Molecular mimicry of host structures by bacterial lipopolysaccharides and its contribution to disease.

The core oligosaccharides of low-molecular-weight lipopolysaccharide (LPS), also termed lipooligosaccharide (LOS), of pathogenic Neisseria spp. mimic the carbohydrate moieties of glycosphingolipids present on human cells. Such mimicry may serve to camouflage the bacterial surface from the host. The LOS component is antigenically and/or chemically identical to lactoneoseries glycosphingolipids and can become sialylated in Neisseria gonorrhoeae when the bacterium is grown in the presence of cytidine 5'-monophospho-N-acetylneuraminic acid, the nucleotide sugar of sialic acid. Strains of Neisseria meningitidis and Haemophilus influenzae also express similarly sialylated LPS. Sialylation of the LOS influences susceptibility to bactericidal antibody, may decrease or prevent phagocytosis, cause down-regulation of complement activation, and decrease adherence to neutrophils and the subsequent oxidative burst response. The core oligosaccharides of LPS of Campylobacter jejuni serotypes which are associated with the development of the neurological disorder, Guillain-Barré syndrome (GBS), exhibit mimicry of gangliosides. Cross-reactive antibodies between C. jejuni LPS and gangliosides are considered to play an important role in GBS pathogenesis. In contrast, the O-chain of a number of Helicobacter pylori strains exhibit mimicry of Lewis(x) and Lewis(y) blood group antigens. The role of this mimicry remains to be investigated, but may play a role in bacterial camouflage, the induction of autoimmunity and immune suppression in H. pylori-associated disease.


Lipopolysaccharides
(LPS) are a family of toxic glycolipids present in the outer membrane of Gramnegative bacteria and are essential for the physical integrity and functioning of that membrane [ 1,2].

Neisseria meningitidis and Haemophilus inji'uenzae
has been the most intensively studied [4]. However, studies within the last half-decade show that LPS of other medically important bacteria also mimic host structures. Campylobacter jejuni which is a common cause of acute gastroenteritis in humans [5] and Helicobacter pylori which is the causal agent of active chronic gastritis and a cofactor in the development of peptic ulcers and gastric cancer [6-81 exhibit mimicry in their LPS which contributes to virulence in these infections. The aim of this review is to discuss the basis within LPS for the molecular mimicries exhibited by the various bacterial species. Also, the contribution of molecular mimicry to the outcome of infection and disease development will be addressed.

Structure of LPS
Chemically LPS, as characterized by enterobacterial LPS, is composed of a poly-or oligosaccharide covalently linked to a lipid component, termed lipid A ( Fig. 1) [2,3]. High-molecular weight (M,) smooth-form LPS consist of an O-specific polysaccharide chain, which is a polymer of repeating oligosaccharide units, a core oligosaccharide, and lipid A; whereas low-M, rough-form LPS lack the O-chain [3]. Like wild-type strains of the Enterobac- structurally distinct low-M, LPS resembling those of Neisseria and Haemophilus spp., or both [12,13] ( Fig. 1 A, B). 3. N. gonorrhoeae, N. meningitidis and H. influenzue LPS

Molecular mimicry of glycolipids and glycosphingolipids
The first reported structure of an oligosaccharide (OS) moiety of neisserial LOS was that of N. meningitidis L3,7,9 (Table 1, 2B). With an anti-Pk monoclonal antibody (Mab), a Pk-like antigen was found on 10% and 50% of meningococcal and gonococcal strains, and on nontypable and type b H. influenzae [19].
Anti-lactoneoseries monoclonal antibodies bind to strains of Haemophilus ducreyi, the causative agent of chancroid in humans, and the mimicry in the LOS has been verified structurally [4,21,22]. These same antibodies bind to non-typable and type b H. injluenzae LOS and the terminal OS of both H. ducreyi and H. influenzae LOS may be sialylated endogenously [23-251.

