JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2002, p. 547–552 0095-1137/02/$04.00⫹0 DOI: 10.1128/JCM.40.2.547–552.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 40, No. 2
Novel Antigens of Helicobacter pylori Correspond to Ulcer-Related Antibody Pattern of Sera from Infected Patients Christo Atanassov,1* Leon Pezennec,1 Jacques d’Alayer,2 Ghislaine Grollier,1 Bertrand Picard,3 and Jean-Louis Fauche`re1 Department of Microbiology A, IFR 59, University Hospital Center, 86021 Poitiers,1 Laboratory of Protein Microsequencing, Institut Pasteur, 75724 Paris,2 and Laboratory of Microbiology, Faculty of Medicine, 29200 Brest,3 France Received 15 June 2001/Returned for modification 24 September 2001/Accepted 18 November 2001
Recently, we reported that the patterns of antibodies to Helicobacter pylori protein antigens in serum may be useful for screening patients at high risk for ulcers (P. Aucher et al., J. Clin. Microbiol. 36:931–936, 1998). Here we report the identification, by a combination of electrophoretic, immunochemical, and protein sequencing methods, of five antigens that correspond to this antibody pattern: groEL, catalase A, flagellin A, beta-ketoacylacyl carrier protein synthase I (-ketoacyl-ACP S), and peptidyl prolyl cis-trans isomerase (PPiase). -Ketoacyl-ACP S and PPiase are reported for the first time as antigens of diagnostic interest in infections by H. pylori. The antigenicity of the five antigens, together with those of CagA and VacA, was tested in an immunoblot assay with water-soluble protein extracts from two H. pylori pathogenic strains (HP 141 and ATCC 43579) and panels of sera from H. pylori-positive patients with gastroduodenal ulcers (GDU), nonulcer dyspepsia (NUD), as well as sera from H. pylori-negative healthy volunteers. For catalase A, groEL, and flagellin A antigens, no overall statistically important values were found making it possible to discriminate between patients with GDU and NUD. For both H. pylori strains, the mean performance indices (MPI) presenting percentages of correctly classified patients with GDU and NUD showed that the most significant antibody patterns were as follows: anti-VacA ⴙ anti--ketoacyl-ACP S (MPI ⴝ 76.1), anti-VacA ⴙ anti-PPiase (MPI ⴝ 71.8), and anti-CagA ⴙ anti-VacA ⴙ anti--ketoacyl-ACP S (MPI ⴝ 70.5). Antibody patterns detected with these antigen profiles may therefore be useful in developing a diagnostic test designed to predict the clinical severity of the H. pylori infection within the adult population of France. Serological tests and assays for the diagnosis of Helicobacter pylori infection are included among the noninvasive methods recommended by the European Helicobacter pylori Study Group (7). Evaluation of the humoral immune response to H. pylori antigens by immunoblotting is a valuable alternative and complement to the more routinely used enzyme-linked immunosorbent assay (ELISA) tests (8, 13, 14, 19, 25). Immunoblotting appears to be sometimes more sensitive and useful for detecting low-abundance antibodies and antibodies directed toward nonconformational epitopes of immunodominant antigens. By this method, we recently reported that several antigens of H. pylori are preferentially recognized by the serum antibodies of adult French patients with gastroduodenal ulcers (GDU) that had been infected by this gastric pathogen. We therefore postulated that the five antigens corresponding to this antibody pattern could be useful for differentiating patients at high risk of GDU from patients with nonulcer dyspepsias (NUD) (2). Under the conditions of an immunoblot assay, two proteins of this antigenic profile were identified as CagA (125 kDa) and VacA (87 kDa), while the three other proteins were assigned only as immunoreactive bands with approximate molecular masses of 54, 42, and 35 kDa (denoted, respectively, p54, p42, and p35). Knowledge of the exact primary structure of p54, p42, and p35 is thus an essential prerequisite for preparing and further analyzing the antigenic
preparation designed for the development of a test predicting the clinical outcome of the H. pylori infection. Our initial aim was to purify the three unidentified antigens in order to determine their amino acid sequences. After optimizing the electrophoretic separation of these antigens, we differentiated three antigens within the zone of p54. This prompted us to reinvestigate all members of antigenic profile by an immunoblot assay by using panels of sera from H. pyloripositive patients with GDU and NUD, as well as with the control sera of H. pylori-negative healthy volunteers.
