Analysis of 7-Substituted Sialic Acid in Some Enterobacterial Lipopolysaccharides

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JOURNAL OF BACTERIOLOGY, Mar. 1993, p. 1508-1513 0021-9193/93/051508-06$02.00/0 Copyright 1993, American Society for Microbiology Vol. 175, No.

1 JOURNAL OF BACTERIOLOGY, Mar. 1993, p /93/ $02.00/0 Copyright 1993, American Society for Microbiology Vol. 175, No. 5 Analysis of 7-Substituted Sialic Acid in Some Enterobacterial Lipopolysaccharides ANDRZEJ GAMIANl* AND LENNART KENNE2 Ludwik irszfeld Institute of Immunology and Experimental Therapy, Czerska 12, Wroclaw, Poland,' and Department of Chemistry, Swedish University ofagricultural Sciences, S Uppsala, Sweden2 Received 27 July 1992/Accepted 14 December 1992 Sialic acid-containing lipopolysaccharides (LPS) were isolated from six bacterial strains of the family Enterobacteriaceae. Sialic acid was released from permethylated LPS by methanolysis, and partially 0-methylated N-acetyl-N-methyl-neuraminic acid methyl ester methyl glycosides were analyzed by gas-liquid chromatography-electron ionization mass spectrometry. It was proved that all LPS contain N-acetylneuraminic acid (NeuAc). The occurrence of 7-substituted NeuAc in Escherichia coli serotypes 024 and 056 and in Citrobacterfreundii 037 LPS was documented. The LPS preparations also contained terminal NeuAc. LPS of E. coli 0104 had exclusively 4-substituted sialic acid. The remaining LPS studied, namely, from Salmonella toucra 048 and afnia alvei 2, had 4-linked and terminally localized NeuAc residues. In bacterial endotoxic lipopolysaccharides (LPS), the polysaccharide moiety is built up of oligosaccharide repeating units, which in some strains contain sialic acid (11). Sialic acid residues in bacteria may occupy a terminal or an internal position within a carbohydrate chain. Internally located sialic acid was found as 8-, 9-, or 4-linked N-acetylneuraminic acid (NeuAc) (1, 7, 8). A trisaccharide with 7-linked sialic acid was isolated recently from a Rhodobacter strain (10). Studies on sialic acid-containing LPS are necessary to understand the role of sialic acid in the interaction of procaryotes with hosts. The structure of an 0-specific polysaccharide with sialic acid was described for the first time in afnia alvei 2 (3). This investigation was performed to determine whether terminal sialic acid is present in intact LPS and to determine the substitution of internal sialic acid. MATERIALS AND METODS Strains, cultivation, and LPS preparations. Escherichia coli 024 strain E41a, E. coli 056 strain Su , and E. coli 0104 strain 519 were kindly provided by F. 0rskov, TABLE 1. Statens Seruminstitut, Copenhagen, Denmark. Salmonella toucra 048 strain KOS 1386 was from the National Salmonella Centre, Gdansk, Poland;. alvei 2 (PCM 2386) was from the Collection of the Institute of Immunology and Experimental Therapy, Wroclaw, Poland; and Citrobacter freundii 037 strain Bell5/66 was from the Czechoslovak National Collection of Type Cultures, Prague. The growth of bacteria in liquid medium, isolation with phenol water, and purification of LPS were carried out as described previously (2, 4, 13). The O-specific polysaccharide was prepared from LPS by treatment with 1% acetic acid, as described previously (3, 4). Methylation analysis of LPS. Samples of dried LPS preparations were methylated by the akomori method (6) and purified by dialysis into water. Dried samples were subjected to methanolysis with 1 M Cl in methanol for 4 h at 80'C. Methanol was replaced once by 2-labeled methanol. Further acetylation was carried out with acetic anhydridepyridine (1:1, vol/vol) for 30 min at 1000C. A parallel sample was silylated with Sigma A reagent (40 A.l; Sigma Chemical Co.) for 30 min at 100'C. For deuterioacetylation, the acetic anhydride was replaced with 2-labeled acetic anhydride. Relative retention times and proportions of sialic acid methyl ester methyl glycoside derivatives analyzed on a fused-silica capillary P-1 column (0.22 mm by 12 m) at 150 to 270'C (80C/min) Compound Relative molar ratio from LPS of: R, on No. Name P-1. alvei 2 E. coli 0104 S. 048 toucra E. coli 024 E. coli 056 C freundii Neu5Ac4,5,7,8,9Me (NT) _ b 30.9 (25.8) 20.1 (12.8) 36.8 (23.7) 48.1 (34.3) 2 Neu5Ac4,5,8,9Me (4.9) 23.3 (7.7) 32.5 (5.6) 3 Neu5Ac4,5,8,9Me47Ac Neu5Ac4,5,8,9Me47TMS 1.03 (82.3) (68.6) (60.1) 5 Neu5Ac5,7,8,9Me44Ac Neu5Ac5,7,8,9Me44TMS 1.06 (NT) (100) (74.2) 7 Neu5Ac4,5,7,9Me48Acc Neu5Ac4,5,7,9Me48TMSC Neu5Ac4,5,7,8Me49Acc Neu5Ac4,5,7,8Me49TMSC 1.10 a NT, not tested. Values in parentheses are for derivatives from samples subjected to silylation. b _, compound not present. c Compounds 7 to 10 derive from the polysaccharides used as standards. * Corresponding author. 1508

