Structure and gene cluster of the O-antigen of Escherichia coli O54

Carbohydrate Research 462 (2018) 34–38

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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres

Structure and gene cluster of the O-antigen of Escherichia coli O54 a,d

b

a

c

T a,∗

Olesya I. Naumenko , Xi Guo , Sof'ya N. Senchenkova , Peng Geng , Andrei V. Perepelov , Alexander S. Shashkova, Bin Liub, Yuriy A. Knirela a

N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991, Moscow, Russian Federation TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, 300457, Tianjin, PR China School of Basic Medical Sciences, Tianjin Medical University, Heping District, Tianjin, 300070, PR China d Higher Chemical College of the Russian Academy of Sciences, D. I. Mendeleev University of Chemical Technology of Russia, Moscow, Russia b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Escherichia coli O-polysaccharide O-antigen Bacterial polysaccharide structure O-antigen gene cluster

Mild acid hydrolysis of the lipopolysaccharide of Escherichia coli O54 afforded an O-polysaccharide, which was studied by sugar analysis, solvolysis with anhydrous trifluoroacetic acid, and 1H and 13C NMR spectroscopy. Solvolysis cleaved predominantly the linkage of β-D-Ribf and, to a lesser extent, that of β-D-GlcpNAc, whereas the other linkages, including the linkage of α-L-Rhap, were stable under selected conditions (40 °C, 5 h). The following structure of the O-polysaccharide was established: →4)-α-D-GalpA-(1 → 2)-α-L-Rhap-(1 → 2)-β-D-Ribf-(1 → 4)-β-D-Galp-(1 → 3)-β-D-GlcpNAc-(1→ The O-antigen gene cluster of E. coli O54 was analyzed and found to be consistent in general with the Opolysaccharide structure established but there were two exceptions: i) in the cluster, there were genes for phosphoserine phosphatase and serine transferase, which have no apparent role in the O-polysaccharide synthesis, and ii) no ribofuranosyltransferase gene was present in the cluster. Both uncommon features are shared by some other enteric bacteria.

1. Introduction Escherichia coli is the predominant facultative anaerobe of the colonic flora of many mammals, including humans, and has both commensal and pathogenic forms [1]. The O-antigen (O-polysaccharide) is a part of the lipopolysaccharide (LPS) embedded into the outer membrane of Gram-negative bacteria. It usually consists of many oligosaccharide repeats (O-units) containing two to eight residues from a broad range of common or rarely occurring sugars and their derivatives. The O-antigen is one of the most variable cell constituents, with variation in the types of sugars present, their arrangement within the Ounit, and the linkages within and between O-units, providing the basis of serotyping. Being exposed on the cell surface, the O-antigen is highly immunogenic and, therefore, subject to intense selection by the host immune system and bacteriophages [2]. Genes involved in the O-antigen synthesis of E. coli and some related bacteria are usually clustered in a chromosomal locus localized between the housekeeping genes galF and gnd. Most of these genes fall into one of three major classes: nucleotide sugar synthesis genes, glycosyltransferase genes, and O-unit processing genes, including flippase for translocation of the lipid-linked O-unit across the inner membrane and O-antigen polymerase [3]. Up to date, there are 184 internationally recognized O serogroups of E.

∗

Corresponding author. E-mail address: [email protected] (A.V. Perepelov).

https://doi.org/10.1016/j.carres.2018.04.001 Received 27 February 2018; Received in revised form 28 March 2018; Accepted 4 April 2018 Available online 05 April 2018 0008-6215/ © 2018 Elsevier Ltd. All rights reserved.

coli, with all O-antigen gene clusters being sequenced [4]. O-polysaccharide structures have been established for most O serogroups (http:// nevyn.organ.su.se/ECODAB/; http://csdb.glycoscience.ru/bacterial/) but a few remain unknown. In this work, we established the structure and analyzed the gene cluster of the O-antigen of E. coli O54. 2. Results and discussion 2.1. Structure elucidation of the O-polysaccharide LPS was obtained from E. coli O54 cells by the phenol-water method [5] and degraded with dilute HOAc to give an O-polysaccharide. Sugar analysis using GLC of the alditol acetates derived after full acid hydrolysis of the O-polysaccharide revealed Rib, Rha, Gal, and GlcNAc in the ratios ∼1:1.3:1.4:1.2 (detector response), respectively. Further studies showed that GalA also was present. GLC analysis of the acetylated glycosides with (S)-2-octanol indicated that Rha is L and Gal is D [6]. The D configuration of the other monosaccharides was confirmed by analysis of the 13C NMR chemical shifts of the O-polysaccharide and derived oligosaccharides taking into account known regularities in glycosylation effects [7], combined with analysis of genes in the Oantigen gene cluster (see below).

