Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada
Received on May 13, 2003; revised on July 17, 2003; accepted on July 23, 2003
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Abstract |
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Key words: colonization / Helicobacter pylori / Lewis antigen / lipopolysaccharide
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Introduction |
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It has been suggested that there is a correlation between the lack of Le antigen expression and the lack of colonization and pathology of disease (Sakamoto et al., 1989). Previous surveys of H. pylori isolates suggest that a strong expression of Lex and Ley antigens by cagA-positive isolates contributes to inflammatory activities and persistence (Wirth et al., 1996
). More recently, it was shown that Le expression is not necessary for successful mouse gastric colonization or for H. pylori adherence to epithelial cells (Monteiro et al., 2001
; Takata et al., 2002
). We report on the isolation and structural analysis of the LPS from a clinical isolate, strain PJ2, that is devoid of both the Le antigens and the DD-heptoglycan structures but is still capable of colonization. Its ability to colonize the murine stomach was essentially identical to that of DD-heptoglycan and Le antigenexpressing H. pylori strains.
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Results |
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Nuclear magnetic resonance (NMR) analysis
LPS was delipidated with 0.1 M sodium acetate buffer (pH 4.5) and subjected to gel filtration chromatography on a Bio-Gel P-2 column, affording a single fraction eluting in the void volume of the column. The 1H-NMR spectrum of the delipidated aqueous-phase LPS showed a predominant proton resonance at 4.99 ppm belonging to H-1 of
1,6-linked glucan. Based on the integration of proton resonances, the average length of
1,6-linked glucan corresponded to approximately eight glucose residues (Figure 2). As seen in other H. pylori LPS structures (Monteiro et al., 2000
), anomeric resonances characteristic of
-DD- and
-LD-Hep were observed in the
5.15.45 ppm region together with anomeric resonances at
4.6 ppm and
4.5 ppm corresponding to ß-GlcNAc and ß-Gal respectively, a signal at
2.05 ppm corresponding to the CH3 of N-acetyl group of ß-GlcNAc and a signal at
1.18 ppm corresponding to the methyl group of
-Fuc.
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Mouse colonization experiments
The ability of this nontypable H. pylori strain PJ2, which expresses only an 1,6-glucan chain linked to the conserved core structure, to colonize the murine stomach was investigated. Colonization levels of this isolate were compared with the colonization levels achieved for the SS1 strain expressing Le O antigen and PJ1 strain expressing DD-heptoglycan (Table III). Although the Le antigenexpressing strain SS1 was shown to be a good colonizer, there was a 10-fold difference in numbers of SS1 and PJ2 bacteria recovered at 4 weeks postchallenge. However, the colonization levels of H. pylori strain PJ2 were still significant (4.667 ± 0.186 log10 cfu of bacteria recovered) and were practically identical to the ones achieved using a second nontypable clinical isolate H. pylori strain PJ1 with extended DD-heptoglycan structure (4.799 ± 0.572 log10 cfu of bacteria recovered).
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Discussion |
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Previous studies have demonstrated that H. pylori strains with mutations in both 1,3-fucosyltransferase genes were able to colonize mouse stomach, suggesting that Lex and Ley antigen expression was not required for gastric colonization (Takata et al., 2002). Suresh and colleagues (2000)
have also noted colonization of Balb/c and C57 mice by Le antigennegative H. pylori strains. However, in these cases, the LPS structure presented on the cell surface remained uncharacterized. We now extend this work to show that the presence of neither O-chain polylactosamine backbone nor an extended DD-heptoglycan is a prerequisite for colonization in a murine model. This study indicates that the presence of a long glycan alone could be sufficient for successful gastric colonization and that nontypable strains of H. pylori expressing extended
1,6-glucans can colonize the murine stomach at a comparable level to Le antigenproducing strains. Whether these studies point to features of the H. pylori cell critical for colonization of human gastric epithelium remains to be established.
