the encapsulated form of the organism. This is compatible with the hypothesis (25) that anticapsular antibodies play an important role in complement-mediated bacterial killing and in protection against disease. Prior studies from our laboratory and from other investigators have shown that the capsule is a critical virulence factor for V. vulnificus (36, 45, 47). Our data on capsule composition and structure provide a necessary basis for analysis of CPS genetics and expression; these data, in turn, should help to improve our understanding of the pathogenesis of this highly virulent organism.
VULNIFICUS M06-24 CAPSULAR POLYSACCHARIDE
2629
ACKNOWLEDGMENTS We thank Shunji Kaya and Tord Holme for their generous donation of purified polysaccharide standards. Sarvamangala Devi provided data on the partition coefficient of the polysaccharide. Support for these studies was provided in part by a Designated Research Initiative Fund grant from the University of Maryland at Baltimore (to J.G.M.), by a grant from the Department of Veterans Affairs (to J.G.M.), and by Public Health Service grant DE-09445 from the National Institutes of Health (to C.A.B.). REFERENCES 1. Abeygunawardana, C., C. A. Bush, and J. 0. Cisar. 1990. Complete structure of the polysaccharide from Streptococcus sanguis J22. Biochemistry 29:234-248. 2. Abeygunawardana, C., C. A. Bush, and J. 0. Cisar. 1991. Complete structure of the cell surface polysaccharide of Streptococcus oralis ATCC 10557: a receptor for lectin-mediated interbacterial adherence. Biochemistry 30:6528-6540.
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