MATERIALS AND METHODS Patients and patient sera. A total of 115 individual sera were collected in the university hospitals of Poitiers (southwest), Nancy (east), and Brest (northwest). These cities represent three geographical locations in France situated at a distance of more than 700 km from one another. Eighty-five sera were obtained from patients attending the departments of hepato-gastroenterology from 1995 to 2000. All patients underwent upper gastroduodenal endoscopy with multiple antral and fundic biopsies that were processed for histology. These subjects had received neither antimicrobial nor antiacid therapies during the previous 3 months. As determined by in-house ELISA (29), all patients were seropositive for H. pylori. Of the 85 patients, 55 (43 males and 12 females) had GDU, and 30 (17 males and 13 females) showed signs of gastritis or a dyspeptic symptoms but were ulcer-free (NUD). For the GDU patient group, the median ages were 60.8 years (range, 26 to 89 years) and 69.5 years (range, 51 to 93 years) for males and females, respectively; the mean ELISA index was 0.89. (The threshold ELISA index for a positive result was 0.3.) For of NUD patient group, the median ages were 45.4 years (range, 24 to 77 years) and 64 years (range, 27 to 86 years) for males and females, respectively; the mean ELISA index was 0.63. Thirty control sera were collected from healthy blood donors (16 males and 14 females). The median ages were 40.8 years (range, 28 to 51 years) and 35.8 years (range, 21 to 46 years) for males and females, respectively. All persons within this control
* Corresponding author. Mailing address: Laboratoire de Microbiologie A, Centre Hospitalier Universitaire–La Miletrie, BP 577, 86021 Poitiers, France. Phone: 33-5-49-44-43-53. Fax: 33-5-49-44-38-88. Email:
[email protected]. 547
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J. CLIN. MICROBIOL. TABLE 1. Characteristics of identified protein antigens of H. pylori HP 141
Antigen
Catalase A subunit groEL Flagellin A subunit -Ketoacyl-ACP S PPiase
Classification (J99/26695)
JHP JHP JHP JHP JHP
809/HP 008/HP 548/HP 505/HP 161/HP
875 010 601 558 175
Pairwise protein identity (%)
Mol mass (kDa) (J99/ 26695)
Protein length (aa)a (J99/26695)
Predicted pI (J99/26695)
Sequenced fragment (strain HP 141)b
Identity of HP 141 sequences with J99/26695 (%)
N-terminal position on J99/26695
97.4 99.6 100 97.3 97
58.63/58.53 58.26/58.24 53.28/53.28 43.60/43.56 34.03/34.04
505/505 546/546 510/510 412/412 299/299
9.04/8.89 5.48/5.46 6.32/6.34 6.63/6.52 9.96/10.04
MVNKDV AKEIKFS AFQVNTN VEVGYVN KPAHNANNATHNTKK
100/100 100/100 100/100 100/87c 100/87c
1–6 2–8 2–8 296–302 22–36d
a
aa, amino acids. Sequenced fragments of catalase A subunit, groEL, flagellin A subunit, and PPiase were obtained by N-terminal Edman degradation; the fragment of -ketoacylACP S was obtained by internal microsequencing of major HPLC peak 25 (see Materials and Methods). c 100% homology with respective sequences VEIGYVN of TIGR HP 558 (beta-ketoacyl-ACP synthase II), and KPAHNANNSTHNTKE of TIGR HP 175 (cell binding factor 2). Nonidentical amino acids are underlined. d Corresponds to the second (by importance) most likely cleavage site between positions 21 and 22 (LMA-KP) producing N-terminal signal peptide. Determined by using SignalP predictions based on neural networks and hidden Markov models of gram-negative bacteria (Nielsen et al. [18]; SignalIP server [http://www.cbs.dtu.dk/ services/SignalP-2.0/]). b
group were seronegative for H. pylori; the mean ELISA index was 0.023. Handling, storage, and processing of these sera were as described previously (2). H. pylori strains and extracts. Two H. pylori strains were used: ATCC 43579 and HP 141. Both strains express CagA and VacA in an immunogenic form. H. pylori ATCC 43579 was used in our previous study (2). H. pylori HP 141 (CagA⫹ VacA⫹ s1m1) was isolated and characterized at the University Hospital of Poitiers in 1997 from a 22-year-old woman suffering from severe gastritis. Watersoluble proteins of the saline extract from both H. pylori strains were obtained according to a previously described protocol of extraction (2). Electrophoresis, immunoblot assay, and protein