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Structure elucidation and analysis of biosynthesis genes of the O-antigen of Escherichia coli O131 containing N-acetylneuraminic acid
Carbohydrate Research 436 (2016) 41e44
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Structure elucidation and analysis of biosynthesis genes of the O-antigen of Escherichia coli O131 containing N-acetylneuraminic acid Andrei V. Perepelov a, *, Xi Guo b, Sof'ya N. Senchenkova a, Alexander S. Shashkov a, Bin Liu b, Yuriy A. Knirel a a
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991, Moscow, Russian Federation Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 September 2016 Received in revised form 3 November 2016 Accepted 3 November 2016 Available online 4 November 2016
The O-polysaccharide (O-antigen) of Escherichia coli O131 was studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established: /8)-a-Neup5Ac-(2 / 6)-b-D-Galp-(1 / 6)-b-D-Galp-(1 / 3)-b-D-GalpNAc-(1/ The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in agreement with the E. coli O131-antigen structure. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Escherichia coli Bacterial polysaccharide structure O-polysaccharide structure N-acetylneuraminic acid Lipopolysaccharide O-antigen gene cluster
Escherichia coli is the predominant facultative anaerobe of the colonic flora of many mammals, including humans, and has both commensal and pathogenic forms [1]. O-polysaccharide, or O-antigen, which contains a number of oligosaccharide repeats (Ounits), is a part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. Variations in sugar composition, arrangement of monosaccharide residues, and linkages within and between the O-units make the O-antigen the most variable constituents on the cell surface and provides the basis for serotyping of many Gram-negative bacteria [2]. Genes involved in the O-antigen synthesis are generally combined in a cluster; in E. coli this is usually located 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 also present in other bacterial structures or involved in metabolism usually map at other loci. A high diversity of the O-antigen forms is mainly due to genetic variations in the Oantigen gene cluster [3]. Currently, >180 O-serogroups of E. coli are
* Corresponding author. E-mail address: [email protected] (A.V. Perepelov). http://dx.doi.org/10.1016/j.carres.2016.11.003 0008-6215/© 2016 Elsevier Ltd. All rights reserved.
internationally recognized, and sequences of their O-antigen gene clusters have been reported [4]. Most E. coli O-antigen structures have been determined (http://nevyn.organ.su.se/ECODAB/) but some remain unknown. In this work, we established the structure of the O-polysaccharide of E. coli O131, which has been reported to be associated with pigs with post-weaning diarrhea [5]. The O131-antigen gene cluster was analyzed and found to be consistent with the O-polysaccharide structure. Structure elucidtaion of the O-polysaccharide. LPS was isolated from E. coli O131 cells by extraction with hot phenol-water. On degradation with dilute acetic acid, the LPS afforded no expected high-molecular mass polysaccharide but an oligosaccharide (OS), most likely, owing to a cleavage of an acid-labile glycosidic linkage of an O-polysaccharide component. Further studies showed that this component was N-acetylneuraminic acid (Neu5Ac). Sugar analysis using GLC of the alditol acetates derived after full acid hydrolysis of the OS revealed GalN and Gal in the ratio ~0.5:1.0. The 13C NMR spectrum of the OS (Fig. 1, A) contained signals for four anomeric carbons at d 97.4 (quaternary carbon; low intense signal identified as C-2 of Neu5Ac), 101.8, 104.8, and 105.9, four HOCH2eC groups (C-6 of Gal and GalN residues, C-9 of Neu5Ac) at
42
A.V. Perepelov et al. / Carbohydrate Research 436 (2016) 41e44
Fig. 1.
13
C NMR spectra of the OS (top) and LPSOH (bottom) from E. coli O131. Numbers refer to carbons in sugar residues denoted by letters as shown in Table 1.
