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Helicobacter pylori is a gram-negative bacterium that colonizes the gastric mucosa of humans. The mechanisms by which H. pylori elicits an inflammatory response and persists for decades in the gastric mucosa remain incompletely understood (4, 14). Secreted proteins play an important role in the pathogenesis of many bacterial infections (20), and therefore, in this study we sought to characterize proteins that are released into the extracellular space by H. pylori during growth in vitro.

H. pylori 60190 (ATCC 49503) was cultured with constant agitation in sulfite-free brucella broth (9) containing 0.5% charcoal at 37°C in ambient air supplemented with 6% CO₂. After 48 h, cultures were centrifuged to remove intact bacteria, and proteins in the culture supernatant were concentrated by precipitation with a 50% saturated solution of ammonium sulfate (3). The concentrated culture supernatant then was used to immunize a New Zealand White rabbit. Immunoblot analysis indicated that the antiserum recognized at least 12 different bands in H. pylori broth culture supernatant. When H. pylori broth culture supernatant proteins were fractionated by gel filtration chromatography with a Superose 6 HR 16/50 column (Pharmacia), multiple immunoreactive bands were identified in fraction 19, which corresponds to the void volume of the column (Fig. 1). Based on the high-molecular-mass distribution of these proteins, we speculate that these represent either (i) membranous vesicles or blebs that are released from the surface of H. pylori (12, 13), (ii) membrane fragments from lysed organisms, or (iii) aggregated protein species. Several immunoreactive bands in other fractions corresponded to high-molecular-mass oligomeric H. pylori proteins that are known to be present in broth culture supernatant (3, 5, 25). Thus, an 87-kDa band in fractions 25 to 29 represented vacuolating cytotoxin (VacA) (3, 5), 66- and 31-kDa bands in fractions 29 to 34 represented two urease subunits (2, 5), and a 56-kDa band in fractions 29 to 34 represented HspB (a GroEL heat shock protein homolog) (2, 5). These bands were recognized by anti-VacA, antiurease, and anti-HspB sera, respectively (data not shown). Finally, multiple immunoreactive bands ranging in size from about 20 to 100 kDa were identified in fractions 35 to 45. 

View larger version (37K): [in this window] [in a new window]   FIG. 1.   Analysis of antigenic proteins in broth culture supernatant from H. pylori 60190. Proteins in broth culture supernatant from H. pylori 60190 were precipitated with a 50%-saturated solution of ammonium sulfate, resuspended, passed through a 0.2-µm-cutoff filter, and then fractionated by passage over a Superose 6 HR 16/50 gel filtration column. (A) Tracing of absorbance at 280 nm. Fraction numbers are indicated underneath; each fraction represents 2 ml. (B) Immunoblot analysis of proteins in selected fractions. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12% acrylamide gel and transfer to nitrocellulose paper, the proteins were immunoblotted with rabbit antiserum to supernatant proteins (1:300 dilution of antiserum). Fraction 19 corresponds to the void volume of the column.

To characterize further the antigenic proteins found in H. pylori broth culture supernatant, a ZAPII library of chromosomal fragments from H. pylori 60190 was screened with the rabbit antiserum by methods described previously (24). A total of 17 reactive plaques were selected and purified, and the H. pylori DNA fragments were subcloned into pBluescript vectors and transformed into Escherichia coli XL1-Blue. In an immunoblot analysis of these 17 clones, six different patterns of recombinant antigen expression were identified (Fig. 2). The high rate of redundancy suggests that (i) these may be immunodominant H. pylori antigens, (ii) these genes may have been selected based on codon usage or promoter sequences that allow high levels of expression in E. coli, or (iii) these antigens may be released into the supernatant via specific and selective secretion mechanisms (25). Six representative clones were chosen (Table 1), and the nucleotide sequences corresponding to the two ends of each insert were determined by using vector-derived primers for the sequencing reactions. These sequences were then aligned with the complete genome sequence of H. pylori 26695 (23). The estimated sizes of the cloned DNA fragments from H. pylori 60190, determined by restriction mapping, correlated favorably with the sizes of the corresponding chromosomal regions from H. pylori 26695, which indicates that the chromosomal organizations of H. pylori 60190 and 26695 are similar in these regions.

View larger version (90K): [in this window] [in a new window]   FIG. 2.   Immunoblot analysis of E. coli DH5MCR expressing recombinant H. pylori antigens. E. coli containing the designated plasmids were grown in Luria-Bertani broth containing ampicillin (50 µg/ml), and the bacterial pellets were analyzed by immunoblotting with rabbit antiserum to supernatant proteins (1:1,000 dilution). Plasmid designations and the names of the expressed H. pylori antigens are as follows: pBluescript (a), pH102 (UreA and UreB) (b), pH109 (HspB) (c), pH113 (DnaK) (d), pH105 (MsrA) (e), pH103 (HcpA) (f), and pH120 (Lpp20) (g).

