Expression of histo-blood group antigens by lipopolysaccharides of Helicobacter pylori strains from Asian hosts: the propensity to express type 1 blood-group antigens

Mario A. Monteiro1, Peng-yuan Zheng2, Bow Ho2, Shin-ichi Yokota3, Ken-ichi Amano3, Zhi-jun Pan4, Douglas E. Berg4, Kenneth H. Chan, Leann L. MacLean and Malcolm B. Perry

Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada, 2Department of Microbiology, National University of Singapore, Singapore, 3Central Research Laboratory, Akita University School of Medicine, Akita, Japan, and 4Departments of Molecular Microbiology and Genetics, Washington University School of Medicine, St. Louis, MO 63130, USA

Received on November 16, 1999; revised on January 18, 2000; accepted on January 22, 2000.


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Past studies have shown that the cell surface lipopolysaccharides (LPSs) of the ubiquitous human gastric pathogen Helicobacter pylori (a type 1 carcinogen) isolated from people residing in Europe and North America express predominantly type 2 Lewis x (Lex) and Ley epitopes and, infrequently, type 1 Lea, Leb, and Led antigens. This production of Lewis blood-group structures by H. pylori LPSs, similar to those found in the surfaces of human gastric cells, allows the bacterium to mimic its human niche. In this study, LPSs of H.pylori strains extracted from patients living in China, Japan, and Singapore were chemically and serologically analyzed. When compared with Western H.pylori LPSs, these Asian strains showed a stronger tendency to produce type 1 blood groups. Of particular interest, and novel observations in H.pylori, the O-chain regions of strains F-58C and R-58A carried type 1 Lea without the presence of type 2 Lex, strains R-7A and H607 were shown to have the capability of producing the type 1 blood group A antigen, and strains CA2, H507, and H428 expressed simultaneously the difucosyl isomeric antigens, type 1 Leb and type 2 Ley. The apparent proclivity for the production of type 1 histo-blood group antigens in Asian H.pylori LPSs, as compared with Western strains, may be an adaptive evolutionary effect in that differences in the gastric cell surfaces of the respective hosts might be significantly dissimilar to select for the formation of different LPS structures on the resident H.pylori strain.

Key words: Helicobacter pylori/lipopolysaccharides/histo-blood groups/structural determination/molecular mimicry


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
In humans, the initiation of various gastric diseases and ailments are attributed to infection by the Gram-negative bacterium Helicobacter pylori, a type 1 carcinogen (Marshall and Warren, 1984Go; Dunn et al., 1997Go). The cell surface lipopolysaccharides (LPSs; O-chain->core->lipid A) elaborated by H.pylori strains isolated from North American and European populations, have been shown to possess the cunning ability to mimic host gastric-cell surface glycan molecules by expressing in their O-antigen chain (O-chain) region predominantly type 2 Lewis x (Lex) {ßDGalp-(1->4)-[{alpha}LFucp-(1->3)]-ßDGlcpNAc} and Ley {{alpha}LFucp-(1->2)-ßDGalp-(1->4)-[{alpha}LFucp-(1->3)]-ßDGlcpNAc} antigens in monomeric and/or polymeric forms (Aspinall et al., 1994Go, 1996, 1997; Aspinall and Monteiro, 1996Go; Monteiro et al., 2000Go) (Figure 1); and, seldom, some strains were shown to carry type 1 Lea {ßDGalp-(1->3)-[{alpha}LFucp-(1->4)]-ßDGlcpNAc}, Leb {{alpha}LFucp-(1->2)-ßDGalp-(1->3)-[{alpha}LFucp-(1->4)]-ßDGlcpNAc} and/or Led {{alpha}LFucp-(1->2)-ßDGalp-(1->3)-ßDGlcpNAc} determinants in addition to type 2 antigens (Monteiro et al., 1998aGo, 2000). Two other types of H.pylori O-chains carried type-2 N-acetyl-polylactosamine polymers that were glucosylated (Monteiro et al., 1998bGo) or galactosylated (Aspinall et al., 1999Go) at the O-6 position of the backbone GlcNAc residues, {-[->3)-ßDGalp-(1->3)[{alpha}DGlc/Gal-(1->6)]-ßDGlcpNAc-(1-]n->}. The LPS of the Helicobacter species that colonizes the gastric mucosa of ferrets, H.mustelae, also mimics its particular niche by producing the type 1 blood-group A antigen {{alpha}DGalpNAc-(1->3)-[{alpha}LFucp-(1->2)]-ßDGalp-(1->3)-ßDGlcpNAc} (Monteiro et al., 1997Go; O'Croinin et al., 1998Go).



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Fig. 1. Structure the O-chain and core oligosaccharide LPS regions of Western H.pylori strains. The LPS from most Western H.pylori strains express type 2 Lex and Ley blood-group antigens.

 
The LPSs of H.pylori strains thus far examined were found to express a conserved core oligosaccharide structure based on a linear phosphorylated (by a monoester phosphate or a by a 2-aminoethylphosphate) backbone, {alpha}DGlcp-(1->3)-{alpha}DGlcp-(1->4)-ßDGalp-(1->7)-D{alpha}DHepp-(1->2)-L{alpha}DHepp-(1->3) [P/AEP->7]-L{alpha}DHepp-(1->5)-Kdo, that is extended by an additional side-branch D{alpha}DHepp residue appended to the O-2 position of the distal D{alpha}DHepp of the backbone glycan (Figure 1) (Aspinall and Monteiro, 1996Go; Aspinall et al., 1996Go, 1997; Monteiro et al., 1998aGo, 2000). A few H.pylori core oligosaccharides also contained 2- and 6-substituted DDHep units as members of the DDHep side-branch (Aspinall and Monteiro, 1996Go; Monteiro et al., 2000Go). In some H.pylori strains, a (1->6)-{alpha}-glucan antenna, {alpha}DGlcp-1-[->6-{alpha}DGlcp-1]n->, was found to be connected to the O-2 site of the side-branch D{alpha}DHepp (Figure 1).