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Fig. 1. 13C NMR spectrum of the O-polysaccharide from E. coli O54. Arabic numerals refer to carbons in sugar residues denoted by letters as shown in Chart 1 and Table 1.

The 1H and 13C NMR spectra of the O-polysaccharide (Fig. 1) showed signals for five monosaccharide residues, including five anomeric atoms at δH 4.44–5.44 and δC 99.3–108.1, one CH3-C group (C-6 of Rha) at δH 1.30, δC 17.8, three OCH2-C groups (C-5 of Rib, C-6 of Gal and GlcNAc) at δC 62.5–63.8, one nitrogen-bearing carbon (C-2 of GlcNAc) at δC 56.1, one N-acetyl group at δH 2.03, δC 23.7, two CO groups (NAc and C-6 of GalA) at δC 174.5 and 176.1, and other signals at δH 3.38–4.88 and δC 69.9–83.9. The 1H and 13C NMR spectra of the O-polysaccharide were assigned using 2D 1H,1H COSY, TOCSY, and 1H,13C HSQC experiments (Table 1). Based on 3JH,H coupling constants and 1H and 13CNMR chemical shifts, spin systems for one residue each of β-GlcNAc (unit A), β-Galp (unit B), β-Ribf (unit C), α-Rhap (unit D), and α-GalpA (unit E) were identified. The GlcNAc residue was confirmed by correlations between the proton at the nitrogen-bearing carbon (H-2) and the corresponding carbon (C2) at δ 3.80/56.1 in the 1H,13C HSQC spectrum. Linkage and sequence analysis of the O-polysaccharide was performed using the 2D 1H,1H ROESY (Fig. 2) and 1H,13C HMBC 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-2 of units C and D, C-3 of unit A, and C-4 of units B and E (Table 1), as compared with their positions in the spectra of the corresponding non-substituted monosaccharides [8–10]. The O-polysaccharide was cleaved by anhydrous trifluoroacetic acid at 40 °C for 5 h, and the products were fractionated by GPC on Fractogel TSK-HW 40S to give fractions I-V. Fractions III and IV contained oligosaccharides, which were studied by high-resolution ESI MS (Table 3) and 1H and 13C NMR spectroscopy (Table 1). Fraction III contained pentasaccharide 1 with ribose at the reducing end (δH-1/C-1 4.98/93.7, J1,2 6.7 Hz for the major β-D-Ribp form). Fraction IV consisted of disaccharide 2 and trisaccharide 3 with GlcNAc (δH-1/C-1 5.19/92.7, J1,2 3.5 Hz and δH-1/C-1 4.76/96.3, J1,2 7.8 Hz for the α- and β-anomers, respectively) and ribose (δH-1/C-1 5.00/94.1, J1,2 6.3 Hz for β-D-Ribp) at the reducing end, respectively (Chart 1). Therefore, these oligosaccharides resulted from the selective cleavage of the linkages of Rib (1) or both Rib and GlcNAc (2 and 3). Identification of oligosaccharides 1–3 confirmed the O-polysaccharide structure established independently by NMR spectroscopy and pentasaccharide 1 did correspond to the full O-unit of the O54-polysaccharide. Recently, it has been demonstrated that solvolysis with CF3CO2H

Table 1 1 H and 13C NMR chemical shifts (δ, ppm) of the O-polysaccharide of E. coli O54 and pentasaccharide 1. Sugar residue O-polysaccharide →4)-α-D-GalpA(1→ E →2)-α-L-Rhap(1→ D →2)-β-D-Ribf(1→ C →4)-β-D-Galp(1→ B →3)-β-DGlcpNAc-(1→ A Pentasaccharide 1 β-D-Galp-(1→ B →3)-β-DGlcpNAc-(1→ A →4)-α-D-GalpA(1→ E →2)-α-L-Rhap(1→ D →2)-β-D-Ribp C

