Pseudomonas aeruginosa isolates, and they separated a rhamnose-rich polysaccharide as the probable antigen for MAb E87 from P. aeruginosa IFO 3080 (S.
Vol. 172, No. 10
JOURNAL OF BACTERIOLOGY, OCt. 1990, p. 6162-6164
0021-9193/90/106162-03$02.00/0 Copyright © 1990, American Society for Microbiology
Occurrence of D-Rhamnan as the Common Antigen Reactive against Monoclonal Antibody E87 in Pseudomonas aeruginosa IFO 3080 and Other Strains SHIN-ICHI YOKOTA,lt SHUNJI KAYA,1* YOSHIO ARAKI,1 EIJI ITO,1 TAKASHI KAWAMURA,2 AND SHUZO SAWADA2
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060,1 and Teijin Institute for Biomedical Research, Hino Tokyo 191,2 Japan Received 12 March 1990/Accepted 26 July 1990
S. Sawada and co-workers reported that a monoclonal antibody (MAb), E87, interacted with about 80% of Pseudomonas aeruginosa isolates, and they separated a rhamnose-rich polysaccharide as the probable antigen for MAb E87 from P. aeruginosa IFO 3080 (S. Sawada, T. Kawamura, Y. Masuho, and K. Tomibe, J. Infec. Dis. 152:12901299, 1985). In the present study, the rhamnose-rich polysaccharide was shown to be structurally and immunologically identical to the D-rhamnan of P. aeruginosa HD 1008 (S. Yokota, S. Kaya, S. Sawada, T. Kawamura, Y. Araki, and E. Ito, Eur. J. Biochem. 167:203-209, 1987). Furthermore, a set of enzymes responsible for the formation of GDP-rhamnose (probably in a D-form) from GDP-D-mannose was found in the 100,000 x g supernatant fractions obtained from all of nine P. aeruginosa strains reactive against MAb E87. The result strongly supports a possibility that lipopolysaccharides having a D-rhamnan chain widely occur as the common antigen among various P. aeruginosa isolates.
2:1:1, and the D-ribose residue was presumed to be a possible antigenic epitope for MAb E87. At the present time, why the MAb E87 preparation reacts with their L-rhamnoserich polysaccharide as well as with our D-rhamnan can not be well explained, because both of our D-rhamnan preparations isolated from P. aeruginosa IID 1008 (16) and IFO 3080 (in the present study) did not contain ribose in a detectable amount. The conflicting results may arise from the assay methods used, competitive enzyme-linked immunosorbent assay (ELISA) versus immunoblotting analysis. In the 1H-NMR spectroscopy, the rhamnose-rich polysaccharide of strain IFO 3080 (Fig. 1A) and the D-rhamnan of strain IID 1008 (Fig. 1B) exhibited similar signals. Three pseudo-singlet signals (4.96, 5.02, and 5.20 ppm; 1H each) were ascribable to anomeric protons of the rhamnose residues substituted at C3 and C2. As judged from the chemical shifts of these signals in the down-field region of 4.9 ppm, all the rhamnose residues were presumed to be a-linked and in a "C1 conformation (1, 3, 13). Taking into account the similarity between the 'H-NMR spectra of the rhamnoserich polysaccharide of strain IFO 3080 and the D-rhamnan of strain IID 1008, the main polymer chain of the rhamnose-rich polysaccharide seemed to consist of repeating 43)Rha(al-*3)Rha(a1-*2)Rha(al1- units, just like that of D-rhamnan of strain IID 1008 (16). But the integral values for signals at 5.13 ppm (probably ascribable to the anomeric proton of 3-0methyl-6-deoxyhexose) and 3.45 ppm (the methyl proton of methylether group in the same sugar residue) considerably differed in the 'H-NMR spectra; both values for the rhamnose-rich polysaccharide of strain IFO 3080 were smaller than those for the D-rhamnan of strain IID 1008. Thus, the contents of 3-O-methyl-6-deoxyhexose in both preparations differed from each other. The value of the optical rotation ([a]D) observed with the rhamnose-rich polysaccharide of strain IFO 3080, +84°, was in agreement with the value reported with the D-rhamnan of strain IID 1008, +750 (16), as well as the calculated value for a-D-rhamnan, +820 (5, 13). The result suggested that the