$VOL. 175, 1993 7-SUBSTITUTED SIALIC ACID IN ENTEROBACTERIAL LPS 1509 5,0 j R 2 AL -ic OR \ COR7 CORB C2ORg OOR1 1 R1=R2=R4=R5.=R7=R8=R9=C3; R5=COC3 2 R1R2=R4R5.$

2 VOL. 175, SUBSTITUTED SIALIC ACID IN ENTEROBACTERIAL LPS ,0 j R 2 AL -ic OR \ COR7 CORB C2ORg OOR1 1 R1=R2=R4=R5.=R7=R8=R9=C3; R5=COC3 2 R1R2=R4R5.=R8=R9=C3; R7=; R5=COC3 3 R 1R2=R4=R5,=R8=R9=C3; R5=R7=COC3 4 R1R2=R4=R5=R8=R9=C3; R5=COC3; R7=Si(C3)3 5 R1=R2=R5,=R7=R8=R9=C3; R4=R5=COC3 6 R1R2R5 =R7R8=R9=C3; R5=COC3; R4=Si (C3)3 7 R1=R2=R4=R5)=R7=R=9C3; R5 R8=COC3 8 R1=R2=R4=R5,=R7=R9=C3; R5=COC3; R8=Si(C3)3 9 R1=R2=R4=R5,=R7=R8=C33; R5=R 9=COC3 R1 2 4=R5,=R7=R8=C3; R5=COC3; Rg=Si(C3)3 R5.s 0 OCR7 OR2 R8 ' < ' COOR1 ORI 11 R1R2=R4=R5 =C3; R5=R7=COC3; R8=C2Oc3 12 R1=R2=R4=R5 =C3; R5=COC3; R7=COCD3; R8=C2OC3 13 R1=R2=R4=R5 =C3; R5=COC3; R7=Si (C3)3; R8=C2OC3 1 R2 4=R5=C3; R5=COC3; R7=Si(C3)3; R8=CDOC3 15 R1 2 4=R5 =C3; R5=COC3: R7=; R8=CDOC3 1 2 D3; R4 5 C3; R5--COC3; R7=COC3; R8=C2oc3 FIG. 1. Chemical derivatizations of NeuAc. Fragment TABLE 2. After derivatization, the samples were used for NeuAc analysis by gas chromatography-mass spectrometry (GC- MS). Glycophorin A (gift from E. Lisowska), meningococcal group C polysaccharide (gift from. J. Jennings), and colominic acid (Sigma) were used as reference samples. Smith degradation of O-specific polysaccharide (5). The polysaccharide (18 mg) was oxidized with 0.1 M NaIO4 (2 ml) for 48 h at 4 C in the dark. One part of the oxidized product was reduced with NaB4 (40 mg), and another was reduced with 40 mg of NaBD4; they were acidified with acetic acid and dialyzed into water. The product was hydrolyzed with 0.5 M trifluoroacetic acid at room temperature for 24 h. Permethylation, methanolysis, and further acetylation, deuterioacetylation, or silylation were all performed under the conditions described above. GC-MS. Combined GC-MS was performed on a ewlett- Packard 5891A gas chromatograph-mass selective detector, using a fused-silica capillary column (P-1; 12 m by 0.22-mm inside diameter). The oven was programmed to hold at 150 C for 3 min, which was followed by an 8 C/min rise to 270 C. RESULTS AND DISCUSSION The acid lability of polysaccharides containing sialic acid, under the conditions used for delipidation of LPS (1% C3COO, 1 h, 100 C), normally causes the polysaccharide obtained to be partially depolymerized. With. alvei 2 LPS, complete depolymerization occurred under the conditions used (3). These circumstances prompted us to investigate the intact LPS instead for the presence of terminal or substituted sialic acid in the 0 antigen of some strains of the family Enterobacteriaceae. Methylation analysis was used to determine terminal and internal sialic acid residues in the LPS, using methanolysis for releasing individual sugar residues. The partially O-methylated N-acetyl-N-methyl-neuraminic acid methyl ester methyl glycosides obtained were acetylated, deuterioacetylated, or silylated at the free hydroxyl groups prior to GC-MS analysis. The methanolysis used (1 M Cl, 80 C, 4 h) results in incomplete release of the glycosidically substituted (internal) sialic acid but should release the terminal sialic acid groups in good yield. The conditions used should Mass/charge ratios of characteristic fragment ions obtained from acetylated or trimethylsilylated and methylated sialic acid derivatives m/z (intensity) of sialic acid derivative: A 392 (1.5) 378 (1.3) 420 (4.0) 450 (7.9) 376 (2.4) 379 (4.1) 406 (6.6) 407 (16.5) 335 (2.1) 379 (5.4) M-OC3 376 (3.4) 362 (2.1) 404 (5.8) 434 (1.3) 360 (2.1) 390 (1.0) 391 (4.4) 319 (4.7) 366 (5.5) ' 362 (2.7) 348 (1.0) 390 (7.3) 420 (1.0) B 348 (54.6) 334 (30.7) 376 (57.1) 406 (21.2) 332 (39.0) 335 (45.1) 362 (24.1) 363 (58.0) 291 (93.8) 335 (39.0) M-OC3, C (8.1) 330 (5.6) 372 (14.6) 402 (3.9) 328 (2.7) 331 (3.9) 358 (3.9) 359 (8.7) 287 (11.9) 334 (3.5) M-R70, NR5R5 258 (43.0) 258 (50.9) 258 (47.4) 259 (59.7) 264 (33.0) C 318 (58.5) 304 (12.3) 376 (27.4) 376 (4.6) 376 (11.8) 304 (13.9) 298 (89.3) 298 (36.4) 298 (66.5) 298 (65.8) C-R (8.7) 272 (40.0) 344 (14.6) 344 (5.4) 344 (12.1) 272 (32.8) E' 274 (28.1) 274 (9.2) 274 (19.7) 274 (17.9) 274 (6.2) 274 (13.3) 274 (7.8) D 254 (80.3) 240 (33.5) 312 (64.4) 312 (20.4) 312 (45.7) 240 (17.6) E'-C (14.9) 242 (15.1) 242 (18.7) 242 (8.7) 242 (5.8) 242 (9.2) 242 (8.1) 242 (12.1) E 201 (35.3) 201 (39.6) 201 (45.9) 201 (32.2) 201 (27.3) 201 (26.0) 201 (62.7) 201 (95.4) 201 (60.2) 207 (30.6) E-C (31.1) 169 (40.1) 169 (65.3) 169 (17.0) 169 (28.0) 169 (19.9) 169 (33.6) 169 (39.2) 169 (53.6) 172 (25.4) R5-N-R5=C- 142 (62.1) 128 (34.2) 170 (41.4) 200 (32.0) 128 (51.8) 131 (12.1) 158 (23.8) 158 (26.9) 128 (63.1) 134 (12.6) C=COR7 G 129 (100) 129 (100) 129 (100) 129 (100) 129 (100) 129 (100) 129 (100) 129 (100) 129 (94.3) 129 (75.3) F 89 (19.1) 89 (27.2) 89 (21.4) 89 (52.2) 45 (31.1) 45 (24.6) 45 (31.1) 46 (13.4) 46 (36.9) 45 (47.6)

