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

FEMS Microbiology Letters 154 (1997) 103^109

The lipopolysaccharide of Helicobacter mustelae type strain ATCC 43772 expresses the monofucosyl A type 1 histo-blood group epitope Mario A. Monteiro a *, P.Y. Zheng ;

a

b ;c

, Ben J. Appelmelk b , Malcolm B. Perry

a

Canadian Bacterial Diseases Network, Institute for Biological Sciences, National Research Council Canada, 100 Sussex Drive, Ottawa, Ont. K1A OR6, Canada

b c

Department of Medical Microbiology, Vrije Universiteit Medical School, Amsterdam, The Netherlands Department of Gastroenterology, 2nd A¤liated Hospital, Henan Medical University, Zhengzhou, China

Received 21 March 1997; revised 3 June 1997; accepted 7 June 1997

Abstract

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, K-D-GalNAc-(1 3)-[K-L-Fuc-(1 2]-L-D-Gal-(1 3)-L-D-GlcNAc, blood group determinant, a mimic of animal cell surface glycolipids and glycoproteins.

C

Keywords :

Blood group A type 1;

C

Helicobacter mustelae

C

; 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 ¢ve strains of H. pylori have * Corresponding author. Tel.: +1 (613) 990-0832; Fax: +1 (613) 941-1327; E-mail: [email protected]

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been shown to possess Lewis X {L-D-Gal-(1 4)[K-L-Fuc-(1 3)]-L-D-GlcNAc} and/or Lewis Y {KL-Fuc-(1 2) -L-D-Gal- (1 4) -[K-L-Fuc-(1 3)]-L-DGlcNAc} blood group antigens in mimicry of human cell surface glycoconjugates [2^5]. 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

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0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 3 0 9 - 1

FEMSLE 7737 25-10-97

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M.A. Monteiro et al. / FEMS Microbiology Letters 154 (1997) 103^109

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 animalspeci¢c 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

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 100³C for 1 h and subsequent removal of the insoluble lipid A was achieved by centrifugation (5000Ug). The supernatant was puri¢ed 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 phenolsulfuric acid assay method [3]. Defucosylated glycan, OS-2, was obtained by treating OS-1 with 10% acetic acid at 100³C for 1 h followed by fractionation on Bio-Gel P-2. H. mustelae

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 tri£uoroacetic acid at 100³C for 4 h followed by reduction in H2 O 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 massspectrometry (GLC-MS) using a Hewlett-Packard chromatograph equipped with a 30 m DB-17 capillary column [210³C (30 min)^240³C at 2³C/min] and

MS in the electron impact (EI) mode was recorded using a Varian Saturn II mass spectrometer. Enantiomeric con¢gurations 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 190³C for 60 min). A fraction (V1/4) of the permethylated sample was used for positive ion fast atom bombardmentmass 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 1 H 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 D2 O (99.9%). The HOD peak was used as the internal reference at NH 4.786. 2D homonuclear correlation (COSY), total correlation (TOCSY) and nuclear Overhauser e¡ect (NOESY) experiments were done using standard parameters [3]. The mixing times for the TOCSY and NOESY experiments were 35 and 400 ms, respectively. 31 P NMR spectra was recorded at 202.5 MHz with ortho-phosphoric acid as the external reference (NP 0.0). 2.4. Serological procedures

Monoclonal antibody (MAb) 3-3a, speci¢c for blood group A [13], was obtained from Bioprobe, Amsterdam, The Netherlands. TMAbs 1E52 and 6H3, speci¢c for Lewis Y and X, respectively, were obtained from R. Negrini, Brescia, Italy. TMAb 54.4 F6A, speci¢c 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

FEMSLE 7737 25-10-97

M.A. Monteiro et al. / FEMS Microbiology Letters 154 (1997) 103^109

105

V

bu¡er system [14]. Part of the gel was silver stained

ratio of 0.6 :2 :3 :1.5 :1 :1. Methylation linkage analy-

and part of it electroblotted to nitrocellulose. The

sis of OS-1 (

blot was incubated with MAb 54.4F6a (anti-Lewis

be in the pyranose ring form and furnished the fol-

X) or 3-3a (anti-blood group A), and subsequently

lowing permethylated alditol acetate derivatives as

developed.

