J Med Microbiol 57 (2008), 585-591; DOI: 10.1099/jmm.0.47783-0
© 2008 Society for General Microbiology
ISSN 1473-5644
Analysis of Helicobacter pylori isolates from Chile: occurrence of selective type 1 Lewis b antigen expression in lipopolysaccharide
Eleonora Altman1,
Heriberto Fernández2,
Vandana Chandan1,
Blair A. Harrison1,
Myra Wilson Schuster2,
Laura Otth Rademacher2 and
Claudio Toledo3
1 Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON K1A 0R6, Canada
2 Instituto de Microbiología Clínica, Edificio de Ciencias Biomédicas, Facultad de Medicina, Universidad Austral de Chile, PO Box 567, Valdivia, Chile
3 Instituto de Medicina, Facultad de Medicina, Universidad Austral de Chile, PO Box 567, Valdivia, Chile
Correspondence
Eleonora Altman
Eleonora.altman{at}nrc-cnrc.gc.ca
Received 27 November 2007
Accepted 19 January 2008
Previous studies have shown that the LPS of Helicobacter pylori isolated from North American and European hosts predominantly expresses type 2 Lewis x (Lex) and Ley epitopes, whilst the LPS from Asian strains has the capacity to express type 1 Lea and Leb structures. The aim of this study was to evaluate the expression of Le antigens and the cytotoxin-associated antigen (CagA) by H. pylori isolates from Chile. A total of 38 isolates were screened. The expression of Le antigens and CagA was determined by whole-cell indirect ELISA, using commercially available monoclonal anti-Le and polyclonal anti-CagA antibodies. LPS profiles of H. pylori isolates were assessed by gel electrophoresis and Western blotting. Expression of Lex and/or Ley epitopes was confirmed in 32/38 isolates (84 %), whilst 9/38 isolates (24 %) expressed type 1 Leb blood group determinants, in addition to type 2 Lex and Ley structures. Six strains (16 %) were non-typeable. The majority of H. pylori strains examined were CagA-positive (83.3 %).
Abbreviations: CagA, cytotoxin-associated antigen; HRP, horseradish peroxidase; Le, Lewis; WCE, whole-cell indirect ELISA.
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INTRODUCTION
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Helicobacter pylori is recognized as the most common bacterial pathogen associated with chronic gastritis and peptic ulcers in humans and with increased risk of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. It has been estimated to infect the gastric mucosa of more than 60 % of adults over the age of 60 in industrialized countries (Dunn et al., 1997). Recent studies suggest that, in Latin America, H. pylori infection is most commonly acquired in childhood. The prevalence rates of H. pylori vary from 30 to 90 %, depending on the socioeconomic status of a given population, with most children being infected by 10 years of age (Coelho et al., 2000).
H. pylori produces several putative colonization factors, including urease, adhesin, flagella, cytotoxin-associated antigen (CagA), VacA and LPS (Covacci et al., 1999). H. pylori LPS from a number of strains from North American and European populations expresses type 2 Lewis x (Lex) and Ley blood group epitopes that mimic cell-surface fucose polylactosamine-containing structures present on human gastric and tumour cells (Monteiro, 2001) (Fig. 1
). Previous studies have shown a strong correlation between the expression of Lex and Ley antigens and CagA by H. pylori isolates contributing to inflammation and persistence (Wirth et al., 1996). More recently, the detection of type 1 Lea and Leb structures in the LPS of H. pylori strains obtained from Asian symptomatic hosts compared with LPS from Western strains was reported (Monteiro et al., 2000) (Fig. 1). Studies on host gastric Le expression of Taiwanese H. pylori-infected patients have provided evidence for a role of gastric Leb and Lex determinants in the bacterial density of H. pylori in the stomach that could be correlated with clinicohistological outcome (Sheu et al., 2003). In this study, we report on the distribution of Le antigens and CagA in Chilean clinical isolates of H. pylori and the occurrence of Leb blood group antigen in the LPS of these strains.
