Characterization and Immunogenicity of the CagF Protein of the cag Pathogenicity Island of Helicobacter pylori

Purification and antibody production of CagF.

CagF was overproduced and purified as a glutathione S-transferase (GST) recombinant protein. The cagF gene was amplified by PCR from isolated H. pylori G27 DNA (3), by using the primers 5′-ACGCGTCGACAAACAAAATTTGCGTGAACAAAAAT-3′ (forward, SalI site underlined) and 5′-AGAATGCGGCCGCTCAATCGTTATTTTTGTTTTGATT-3′ (reverse, NotI site underlined), and was cloned into the vector pGEX-4T-3. The subsequent expression and purification of the protein were carried out essentially following the manufacturer's recommendations (Pharmacia Biotech). Briefly, Escherichia coli XL1-blue, carrying the GST-CagF-encoding plasmid, was induced with 1 mM isopropyl-β-d-thiogalactopyranoside for 5 h at 30°C. The bacteria were harvested by centrifugation, resuspended, and lysed by two passages through a French press at high pressure (1.038 × 10⁸ Pa). The lysate was centrifuged to remove cell debris and was incubated for 1 h with glutathione-Sepharose. The resin was washed extensively, and CagF was eluted by digestion with thrombin for 1 h at room temperature. The protein thus purified yielded a single band with an apparent molecular mass of 31 kDa (Fig. 1A), which is in good agreement with its calculated molecular mass (30,279 Da). The circular dichroic spectrum of purified recombinant CagF indicates that its secondary structure contains about 50% α-helix and about 25% of β structure (data not shown).

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FIG. 1.

Purification and membrane localization of CagF. (A) A sample of recombinant CagF after purification was subjected to SDS-PAGE (10% acrylamide) and stained with Coomassie blue. (B) Membranes prepared from H. pylori G27 (lane M) contain the majority of CagF when compared to the content of the supernatant representing the cyto- and periplasm (lane SN). The specific solubilization of CagF when membranes were treated with different detergents indicates an outer membrane localization (remaining lanes). M, membranes; SP, soluble proteins; Sarc., β-lauryl-sarcosyl; Tween, Tween 20; and Triton, Triton X-100. (C) Whole H. pylori G27 was treated with various amounts of trypsin (0, 0.05, 0.1, 0.25, 0.5, and 1 μg/μl, respectively). Proteins were separated by SDS-PAGE and CagF, and its fragment was detected with CagF-specific polyclonal antibodies. Δ, H. pylori G27 Δcag PAI.

Polyclonal rabbit antibodies were raised against the purified recombinant CagF protein according to standard methods (8) and showed a strong specific signal in Western blots.

Expression and membrane localization of CagF in H. pylori.

The subcellular localization of CagF was determined by differential solubilization of membranes with specific detergents. H. pylori membranes were prepared from 3-day-old cultures lysed by two passages through a French press. Lysates were subjected to a low-speed centrifugation to eliminate cell debris, and membranes were collected by ultracentrifugation for 1 h at 120,000 × g. The proteins were solubilized from the resuspended membranes upon incubation on ice with one of the following detergents to a final concentration of 2% (wt/vol): Tween 20, Triton X-100, and β-lauryl-sarcosyl; the first two mainly solubilize the cytoplasmic membrane proteins, and the third preferentially solubilizes outer membrane proteins (6). Samples were centrifuged, and the soluble (supernatant) and insoluble (pellet) fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting. CagF was found almost entirely in the pelleted membrane fraction (Fig. 1B, lane 1) and could best be solubilized by β-lauryl-sarcosyl (Fig. 1B, lanes 3 and 4), which indicates an outer membrane localization. Membrane treatment with 6 M urea revealed only a weak detachment of CagF, indicating that this localization was not the result of an unspecific membrane aggregation. Here we found that CagF is always expressed in H. pylori under standard culture conditions, i.e., without infection of host cells. In addition to this, epithelial AGS cells infected with bacteria do not induce an overexpression of CagF, even when close contact between bacteria and cells is established (data not shown).