Recently, two types of monoclonal antibodies (MAbs), E87 (12) and MH-4H7 (17), which can react with about 80 and 60% of Pseudomonas aeruginosa isolates, respectively, have been produced, suggesting the presence of some common antigens in P. aeruginosa. Such an antigen is expected to be a useful vaccine against P. aeruginosa infections. A rhamnose-rich polysaccharide was presumed to be the antigen for MAb E87 (12), while an L-rhamnose residue (or the neighbor structure) in the outer core part of the lipopolysaccharide was presumed to be an antigenic epitope for MAb MH-4H7 (17). However, the actual occurrence of such common antigen(s) in P. aeruginosa is still unclear. Several polysaccharides, such as a rhamnose-rich polysaccharide (12), D-rhamnan (6, 16), L-rhamnose-containing neutral polysaccharide (7), and A-band lipopolysaccharide (8, 10, 11), have been reported as the possible antigens for MAb E87, but no information on the presence of any common structure unit in these polysaccharides was available. Because any structural information.on the rhamnose-rich polysaccharide obtained from strain IFO 3080 of P. aeruginosa (12) had been lacking, we compared it with the D-rhamnan obtained from strain IID 1008 (16) in 1H-nuclear magnetic resonance (NMR) spectroscopic and immunochemical characteristics. Analysis by gas-liquid chromatography showed that the rhamnose-rich polysaccharide contains mainly rhamnose, along with small amounts of 3-0-methyl6-deoxyhexose, glucose, and xylose. However, ribose, which was previously reported to be present in this polysaccharide (12), was not detected in a significant amount. On the other hand, Kocharova et al. (7) reported that an L-rhamnose-rich polysaccharide which occurred widely in P. aeruginosa isolates and reacted against MAb E87 consists of L-rhamnose, D-glucose, and D-ribose in a molar ratio of * Corresponding author. t Present address: Laboratory of Biotechnology, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Takarazuka, Hyogo 665, Japan. 6162
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A
10.8
1
w
0.4
co
0
6
5
3
4
2
0
ppm
FIG. 1. 'H-NMR spectrum of a rhamnose-rich polysaccharide. (A) The rhamnose-rich polysaccharide was isolated from defatted cells of P. aeruginosa IFO 3080 by the method of Darveau and Hancock (2), and its 'H-NMR spectrum was measured in 99.96% D20 with a Jeol FX-500 spectrometer. (B) The 'H-NMR spectrum of reference, D-rhamnan, which was isolated from P. aeruginosa IID 1008 by the hot aqueous phenol method (14). Chemical shifts were given with sodium 3-trimethylsilylpropane sulfonate as an internal standard (6 = 0.00 ppm).
major sugar, rhamnose, of the rhamnose-rich polysaccharide is in a D-configuration. Thus, in the sugar composition, 1H-NMR spectrometry, and optical rotatation, the rhamnose-rich polysaccharide of strain IFO 3080 and the D-rhamnan of strain IID 1008 are almost structually identical. To confirm the immunological similarity between the polysaccharide preparations obtained from P. aeruginosa IFO 3080 and IID 1008, competitive ELISA was carried out. As shown in Fig. 2, the rhamnose-rich polysaccharide of strain IFO 3080 and the D-rhamnan of strain IID 1008 inhibited the binding of MAb E87 to antigenic cells of P. aeruginosa PAO 1 to almost the same extent. The result suggested that both polysaccharides are specifically bound to MAb E87 and that the rhamnose-rich polysaccharide of strain IFO 3080 cannot be immunologically distinguished from the D-rhamnan of strain IID 1008. In addition, the monosaccharide, L-rhamnose, used in the assay did not inhibit binding (Fig. 2). However, we could not determine whether D-rhamnose inhibits the assay, since this sugar is not commercially available. Previously, GDP-D-rhamnose and GDP-D-talomethylose were shown to be formed from GDP-D-mannose via GDP-4keto-D-rhamnose by a nonstereoselective ketoreductase in a
4 8 6 2 Dilution of samples (2n)
FIG. 2. Competitive ELISA of rhamnose-rich polysaccharide and D-rhamnan. Competitors, rhamnose-rich polysaccharide (0) (0.1 mM in the amount of rhamnose), D-rhamnan (0) (0.1 mM in the amount of rhamnose), and L-rhamnose (i\) (10 mM), were assayed. The original competitor solutions were diluted in serial twofold dilutions. After incubation with MAb E87 (10 ,ul/ml) at 25°C for 1 h, the serial samples were applied to 96-well microplates coated with antigenic cells of P. aeruginosa PAO 1. ELISA assay was carried out by using alkaline phosphatase-conjugated goat anti-mouse immunoglobulin antibody (Tago, Inc., Burlingame, Calif.) and p-nitrophenylphosphate as the second antibody and substrate.