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Fig. 1. Structure of the O-chain and core oligosaccharide LPS regions of Western H. pylori strains. Reproduced from Monteiro et al. (2000) by permission of Oxford University Press. The O-chain is covalently linked to the core oligosaccharide through a side chain DD-Hep. Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; AEP, 2-aminoethylphosphate; P, phosphate.
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METHODS
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Patients.
H. pylori was cultured from two biopsies each taken for culture from the antrum and corpus during endoscopy performed at the County Hospital and at a private clinic in Valdivia, Chile (by C. T.). Written informed consent to participate in this study was obtained from all patients. The Ethics Committee of the Facultad de Medicina, Universidad Austral de Chile, approved this study. Of the 196 biopsy samples collected, 59 were identified as H. pylori (isolation frequency 30.1 %). The median age of patients was 51.4 years (range 17–87 years) with a male-to-female ratio of 0.51 (20 males and 39 females). Thirteen adult patients had gastritis, ten had gastric ulcers, six had oesophagitis, two had reflux and five had duodenal ulcers. Fourteen isolates were from patients without lesions and nine isolates were from patients with no registered symptoms. A total of 38 H. pylori isolates were chosen at random and examined in the present study.
Culture.
Biopsy samples were homogenized and inoculated onto antibiotic-supplemented (Oxoid Dent Supplement) blood agar plates and incubated at 37 °C under microaerophilic conditions for 4–5 days. Characteristic colonies were tested for culture purity by a urease assay and Gram staining. Single colonies were propagated further, and frozen stocks were prepared using brain heart infusion broth containing 20 % glycerol. They were stored at –80 °C. H. pylori isolates were cultured at 37 °C on Columbia blood agar (Difco Laboratories) containing 7 % defibrinated horse blood in a microaerophilic environment for 48 h.
Whole-cell indirect ELISA (WCE).
H. pylori strains were cultivated on plates as described previously (Logan et al., 2000). Cells were harvested and washed with 5 ml 10 mM PBS (pH 7.4) per plate. Following centrifugation (5000 g, 5 min, 4 °C), pellets were suspended in 25 ml PBS to give a final concentration of 108 cells ml–1. Microtitre plates (ICN) were coated with 100 µl bacterial suspension and incubated overnight at 4 °C. The bacteria were removed and the wells were fixed for 5 min with methanol and then dried for 15 min. The wells were blocked with 200 µl milk diluent/blocking solution (MDB; KPL) for 2 h at 37 °C. Subsequently, the wells were incubated for 2 h at 37 °C with 100 µl anti-Lea, -Leb, -Lex or -Ley monoclonal antibody (mAb) solution (Signet Laboratories) diluted 1 : 200 in MDB or rabbit anti-CagA antibody (Austral Biologicals) diluted 1 : 100 in MDB. Corresponding secondary antibody, either horseradish peroxidase (HRP)-conjugated anti-mouse IgG+IgM or HRP-conjugated anti-rabbit IgG (Cedarlane Laboratories) diluted 1 : 1000 in MDB, was added and the plates were incubated for 1 h at room temperature. Between incubation steps, the wells were washed four times with PBS. After a final washing step, the substrate 3,3',5,5'-tetramethylbenzidene (KPL) was added and the reaction was stopped with 1 M phosphoric acid. The A450 was determined using a microtitre plate reader (Dynatech). Non-specific background values were determined as A450 of the negative-control wells containing bacterial cells, secondary antibody conjugate and substrate. These A450 values were 
0.2. Values of A450 <0.2 were classified as negative and values of A450 
0.2 were classified as positive reactions. To ensure plate-to-plate consistency, H. pylori strain 26695 cells were used as a positive control for Lex, Ley and CagA. Assays did not vary by more than 10 %.
Electrophoresis and Western blotting.