d 62.2 (signal of triple integral intense) and 70.5 (data of a DEPT experiment), two nitrogen-bearing carbons (C-2 of GalN and C-5 of Neu5Ac) at d 53.0 and 53.6, respectively, one CeCH2eC group (C-3 of Neu5Ac) at d 40.6, 15 oxygen-bearing sugar ring carbons in the region d 68.6e81.2, two N-acetyl groups at d 23.4 and 23.6 (both CH3), and CO groups of NAc and C-1 of Neu5Ac at d 176.0e176.1. The 1H NMR spectrum of the OS showed signals for three anomeric protons of b-linked pyranosides (H-1 of Gal and GalNAc) at d 4.41, 4.43, and 4.64 (all doublets, J ~8 Hz), one CHeCH2eC group (H-3 of Neu5Ac) at d 1.82 (H-3ax, triplet, J ~11 Hz) and d 2.24 (H3eq, doublet of doublets, J ~5 and ~13 Hz), and two N-acetyl group at d 2.02 and 2.04 (both singlets). A relatively small difference between the H-3ax and H-3eq chemical shifts of (0.43 ppm) demonstrated the b-configuration of Neu5Ac [6]. Therefore, the Ounit includes two b-Gal residues and one residue each of b-GalNAc and b-Neu5Ac. The 1H and 13C NMR spectra of the OS were assigned using 2D 1 1 H, H COSY, 1H,1H TOCSY, 1H,1H ROESY, 1H,13C HSQC, and 1H,13C HMBC experiments (Table 1), and spin systems for two Gal residues (units A and B), GalNAc (C), and Neu5Ac (D) were recognized. Significant downfield displacements, due to a-effects of glycosylation, of the signals for C-6 of B, C-3 of C, and C-8 of D to d 70.5, 81.2, and 79.2, respectively, as compared with their positions in the corresponding non-substituted monosaccharides at d 62.2, 72.4 [7], and 71.6 [6], respectively, indicated the modes of substitution of the sugar residues. The ROESY spectrum of the OS showed the following correlations between anomeric protons and protons at the linkage carbons: A H-1/B H-6a, H-6b; B H-1/C H-3, and C H-1/D H-8 at d 4.41/3.88, 4.01; 4.43/3.87, and 4.64/3.92, 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 vice versa (data not shown). These data demonstrated that the OS is a linear tetrasaccharide with b-Neup5Ac (unit D) at the reducing end:
To confirm that the OS represents the O-unit of the O-polysaccharide and to establish the mode of the linkage between the Ounits, the LPS was O-deacylated with aqueous ammonia and the resultant polymer (LPSOH) was studied by 1H NMR and 13C NMR spectroscopy (Fig. 1, B) as described above for the OS. In the LPSOH, the same three sugar residues as in OS were found (for the assignment of the spectra see Table 1). A down-field displacement of the signal for C-6 of unit A from d 62.2 in the OS to d 64.6 in the LPSOH indicated substitution of this monosaccharide in the LPSOH with a keto sugar (Neu5Ac) at position 6. This conclusion was confirmed by a 2D 1H,13C HMBC experiment, which, inter alia, showed a correlation between C-2 of D and H-6a,6b of A at d 102.1/ 3.67, 3.92. A relatively large difference between the H-3ax and H3eq chemical shifts of unit D (0.97 ppm) demonstrated the aconfiguration of Neup5Ac [6]. Therefore, the O-polysaccharide of E. coli O131 has the following structure:
To our knowledge, it is unique among known bacterial polysaccharide structures (Bacterial Carbohydrate Structure Database at http://csdb.glycoscience.ru/bacterial/). In the other E. coli O-polysaccharides that contain Neu5Ac (O24, O56, O104, O145, O171), this sugar is substituted at position either 4 or 7 (http://nevyn.organ.su. se/ECODAB/) rather than 8 as in the O131 polysaccharide. Analysis of O-antigen biosynthesis genes. The O-antigen gene cluster of E. coli O131 between the conserved genes galF and gnd has been sequenced (GenBank accession number JX501336). It contains nine ORFs having the same transcription direction from galF and gnd (Fig. 2). D-Gal existing in the O131 polysaccharide is a common sugar in bacteria, and genes for synthesis of its nucleotide precursors are usually located outside the O-antigen gene cluster [8]. Hence, no such genes were found between galF and gnd in E. coli O131 too.