Emanating from the interest in examining LPSs of H.pylori strains from different regions of the globe, for an overall structural comparison and specifically to determine if H.pylori strains of different origins followed the paradigm of Western strains in producing "Lewis blood-group" O-chains, H.pylori LPSs isolated from four strains of patients residing in China, five strains isolated from hosts living in Japan and three strains extracted from hosts living in Singapore were investigated by detailed chemical and serological analyses. The intact LPS molecules, removed by water-phenol extractions of bacterial cells, were used for chemical, mass-spectrometry, and serological analyses, and the lipid-free polysaccharides were used for nuclear magnetic resonance studies. Due to intra-strain molecular heterogeneity and/or insufficient quantities of material (H.pylori is an extremely fastidious grower in vitro), the complete assignment of sugar anomeric configurations by nuclear magnetic resonance was not possible and thus in most instances the data obtained by serology, using well-characterized monoclonal antibodies (mAbs) specific for histo-blood group antigens, aided in confirming the sugar ring configurations. The results obtained in this study unveiled structural information on Asian H.pylori LPSs and provided the basis for a comparison with already elucidated LPS structures from Western strains. The following results, with each section describing strains that express similar antigens, will shed light on the role(s) of LPS in H.pylori pathogenesis, and will assist in understanding the LPS biosynthetic pathways that involve the histo-blood group glycosyltransferases of H.pylori.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Lipopolysaccharides of H.pylori Chinese strains F-58C and R-58A
The alditol acetates and the butyl chiral-glycosides derivatives, obtained respectively from monosaccharide composition and enantiomeric analyses, of H.pylori strain F-58C LPS (Figure 2) showed that this glycan molecule was composed of 6-deoxy-L-galactose [L-fucose (LFuc)], D-glucose (DGlc), D-galactose (DGal), N-Acetyl-D-glucosamine (DGlcNAc), D-glycero-D-manno-heptose (DDHep), and L-glycero-D-manno-heptose (LDHep) with the respective gas-liquid-chromatogram (GLC) responses 17.3, 5.2, 38.4, 26.5, 5.0, and 4.5. The sugar linkage types present in the LPS of H.pylori F-58C were determined by methylation linkage analysis performed on the intact LPS, and it showed (Table I) that this LPS was composed of Fuc, Glc, and Gal end-groups, 3-substituted Glc and Gal, 4-substituted Gal, 7- and 2,7-substituted DDHep, 2- and 3,7-substituted (traces) LDHep, and 3-, 4-, and 3,4-substituted GlcNAc units. Sugar sequence analysis of the LPS peripheral O-chain region (nonreducing end of the LPS molecule) was obtained by a fast atom bombardment–mass spectrometry (FAB-MS) experiment, along with product ion scan experiments performed on primary ions, carried out on the methylated F-58C intact LPS (Figure 3, Table II), where two strong A type primary glycosyl oxonium ions, originating from the preferred cleavage at the reducing GlcNAc and Hep (Dell et al., 1990Go; Pohlentz et al., 1998Go), were observed at m/z 638 [Fuc, Hex, GlcNAc]+ and m/z 886 [Fuc, Hex, GlcNAc, Hep]+. A product ion scan on m/z 638 showed a secondary ion, arising through ß-elimination (displacement of the residue linked to O-3 of the reducing GlcNAc) of 236 atomic mass units (amu), at m/z 402[638–236(HexOH)], which revealed the presence of one hexose end-group at position O-3 of the 3,4-disubstituted GlcNAc residue {Hex-(1->3)-[Fuc-(1->4)]-GlcNAc}. The combination of terminal Fuc and Gal, and 3,4-disubstituted GlcNAc (Table I) and the FAB-MS fragmentation ion pattern of m/z 638->402 was representative of the type 1 Lea blood-group {ßDGalp-(1->3)-[{alpha}LFucp-(1->4)]-ßDGlcpNAc} (Egge and Peter-Katalinic, 1987Go) in H.pylori F-58C LPS (Figure 4). The other possibility stemming from the presence of m/z 638 would be the presence of Lex {ßDGalp-(1->4)-[{alpha}LFucp-(1->3)]-ßDGlcpNAc}, but no secondary ion at m/z 432 was observed and thus the presence of Lex was ruled out. Also, an enzyme-linked immunosorbent assay (ELISA) experiment performed on whole cells of strain F-58C and employing the Lex mAb BG-7 did not detect the Lex antigen, but a similar experiment with Lea mAb BG-6 confirmed the presence of Lea (OD > 2.0) thus supporting the results obtained by FAB-MS and chemical analyses. Ion m/z 886 embodied the just-described Lea trisaccharide (638 amu) and a monosubstituted heptose (248 amu) from the outer core region [Lea->Hep]+ (Figure 4). Additional ions (Table II) were observed at m/z 668 (primary ion) and at m/z 228, from a product ion scan on m/z 668 [668–410(Hex-HexOH)] (Egge and Peter-Katalinic, 1987Go) that showed the presence of Hex->Hex-(1->3)-GlcNAc. Since 4-substituted Gal and terminal Glc belong to the core oligosaccharide architecture (Figure 1), this Hex->Hex moiety was tentatively assigned, in corroboration with the presence of terminal and 3-substituted Gal in the linkage analysis data (Table I), to denote a Gal-(1->3)-Gal disaccharide in a type 1 linear B blood-group antigen {Gal-(1->3)-Gal-(1->3)-GlcNAc} (Hanfland et al., 1988Go) (Figure 4). The FAB-MS spectrum (Figure 3) of the methylated F-58C LPS also showed a primary ion at m/z 916 representing the just-described Gal-Gal-GlcNAc antigen (668 amu) and one linear heptose (248 amu) from the outer core region [linear B blood-group->DDHep]+.



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Fig. 2. Gas-liquid chromatogram of alditol acetate derivatives from H.pylori F-58C LPS. The profile shows the presence of Fuc, Glc, Gal, GlcNAc, DDHep, and LDHep.