SDS-PAGE was performed with a mini-slab apparatus (Bio-Rad) using the Laemmli method. LPS samples were prepared from whole cells according to a previously described method (Logan & Trust, 1984) and equivalent amounts were loaded in each lane and stained according to Tsai & Frasch (1982) or transferred to nitrocellulose for immunological detection with anti-Lex and anti-Ley mAbs as described previously (Logan et al., 2000). To ensure consistency, H. pylori strain 26695 was used as a positive control for anti-Lex and anti-Ley mAbs. For detection of Leb in bacterial whole-cell samples, nitrocellulose membranes were blocked with 1 % skimmed milk in 10 mM PBS (pH 7.4) overnight at 4 °C. Subsequently, membranes were incubated with anti-Leb mAb solution diluted 1 : 100 in 10 mM PBS (pH 7.4) overnight at 4 °C. Corresponding secondary antibody, HRP-conjugated anti-mouse IgG+IgM diluted 1 : 3000 in 10 mM PBS (pH 7.4), was added and the nitrocellulose membranes were incubated for 1 h at room temperature. The membranes were washed three times with 10 mM PBS (pH 7.4) between the incubation steps and developed using SuperSignal West Pico Chemiluminescent substrate (Pierce) following the manufacturer's instructions. The synthetic Leb antigen Leb–hexasaccharide–BSA (lacto-N-difucohexaose I–BSA or LNDFHI–BSA; V-Labs) was used as a positive control. Whole cells of the Ley antigen-expressing H. pylori Sydney (SS1) strain were used as a negative control in Western blots to ensure specificity of anti-Leb mAbs. In no case did we find any cross-reactivity with Ley for the Leb-specific mAbs under Western blot conditions.
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RESULTS AND DISCUSSION
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In the present study, we examined H. pylori isolates from Chile by WCE, gel electrophoresis and Western blotting. Similar approaches have been applied previously to the analysis of H. pylori isolates from gastric biopsies (Rasko et al., 2000) and typing of H. pylori isolates from different geographical regions (Simoons-Smit et al., 1996). However, the distribution of Le antigens in H. pylori strains from Latin America has not been examined.
Screening of 38 Chilean isolates of H. pylori by WCE resulted in the detection of Lex and/or Ley epitopes in 32 strains. Co-expression of Lex and Ley antigens was detected in 14 strains. Three isolates expressed only Lex antigen (7.9 %), whilst exclusive Ley expression was identified in six isolates (15.8 %). Leb antigen was identified in nine isolates (23.7 %), either co-expressed with Lex and Ley (four isolates) or just with Ley (five isolates) (Table 1). No isolate expressing Lea antigen was identified. Expression of the CagA protein was detected in 83.3 % of isolates studied. Six isolates (15.8 %) contained no identifiable Le antigen and were non-typeable. A previous survey of H. pylori isolates from different geographical regions by Simoons-Smit et al. (1996) reported a similar distribution of typeable (85 %) and non-typeable strains (15 %), with most of the non-typeable isolates originating from China.
We were unable to demonstrate a significant correlation between the expressed Le antigen type and the associated disease (Table 1
). Previous studies by Wirth et al. (1996) suggested that Le expression appeared to be more common among H. pylori isolates from patients with ulcer disease. In the present study, 9/10 analysed isolates from patients with ulcer disease (five gastric ulcer isolates and five duodenal ulcer isolates) tested positive for at least one Le determinant. However, the expression of at least one Le determinant was also confirmed in 24/28 (85.7 %) surveyed isolates of non-ulcer origin (16 dyspepsia isolates, six gastritis isolates, four oesophagitis isolates, one antro-pyloric deformation isolate and one gastro-oesophagic reflux isolate). Interestingly, four of the nine Leb-expressing isolates identified in this study originated from dyspepsia patients (Table 1). Four of six non-typeable strains also originated from dyspepsia patients. The prognostic significance of these findings remains to be established. Leb expression was also found in two isolates from patients with ulcer disease (one duodenal ulcer and one gastric ulcer isolate) in combination with either type 2 Lex and/or Ley expression. Exclusive Lex expression was confirmed in 2/4 isolates from patients with oesophagitis and one gastric ulcer isolate, whilst exclusive Ley expression was established in six isolates (6/38, 15.8 %) from patients with diverse clinical symptoms and associated diseases (Table 1).