microsequencing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 100-g portions of water-soluble proteins was carried out on 4% stacking–10% resolving gels and 1-mm-thick polyacrylamide gels, with a 16-cm net distance of migration within the resolving gel, by using a Maxi-Gel apparatus (Bio-Rad) under a constant current of 12 mA for 16 h. The electrophoresis buffer in the upper chamber was Tris-glycine (pH 8.3) containing 0.1 mM thioglycolate. After the end of electrophoresis, slab gels were briefly rinsed in Towbin buffer containing 0.1 mM thioglycolate and then subjected to Western transfer onto polyvinylidene difluoride (PVDF) Sequi-Blot membranes (Bio-Rad) at a constant current of 1.56 mA/cm2 of membrane for 1 h. For controlling transfer quality, some PVDF strips from each Western blot were processed with different stains (i.e., Amidoblack 10B or CBB); the remaining PVDF strips were used in an immunoblot assay as described previously (2). Strips developed with antibodies were mounted together with the part of the PVDF membrane that had been stained either with Amidoblack 10B or CBB. This allowed the exact localization of immunoreactive proteins of interest to be N-terminally sequenced. For this purpose, stained PVDF bands that corresponded by localization to the respective band on the immunoblot were excised and subjected to Edman degradation on an Applied Biosystems 473A or 494 sequencer. Sequencing was terminated when 100% identity between the sequenced fragment and the respective H. pylori protein belonging to one or both strains with known complete genome (i.e., ATCC 26695 and J99) was achieved and at a probability score that would exclude the possibility that other proteins might belong to this bacterial species. In order to determine the primary structure of N-terminally blocked proteins, a hydrosoluble extract of H. pylori HP 141 was subjected to preparative SDS-PAGE on a 1-mm-thick slab gel and resolved proteins were stained with 0.25% CBB in 40% methanol–10% acetic acid. A control strip of unstained gel was excised, electroblotted onto a PVDF membrane, under the above-mentioned conditions, and further processed with sera that allowed us to immunolocalize bands of interest. At the end of Western transfer, the exact height of the gel strip was measured and used to adjust to the height of the scanned CBB-stained gel. The PVDF immunoblot strips were then mounted together with the scan of the gel. The CBB-stained bands in the PPA that corresponded to immunolocalized antigens were excised and subjected to internal microsequencing. They were digested twice for 18 h at 35°C with 0.8 g of endolysine C in 500 l of 50 mM Tris-HCl (pH 8.6) containing 0.01% Tween 20. The resulting peptides were fractionated on a high-pressure liquid chromatography (HPLC) DEAE-C18 columns, 1 mm in diameter, at an elution gradient of 2 to 70% of acetonitrile in 0.1% trifluoroacetic acid. Major HPLC peaks were further subjected to Edman degradation. The resulting N-terminal or internal sequences were compared to the existing protein sequence database for H. pylori (Astra; The Institute for
Genome Research [TIGR]) and to the National Institutes of Health database (BLAST). Data analysis of antibody patterns. For factorial analysis of correspondence (FAC), data were summarized as “height 2” tables corresponding to the antibody patterns obtained with antigens from HP 141 and from ATCC 43579 H. pylori strains. Each table contained one row for all individual 115 tested sera and a number of columns corresponding to the number of antibodies tested in each combination. In each column, the presence of the corresponding antibody was coded in a binary manner (“2” for presence and “1” for absence of antibody). FAC was then systematically applied on all tables through by using SPAD.N software (CISA Cerestia, Saint-Mandé, France [15]) The chi-square test, together with the Yates correction coefficient when required, was used for comparisons of frequencies of antibody patterns between the different groups of sera. To assess the ability of the antibody patterns to predict for GDU, the performance index (i.e., the ratio of the number of truepositive results plus the number of true-negative results to the number of sera tested) was used.