 

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Table I. Sugar linkage types of LPSs from H.pylori strains investigated in this study
 


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Fig. 3. The complete FAB-MS spectrum of methylated F-58C LPS. The spectrum reveals the presence of type 1 Lea (m/z 638->402), type 1 linear B blood-group (m/z 668->228), and the connection of Lea and linear B antigens to the core region via the 7-substituted DDHep unit (m/z 886 and 916).

 

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Table II. Interpretation of m/z ions in the FAB-MS spectrums of the methylated intact LPSs from H.pylori F-58C, R-58A, F-15A, R-7A, CA2, CA4, CA5, CA6, GU2, H428, and H507 strains*
 


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Fig. 4. Structure the O-chain and core oligosaccharide LPS regions of H.pylori F-58C and R-58A. The LPS is composed of the type 1 Lea blood-group antigen (O-chain region) covalently connected to a core oligosaccharide. The O-chain section of H.pylori F-58C also expresses the type 1 linear B blood-group determinant.

 
The composition analysis of H.pylori R-58A LPS resembled that of strain F-58C in giving LFuc, DGlc, DGal, DGlcNAc, DDHep, and LDHep in approximate equimolar ratios. Table I shows the sugar linkage types found to be present in R-58A LPS by methylation linkage analysis. Terminal Fuc and Gal and 3,4-substituted GlcNAc were the representative units of O-chain Lewis blood-group structure(s). These glycose units were indeed observed in the FAB-MS spectra of the methylated R-58A LPS which showed the primary ion m/z 638 and its secondary ion m/z 402 [from a product ion scan on m/z 638(638–236)] showing the presence of the type 1 Lea blood-group determinant in this LPS (Figure 4) as in strain F-58C LPS (Figure 3). The same R-58A LPS FAB-MS spectrum also yielded the primary ion m/z 886 [Lea->Hep]+ (Table II) that represented the covalent connection of the O-chain Lea epitope to the core region through a mono-substituted heptose unit. As with strain F-58C, the sole linear heptose detected, and that was located in the outer-core area, was a 7-substituted DDHep residue (Table I), and thus it was the heptose responsible for the O-chain to core connection in ion m/z 886 [Lea->7-DDHep]+ and in m/z 916 [linear B blood-group->7-DDHep]+ (Table II, Figure 3).

The non-histo blood group monosaccharide components detected in the methylation linkage analysis of F-58C and R-58A LPSs (Table I) were placed in the context of the H.pylori core region. All the sugar units that compose a typical H.pylori core hexasaccharide backbone with an appended DDHep unit, Glc-(1->3)-Glc-(1->4)-Gal-(1->7)-[DDHep-(1->2)]-DDHep-(1->2)-LDHep-(1->3)-[PO4->7]-LDHep (Figure 1), were attended to in the chemical linkage analysis of F-58C and R-58A LPSs (Figure 4, Table I). H.pylori F-58C and R-58A LPSs did not carry any 6-substituted Glc units which, in line with the equimolar concentrations of 7 and 2,7-substituted DDHep, ruled out the presence of the side-branch (1->6)-glucan antenna that is present in some Western H.pylori LPSs (Figure 1). The distal side-branch 7-substituted DDHep residue was thus the only plausible unit responsible for bridging the Lewis blood-group determinant of the O-chain and the core part of the LPS represented in the FAB-MS spectrum by the primary glycosyl oxonium ions m/z 886 {Lea->7-DDHep} and m/z 916 {linear B blood-group->7-DDHep} discussed above (Figures 3, 4). The 31P NMR spectrums of the lipid-free LPSs of F-58C and R-58A each showed one resonance at {delta} 3.00 and 3.03, respectively, belonging to a monoester phosphate (PO4->sugar) that which is attached to the inmost 3,7-substituted LDHep (Table I, Figure 1 and 4).

The LPSs of H.pylori F-58C and R-58A were determined to contain an O-chain region composed of a single type 1 Lea blood-group antigen covalently connected to the core oligosaccharide by a linear 7-substituted DDHep unit (Figure 4). The presence of a sole type 1 Lea determinant (without the usual concomitant presence of type 2 Lex) in strains F-58C and R-58A is a new finding in H.pylori LPSs. The O-chain region of strain F-58C also contained the type 1 linear B blood group antigen (Figure 4). The sugar linkage types of the core oligosaccharide regions of H.pylori F-58C and R-58A LPSs (Figure 4) were analogous and expressed the typical H.pylori core-backbone structural units (Table I, Figures 1 and 4). No side-branch (1->6)-{alpha}-glucan and no type 2 Lex and Ley blood-group epitopes were detected in H.pylori F-58C nor R-58A LPS, and no data was obtained that would be indicative of long fucosylated polyLacNAc O-chains.

