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Structures of the O-polysaccharides of Salmonella enterica O59 and Escherichia coli O15
Carbohydrate Research 346 (2011) 381–383
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Note
Structures of the O-polysaccharides of Salmonella enterica O59 and Escherichia coli O15 Andrei V. Perepelov a,⇑, Bin Liu b,c, Sof’ya N. Senchenkova a, Alexander S. Shashkov a, Dan Guo b,c, Lu Feng b,c, Yuriy A. Knirel a, Lei Wang b,c a
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin 300457, PR China c Tianjin Key Laboratory for Microbial Functional Genomics, TEDA College, Nankai University, TEDA, Tianjin 300457, PR China b
a r t i c l e
i n f o
Article history: Received 13 August 2010 Received in revised form 7 November 2010 Accepted 16 November 2010 Available online 20 November 2010 Keywords: Salmonella enterica Escherichia coli Bacterial polysaccharide structure O-Antigen gene cluster Lipopolysaccharide
a b s t r a c t The O-polysaccharide of Salmonella enterica O59 was studied using sugar analysis and 2D 1H and 13C NMR spectroscopy, and the following structure of the tetrasaccharide repeating unit was established: ?2)-b-D-Galp-(1?3)-a-D-GlcpNAc-(1?4)-a-L-Rhap-(1?3)-b-D-GlcpNAc-(1? Accordingly, the O-antigen gene cluster of S. enterica O59 includes all genes necessary for the synthesis of this O-polysaccharide. Earlier, another structure has been reported for the O-polysaccharide of Salmonella arizonae (S. enterica IIIb) O59, which later was found to be identical to that of Citrobacter (Citrobacter braakii) O35 and, in this work, also to the O-polysaccharide of Escherichia coli O15. Ó 2010 Elsevier Ltd. All rights reserved.
Salmonella enterica is recognized as a major pathogen of animals and humans. Estimated 1.4 million cases of salmonellosis and 556 estimated food-related deaths (31% of the total) are attributed to S. enterica annually in the United States.1 Based on the O-antigens (O-polysaccharides), strains of S. enterica are classified into 46 O-serogroups. Serogrouping has proven extremely useful for understanding the host range and disease spectrum of this pathogen. Genetic variations in the O-antigen gene clusters contribute to the major difference between the diverse O-antigen forms. O-Polysaccharide structures have been elucidated in many but not all S. enterica O-serogroups (Refs. 2–4; see also Bacterial Carbohydrate Structure Data Base at http://www.glyco.ac.ru/bcsdb3). In this work, we established the O-polysaccharide structure and characterized the O-antigen gene cluster of S. enterica O59, belonging to the Salmonella subspecies II. We also showed that another O-polysaccharide structure reported earlier for Salmonella arizonae (S. enterica IIIb) O595 and Citrobacter (Citrobacter braakii) O356 is shared by Escherichia coli O15. A high-molecular mass O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide isolated from dried bacterial cells of S. enterica O59 by the phenol–water procedure. Sugar analysis by GLC of the alditol acetates derived after full acid hydrolysis of the O-polysaccharide revealed rhamnose (Rha), Gal, and ⇑ Corresponding author. Tel.: +7 499 1376148; fax: +7 499 1355328. E-mail address: [email protected] (A.V. Perepelov). 0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2010.11.017
GlcNAc in the ratio 1:1.2:0.9 (detector response). GLC analysis of the acetylated (S)-2-octyl glycosides demonstrated that Rha is L whereas Gal and GlcNAc have the D configuration. The 13C NMR spectrum of the O-polysaccharide (Fig. 1A) showed signals for four anomeric carbons in the region d 99.2– 102.5, one CH3–C group (C-6 of Rha) at d 17.9, three HOCH2–C groups (C-6 of Gal and GlcNAc) at d 61.4–62.3, two nitrogen-bearing carbons (C-2 of GlcNAc) at d 53.9 and 56.8, 14 oxygen-bearing non-anomeric sugar ring carbons in the region d 69.1–82.8, and two N-acetyl groups at d 23.4, 23.6 (both CH3), 175.0, and 175.6 (both CO). Accordingly, the 1H NMR spectrum of the O-polysaccharide contained signals for four anomeric protons at d 4.56–4.99, one CH3–C group (H-6 of Rha) at d 1.21, other sugar protons in the region d 3.40–4.09, and two N-acetyl groups at d 2.07 and 2.09. Therefore, the O-polysaccharide has a tetrasaccharide repeat (O-unit) containing one residue each of L-Rha and D-Gal and two residues of D-GlcNAc. The 1H and 13C NMR spectra of the O-polysaccharide were analyzed using 2D homonuclear 1H, 1H COSY, TOCSY, ROESY, and heteronuclear 1H, 13C HSQC experiments (Table 1). Based on 3JH,H coupling constants estimated from the 2D NMR spectra, spin systems were assigned to four constituent monosaccharides, all being in the pyranose form. The spin systems for GlcNAc were distinguished by correlations between protons at the nitrogen-bearing carbons (H-2) and the corresponding carbons (C-2) at d 4.06/53.9 and 3.82/56.8. J1,2 coupling constants of 7–8 Hz showed that Gal
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A. V. Perepelov et al. / Carbohydrate Research 346 (2011) 381–383
Figure 1.