To further assess the location of CagF in H. pylori, we tested if the protein is accessible to proteolysis by trypsin added to intact bacteria. H. pylori was grown for a day, harvested by centrifugation, washed, and resuspended in 0.1 volume of 25 mM Tris, pH 7.9, and 1 mM EDTA. Aliquots were incubated for 30 min on ice with increasing amounts of trypsin (final concentration, 0 to 1 μg/μl). Protease inhibitors were added to stop the reaction, and samples were examined by SDS-PAGE immunoblotting. The trypsin treatment of whole bacteria showed that CagF was accessible to proteolysis and that a 14-kDa fragment resisted degradation (Fig. 1C) while the remaining part of the 31-kDa protein was presumably digested into small peptides, indicating that a major part of the CagF protein is exposed on the bacterial surface.

CagF in different H. pylori strains.

To assess the expression of CagF in different strains, we cultivated different H. pylori strains (strains G27, G39, CCUG 17874, and 342 were provided as cag PAI-containing strains by A. Covacci, Chiron, Siena, Italy, and strains RHP901a, SS1, MI355, 1811a, 5060d, 2a, 326, 593, 346, 503, 506, 512, 561, 685, 798, and 872 were provided by W. Dundon [University of Padua]). After cultivation, the bacterial raw extracts were subjected to SDS-PAGE, immunoblotted with anti-CagF-specific rabbit polyclonal antibodies, and developed with enhanced chemiluminescence (ECL; Amersham). Our results show that CagF is expressed in all the strains tested and exhibits a limited range of variation in electrophoretic mobility (Fig. 2). These differences could result from polymorphisms such as short sequence repetitions, deletions, or sequence variations, as has been demonstrated for several H. pylori proteins, including CagA (5). Indeed, gene sequencing of the strains analyzed so far has revealed both silent and amino acid changes.

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FIG. 2.

Expression of CagF in different H. pylori strains. The H. pylori strains 342, 346, MI355, 980, 872, 798, 685, 561, 512, 506, 503, 5060d, 1811a, SS1, 593, 326, 2a, RHP901a, CCUG 17874, G39, and G27, containing the cag PAI, and strain G27 not containing the cag PAI (G27Δcag) were separated by SDS-10% PAGE, and each lane was loaded with the same amount of bacterial raw extract. The subsequent immunoblot was developed with a polyclonal CagF-specific antibody.

Immunogenicity of CagF in infected humans.

To test the immunogenicity of CagF in chronically infected human patients, we selected 30 serum samples positive for antibodies against H. pylori. The samples were initially selected based on urea-breath positivity, and further analysis with enzyme-linked immunosorbent assay (ELISA) gave high titers using whole-cell bacterial extracts, confirming the infection to be H. pylori. Twenty of these sera were also positive for immunoglobulin G antibodies against CagA. All serum were tested with the ELISA by using the recombinant, purified CagF protein as the solid phase following a procedure similar to that used for CagA (13). As shown in Table 1, 15 of the 30 sera (50%) were positive for immunoglobulin G antibodies against CagF. It is noteworthy that half of the samples positive for anti-CagA antibodies reacted with CagF and that half of the samples negative for anti-CagA reacted with CagF. These data show that CagF is immunogenic in individuals chronically infected with H. pylori and that immune responses to proteins of the Cag PAI can be induced by the infection, even in the absence of detectable responses to CagA. These data strongly suggest that CagF in particular, and Cag PAI proteins in general, may play an important role in a better dissection of the immune response to this microorganism and that individuals infected with Cag-positive strains may develop immune responses to Cag proteins other than CagA.

In summary, our results suggest that CagF, despite controversial results concerning its requirement in interleukin-8 and/or CagA secretion (7, 16), may play a role as an outer membrane protein in the assembly of the secretion machinery and that CagF may be exploitable for diagnostic tests on the basis of its immunogenicity.

Nucleotide sequence accession number.

The following sequences have been deposited: MI355, 1811a, 326, 5060d, 593, 2a, G27, G39, RHP901a, and SS1 (GenBank accession numbers AY136637 to AY136646 ).