Potential role in camouflage and resistance to the bactericidal effect of serum and to phagocytosis
The expression of structures within LOS resembling those of the host may serve to camouflage the bacterial surface from the host in a similar way to that described for capsular polysaccharides [26]. For example, the Pk antigen ( Fig. 2B) is present in a number of human tissues or cells relevant to infection by mucosal pathmogens including Neisseria spp., such as uroepithelial cells [27]. Also, the ganglioseries-like LOS of N. gonorrhoeae are of interest since human glycoconjugates with similar terminal OS serve as precursors for synthesis of carbohydrates in human cervical mucins [28].
Fresh clinical isolates of gonococci from urethral exudates possess a virulence factor that is lost upon subculturing strains in vitro [29] but when such strains are incubated in human fluids and secretions, the gonococci can be converted from serum-sensitivity to resistance which is associated with a change in LOS structure [30]. The inducing factor is concentrated in an environment relevant to N. gonorrhoeae infection since Pate1 et al.
[31] found a higher resistance-inducing capacity in leukocytes than in whole blood. Both a high-and low-M, resistance-inducing factor have been detected but the low-M, resistanceinducing factor was identified as cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuSAc), the nucleotide sugar of N-acetylneuraminic (sialic) acid [32]. Furthermore, studies suggested that the enzymes for sialylation were in the outer bacterial layer, that sufficient concentrations of CMP-Neu5Ac might be present in vivo to sialylate LOS and thus induce serum resistance [4]. The major LOS component modified by sialylation was identified as a 4.5 kDa component that binds a Mab which recognizes the lactoneoseries of glycolipids including lacto-hrneotetraose and lactotriaose [33]. This LOS molecule is expressed in greater quantities in anaerobically grown than in aerobically grown gonococci [34]. Most strains of meningococci that synthesize a capsule containing sialic acid (serogroups B, C, W and Y) and a 4.5 kDa LOS also sialylate their LOS endogenously 14,351. Determination of the chemical structure of sialylated N. meningitidis L3 has confirmed the presence of terminal sialic acid on a lacto-N-neotetraose-like OS [36] and the CMP-NeuSAc synthetase of group B meningococci has been cloned and sequenced [37]. The potential 4.5 kDa LOS sialic acid-acceptor is present on type B and non-typable H. influenzae and H. ducreyi [23,25] and H. injluenzae strains are endogenously sialylated as described above. In contrast, meningococci that cannot synthesize sialic acid (e.g., serogroups A and X) and non-pathogenic Neisseria spp., although they may express the 4.5 kDa LOS, do not endogenously sialylate LOS [4]. Moreover, all strains of pathogenic Neisseria have an LOS-specific sialyltransferase [4,38].
Although some N. gonorrhoeae strains remain serum-resistant in vitro without the addition of CMP-NeuSAc whereas others remain serum-sensitive after growth in CMP-NeuSAc, the primary role of LOS sialylation may be somewhat different than protecting strains from the lytic effects of serum antibody and complement [4,39]. Sialylation of LOS may decrease or prevent, phagocytosis of gonococci and meningococci by human neutrophils [40,41].
Furthermore, bacterial surface sialylation can cause a down-regulation of complement activation, e.g., the C3 convertase of the alternative pathway is downregulated by the presence of sialic acid-containing structures [42]. Compared to non-sialylated gonococci, sialylated strains have a decreased ability to induce an oxidative burst in neutrophils, and to adhere to neutrophils in the absence of complement and specific antibody [40]. Therefore, the influence of LOS sialylation may play a pivotal role in the survival of Neisseria spp. in the mucosal ecological niche.