13
C NMR spectrum of the O-polysaccharide of S. enterica O59. Numbers refer to carbons in sugar residues denoted as shown in Table 1.
Table 1 1 H and 13C NMR chemical shifts (d, ppm) of the O-polysaccharide of S. enterica O59. Sugar residue
?2)-b-D-Galp-(1? A ?3)-a-D-GlcpNAc-(1? B ?4)-a-L-Rhap-(1? C ?3)-b-D-GlcpNAc-(1? D
H-1 C-1
H-2 C-2
H-3 C-3
H-4 C-4
H-5 C-5
H-6 (6a, 6b) C-6
4.56 102.2 4.99 99.2 4.87 102.2 4.80 102.5
3.65 79.7 4.06 53.9 3.78 72.1 3.82 56.8
3.71 75.1 3.93 81.1 3.82 70.2 3.61 82.8
3.88 70.1 3.64 69.3 3.44 82.0 3.47 69.8
3.69 76.3 4.09 72.7 4.09 69.1 3.40 77.1
3.76, 3.76 62.0 3.83, 3.83 61.4 1.21 17.9 3.73, 3.92 62.3
and one of the GlcNAc residues are b-linked, whereas a significantly smaller value of 3 Hz indicated the a-linkage of the other GlcNAc residue. The position of the signal for C-5 at d 69.1 indicated that Rha is a-linked (compare published data d 70.0 and 73.2 for a- and b-Rhap, respectively7). The signals for C-2 of b-Gal, C-3 of a-GlcNAc and b-GlcNAc, and C-4 of a-Rha, were shifted downfield by 6–9 ppm, as compared with their positions in the corresponding non-substituted monosaccharides.7 These data showed that the O-polysaccharide is linear and defined the glycosylation pattern in the O-unit. The ROESY spectrum of the O-polysaccharide showed the following correlations between anomeric protons and protons at the linkage carbons: b-Gal H-1,a-GlcNAc H-3; a-GlcNAc H-1,aRha H-4; a-Rha H-1,b-GlcNAc H-3; and b-GlcNAc H-1,b-Gal H-2 at d 4.56/3.93; 4.99/3.44; 4.87/3.61; and 4.80/3.65, respectively. The monosaccharide sequence thus defined was confirmed by a heteronuclear 1H, 13C HMBC experiment, which showed correlations between anomeric protons and linkage carbons and contrariwise (data not shown). Therefore, the O-polysaccharide of S. enterica O59 has the structure shown in Chart 1, which is unique among the known bacterial polysaccharide structures.
→2)-β-D-Galp-(1→3)-α-D-GlcpNAc-(1→4)-α-L-Rhap-(1→3)-β-D-GlcpNAc-(1→ S. enterica O59 →2)-β-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-β-D-GlcpNAc-(1→ S. arizonae (S, enterica IIIb) O59,5 Citrobacter (C. braakii) O35,6 E. coli O15 Chart 1. Structures of the O-polysaccharides.
