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Structure and gene cluster of the O-antigen of Escherichia coli O156 containing a pyruvic acid acetal
Carbohydrate Research 430 (2016) 24–28
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Carbohydrate Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a r r e s
Note
Structure and gene cluster of the O-antigen of Escherichia coli O156 containing a pyruvic acid acetal Zhifeng Duan a, Sof’ya N. Senchenkova b, Xi Guo a, Andrei V. Perepelov b, Alexander S. Shashkov b, Bin Liu a, Yuriy A. Knirel b,* a Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457 Tianjin, China b N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation
A R T I C L E
I N F O
Article history: Received 5 April 2016 Received in revised form 26 April 2016 Accepted 27 April 2016 Available online 29 April 2016 Keywords: Escherichia coli O-polysaccharide O-antigen Bacterial polysaccharide structure O-antigen gene cluster
A B S T R A C T
The lipopolysaccharide of Escherichia coli O156 was degraded under mild acidic and alkaline conditions and the resulting polysaccharides were studied by sugar analysis and 1H and 13C NMR spectroscopy. The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established:
where Rpyr indicates R-configurated pyruvic acid acetal. Minor O-acetyl groups also were present and tentatively localized on the Gal residues. The gene cluster for biosynthesis of the O-antigen of E. coli O156 was analyzed and shown to be consistent with the O-polysaccharide structure. © 2016 Elsevier Ltd. All rights reserved.
Escherichia coli is the predominant facultative anaerobe of the colonic flora of many mammals, including humans, and has both commensal and pathogenic forms. Pathogenic strains causing diarrhea and other disorders belong to 6 categories: enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), and diffusely adherent (DAEC) E. coli.1 O-polysaccharide (O-antigen) is a part of the lipopolysaccharide (LPS) on the outer membrane of gram-negative bacteria and consists usually of oligosaccharide repeats (O-units) containing two to eight residues from a broad range of common and rarely occurring sugars and their derivatives. The O-antigen is the most variable cell constituent with variations in the types of sugars present, their arrangement, and the linkages within and between O-units.2 Genes involved in the O-antigen synthesis are generally combined in a cluster; in E. coli it is usually localized between conserved galF and gnd genes. Most of the genes fall into one of three major classes: sugar nucleotide synthesis genes, glycosyl transferase genes, and O-unit processing genes. Genes for synthesis of sugars that are
* Corresponding author. N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation. Tel.: +7 499 1376148; fax: +7 499 1355328. E-mail address: [email protected] (Y.A. Knirel). http://dx.doi.org/10.1016/j.carres.2016.04.025 0008-6215/© 2016 Elsevier Ltd. All rights reserved.
also present in other bacterial structures or involved in metabolism usually map at other loci. Most variations among diverse O-antigen forms are due to polymorphism of the O-antigen gene cluster.3 Currently, >180 O-serogroups of E. coli are internationally recognized. Sequences of their O-antigen gene clusters have been reported4 and >140 O-antigen structures have been determined (http://nevyn.organ.su.se/ECODAB/). In this work, we report the structure of the O-polysaccharide of E. coli O156, which belongs to Shiga toxin-producing E. coli (STEC) and has been reported to be associated with EHEC-related disease in humans.5 In addition, the O156antigen gene cluster was analyzed and putative gene functions were assigned based on their homology to genes in available databases. Structure elucidation of the O-polysaccharide. Mild acid degradation of LPS obtained from E. coli O156 cells by phenol–water extraction afforded a polysaccharide (PS) isolated by GPC on Sephadex G-50. NMR analysis revealed an irregularity in the PS structure, owing to the presence of O-acetyl groups (δH 2.18–2.19, δC 21.6– 21.8 for Me) and pyruvic acid acetal (δH 1.50, δC 26.3 for Me) in nonstoichiometric amounts. Therefore, LPS was treated with alkali under mild conditions to yield an O-deacylated lipopolysaccharide (LPSOH), with O-polysaccharide chain having a regular structure. Therefore, pyruvic acid was partially cleaved in the course of mild acid hydrolysis of LPS. Indeed, a longer hydrolysis under the same acidic conditions (4 h versus 1 h) resulted in a modified polysaccharide
Z. Duan et al. / Carbohydrate Research 430 (2016) 24–28
25
Table 1 1 H and 13C NMR chemical shifts (δ, ppm) Sugar residue LPSOH (O-polysaccharide chain) →3)-β-d-GlcpNAc-(1→a A →3)-α-l-Fucp-(1→ B →6)-α-d-Galp-(1→ C →3,4)-α-l-Fucp-(1→ D α-d-Galp4,6Rpyr-(1→ E DPSOH →3)-β-d-GlcpNAc-(1→b A →3)-α-l-Fucp-(1→ B →6)-α-d-Galp-(1→ C →3,4)-α-l-Fucp-(1→ D α-d-Galp-(1→ E
Nucleus
1
2
3
4
5
6
1H
4.63 102.1 5.02 100.7 5.19 101.8 4.88 99.8 5.32 101.5
3.84 57.1 3.91 68.4 3.83 69.9.0 4.02 69.3 3.91 69.6
3.69 81.3 3.90 79.7 3.94 70.5 3.96 75.4 4.02 69.1
3.52 70.0 3.95 72.9 4.05 70.5 4.03 78.9 4.26 72.9
3.39 77.5 4.33 68.1 4.25 70.7 4.06 68.3 3.96 63.94
3.67, 3.87 62.3 1.17 16.6 3.57, 3.85 67.6 1.24 17.1 3.90, 4.04 66.3
4.66 102.4 5.04 101.0 4.90 99.9 5.21 101.8 5.31 101.0
3.91c 57.1b 3.91 68.4 3.83 70.0 4.05 69.3 3.81 69.9
3.70 81.2 3.91 79.4 3.96 70.5 4.04 74.7 3.96 70.7
3.59 69.8 3.98 72.9 4.06 70.5 4.14 79.4 4.02 70.8
3.46 77.4 4.36 68.1 4.27 70.7 4.10 68.5 4.21 72.7
3.76, 3.91 62.2 1.17 16.6 3.57, 3.87 67.3 1.27 17.2 3.73, 3.78 62.7
13C 1
H C 1H 13C 1 H 13 C 1H 13C 13
1H 13C 1H 13
C
1H 13C 1H 13
C
1H 13C
Additional chemical shifts for the N-acetyl group are aδH 2.01, δC 23.6 (CH3) and 175.5 (CO); for pyr δH 1.50, δC 26.3 (C-3), 100.9 (C-2) and 175.7 (C-1); bδH 2.04, δC 23.5 (CH3) and 175.7 (CO).