RESULTS Identification of protein antigens. In an initial experiment, the electrophoretically separated proteins of both strains HP 141 and ATCC 43579 displayed very similar band profiles within the zones of interest encompassing silver-stained p54, p42, and p35. However, the protein bands of HP 141 extract within the zone of p54 were better differentiated than those of ATCC 43579 (data not shown). Strain HP 141 was therefore used as the sole source for further protein purification and sequence analysis. We next optimized the electrophoretic separations of proteins in terms of migration time and protein concentations to be loaded, conditions of Western transfer, and immunolocalization of protein bands to be sequenced. Four proteins were successfully sequenced by N-terminal Edman degradation: three proteins within the zone of p54 and one protein within the zone of p35. The antigenic protein within the zone of p42 turned out to be blocked at its N terminus, and its sequence was determined by internal microsequencing. As presented in Table 1, comparisons of determined sequences of HP 141 strain with those of both H. pylori strains with known complete genomes (26695 and J99) made it possible to identify five antigenic proteins, namely, catalase A subunit (catalase A), groEL (other common or recent denominations of this protein are HspB and HSP60), flagellin A subunit (flagellin A), beta-ketoacyl-acyl carrier protein syn-
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TABLE 2. Absolute and relative (%) numbers of immunoreactive bandsa and the performance indexb as detected by immunoblot assay in the sera of patients with GDU and NUD and of clinically healthy controls No. of bands (%) Antigen
CagA VacA Catalase A subunit groEL Flagellin A subunit -Ketoacyl-ACP S PPiase
Patients with GDU (n ⫽ 55)
Patients with NUD (n ⫽ 30)
Performance index (%) (n ⫽ 115)
Healthy volunteers (n ⫽ 30)
HP 141
ATCC 43579
HP 141
ATCC 43579
HP 141
ATCC 43579
HP141
ATCC 43579
Meanc
41 (75) 49 (89) 51 (93) 51 (93) 54 (98) 50 (91) 33 (60)
40 (73) 45 (82) 54 (98) 46 (84) 48 (87) 33 (60) 30 (55)
19 (63) 16 (53) 20 (67) 23 (77) 30 (100) 18 (60) 8 (27)
20 (67) 20 (67) 28 (93) 28 (93) 28 (93) 7 (23) 7 (23)
12 (40) 1 (3) 22 (73) 20 (67) 29 (97) 1 (3) 0 (0)
10 (33) 0 (0) 21 (70) 22 (73) 23 (77) 1 (3) 0 (0)
60.9 80.0 60.0 59.1 47.8 79.1 82.6
60.9 73.9 56.5 48.7 49.6 73.9 72.2
60.9 76.9 58.2 53.9 48.7 76.5 77.4
a Antibody specificities more frequent in patients with GDU than in patients with NUD concern antigens VacA and catalase A of strain HP 141 and -ketoacyl-ACP S and PPiase of both H. pylori strains (as determined by chi-square test; P ⱕ 0.05). b That is, the percentage of patients with ulcers that are correctly classified (i.e., The number of true positive plus the number of negative immunoblot controls/the total number), where the total number is 115. c MPI for both H. pylori strains.
thase I (-ketoacyl-ACP S [EC 1.11.1.6]), and peptidyl prolyl cis-trans isomerase (PPiase [EC 5.2.1.8]). Antibody frequencies to single antigens. After electrophoretically differentiating catalase A, groEL, and flagellin A subunit within the zone of p54 from strain HP 141 and then performing corresponding immunoblots with 15 positive sera (mean ELISA of index 1.1) from patients with GDU, we found that all three bands remained immunoreactive. This result was in contrast to our previous study with ATCC 43579 extract separated on a gradient PAA gel, where we observed only one immunoreactive p54 band that was supposed to be HspB (2). For this reason, we reinvestigated the antigenicity of the proteins to be sequenced by immunoblotting with extracts of both H. pylori HP 141 and H. pylori ATCC 43579, with three panels of sera from H. pylori-positive patients with GDU and NUD, and with control sera from H. pylori-negative healthy volunteers. Table 2 shows that the frequencies of positive sera reacting with protein antigens from both strains HP 141 and ATCC 43579 are quite similar with respect to antigens CagA, VacA, groEL, flagellin A, and PPiase, and this concerns both groups of patients with GDU and NUD. Within the group of patients with NUD, the percentage