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Infection and Immunity, June 2004, p. 3429-3435, Vol. 72, No. 6
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.6.3429-3435.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Heterogeneity among Helicobacter pylori Strains in Expression of the Outer Membrane Protein BabA

Ewa E. Hennig,¹ Ray Mernaugh,² Jennifer Edl,² Ping Cao,³ and Timothy L. Cover^3,4,5*

Departments of Medicine,³ Microbiology and Immunology,⁴ Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232,² Veterans Affairs Medical Center, Nashville, Tennessee 37212,⁵ Department of Gastroenterology, Medical Center for Postgraduate Education, Cancer Center, 02-781 Warsaw, Poland¹

Received 24 October 2003/ Returned for modification 12 December 2003/ Accepted 16 February 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
The BabA adhesin of Helicobacter pylori is an outer membrane protein that binds to the fucosylated Lewis b histo-blood group antigen on the surface of gastric epithelial cells. We screened a phage-displayed ScFv (single-chain fragment variable) recombinant antibody library for antibodies reactive with a recombinant BabA fragment and identified two such antibodies. Each antibody recognized an 75-kDa protein present in wild-type H. pylori strain J99 but absent from an isogenic babA mutant strain. An immunoreactive BabA protein was detected by at least one of the antibodies in 18 (46%) of 39 different wild-type H. pylori strains and was detected more commonly in cagA-positive strains than in cagA-negative strains. Numerous amino acid polymorphisms were detected among BabA proteins expressed by different strains, with the greatest diversity occurring in the middle region of the proteins. Among the 18 strains that expressed a detectable BabA protein, there was considerable variation in the level of binding to Lewis b in vitro. Heterogeneity among H. pylori strains in expression of the BabA protein may be a factor that contributes to differing clinical outcomes among H. pylori-infected humans.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Helicobacter pylori is a gram-negative bacterial organism that persistently colonizes the human gastric mucosa. Most H. pylori-infected humans tolerate the presence of this organism relatively well and never develop symptomatic gastroduodenal pathology. However, H. pylori infection is a risk factor for the development of peptic ulcer disease and distal gastric adenocarcinoma (8, 11, 27).

Within the gastric mucosa, H. pylori lives within the mucus layer and may also attach to gastric epithelial cells. At least five different putative H. pylori adhesins (designated BabA, SabA, AlpA, AlpB, and HopZ) have been identified (16-19). Of these, the BabA adhesin has been investigated in the most detail thus far. The H. pylori BabA adhesin mediates binding of H. pylori to the fucosylated Lewis b histo-blood group antigen present on the surface of gastric epithelial cells (5, 16). In an animal model, Lewis b-dependent attachment of H. pylori to gastric epithelial cells is accompanied by increased severity of inflammation, development of parietal cell autoantibodies, and parietal cell loss (12, 15).

There is a high level of genetic diversity among H. pylori isolates from different humans (4). Consistent with this observation, there is variation among H. pylori isolates in the capacity to bind to Lewis b (7, 16, 23, 28). One study reported that 63 (66%) of 95 H. pylori isolates bound to Lewis b (16). The molecular basis for variation among strains in Lewis b-binding capacity has not yet been investigated in any detail.

In the present study, we sought to investigate diversity among H. pylori strains in expression of the BabA protein. We report here the development of methodology for detecting expression of the BabA protein and demonstrate that only about half of the H. pylori strains tested produce a detectable BabA protein. Among H. pylori strains that produce a detectable BabA protein, there is considerable variation in binding to Lewis b in vitro.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains. H. pylori J99 and 26695 are reference strains for which the entire genome sequences are known (2, 29). An isogenic babA2 mutant derivative of H. pylori J99 was obtained from David Pride and Martin Blaser (23). The other strains utilized in the present study were isolated from patients in Denver, Colo., or Nashville, Tenn. The cagA and vacA genotypes of these strains have been reported previously (3, 6, 9, 30). The term "cagA positive" indicates that cagA sequences are detectable by either PCR or colony hybridization, and the term "cagA negative" indicates that cagA sequences are not detectable by these methods (providing evidence that the cagA gene is absent). H. pylori strains were cultured at 37°C on Trypticase soy agar plates containing 5% sheep blood in ambient air containing 5% CO₂.