Nucleus

1

2

3

4

5 (5a, 5b)

1

5.05

3.72

4.05

4.42

4.88

99.3 5.07

69.4 4.07

70.4 3.89

80.0 3.47

72.1 3.77

174.5 1.30

99.7 5.44

77.7 4.22

70.6 4.24

73.2 4.01

70.7 3.66, 3.83

17.8

108.1 4.44

82.2 3.50

71.3 3.74

83.9 3.99

63.8 3.71

3.73, 3.73

104.7 4.70

72.1 3.80

74.1 3.78

76.8 3.52

76.0 3.38

62.5 3.73, 3.86

C

103.1

56.1

83.6

69.9

76.3

62.3

H C 1 H

4.45 104.7 4.68

3.53 72.0 3.85

3.65 73.7 3.79

3.92 69.8 3.50

3.71 76.5 3.39

3.76, 3.88 62.3 3.71, 3.86

13

103.2 5.07

56.2 3.73

83.5 4.06

70.1 4.38

76.4 4.65

62.5

98.8 5.08

69.6 4.06

70.9 3.91

80.7 3.50

73.0 3.92

176.6 1.29

96.9 4.98 93.7

77.2 3.58 77.2

70.7 4.29 67.8

72.9 3.86 68.1

70.7 3.71, 3.84 64.1

17.7

H

13 1

C H

13 1

C H

13 1

C H

13 1

C H

13

1

13

1

C H

13 1

C H

13

C H 13 C 1

6 (6a, 6b)

Chemical shifts for the N-acetyl group are δH 2.03–2.05, δС 23.7 (Me) and 176.1–176.2 (CO).

cleaved selectively pyranosidic linkages of 6-deoxy-α-hexoses and Nacetyl-β-hexosamines [11]. As expected, β-galactofuranosidic linkages also were unstable towards solvolysis [12]. In the O-polysaccharide of E. coli O54, the linkages of β-ribofuranose and N-acetyl-β-glucosamine were cleaved selectively by anhydrous CF3CO2H, the former being least stable. The α-rhamnosidic linkages in this O-polysaccharide were more stable, and no products due to their cleavage were obtained under the selected conditions. Based on the data obtained, it was concluded that the O-polysaccharide of E. coli O54 has the structure shown in Chart 1, which, to 35

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Table 3 Positive and negative ion mode high-resolution ESI MS data of CF3CO2H solvolysis products from the O-polysaccharide. Sugar sequences were inferred from the oligosaccharide structures shown on Chart 1. Oligosaccharide

Fraction III 1

Fraction IV 2 3

Sugar composition and sequence

Molecular mass, Da

[M+Na]+/[M-H]− ion peaks at m/z experimental

calculated

Hex-HexNAcHexA-6dHexPen

837.2750

860.2708/ 836.2678

860.2642/ 836.2777

Hex-HexNAc HexA-6dHexPen

383.1428 472.1428

406.1337 495.1331/ 471.1365

406.1320 495.1320/ 471.1355

Pen, Hex, 6dHex, HexA, and HexNAc indicate pentose, hexose, 6-deoxyhexose, hexuronic acid, and N-acetylhexosamine, respectively.