A.V. Perepelov et al. / Carbohydrate Research 436 (2016) 41e44
43
Table 1 1 H and 13C NMR chemical shifts of the OS and LPSOH from E. coli O131 (d, ppm). Sugar residue OSa b-D-Galp-(1/ A /6)-b-D-Galp-(1/ B /3)-b-D-GalpNAc-(1/ С /8)-b-Neup5Ac D LPSOHb /6)-b-D-Galp-(1/ A /6)-b-D-Galp-(1/ B /3)-b-D-GalpNAc-(1/ С /8)-a-Neup5Ac D
Nucleus
1
2
3 (3ax, 3eq)
4
5
6 (6a, 6b)
7
8
9 (9a, 9b)
1
H C 1 H 13 C 1 H 13 C 1 H 13 C
4.41 104.8 4.43 105.9 4.64 101.8
3.51 72.0 3.52 71.8 3.95 53.0
176.0
97.4
3.62 74.1 3.61 73.6 3.87 81.2 1.82, 2.24 40.6
3.92 69.9 3.93 69.9 4.15 69.2 3.99 68.6
3.67 76.3 3.83 74.9 3.66 75.9 3.88 53.6
3.76, 3.76 62.2 3.88, 4.01 70.5 3.76, 3.76 62.2 4.10 71.1
3.68 68.4
3.92 79.2
3.73, 3.83 62.2
1
4.42 104.9 4.47 105.9 4.74 103.0
3.53 72.0 3.55 71.9 4.01 53.0
174.5
102.1
3.64 73.9 3.64 73.6 3.92 81.1 1.68, 2.65 41.1
3.95 69.9 4.95 69.7 4.19 69.3 3.66 69.2
3.77 74.9 3.84 75.1 3.73 76.1 3.85 53.6
3.67, 3.92 64.6 3.88, 4.03 70.8 3.79, 3.79 62.3 3.91 74.5
3.87 70.7
3.98 84.0
3.70 4.00 62.5
13
H C H 13 C 1 H 13 C 1 H 13 C 13 1
Сhemical shifts for the N-acetyl groups are: a dH 2.02 and 2.04; dС 23.4, 23.6 (both Me), and 176.1 (2 CO). b dH 2.04 and 2.05; dС 23.5, 23.6 (both Me), 176.2, and 176.3 (both CO).
Fig. 2. Schematic of the E. coli O131-antigen gene cluster.
GlcNAc or GalNAc is the first sugar of the O-unit in almost all E. coli O-serogroups. wecA gene responsible for the transfer of GlcNAc-P from UDP-GlcNAc to undecaprenyl phosphate (Und-P) to give UndPP-D-GlcNAc is located in the enterobacterial common antigen gene cluster [9] and is not duplicated in the O-antigen gene cluster. When the first sugar is GalNAc, 4-epimerase Gnu converts UndPPD-GlcNAc to UndPP-D-GalNAc [10,11], and the gnu gene is present upstream of the O131 antigen gene cluster next to galF (Fig. 2). orf1-4 of the O131 were identified as wckD (nnaD), nnaB, nnaC, and nnaA, respectively, and designated accordingly (Fig. 2). Their products have been proposed for the synthesis of CMP-Neu5Ac, the nucleotide precursor of Neu5Ac (ref. [12] and refs. cited therein), which is a component of the O131 antigen. Briefly, NnaA is GlcNAc 2-epimerase that converts GlcNAc to ManNAc. Neu5Ac synthetase NnaB has a role in the synthesis of Neu5Ac by condensation of ManNAc with phosphoenolpyruvate. Neu5Ac cytidylyltransferase NnaC activates the sugar before it is linking to a growing oligosaccharide. Finally, NnaD plays a role in this pathway by interacting with NnaB. There were three glycosyltransferase genes, orf6, orf8, and orf9, in the O131 antigen gene cluster. BLAST research enabled annotation of orf6 as the a-2,3-sialyltransferase gene lst. In E. coli O104 and Haemophilus ducreyi, WbwA or Lst has been characterized as a Neu5Ac transferase for making the a-Neup5Ac-(2 / 3)-D-Galp linkage [13]. However, the lst product of the O131 shows no obvious identity to those of O104 or H. ducreyi, and the corresponding linkage in the O131 is a-Neup5Ac-(2 / 6)-D-Galp. On the other hand, Lst of the O131 shares 29% identity to the glycosyltransferase WfdL of Shigella bodyii type 7, which was proposed for the transfer of Pse5Ac7RHb (where Pse indicates pseudaminic acid and Hb