(DPS) that contained only a trace amount of pyruvic acid. Mild alkaline hydrolysis of DPS resulted in an O-deacetylated polysaccharide (DPSOH). The 13C NMR spectrum of LPSOH showed signals for five monosaccharide residues, including five anomeric atoms at δH 4.63– 5.32 and δC 99.8–102.1; two CH3-C groups (C-6 of Fuc) at δH 1.17 and 1.24, δC 16.6 and 17.1; three OCH2-C groups (C-6 of hexoses) at δC 62.3 (non-substituted), 66.3 and 67.6 (both O-substituted); one nitrogen-bearing carbon (C-2 of GlcNAc) at δC 57.1; one acetallinked pyruvic acid residue (pyr) at δH 1.50, δC 26.3 (C-3, Me), 100.9 (C-2) and 175.7 (C-1, CO2H); one N-acetyl group at δH 2.01, δC 23.6 (Me) and 175.5 (CO); and other signals at δH 3.39–4.33 and δC 63.9– 81.3 (Table 1). Signals for OCH2-C and CO groups were assigned using multiplicity-edited 1H,13C HSQC and 1H,13C HMBC experiments, respectively. Sugar analysis of LPSOH using GLC of the alditol acetates after full acid hydrolysis revealed Fuc, Gal, and GlcNAc in the ratios 1.2 : 1.0 : 0.4. GLC analysis of the acetylated glycosides with (+)-2-octanol indicated that Fuc has the l configuration and Gal and GlcNAc have the d configuration. The 1H and 13C NMR spectra of LPSOH were assigned using 2D 1 1 H, H COSY, TOCSY, and 1H,13C HSQC experiments (Table 1). Based on 3JH,H coupling constants and 1H and 13CNMR chemical shifts, spin systems for two residues each of α-Galp (units C and E) and α-Fucp (units B and D), and one residue of β-GlcpNAc (unit A) were identified. The GlcNAc residue was confirmed by correlations between the proton at the nitrogen-bearing carbon (H-2) and the corresponding carbon (C-2) at δ 3.84/57.1 in the 1H,13C HSQC spectrum. Linkage and sequence analysis of LPSOH was performed using the 2D 1H,1H ROESY (Fig. 1) and 1H,13C HMBC (Fig. 2) experiments, which showed correlations between anomeric protons and protons at the linkage carbons (ROESY) or between anomeric protons and linkage carbons and vice versa (HMBC) (Table 2). The sugar substitution pattern in the O-polysaccharide repeat thus defined (Chart 1) was confirmed by significantly downfield displacements due to glycosylation of the signals for C-3 of units A and B, C–3 and C-4 of unit D, and C-6 of unit C (Table 1), as compared with their positions in the spectra of the corresponding non-substituted monosaccharides.6
Similarly, a structure of DPSOH (Chart 1) was established by 2D NMR spectroscopy (for assigned 1H and 13C NMR chemical shifts of DPSOH, see Table 1) and found to be in full agreement with the data of LPSOH. A comparison of the 13C NMR and 2D 1H,13C HSQC spectra of DPSOH and LPSOH revealed downfield displacements of the signals for C-4 and C-6 of unit E from δ 70.8 and 62.7 in DPSOH to δ 72.9 and 66.3 in LPSOH and an upfield displacement of the signal for C-5 of unit E from δ 72.7 to δ 63.9. This finding indicated that pyr is localized at positions 4 and 6 of unit E. The chemical shifts of the methyl group δ H 1.50 and δ C 26.3 showed that pyr has the R configuration.7 Therefore, the polysaccharide chain in LPS OH includes d-Galp4,6Rpyr and has the structure shown in Chart 1. Earlier, d-GlcpNAc4,6Spyr has been found in the O-polysaccharides of E. coli O112ac8 and O149,9 whereas d-Galp4,6Rpyr is identified in E. coli for the first time. A comparison of the NMR spectra of DPSOH and DPS revealed a displacement of a minor part of the H-1/C-1 cross-peak from δ 5.29/ 101.0 in the former to δ 5.34/98.6 in the latter. This displacement was evidently due to a β-effect of O-acetylation and showed that one of the O-acetyl groups occurs at position 2 of unit E. As judged by relative intensities of the signals for H-1 of the O-acetylated and non-acetylated unit E in DPS, the degree of 2-O-acetylation was ~35%. Positions of other minor O-acetyl groups could not be unambiguously determined by NMR spectroscopy. However, signals for units A, B, and D were essentially the same in the spectra of DPSOH and DPS, and hence these units were not O-acetylated. Therefore, the minor O-acetyl groups could be localized on a Gal residue(s) at position 3 (unit C or/and E) or/and position 4 (unit C). Characterization of the O-antigen gene cluster. The O-antigen gene cluster of E. coli O156 between the housekeeping genes galF and gnd has been sequenced (GenBank accession number AB812065).4 It contains 14 ORFs having the same transcription direction from galF to gnd (Fig. 3). d-GlcNAc and d-Gal that are contained in the O156-polysaccharide are common sugars in bacteria, and genes for synthesis of their nucleotide precursors are usually located outside the O-antigen gene cluster. 