Role in the development of Guillain-Barr6 syndrome
Guillain-Barre syndrome (GBS) is an acute, inflammatory and demyelinating paralytic disease of the peripheral nervous system, and although of infrequent occurrence, is now the most common cause of generalized paralysis. In up to 75% of GBS cases, the syndrome develops following various respiratory and gastrointestinal infections [43]. C. jejuni has been identified as the most common single pathogen associated with the development of GBS (20-50% of patients), particularly with a severe form of the disease [43-451.
With the heat-stable antigen serotyping system of C. jejuni which is based on LPS [ 12,131, a predominance of C. jejuni 0: 19, an uncommon serotype in gastroenteritis patients, has been found in Japanese GBS patients [46], and has even been reported in a familial outbreak of GBS  In concurrence with these findings, structural analyses have shown that the core OS of C. jejuni 0:19 LPS, including those from GBS-associated isolates, contain tetra-and pentasaccharide moieties identical to those of GM, and GD,, gangliosides, respectively [61-631 (Fig. 4A). Molecular mimicry of GT,, and GD, gangliosides by the terminal hexasaccharides and trisaccharides, respectively, of LPS of some GBS-associated C. jejuni 0: 19 isolates has also been reported [62] (Fig. 4B, C). Analysis with anti-ganglioside antibodies has verified the presence of GMl-, GTla-and GD3-like epitopes in LPS of GBS-associated C. jejuni 0:19 strains [64].
In addition to C. jejuni 0:19, other serotypes possess sialylated LPS [65,66]. Chemical structural A r__~~~~~'_____-'~~~~~~~___---~_~~-, I r l! studies on LPS have shown that the terminal pentasaccharide of the core OS of C. jejuni serostrain (serotype-reference strain) 0:4 mimics that of GD,, ganglioside [67]. The presence of both GD,,-and GM,-like epitopes in this LPS has been shown serologically [60]. LPS of C. jejuni 0:41 GBS-associated isolates is sialylated and bears a GM,-like epitope (M.M. Prendergast, unpublished results). Other forms of ganglioside mimicry are exhibited by C. jejuni serostrains. The non-reducing ends of the core OS of LPS of C. jejuni 0:23 and 0:36 are composed of the same tetrasaccharide as that present in GM, ganglioside (Fig. 3B), whereas in LPS of C. jejuni serostrain 0:l the extent of mimicry of GM, ganglioside is limited to a terminal trisaccharide [67]. Although C. jejuni 0:2 is associated with the development of GBS, mimicry of gangliosides is limited to that of a  terminal disaccharide (NeuSAc (Y 2-3Gal) [68] which is present in GM, ganglioside (Fig. 3C), but also in other gangliosides (e.g., GD,,, GT,, and GM,). Despite this, asialoGMl-antibody can bind to both 0:2 and 0:19 LPS indicating a shared epitope, not dependent on ganglioside mimicry 1.541. The latter shared epitope may, therefore, explain the involvement of C. jejclni 0:2 in GBS pathogenesis. This should be emphasized since C. jejuni 0:3 has not been associated with the induction of GBS, and whose LPS has been used as a negative control in experiments with anti-ganglioside antibodies [54] is not sialylated [65,66] and does not exhibit any mimicry of gangliosides [69].
On the other hand, mimicry of gangliosides by the core OS of C. jejk 0:19 LPS, is not limited to strains that are associated with GBS but is also present in strains that are isolated from individuals with enteritis but who do not develop GBS [63]. Some other attribute of the host and/or of the bacterium, in addition to mimicry of gangliosides, may contribute to the development of GBS. The LPS of the 0: 19 serostrain and GBS-associated 0: 19 isolates contain identical O-specific polysaccharide chains of a hyaluronic acid-like polymer [70]. This polymer consists of disaccharide-repeating units containing residues of /3-D-glucuronic acid amidated with 2-amino-2-deoxyglycerol and linked to P-D-Nacetylglucosamine (Fig. 5). Expression of this hyaluronic acid-like O-chain is greater in GBS-associated 0: 19 isolates than in isolates from patients with enteritis alone [63]. The finding that the O-chain reflects in structure hyaluronic acid, a common human tissue component, indicates that it would be imprudent to preclude the O-chain as a contributing factor in the development of GBS until further investigations are undertaken. Conversely, an association has been reported between certain human leukocyte antigen types, e.g. HLA-B35 [71], and the develop-ment of GBS after C. jejuni infection. Thus, it can be speculated that infection with C. jejuni strains (e.g., 0: 19) bearing ganglioside-like epitopes in their LPS might induce high production of anti-ganglioside autoantibodies in patients with certain immunogenetic backgrounds, such as the HLA-B35 antigen, due to the abolition of tolerance [72].

Molecular mimicry of Lewis blood group antigens
Compositional analysis has revealed that LPS of a number of H. pylori strains are fucosylated [9]. The O-specific polysaccharide chains of H. pylori LPS have been found upon structural analysis to exhibit mimicry of fucosylated LewisX (Le") or LewisY (LeY) blood group antigens [73,74].
The structure of the O-chain of LPS of H. pylori NCTC 11637 (ATCC 43504), the type strain of H. pylori, was the first to be established [75] and is composed of repeating units that exhibit mimicry of LeX [75,76] (Fig. 6A) Cl, also exhibits mimicry of Le" units [77]. However, antigenic differences between the LPS from these two strains has been detected by immunoblotting and passive haemagglutination assays [78] and may reflect the presence of structurally different core LPS regions [77]. The 0-polysaccharide of another strain, P466, differs from that of the type strain in termination by a LeY determinant, but also has internal Le" determinants [79] (Fig. 6B). In contrast the O-chain of H. pylori MO19 consists of a single LeY unit [73,79] (Fig. 6C). Thus, the expression of Le" or LeY determinants and the degree of 0 chain extension may vary in LPS of different H. pylori strains. Nevertheless, analysis of H. pylori strains with Mab against Lewis blood group antigens has demonstrated the presence of Le" and/or LeY determinants on 81% of the strains examined [80].