Lewis b-binding assay. H. pylori strains were tested for capacity to bind Lewis b by using a modification of a previously described immunoassay (23). H. pylori were cultured for 48 h on solid medium as described above and then harvested and suspended in 50 mM carbonate buffer (pH 9.6) to an optical density (OD) at 600 nm of ca. 0.8. H. pylori were bound to wells of Immunolon-2HB immunoassay plates (Thermo Labsystems) overnight at 4°C. Nonadherent bacteria were removed, and wells were blocked with phosphate-buffered saline (PBS)-0.05% Tween 20 (PBS-Tween). Replicate wells were then incubated for 1 h with either PBS containing 1 µg of Lewis b-human serum albumin glycoconjugate (Isosep, Tullinge, Sweden)/ml and 0.5% bovine serum albumin or PBS-0.5% albumin without Lewis b. After three washes with PBS-Tween, wells were incubated for 1 h at room temperature with anti-Lewis b murine monoclonal antibody BG-6 (Signet Pathology Laboratories, Inc., Dedham, Mass.), diluted 1:500 in PBS, washed again, and then incubated for 1 h at room temperature with anti-mouse immunoglobulin M-horseradish peroxidase (HRP) conjugate (ICN) diluted 1:1,000 in PBS. Wells were washed five times with PBS-Tween, and then the color was developed by the addition of ABTS [2,2'azinobis(3-ethylbenzthiazolinesulfonic acid)] and H₂O₂ in a phosphate-citric acid buffer. OD values were determined by using an enzyme-linked immunosorbent assay (ELISA) reader at 410 nm. The results are expressed in relative OD units, calculated as the OD difference between bacteria incubated with Lewis b and bacteria incubated with an albumin-containing buffer control without Lewis b (OD x 1,000). Duplicate assays for each strain were performed on at least two different days.

Expression of a BabA-GST fusion protein. The oligonucleotides 5'-CCCGGGTAACGCCAATGGTCAAAA (forward) and 5'-CTCGAGGGCGTTAGCCTCACTACTA (reverse), with recognition sequences (underlined) for SmaI and XhoI, respectively, were used to PCR amplify an 550-bp fragment of babA (jhp0833) from H. pylori strain J99 (2). The PCR product was ligated into pGEM-T Easy vector (Promega) and sequence analysis confirmed that the cloned fragment corresponded to nucleotides encoding amino acids 128 to 310 of the 744-amino-acid BabA protein (GenBank accession number AAD06409). This fragment was subcloned into pGEX-6P-1 vector (Amersham Biosciences), previously digested with SmaI and XhoI, and then transformed into Escherichia coli BL21. After induction with IPTG (isopropyl-ß-D-thiogalactopyranoside), a BabA-glutathione S-transferase (GST) fusion protein of 47 kDa was successfully expressed in a soluble form. The fusion protein was then purified by adsorption to glutathione-Sepharose 4B beads (Amersham Biosciences) and eluted with 20 mM reduced glutathione in 50 mM Tris buffer containing 100 mM NaCl and 0.5% NP-40. The purified BabA-GST fusion protein then was dialyzed in Slide-A-Lyzer dialysis cassettes with a 10-kDa cutoff membrane (Pierce) against 150 mM NaCl and 20% glycerol.

Selection of BabA-specific antibodies from a phage-displayed ScFv recombinant antibody library. Spleens from newborn and/or 3- to 4-week-old ICR, NSA, MF1, NIH Swiss Webster, and ND4 Swiss Webster mice and Wistar Hanover, Long Evans, LBNF1, SD, WIS, and F/344 rats were used as a source of genetic starting material to produce a large (2.9 x 10⁹ members) phage-displayed ScFv (single-chain fragment variable) recombinant antibody library, by using modifications of a previously published protocol (21). To analyze the diversity and complexity of the phage antibody library, 172 randomly picked, individual colonies were selected for analysis. BstNI digestion of PCR products (21) from these colonies indicated that 167 (97%) contained inserts (750 bp) typical of full-length ScFv gene fragments. Forty-three of the ScFv PCR products were subsequently subjected to digestion with BstNI and analysis by agarose gel electrophoresis. The DNA fingerprints of digested fragments from each of the 43 different PCR products were unique and suggested that the library consisted mainly of full-length, diverse ScFv recombinant antibody clones. Antibodies are encoded within the pCANTAB5E phagemid vector (Amersham Biosciences), which contains an ampicillin resistance gene to select for bacterial clones that contain the ScFv-encoding phagemid. ScFv were expressed as phage gene 3 fusion proteins (for phage display purposes and selections on antigens) and as epitope E-tagged ScFv for immunoassays.