10 Hence, no such genes were found between galF and gnd in O156 too. The biosynthesis pathway of GDP-l-Fuc, the nucleotide precursor of l-Fuc, has been
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Fig. 1. Part of a 2D ROESY spectrum of the alkali-treated lipopolysaccharide (LPSOH) from E. coli O156. The corresponding parts of the 1H NMR spectrum are displayed along the axes. Arabic numerals refer to protons in sugar residues denoted by letters as shown in Table 1 and Chart 1.
Fig. 2. Part of a 2D 1H,13C HMBC spectrum of the alkali-treated lipopolysaccharide (LPSOH) form E. coli O156. The corresponding parts of the 1H and 13C NMR spectra are displayed along the horizontal and vertical axis, respectively. Arabic numerals before and after oblique stroke refer to protons and carbons, respectively, in sugar residues denoted by letters as shown in Table 1 and Chart 1.
Z. Duan et al. / Carbohydrate Research 430 (2016) 24–28
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Table 2 Correlations for H-1 and C-1 in the 2D 1H,1H ROESY and 1H,13C HMBC spectra Atom in sugar residue (δ)
A H–1 (4.63) A S–1 (102.1) B H–1 (5.02) B S–1 (100.7) C H–1 (5.19) C S–1 (101.8) D H–1 (4.88) D S–1 (99.8) E H–1 (5.32) E S–1 (101.5)
Correlations to atom in sugar residue (δ) ROESY
HMBC
D H–4 (4.03), A H–3 (3.69), A H–5 (3.39)
D C–4 (78.9) D H–4 (4.03), A H-2 (3.84), A H–5 (3.39) A C–3 (81.3), B C–3 (79.7), B C–5 (68.1) A H–3 (3.69), B H-2 (3.91) B C–3 (79.7), C C–3,5 (70.5–70.7) C H-3 (3.94), C H-5 (4.25) D C–3 (75.4) , D C–5 (68.3) D H–6a (3.57), D H–6b (3.85), D H-5 (4.06) D C–3 (75.4), E C–3 (69.1) , E C–5 (63.9) D H–3 and/or E H-5 (3.96)
A H–3 (3.69) B H–3 (3.90), C H–2 (3.83), C H–3 (3.94) C H–6a (3.57) D H–3 (3.96), E H–2 (3.81), E H–3 (4.02)
elucidated.11 It starts from fructose 6-phosphate and involves sequential reactions catalyzed by phosphomannose isomerase (ManA), phosphomannomutase (ManB), mannose-1-phosphate guanylyltransferase (ManC), GDP-d-mannose 4,6-dehydratase (Gmd), and GDP-l-fucose synthetase (Fcl). The corresponding manB, manC, gmd, and fcl genes were identified in the O156 gene cluster. As in other E. coli clones, manA gene was located outside the O-antigen gene cluster, and gmm gene was present downstream of fcl. The product of the latter catalyzes hydrolysis of GDP-d-Man into GDP and d-Man, and participates in the cell wall biosynthesis by regulation of the concentration of GDP-d-Man.12 As in many other E. coli serogroups, GlcNAc is evidently the first sugar of the O-unit in O156. The wecA gene, which is responsible for the transfer of d-GlcNAc 1-phosphate from UDP-d-GlcNAc to undecaprenyl phosphate (Und-P) to give UndPP-d-GlcNAc, is localized in the enterobacterial common antigen gene cluster13 and is not duplicated in the O-antigen gene cluster. There were four putative glycosyltransferase genes (orfs5, 7, 8, 13) in the O156antigen gene cluster, conforming with the requirement of the assembly of the pentasaccharide O-unit. Orf13 of E. coli O156 and WfeY of E. coli O168 share 99% identity, and the O-polysaccharides of both bacteria have the α-l-Fucp-(1→3)-d-GlcpNAc linkage in common. Therefore, it is suggested that orf13 be named wefY and the product of both genes be responsible for the formation of this linkage. The exact functions of orf5, 7, and 8 could not be inferred by homology as no significant similarity was observed between them and other glycosyltransferases with assigned functions. Orf4 was predicted to be pyruvyl transferase that is responsible for formation of the pyruvic acid acetal on the side-chain Gal residue. No expected acetyltransferase gene that would account for either 2-O-acetylation of the side-chain Gal residue or minor O-acetylation at other sites was identified in the O156 gene cluster,