Lipopolysaccharide of H.pylori Chinese strain F-15A
The alditol acetates and butyl chiral-glycoside derivatives (approximate molar ratios in brackets) of LFuc (2), DGlc (2), DGal (2), DGlcNAc (1), DDHep (2), and LDHep (2) were found in the analyses of the LPS isolated from H.pylori F-15A. The permethylated alditol acetate derivatives derived from the LPS of strain F-15A (Table I), showed the characteristic Fuc (terminal Fuc), Gal (terminal, 2- and 3-substituted Gal), and GlcNAc (3-, 4-, and 3,4-substituted GlcNAc) derivatives, characteristic of histo-blood group epitopes, which pointed to the possible presence of Lewis antigens in this LPS. In addition, the methylation linkage analysis (Table I) also indicated that this F-15A LPS also contained 3-substituted Fuc, terminal and 3-substituted Glc, 4-substituted Gal, 7- and 2,7-substituted DDHep, and 2- and 3,7-substituted (traces) LDHep. The complete FAB-MS spectrum, and spectra of selected product ion scan experiments, of the methylated intact F-15A LPS yielded primary and secondary ions (Table II) at m/z 608->576 [608–32(MeOH)] (Figure 5) for a difucosyl antigen that contained one GlcNAc unit substituted at O-4 by a Fuc-(1->3)-Fuc fucobiose structure in a fucosylated type 1 Lewis disaccharide structure Fuc-(1->3)-Fuc-(1->4)-GlcNAc (Lewis disaccharide = Fuc-(1->4)-GlcNAc), m/z 668->228 [668–410(Gal-GalOH)] and m/z 668->636 [668–32(MeOH)] for a type 1 [Gal(1->3)Gal(1->3)GlcNAc] and type 2 [Gal(1->3)Gal(1->4)GlcNAc] linear B blood group determinants, respectively, m/z 638->402 [638–236(GalOH)] represented a type 1 Lea antigen, m/z 886->854 [886–32(MeOH)] incorporated a Lea and one outer core-related heptose, and m/z 812->606 [812–206(FucOH)] for a type 2 Ley epitope. A positive reaction in an ELISA experiment with F-15A whole cells and the Ley mAb BG-8 and Lea mAb BG-6 confirmed the presence of Ley (OD > 1.5) and Lea (OD > 2.0) in F-15A, but, as expected, no reaction was seen with the Lex mAb BG-7. The methylation linkage analysis furnished the permethylated alditol acetate derivatives (Table I) usually found in the core oligosaccharide of H.pylori LPS (Figure 1). However no 6-substituted Glc derivative, indicatory of the side-branch (1->6)-{alpha}-glucan (Figure 1), was detected. The 31P NMR spectrum of delipidated F-15A LPS showed a resonance at {delta} 3.02 belonging to a monoester phosphate (PO4->sugar) attached to the 3,7-substituted inner-core LDHep (Table I).



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Fig. 5. Product ion scan spectrum of methylated H.pylori F-15A LPS. The A type primary ion m/z 608 [two Fuc units and one GlcNAc] looses methanol (32 a.m.u.) through ß-elimination to yield m/z 576 which indicated the presence of a type 1 fucosylated Lewis disaccharide [Fuc-(1->3)-Fuc-(1->4)-GlcNAc].

 
The O-chain region of H.pylori F-15A was composed of a type 1 Lea blood-group antigen, a type 1 fucosylated Lewis disaccharide, type 1 and 2 linear B blood group antigens, and also a type 2 Ley epitope (Table II). H.pylori F-15A produced the same core oligosaccharide glycans emblematic of the core region of H.pylori LPS (Figure 1).

Lipopolysaccharides of H.pylori Chinese strain R-7A and Singaporean strain H607
LFuc, Dgal, and DGlcNAc were the dominant monosaccharide units detected in the composition analyses of H.pylori R-7A LPS and, with lower intensity, DGlc, DGal, DDHep, LDHep, and DGalNAc were also observed. Table I displays the sugar linkage types detected by a methylation linkage analysis performed on the intact R-7A LPS. The detection of histo-blood group related units terminal Fuc, 2- and 3-substituted Gal, terminal GalNAc, 3-,4-, and 3,4-substituted GlcNAc suggested the presence of Lewis determinants in this LPS, and in particular it pointed towards histo-blood group structures containing terminal GalNAc (the A blood-group family). No terminal GlcNAc derivative was detected. The FAB-MS spectrum (Figure 6) of the intact methylated R-7A LPS furnished primary glycosyl oxonium ions and corresponding secondary ions (from product ion scan experiments) emanating from the O-chain region (Table II) at m/z 260->228 [260–32(MeOH)] {HexNAc}+ for terminal GalNAc, m/z 464->432 [464–32(MeOH)] {Gal-(1->4)-GlcNAc}+ showed a terminal type 2 LacNAc moiety, m/z 638->432 [638–206(FucOH)] {Gal-(1->4)-[Fuc-(1->3)]-GlcNAc}+ represented a type 2 Lex, m/z 883->228 [883–655(GalNAc-[Fuc-]-GalOH)] {GalNAc-(1->3)-[Fuc-(1->2)]-Gal-(1->3)-GlcNAc}+ indicated the presence of a monofucosyl type 1 A blood-group antigen and accounted for the detection of a terminal GalNAc in the methylation linkage analysis and FAB-MS (Table I and II), m/z ion 709, although of weak intensity, represented a defucosylated A blood-group structure {GalNAc-(1->3)-Gal-(1->3)-Glc NAc}+, m/z 886 [Lex->7-DDHep]+ included the Lex and the outer-core heptose unit, m/z 1057 {GalNAc-(1->3)-[Fuc-(1->2)]-Gal-(1->3)-[Fuc-(1->4)]-GlcNAc}+ showed that a difucosyl type 1 A blood-group epitope was also present, m/z 1087->1055[1087–32(MeOH)] {Lex->LacNAc}+ and 1087-> 881 [1087–206(FucOH)] {LacNAc->Lex}+ represented a chain containing one Lex and one LacNAc epitope, m/z 1261->1055 [1261–206(FucOH)] {Lex->Lex}+ placed two consecutive Lex repeats in a linear chain, m/z 1305 [difucosyl A blood-group->7-DDHep]+ showed the connection of the A blood-group epitope to the core region via the 7-substituted heptose, m/z 1332 stood for a monofucosyl A blood-group (883 amu) attached to an internal LacNAc (449 amu) moiety {monofucosyl A blood-group->LacNAc}+, and m/z 1335 showed the connection of Lex->LacNAc and/or LacNAc->Lex to the 7-substituted DDHep core residue. An ELISA experiment with R-7A LPS and the Lex mAb BG-7 showed a positive reaction (OD > 2.6) confirming the expression of the Lex antigen by this LPS. A similar experiment with a blood-group A specific mAb, 3–3a, also showed the presence of this epitope in the LPS of R-7A (OD > 2.0) and also in strain H607 (OD > 1.5). A methylation linkage analysis of H607 LPS indeed revealed the presence of terminal GalNAc, 2,3-substituted Gal and terminal Fuc underlining the presence of the blood-group A [GalNAc-(1->3)-[Fuc-(1->2)]-Gal] in the LPS of strain H607 also (Table I).