indicates 3-hydroxybutanoyl) from CMP-Pse5Ac7RHb, an analogue of CMP-Neu5Ac, to D-Gal, making the b-Psep5Ac7RHb-(2 / 6)-Galp linkage [8]. It seems that Lst of the O131 recognizes similar sugar donor and acceptor compared to its WfdL counterpart but shows a stringent specificity on the attended mode. Therefore, lst in the O131 antigen gene cluster may represent a new class of sialyltransferases. The orf8 and orf9 glycosyltransferase genes were designated as wepN and wclG, respectively. WclG shares 57% identity to WbwC, which was proposed for the formation of b-Galp-(1 / 3)-GalpNAc linkage in the biosynthesis of the E. coli O104 antigen [13]. Hence, it was suggested that WclG forms the same linkage in the O131 antigen. The remaining glycosyltransferase WepN should be responsible for the formation of the third linkage in the O-unit, namely bD-Galp-(1 / 6)-D-Galp. Finally, the O131 gene cluster includes the wzx and wzy genes, which encode O-antigen processing proteins: flippase and O-antigen polymerase, respectively. The presence of these genes indicates that the O131 antigen is synthesized by the Wzx/Wzy-dependent pathway [14]. 1. Experimental 1.1. Bacterial strain and isolation of the lipopolysaccharide E. coli O131 type strain F8188-41 (laboratory stock number G2809) was 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
44
A.V. Perepelov et al. / Carbohydrate Research 436 (2016) 41e44
and pH 7.0. Bacterial cells were washed and dried as described [15]. A LPS sample was isolated in a yield 10.6% from bacterial cells by the phenol-water method [16], the crude extract was dialyzed without separation of the layers and freed from nucleic acids and proteins by treatment with 50% aq CCl3CO2H at pH 2. The supernatant was dialyzed and lyophilized. 1.2. Acid and alkaline degradations of the lipopolysaccharide Mild acid degradation of a LPS sample (108 mg) was performed with aq 2% HOAc at 100 C until precipitation of a lipid (2.5 h). The precipitate was removed by centrifugation (13,000g, 20 min) and the supernatant was fractionated by GPC sequentially on a column (56 2.6 cm) of Sephadex G-50 Superfine (Amersham Biosciences, Sweden) in pyridinium acetate buffer (4 mL pyridine and 10 mL HOAc in 1 L water) and a column (80 2.5 cm) of TSK HW-40 (S) (Merck, Germany) in aq 1% HOAc. Eluates were monitored with a differential refractometer (Knauer, Germany) to give an OS sample in a yield 24% of the LPS mass. O-Deacylation of a LPS sample (73 mg) was performed with aq 12.5% ammonia (37 C, 16 h), the precipitate was removed by centrifugation (13,000g, 20 min), and the supernatant was fractionated by GPC on TSK HW-40 (S) as above to give an O-deacylated LPS sample in a yield 57% of the LPS mass from.
from 99.9% D2O and then examined as solutions in 99.95% D2O. NMR spectra were recorded on a Bruker Avance II 600 spectrometer C (Germany) at 40 using internal sodium 3trimethylsilylpropanoate-2,2,3,3-d4 (dH 0, dC 1.6) as reference for calibration. 2D NMR spectra were obtained using standard Bruker software, and Bruker TopSpin 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 This work was supported by the Russian Foundation for Basic Research (project 14-04-01709-а). References [1] [2] [3] [4] [5] [6]
[7]
1.3. Monosaccharide analysis
[8]
An OS 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 at 7 C min 1.