thus indicating its location elsewhere in the genome. Finally, the O-antigen gene cluster includes wzx and wzy genes, which encode O-antigen processing proteins: flippase and O-antigen polymerase, respectively. The presence of these genes indicates that the O156-polysaccharide is synthesized by the Wzx/Wzy-dependent pathway.14 1. Experimental 1.1. Cultivation of bacteria and isolation of the lipopolysaccharide E. coli O156 type strain (laboratory stock number G2315) was obtained from the Institute of Medical and Veterinary Science, Adelaide, Australia (IMVS). Bacteria were grown to late log phase in 8 L of LB using a 10-L fermentor (BIOSTAT C-10, B. Braun Biotech International, Germany) under constant aeration at 37 °C and pH 7.0. Bacterial cells were washed and dried as described.15 The lipopolysaccharide was isolated in a yield 6.8% from dried cells by the phenol–water method16; the crude extract was dialyzed without separation of the layers and freed from nucleic acids and proteins by treatment with 50% aq CCl3CO2H to pH 2 at 4 °C. The supernatant was dialyzed and lyophilized. 1.2. Isolation of polysaccharides Mild alkaline degradation of the LPS (85 mg) was performed with aq 12.5% ammonia at 37 °C (2.5 h) and LPSOH (4 5 mg) was isolated 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 using a differential refractometer (Knauer, Germany).
Chart 1. Structure of the polysaccharide chain in LPSOH (top) and the modified polysaccharide DPSOH (bottom) from E. coli O156. Rpyr indicates (R)-1-carboxyethylidene (pyruvic acid acetal).
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d4 (δH 0, δC −1.6) as reference for calibration. 2D NMR spectra were obtained using standard Bruker software, and Bruker TopSpin 2.1 program was used to acquire and process the NMR data. A mixing time of 100 and 150 ms was used in TOCSY and ROESY experiments, respectively. Acknowledgements Fig. 3. Organization and content of the O-antigen gene cluster of E. coli O156.
Mild acid degradation of LPS samples (100 mg each) was performed with aq 2% HOAc at 100 °C until precipitation of lipid (1 or 4 h). The precipitate was removed by centrifugation (13,000 × g, 20 min), and the supernatant was fractionated by GPC on Sephadex G-50 Superfine as described above to give PS or DPS (22 and 17 mg, respectively). A DPS sample (17 mg) was treated with 12.5% aqueous ammonia (37 °C, 16 h); ammonia was removed in a stream of air, and DPSOH (14 mg) was isolated by GPC on TSK HW-40 (S) (Toyo Pearl) in 1% HOAc/H2O monitored using a differential refractometer (Knauer). 1.3. Sugar analyses A LPSOH sample (0.5 mg) was hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Monosaccharides were identified by GLC of the alditol acetates on a Maestro (Agilent 7820) chromatograph (Interlab, Russia) equipped with an HP-5 column (0.32 mm × 30 m) and a temperature program of 160 °C (1 min) to 290 °C at 7 °C min−1. The absolute configurations of the monosaccharides were determined by GLC of the acetylated (S)-2-octyl glycosides as described.17
This work was supported by the Russian Science Foundation (grant 14-14-01042 for Y. A. K.). Z.D., X.G., and B.L. were supported by the International Science & Technology Cooperation Program of China (2012DFG31680 and 2013DFR30640), the National Key Program for Infectious Diseases of China (2013ZX10004216-001-001 and 2013ZX10004221-003), the National Natural Science Foundation of China (NSFC) Program (31371259 and 81471904), and the Research Project of Chinese Ministry of Education (No. 113015A). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
1.4. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying from 99.9% D2O and then examined as solution in 99.95% D2O. NMR spectra were recorded on a Bruker Avance II 600 MHz spectrometer (Germany) at 40 °C using internal sodium 3-trimethylsilylpropanoate-2,2,3,3-
11. 12. 13. 14. 15. 16. 17.
