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The lipopolysaccharide of Helicobacter mustelae type strain ATCC 43772 expresses the monofucosyl A type 1 histo-blood group epitope

Mario A Monteiro, P.Y Zheng, Ben J Appelmelk, Malcolm B Perry
DOI: http://dx.doi.org/10.1111/j.1574-6968.1997.tb12630.x 103-109 First published online: 1 September 1997


The lipopolysaccharide of Helicobacter mustelae type strain ATCC 43772 was obtained by phenol-water extraction of bacterial cells. Structural investigations were made on the lipid A free saccharide moiety released from the lipopolysaccharide by mild acetic acid hydrolysis. Nuclear magnetic resonance, gas liquid chromatography-mass spectrometry and fast atom bombardment-mass spectrometry were employed in the characterization of products from chemical manipulations. A monoclonal antibody specific for blood group A reacted strongly with lipopolysaccharide of H. mustelae. Chemical and serological data showed that the outer core region of the lipopolysaccharide from H. mustelae ATCC 43772 expresses the monofucosyl A type 1, α-d-GalNAc-(1→3)-[α-l-Fuc-(1→2]-β-d-Gal-(1→3)-β-d-GlcNAc, blood group determinant, a mimic of animal cell surface glycolipids and glycoproteins.

  • Blood group A type 1
  • Helicobacter mustelae
  • Lipopolysaccharide
  • Molecular mimicry
  • Structural determination

1 Introduction

Lipopolysaccharides (LPSs) are bacterial cell surface glycan molecules responsible for bacteria-host interactions. It is now well established that infection in humans caused by the Gram-negative bacteria Helicobacter pylori may lead to the onset of gastritis, gastric and duodenal ulcers and gastric carcinoma [1]. The LPSs from five strains of H. pylori have been shown to possess Lewis X {β-d-Gal-(1→4)-[α-l-Fuc-(1→3)]-β-d-GlcNAc} and/or Lewis Y {α-l-Fuc-(1→2)-β-d-Gal-(1→4)-[α-l-Fuc-(1→3)]-β-d-GlcNAc} blood group antigens in mimicry of human cell surface glycoconjugates [25]. The presence of these blood group epitopes in the LPSs are now a basis for a serotyping system for H. pylori[6]. Also, this molecular mimicry has been implicated in autoimmunity associated with H. pylori infections [7]. Various attempts to generate an animal model using H. pylori from humans have not been generally successful [8]. To circumvent this, Helicobacter isolates have been isolated from experimental animals [1] such as ferrets, cats and dogs, and are now being employed as animal models of Helicobacter infection [9]. Thus, we decided it would be of interest to investigate the LPS structures of some of these animal-specific Helicobacter species for comparison with those of H. pylori. The present work describes the structure of the outer core region of the LPS from H. mustelae type strain ATCC 43772, isolated from the gastric mucosa of ferrets, which gives rise to gastritis-like symptoms in ferrets [10].

2 Materials and methods

2.1 Generation of lipopolysaccharide and oligosaccharides OS-1 and OS-2

H. mustelae ATCC 43772 cells were grown in the laboratory of Dr. John L. Penner at the University of Toronto in the same manner as H. pylori cells [11] and the LPS was extracted by the phenol-water method as described previously [11]. The LPS preparation was treated with 1% acetic acid at 100°C for 1 h and subsequent removal of the insoluble lipid A was achieved by centrifugation (5000×g). The supernatant was purified on a column of Bio-Gel P-2 with water (1 ml/tube) as the eluent to give glycan OS-1. Glycan fractions were scanned for by the phenol-sulfuric acid assay method [3]. Defucosylated glycan, OS-2, was obtained by treating OS-1 with 10% acetic acid at 100°C for 1 h followed by fractionation on Bio-Gel P-2.