However, recent studies have addressed the role of H. pylori LPS, and in particular Le antigen expression, in adherence and internalization using in vitro and in vivo human tissue models (AGS cells, human xenografts in nude mice, and binding to human gastric sections) (Edwards et al., 2000; Lozniewski et al., 2003
; Mahdavi et al., 2003
). Studies on bacterial internalization in AGS cells by Lozniewski et al. (2003)
provide evidence for a role of Le antigens in the internalization process for strains 26695, UA948, and UA1111. In contrast, a second study examining the role of Le antigens in bacterial adherence using human gastric epithelial cells indicated that they played only a minor role in adhesion and that adherence in this system was more likely related to the presence of the babA adhesin (Mahdavi et al., 2003
). The ability to examine a natural H. pylori isolate devoid of fucosylated or nonfucosylated polylactosamine chains in the two aforementioned systems will be invaluable in determining the role of Le antigens in both adhesion and internalization processes in human gastric epithelial cells.
It has been shown that 80% of cagA+ H. pylori strains express Lex or Ley and that half of them express both (Wirth et al., 1996). Heneghan et al. (2000)
reported that in patients with H. pylorirelated chronic gastritis, a significant relationship was found between the amount of bacterial Lex and Ley expression and the neutrophil and lymphocyte infiltration. Other studies have shown that neutrophils are a potential target recognized by anti-Lex antibodies (Appelmelk et al., 1996
). It was shown in this analysis that PJ2 LPS was unable to express Lex and Ley epitopes; correspondingly, PCR analysis confirmed that the strain also appears to lack the cagA gene or that its sequence was significantly divergent. Because cagA+ isolates induce more inflammation than do cagA- isolates (Peek et al., 1996
), it is plausible that the presence of Le antigens could be important for long-term colonization and pathology and that nontypable H. pylori strains expressing only an
1,6-glucan may indeed be unable to induce significant pathology yet are still capable of colonization. We are currently investigating if this is indeed the case for the PJ2 strain characterized in this study by utilizing the SCID mouse model of disease (Eaton et al., 1999
).
Previous structural studies have shown that 1,6-glucan is produced by a majority of H. pylori strains expressing type 2 Lex and Ley antigens with an average reported glucan chain corresponding to three to four glucoses (Monteiro, 2001
). Some strains are also capable of producing a longer
1,6-glucan chain (Altman, unpublished data). This study indicates that the ability to synthesize
1,6-glucan by H. pylori strains could be an important structural feature contributing to the successful colonization of the gastric mucosa by all H. pylori isolates, and the presence of other additional LPS structures contributes to the ensuing pathology of disease.
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Materials and methods |
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Electrophoresis and western blotting
SDSPAGE was performed with a mini-slab gel apparatus (Bio-Rad, Hercules, CA) by the method of Laemmli (1970). LPS samples were loaded in each lane and stained according to Tsai and Frasch (1982)
or transferred to nitrocellulose for immunological detection with anti-Le monoclonal antibodies (Signet Laboratories, Dedham, MA) as previously described (Logan et al., 2000
).
LPS isolation and delipidation
LPS was isolated by the hot phenol-water extraction procedure (Westphal et al., 1965) and, following the removal of insoluble material by low-speed centrifugation, purified by ultracentrifugation (105,000 x g, 4°C, 12 h).
Purified LPS (20 mg) was hydrolyzed in 0.1 M sodium acetate buffer, pH 4.2, for 2 h at 100°C; the solution was cooled; and the precipitated lipid A was removed by low-speed centrifugation. The supernatant solution was lyophilized, and water-soluble components were fractionated by gel filtration on a Bio-Gel P-2 column (1.6 cm x 95 cm) equilibrated with pyridinium acetate (0.02 M, pH 5.4). Elution was performed with pyridinium acetate (0.02 M, pH 5.4). The fractions (1 ml) were monitored for neutral glycoses (Dubois et al., 1956) and those giving positive reaction were combined and lyophilized.
Sugar composition and methylation analyses
Sugar composition analysis was performed by the alditol acetate method (Sawardeker et al., 1967). The hydrolysis was done in 4 M trifluoroacetic acid at 100°C for 4 h or 2 M trifluoroacetic acid at 100°C for 16 h followed by reduction in H2O with NaBH4 and subsequent acetylation with acetic anhydride/pyridine. Alditol acetate derivatives were analyzed by gas-liquid chromatography (GLC) MS using a Hewlett-Packard chromatograph equipped with a 30 m DB-17 capillary column [210°C (30 min) to 240°C at 2°C/min] and MS spectra in the electron impact mode were recorded using a Varian Saturn II mass spectrometer. Methylation linkage analysis was carried out by the NaOH/dimethyl sulfoxide/CH3I procedure (Ciucanu and Kerek, 1984
) and with characterization of permethylated alditol acetate derivatives by GLC-MS in the electron impact mode (DB-17 column, isothermally at 190°C for 60 min).