NAc CH3
CO
2.09 23.6
175.6
2.07 23.4
175.0
The O-antigen gene cluster of S. enterica O59 was found between the housekeeping genes galF and gnd. Sequencing revealed nine open reading frames (orfs), excluding galF and gnd, all of which have the same transcriptional direction from galF to gnd. The functions were tentatively assigned to all orfs based on their similarity to related genes from the available databases and taking into account the S. enterica O59 antigen structure. orfs1–4 were identified as rmlB, rmlD, rmlA, and rmlC, respectively. The products of the rmlBDAC genes are known to be responsible for the four-step synthesis of dTDP-L-Rha from glucose 1-phosphate.8 Genes for the synthesis of the nucleotide precursors of common sugars, including Gal and GlcNAc, are located outside the O-antigen gene cluster.8 In many S. enterica and E. coli strains, WecA initiates the O-unit synthesis by transfer of GlcNAc 1-phosphate or GalNAc 1-phosphate to an undecaprenol phosphate carrier. The wecA gene maps outside the O-antigen gene cluster.9 Three additional glycosyl transferase genes (wdcF, wdcG, and wdcH), which are responsible for the transfer of three other sugar residues to assemble the full O-unit, were found within the O59-antigen cluster. orf5 and orf9 were identified as wzy and wzx, the O-antigen processing genes that encode O-antigen polymerase and O-unit flippase, respectively. Therefore, the O-antigen gene cluster is in full agreement with the O-antigen structure of S. enterica O59 established in this work. Earlier, another structure has been reported for the O-polysaccharide of S. arizonae (S. enterica IIIb) O59.5 It has a trisaccharide O-unit containing one residue each of D-Gal, D-GlcNAc, and L-FucpNAc (Chart 1). Later, it was found that the O-antigen of Citrobacter (C. braakii) O35 possesses the same structure.6 There are two possible explanations for a discrepancy between the Salmonella O59 antigen structures established earlier5 and in this work. Both O-antigens include a ?3)-b-D-GlcpNAc-(1?2)-b-D-Galp-(1? dis-
A. V. Perepelov et al. / Carbohydrate Research 346 (2011) 381–383
accharide fragment, which could provide a common epitope responsible for the serological relatedness. To prove or disprove this suggestion, a detailed serological study is necessary, which may result in reclassification of one of the strains to a new Salmonella serogroup. Alternatively, the strain studied earlier5 could be classified to the Salmonella O59 serogroup erroneously and, in fact, might be another bacterium, e.g. a strain of C. braakii O35. In our studies of the O-antigens of E. coli, we noticed that the O-polysaccharide of E. coli O15 showed essentially the same 13C NMR spectrum as that of S. arizonae (S. enterica IIIb) O59 reported earlier.5 In this work, we analyzed the former in more detail using 2D 1H and 13C NMR spectroscopy as described above (data not shown) and confirmed that the O-polysaccharides of the two bacteria have the identical structure too (Chart 1). The O-antigen gene cluster of E. coli O15 has been characterized earlier.10 It includes three genes for the synthesis of L-FucNAc (fnlA, fnlB, and fnlC), two glycosyl transferase genes (wbuB and wbuS), and two O-antigen processing genes (wzx for flippase and wzy for polymerase), and is thus in agreement with the O-polysaccharide structure.
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gradient of 160–290 °C at 5 °C min 1. The absolute configurations of the monosaccharides were determined by GLC of the acetylated (S)-2-octyl glycosides as described.14 1.4. NMR spectroscopy O-Polysaccharide samples were deuterium-exchanged by freeze-drying from D2O and then examined as solutions in 99.95% D2O. NMR spectra were recorded on a Bruker Avance 600 spectrometer (Germany) using internal TSP (dH 0) and external acetone (dC 31.45) as references. 2D NMR spectra were obtained using standard Bruker software, and Bruker TOPSPIN program was used to acquire and process the NMR data. Mixing times of 100 and 150 ms were used in TOCSY and ROESY experiments, respectively. 1.5. Sequencing and analysis of genes Sequencing of the chromosome region between galF and gnd, analysis of genes in the O-antigen gene cluster of S. enterica O59, and search of databases for possible gene functions were performed as described.15