Sequence analysis of plasmid pH102 indicated that it contained the portion of the H. pylori urease operon extending from ureC to ureF. Similar analysis of plasmid pH109 indicated that it contained the entire H. pylori hspB gene. Thus, the production of 66- and 31-kDa antigens by plasmid pH102 and of a 56-kDa antigen by plasmid pH109 was consistent with the expression of UreB, UreA, and HspB, respectively. Plasmid pH113 encoded DnaK, a molecular chaperone belonging to the Hsp70 family of heat shock proteins. DnaK homologs are highly conserved proteins that are known to be major antigens of several pathogenic bacterial species (22).

For the remaining three plasmids, the open reading frames that encoded the antigens of interest were isolated by a combination of restriction endonuclease digestions and PCR amplifications, and the complete nucleotide sequences of these three open reading frames were determined for both strands. The antigen encoded by plasmid pH120 was a previously characterized H. pylori lipoprotein (Lpp20) (13). The antigen encoded by plasmid pH105 had a predicted molecular mass of 41.3 kDa and was a homolog of peptide methionine sulfoxide reductases (MsrA proteins) from Streptococcus pneumoniae (26), Neisseria gonorrhoeae (where the reductase is also designated PilB, a fimbrial transcription repressor) (21, 26), and Haemophilus influenzae (6). Peptide methionine sulfoxide reductases have been recently shown to play a role in the adherence of several mucosal pathogens to host tissue (26).

Plasmid pH103 encoded a novel H. pylori antigen that we designate HcpA (H. pylori cysteine-rich protein A). The predicted hcpA product is 27.3 kDa in size and contains 250 amino acids, of which 14 (5.6%) are cysteines. Analysis of the genome sequence from H. pylori 26695 (23) indicates that hcpA belongs to a family of seven paralogous genes that are scattered throughout the genome (Fig. 3). A database search failed to identify any closely related proteins in other bacterial species, which suggests that this family of genes may be found exclusively in the genus Helicobacter.

View larger version (63K): [in this window] [in a new window]   FIG. 3.   Alignment of the deduced HcpA amino acid sequence from H. pylori 60190 with deduced amino acid sequences of seven members of a paralogous gene family from H. pylori 26695 (23). Amino acid residues that are conserved in all eight sequences are indicated by asterisks, and conserved cysteine residues are indicated by plus signs.

Several lines of evidence suggest that there is a relatively high level of genetic diversity among H. pylori strains (1, 7, 8, 10, 11). To investigate this phenomenon, we compared the nucleotide sequences of msrA, lpp20, and hcpA from H. pylori 60190 with the corresponding sequences from H. pylori 26695 (23). The levels of nucleotide identity for these sequences were 95.4, 96.0, and 97.3%, respectively, which are consistent with results that have been reported for several other H. pylori genes (7, 11).

To investigate potential mechanisms whereby these six antigens might enter the extracellular space, each of the sequences was analyzed with the SignalP World Wide Web program (18). MsrA and HcpA are predicted to contain N-terminal signal sequences recognized by the Sec family of proteins, and Lpp20 contains a classical lipoprotein signal sequence (13). In contrast, DnaK, HspB, UreA, and UreB are predicted to lack N-terminal signal sequences. Moreover, urease and HspB are large oligomeric structures (2, 5, 17) that are typically found exclusively in the cytoplasm of bacteria and that would not be expected to cross the bacterial outer membrane.

Autolysis is one mechanism that could account for the appearance of a wide assortment of bacterial proteins in H. pylori broth culture supernatant (19). Alternatively, a subset of proteins could be released into the extracellular space either via specific and selective secretion mechanisms (25) or in the form of membranous vesicles (12, 13). Lpp20 has been reported to be present in vesicles from H. pylori (13), and in agreement with that observation, the present study demonstrated that an immunodominant 20-kDa antigen was found in the very high-molecular-mass void volume fraction (Fig. 1).

The extracellular release of proteins could potentially be a phenomenon that occurs only during growth of H. pylori in vitro. However, the presence of urease in the lamina propria of H. pylori-infected humans (15) suggests that similar release also occurs in vivo. The entry of soluble H. pylori proteins into the gastric mucosa may have important functional consequences, including a potential role in inciting a gastric mucosal inflammatory response (16). In addition, the extracellular release of H. pylori proteins may represent a bacterial strategy for diverting an effective local immune response.

Nucleotide sequence accession numbers. The nucleotide sequences of lpp20, msrA, and hcpA from H. pylori 60190 have been submitted to the GenBank/EMBL Data Bank and have been assigned accession numbers AF053710, AF053709, and AF053708, respectively.