Kaper JB, Nataro JP, Mobley HL. Nat Rev Microbiol 2004;2:123–40. Reeves PR, Wang L. Curr Top Microbiol Immunol 2002;264:109–35. Samuel G, Reeves PR. Carbohydr Res 2003;338:2503–19. Iguchi A, Iyoda S, Kikuchi T, Ogura Y, Katsura K, Ohnishi M, et al. DNA Res 2015;22:101–7. Geue L, Schares S, Mintel B, Conraths FJ, Müller E, Ehricht R. Appl Environ Microbiol 2010;76:5510–9. Jansson PE, Kenne L, Widmalm G. Carbohydr Res 1989;188:169–91. Garegg PJ, Jansson P-E, Lindberg B, Lindh F, Lönngren J, Kvarnström I, et al. Carbohydr Res 1980;78:127–32. Perepelov AV, Weintraub A, Liu B, Senchenkova SN, Shashkov AS, Feng L, et al. Carbohydr Res 2008;343:977–81. Adeyeye A, Jansson PE, Lindberg B, Abaas S, Svenson SB. Carbohydr Res 1988;176:231–6. Liu B, Knirel YA, Feng L, Perepelov AV, Senchenkova SN, Wang Q, et al. FEMS Microbiol Rev 2008;32:627–53. Ginsburg V. J Biol Chem 1961;236:2389–93. Frick DN, Townsend BD, Bessman MJ. J Biol Chem 1995;270:24086–91. Lehrer J, Vigeant KA, Tatar LD, Valvano MA. J Bacteriol 2007;189:2618–28. Raetz CR, Whitfield C. Annu Rev Biochem 2002;71:635–700. Robbins PW, Uchida T. Biochemistry 1962;1:323–35. Westphal O, Jann K. Methods Carbohydr Chem 1965;5:83–91. Leontein K, Lönngren J. Methods Carbohydr Chem 1993;9:87–9.
Contents lists available at ScienceDirect
Carbohydrate Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a r r e s
Note
Structure and gene cluster of the O-antigen of Escherichia coli O156 containing a pyruvic acid acetal Zhifeng Duan a, Sof’ya N. Senchenkova b, Xi Guo a, Andrei V. Perepelov b, Alexander S. Shashkov b, Bin Liu a, Yuriy A. Knirel b,* a Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457 Tianjin, China b N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation
A R T I C L E
I N F O
Article history: Received 5 April 2016 Received in revised form 26 April 2016 Accepted 27 April 2016 Available online 29 April 2016 Keywords: Escherichia coli O-polysaccharide O-antigen Bacterial polysaccharide structure O-antigen gene cluster
A B S T R A C T
The lipopolysaccharide of Escherichia coli O156 was degraded under mild acidic and alkaline conditions and the resulting polysaccharides were studied by sugar analysis and 1H and 13C NMR spectroscopy. The following structure of the pentasaccharide repeating unit of the O-polysaccharide was established:
where Rpyr indicates R-configurated pyruvic acid acetal. Minor O-acetyl groups also were present and tentatively localized on the Gal residues. The gene cluster for biosynthesis of the O-antigen of E. coli O156 was analyzed and shown to be consistent with the O-polysaccharide structure. © 2016 Elsevier Ltd. All rights reserved.
Escherichia coli is the predominant facultative anaerobe of the colonic flora of many mammals, including humans, and has both commensal and pathogenic forms. Pathogenic strains causing diarrhea and other disorders belong to 6 categories: enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), and diffusely adherent (DAEC) E. coli.1 O-polysaccharide (O-antigen) is a part of the lipopolysaccharide (LPS) on the outer membrane of gram-negative bacteria and consists usually of oligosaccharide repeats (O-units) containing two to eight residues from a broad range of common and rarely occurring sugars and their derivatives. The O-antigen is the most variable cell constituent with variations in the types of sugars present, their arrangement, and the linkages within and between O-units.2 Genes involved in the O-antigen synthesis are generally combined in a cluster; in E. coli it is usually localized between conserved galF and gnd genes. Most of the genes fall into one of three major classes: sugar nucleotide synthesis genes, glycosyl transferase genes, and O-unit processing genes. Genes for synthesis of sugars that are
* Corresponding author. N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation. Tel.: +7 499 1376148; fax: +7 499 1355328. E-mail address: [email protected] (Y.A. Knirel). http://dx.doi.org/10.1016/j.carres.2016.04.025 0008-6215/© 2016 Elsevier Ltd. All rights reserved.
also present in other bacterial structures or involved in metabolism usually map at other loci. Most variations among diverse O-antigen forms are due to polymorphism of the O-antigen gene cluster.3 Currently, >180 O-serogroups of E. coli are internationally recognized. Sequences of their O-antigen gene clusters have been reported4 and >140 O-antigen structures have been determined (http://nevyn.organ.su.se/ECODAB/). In this work, we report the structure of the O-polysaccharide of E. coli O156, which belongs to Shiga toxin-producing E. coli (STEC) and has been reported to be associated with EHEC-related disease in humans.5 In addition, the O156antigen gene cluster was analyzed and putative gene functions were assigned based on their homology to genes in available