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Fig. 6. The complete FAB-MS spectrum of methylated R-7A LPS. The spectrum reveals the presence of type 1 monofucosyl (m/z 883->228) and difucosyl (m/z 1057->402) A blood-group. Please see Table III for the other structures showed by this FAB-MS spectrum.

 
The presence of terminal and 3-substituted Glc, 4-substituted Gal, 7- and 2,7-substituted DDHep, and 2-, and 3,6- (traces) and 3,7-substituted (traces) LDHep units that make up the core oligosaccharide of H.pylori R-7A and H607 LPSs (Table I) reflected the classic core structural regions of H.pylori LPS. The 31P NMR spectrum of R-7A PS showed one resonance at {delta}P 0.06 for a phosphodiester moeity. The 1H NMR spectrum of R-7A polysaccharide also pointed to the presence of a phosphodiester, that being, a 2-aminoethylphosphate (AEP: NH2-CH2-CH2-O-PO3->sugar) residue by virtue of the characteristic resonances of the methylene protons at {delta}H 2.92 and 3.90. The presence of this phosphodiester unit thus explains the presence, albeit in trace amounts, of 3,6- and 3,7-substituted LDHep (inmost LDHep) (Table I) an effect of intra-ring (between positions O-6 and O-7) diesterphosphate migration due to the alkaline conditions employed in the methylation procedure. The core of H.pylori R-7A LPS was thus phosphorylated by a 2-aminoethylphosphate at the inmost LDHep residue.

The LPS of H.pylori R-7A possessed a very distinctive O-chain region in that it carried the type 1 A blood-group determinant in the monofucosylated {{alpha}DGalNAc-(1->3)-[{alpha}LFuc-(1->2)]-ßDGal-(1->3)-ßDGalNAc} and difucosylated {{alpha}DGalNAc-(1->3)-[{alpha}LFuc-(1->2)]-ßDGal-(1->3)-[{alpha}LFuc-(1->4)]-ßDGalNAc} forms (Table II). Strain H607 was also shown to express the blood-group A in its LPS. This study has shown for the first time the presence of a member of the blood-group A family in H.pylori LPS. In addition to the A blood-group antigen, the O-chain of R-7A LPS also expressed the type 2 Lex and fucose-free LacNAc, in monomeric and dimeric arrangements (Table II). The O-chain histo-blood group antigens present in R-7A LPS were shown to be covalently linked to a typical H.pylori core oligosaccharide that was phosphorylated by a 2-aminoethylphosphate (Table II).

Lipopolysaccharides of H.pylori Japanese strain CA2 and Singaporean strains H507 and H428
The LPS of H.pylori CA2 was composed (approximate molar ratios in brackets) of LFuc (4), DGlc (2), DGal (6), DGlcNAc (4), DDHep (2), and LDHep (2). The methylation linkage analysis performed on the intact CA2 LPS showed the following Lewis-related sugar derivatives (Table I): terminal Fuc, 2- and 3-substituted Gal, 4- and 3,4-substituted GlcNAc units. The FAB-MS spectrum (in combination with selective product ion scan experiments) of the methylated intact H.pylori CA2 LPS showed the presence of the primary glycosyl oxonium ion at m/z 812 [Fuc2, Gal, GlcNAc]+ (Table II, Figure 7) from which secondary ions at m/z 402 [812–410(Fuc-GalOH)] for type 1 Leb, and m/z 606 [812–206(FucOH)] for type 2 Ley were observed (Egge and Peter-Katalinic, 1987Go). In the higher m/z range, m/z 1261 and 1229 [1261–32(MeOH)] represented a Leb/y->LacNAc chain, m/z 1435 and 1229 [1435–206(FucOH)] stood for a Leb/y->Lex structure, and m/z 1884 showed the presence of Leb/y->Lex->LacNAc or Leb/y->LacNAc->Lex chains (Table II). An ELISA experiment with whole cells of CA2 and Leb mAb BG-6 and Ley mAb BG-8 confirmed the presence of the respective type 1 Leb and type 2 Ley determinants in strain CA2 (OD > 2.0). Whole cells of strains H507 and H428 were also recognized (OD > 2.3) by the Leb mAb 225Le and the Ley mAb 1E52, and the FAB-MS spectra of methylated H507 and H428 LPSs indeed showed the presence of these difucosyl antigens by virtue of the presence of the A-type ion m/z 812 (Table II), which complemented the presence of 2-substituted Gal, 3,4-substituted GlcNAc and terminal Fuc in the linkage analyses of H507 and H428 LPSs (Table I). The sugar derivatives observed in the methylation linkage analysis of these 3 strains (Table I), that form the core region, were of the same type as those found in the typical H.pylori core oligosaccharide (Figure 1 and 4). The 31P NMR spectrum of the lipid-free CA2 LPS showed a resonance at {delta}P 3.00 suggesting that the core’s inmost 3,7-substituted LDHep (Table I) was phosphorylated by a monoester phosphate.



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Fig. 7. Product ion scan spectrum of methylated H.pylori CA2 LPS. The A type primary ion m/z 812 looses Fuc-OH (206 a.m.u.) and Fuc-Gal-OH (410 a.m.u.) through ß-elimination to yield m/z 606 and m/z 402, which indicated the presence of a type 2 Ley and type 1 Leb, respectively.

 
The O-chains of H.pylori CA2, H507, and H428 LPSs were found to express the type 1 Leb determinant, a rare antigen in Western H.pylori LPSs, and also the type 2 isomer Ley which is a common antigen in Western H.pylori LPS molecules (Table III). O-Chains composed of Leb/y->LacNAc, Leb/y ->Lex, Leb/y->Lex->LacNAc and Leb/y->LacNAc->Lex moieties were also detected in H.pylori CA2 LPS. The core of H.pylori CA2, H428, and H507 LPSs were composed of the typical H.pylori core units (Figure 1).