A Nunc Maxisorb tube was coated for 1 h at room temperature with 1 ml of BabA-GST diluted to 100 µg/ml in PBS. The tube was blocked with PBS containing 0.1% Tween 20 (PBS-T) for 1 to 2 h to prevent phage from binding nonspecifically to the coated tube. An aliquot (ca. 10¹² to 10¹³ CFU) of the phage antibody library was preblocked with 0.1% Tween 20 for 1 to 2 h, added to the BabA-GST coated tube, and allowed to interact with BabA-GST for 2 h at room temperature. The tube was washed six times with PBS-T to remove unbound phage. Phage bound to BabA-GST were eluted with 100 mM triethylamine for 10 min, and then the pH was adjusted to near neutrality with 1 M Tris-HCl (pH 7.6). Eluted phage were used to infect E. coli TG1 cells. Infected E. coli were plated onto 2xYTAG agar medium, which contains 17 g of Bacto Tryptone, 10 g of Bacto Yeast Extract, 5 g of sodium chloride, 15 g of agar, 20 g of glucose, and 100 mg of ampicillin per liter of medium. Infected E. coli cultures were incubated overnight at 30°C and then rescued with M13KO7 helper virus to obtain an enriched population of phage. The enriched phage were used for a second round of selection on BabA-GST, as described above. Bacterial colonies stemming from the second round of selection were picked from 2xYTAG agar plates to microtiter wells containing 100 µl of 2xYT with ampicillin and 1 mM IPTG (2xYTAI) and induced to express soluble E-tagged ScFv by incubation overnight at 30°C. Microtiter plates containing individual bacterial isolates were centrifuged at 1,000 x g for 10 min to pellet bacterial cells. Cell pellets were resuspended in 40 µl of TES (0.2 M Tris-HCl [pH 8.0], 0.5 mM EDTA, 0.5 M sucrose) and 60 µl of 1/5xTES (TES diluted 1:5 with distilled water) to yield a final volume of 100 µl. Resuspended cells were incubated on ice for 1 h to release soluble E-tagged ScFv from the bacterial periplasmic space. ScFv from bacterial periplasmic extracts were assayed against BabA-GST or GST by an immune complex ELISA (ICELISA), as described below.

ICELISA for detection of BabA-specific ScFv. The ICELISA protocol accompanying the HRP-Anti-E Tag conjugate (Amersham Biosciences) was used to detect E-tagged soluble ScFv reactive with BabA. Individual wells of 384-well microtiter plates were coated for 1 h at room temperature with 50 µl of BabA-GST or GST diluted to 5 µg/ml of PBS and then blocked with PBS-T for 15 to 30 min at room temperature. Wells were emptied, and 25 µl of HRP-Anti-E Tag diluted to 1:4,000 in PBS-T was added to each well. Then, 25 µl of periplasmic extract from each bacterial isolate was added to separate wells of the microtiter plates coated with either BabA-GST or GST. The microtiter plates were incubated for 1 h at room temperature and then washed six times with PBS-T. Next, 50 µl of ABTS containing hydrogen peroxide was added to each well for color development and, after incubation at room temperature for 10 to 30 min, the OD values were determined by using a BioTek Elx800NB plate reader at 405 nm.

Preparation of periplasmic extracts from ScFv-expressing E. coli. Bacterial clones producing antibodies reactive with BabA-GST in ELISA were used to inoculate 250 ml of 2xYTAG. Cultures were incubated overnight at 30°C with shaking at 100 rpm and then centrifuged to pellet the cells. Cell pellets were resuspended in 250 ml of 2xYTAI, incubated overnight at 30°C with shaking, and centrifuged. Cell pellets were resuspended in 10 ml of TES and 15 ml of 1/5xTES to yield a final volume of 25 ml, placed on ice for 1 h, and then centrifuged. The supernatant, containing soluble ScFv in periplasmic extract, was frozen at –70°C until needed.

Western blot analysis of BabA expression in H. pylori strains. H. pylori strains were grown on Trypticase soy agar plates containing 5% sheep blood. Standardized inocula of H. pylori were suspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) lysis buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked with PBS-T for 30 to 60 min and then probed for 1 h with ScFv in periplasmic extracts diluted with an equal volume of PBS that contained 1% nonfat dry milk, 0.1% Tween 20, and a 1:4,000 dilution of HRP-Anti-E Tag conjugate. Of several different blocking conditions that were tested, this set of conditions permitted the most specific detection of BabA. Membranes were washed for 30 to 60 min with PBS-T to remove unbound antibody and then developed by using the SuperSignal West Pico chemiluminescence substrate (Pierce) and film.