2.2 Sugar composition and methylation linkage analysis

Sugar composition analysis was done by the alditol acetate method ([3] and references therein). The hydrolysis was done in 4 M trifluoroacetic acid at 100°C for 4 h followed by reduction in H2O with NaBD4 and subsequent acetylation with acetic anhydride and with residual sodium acetate as the catalyst. Characterization of the alditol acetate derivatives was done by gas-liquid-chromatography mass-spectrometry (GLC-MS) using a Hewlett-Packard chromatograph equipped with a 30 m DB-17 capillary column [210°C (30 min)–240°C at 2°C/min] and MS in the electron impact (EI) mode was recorded using a Varian Saturn II mass spectrometer. Enantiomeric configurations of the individual sugars were determined by the formation of the respective 2-(S)- and 2-(R)-butyl glycosides [12]. Methylation linkage analysis was carried out by the Ciucanu and Kerek (NaOH/DMSO/MeI) procedure ([3] and references therein) and with full characterization of permethylated alditol acetate derivatives by GLC-MS in the EI mode (DB-17, isothermally at 190°C for 60 min). A fraction (∼1/4) of the permethylated sample was used for positive ion fast atom bombardment-mass spectrometry (FAB-MS) which was carried out on a Jeol JMS-AX505H mass spectrometer with glycerol(1):thioglycerol(3) as the matrix and 3 kV as the tip voltage.

2.3 Nuclear magnetic resonance spectroscopy (NMR)

1-Dimensional (1D) and 2D 1H NMR experiments were recorded on a Bruker AMX 500 spectrometer at 300 K using standard Bruker software. Prior to performing the NMR experiments the samples were lyophilized thrice with D2O (99.9%). The HOD peak was used as the internal reference at δH 4.786. 2D homonuclear correlation (COSY), total correlation (TOCSY) and nuclear Overhauser effect (NOESY) experiments were done using standard parameters [3]. The mixing times for the TOCSY and NOESY experiments were 35 and 400 ms, respectively. 31P NMR spectra was recorded at 202.5 MHz with ortho-phosphoric acid as the external reference (δP 0.0).

2.4 Serological procedures

Monoclonal antibody (MAb) 3-3a, specific for blood group A [13], was obtained from Bioprobe, Amsterdam, The Netherlands. TMAbs 1E52 and 6H3, specific for Lewis Y and X, respectively, were obtained from R. Negrini, Brescia, Italy. TMAb 54.4 F6A, specific for polymeric Lewis X, was obtained from G. Van Dam, Leiden, The Netherlands. H. pylori cells served as positive controls for the anti-Lewis X and Y MAbs. Bacterial cells of H. mustelae and H. pylori were digested with proteinase K and electrophoresed in a 12% gel using the tricine buffer system [14]. Part of the gel was silver stained and part of it electroblotted to nitrocellulose. The blot was incubated with MAb 54.4F6a (anti-Lewis X) or 3-3a (anti-blood group A), and subsequently developed.

3 Results and discussion

Cells from H. mustelae ATCC 43772 proved to be very fastidious to grow and the phenol-water extraction of bacterial cells gave very low amounts of LPS. The water-insoluble LPS was heated in mild acetic acid to cleave the acid-sensitive ketosidic linkage of the assumed present 3-deoxy-d-manno-octulosonic acid (Kdo) and purification on a column of Bio-Gel P-2 gave lipid A-free OS-1, which eluted after the void volume. Traces of free fucose were also detected presumably arising from inadvertent hydrolytic release during the mild acetic acid treatment.