3 1510 GAMIAN AND KENNEJ.ATEI. BACTERIOL. Wbundan ce Scan 816 ( min) b /Z -> ~~~~~~~~ ~~~~~~~~~~~~~~~5 LI ka ~~~~~~18 47 TI FIG. 2. Mass spectra of NeuAc derivatives 2 (a) and 4 (b). minimize de-n-acylation, however. The retention times and the yields of the partially methylated sialic acid methyl ester methyl glycosides obtained from the original LPS are presented in Table 1. The electron ionization spectra of sialic acid derivatives were interpreted in accordance with data in the literature (9, 12) and by using deuterium-labeling techniques. Permethylated methyl ester methyl glycoside of NeuAc, which derived from terminal sialic acid, was present in all LPS preparations except E. coli The E. coli 0104 LPS yielded a derivative of 4-substituted NeuAc (compound 5) exclusively. Characteristic fragments (9) of 4-O-acetyl Neu5Ac5,7,8,9Me4 methyl ester methyl glycoside (coinpound 5) were m/z 420 (fragment A), 376 (B), 346 (C), 254 (D), 89 (F), 157 (G), and 289 (), with fragment E missing and the expected increase when the sample was deuterium labeled. The 4-linked sialic acid was present also in LPS of S. toucra 048 and. alvei 2. The fact that. alvei 2 LPS contains terminal sialic acid (18% of the total yielded NeuAc derivative) (Table 1) indicates that a biological 0-specific repeating unit in the original LPS is terminated at the nonreducing end by sialic acid. The chemical repeating unit is a branched octasaccharide with 4-substituted NeuAc (3). The LPS from E. coli 024, E. coli 056, and C. freundii 037 strains all gave the same kind of derivatives; namely,

4 kbundanace Scan 749 ( min) '0 4000'0 3500'0 3000'O ) ,0 19 a 10' ~~~~~~~~~~~~~ ~~~ M/z -> n ir hbundance Scan 752 ( min) l M/ ~~~~ II~ Aundance Scan 714 ( min) IOLIJh~ Ai~~~~~13I ~~ ~~~~~ II ~~~15927 liii ~ ~ ~ ~~~ k/z -> , ' 350 FIG. 3. Mass spectra of derivatives 11 (a), 13 (b), and 15 (c). 1511