soil bacterium, strain GS (ATCC 19241) (9, 15). Thus, we tried to detect such enzyme activity. When GDP-D-mannose was incubated with the 100,000 x g supernatants obtained from the cell homogenate of P. aeruginosa IID 1008 (Homma serotype G) (4) and the sugar moieties liberated from nucleotide sugars by mild acid hydrolysis (0.02 M HC1, 100°C, 15 min) were subjected to paper chromatography in 1-butanol-pyridine-water (6:4:3 [vol/vol/vol]), only a radioactive rhamnose was formed, in addition to mannose derived from the starting substrate. Thus, in P. aeruginosa IID 1008, GDP-rhamnose (probably in a D-form) may be formed from GDP-D-mannose by a GDP-rhamnose synthetase system similar to the above system. However, talomethylose (4epimer form of rhamnose) or other 6-deoxyhexoses could not be detected, indicating that in this enzyme system, a stereospecific conversion may occur. Taking into account the occurrence of D-rhamnan in this strain (16), it is most likely that the possible reaction product, GDP-rhamnose, may be involved in the synthesis of a D-rhamnan chain (or other D-rhamnose-containing polysaccharides). To know the relationship between the distribution of GDP-rhamnose synthetase and the spectrum of binding to MAb E87, 12 other strains (Homma serotypes A to F and H to M) of P. aeruginosa were tested under the same reaction conditions. As shown in Table 1, the enzyme activity of GDP-rhamnose synthetase was found in high levels in six strains of P. aeruginosa belonging to Homma serotypes D, G, I, K, L, and M. Three serotypes, A, F, and H, showed weak enzyme activities. The other strains had undetectable enzyme activity. Thus, within 13 serotypes of P. aeruginosa
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TABLE 1. Distribution of GDP-rhamnose synthetasea among various serotypes of P. aeruginosa and the binding between MAb E87 and these bacteria P. aeruginosa
Serotype
1001 (ATCC 27577) 1002 (ATCC 27588) 1003 (ATCC 27579) 1004 (ATCC 27580) JID 1005 (ATCC 27581) IID 1006 (ATCC 27582) IID 1008 (ATCC 27584) IID 1009 (ATCC 27585) IID 1010 (ATCC 27586) IID 1011 (ATCC 27587) IID 1012 (ATCC 27588) IID 1014 (ATCC 27590) IFO 3080
A B C D E F G H I J K L M
IID IID IID IID
Enzymeofactivity' (mU/mg protein) 26 NDd ND 110 ND 20 410 25 610 ND 210 540 380
We thank Masato Nagaoka for providing authentic talomethylose. LITERATURE CITED
Bindingc (A405)
1. Bebault, G. M., G. G. S. Dutton, N. A. Funnell, and K. L.
0.3 0.1 0 0.8 0 0.4 0.3 0.5 0.5 0 0.4 0.1 1.5
K32 polysaccharide. Carbohydr. Res. 63:183-192. 2. Darveau, R. P., and R. E. W. Hancock. 1983. Procedure for isolation of bacterial lipopolysaccharides from smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J. Bacteriol. 155:831-838. 3. Dutton, G. G. S., and K. L. Mackie. 1977. Structural investigation of Klebsiella serotype K36 polysaccharide. Carbohydr. Res. 55:49-63. 4. Homma, J. Y. 1976. A new antigenic schema and live-cell slide-agglutination procedure for the infrasubspecific serologic classification of Pseudomonas aeruginosa. Jpn. J. Exp. Med. 46:329-336. 5. Klyne, W. 1950. The configuration of the anomeric carbon atoms in some cardic glycosides. Biochem. J. 47:xii-xiii. 6. Kocharova, N. A., Y. A. Knirel, N. K. Kochetokov, and E. S. Stanislavsky. 1988. Characterization of a D-rhamnan derived from preparation of Pseudomonas aeruginosa lipopolysaccharides. Bioorg. Khim. 14:701-703. 7. Kocharova, N. A., Y. A. Knirel, A. S. Shashkov, N. K. Kochetokov, and G. B. Pier. 1988. Structure of an extracellular crossreactive polysaccharide from Pseudomonas aeruginosa immunotype 4. J. Biol. Chem. 263:11291-11295. 8. Lam, M. Y. C., E. J. McGroarty, A. M. Kropnski, L. A. MacDonald, S. S. Pedersen, N. Hoiby, and J. S. Lam. 1989. Occurrence of a common lipopolysaccharide antigen in standard strain and clincal strains of Pseudomonas aeruginosa. J. Clin. Microbiol. 27:962-967. 9. Markovitz, A. 1964. Biosynthesis of guanosine diphosphate D-rhamnose and guanosine diphosphate D-talomethylose from guanosine diphosphate a-D-mannose. J. Biol. Chem. 239:20912098. 10. Rivera, M., L. E. Bryan, R. E. W. Hankock, and E. J. McGroarty. 1988. Heterogeneity of lipopolysaccharides from Pseudomonas aeruginosa: analysis of lipopolysaccharide chain length. J. Bacteriol. 170:512-521. 11. Rivera, M., and E. J. McGroarty. 1989. Analysis of a commonantigen lipopolysaccharide from Pseudomonas aeruginosa. J. Bacteriol. 171:2244-2248. 12. Sawada, S., T. Kawamura, Y. Masuho, and K. Tomibe. 1985. A new common polysaccharide antigen of strains of Pseudomonas aeruginosa detected with a monoclonal antibody. J. Infect. Dis.