Nucleotide sequence analysis of babA alleles. babA alleles. babA alleles and flanking DNA were PCR amplified from H. pylori genomic DNA with the primers 5'-GTATTTTGTGTAGTCTTTGTTGGTGG and 5'-GCAGTTGCATGGTCAGCTCTGAGG. These two primers were derived from the sequences of two genes (jhp0832 and jhp0835, respectively), which flank babA (jhp0833) in a reference strain (H. pylori J99) for which the entire genome sequence is known (2). The PCR products were purified by using a purification kit (Qiagen), and the nucleotide sequences of babA alleles were determined on both strands by using a series of appropriate primers.

Nucleotide sequence accession numbers. babA nucleotide sequences have been deposited in GenBank (accession numbers AY549174 to AY549178).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Selection of BabA-specific ScFv antibodies. To analyze expression of the H. pylori BabA protein, we sought to develop specific anti-BabA antibodies. BabA belongs to a large family of closely related H. pylori outer membrane proteins (1, 22, 23) and, consequently, there was a high probability that a polyclonal BabA antiserum might cross-react with multiple H. pylori outer membrane proteins. Therefore, we undertook efforts to isolate an epitope-specific antibody that specifically recognized BabA. As a first step, a BabA-GST fusion protein was expressed in E. coli and purified as described in Materials and Methods. This fusion protein was derived from a region of BabA (jhp0833) from H. pylori strain J99 (amino acids 128 to 310) that exhibits relatively little relatedness to paralogous H. pylori outer membrane proteins, including BabB. The purified BabA-GST fusion protein (Fig. 1) was then used to select phage encoding BabA-specific ScFv antibodies from a large phage-displayed ScFv (single-chain fragment variable) recombinant antibody library, as described in Materials and Methods. Eluted phage were used to infect E. coli, and E. coli clones were induced to express soluble epitope-E-tagged ScFv. ScFv from bacterial periplasmic extracts were then assayed for reactivity with BabA-GST by ELISA. Thirty clones were identified that reacted more strongly with the BabA-GST fusion protein than with a GST control protein. When analyzed by immunoblotting, 2 of the 30 antibodies reacted strongly with the BabA-GST fusion protein but not with GST alone (data not shown). These two antibodies (designated K15 and K22) were selected for further study.

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FIG. 1. Expression and purification of recombinant BabA-GST. A BabA-GST fusion protein was expressed and purified as described in Materials and Methods. A Coomassie blue-stained SDS-PAGE gel is shown. Lanes: a, lysate from an E. coli strain expressing BabA-GST; b, lysate from a control E. coli strain expressing GST alone; c, purified BabA-GST; d, purified GST.

Detection of BabA expression in H. pylori strain J99. To determine whether the recombinant ScFv antibodies could detect expression of BabA produced by H. pylori, we tested the reactivity of these antibodies with lysates from H. pylori wild-type strain J99 (a strain known to produce BabA) (16, 23). In an immunoblot assay, both the K15 and K22 antibodies recognized a single 75-kDa band (consistent with the expected mass of BabA) in lysate of wild-type strain J99 (Fig. 2). In contrast, neither antibody recognized a band of this mass in lysate from an isogenic J99 babA-null mutant strain, in which the babA gene was disrupted by insertion of a kanamycin cassette (Fig. 2). These data indicate that the K15 and K22 antibodies specifically recognize not only the recombinant BabA fragment produced in E. coli but also the BabA protein produced by H. pylori.

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FIG. 2. Immunoblot detection of BabA expression in H. pylori strain J99. Proteins in lysates of H. pylori strain J99 (lanes a) and an isogenic babA mutant strain (lanes b) were separated by SDS-PAGE, transferred to nitrocellulose membrane, and then immunoblotted with periplasmic extracts from either E. coli K15 (left panel) or K22 (right panel). Binding of anti-BabA ScFv antibodies was detected as described in Materials and Methods. Each of these recombinant ScFv antibodies recognized an 75-kDa band produced by strain J99 but not the isogenic babA mutant strain.