of positive sera reacting with catalase A and -ketoacyl-ACP S varies ca. 30% with respect to the used H. pylori strain. Regarding catalase A, groEL, and flagellin A antigens, there are no overall statistically important values that make it possible to discriminate between the three groups of patients, i.e., those with GDU or NUD and the controls. However, performance indices presenting percentages of correctly classified patients with GDU and NUD do confirm the closely comparative values of CagA, flagellin A, catalase A, -ketoacyl-ACP S, and VacA for both H. pylori strains. In this regard, the antigens groEL and PPiase were more immunologically detectable when they originated from HP 141. The highest mean performance indices (MPI), which represent the mean of the performance indices obtained with strains HP 141 and ATCC 43579, are those of PPiase (MPI ⫽ 77.4), VacA (MPI ⫽ 76.9), and -ketoacyl-ACP S (MPI ⫽ 76.5). Antibody patterns. Another interpretation of our results takes into consideration antibody patterns that provide statis-
tical differences between the groups of patients with GDU or NUD and the control subjects. The antibody pattern of a given serum has been defined as the combination of antibodies present in this serum and immunoreacting with two or more of the seven antigens targeted. We looked for antibody patterns characterizing particular clinical categories of patients. Because the number of expected antibody patterns is relatively high, we used FAC, a one-step computed analysis, allowing us to gather sera according to their antibody patterns and clinical categories. Figure 1 shows the results of the FAC analysis for the antibody pattern defined by the presence of antibodies to CagA, VacA, and -ketoacyl-ACP S with the HP 141 strain. In this case, the plane F1/F2, which accounted for 89.1% of the total variance, clearly discriminated the sera from healthy volonteers from the GDU sera. These two categories of sera are graphically concentrated on both sides of a virtual diagonal line drawn on Fig. 1. The sera from the patients with NUD appeared intersperced in the factorial analysis plane. This was also valid for five other antibody patterns selected (data not shown). The application of another analysis (i.e., chi-square test and calculation of the performance indices) on the six selected antibody patterns made it possible to quantitatively evaluate their discriminating potential with respect to the patients with GDU and NUD (Table 3).) By using the antigens of both strains, the most discriminant patterns were those that included antibodies to VacA, -ketoacyl-ACP-S, and PPiase, as follows: anti-VacA plus anti--ketoacyl-ACP-S (MPI ⫽ 76.1%) and anti-VacA plus anti-PPiase (MPI ⫽ 71.8%). DISCUSSION The antigenic material used in our study was obtained by a method that is considered to be optimized for the extraction of water-soluble proteins from the outer membrane of viable bacteria. However, only one identified antigen (i.e., flagellin A) meets the criteria for such proteins. At least three proteins (catalase A, groEL, and -ketoacyl-ACP S) are cytoplasmic and theoretically should not be present in the extract. This apparent methodological discrepancy may be explained in view of the existing data on membrane expression of groEL and urease by a specific secretory system (28) or by genetically
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FIG. 1. Factorial analysis of the correspondence of the antibody pattern defined by the presence of antibodies to CagA, VacA, and -ketoacylACP S by using H. pylori HP 141. The different areas of the circles are proportional to the number of sera of each clinical category: clear areas show the projection of 30 seronegative healthy volunteers, open areas with black dots show the projection of 30 patients with NUD, and dark areas with white dots show the projection of 55 patients with GDU. With the exception of one case, all of the GDU patients are situated on the right side of the diagonal line drawn, and they are mainly gathered in a single group. The healthy volunteers are gathered on the left. The NUD are interspersed over the factorial analysis plane.