databases. Structure elucidation of the O-polysaccharide. Mild acid degradation of LPS obtained from E. coli O156 cells by phenol–water extraction afforded a polysaccharide (PS) isolated by GPC on Sephadex G-50. NMR analysis revealed an irregularity in the PS structure, owing to the presence of O-acetyl groups (δH 2.18–2.19, δC 21.6– 21.8 for Me) and pyruvic acid acetal (δH 1.50, δC 26.3 for Me) in nonstoichiometric amounts. Therefore, LPS was treated with alkali under mild conditions to yield an O-deacylated lipopolysaccharide (LPSOH), with O-polysaccharide chain having a regular structure. Therefore, pyruvic acid was partially cleaved in the course of mild acid hydrolysis of LPS. Indeed, a longer hydrolysis under the same acidic conditions (4 h versus 1 h) resulted in a modified polysaccharide
Z. Duan et al. / Carbohydrate Research 430 (2016) 24–28
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Table 1 1 H and 13C NMR chemical shifts (δ, ppm) Sugar residue LPSOH (O-polysaccharide chain) →3)-β-d-GlcpNAc-(1→a A →3)-α-l-Fucp-(1→ B →6)-α-d-Galp-(1→ C →3,4)-α-l-Fucp-(1→ D α-d-Galp4,6Rpyr-(1→ E DPSOH →3)-β-d-GlcpNAc-(1→b A →3)-α-l-Fucp-(1→ B →6)-α-d-Galp-(1→ C →3,4)-α-l-Fucp-(1→ D α-d-Galp-(1→ E
Nucleus
1
2
3
4
5
6
1H
4.63 102.1 5.02 100.7 5.19 101.8 4.88 99.8 5.32 101.5
3.84 57.1 3.91 68.4 3.83 69.9.0 4.02 69.3 3.91 69.6
3.69 81.3 3.90 79.7 3.94 70.5 3.96 75.4 4.02 69.1
3.52 70.0 3.95 72.9 4.05 70.5 4.03 78.9 4.26 72.9
3.39 77.5 4.33 68.1 4.25 70.7 4.06 68.3 3.96 63.94
3.67, 3.87 62.3 1.17 16.6 3.57, 3.85 67.6 1.24 17.1 3.90, 4.04 66.3
4.66 102.4 5.04 101.0 4.90 99.9 5.21 101.8 5.31 101.0
3.91c 57.1b 3.91 68.4 3.83 70.0 4.05 69.3 3.81 69.9
3.70 81.2 3.91 79.4 3.96 70.5 4.04 74.7 3.96 70.7
3.59 69.8 3.98 72.9 4.06 70.5 4.14 79.4 4.02 70.8
3.46 77.4 4.36 68.1 4.27 70.7 4.10 68.5 4.21 72.7
3.76, 3.91 62.2 1.17 16.6 3.57, 3.87 67.3 1.27 17.2 3.73, 3.78 62.7
13C 1
H C 1H 13C 1 H 13 C 1H 13C 13
1H 13C 1H 13
C
1H 13C 1H 13
C
1H 13C
Additional chemical shifts for the N-acetyl group are aδH 2.01, δC 23.6 (CH3) and 175.5 (CO); for pyr δH 1.50, δC 26.3 (C-3), 100.9 (C-2) and 175.7 (C-1); bδH 2.04, δC 23.5 (CH3) and 175.7 (CO).
(DPS) that contained only a trace amount of pyruvic acid. Mild alkaline hydrolysis of DPS resulted in an O-deacetylated polysaccharide (DPSOH). The 13C NMR spectrum of LPSOH showed signals for five monosaccharide residues, including five anomeric atoms at δH 4.63– 5.32 and δC 99.8–102.1; two CH3-C groups (C-6 of Fuc) at δH 1.17 and 1.24, δC 16.6 and 17.1; three OCH2-C groups (C-6 of hexoses) at δC 62.3 (non-substituted), 66.3 and 67.6 (both O-substituted); one nitrogen-bearing carbon (C-2 of GlcNAc) at δC 57.1; one acetallinked pyruvic acid residue (pyr) at δH 1.50, δC 26.3 (C-3, Me), 100.9 (C-2) and 175.7 (C-1, CO2H); one N-acetyl group at δH 2.01, δC 23.6 (Me) and 175.5 (CO); and other signals at δH 3.39–4.33 and δC 63.9– 81.3 (Table 1). Signals for OCH2-C and CO groups were assigned using multiplicity-edited 1H,13C HSQC and 1H,13C HMBC experiments, respectively. Sugar analysis of LPSOH using GLC of the alditol acetates after full acid hydrolysis revealed Fuc, Gal, and GlcNAc in the ratios 1.2 : 1.0 : 0.4. GLC analysis of the acetylated glycosides with (+)-2-octanol indicated that Fuc has the l configuration and Gal and GlcNAc have the d configuration. The 1H and 13C NMR spectra of LPSOH were assigned using 2D 1 1 H, H COSY, TOCSY, and 1H,13C HSQC experiments (Table 1). Based on 3JH,H coupling constants and 1H and 13CNMR chemical shifts, spin systems for two residues each of α-Galp (units C and E) and α-Fucp (units B and D), and one residue of β-GlcpNAc (unit A) were identified. The GlcNAc residue was confirmed by correlations between the proton at the nitrogen-bearing carbon (H-2) and the corresponding carbon (C-2) at δ 3.84/57.1 in the 1H,13C HSQC spectrum. Linkage and sequence analysis of LPSOH was performed using the 2D 1H,1H ROESY (Fig. 1) and 1H,13C HMBC (Fig. 2) experiments, which showed correlations between anomeric protons and protons at the linkage carbons (ROESY) or between anomeric protons and linkage carbons and vice versa (HMBC) (Table 2). The sugar substitution pattern in the O-polysaccharide repeat thus defined (Chart 1) was confirmed by significantly downfield displacements due to glycosylation of the signals for C-3 of units A and B, C–3 and C-4 of unit D, and C-6 of unit C (Table 1), as compared with their positions in the spectra of the corresponding non-substituted monosaccharides.6