WCE findings were corroborated by SDS-PAGE and Western blot analyses with anti-Lex, anti-Ley and anti-Leb mAbs. As expected, most typeable isolates exhibited typical high-molecular-mass ladder-like patterns indicative of smooth-form LPS (Table 1, Figs 2 and 3). Both WCE (Table 1) and silver staining revealed that strains 104CL (Fig. 2a, panel iii), 153CL (Fig. 2a, panel iv) and 217CL (Fig. 2a, panel iv) possessed an O-chain polysaccharide expressing Ley and/or Lex antigens, although their presence could not be detected by Western blotting with their respective anti-Le mAbs. The detection of a high-molecular-mass ladder-like pattern by silver staining in strains 018CL (Fig. 2a, panel i) and 160CL (Fig. 2a, panel iv), which tested negative for the presence of Le antigens by WCE and Western blotting, was indicative of the expression of an additional antigen that did not react with anti-Le mAbs (Table 1, Fig. 2).
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Fig. 2. Silver-staining and Western blots of proteinase K-treated whole cells of H. pylori. (a) Silver-stained 12.5 % SDS-polyacrylamide gels. H. pylori strain 26695 was used as a positive control; 002 denotes strain 002CL, etc. (b, c) Corresponding Western blots using anti-Lex mAb at a 1 : 150 dilution (b) and anti-Ley mAb at a 1 : 500 dilution (c).
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Fig. 3. Silver-stained 12.5 % SDS-polyacrylamide gels (a) and Western blots (b) of proteinase K-treated whole cells of H. pylori Leb-containing isolates. The Western blot used anti-Leb mAb at a 1 : 100 dilution. Std, Leb–hexasaccharide–BSA used as a positive control; SS1, H. pylori strain SS1 used as a negative control; 016 denotes 016CL, etc. (see Methods for details).
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Previous attempts to carry out Western blotting experiments with anti-Leb mAbs on H. pylori whole-cell samples were unsuccessful, and it was assumed that these antibodies did not react under Western blot conditions (Monteiro et al., 1998). However, Holgersson & Löfling (2006) have shown recently that the detection of Leb antigens on glycoconjugates can be achieved with anti-Leb mAbs in a Western blot format when a chemiluminescent substrate is utilized. By adopting a similar approach, we were able to demonstrate the recognition of Leb epitope by anti-Leb mAbs under Western blot conditions in 6/9 H. pylori isolates tested. Three Leb-containing H. pylori isolates, 049CL, 104CL and 240CL, did not react in the Western blot format but were positive for the presence of Leb antigen in WCE (Table 1). It is possible that in these three strains Leb epitope is linked directly to the core region of LPS and is conformationally inaccessible. The silver-staining pattern of Leb-containing H. pylori strain 240CL (Fig. 2a, panel iv) was consistent with the presence of a high-molecular-mass non-Le structure, as this strain had a low content of Ley and Lex antigens according to the WCE results and tested negative for Ley and Lex antigens by Western blotting (Fig. 2b, panel iv, and Fig. 2c, panel iv). The presence of the O-chain in two other Leb-containing strains, 049CL (Fig. 2a, panel ii) and 104CL (Fig. 2a, panel iii), was confirmed by silver staining although the signal was faint. Only one of these strains, 049CL, tested positive for the presence of Ley antigen by Western blotting (Fig. 2c, panel ii). It is plausible that O-chain polysaccharide in the LPS of these strains is composed of non-fucosylated or partially fucosylated type 2 N-acetyllactosamine repeating units capped by Ley and/or Lex antigens (Fig. 1). These structures were previously found to be co-expressed with type 1 Leb antigens in H. pylori LPS from Asian hosts (Monteiro et al., 2000). Further structural studies are needed to confirm these findings.