1. Experimental Acknowledgments 1.1. Bacterial strains and isolation of lipopolysaccharides S. enterica O59 strain G1463 and E. coli O15 strain G1201 were obtained from the Institute of Medical and Veterinary Science (Adelaide, Australia). Bacteria were grown to late log phase in 8 L of Luria–Bertani broth using a 10-L BIOSTAT C-10 fermentor (B. Braun Biotech Int., Germany) under constant aeration at 37 °C and pH 7.0. Bacterial cells were washed and dried as described.11 The lipopolysaccharides in yields 10% and 6%, respectively, were isolated from dried bacterial cells by the phenol–water method,12 the crude extract was dialyzed without separation of layers and freed from nucleic acids and proteins by treatment with aq 50% CCl3CO2H at pH 2 at 4 °C.13 The supernatant was dialyzed and lyophilized. 1.2. Isolation of O-polysaccharides Delipidation of the lipopolysaccharides was performed with aq 2% HOAc at 100 °C until the precipitation of lipid A. The precipitate was removed by centrifugation (13,000g, 20 min), and the supernatant was fractionated by GPC on a column (56 2.6 cm) of Sephadex G-50 Superfine (Amersham Biosciences, Sweden) in 0.05 M pyridinium acetate buffer pH 4.5 monitored with a differential refractometer (Knauer, Germany). High-molecular-mass Opolysaccharides were obtained in yields 30% and 45% of the lipopolysaccharide mass, respectively. 1.3. Monosaccharide analysis The O-polysaccharide from S. enterica O59 was hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Monosaccharides were identified by GLC of the alditol acetates on a Hewlett-Packard 5890 chromatograph (USA) equipped with an Ultra-1 column using a temperature
This work was supported by the Russian Foundation for Basic Research (projects 08-04-01205 and 08-04-92225), the Chinese National Science Fund for Distinguished Young Scholars (30788001), NSFC General Program Grant 30670038, 30870070, 30870078, 30771175 and 30900041, Tianjin Research Program of Application Foundation and Advanced Technology (10JCYBJC 10000), the National 863 program of China grants 2006AA020703 and 2006AA06Z409, the National 973 program of China grant 2009CB522603, and National Key Programs for Infectious Diseases of China 2009ZX10004-108 and 2008ZX10003-005. References 1. Mead, P. S.; Slutsker, L.; Dietz, V.; McCaig, L. F.; Bresee, J. S.; Shapiro, C.; Griffin, P. M.; Tauxe, R. V. Emerg. Infect. Dis. 1999, 5, 607–625. 2. Knirel, Y. A.; Kochetkov, N. K. Biochemistry (Moscow) 1994, 59, 1325–1383. 3. Jansson, P.-E. In Endotoxin in Health and Disease; Brade, H., Opal, S. M., Vogel, S. N., Morrison, D. C., Eds.; Marcel Dekker: NY, 1999; pp 155–178. 4. Gajdus, J.; Glosnicka, R.; Szafranek, J. Wiadomosci. Chem. 2006, 60, 621–653. 5. Vinogradov, E. V.; Knirel, Y. A.; Lipkind, G. M.; Shashkov, A. S.; Kochetkov, N. K.; Stanislavsky, E. S.; Kholodkova, E. V. Bioorg. Khim. 1987, 13, 1275–1281. 6. Kocharova, N. A.; Knirel, Y. A.; Stanislavsky, E. S.; Kholodkova, E. V.; Lugowski, C.; Jachymek, W.; Romanowska, E. FEMS Immunol. Med. Microbiol. 1996, 13, 1–8. 7. Lipkind, G. M.; Shashkov, A. S.; Knirel, Y. A.; Vinogradov, E. V.; Kochetkov, N. K. Carbohydr. Res. 1988, 175, 59–75. 8. Samuel, G.; Reeves, P. Carbohydr. Res. 2003, 338, 2503–2519. 9. Reeves, P. R.; Wang, L. Curr. Top. Microbiol. Immunol. 2002, 264, 109–135. 10. Beutin, L.; Tao, J.; Feng, L.; Krause, G.; Zimmermann, S.; Gleier, K.; Xia, Q.; Wang, L. J. Clin. Microbiol. 2005, 43, 703–710. 11. Robbins, P. W.; Uchida, T. Biochemistry 1962, 1, 323–335. 12. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83–91. 13. Zych, K.; Toukach, F. V.; Arbatsky, N. P.; Kolodziejska, K.; Senchenkova, S. N.; Shashkov, A. S.; Knirel, Y. A.; Sidorczyk, Z. Eur. J. Biochem. 2001, 268, 4346– 4351. 14. Leontein, K.; Lönngren, J. Methods Carbohydr. Chem. 1993, 9, 87–89. 15. Feng, L.; Senchenkova, S. N.; Yang, J.; Shashkov, A. S.; Tao, J.; Guo, H.; Cheng, J.; Ren, Y.; Knirel, Y. A.; Reeves, P.; Wang, L. J. Bacteriol. 2004, 186, 4510–4519.