and gnd genes has been sequenced [4] (GenBank accession number AB812085). It contains 11 genes having the same transcriptional direction from galF to gnd (Fig. 3). The cluster includes the wzx and wzy genes (orf4 and orf10), which encode O-antigen processing proteins, flippase and O-antigen polymerase, for translocation of the preassembled O-unit through the plasma membrane and its polymerization, respectively. The presence of these genes indicates that the Oantigen of E. coli O54 is synthesized by the Wzx/Wzy-dependent pathway [18]. D-GlcNAc, D-Gal, and D-Rib are common sugars in bacteria, and genes for synthesis of their nucleotide precursors are usually located outside the O-antigen gene cluster. Typically of D-GalA-containing E. coli O-antigens [4,19], ugd and gla genes are involved in the synthesis of the D-GalA nucleotide precursor, UDP-D-GalA, from UDP-D-Glc [3]. These genes map near the gene cluster just downstream of gnd. They are followed by the wzz gene encoding a protein that is responsible for the O-antigen chain length regulation [20]. Synthesis of the nucleotide precursor of L-Rha, dTDP-L-Rha, requires four proteins RmlABCD [3], and a set of the corresponding genes in the order rmlBDAC are usually localized at the 5′ end of the O-antigen gene cluster [4]. In E. coli O54, the first three genes are found in their usual location whereas the rmlC gene maps at the 3′ end of the cluster (Fig. 3). In most E. coli serotypes, the first sugar of an O-unit is either DGlcNAc or D-GalNAc. WecA initiates the assembly of an O-unit by transfer of D-GlcNAc 1-phosphate from UDP-D-GlcNAc to undecaprenyl phosphate (UndP) to give UndPP-D-GlcNAc [18]. The wecA gene is localized in the enterobacterial common antigen gene cluster [21] and is not duplicated in the O-antigen gene cluster. Then, glycosyltransferases act to transfer sequentially other sugar components of the O-unit on to the first UndPP-linked monosaccharide. The E. coli O54 gene cluster includes three glycosyltransferase genes: orf5, orf6, and orf9, whereas four genes are expected for the assembly of the pentasaccharide O-unit. Predicted Orf5 shares 49% identity and 65% similarity with WbuP of E. coli O114, which has been demonstrated to form the β-D-Galp(1 → 3)-D-GlcNAc linkage [22]. This linkage is also present in E. coli O54, and Orf5 was assigned accordingly. Predicted Orf6 is 34% identical and 55% similar to WcnX of E. coli O35, which could be assigned to

Fig. 2. Part of a 2D 1H,1H ROESY spectrum of the O-polysaccharide from E. coli O54. 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 Chart 1 and Table 1.

Table 2 Correlations for H-1 and C-1 in the 2D 1H,1H ROESY and 1H,13C HMBC spectra of the O-polysaccharide of E. coli O54. Atom in sugar residue (δ)

A H-1 (4.70) A С-1 (103.1) B H-1 (4.44) B С-1 (104.7) C H-1 (5.44) C С-1 (108.1) D H-1 (5.07) D С-1 (99.7) E H-1 (5.05) E С-1 (99.3)

Correlations to atom in sugar residue (δ) ROESY

HMBC

E H-4 (4.42), A H-3 (3.78), A H-5 (3.38)

E C-4 (80.0)

A H-3 (3.78), B H-2 (3.50), B H-3 (3.74), B H-5 (3.71) B H-4 (3.99), C H-2 (4.22), D H-5 (3.77) C H-2 (4.22), D H-2 (4.07)

D H-2 (4.07), E H-2 (3.72)

E H-4 (4.42), A H-2 (3.80) A C-3 (83.6) A H-4 (3.77), B H-2 (3.51) B C-4 (76.8), C C-2 (82.2), C C-3 (71.3), C C-4 (83.9) B H-4 (3.99) C C-2 (82.2), D C-2 (77.7), D C-3 or/and C-5 (70.6) C H-2 (4.22) E C-3 (70.4), E C-5 (72.1) D H-2 (4.07), E H-5 (4.88)

our knowledge, is unique among known bacterial polysaccharide structures. Remarkably, it shares a β-D-Ribf-(1 → 4)-β-D-Galp-(1 → 3)-DGlcNAc trisaccharide fragment with E. coli O114 [13], which is similar to trisaccharide fragments β-D-Ribf-(1 → 4)-β-D-Galp-(1 → 3)-D-GalNAc of E. coli O5 [14], O178 [15], O185 [16], and β-D-Ribf-(1 → 4)-α-DGalpNAc-(1 → 3)-D-GlcNAc of Salmonella enterica O56 [17].

2.2. Analysis of the O-antigen gene cluster The O-antigen gene cluster of E. coli O54 between conserved galF

Chart 1. Structure of the O-polysaccharide of E. coli O54 (top) and oligosaccharides 1–3 derived from the O-polysaccharide by solvolysis with CF3CO2H.