Silver staining and Western blotting with anti-Leb mAbs revealed significant variations in the O-chain length of Leb-containing isolates (Fig. 3). In H. pylori strains 016CL, 099CL and 109CL, the Leb epitope was associated with short O-chains, whilst in strains 048CL and 153CL, the Leb structure was capping longer O-chains. One strain, 026CL, appeared to produce a very long O-chain capped by Leb antigen (Fig. 3).
Previous studies have addressed the distribution of Le antigens in H. pylori isolates from different geographical regions, including North America, Europe and Asia. These investigations have identified type 2 Lex and Ley antigens as the most frequently encountered antigens in H. pylori LPS (Simoons-Smit et al., 1996; Wirth et al., 1996). Recently, expression of type 1 Lea and Leb antigens in LPS from Asian H. pylori isolates has been demonstrated, either solely or in combination with type 2 Le and/or N-acetyllactosamine epitopes (Monteiro et al., 2000). The tendency for expression of type 1 Le blood group antigens in H. pylori LPS from Asian hosts compared with Western populations is not well understood but is thought to be related to the host Le phenotype (Wirth et al., 1997).
We were able to demonstrate the presence of Leb antigen in the LPS of H. pylori strains from Chile by two methods, WCE and Western blotting. The results of the Western blot analysis suggested further structural diversity among type 1 Leb antigen-containing H. pylori strains associated with the presence of capping Leb epitopes on either short or long O-chain LPSs. To our knowledge, this is the first instance in which this blood group epitope has been identified in H. pylori strains from Latin America. Previous studies of H. pylori isolates obtained from various geographical regions have shown type 1 Leb antigen expression in 13 % of isolates (Wirth et al., 1996). Of these, only three strains were from Latin America (Peru), and a correlation between geographical origin and observed Le antigen type was not reported.
Our study examined the correlation between CagA expression and Le status. H. pylori strains isolated from both symptomatic and asymptomatic patients were examined. In a previous study by Cover et al. (1995), the proportion of CagA+ H. pylori isolates from duodenal ulcer patients was found to be 87.5 % based on combined bacteriological and serological testing. Wirth et al. (1996) found that cagA was expressed by 97 % of isolates, of which 75.7 % were CagA+ as determined by WCE. In the present study, we observed a higher number of CagA+ strains. No correlation between the absence of CagA expression and Le antigen type was found.
It has been shown that, whilst H. pylori infection is common among the Chilean adult population, seroconversion occurs early in life and is related directly to socioeconomic status (Hopkins et al., 1993). Increased seroprevalence of H. pylori in children of lower socioeconomic groups has been reported in other Latin American countries, such as Brazil (Oliveira et al., 1994), Peru (Klein et al., 1994), Colombia (Goodman et al., 1996) and Mexico (Castillo-Rojas et al., 2004). Consumption of uncooked vegetables and shellfish has been found to correlate with H. pylori seropositivity, as have modes of vegetable washing and swimming near contaminated beaches (Hopkins et al., 1993). Evaluation of the Le antigen distribution in H. pylori isolates from Chile could be applied to general epidemiological and population studies to assess the effectiveness of H. pylori prevention and eradication strategies.
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ACKNOWLEDGEMENTS
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The authors thank Dr Jean-Marc Gabastou from the Pan American Health Organization (PAHO) for support and encouragement of these studies. We thank Tom Devecseri for assistance with photography. Financial support from the Canadian International Development Agency (CIDA) in collaboration with the International Development Research Centre (IDRC), the Canadian Institutes of Health Research (CIHR) and Health Canada (HC) through the Global Health Research Initiative (GHRI) operational research grants for the Canadian International Immunization Initiative (CIII2) (File: 102172-006) (to E. A. and H. F.) and the Research and Development Bureau-Universidad Austral de Chile (Grant DID-UACH S-2006-25) (to L. O. R., M. W. S. and H. F.) is gratefully acknowledged.
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Type 1 and 2 Lewis antigens of Helicobacter pylori - a potential marker of the human geographical distribution
J. Med. Microbiol.,
May 1, 2008;
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543 - 544.
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INTRODUCTION
METHODS