BabA expression in a panel of H. pylori strains. We next used immunoblotting methodology to analyze expression of BabA in a panel of 39 different H. pylori strains, originally isolated from 39 different patients. Antibody K15 recognized a band of the expected size in 18 of the 39 strains, and antibody K22 recognized a band of the expected size in 11 of the 39 strains (Fig. 3 and Table 1). The BabA protein in 11 strains was recognized by both antibodies, and the BabA protein in seven strains was recognized by only one antibody (K15). The immunoreactive BabA proteins produced by different strains all appeared to have similar molecular masses, except that the reactive band recognized by antibody K15 in one strain (i.e., strain 87-81) was slightly smaller in mass than the corresponding reactive bands in the other 17 strains (data not shown). As illustrated in Fig. 3, it seems likely that antibodies K15 and K22 recognize different BabA epitopes and that the use of antibody K15 is a more sensitive approach for detecting BabA expression compared to use of antibody K22. Of the 39 H. pylori strains analyzed in the present study, 25 (64%) contained the cagA gene and 14 (36%) did not (3, 6, 9, 30). BabA expression was detected by immunoblot analysis in 15 (60%) of 25 cagA-positive strains versus 3 (21%) of 14 cagA-negative strains (P = 0.04, Fisher exact test).

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FIG. 3. Immunoblot analysis of selected H. pylori strains. Lysates from nine different H. pylori strains were immunoblotted with periplasmic extracts from either E. coli K15 or K22. Binding of anti-BabA ScFv antibodies was detected as described in Materials and Methods. The K15 anti-BabA ScFv antibody recognized an 75-kDa band produced by six of the nine strains (top panel), and the K-22 anti-BabA ScFv antibody recognized BabA proteins produced by two of the nine strains (bottom panel). Lanes: a, H. pylori strain J99; b, J258; c, 87-33; d, 87-29; e, 87-199; f, Tx30a; g, J190; h, J63; i, 86-313.

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TABLE 1. Expression of BabA by H. pylori strains

Binding of H. pylori strains to Lewis b. H. pylori strains next were tested for the capacity to bind Lewis b by using an in vitro binding assay. In brief, H. pylori strains were incubated with a Lewis b-human serum albumin glycoconjugate or with albumin as a control, and then binding of Lewis b to the bacteria was detected by using an anti-Lewis b antibody, as described in Materials and Methods. In agreement with previous reports (16, 23), the wild-type strain J99 bound Lewis b, whereas an isogenic babA mutant strain did not (mean OD units ± the standard error of the mean of 235 ± 15 versus 4 ± 1, respectively, P < 0.001). Also in agreement with a previous report (16), there was no detectable binding of H. pylori strain 26695 to Lewis b. We then tested the entire panel of 39 H. pylori strains for their capacity to bind Lewis b. Four strains (J262, 87-230, 87-75, and J223) yielded very high OD values in the ELISA after incubation with either exogenous Lewis b or the albumin control, which suggests that these strains may express Lewis b as part of their lipopolysaccharide structures (33). Because the capacity of these four strains to bind exogenous Lewis b could not be assessed, these strains were excluded from further analysis. Among the remaining 35 strains, the level of Lewis b binding in the ELISA was higher for 17 BabA-positive strains than for 18 BabA-negative strains (P = 0.004; Fig. 4). The seven H. pylori strains that bound Lewis b at the highest levels (strains J99, 87-81, J87, J133, 87-33, 87-29, and 92-25) each expressed a detectable BabA protein. Notably, among strains that expressed BabA, there was considerable variation in the level of binding to Lewis b (Fig. 4).

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FIG. 4. Binding of Lewis b antigen by H. pylori strains. H. pylori strains were tested for capacity to bind Lewis b antigen, by using an ELISA as described in Materials and Methods. The results are expressed in relative OD units. H. pylori strains that expressed a BabA protein detectable by immunoblot analysis (n = 17) bound significantly higher levels of Lewis b than did strains that failed to express a detectable BabA protein (n = 18; mean OD units ± the standard errors of the mean of 84 ± 21 versus 13 ± 4, respectively; P = 0.004, Student t test). The seven H. pylori strains (J99, 87-81, J87, J133, 87-33, 87-29, and 92-25) that bound the highest levels of Lewis b (>70 OD units in the Lewis b binding assay) each expressed a detectable BabA protein.