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Table III . The glycotype families of H.pylori lipopolysaccharides
 
Lipopolysaccharides of H.pylori Japanese strains CA4, CA5, CA6, and GU2
The LPSs of H.pylori CA5 and CA4 were determined to be composed of LFuc (3), D-Glc (2), DGal (4), DGlcNAc (3), DDHep (2), and LDHep (2). The sugar linkage types observed in the methylation linkage analyses that were performed on the intact CA4 and CA5 LPSs are listed in Table I. The complete FAB-MS spectrum, and corresponding product ion scan experiments, of the methylated H.pylori CA5 LPS showed primary glycosyl oxonium ions and the corresponding secondary ions (Table II) at m/z 638->432 [Gal-(1->4)[Fuc-(1->3)]-GlcNAc]+ for a type 2 Lex, m/z 638->402 [Gal-(1->3)[Fuc-(1->4)]-GlcNAc]+ for a type 1 Lea and also a weaker m/z 638->228 [Fuc-(1->2)-Gal-(1->3)-GlcNAc]+ belonging to a type 1 Led (H-type 1) determinant, ions m/z 1261->1055 represented a Lex/a->Lex chain, and m/z 1710->1688 belonged to a longer Lex/a->Lex->LacNAc chain (Table II). The FAB-MS spectrum (in combination with product ion scan) of H.pylori CA4 methylated LPS (Figure 8, Table II) revealed the presence of type 1 Led [m/z 638->228], type 1 Lea [m/z 638->402] and type 2 Lex [m/z 638->432]. Polymeric Lewis chains were also confirmed to be present in this LPS by various high mass m/z ions (Table II), those being m/z 1087->1055 [Led/a/x ->LacNAc]+, m/z 1261->1055 [Led/a/x->Lex]+, m/z 1710->1504 [Led/a/x->LacNAc->Lex]+, and m/z 1884->1688 [Led/a/x->Lex->Lex]+. An ELISA experiment performed with whole cells of strains CA4 and CA5 and employing Lex mAb BG-7 and Lea mAb 2108 underlined the presence of the Lex (OD > 2.0) and Lea (OD > 1.5) determinants in H.pylori strains CA4 and CA5.



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Fig. 8. Product ion scan spectrum of methylated H.pylori CA4 LPS. The A type primary ion m/z 638 and secondary ions m/z 228, 402, and 432 indicated the presence of a type 1 Led, type 1 Lea, and type 2 Lex, respectively.

 
The LPSs of H.pylori CA6 and GU2 were determined to be composed of LFuc (2 and 4), DGlc (2), DGal (3 and 5), DGlcNAc (2 and 4, respectively), DDHep (2), and LDHep (2). Table I lists the sugar linkage types of H.pylori CA6 and GU2 LPSs. The Lewis blood groups type 1 Lea [m/z 638->402(638–236)] and type 2 Lex [m/z 638->432(638–206)] were observed in the FAB-MS spectrums of H.pylori CA6 and GU2 methylated LPSs (Table II). The FAB-MS spectrum of H.pylori GU2 methylated LPS also showed m/z 1261->1055 for a Lea/x->Lex chain. An ELISA experiment using intact LPSs and employing Lex mAb BG-7 and Lea mAb 2108 also showed (OD > 2.2) the presence of Lex and Lea antigens in strains CA6 and GU2.

H.pylori strains CA5 and CA4 LPSs carried O-chains expressing at the nonreducing terminus three monofucosyl antigens, the type 1 Led (H-type 1), type 1 Lea, and type 2 Lex. The LPSs of H.pylori CA4 and CA5 also elaborated polymeric forms involving the above listed Lewis determinants (Table II). The O-chain regions of H.pylori CA6 and GU2 LPSs expressed the type 1 Lea and type 2 Lex blood-group antigens (Table II). H.pylori GU2 also carried a dimeric Lewis linear chain (Table II). The core of H.pylori CA4, CA5, CA6 and GU2 had invariable structural regions (Table I) and were similar to those of other H.pylori strains. However, the methylation linkage analysis of H.pylori CA6 LPS revealed the presence of 6-substituted Glc, which pointed to the presence of CA6 LPS molecules possessing a side-branch (1->6)-glucan (approximately two units) as found in the type strain (Figure 1).

Fatty-acid analyses were performed on the lipid A molecules of the H.pylori strains described above and C18 was found to be the dominant component. Traces of C14 and C16 were also detected in all the H.pylori lipid A structures.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
This study unveiled the chemical structures of LPS molecules from H.pylori strains obtained from Chinese, Japanese, and Singaporean symptomatic hosts. The new pivotal findings obtained by this investigation were: (1) the prevalent production of type 1 blood-group determinants (Table II) in LPSs of Asian H.pylori strains, in contrast to Western H.pylori LPSs, and, in particular, the sole expression of type 1 Lea in strains F-58C and R-58A without the accompanying type 2 Lex; (2) strain R-7A and H607 were shown to have the capability of producing the type 1 blood-group A antigen; and (3) strains CA2, H507 and H428 expressed simultaneously the difucosyl isomeric antigens, type 1 Leb and type 2 Ley.