the major components (linkage types and approxi-

0.8 mg) showed all sugar residues to

mate ratios in brackets) : 1,5-di-O-acetyl-2,3,4-tri-O-

methyl-fucitol [terminal Fuc (0.65)], 1,5-di-O-acetyl2,3,4,6-tetra-O-methyl-glucitol

3. Results and discussion

Cells from

H. mustelae

[terminal

Glc(1.8)],

1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-galactitol [terATCC 43772 proved to be

very fastidious to grow and the phenol-water extraction of bacterial cells gave very low amounts of LPS.

minal 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-acetyl2,3,4-tri-O-methyl-galactitol

[6-linked

Gal

(0.7)],

The water-insoluble LPS was heated in mild acetic

1,2,3,5-tetra-O-acetyl-4,6-di-O-methyl-galactitol [2,3-

acid to cleave the acid-sensitive ketosidic linkage of

linked

the

assumed

present

3-deoxy-D-manno-octulosonic

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-de-

acid (Kdo) and puri¢cation on a column of Bio-

oxy-1,5-di-O-acetyl-3,4,6-tri-O-methyl-2-(N-methyla-

Gel P-2 gave lipid A-free OS-1, which eluted after

cetamido)-galactitol [terminal GalNAc (0.9)] and 2-

the void volume. Traces of free fucose were also

deoxy -1,3,5-tri-O- acetyl-4,6- di-O-methyl-2-(N-meth-

detected presumably arising from inadvertent hydro-

ylacetamido)-glucitol [3-linked GlcNAc (0.9)]. Also

V

lytic release during the mild acetic acid treatment. Sugar composition analysis of OS-1 ( revealed the presence of (Glc),

D-galactose

(LD-Hep),

(Gal),

L-fucose

(Fuc),

L-glycero-D-

N-acetyl-D-glucosamine

0.1 mg)

D-glucose

manno-heptose N-

(GlcNAc) and

acetyl-D-galactosamine (GalNAc) in an approximate

Fig. 1. FAB-MS spectrum of the permethylated

H. mustelae

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 de¢ned composition, at

m/z

260 [HexNAc],

ATCC 43772 OS-1 showing the primary glycosyl oxonium ions at

709 and 883 that originate from preferential cleavage at the HexNAc units.

FEMSLE 7737 25-10-97

m/z

260,

106

M.A. Monteiro et al. / FEMS Microbiology Letters 154 (1997) 103^109

Fig. 2. Interpretation of the primary glycosyl oxonium m/z ions obtained from the FAB-MS of the permethylated H. mustelae ATCC 43772 OS-1.

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, 2,3linked Gal and terminal Fuc in the arrangement m/z

shown in Fig. 2. The m/z 709 ion arises from the defucosylated trisaccharide GalNAcCGalCGlcNAc. The secondary ion at m/z 228 may arise from loss of 32 amu [CH3 OH] 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 L-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 con¢rmed by achieving complete defucosylation and analyzing the linkage types after the removal of Fuc. Selective defucosylation of OS-1 with 10% acetic acid at 100³C 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

Fig. 3. 1 H NMR spectrum of H. mustelae ATCC 43772 OS-1 in D2 O at 300 K showing the down¢eld anomeric resonances for K-Fuc (N 5.28, a), for K-GalNAc (N 5.19, b), and for the 2,3-linked L-Gal (N 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 L-anomeric signals stemming from the remaining units, 3-linked GlcNAc, 3-linked Gal, 6-linked Gal and terminal Glcs. In the up¢eld region there can be seen the H-6,6P deoxy signals of Fuc (N 1.22) and the singlet from the acetamido [-CH3 ] protons of the N-acetyl-hexosamines (N 2.08).

FEMSLE 7737 25-10-97

M.A. Monteiro et al. / FEMS Microbiology Letters 154 (1997) 103^109

107

ratio of 3-linked Gal had signi¢cantly increased. The other linkage types had the same ratios as in OS-1.

O

Hence, the terminal Fuc can be placed at the 2position of the 2,3-linked Gal unit.