Potential role in immune suppression and autoimmunity
Although it has been known for some years that A, B, and H blood group structures are present on certain Gram-negative bacteria [8 1,821, the expression of Lewis blood group antigens on the surface of H. pylori is a novel property of the bacterium and has important biological implications.
Lewis blood group antigens are expressed in normal human gastric mucosa and human gastric carcinoma [83]. The expression of host structures on the bacterial surface may camouflage H. pylori for a period after infection, thereby aiding survival of the bacterium in the gastric mucosa [74]. .Moreover, it could be considered that this bacterium may have evolved this characteristic during its evolution as a successful human colonizer. This is partly supported by the report of Chan et al. [84] who studied the biosynthetic pathways involved in the expression of Le" on the surface of the bacterium. With acceptor molecules for fucosylation, H. pylori was shown to contain (Y 1,3-fucosyltransferase and /3 1,4-galactosyltransferase activities, and analysis of the enzyme reaction products showed that biosynthesis of Le" in H. pylori is strictly ordered, i.e. addition of galactose is followed by that of fucose, and is therefore identical to that found in humans. Although we independently detected CY 1,3-fucosyltransferase activity in H. pylori strains expressing Le', (Y 1,2-fucosyltransferase activity was not detectable in strains expressing Ley [85]. The most general acceptor molecule known for cu 1,2_fucosyltransferase activity was used in our studies, and therefore it may be possible that the enzyme in H. pylori is a rare class of fucosyltransferase.
During the course of prolonged infection, seropositivity against H. pylori has been found to correlate with the presence of autoantibodies against human antral gastric mucosa, and antibodies cross-reacting with antigens of the gastric mucosa have been demonstrated in mice immunized with H. pylori [86]. Therefore, with the progression of infection and the induction of an immune response, the presence of Le"-bearing structures on H. pylori might explain the development of autoantibodies.
Supporting this, anti-Lex and -LeY have been found in patients infected with H. pylori [87]. Moreover, anti-Lex antibodies have been induced post-infection in a. H. pylori mouse model with a strain bearing the Le" determinant, and H. pylori-induced anti-Le" and -LeY Mab reacted with the gastric mucosa [85].
Chronic gastritis has been classified as two major types: chronic superficial gastritis without glandular loss and chronic atrophic gastritis with destruction of the glands [88]. On the basis of fhe prevalence of these two types of gastritis in different age groups, it was deduced that the superficial form may develop to the atrophic form [89,90]. H. pylori is the primary cause of chronic superficial gastritis [6], whereas the pathogenic mechanisms triggering chronic atrophic gastritis include autoimmunity [91]. Further studies, therefore, are required to determine the role of Lewis antigen mimicry by LPS in the induction of autoimmunity in H. pylori-associated disease. Another and less controversial consequence of Le" expression by H. pylori concerns the down-regulation of the T-lymphocyte response in H. pylori gastritis, T-helper lymphocytes (T,, cells) can be subdivided into T,, and T,, cells, and suppressed T,, and enhanced T,, responses in H. pylori infection may be involved in the immunopathogenesis of this chronic infection [92]. Le" is also found as a surface antigen on the eggs of the parasitic worm Schistosoma mansonii which also down-regulates the T-lymphocyte response during infection [93]. The eggs of S. mansonii trigger an alteration in the T-lymphocyte response, shifting immunity from cell-mediated to antibody-mediated which, as in H. pylori infection, is ineffective in combating S. mansonii infection [94]. The polymeric Le"-containing structure of S. mansonii stimulates a B-lymphocyteenriched population of spleen cells to produce large amounts of interleukin 10 and prostaglandin E,, which down-regulate T,, responses, thereby suppressing cell-mediated immunity [93]. Whether a similar phenomenon occurs in H. pylori-strains expressing Le" requires investigation.