In contrast to LPSs of H.pylori strains isolated from hosts residing in Western countries (Figure 1), which, overwhelmingly, have been found to express type 2 Lex and Ley blood-group determinants (Aspinall et al., 1994Go, 1996, 1997; Aspinall and Monteiro, 1996Go; Monteiro et al., 2000Go), the H.pylori LPSs investigated here showed a stronger tendency to produce type 1 histo-blood group determinants. The H.pylori F-58C and R-58A LPSs did not express type 2 Lex nor Ley determinants and the sole presence of type 1 Lea these strains, without the concurrent expression of type 2 Lex, is a new characteristic observed in H.pylori LPS molecules. The LPS O-chain of H.pylori F-15A followed the propensity of strains F-58C and R-58A in that it was also shown to possess the type 1 Lea blood-group antigen (Table II), but in addition, strain F-15A also carried LPSs molecules with the type 1 fucosylated Lewis disaccharide, type 1 and type 2 linear B blood-group (as did strain F-58C), and type 2 Ley structures in the O-chain region (Table II). The most complex O-chain molecules were found to be present in the LPS of H.pylori strain R-7A where an array of histo-blood group glycoforms were observed (Table II); for the first time, a LPS of H.pylori (strains R-7A and H607) was shown to express the histo-blood group A. Both blood-group A type 1 forms, monofucosyl and difucosyl, were detected (Table II, Figure 6) in H.pylori R-7A LPS, and it was shown that the type 1 A blood-group was connected to a type 2 LacNAc backbone, an uniqueness in histo-blood group chemistry. Moreover, the R-7A strain also furnished a string of type 2 Lex and LacNAc epitopes as terminal entities and as members of elongated linear chains (Table II). The type 1 fucosylated Lewis disaccharide and a defucosylated blood-group A were also present in the LPS of R-7A (Table II).

For the first time, LPSs of H.pylori were found to carry both difucosyl type 1 Leb and type 2 Ley structures (Table II), that of strains CA2, H507, and H428. The coexistent expression of the structural isomers Leb and Ley indicated that a variety of glycosylations (i.e., fucosylation and galactosylation at O-3 or O-4 of GlcNAc) were available at any instance in these LPS biosyntheses, and may be dictated by an array of internal and/or external factors. The end products (Leb and Ley in this case) may also have different functions in H.pylori pathogenesis, that is, they may perform particular physical roles in endogenous (bacterium–bacterium) or exogenous (bacterium–host) recognition/adhesion, or may be intrinsic players in the host’s immune response pathways. Similar roles may also apply to the other H.pylori LPS blood-group antigens.

H.pylori F-15A carried a LPS with a difucosyl moiety in the O-chain region, a fucosylated type 1 Lewis disaccharide {{alpha}LFuc-(1->3)-{alpha}LFuc-(1->4)-ßDGlcNAc} (Figure 5 and Table II). The presence of the fucobiose structure, Fuc-1->3-Fuc, implies the significant activity, in this strain, of a fucosyltransferase with the ability of appending a Fuc unit to another Fuc residue of a type 1 Lewis disaccharide {Fuc-1->4-GlcNAc}. The genome sequences of H.pylori strains 26695 (Tomb et al., 1997Go) and J99 (Alm et al., 1999Go) were found to include at least two (1->3)-{alpha}-L-fucosyltransferases and it may be that both fucosyltransferases are involved in the biosynthesis of this fucosylated Lewis disaccharide. In strains CA4 and CA5, fucosylation at O-2 of a terminal Gal of a Gal-1->3-GlcNAc structure yielded the type 1 Led, Fuc-(1->2)-Gal-(1->3)-GlcNAc, also commonly know as the H-type 1 antigen. The presence of three different monofucosylated Lewis determinants, Lea, Lex, and Led, in H.pylori strains CA5 and CA4 reflect three possibly self-regulating fucosylation pathways [Fuc-(1->4), Fuc-(1->3), and Fuc-(1->2)] in the biosynthesis of CA5 and CA4 LPSs.

In some of the above H.pylori LPSs, a covalent connection between the O-chain (composed of histo-blood groups) and the core was shown to be formed by a ligation between the reducing-end GlcNAc unit of the O-chain and an outer core 7-substituted DDHep residue. In H.pylori strains F-58C, R-58A, and F-15A, ion m/z 886 showed the linkage of the type 1 Lea epitope to the O-7 position of the outer core DDHep (Table II), and a similar ion also showed the same type of connection between the type 2 Lex and the core-related DDHep unit in strain R-7A. Several higher m/z in the FAB-MS of the methylated R-7A LPS (Table II), of defined composition and that included the linear 7-substituted DDHep of the outer core region, yielded important evidence for the connection of the type 1 difucosyl blood-group A to core (m/z 1305), and of the elongated type 2 Lex->LacNAc and LacNAc->Lex to the core (m/z 1335). These structural interpretations, that showed the covalent linkage between the O-chain region and the core, offered unequivocal evidence that these H.pylori cell surface glycan molecules were indeed LPSs and were not capsular- nor exo-polysaccharides. However, it is possible that some high molecular weight type-2 fucosylated poly(LacNAc) chains found in some H.pylori strains may be present as non-LPS molecules, since it is not always possible to prove the covalent connection of O-chain to core.

With the exception of some H.pylori strains, the LPS biosynthetic mechanism afforded polymeric Lewis chains (Table II); these elongated chains may act as a bacterial arm to display the terminal histo-blood group antigen in order to allow an easier contact and recognition by the host or by other H.pylori cells and/or may have a more detailed biochemical role in H.pylori pathogenesis as similar elongated molecules have in cancerous cells (Hakomori, 1989Go). The heptoglycan domain observed in some H.pylori LPSs (Aspinall and Monteiro, 1996Go; Aspinall et al., 1997Go) located between an O-chain region containing a monomeric Lewis antigen and the core oligosaccharide (Table III) may also serve as an "arm" to introduce the sole Lewis blood-group to intra- or inter-receptors.

The core oligosaccharide derivatives (non histo-blood group units) obtained from chemical linkage analysis of these H.pylori LPSs (Table I) were similar and were of the same type as found in the core structures of previously investigated H.pylori strains (Figure 1). All core residues detected fell in line with the typical core structural regions of H.pylori (Figures 1, 4): Glc-(1->3)-Glc-(1->4)-Gal-(1->7)-[DDHep-(1->2)]-DDHep-(1->2)-LDHep-(1->3)-[P or AEP->7]-LDHep. The 31P NMR spectrums and the linkage analyses data suggested the presence of a monoester phosphate (PO4) at the O-7 position of the inmost LDHep of the strains studied here, except in strain R-7A, where a 2-aminoethylphosphate was shown to replace the monoester phosphate.