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Note
Structures of the O-polysaccharides of Salmonella enterica O59 and Escherichia coli O15 Andrei V. Perepelov a,⇑, Bin Liu b,c, Sof’ya N. Senchenkova a, Alexander S. Shashkov a, Dan Guo b,c, Lu Feng b,c, Yuriy A. Knirel a, Lei Wang b,c a
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation TEDA School of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin 300457, PR China c Tianjin Key Laboratory for Microbial Functional Genomics, TEDA College, Nankai University, TEDA, Tianjin 300457, PR China b
a r t i c l e
i n f o
Article history: Received 13 August 2010 Received in revised form 7 November 2010 Accepted 16 November 2010 Available online 20 November 2010 Keywords: Salmonella enterica Escherichia coli Bacterial polysaccharide structure O-Antigen gene cluster Lipopolysaccharide
a b s t r a c t The O-polysaccharide of Salmonella enterica O59 was studied using sugar analysis and 2D 1H and 13C NMR spectroscopy, and the following structure of the tetrasaccharide repeating unit was established: ?2)-b-D-Galp-(1?3)-a-D-GlcpNAc-(1?4)-a-L-Rhap-(1?3)-b-D-GlcpNAc-(1? Accordingly, the O-antigen gene cluster of S. enterica O59 includes all genes necessary for the synthesis of this O-polysaccharide. Earlier, another structure has been reported for the O-polysaccharide of Salmonella arizonae (S. enterica IIIb) O59, which later was found to be identical to that of Citrobacter (Citrobacter braakii) O35 and, in this work, also to the O-polysaccharide of Escherichia coli O15. Ó 2010 Elsevier Ltd. All rights reserved.
Salmonella enterica is recognized as a major pathogen of animals and humans. Estimated 1.4 million cases of salmonellosis and 556 estimated food-related deaths (31% of the total) are attributed to S. enterica annually in the United States.1 Based on the O-antigens (O-polysaccharides), strains of S. enterica are classified into 46 O-serogroups. Serogrouping has proven extremely useful for understanding the host range and disease spectrum of this pathogen. Genetic variations in the O-antigen gene clusters contribute to the major difference between the diverse O-antigen forms. O-Polysaccharide structures have been elucidated in many but not all S. enterica O-serogroups (Refs. 2–4; see also Bacterial Carbohydrate Structure Data Base at http://www.glyco.ac.ru/bcsdb3). In this work, we established the O-polysaccharide structure and characterized the O-antigen gene cluster of S. enterica O59, belonging to the Salmonella subspecies II. We also showed that another O-polysaccharide structure reported earlier for Salmonella arizonae (S. enterica IIIb) O595 and Citrobacter (Citrobacter braakii) O356 is shared by Escherichia coli O15. A high-molecular mass O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide isolated from dried bacterial cells of S. enterica O59 by the phenol–water procedure. Sugar analysis by GLC of the alditol acetates derived after full acid hydrolysis of the O-polysaccharide revealed rhamnose (Rha), Gal, and ⇑ Corresponding author. Tel.: +7 499 1376148; fax: +7 499 1355328. E-mail address: [email protected] (A.V. Perepelov). 0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2010.11.017
GlcNAc in the ratio 1:1.2:0.9 (detector response). GLC analysis of the acetylated (S)-2-octyl glycosides demonstrated that Rha is L whereas Gal and GlcNAc have the D configuration. The 13C NMR spectrum of the O-polysaccharide (Fig. 1A) showed signals for four anomeric carbons in the region d 99.2– 102.5, one CH3–C group (C-6 of Rha) at d 17.9, three HOCH2–C groups (C-6 of Gal and GlcNAc) at d 61.4–62.3, two nitrogen-bearing carbons (C-2 of GlcNAc) at d 53.9 and 56.8, 14 oxygen-bearing non-anomeric sugar ring carbons in the region d 69.1–82.8, and two N-acetyl groups at d 23.4, 23.6 (both CH3), 175.0, and 175.6 (both CO). Accordingly, the 1H NMR spectrum of the O-polysaccharide contained signals for four anomeric protons at d 4.56–4.99, one CH3–C group (H-6 of Rha) at d 1.21, other sugar protons in the region d 3.40–4.09, and two N-acetyl groups at d 2.07 and 2.09. Therefore, the O-polysaccharide has a tetrasaccharide repeat (O-unit) containing one residue each of L-Rha and D-Gal and two residues of D-GlcNAc. The 1H and 13C NMR spectra of the O-polysaccharide were analyzed using 2D homonuclear 1H, 1H COSY, TOCSY, ROESY, and heteronuclear 1H, 13C HSQC experiments (Table 1). Based on 3JH,H coupling constants estimated from the 2D NMR spectra, spin systems were assigned to four constituent monosaccharides, all being in the pyranose form. The spin systems for GlcNAc were distinguished by correlations between protons at the nitrogen-bearing carbons (H-2) and the corresponding carbons (C-2) at d 4.06/53.9 and 3.82/56.8. J1,2 coupling constants of 7–8 Hz showed that Gal
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A. V. Perepelov et al. / Carbohydrate Research 346 (2011) 381–383
Figure 1.