Sugar composition analysis of OS-1 (∼0.1 mg) revealed the presence of l-fucose (Fuc), d-glucose (Glc), d-galactose (Gal), l-glycero-d-manno-heptose (ld-Hep), N-acetyl-d-glucosamine (GlcNAc) and N-acetyl-d-galactosamine (GalNAc) in an approximate ratio of 0.6:2:3:1.5:1:1. Methylation linkage analysis of OS-1 (∼0.8 mg) showed all sugar residues to be in the pyranose ring form and furnished the following permethylated alditol acetate derivatives as the major components (linkage types and approximate ratios in brackets): 1,5-di-O-acetyl-2,3,4-tri-O-methyl-fucitol [terminal Fuc (0.65)], 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-glucitol [terminal Glc(1.8)], 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-galactitol [terminal Gal (0.4)], 1,3,5-tri-O-acetyl-2,4,6-tri-O-methyl-galactitol [3-linked Gal (1.3)], 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-galactitol [6-linked Gal (0.7)], 1,2,3,5-tetra-O-acetyl-4,6-di-O-methyl-galactitol [2,3-linked galactose (0.6)], 1,2,3,5-tetra-O-acetyl-4,6,7-tri-O-methyl-heptitol [2,3-linked ld-Hep (1.0)], 2-deoxy-1,5-di-O-acetyl-3,4,6-tri-O-methyl-2-(N-methylacetamido)-galactitol [terminal GalNAc (0.9)] and 2-deoxy-1,3,5-tri-O-acetyl-4,6-di-O-methyl-2-(N-methylacetamido)-glucitol [3-linked GlcNAc (0.9)]. Also detected, but in trace amounts, was 1,2,3,5,7-penta-O-acetyl-4,6-di-O-methyl-heptitol [2,3,7-linked ld-Hep]. The FAB-MS spectrum (Fig. 1) of the permethylated OS-1 showed primary glycosyl oxonium ions, from preferential cleavage at HexNAc residues, and of defined composition, at m/z 260 [HexNAc], m/z 709 [HexNAc, Hex, HexNAc] and at m/z 883 [HexNAc, Hex, HexNAc, deoxy-Hex]. Fig. 2 shows the interpretation of the m/z ions obtained from FAB-MS. M/z 260 belongs to the terminal GalNAc and, taken together with the linkage analysis data, m/z 883 must emanate from a tetrasaccharide unit containing terminal GalNAc, 3-linked GlcNAc, 2,3-linked Gal and terminal Fuc in the arrangement shown in Fig. 2. The m/z 709 ion arises from the defucosylated trisaccharide GalNAc→Gal→GlcNAc. The secondary ion at m/z 228 may arise from loss of 32 amu [CH3OH] from m/z 260, from loss of 481 amu [HexNAc, Hex, OH] from m/z 709, or from loss of 655 amu [HexNAc, Hex, deoxy-Hex, OH] from m/z 883, all losses due to β-elimination of the residues attached at O-3 of HexNAc units. The ion at m/z 411 could not be assigned and it is most likely a contaminant. These FAB-MS data give no information about the precise location of the Fuc unit on the 2,3-linked Gal. However, the equimolar ratios of terminal Fuc and 2,3-linked Gal and the greater than 1 ratio for 3-linked Gal pointed to the Fuc being attached at the 2-O position of the Gal. This fact was confirmed by achieving complete defucosylation and analyzing the linkage types after the removal of Fuc.

Figure 1

FAB-MS spectrum of the permethylated H. mustelae ATCC 43772 OS-1 showing the primary glycosyl oxonium ions at m/z 260, 709 and 883 that originate from preferential cleavage at the HexNAc units.

Figure 2

Interpretation of the primary glycosyl oxonium m/z ions obtained from the FAB-MS of the permethylated H. mustelae ATCC 43772 OS-1.

Selective defucosylation of OS-1 with 10% acetic acid at 100°C for 1 h gave oligosaccharide OS-2. Methylation linkage analysis on OS-2 revealed that the 2,3-linked Gal was no longer present and that the ratio of 3-linked Gal had significantly increased. The other linkage types had the same ratios as in OS-1. Hence, the terminal Fuc can be placed at the 2-O position of the 2,3-linked Gal unit.