Similarly, a structure of DPSOH (Chart 1) was established by 2D NMR spectroscopy (for assigned 1H and 13C NMR chemical shifts of DPSOH, see Table 1) and found to be in full agreement with the data of LPSOH. A comparison of the 13C NMR and 2D 1H,13C HSQC spectra of DPSOH and LPSOH revealed downfield displacements of the signals for C-4 and C-6 of unit E from δ 70.8 and 62.7 in DPSOH to δ 72.9 and 66.3 in LPSOH and an upfield displacement of the signal for C-5 of unit E from δ 72.7 to δ 63.9. This finding indicated that pyr is localized at positions 4 and 6 of unit E. The chemical shifts of the methyl group δ H 1.50 and δ C 26.3 showed that pyr has the R configuration.7 Therefore, the polysaccharide chain in LPS OH includes d-Galp4,6Rpyr and has the structure shown in Chart 1. Earlier, d-GlcpNAc4,6Spyr has been found in the O-polysaccharides of E. coli O112ac8 and O149,9 whereas d-Galp4,6Rpyr is identified in E. coli for the first time. A comparison of the NMR spectra of DPSOH and DPS revealed a displacement of a minor part of the H-1/C-1 cross-peak from δ 5.29/ 101.0 in the former to δ 5.34/98.6 in the latter. This displacement was evidently due to a β-effect of O-acetylation and showed that one of the O-acetyl groups occurs at position 2 of unit E. As judged by relative intensities of the signals for H-1 of the O-acetylated and non-acetylated unit E in DPS, the degree of 2-O-acetylation was ~35%. Positions of other minor O-acetyl groups could not be unambiguously determined by NMR spectroscopy. However, signals for units A, B, and D were essentially the same in the spectra of DPSOH and DPS, and hence these units were not O-acetylated. Therefore, the minor O-acetyl groups could be localized on a Gal residue(s) at position 3 (unit C or/and E) or/and position 4 (unit C). Characterization of the O-antigen gene cluster. The O-antigen gene cluster of E. coli O156 between the housekeeping genes galF and gnd has been sequenced (GenBank accession number AB812065).4 It contains 14 ORFs having the same transcription direction from galF to gnd (Fig. 3). d-GlcNAc and d-Gal that are contained in the O156-polysaccharide are common sugars in bacteria, and genes for synthesis of their nucleotide precursors are usually located outside the O-antigen gene cluster. 10 Hence, no such genes were found between galF and gnd in O156 too. The biosynthesis pathway of GDP-l-Fuc, the nucleotide precursor of l-Fuc, has been
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Fig. 1. Part of a 2D ROESY spectrum of the alkali-treated lipopolysaccharide (LPSOH) from E. coli O156. The corresponding parts of the 1H NMR spectrum are displayed along the axes. Arabic numerals refer to protons in sugar residues denoted by letters as shown in Table 1 and Chart 1.
Fig. 2. Part of a 2D 1H,13C HMBC spectrum of the alkali-treated lipopolysaccharide (LPSOH) form E. coli O156. The corresponding parts of the 1H and 13C NMR spectra are displayed along the horizontal and vertical axis, respectively. Arabic numerals before and after oblique stroke refer to protons and carbons, respectively, in sugar residues denoted by letters as shown in Table 1 and Chart 1.
Z. Duan et al. / Carbohydrate Research 430 (2016) 24–28
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Table 2 Correlations for H-1 and C-1 in the 2D 1H,1H ROESY and 1H,13C HMBC spectra Atom in sugar residue (δ)
A H–1 (4.63) A S–1 (102.1) B H–1 (5.02) B S–1 (100.7) C H–1 (5.19) C S–1 (101.8) D H–1 (4.88) D S–1 (99.8) E H–1 (5.32) E S–1 (101.5)
Correlations to atom in sugar residue (δ) ROESY
HMBC
D H–4 (4.03), A H–3 (3.69), A H–5 (3.39)
D C–4 (78.9) D H–4 (4.03), A H-2 (3.84), A H–5 (3.39) A C–3 (81.3), B C–3 (79.7), B C–5 (68.1) A H–3 (3.69), B H-2 (3.91) B C–3 (79.7), C C–3,5 (70.5–70.7) C H-3 (3.94), C H-5 (4.25) D C–3 (75.4) , D C–5 (68.3) D H–6a (3.57), D H–6b (3.85), D H-5 (4.06) D C–3 (75.4), E C–3 (69.1) , E C–5 (63.9) D H–3 and/or E H-5 (3.96)
A H–3 (3.69) B H–3 (3.90), C H–2 (3.83), C H–3 (3.94) C H–6a (3.57) D H–3 (3.96), E H–2 (3.81), E H–3 (4.02)
elucidated.11 It starts from fructose 6-phosphate and involves sequential reactions catalyzed by phosphomannose isomerase (ManA), phosphomannomutase (ManB), mannose-1-phosphate guanylyltransferase (ManC), GDP-d-mannose 4,6-dehydratase (Gmd), and GDP-l-fucose synthetase (Fcl). The corresponding manB, manC, gmd, and fcl genes were identified in the O156 gene cluster. As in other E. coli clones, manA gene was located outside the O-antigen gene cluster, and gmm gene was present downstream of fcl. The product of the latter catalyzes hydrolysis of GDP-d-Man into GDP and d-Man, and participates in the cell wall biosynthesis by regulation of the concentration of GDP-d-Man.12 As in many other E. coli serogroups, GlcNAc is evidently the first sugar of the O-unit in O156. The wecA gene, which is responsible for the transfer of d-GlcNAc 1-phosphate from UDP-d-GlcNAc to undecaprenyl phosphate (Und-P) to give UndPP-d-GlcNAc, is localized in the enterobacterial common antigen gene cluster13 and is not duplicated in the O-antigen gene cluster. There were four putative glycosyltransferase genes (orfs5, 7, 8, 13) in the O156antigen gene