FAB-MS
A fraction of the methylated sample was used for positive ion FAB-MS performed on a JEOL JMS-AX505H mass spectrometer with glycerol-thioglycerol (1:3) as the matrix. A 6 kV xenon beam was used to produce pseudo-molecular ions that were then accelerated to 3 kV and their mass analyzed. Product ion scan (B/E) and precursor ion scan (B2/E) were performed on metastable ions created in the first free field with a source pressure of 5 x 10-5 torr.
ES-MS
Samples were analyzed on a crystal Model 310 CE instrument (ATI Unicam, Boston, MA) coupled to an API 3000 mass spectrometer (Applied Biosystems/Sciex, Concord, ON) via a micro-ionspray interface. A sheath solution (isopropanol-methanol, 2:1) was delivered at a flow rate of 1 µl/min to a low dead volume tee (250 µm ID, Chromatographic Specialties, Brockville, Canada). All aqueous solutions were filtered through a 0.45-µm filter (Millipore, Bedford, MA) before use. An electrospray stainless steel needle (27-gauge) was butted against the low dead volume tee and enabled the delivery of the sheath solution to the end of the capillary column. The separations were obtained on about 90 cm length bare fused-silica capillary using 10 mM ammonium acetate/ammonium hydroxide in deionized water, pH 9.0, containing 5% methanol. A voltage of 25 kV was typically applied at the injection. The outlet of the capillary was tapered to 15 µm ID using a laser puller (Sutter Instruments, Novato, CA). Mass spectra were acquired with dwell times of 3.0 ms per step of 1 m/z unit in full-mass scan mode. Accurate masses were determined using internal standartization on a Q-Star quadropole/time-of-flight instrument (Applied Biosystems/Sciex). The mass spectra resolution (half-height definition) was 7,000.
PCR analysis of cagA and vacA
Amplifications were performed on a 9600 Thermocycler (Perkin Elmer, Canada) using Pwo polymerase (Roche) with the following cycling profiles: for cagA DZ3 and R009, 94°C for 3 min, 94°C for 30 s, 50°C for 30 s, 72°C for 60 s, for 30 cycles, followed by an extension at 72°C for 10 min 30 s; for cagA D008 and R008: 94°C for 3 min, 94°C for 30 s, 60°C for 30 s, 72°C for 60 s for 30 cycles, followed by an extension at 72°C for 10 min; for vacA 1-F and 1-R: 94°C for 3 min, 94°C for 30 s, 53°C for 30 s, 72°C for 60 s for 30 cycles, followed by an extension at 72°C for 10 min.
Mouse colonization
Specific pathogen-free female CD1 mice were purchased from Charles Rivers Laboratories (Montreal, Quebec) when they were 68 weeks old. Mice were maintained and used in accordance with the recommendations of the Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals (1993). Mice were inoculated with bacteria harvested from 36 h broth culture. Aliquots of 0.2 ml, containing approximately 108 bacteria resuspended in phosphate buffered saline (PBS) were given by gavage directly into the gastric lumen using a 20 g gavage needle. Three inocula were given over a period of 6 days. No attempt was made to neutralize gastric acidity prior to inoculation. To recover viable bacteria from the stomach, mice were killed by CO2 asphyxiation, and their stomachs were removed whole. Stomachs were cut open along the greater curvature, and the exposed lumenal surface was gently irrigated with 10 ml sterile PBS, delivered via a syringe fitted with a 20 g gavage needle, to dislodge the loosely adherent stomach contents. This step effectively diminished the small numbers of ubiquitous contaminating bacteria that otherwise overgrow on GSS agar to thereby mask the presence of the slower-growing H. pylori organisms. The washed stomach tissue was then homogenized, and serial dilutions plated on GSS agar. H. pylori colonies were counted following 36 days incubation.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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