[9] [10] [11] [12] [13]
1.4. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying twice
[14] [15] [16]
J.B. Kaper, J.P. Nataro, H.L. Mobley, Nat. Rev. Microbiol. 2 (2004) 123e140. P.R. Reeves, L. Wang, Curr. Top. Microbiol. Immunol. 264 (2002) 109e135. G. Samuel, P.R. Reeves, Carbohydr. Res. 338 (2003) 2503e2519. A. Iguchi, S. Iyoda, T. Kikuchi, Y. Ogura, K. Katsura, M. Ohnishi, T. Hayashi, N.R. Thomson, DNA Res. 22 (2015) 101e107. X. Chen, S. Gao, X. Jiao, X.F. Liu, Vet. Microbiol. 103 (2004) 13e20. J.F.G. Vliegenthart, L. Dorland, H. van Halbeek, J. Haverkamp, NMR spectroscopy of sialic acids, in: R. Schauer (Ed.), Sialic Acids: Chemistry, Metabolism and Function, Springer Verlag, Wien, 1982, pp. 127e172. G.M. Lipkind, A.S. Shashkov, Y.A. Knirel, E.V. Vinogradov, N.K. Kochetkov, Carbohydr. Res. 175 (1988) 59e75. B. Liu, Y.A. Knirel, L. Feng, A.V. Perepelov, S.N. Senchenkova, Q. Wang, P.R. Reeves, L. Wang, FEMS Microbiol. Rev. 32 (2008) 627e653. U. Meier-Dieter, K. Barr, R. Starman, L. Hatch, P.D. Rick, J. Biol. Chem. 267 (1992) 746e753. J.S. Rush, C. Alaimo, R. Robbiani, M. Wacker, C.J. Waechter, J. Biol. Chem. 285 (2010) 1671e1680. M.M. Cunneen, B. Liu, L. Wang, P.R. Reeves, PLoS One 14 (2013) e67646. L. Feng, S.N. Senchenkova, J. Tao, A.S. Shashkov, B. Liu, S.D. Shevelev, P.R. Reeves, J. Xu, Y.A. Knirel, L. Wang, J. Bacteriol. 187 (2005) 758e764. L. Wang, C.E. Briggs, D. Rothemund, P. Fratamico, J.B. Luchansky, P.R. Reeves, Gene 270 (2001) 231e236. C.R. Raetz, C. Whitfield, Annu. Rev. Biochem. 71 (2002) 635e700. P.W. Robbins, T. Uchida, Biochemistry 1 (1962) 323e335. O. Westphal, K. Jann, Methods Carbohydr. Chem. 5 (1965) 83e91.
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Structure elucidation and analysis of biosynthesis genes of the O-antigen of Escherichia coli O131 containing N-acetylneuraminic acid Andrei V. Perepelov a, *, Xi Guo b, Sof'ya N. Senchenkova a, Alexander S. Shashkov a, Bin Liu b, Yuriy A. Knirel a a
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991, Moscow, Russian Federation Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin, China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 September 2016 Received in revised form 3 November 2016 Accepted 3 November 2016 Available online 4 November 2016
The O-polysaccharide (O-antigen) of Escherichia coli O131 was studied by sugar analysis along with 1D and 2D 1H and 13C NMR spectroscopy. The following structure of the linear tetrasaccharide repeating unit of the polysaccharide was established: /8)-a-Neup5Ac-(2 / 6)-b-D-Galp-(1 / 6)-b-D-Galp-(1 / 3)-b-D-GalpNAc-(1/ The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in agreement with the E. coli O131-antigen structure. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Escherichia coli Bacterial polysaccharide structure O-polysaccharide structure N-acetylneuraminic acid Lipopolysaccharide O-antigen gene cluster
Escherichia coli is the predominant facultative anaerobe of the colonic flora of many mammals, including humans, and has both commensal and pathogenic forms [1]. O-polysaccharide, or O-antigen, which contains a number of oligosaccharide repeats (Ounits), is a part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. Variations in sugar composition, arrangement of monosaccharide residues, and linkages within and between the O-units make the O-antigen the most variable constituents on the cell surface and provides the basis for serotyping of many Gram-negative bacteria [2]. Genes involved in the O-antigen synthesis are generally combined in a cluster; in E. coli this is usually located 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 also present in other bacterial structures or involved in metabolism usually map at other loci. A high diversity of the O-antigen forms is mainly due to genetic variations in the Oantigen gene cluster [3]. Currently, >180 O-serogroups of E. coli are
* Corresponding author. E-mail address: [email protected] (A.V. Perepelov). http://dx.doi.org/10.1016/j.carres.2016.11.003 0008-6215/© 2016 Elsevier Ltd. All rights reserved.
internationally recognized, and sequences of their O-antigen gene clusters have been reported [4]. Most E. coli O-antigen structures have been determined (http://nevyn.organ.su.se/ECODAB/) but some remain unknown. In this work, we established the structure of the O-polysaccharide of E. coli O131, which has been reported to be associated with pigs with post-weaning diarrhea [5]. The O131-antigen gene cluster was analyzed and found to be consistent with the O-polysaccharide structure. Structure elucidtaion of the O-polysaccharide. LPS was isolated from E. coli O131 cells by extraction with hot phenol-water. On degradation with dilute acetic acid, the LPS afforded no expected high-molecular mass polysaccharide but an oligosaccharide (OS), most likely, owing to a cleavage of an acid-labile glycosidic linkage of an O-polysaccharide component. Further studies showed that this component was N-acetylneuraminic acid (Neu5Ac). Sugar analysis using GLC of the alditol acetates derived after full acid hydrolysis of the OS revealed GalN and Gal in the ratio ~0.5:1.0. The 13C NMR spectrum of the OS (Fig. 1, A) contained signals for four anomeric carbons at d 97.4 (quaternary carbon; low intense signal identified as C-2 of Neu5Ac), 101.8, 104.8, and 105.9, four HOCH2eC groups (C-6 of Gal and GalN residues, C-9 of Neu5Ac) at
42
A.V. Perepelov et al. / Carbohydrate Research 436 (2016) 41e44
Fig. 1.