1

The

H NMR (Fig. 3) of OS-1 (taken from the

¢rst tube for homogeneity), with aid of assignments from 2D COSY and TOCSY experiments, showed

NHÿ6 60

up¢eld resonances belonging to Fuc at (d) (

J5 6

from the

N-acetyl

hexosamine

K-anomeric 5.19 (

J3 4 ;

J1 2 ;

V

Gal

In

the

N

resonances at

anomeric 5.28 (

1.22

;

2.08 stemming

N-acetyl-

methyl protons of the

units.

region,

J1 2

two

3.4 Hz) and

;

V

3.1 Hz) were assigned to sugars with the

con¢guration 4

N

6.1 Hz) and a singlet at

;

Hz).

The

(both

J2 3

having

K-anomeric

;

10

resonance

Hz

N

at

and 5.28

Fig. 4. a : Silver stain. b : Blot probed with anti-Lewis X MAb. c:

Blot

lanes :

was assigned to the Fuc residue by total correlation

3.75, H-3

N

N

3.58, H-4

3.75, H-5

N

4.25, H-6,6P

inter-

1.22]. A 2D NOESY experiment showed an

V

V

space nOe connectivity between H-1 of Fuc and H-2 (

N

L-Gal

3.87) of a

J2 3

(

;

10 Hz and

J3 4 ;

N 4.69 K-Fuc-(1

C

whose anomeric resonance was at Hz) revealing the presence of a

4 Hz)

(

J1 2

inter-space

(

N

C

7.8

;

L-Gal N 5.19

2)-

sequence. In addition, the anomeric H-1 at also showed an

nOe connectivity to H-3

L-Gal indicating a K-GalNAcL-Gal connection. The L-anomeric resonance

3.92) of the same

(1

N

at

3)-

4.69 was then assigned to the 2,3-linked Gal

residue

and

K-anomeric

the

resonance

N

at

5.19

N

was assigned to the terminal GalNAc unit [H-1 5.19, H-2

with and

anti-blood

H. mustelae,

group

N 4.21, H-3 N 3.93]. The anomeric resonan-

A

MAb.

Left

and

right

respectively.

N 5.28, H-2

of ring protons from H-1 to H-6,6P [H-1

N N

probed

H. pylori

H. mustelae

acted strongly with cells of

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 molecular mass

lae

O-antigen

H. pylori

possesses high

chains whereas

H. muste-

does not (Fig. 4). The tricine bu¡er 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

ces belonging to the 3-linked GlcNAc, terminal Glc

bu¡er did not give a good resolution of the `lad-

and

der-like pattern'

Gal

units,

3-

and

6-linked

Gal

could

not

be

unambiguously assigned due to overlapping of reso-

O-antigen

bands.

The structural information obtained from GLC-

K-anomerics

MS of sugar derivatives, FAB-MS, NMR and sero-

belonging to Fuc and GalNAc, and to sugars with

logical studies shows that part of the LPS structure,

nances. However, since there are only

the

manno-pyranose

blets)

from

including

L-anomeric chemical

LD-Hep

the

the

con¢guration (unresolved dou-

3-linked

units,

all

remaining

GlcNAc,

must

units,

have

con¢guration. The data obtained from

analysis

C

and

NMR

C

pointed

towards

K-

3)[

L-Fuc-(1

GlcNAc, in the LPS of

L-

2]-

C

D-Gal-(1

H. mustelae

the

monofucosyl A type 1 histo-blood group determinant [15], that being :

KÿdÿGalNAcÿ…1!3†ÿLÿdÿGalÿ…1!3†ÿLÿdÿGlcNAcÿ…1!:::

K3)-L-

presence of the blood group A type 1 epitope, GalNAc-(1

the

more precisely, the outer core region, expresses the

2

j

D-

D-

1

KÿlÿFuc:

type strain.