The complete genomes of two Western H.pylori strains, 26695 (Tomb et al., 1997Go) and J99 (Alm et al., 1999Go), have been determined to be similar. Chemical analyses have shown that the O-chain region of H.pylori 26695 and J99 LPSs were composed of type 2 Lex and/or Ley antigens and no type 1 Lea, Leb, or blood-group A epitopes were observed (Monteiro et al., 2000Go). The LPS structural differences between the Asian and Western H.pylori strains observed in this study, based on a more prominent expression of type 1 histo-blood group antigens in the O-chain sections of Asian strains, suggest that some significant differences may be encountered between the genomes of Asian and Western H.pylori strains. The fucosylation of GlcNAc at O-3 in Lex and at O-4 in Lea, and the galactosylation at O-4 in Lex and at O-3 in Lea, may be performed by a single fucosyl- and galactosyltransferase or, alternatively, by different glycosyltransferases. In Leb, Ley, and Led, fucosylation at O-2 of terminal Gal is most likely performed by the same fucosyltransferase. In the biosynthesis of the blood-group A in H.pylori R-7A (Table II), the presence of fucose-free GalNAc(1->3)Gal(1->3)GlcNAc, implies that the addition of the terminal GalNAc does not require fucosylation at O-2 of Gal in the formation of the blood-group A antigen, and thus points to either a sugar-by-sugar biosynthetic pathway or to the addition of an incomplete O-chain block to the core region.

The LPSs of these H.pylori strains were placed into their respective glycotype families (Table III) (Monteiro et al., 1998aGo, 2000): strains R-7A, CA2, CA5, CA4, GU2, H428, and H507 were incorporated in the glycotype F family (strains that possess smooth-form LPS expressing type 1 and type 2 blood-group antigens); strains F-15A, CA9, and CA6 were placed in glycotype G family (strains that possess semi-rough form LPS expressing type 1 and type 2 blood-group antigens); and strains F-58C and R-58A were incorporated in glycotype H family (strains that possess LPS expressing solely type 1 blood-group antigens). The H.pylori niche in humans, the gastric epithelium (gastric superficial and glandular epithelium expresses type 1 and type 2 blood-group antigens in its cells surfaces (Mollicone et al., 1985Go; Davidson and Triadafilopoulos, 1992Go; Kobayashi et al., 1993Go); thus, these Asian H.pylori strains, by expressing a variety of histo-blood groups in their LPSs, mimic their immediate surrounding environment with the possible intent of avoiding the host’s immune defense system. The apparent strong inclination for the production of type 1 histo-blood group antigens in Asian H.pylori LPSs (Tables II, III), when compared with Western strains, may be an adaptive evolutionary effect in that differences in the gastric cell surfaces of the respective hosts might be significantly dissimilar to select for the formation of different LPS structures on the resident H.pylori strain. A large-scale serological experiment using blood-group mAbs is needed to come to a definite conclusion regarding the extent to which Asian H.pylori strains express type 1 histo-blood group antigens.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Origin and cell production of H.pylori strains
All H.pylori were clinical isolates extracted from the lesion biopsy specimens obtained from hosts with gastric ulcer or gastric cancer. Every one of these strains carried the cag pathogenicity island and contained at least potentially toxigenic (s1) alleles of the vacuolating cytotoxin gene. These H.pylori strains were frozen immediately after isolation (–70°C). Cultures were subsequently plated onto brain heart infusion agar (1.2% w/v) plates supplemented with 0.5% (w/v) yeast extract and 0.5% (v/v) fetal bovine sera (Hyclone, Logan, Utah, USA).

Isolation of lipopolysaccharides
The LPSs were extracted from cells by the hot phenol-water extraction procedure (Westphal and Jann, 1965Go). The water-soluble LPSs were purified by gel-permeation-chromatography on a column of Bio-Gel P-2 (1m x 1cm). In all cases, only one carbohydrate positive fraction was obtained which eluted in the high Mr range (Dubois et al., 1956Go). These intact H.pylori LPSs were used for chemical and serological analyses. Lipid-free polysaccharides were obtained by mild acid hydrolysis of LPS with 1% acetic acid at 100°C for 1 h.

Sugar composition and methylation linkage analyses
Sugar composition analysis was performed by the alditol acetate method (Gunner et al., 1961Go; Sawardeker et al., 1965Go). The alditol acetate derivatives were analyzed 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 chiral glycosides (Leotein et al., 1978Go). Methylation linkage analysis was carried out by the NaOH/DMSO/CH3I procedure (Ciucanu and Kerek, 1984Go) and with characterization of permethylated alditol acetate derivatives by GLC-MS in the EI mode (DB-17 column, isothermally at 190°C for 60 min).

Fast atom bombardment-mass spectrometry (FAB-MS)
A fraction of the methylated sample was used for positive ion fast atom bombardment-mass spectrometry (FAB-MS) which was performed on a Jeol JMS-AX505H mass spectrometer with glycerol(1):thioglycerol(3) as the matrix. A 6 kV xenon beam was used to produce pseudo molecular ions which were then accelerated to 3 kV and their mass analyzed. Product ion scan (B/E) experiments were preformed on metastable ions created in the first free field with a source pressure of 5 x 10–5 torr. The interpretations of positive ion mass spectra of the permethylated LPS derivatives were as described previously by Dell et al. (1990)Go).

Nuclear magnetic resonance (NMR) spectroscopy
1H and 31P NMR spectra of the polysaccharides 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 three times with D2O (99.9%). Acetone and orthophosphoric acid were used as the references at {delta}H 2.225 and {delta}P 0.0.

Serological procedures
For ELISA experiments, the same protocols were employed as described in Monteiro et al. (1998a)Go and Yokota et al. (1998)Go.


    Acknowledgements
 
Z-j. P. and D.E.B. were supported by NIH grants AI38166 and DK 48029.


    Footnotes
 
1 To whom correspondence should be addressed at: Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Ottawa, Ontario, K1A 0R6, Canada Back


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 Materials and methods
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