13
C NMR spectra of the OS (top) and LPSOH (bottom) from E. coli O131. Numbers refer to carbons in sugar residues denoted by letters as shown in Table 1.
d 62.2 (signal of triple integral intense) and 70.5 (data of a DEPT experiment), two nitrogen-bearing carbons (C-2 of GalN and C-5 of Neu5Ac) at d 53.0 and 53.6, respectively, one CeCH2eC group (C-3 of Neu5Ac) at d 40.6, 15 oxygen-bearing sugar ring carbons in the region d 68.6e81.2, two N-acetyl groups at d 23.4 and 23.6 (both CH3), and CO groups of NAc and C-1 of Neu5Ac at d 176.0e176.1. The 1H NMR spectrum of the OS showed signals for three anomeric protons of b-linked pyranosides (H-1 of Gal and GalNAc) at d 4.41, 4.43, and 4.64 (all doublets, J ~8 Hz), one CHeCH2eC group (H-3 of Neu5Ac) at d 1.82 (H-3ax, triplet, J ~11 Hz) and d 2.24 (H3eq, doublet of doublets, J ~5 and ~13 Hz), and two N-acetyl group at d 2.02 and 2.04 (both singlets). A relatively small difference between the H-3ax and H-3eq chemical shifts of (0.43 ppm) demonstrated the b-configuration of Neu5Ac [6]. Therefore, the Ounit includes two b-Gal residues and one residue each of b-GalNAc and b-Neu5Ac. The 1H and 13C NMR spectra of the OS were assigned using 2D 1 1 H, H COSY, 1H,1H TOCSY, 1H,1H ROESY, 1H,13C HSQC, and 1H,13C HMBC experiments (Table 1), and spin systems for two Gal residues (units A and B), GalNAc (C), and Neu5Ac (D) were recognized. Significant downfield displacements, due to a-effects of glycosylation, of the signals for C-6 of B, C-3 of C, and C-8 of D to d 70.5, 81.2, and 79.2, respectively, as compared with their positions in the corresponding non-substituted monosaccharides at d 62.2, 72.4 [7], and 71.6 [6], respectively, indicated the modes of substitution of the sugar residues. The ROESY spectrum of the OS showed the following correlations between anomeric protons and protons at the linkage carbons: A H-1/B H-6a, H-6b; B H-1/C H-3, and C H-1/D H-8 at d 4.41/3.88, 4.01; 4.43/3.87, and 4.64/3.92, 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 vice versa (data not shown). These data demonstrated that the OS is a linear tetrasaccharide with b-Neup5Ac (unit D) at the reducing end:
To confirm that the OS represents the O-unit of the O-polysaccharide and to establish the mode of the linkage between the Ounits, the LPS was O-deacylated with aqueous ammonia and the resultant polymer (LPSOH) was studied by 1H NMR and 13C NMR spectroscopy (Fig. 1, B) as described above for the OS. In the LPSOH, the same three sugar residues as in OS were found (for the assignment of the spectra see Table 1). A down-field displacement of the signal for C-6 of unit A from d 62.2 in the OS to d 64.6 in the LPSOH indicated substitution of this monosaccharide in the LPSOH with a keto sugar (Neu5Ac) at position 6. This conclusion was confirmed by a 2D 1H,13C HMBC experiment, which, inter alia, showed a correlation between C-2 of D and H-6a,6b of A at d 102.1/ 3.67, 3.92. A relatively large difference between the H-3ax and H3eq chemical shifts of unit D (0.97 ppm) demonstrated the aconfiguration of Neup5Ac [6]. Therefore, the O-polysaccharide of E. coli O131 has the following structure:
To our knowledge, it is unique among known bacterial polysaccharide structures (Bacterial Carbohydrate Structure Database at http://csdb.glycoscience.ru/bacterial/). In the other E. coli O-polysaccharides that contain Neu5Ac (O24, O56, O104, O145, O171), this sugar is substituted at position either 4 or 7 (http://nevyn.organ.su. se/ECODAB/) rather than 8 as in the O131 polysaccharide. Analysis of O-antigen biosynthesis genes. The O-antigen gene cluster of E. coli O131 between the conserved genes galF and gnd has been sequenced (GenBank accession number JX501336). It contains nine ORFs having the same transcription direction from galF and gnd (Fig. 2). D-Gal existing in the O131 polysaccharide is a common sugar in bacteria, and genes for synthesis of its nucleotide precursors are usually located outside the O-antigen gene cluster [8]. Hence, no such genes were found between galF and gnd in E. coli O131 too.