Diversity among BabA proteins. To analyze potential amino acid sequence diversity among BabA proteins produced by different H. pylori strains, we sought to determine the nucleotide sequences of babA alleles from multiple strains. Six H. pylori strains that each produced a detectable BabA protein (based on immunoblotting assays) were selected for analysis of babA nucleotide sequences. In order to PCR amplify full-length babA alleles, primers were chosen based on the sequences of genes that flank babA in H. pylori J99 as described in Materials and Methods. Using this approach, PCR products containing babA were successfully amplified from each of the six strains.

Nucleotide sequence analysis of the amplified babA alleles indicated that the sizes of the babA open reading frames ranged from 2,211 to 2,232 nucleotides in length (encoding 737 to 744 amino acids). The predicted molecular masses of the encoded proteins ranged from 79.7 to 80.6 kDa. An alignment of the six deduced BabA amino acid sequences is shown in Fig. 5. Amino acid polymorphisms are found throughout these BabA proteins but are relatively uncommon in the C-terminal region. The region of maximum diversity corresponds to amino acids 160 to 361 in the BabA sequence of reference strain J99. Potentially, this constitutes a surface-exposed region of the BabA outer membrane protein. The recombinant BabA peptide used for generation of the anti-BabA antibodies in the present study comprised amino acids 128 to 310 of the BabA sequence from strain J99, i.e., a region of relatively high diversity.

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FIG. 5. Alignment of deduced BabA amino acid sequences from six different H. pylori strains. The predicted N-terminal signal sequence corresponds to amino acids 1 to 20 of BabA from strain J99. Three of these H. pylori strains (J99, 87-33, and 87-29) consistently bound relatively high levels of Lewis b in vitro, whereas the other three strains (J116, 92-26, and 92-18) did not exhibit detectable binding.

Among the six BabA-expressing H. pylori strains selected for analysis in Fig. 5, three strains (J99, 87-33, and 87-29) bound relatively high levels of Lewis b in vitro, whereas the other three strains (J116, 92-26, and 92-18) did not exhibit detectable binding or perhaps bound very low levels of Lewis b. One possible explanation for variation in levels of Lewis b binding among different strains is that strain-specific variation in the amino acid sequences of BabA proteins might result in different binding properties. However, an examination of the alignment shown in Fig. 5 did not reveal any BabA amino acid sequences that are highly conserved among the three strains that bind Lewis b and divergent among the three strains that do not exhibit detectable binding.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Analyses of the complete genome sequences of two H. pylori strains have revealed the existence of a large family of paralogous genes (hop genes) that are predicted to encode outer membrane proteins (1, 2, 29). Several of these outer membrane proteins function as porins, and others, including BabA, SabA, AlpA, AlpB, and HopZ, have been reported to contribute to the binding of H. pylori to epithelial cells (16-19).

Of the H. pylori adhesins identified thus far, BabA has been investigated in the most detail. The H. pylori strain that was used for initial characterization of BabA (H. pylori strain CCUG 17875) contains two copies of the babA gene (designated babA1 and babA2) (16). In this strain, the babA2 gene encodes a functional BabA outer membrane protein that binds to Lewis b; a 10-nucleotide segment containing the expected translational initiation codon is deleted from the babA1 gene, and therefore this gene is not predicted to be translated. In contrast to H. pylori CCUG 17875, two H. pylori strains for which entire genome sequences are available (strains 26695 and J99) each contain only one copy of babA (designated HP1243 and jhp0833, respectively) (2, 29). Using a PCR assay designed to amplify babA2 genes but not babA1 genes, one study reported detection of a babA2 genotype in 82 (71.9%) of 114 H. pylori isolates (14). Reverse transcription-PCR analyses indicated that the babA2 gene was transcribed in each of the strains that contained a babA2 gene (14). Moreover, 28 of 31 babA2-positive H. pylori strains were reported to bind to Lewis b compared to none of 23 babA2-negative strains (14).

In most previous studies, BabA protein expression has been detected indirectly, by using assays that test the capacity of H. pylori strains to bind to the Lewis b antigen (7, 14, 16, 23, 28). One recent study reported the generation of a polyclonal BabA antiserum (35), but experiments to evaluate the specificity of this antiserum were not presented. The use of anti-BabA ScFv antibodies, as described here, represents a highly specific method for detecting expression of the H. pylori BabA protein. Use of the K15 antibody in immunoblot assays permitted detection of BabA in all H. pylori strains that exhibited relatively high levels of binding to Lewis b (Fig. 4), which suggests that this antibody recognizes a BabA epitope that is well conserved among different H. pylori strains.