13
C NMR spectrum of the O-polysaccharide of S. enterica O59. Numbers refer to carbons in sugar residues denoted as shown in Table 1.
Table 1 1 H and 13C NMR chemical shifts (d, ppm) of the O-polysaccharide of S. enterica O59. Sugar residue
?2)-b-D-Galp-(1? A ?3)-a-D-GlcpNAc-(1? B ?4)-a-L-Rhap-(1? C ?3)-b-D-GlcpNAc-(1? D
H-1 C-1
H-2 C-2
H-3 C-3
H-4 C-4
H-5 C-5
H-6 (6a, 6b) C-6
4.56 102.2 4.99 99.2 4.87 102.2 4.80 102.5
3.65 79.7 4.06 53.9 3.78 72.1 3.82 56.8
3.71 75.1 3.93 81.1 3.82 70.2 3.61 82.8
3.88 70.1 3.64 69.3 3.44 82.0 3.47 69.8
3.69 76.3 4.09 72.7 4.09 69.1 3.40 77.1
3.76, 3.76 62.0 3.83, 3.83 61.4 1.21 17.9 3.73, 3.92 62.3
and one of the GlcNAc residues are b-linked, whereas a significantly smaller value of 3 Hz indicated the a-linkage of the other GlcNAc residue. The position of the signal for C-5 at d 69.1 indicated that Rha is a-linked (compare published data d 70.0 and 73.2 for a- and b-Rhap, respectively7). The signals for C-2 of b-Gal, C-3 of a-GlcNAc and b-GlcNAc, and C-4 of a-Rha, were shifted downfield by 6–9 ppm, as compared with their positions in the corresponding non-substituted monosaccharides.7 These data showed that the O-polysaccharide is linear and defined the glycosylation pattern in the O-unit. The ROESY spectrum of the O-polysaccharide showed the following correlations between anomeric protons and protons at the linkage carbons: b-Gal H-1,a-GlcNAc H-3; a-GlcNAc H-1,aRha H-4; a-Rha H-1,b-GlcNAc H-3; and b-GlcNAc H-1,b-Gal H-2 at d 4.56/3.93; 4.99/3.44; 4.87/3.61; and 4.80/3.65, respectively. The monosaccharide sequence thus defined was confirmed by a heteronuclear 1H, 13C HMBC experiment, which showed correlations between anomeric protons and linkage carbons and contrariwise (data not shown). Therefore, the O-polysaccharide of S. enterica O59 has the structure shown in Chart 1, which is unique among the known bacterial polysaccharide structures.
→2)-β-D-Galp-(1→3)-α-D-GlcpNAc-(1→4)-α-L-Rhap-(1→3)-β-D-GlcpNAc-(1→ S. enterica O59 →2)-β-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-β-D-GlcpNAc-(1→ S. arizonae (S, enterica IIIb) O59,5 Citrobacter (C. braakii) O35,6 E. coli O15 Chart 1. Structures of the O-polysaccharides.