A.V. Perepelov et al. / Carbohydrate Research 436 (2016) 41e44
43
Table 1 1 H and 13C NMR chemical shifts of the OS and LPSOH from E. coli O131 (d, ppm). Sugar residue OSa b-D-Galp-(1/ A /6)-b-D-Galp-(1/ B /3)-b-D-GalpNAc-(1/ С /8)-b-Neup5Ac D LPSOHb /6)-b-D-Galp-(1/ A /6)-b-D-Galp-(1/ B /3)-b-D-GalpNAc-(1/ С /8)-a-Neup5Ac D
Nucleus
1
2
3 (3ax, 3eq)
4
5
6 (6a, 6b)
7
8
9 (9a, 9b)
1
H C 1 H 13 C 1 H 13 C 1 H 13 C
4.41 104.8 4.43 105.9 4.64 101.8
3.51 72.0 3.52 71.8 3.95 53.0
176.0
97.4
3.62 74.1 3.61 73.6 3.87 81.2 1.82, 2.24 40.6
3.92 69.9 3.93 69.9 4.15 69.2 3.99 68.6
3.67 76.3 3.83 74.9 3.66 75.9 3.88 53.6
3.76, 3.76 62.2 3.88, 4.01 70.5 3.76, 3.76 62.2 4.10 71.1
3.68 68.4
3.92 79.2
3.73, 3.83 62.2
1
4.42 104.9 4.47 105.9 4.74 103.0
3.53 72.0 3.55 71.9 4.01 53.0
174.5
102.1
3.64 73.9 3.64 73.6 3.92 81.1 1.68, 2.65 41.1
3.95 69.9 4.95 69.7 4.19 69.3 3.66 69.2
3.77 74.9 3.84 75.1 3.73 76.1 3.85 53.6
3.67, 3.92 64.6 3.88, 4.03 70.8 3.79, 3.79 62.3 3.91 74.5
3.87 70.7
3.98 84.0
3.70 4.00 62.5
13
H C H 13 C 1 H 13 C 1 H 13 C 13 1
Сhemical shifts for the N-acetyl groups are: a dH 2.02 and 2.04; dС 23.4, 23.6 (both Me), and 176.1 (2 CO). b dH 2.04 and 2.05; dС 23.5, 23.6 (both Me), 176.2, and 176.3 (both CO).
Fig. 2. Schematic of the E. coli O131-antigen gene cluster.
GlcNAc or GalNAc is the first sugar of the O-unit in almost all E. coli O-serogroups. wecA gene responsible for the transfer of GlcNAc-P from UDP-GlcNAc to undecaprenyl phosphate (Und-P) to give UndPP-D-GlcNAc is located in the enterobacterial common antigen gene cluster [9] and is not duplicated in the O-antigen gene cluster. When the first sugar is GalNAc, 4-epimerase Gnu converts UndPPD-GlcNAc to UndPP-D-GalNAc [10,11], and the gnu gene is present upstream of the O131 antigen gene cluster next to galF (Fig. 2). orf1-4 of the O131 were identified as wckD (nnaD), nnaB, nnaC, and nnaA, respectively, and designated accordingly (Fig. 2). Their products have been proposed for the synthesis of CMP-Neu5Ac, the nucleotide precursor of Neu5Ac (ref. [12] and refs. cited therein), which is a component of the O131 antigen. Briefly, NnaA is GlcNAc 2-epimerase that converts GlcNAc to ManNAc. Neu5Ac synthetase NnaB has a role in the synthesis of Neu5Ac by condensation of ManNAc with phosphoenolpyruvate. Neu5Ac cytidylyltransferase NnaC activates the sugar before it is linking to a growing oligosaccharide. Finally, NnaD plays a role in this pathway by interacting with NnaB. There were three glycosyltransferase genes, orf6, orf8, and orf9, in the O131 antigen gene cluster. BLAST research enabled annotation of orf6 as the a-2,3-sialyltransferase gene lst. In E. coli O104 and Haemophilus ducreyi, WbwA or Lst has been characterized as a Neu5Ac transferase for making the a-Neup5Ac-(2 / 3)-D-Galp linkage [13]. However, the lst product of the O131 shows no obvious identity to those of O104 or H. ducreyi, and the corresponding linkage in the O131 is a-Neup5Ac-(2 / 6)-D-Galp. On the other hand, Lst of the O131 shares 29% identity to the glycosyltransferase WfdL of Shigella bodyii type 7, which was proposed for the transfer of Pse5Ac7RHb (where Pse indicates pseudaminic acid and Hb