a Each 100,000 x g supernatant (20 to 100 pg of protein) of the cell homogenates was incubated with 0.1 mM GDP-[44C]D-mannose (5,000 cpm/ nmol) (Amersham Corp. Arlington Heights, Ill.; Sigma Chemical Co., St. Louis, Mo.)-3 mM NADPH-1 mM EDTA-and 50 mM Tris hydrochloride buffer (pH 7.8) in a final volume of 30 p.l at 37°C for 30 min. After mild acid hydrolysis, the hydrolysate was deionized by passing through a small column packed with Dowex 1 and Dowex 50. The neutral sugar fraction was separated by paper chromatography, and the radioactive rhamnose was counted. bEnzyme activity is shown in milliunits (a unit of enzyme activity was defined as the formation of 1 nmol of GDP-rhamnose per min) per mg of
protein. c Binding activity was determined by direct ELISA by using 96-well microplates coated with cells of the indicated P. aeruginosa strains. Data are shown in A'405d ND, Not detectable.
tested, 9 serotypes possessed the GDP-rhamnose synthetase. On the other hand, the binding of MAb E87 to P. aeruginosa cells was determined by direct ELISA (Table 1). With the exception of three serotypes, C, E, and J, the cells of the other nine P. aeruginosa strains bound to MAb E87. Although there was no correlation between the strength of the enzyme activity and the binding to MAb E87, the distribution of GDP-rhamnose synthetase and the spectrum of the binding of MAb E87 to P. aeruginosa cells closely correlated with each other. Therefore, it is possible that GDP-rhamnose synthetase forms GDP-D-rhamnose from GDP-D-mannose and that the product is involved in the synthesis of a D-rhamnose chain or D-rhamnose-containing polysaccharides in the above nine P. aeruginosa strains. Moreover, the above results strongly support a possibility that D-rhamnan or other D-rhamnose-containing polysaccharides may occur as the common antigen(s) in a variety of P. aeruginosa strains. Although D-rhamnan has been actually isolated from serotypes G and M of P. aeruginosa, the isolation of D-rhamnan or its related polysaccharides from other Homma serotypes (A, D, F, H, I, K, and L) reactive against MAb E87 must be carried out to confirm the occurrence of the plausible common polysaccharide antigen in wide strains of this bacterium. Moreover, we expect that the structural studies on the A-band lipopolysaccharides, which have been reported by McGroarty and co-workers (8, 10, 11) to react with MAb E87 and to occur widely in 170 P. aeruginosa isolates including strain PAC 1, will provide a great advance concerning the antigenic epitope for MAb E87.
Mackie. 1978. Structural investigation of Klebsiella serotype
152:1290-1299.
13. Smith, A. R. W., S. E. Zamze, S. M. Munro, K. J. Carter, and R. C. Hignett. 1985. Structure of the side chain of lipopolysaccharide from Pseudomonas syringiae pv. morsprunorum C28. Eur. J. Biochem. 149:73-78. 14. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharides: extraction with phenol water and further application of the procedure. Methods Carbohydr. Chem. 5:83-91. 15. Winkler, N. W., and A. Markovitz. 1971. Guanosine phosphate4-keto-D-rhamnose reductase: a non-stereoselective enzyme. J. Biol. Chem. 246:5868-5876. 16. 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. 17. Yokota, S., H. Ochi, H. Ohtsuka, M. Kato, and H. Noguchi. 1989. Heterogeneity of the L-rhamnose residue in the outer core of Pseudomonas aeruginosa lipopolysaccharide, characterized by using human monoclonal antibodies. Infect. Immun. 57: 1691-1696.