The 1H NMR (Fig. 3) of OS-1 (taken from the first tube for homogeneity), with aid of assignments from 2D COSY and TOCSY experiments, showed upfield resonances belonging to Fuc at δH-6,6′ 1.22 (d) (J5,6 6.1 Hz) and a singlet at δ 2.08 stemming from the N-acetyl methyl protons of the N-acetyl-hexosamine units. In the anomeric region, two α-anomeric resonances at δ 5.28 (J1,2 3.4 Hz) and 5.19 (J1,2 3.1 Hz) were assigned to sugars with the Gal configuration (both having J2,3∼10 Hz and J3,4∼4 Hz). The α-anomeric resonance at δ 5.28 was assigned to the Fuc residue by total correlation of ring protons from H-1 to H-6,6′ [H-1 δ 5.28, H-2 δ 3.75, H-3 δ 3.58, H-4 δ 3.75, H-5 δ 4.25, H-6,6′δ 1.22]. A 2D NOESY experiment showed an inter-space nOe connectivity between H-1 of Fuc and H-2 (δ 3.87) of a β-Gal (J2,3∼10 Hz and J3,4∼4 Hz) whose anomeric resonance was at δ 4.69 (J1,2 7.8 Hz) revealing the presence of a α-Fuc-(1→2)-β-Gal sequence. In addition, the anomeric H-1 at δ 5.19 also showed an inter-space nOe connectivity to H-3 (δ 3.92) of the same β-Gal indicating a α-GalNAc-(1→3)-β-Gal connection. The β-anomeric resonance at δ 4.69 was then assigned to the 2,3-linked Gal residue and the α-anomeric resonance at δ 5.19 was assigned to the terminal GalNAc unit [H-1 δ 5.19, H-2 δ 4.21, H-3 δ 3.93]. The anomeric resonances belonging to the 3-linked GlcNAc, terminal Glc and Gal units, 3- and 6-linked Gal could not be unambiguously assigned due to overlapping of resonances. However, since there are only α-anomerics belonging to Fuc and GalNAc, and to sugars with the manno-pyranose configuration (unresolved doublets) from the ld-Hep units, all remaining units, including the 3-linked GlcNAc, must have the β-anomeric configuration. The data obtained from chemical analysis and NMR pointed towards the presence of the blood group A type 1 epitope, α-d-GalNAc-(1→3)[α-l-Fuc-(1→2]-β-d-Gal-(1→3)-β-d-GlcNAc, in the LPS of H. mustelae type strain.

Figure 3

1H NMR spectrum of H. mustelae ATCC 43772 OS-1 in D2O at 300 K showing the downfield anomeric resonances for α-Fuc (δ 5.28, a), for α-GalNAc (δ 5.19, b), and for the 2,3-linked β-Gal (δ 4.69, c). Also present in the anomeric region are the unresolved doublets belonging to ld-Heps (there are more than two ld-Hep anomeric resonances presumably due to structural heterogeneity in the inner core region), and to β-anomeric signals stemming from the remaining units, 3-linked GlcNAc, 3-linked Gal, 6-linked Gal and terminal Glcs. In the upfield region there can be seen the H-6,6′ deoxy signals of Fuc (δ 1.22) and the singlet from the acetamido [-CH3] protons of the N-acetyl-hexosamines (δ 2.08).

The presence of the A type histo-blood group epitope in H. mustelae ATCC 43772 was also detected serologically with a specific MAb. MAb 3-3a [13], specific for the A type blood group determinant, reacted strongly with cells of H. mustelae 43772 (Fig. 4). Anti-Lewis X and anti-Lewis Y MAbs did not react with H. mustelae 43772. The tricine system gave a good separation in the core region and several bands in that region can be seen in silver stain and this experiment showed that H. pylori possesses high molecular mass O-antigen chains whereas H. mustelae does not (Fig. 4). The tricine buffer system was chosen to optimize separation in the core region, and at least two well separated bands can be seen in that region in silver stain (Fig. 4), of which only the higher molecular mass band expressed blood group A. The non-staining lower molecular mass core band most likely represents incomplete core. The tricine buffer did not give a good resolution of the ‘ladder-like pattern’O-antigen bands.

Figure 4

a: Silver stain. b: Blot probed with anti-Lewis X MAb. c: Blot probed with anti-blood group A MAb. Left and right lanes: H. pylori and H. mustelae, respectively.