indicates 3-hydroxybutanoyl) from CMP-Pse5Ac7RHb, an analogue of CMP-Neu5Ac, to D-Gal, making the b-Psep5Ac7RHb-(2 / 6)-Galp linkage [8]. It seems that Lst of the O131 recognizes similar sugar donor and acceptor compared to its WfdL counterpart but shows a stringent specificity on the attended mode. Therefore, lst in the O131 antigen gene cluster may represent a new class of sialyltransferases. The orf8 and orf9 glycosyltransferase genes were designated as wepN and wclG, respectively. WclG shares 57% identity to WbwC, which was proposed for the formation of b-Galp-(1 / 3)-GalpNAc linkage in the biosynthesis of the E. coli O104 antigen [13]. Hence, it was suggested that WclG forms the same linkage in the O131 antigen. The remaining glycosyltransferase WepN should be responsible for the formation of the third linkage in the O-unit, namely bD-Galp-(1 / 6)-D-Galp. Finally, the O131 gene cluster includes the wzx and wzy genes, which encode O-antigen processing proteins: flippase and O-antigen polymerase, respectively. The presence of these genes indicates that the O131 antigen is synthesized by the Wzx/Wzy-dependent pathway [14]. 1. Experimental 1.1. Bacterial strain and isolation of the lipopolysaccharide E. coli O131 type strain F8188-41 (laboratory stock number G2809) was 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
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and pH 7.0. Bacterial cells were washed and dried as described [15]. A LPS sample was isolated in a yield 10.6% from bacterial cells by the phenol-water method [16], the crude extract was dialyzed without separation of the layers and freed from nucleic acids and proteins by treatment with 50% aq CCl3CO2H at pH 2. The supernatant was dialyzed and lyophilized. 1.2. Acid and alkaline degradations of the lipopolysaccharide Mild acid degradation of a LPS sample (108 mg) was performed with aq 2% HOAc at 100 C until precipitation of a lipid (2.5 h). The precipitate was removed by centrifugation (13,000g, 20 min) and the supernatant was fractionated by GPC sequentially on a column (56 2.6 cm) of Sephadex G-50 Superfine (Amersham Biosciences, Sweden) in pyridinium acetate buffer (4 mL pyridine and 10 mL HOAc in 1 L water) and a column (80 2.5 cm) of TSK HW-40 (S) (Merck, Germany) in aq 1% HOAc. Eluates were monitored with a differential refractometer (Knauer, Germany) to give an OS sample in a yield 24% of the LPS mass. O-Deacylation of a LPS sample (73 mg) was performed with aq 12.5% ammonia (37 C, 16 h), the precipitate was removed by centrifugation (13,000g, 20 min), and the supernatant was fractionated by GPC on TSK HW-40 (S) as above to give an O-deacylated LPS sample in a yield 57% of the LPS mass from.
from 99.9% D2O and then examined as solutions in 99.95% D2O. NMR spectra were recorded on a Bruker Avance II 600 spectrometer C (Germany) at 40 using internal sodium 3trimethylsilylpropanoate-2,2,3,3-d4 (dH 0, dC 1.6) as reference for calibration. 2D NMR spectra were obtained using standard Bruker software, and Bruker TopSpin 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 This work was supported by the Russian Foundation for Basic Research (project 14-04-01709-а). References [1] [2] [3] [4] [5] [6]
[7]
1.3. Monosaccharide analysis
[8]
An OS 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 at 7 C min 1.
[9] [10] [11] [12] [13]
1.4. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying twice
[14] [15] [16]
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