NAc CH3
CO
2.09 23.6
175.6
2.07 23.4
175.0
The O-antigen gene cluster of S. enterica O59 was found between the housekeeping genes galF and gnd. Sequencing revealed nine open reading frames (orfs), excluding galF and gnd, all of which have the same transcriptional direction from galF to gnd. The functions were tentatively assigned to all orfs based on their similarity to related genes from the available databases and taking into account the S. enterica O59 antigen structure. orfs1–4 were identified as rmlB, rmlD, rmlA, and rmlC, respectively. The products of the rmlBDAC genes are known to be responsible for the four-step synthesis of dTDP-L-Rha from glucose 1-phosphate.8 Genes for the synthesis of the nucleotide precursors of common sugars, including Gal and GlcNAc, are located outside the O-antigen gene cluster.8 In many S. enterica and E. coli strains, WecA initiates the O-unit synthesis by transfer of GlcNAc 1-phosphate or GalNAc 1-phosphate to an undecaprenol phosphate carrier. The wecA gene maps outside the O-antigen gene cluster.9 Three additional glycosyl transferase genes (wdcF, wdcG, and wdcH), which are responsible for the transfer of three other sugar residues to assemble the full O-unit, were found within the O59-antigen cluster. orf5 and orf9 were identified as wzy and wzx, the O-antigen processing genes that encode O-antigen polymerase and O-unit flippase, respectively. Therefore, the O-antigen gene cluster is in full agreement with the O-antigen structure of S. enterica O59 established in this work. Earlier, another structure has been reported for the O-polysaccharide of S. arizonae (S. enterica IIIb) O59.5 It has a trisaccharide O-unit containing one residue each of D-Gal, D-GlcNAc, and L-FucpNAc (Chart 1). Later, it was found that the O-antigen of Citrobacter (C. braakii) O35 possesses the same structure.6 There are two possible explanations for a discrepancy between the Salmonella O59 antigen structures established earlier5 and in this work. Both O-antigens include a ?3)-b-D-GlcpNAc-(1?2)-b-D-Galp-(1? dis-
A. V. Perepelov et al. / Carbohydrate Research 346 (2011) 381–383
accharide fragment, which could provide a common epitope responsible for the serological relatedness. To prove or disprove this suggestion, a detailed serological study is necessary, which may result in reclassification of one of the strains to a new Salmonella serogroup. Alternatively, the strain studied earlier5 could be classified to the Salmonella O59 serogroup erroneously and, in fact, might be another bacterium, e.g. a strain of C. braakii O35. In our studies of the O-antigens of E. coli, we noticed that the O-polysaccharide of E. coli O15 showed essentially the same 13C NMR spectrum as that of S. arizonae (S. enterica IIIb) O59 reported earlier.5 In this work, we analyzed the former in more detail using 2D 1H and 13C NMR spectroscopy as described above (data not shown) and confirmed that the O-polysaccharides of the two bacteria have the identical structure too (Chart 1). The O-antigen gene cluster of E. coli O15 has been characterized earlier.10 It includes three genes for the synthesis of L-FucNAc (fnlA, fnlB, and fnlC), two glycosyl transferase genes (wbuB and wbuS), and two O-antigen processing genes (wzx for flippase and wzy for polymerase), and is thus in agreement with the O-polysaccharide structure.
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gradient of 160–290 °C at 5 °C min 1. The absolute configurations of the monosaccharides were determined by GLC of the acetylated (S)-2-octyl glycosides as described.14 1.4. NMR spectroscopy O-Polysaccharide samples were deuterium-exchanged by freeze-drying from D2O and then examined as solutions in 99.95% D2O. NMR spectra were recorded on a Bruker Avance 600 spectrometer (Germany) using internal TSP (dH 0) and external acetone (dC 31.45) as references. 2D NMR spectra were obtained using standard Bruker software, and Bruker TOPSPIN program was used to acquire and process the NMR data. Mixing times of 100 and 150 ms were used in TOCSY and ROESY experiments, respectively. 1.5. Sequencing and analysis of genes Sequencing of the chromosome region between galF and gnd, analysis of genes in the O-antigen gene cluster of S. enterica O59, and search of databases for possible gene functions were performed as described.15