5 1512 GAMIAN AND KENNE R S ' t o 7 - R5 Ra -C /8 co7r0 OR2 - R70 0R2 R ~~~~ ṈR5R5- _ COOR1 COOR1 OR4 OR4 m/z 391 m /z 258 FIG. 4. Formation of the m/z 258 fragment. one peak originated from terminal sialic acid (compound 1), and another originated from 7-substituted NeuAc (compound 3) (Table 1). Various derivatizations were performed to prove the 7-substitution of NeuAc (Fig. 1). Mass/charge ratios of characteristic fragment ions obtained from sialic acid derivatives are presented in Table 2. An additional peak present on chromatograms of acetylated samples (compound 2) was ascribed to 7-hydroxy-Neu5Ac4,5,8,9Me4 methyl ester methyl glycoside (Fig. 2). This compound was formed as a result of incomplete acetylation of released permethylated 7-O-NeuAc. This assumption is based on the following observations. Deuterioacetylated, acetylated, and silylated samples gave identical spectra of compound 2. Deuterioacetylated samples had a higher proportion of compound 2 than acetylated samples, while silylated preparations yielded only a small amount of compound 2, which means that the quantity of that compound depends on the method of derivatization. Silylation is more efficient than acetylation. In general, the presence of compounds 2 and 3 supports the existence of 7-substituted NeuAc in the original polysaccharides. Compounds 2 and 3 deriving from the same NeuAc may be combined to calculate the ratio of terminal to internal NeuAc in LPS. Important ions for compound 2 are fragment A (m/z 378), fragment M-OC3 at m/z 362, and fragments B (m/z 334), C (m/z 304), C-C30 (m/z 272), D (m/z 240), (m/z 298), E (m/z 201), G (m/z 129), and F (m/z 89) Ḟurther evidence for the 7-substitution of NeuAc was obtained after Smith degradation (5) of the polysaccharide. This experiment was done on E. coli 024 and 056 polysaccharides, which have in their main chains the other sugar residues that are resistant to periodate oxidation. The polysaccharide subjected to Smith degradation and subsequently to gel filtration on Bio-Gel P-4 was eluted in the void volume, which means that the Smith-degraded product remains polymeric. The Smith-degraded product also is retained in the bag during the dialysis process. If the sialic acid is substituted in the 7 position, vicinal diols at C-8 to C-9 permit oxidation, resulting in a shortage of one carbon. The mass spectrum of peracetylated derivative 11 (Fig. 3a, Table 2) shows characteristic fragment ions of high intensity at m/z 332 (fragment B), 258, and also 376 (fragment A). The appearance of the m/z 258 fragment is associated with elimination of the 7-substituent, because this ion is produced from both acetylated (compounds 11 and 12) and silylated (compound 13) (Fig. 3b) derivatives. In the spectrum of compound 14 in which deuterium was introduced on C-8, the fragment shifts 1 a.m.u. to m/z 259. Moreover, the derivative deuteriomethylated at C-1 and C-2 has a shift of 6 a.m.u. to m/z 264 (Table 2, compound 16), which means that positions C-1 and C-2 are preserved in this fragment. The mechanism proposed for its formation is depicted in Fig. 4. In permethylated NeuAc, abundant cleavage between two methoxy groups occurs between C8OMe and C9OMe, but in permethylated aminodideoxyoctonate, the product of periodate oxidation, this fragmentation does not exist. Therefore, another fragmentation usually not observed occurs (Fig. 4). In general, however, the fragmentation of derivatives 11 to 16 follows the known fragmentation scheme of sialic acids (9, 12) with the expected shifts for the corresponding fragments (Table 2, Fig. 3a to c). Results presented above indicate that internal sialic acid in LPS of E. coli serotypes 024 and 056 and C. freundii 037 is linked through the 7-position. This finding has important biological meaning. As far as we know, such glycosylation of sialic acid has only been found once in nature (10). The partial serological cross-reactivity (2, 11) of the above three serotypes is probably associated with their common feature, the 7-substituted sialic acid. Moreover, the rabbit anti-c. freundii 037 serum agglutinates horse erythrocytes (2). These observations indicate the possibility of the creation of, or participation in, the epitope by the 7-substituted sialic acid. As a result of the present investigations, it was also possible to find the terminal sialic acid in intact LPS. The polysaccharide of LPS is degraded to various degrees during mild acid delipidation due to the lability of the NeuAc linkage. Therefore, the procedure used in this work serves as a method to determine the natural biological unit by searching for the presence of terminal NeuAc in endotoxin to find out whether the biological unit is terminated by sialic acid. Analysis of an original molecule, such as LPS, is helpful, especially when it is difficult to obtain the polysaccharide without degradation. That all LPS studied revealed terminal NeuAc except that from E. coli 0104 is in accordance with gel filtration of the carbohydrate material released from LPS in which free sialic acid was released from all LPS except that of In this case the disaccharide Gal-NeuAc was obtained instead of free NeuAc (4). It is important to prove the presence of sialic acid in endotoxin unequivocally and simply. Terminal sialic acid in bacteria mimics such a molecule in host tissue, thereby contributing to the pathogenicity of bacteria which become unrecognized by the host immune system. Although all LPS studied are antigenic against rabbit sera (2), most likely terminal neuraminic acid does not participate in the respective epitopes; instead, the epitope is formed in the internal part of the polysaccharide. Another mechanism that also should be examined is the biosynthesis of sialic acid-containing, 0-specific polysaccharides. Enzymes which take part in the biosynthesis and metabolism of these 0 antigens may be important virulence factors. Such bacterial enzymes may alter the host structure during infection. ACKNOWLEDGMENT We thank Elzbieta Romanowska for her interest. J. BACTERIOL. REFERENCES 1. Corfield, A. P., and R. Schauer Sialic acids, chemistry, metabolism and function, Cell Biol. Monogr. 10: Gamian, A., A. Romanowska, and E. Romanowska Immunochemical studies on sialic acid-containing lipopolysaccharides from enterobacterial species. FEMS Microbiol. Immunol. 89: Gamian, A., E. Romanowska, U. Dabrowski, and J. Dabrowski Structure of the 0-specific, sialic acid containing polysaccharide chain and its linkage to the core region in lipopolysaccharide from afnia alvei strain 2 as elucidated by chemical methods, gas-liquid chromatography/mass spectrometry and 1 NMR spectroscopy. Biochemistry 30:

VOL. 175, 1993 7-SUBSTITUTED SIALIC ACID IN ENTEROBACTERIAL LPS 1513 4. Gamian, A., E. Romanowska, J. Ulrich, and J. Defaye. 1992.

6 VOL. 175, SUBSTITUTED SIALIC ACID IN ENTEROBACTERIAL LPS Gamian, A., E. Romanowska, J. Ulrich, and J. Defaye The structure of the sialic acid-containing Escherichia coli 0104 O-specific polysaccharide and its linkage to the core region in lipopolysaccharide. Carbohydr. Res. 236: Goldstein, I. J., G. W. ay, B. A. Lewis, and F. Smith Periodate oxidation of polysaccharides. Methods Carbohydr. Chem. 5: akomori, S Rapid permethylation of glycolipids and polysaccharides catalyzed by methylsulfinyl carbanion in dimethylsulfoxide. J. Biochem. 55: Jann, B., and K. Jann Structure and biosynthesis of the capsular antigens of Escherichia coli. Curr. Top. Microbiol. Immunol. 150: Jennings,. J Capsular polysaccharides as vaccine candidates. Curr. Top. Microbiol. Immunol. 150: Kamerling, J. P., and J. F. G. Vliegenthart Carbohydrates, p In A. M. Lawson (ed.), Mass spectrometry. De Gruyter, Berlin. 10. Krauss, J.., K. immelspach, G. Reuter, R. Schauer, and. Mayer Structural analysis of a novel sialic acid-containing trisaccharide from Rhodobacter capsulatus 37b4 lipopolysaccharide. Eur. J. Biochem. 204: rskov, I., F. 0rskov, B. Jann, and K. Jann Serology, chemistry, and genetics of 0 and K antigens of Escherichia coli. Bacteriol. Rev. 41: Van albeek,., J. averkamp, J. P. Kamerling, J. F. G. Vliegenthart, C. Versluis, and R. Schauer Sialic acids in permethylation analysis: preparation and identification of partially 0-methylated derivatives of methyl N-acetyl-N-methyl-,B- D-neuraminate methyl glycoside. Carbohydr. Res. 60: Westphal, O., and K. Jann Bacterial lipopolysaccharides: extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5:83-91.

Gas Chromatography/ Mass Spectrometry

Gas Chromatography/ Mass Spectrometry Edited by H.F. Linskens and J.F. Jackson Contributors R.S. Bandurski G. Combaut A.Ehmann P.Hedden B.Janistyn H.Kameoka H.Kodama D.V.Lynch J.K.MacLeod H.Nyberg L.M.S.Palni