The presence of the A type histo-blood group epitope in

H. mustelae

ATCC 43772 was also detected

H. pyN-acetyl-lactos-

In contrast to LPSs from the human related

serologically with a speci¢c MAb. MAb 3-3a [13],

lori

speci¢c for the A type blood group determinant, re-

aminoglycan

[2^5], no elongated fucosylated

FEMSLE 7737 25-10-97

O-chains

were detected in the LPS of

M.A. Monteiro et al. / FEMS Microbiology Letters 154 (1997) 103^109

108

this

H. mustelae

strain. However, changes in in vitro

H. pylori

cultivation when growing pected

of

being

responsible

for

have been sus-

LPS

modi¢cation

O-chain-de¢cient

from smooth-form LPS to

to

a

strictly

present

linear

H. pylori

architecture

of

the

LD-Heps

LPSs [2^5] and is more akin to

the corresponding region in

C. jejuni

LPSs [19].

rough-

The monofucosyl A type 1 blood group epitope

form LPS [16], and thus, this phenomenon must also

has been found in human erythrocytes, dog intestine,

Helico-

be taken into account when cultivating other

bacters.

H. pylori

As with

hog

and

rat

gastric

mucosa

(for

a

comprehensive

smooth-form LPSs [2^5]

review see [17]), and thus the possibility exists that

which mimic human cell surface glycoconjugates in

it might also be present in ferret gastric mucosa cells.

carrying Lewis X and Lewis Y blood group determi-

The molecular mimicry displayed by the LPS struc-

H. mustelae

nants,

also carries a blood group epi-

tures of

H. pylori (Lewis X and Y blood group H. mustelae (blood group A type 1

tope, the monofucosyl A type 1, in mimicry of hu-

topes) and

man and other animals' cell surface glycolipids and

tope) with mammalian

glycoproteins [17].

H. mustelae

O-chain-type

devoid of an

epiepi-

cell surface molecules may

ATCC 43772 being

have arisen through the adoption of the host's gly-

polysaccharide, character-

cosyltransferases by the bacteria. Also, this molecular

form

molecules may play a role in determining host and

LPSs

which

also

display

molecular

Neisseria gonor-

such as the lipo-oligosaccharides of

rhoeae and

species

the

jejuni

core

which regions

resemble of

LPS

mimicry

paraglobosides from

[18]

Campylobacter

which mimic gangliosides [19]. The immuno-

mimicry

between

Helicobacter

istic of smooth-form LPS, is more akin to rough-

site speci¢city for

Helicobacter

LPSs

and

host

attachment to host

cell surface molecules, in evading the host's immune system and, as with component

might

H. pylori

also

be

[7], an autoimmune

present

in

H. mustelae

H. mustelae

blot and SDS-PAGE-silver stain data (Fig. 4) were

pathogenesis. The fact that

in agreement with these chemical structural results

in the onset of gastritis-like symptoms in ferrets and

by revealing the presence of blood group A and by

mimics a blood group epitope through its LPS struc-

H. mustelae lacks a

typical LPS

O-anti-

The other units present in the

H. mustelae

ATCC

showing that gen chain.

43772, namely, two

resonances (

LD-Heps,

two Gals, two Glcs and

N

1.98 and

N

2.12) in the

C

1

C

structure

(

lipid A). The

O-chain

31

outer

might

be

of

H. pylori, and thus experimental with H. mustelae type strain

ferrets

ATCC 43772 may be a suitable model for mimicking

H. pylori

pathogenesis in man.

C

core

inner

core-

Acknowledgments

P NMR spectrum of OS-1 revealed

the presence of a monoester phosphate at which

infection

H NMR spec-

trum], are placed within the inner core context of LPS

ture, the monofucosyl A type 1, shows striking similarities to human

one Kdo [detected by the characteristic deoxy H-3,3P

is implicated

attached

to

the

NP

0.87

2,3,7-linked

LD-

We thank L.A. Kurjanczyk for growing

lae

H. muste-

cells, R.A.Z. Johnston for running FAB-MS and

Hep, thus explaining the detection of this permeth-

the Canadian Bacterial Diseases Network for fund-

ylated

ing.

alditol

acetate

derivative

in

only

trace

H. pylori studies available of this H. mus-

amounts, a feature also observed in [3]. The limited quantities

telae

LPS at the time were not su¤cient for a de-

References

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following

H. mustelae

di¡erences

between

H. pylori

and

inner-core structures can be stated : (i)

H. pylori strains so far examined have -glycero- -manno-heptose as a core con-

all LPSs from contained

D

D

stituent [2^5], a biosynthetic precursor of this sugar, however, is not present in

LD-Hep,

H. mustelae

type strain ATCC 43772, and (ii) the presence of branched

LD-Hep

units in

H. mustelae

is in contrast

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