Among the H. pylori strains in the present study that produced an immunoreactive BabA protein, there was considerable variation in the levels of binding to Lewis b (Fig. 4). Moreover, there was overlap between the levels of Lewis b bound by several BabA-expressing strains and the levels of Lewis b bound by strains that do not express BabA. Chemiluminescent immunoblotting is likely to be a more sensitive approach for detecting BabA expression than the Lewis b binding ELISA used here. Potentially, the use of a more sensitive Lewis b binding assay would permit detection of Lewis b binding by an increased proportion of BabA-expressing strains.

Several hypotheses can be suggested to explain why there is variation in binding to Lewis b among different H. pylori strains that express BabA. For example, there may be strain-specific differences in levels of BabA expression, differences in BabA export to the outer membrane, or differences in BabA amino acid sequences that contribute to differences in Lewis b binding. To test the latter hypothesis, we analyzed and compared the deduced BabA amino acid sequences of three strains that bound relatively high levels of Lewis b and three strains that did not exhibit detectable binding to Lewis b. Numerous polymorphisms were identified among the six BabA sequences analyzed; this is similar to the diversity that has been described for many other H. pylori proteins. In agreement with a previous report (23), the greatest level of diversity was found in the middle region of BabA. However, the magnitude of diversity among BabA proteins from different strains was less than has been observed among several other surface-exposed or exported H. pylori proteins, such as HopQ and VacA (3, 6). Notably, the comparative analysis of BabA proteins performed here did not permit a definitive identification of any BabA amino acid sequences that are highly conserved among strains that bind Lewis b and consistently divergent among strains that do not exhibit detectable binding.

There is a high level of genetic diversity among H. pylori isolates from different humans, and a large body of evidence suggests that different clinical outcomes of H. pylori infection may be determined, at least in part, by the characteristics of the infecting H. pylori strains. Several strain-specific H. pylori factors that may contribute to disease outcome have been identified. H. pylori strains containing the cag pathogenicity island, type s1 vacA alleles, iceA1 alleles, intact oipA alleles, or type I hopQ alleles each have been associated with an increased risk of severe disease (peptic ulcer disease or gastric carcinoma) compared to strains that lack these genes or allelic types (3, 6, 10, 13, 14, 20, 31, 32, 34). Similarly, strains containing babA2 alleles or exhibiting the Lewis b-binding phenotype have been associated with an increased risk of duodenal ulcer disease and gastric adenocarcinoma compared to strains that lack these characteristics (14, 24-26, 28, 35). We speculate that BabA-expressing strains that bind high levels of Lewis b are associated with a higher risk for serious disease outcomes than are BabA-expressing strains that bind low levels of Lewis b. Further studies, involving larger numbers of patients, will be required to test this hypothesis.

In the present study, we demonstrate that expression of the BabA protein is more common among cagA-positive H. pylori strains than among cagA-negative strains. This finding is consistent with the results of previous studies in which the babA2 gene has been detected more commonly in cagA-positive strains than in cagA-negative strains (14, 16). Similarly, type s1 vacA alleles, type 1 hopQ alleles, and intact oipA alleles are found more commonly in cagA-positive strains than in cagA-negative strains (3, 6, 14, 32, 34). One study reported that H. pylori strains containing multiple markers of virulence ("triple-positive strains" [defined by presence of cagA, type s1 vacA alleles, and babA2 alleles]) are associated with a higher risk of peptic ulcer disease than are strains that contain only one or two of these markers (14). Thus, it seems likely that H. pylori-associated diseases, such as peptic ulcer disease and gastric carcinoma, arise via complex pathways that involve the actions of multiple bacterial virulence factors.

 
We thank Beverly Hosse for technical assistance and thank David Pride and Martin Blaser for providing a babA mutant strain.

This study was funded in part by NIH R01 DK53623, the Department of Veterans Affairs, and grants 501-1-1-09-20/03 CMKP and 6 PO4A 031 15 from the State Committee for Scientific Research of Poland.

 
* Corresponding author. Mailing address: Division of Infectious Diseases, A3310 Medical Center North, Vanderbilt University School of Medicine, Nashville, TN 37232. Phone: (615) 322-2035. Fax: (615) 343-6160. E-mail: timothy.L.cover{at}vanderbilt.edu.

Editor: J. B. Bliska

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, June 2004, p. 3429-3435, Vol. 72, No. 6
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.6.3429-3435.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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