36

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Fig. 3. Content and organization of the O-antigen gene cluster of E. coli O54.

the α-D-GalpNAcA-(1 → 2)-L-Rha linkage as the remaining glycosyltransferases WcnY and WcnZ in E. coli O35 are rhamnosyltransferases (http://nevyn.organ.su.se/ECODAB/). A similar linkage, α-D-GalpA(1 → 2)-L-Rha, is present in E. coli O54, and on this basis, Orf6 was assigned to this linkage. Finally, Orf9 is a homolog of many bacterial rhamnosyltransferases and, therefore, was assigned to the α-L-Rhap(1 → 2)-D-Rib linkage. Therefore, no gene for ribofuranosyltransferase is present in the E. coli O54-antigen gene cluster. Prediced Orf 7 and Orf8 of E. coli O54 are homologs of phosphoserine phosphatase and serine transferase, respectively, which are also encoded in the O-antigen clusters of E. coli O114 [23] and S. enterica O5615 as well as in E. coli O5, O178, and O185.4 It was suggested that these proteins, called WbuN and WbuO in E. coli O114 or WdbY and WdbZ in S. enterica O56, are responsible for the synthesis and transfer of the L-seryl group to dTDP-D-Qui3N or dTDP-D-Qui4N to give, after Nacetylation, dTDP-D-Qui3NSerAc [23] or dTDP-D-Qui4NSerAc [17], respectively. However, the O-polysaccharides of neither E. coli O54 studied in this work nor E. coli O5 [14], O178 [15], and O185 [16] contain serine, and the role of the genes for phosphoserine phosphatase and serine transferase in these bacteria remain unclear. Remarkably, in all cases, the occurrence of these two non-functional genes correlates i) with the presence of the β-D-Ribf-(1 → 4)-D-Galp or, in S. enterica O56, β-D-Ribf-(1 → 4)-D-GalpNAc fragment in the O-polysaccharide and ii) in the serine-lacking bacteria, also with the absence of a ribofuranosyltransferase gene from the O-antigen gene cluster.

3.2. Chemical analyses An O-polysaccharide 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 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)-2octyl glycosides as described [6].

3.3. 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 30 or 40 °C using internal sodium 3-trimethylsilylpropanoate-2,2,3,3-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 spin-lock time of 60 ms and a mixing time of 150 ms were used in TOCSY and ROESY experiments, respectively.

3.4. Mass spectrometry High-resolution positive and negative ion mode ESI mass spectra were measured on a Bruker micrOTOF II instrument. Capillary entrance voltage was set to −4500 and 3200 V, respectively; the drying nitrogen temperature was 180 °C. Samples (∼50 ng μL−1) were dissolved in a 1:1 (v/v) H2O/MeCN mixture and sprayed at a flow rate of 3 μL min−1. Acquisition range was m/z 50–3000, internal calibration was done with Electrospray Calibrant Solution (Fluka).

3. Experimental 3.1. Bacterial strain, isolation of the lipopolysaccharide and Opolysaccharide E. coli O54 type strain (laboratory stock number G3091) was obtained from the Institute of Medical and Veterinary Science, Adelaide, Australia. Bacteria were grown to late log phase in 8 L Luria broth 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 [24]. LPS was isolated in a yield of 8.15% from dried cells by the phenolwater method [5], 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. Delipidation of LPS was performed with aq 2% AcOH at 100 °C until precipitation of lipid A. The precipitate was removed by centrifugation (13,000g, 20 min), and an O-polysaccharide was isolated from the supernatant by GPC on a column (56 × 2.6 cm) of Sephadex G-50 (S) (Amersham Biosciences, Sweden) in 0.05 M pyridinium acetate buffer, pH 4.5, monitored by a differential refractometer (Knauer, Germany).

3.5. Analysis of genes Possible gene functions were assigned using the NCBI BLAST program to screen homologous sequences in the GenBank database.

Acknowledgements Authors thank A.O. Chizhov for help with mass spectrometry. This work was supported by the Russian Science Foundation (project № 1414-01042-P). Work on characterization of the O-antigen gene clusters was also supported by the NSFC General Program Grants 81471904, 81772148, 31470194, 31371259, and the Tianjin Municipal Science and Technology Commission, P. R. China (No. 15JCQNJC44500). 37

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