cluster, conforming with the requirement of the assembly of the pentasaccharide O-unit. Orf13 of E. coli O156 and WfeY of E. coli O168 share 99% identity, and the O-polysaccharides of both bacteria have the α-l-Fucp-(1→3)-d-GlcpNAc linkage in common. Therefore, it is suggested that orf13 be named wefY and the product of both genes be responsible for the formation of this linkage. The exact functions of orf5, 7, and 8 could not be inferred by homology as no significant similarity was observed between them and other glycosyltransferases with assigned functions. Orf4 was predicted to be pyruvyl transferase that is responsible for formation of the pyruvic acid acetal on the side-chain Gal residue. No expected acetyltransferase gene that would account for either 2-O-acetylation of the side-chain Gal residue or minor O-acetylation at other sites was identified in the O156 gene cluster,
thus indicating its location elsewhere in the genome. Finally, the O-antigen gene cluster includes wzx and wzy genes, which encode O-antigen processing proteins: flippase and O-antigen polymerase, respectively. The presence of these genes indicates that the O156-polysaccharide is synthesized by the Wzx/Wzy-dependent pathway.14 1. Experimental 1.1. Cultivation of bacteria and isolation of the lipopolysaccharide E. coli O156 type strain (laboratory stock number G2315) was obtained from the Institute of Medical and Veterinary Science, Adelaide, Australia (IMVS). Bacteria were grown to late log phase in 8 L of LB using a 10-L fermentor (BIOSTAT C-10, B. Braun Biotech International, Germany) under constant aeration at 37 °C and pH 7.0. Bacterial cells were washed and dried as described.15 The lipopolysaccharide was isolated in a yield 6.8% from dried cells by the phenol–water method16; the crude extract was dialyzed without separation of the layers and freed from nucleic acids and proteins by treatment with 50% aq CCl3CO2H to pH 2 at 4 °C. The supernatant was dialyzed and lyophilized. 1.2. Isolation of polysaccharides Mild alkaline degradation of the LPS (85 mg) was performed with aq 12.5% ammonia at 37 °C (2.5 h) and LPSOH (4 5 mg) was isolated 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 using a differential refractometer (Knauer, Germany).
Chart 1. Structure of the polysaccharide chain in LPSOH (top) and the modified polysaccharide DPSOH (bottom) from E. coli O156. Rpyr indicates (R)-1-carboxyethylidene (pyruvic acid acetal).
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d4 (δH 0, δC −1.6) as reference for calibration. 2D NMR spectra were obtained using standard Bruker software, and Bruker TopSpin 2.1 program was used to acquire and process the NMR data. A mixing time of 100 and 150 ms was used in TOCSY and ROESY experiments, respectively. Acknowledgements Fig. 3. Organization and content of the O-antigen gene cluster of E. coli O156.
Mild acid degradation of LPS samples (100 mg each) was performed with aq 2% HOAc at 100 °C until precipitation of lipid (1 or 4 h). The precipitate was removed by centrifugation (13,000 × g, 20 min), and the supernatant was fractionated by GPC on Sephadex G-50 Superfine as described above to give PS or DPS (22 and 17 mg, respectively). A DPS sample (17 mg) was treated with 12.5% aqueous ammonia (37 °C, 16 h); ammonia was removed in a stream of air, and DPSOH (14 mg) was isolated by GPC on TSK HW-40 (S) (Toyo Pearl) in 1% HOAc/H2O monitored using a differential refractometer (Knauer). 1.3. Sugar analyses A LPSOH sample (0.5 mg) was hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Monosaccharides were identified by GLC of the alditol acetates on a Maestro (Agilent 7820) chromatograph (Interlab, Russia) equipped with an HP-5 column (0.32 mm × 30 m) and a temperature program of 160 °C (1 min) to 290 °C at 7 °C min−1. The absolute configurations of the monosaccharides were determined by GLC of the acetylated (S)-2-octyl glycosides as described.17
This work was supported by the Russian Science Foundation (grant 14-14-01042 for Y. A. K.). Z.D., X.G., and B.L. were supported by the International Science & Technology Cooperation Program of China (2012DFG31680 and 2013DFR30640), the National Key Program for Infectious Diseases of China (2013ZX10004216-001-001 and 2013ZX10004221-003), the National Natural Science Foundation of China (NSFC) Program (31371259 and 81471904), and the Research Project of Chinese Ministry of Education (No. 113015A). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
1.4. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying from 99.9% D2O and then examined as solution in 99.95% D2O. NMR spectra were recorded on a Bruker Avance II 600 MHz spectrometer (Germany) at 40 °C using internal sodium 3-trimethylsilylpropanoate-2,2,3,3-
11. 12. 13. 14. 15. 16. 17.
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