1. Experimental Acknowledgments 1.1. Bacterial strains and isolation of lipopolysaccharides S. enterica O59 strain G1463 and E. coli O15 strain G1201 were obtained from the Institute of Medical and Veterinary Science (Adelaide, Australia). Bacteria were grown to late log phase in 8 L of Luria–Bertani broth using a 10-L BIOSTAT C-10 fermentor (B. Braun Biotech Int., Germany) under constant aeration at 37 °C and pH 7.0. Bacterial cells were washed and dried as described.11 The lipopolysaccharides in yields 10% and 6%, respectively, were isolated from dried bacterial cells by the phenol–water method,12 the crude extract was dialyzed without separation of layers and freed from nucleic acids and proteins by treatment with aq 50% CCl3CO2H at pH 2 at 4 °C.13 The supernatant was dialyzed and lyophilized. 1.2. Isolation of O-polysaccharides Delipidation of the lipopolysaccharides was performed with aq 2% HOAc at 100 °C until the precipitation of lipid A. The precipitate was removed by centrifugation (13,000g, 20 min), and the supernatant was fractionated by GPC on a column (56 2.6 cm) of Sephadex G-50 Superfine (Amersham Biosciences, Sweden) in 0.05 M pyridinium acetate buffer pH 4.5 monitored with a differential refractometer (Knauer, Germany). High-molecular-mass Opolysaccharides were obtained in yields 30% and 45% of the lipopolysaccharide mass, respectively. 1.3. Monosaccharide analysis The O-polysaccharide from S. enterica O59 was hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Monosaccharides were identified by GLC of the alditol acetates on a Hewlett-Packard 5890 chromatograph (USA) equipped with an Ultra-1 column using a temperature
This work was supported by the Russian Foundation for Basic Research (projects 08-04-01205 and 08-04-92225), the Chinese National Science Fund for Distinguished Young Scholars (30788001), NSFC General Program Grant 30670038, 30870070, 30870078, 30771175 and 30900041, Tianjin Research Program of Application Foundation and Advanced Technology (10JCYBJC 10000), the National 863 program of China grants 2006AA020703 and 2006AA06Z409, the National 973 program of China grant 2009CB522603, and National Key Programs for Infectious Diseases of China 2009ZX10004-108 and 2008ZX10003-005. References 1. Mead, P. S.; Slutsker, L.; Dietz, V.; McCaig, L. F.; Bresee, J. S.; Shapiro, C.; Griffin, P. M.; Tauxe, R. V. Emerg. Infect. Dis. 1999, 5, 607–625. 2. Knirel, Y. A.; Kochetkov, N. K. Biochemistry (Moscow) 1994, 59, 1325–1383. 3. Jansson, P.-E. In Endotoxin in Health and Disease; Brade, H., Opal, S. M., Vogel, S. N., Morrison, D. C., Eds.; Marcel Dekker: NY, 1999; pp 155–178. 4. Gajdus, J.; Glosnicka, R.; Szafranek, J. Wiadomosci. Chem. 2006, 60, 621–653. 5. Vinogradov, E. V.; Knirel, Y. A.; Lipkind, G. M.; Shashkov, A. S.; Kochetkov, N. K.; Stanislavsky, E. S.; Kholodkova, E. V. Bioorg. Khim. 1987, 13, 1275–1281. 6. Kocharova, N. A.; Knirel, Y. A.; Stanislavsky, E. S.; Kholodkova, E. V.; Lugowski, C.; Jachymek, W.; Romanowska, E. FEMS Immunol. Med. Microbiol. 1996, 13, 1–8. 7. Lipkind, G. M.; Shashkov, A. S.; Knirel, Y. A.; Vinogradov, E. V.; Kochetkov, N. K. Carbohydr. Res. 1988, 175, 59–75. 8. Samuel, G.; Reeves, P. Carbohydr. Res. 2003, 338, 2503–2519. 9. Reeves, P. R.; Wang, L. Curr. Top. Microbiol. Immunol. 2002, 264, 109–135. 10. Beutin, L.; Tao, J.; Feng, L.; Krause, G.; Zimmermann, S.; Gleier, K.; Xia, Q.; Wang, L. J. Clin. Microbiol. 2005, 43, 703–710. 11. Robbins, P. W.; Uchida, T. Biochemistry 1962, 1, 323–335. 12. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83–91. 13. Zych, K.; Toukach, F. V.; Arbatsky, N. P.; Kolodziejska, K.; Senchenkova, S. N.; Shashkov, A. S.; Knirel, Y. A.; Sidorczyk, Z. Eur. J. Biochem. 2001, 268, 4346– 4351. 14. Leontein, K.; Lönngren, J. Methods Carbohydr. Chem. 1993, 9, 87–89. 15. Feng, L.; Senchenkova, S. N.; Yang, J.; Shashkov, A. S.; Tao, J.; Guo, H.; Cheng, J.; Ren, Y.; Knirel, Y. A.; Reeves, P.; Wang, L. J. Bacteriol. 2004, 186, 4510–4519.