The structural information obtained from GLC-MS of sugar derivatives, FAB-MS, NMR and serological studies shows that part of the LPS structure, more precisely, the outer core region, expresses the monofucosyl A type 1 histo-blood group determinant [15], that being: Embedded Image In contrast to LPSs from the human related H. pylori[25], no elongated fucosylated N-acetyl-lactosaminoglycan O-chains were detected in the LPS of this H. mustelae strain. However, changes in in vitro cultivation when growing H. pylori have been suspected of being responsible for LPS modification from smooth-form LPS to O-chain-deficient rough-form LPS [16], and thus, this phenomenon must also be taken into account when cultivating other Helicobacters. As with H. pylori smooth-form LPSs [25] which mimic human cell surface glycoconjugates in carrying Lewis X and Lewis Y blood group determinants, H. mustelae also carries a blood group epitope, the monofucosyl A type 1, in mimicry of human and other animals’ cell surface glycolipids and glycoproteins [17]. H. mustelae ATCC 43772 being devoid of an O-chain-type polysaccharide, characteristic of smooth-form LPS, is more akin to rough-form LPSs which also display molecular mimicry such as the lipo-oligosaccharides of Neisseria gonorrhoeae species which resemble paraglobosides [18] and the core regions of LPS from Campylobacter jejuni which mimic gangliosides [19]. The immunoblot and SDS-PAGE-silver stain data (Fig. 4) were in agreement with these chemical structural results by revealing the presence of blood group A and by showing that H. mustelae lacks a typical LPS O-antigen chain.

The other units present in the H. mustelae ATCC 43772, namely, two ld-Heps, two Gals, two Glcs and one Kdo [detected by the characteristic deoxy H-3,3′ resonances (δ 1.98 and δ 2.12) in the 1H NMR spectrum], are placed within the inner core context of LPS structure (O-chain→outer core→inner core→lipid A). The 31P NMR spectrum of OS-1 revealed the presence of a monoester phosphate at δP 0.87 which might be attached to the 2,3,7-linked ld-Hep, thus explaining the detection of this permethylated alditol acetate derivative in only trace amounts, a feature also observed in H. pylori studies [3]. The limited quantities available of this H. mustelae LPS at the time were not sufficient for a detailed elucidation of inner core structure. However, the following differences between H. pylori and H. mustelae inner-core structures can be stated: (i) all LPSs from H. pylori strains so far examined have contained d-glycero-d-manno-heptose as a core constituent [25], a biosynthetic precursor of ld-Hep, this sugar, however, is not present in H. mustelae type strain ATCC 43772, and (ii) the presence of branched ld-Hep units in H. mustelae is in contrast to a strictly linear architecture of the ld-Heps present H. pylori LPSs [25] and is more akin to the corresponding region in C. jejuni LPSs [19].

The monofucosyl A type 1 blood group epitope has been found in human erythrocytes, dog intestine, hog and rat gastric mucosa (for a comprehensive review see [17]), and thus the possibility exists that it might also be present in ferret gastric mucosa cells. The molecular mimicry displayed by the LPS structures of H. pylori (Lewis X and Y blood group epitopes) and H. mustelae (blood group A type 1 epitope) with mammalian cell surface molecules may have arisen through the adoption of the host's glycosyltransferases by the bacteria. Also, this molecular mimicry between Helicobacter LPSs and host molecules may play a role in determining host and site specificity for Helicobacter attachment to host cell surface molecules, in evading the host's immune system and, as with H. pylori[7], an autoimmune component might also be present in H. mustelae pathogenesis. The fact that H. mustelae is implicated in the onset of gastritis-like symptoms in ferrets and mimics a blood group epitope through its LPS structure, the monofucosyl A type 1, shows striking similarities to human H. pylori, and thus experimental infection of ferrets with H. mustelae type strain ATCC 43772 may be a suitable model for mimicking H. pylori pathogenesis in man.


We thank L.A. Kurjanczyk for growing H. mustelae cells, R.A.Z. Johnston for running FAB-MS and the Canadian Bacterial Diseases Network for funding.


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