programmed bacterial autolysis that occurs in vitro (22), as well in vivo (6). The latter means of membrane expression has also been suggested for catalase (22), and there is evidence that H. pylori catalase may be located both throughout the cytosol and in the periplasmic space (9). In this regard, it is not excluded that, due to unique characteristics of the outer membrane of H. pylori, other cytoplasmic proteins may become absorbed. It is also possible that H. pylori possesses an as-yetunknown mechanism for exporting to the cell surface enzymes
and other molecules that are important for colonizing the stomach. As far as we know, -ketoacyl-ACP S and PPiase are reported here for the first time as H. pylori antigens of clinical relevance. Because of the lack of information about -ketoacyl-ACP S and PPiase in H. pylori, we briefly mention here some aspects of the homologs to these two molecules in other bacterial species. In our opinion, these findings give an idea about the biological importance of these molecules and as
TABLE 3. Frequencies (absolute number and percentage) of seven antibody patternsa to H. pylori in 115 human sera and their abilities to predict GDU and differentiate it from NUD, as measured by the performance indexb No. of patterns (%) Antibody pattern to antigen combination (i.e., “profile”)
CagA ⫹ VacA ⫹ PPiase CagA ⫹ VacA ⫹ -ketoacyl-ACP S VacA ⫹ PPiase VacA ⫹ -ketoacyl-ACP S ⫹ PPiase VacA ⫹ -ketoacyl-ACP S -Ketoacyl-ACP S ⫹ PPiase
Patients with GDU (n ⫽ 55)
Patients with NUD (n ⫽ 30)
Healthy volunteers (n ⫽ 30)
Performance index (%) (n ⫽ 115)
HP 141
ATCC 43579
HP 141
ATCC 43579
HP 141
ATCC 43579
HP 141
ATCC 43579
Meanc
27 (49.0) 37 (67.3) 30 (54.5) 27 (49.0) 45 (81.8) 28 (51.0)
21 (38.2) 21 (38.2) 24 (43.6) 14 (25.4) 28 (50.9) 18 (32.7)
4 (13.3) 10 (33.3) 4 (13.3) 2 (6.7) 11 (36.6) 4 (13.3)
4 (13.3) 6 (20.0) 5 (16.7) 4 (13.3) 7 (23.3) 2 (6.6)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
72.2 75.7 74.8 73.9 81.7 73.0
67.0 65.2 68.7 60.9 70.4 66.9
69.6 70.5 71.8 67.4 76.1 70.0
a The antibody patterns for all antigen profiles were significantly more frequent in patients with GDU than in patients with NUD, except for the antigen combinations CagA ⫹ VacA ⫹ -ketoacyl-ACP S and VacA ⫹ -ketoacyl-ACPS ⫹ PPiase of strain ATCC 43579 (as determined by chi-square test; P ⱕ 0.05). b That is, the percentage of patients with ulcers that are correctly classified (i.e., the number of true positive ⫹ the number of negative immunoblot controls/the total number), where the total number is 115. c MPI for both H. pylori strains.
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potential candidates in developing drug, vaccine, and diagnostic assays. -Ketoacyl-ACP S of H. pylori (gene fabB) belongs to the family of -ketoacyl synthases, key enzymes involved in the biosynthesis of fatty acids and polyketides. At present, -ketoacyl-ACP S from E. coli is being studied structurally as a promising drug target for novel antibiotics to treat tuberculosis, as well as for inhibiting certain cancers (16, 23). Antigenic PPiase from H. pylori HP 141 is evolutionarily related to the cyclophilins, FKBPs (proteins binding to the immunosuppressive drug FK506) and, more particularly, to parvulin (gene ppiC). Recent studies have shown that E. coli PPiase functions independently as an isomerase and as a chaperonin (3, 24). Data dealing with antibodies to five identified antigens of H. pylori are interpreted in terms of the potential value these antigens may have as key compounds of an immunoblot assay that would allow differentiation of GDU disease from NUD. Differentiation of GDU from NUD seems to depend on factors such as age and population. Some reports provide evidence for the usefulness of the immunoblotting diagnosis of GDU in Brazilian children (25). Other authors claim that there is no association of specific H. pylori antigens with antibodies in adult patients suffering from various gastroduodenal pathologies in Germany (14) and Chile (20). Apparently, these contradictory findings reflect both the phenotypic heterogeneity of H. pylori and the specificity of the host immune response within various populations. However, a large-scale comparative immunoblotting study with sera from patients living in countries as geographically distant as the United States, China, and Peru has shown that, despite their diverse origins, all H. pylori strains show several major conserved bands in the range of 25 to 84 kDa (11). Other studies carried out in India and France also give evidence for relatively restricted, although differing, ranges of immunoreactive bands (2, 4). Therefore, it is possible that a pattern of immunoreactive bands may characterize, at least to some extent, the immune response to H. pylori within a given population, and this may be true irrespective of the H. pylori strain. In this latter regard, some 350 genes appear to be specific for H. pylori, but only 6 to 7% of them are considered to be strain specific (1, 27). This implies that some of the remaining 93 to 94% of these genes encode specific H. pylori antigens and/or epitopes that are common to all strains of this microorganism. The fact that we found similar antigen profiles in two H. pylori strains that were recognized by serum antibodies in patients from different regions of France supports this concept, although a prospective study with a greater number of H. pylori strains is needed to confirm our assumption. The results presented in Tables 2 and 3 provide additional support that proteins such as catalase, groEL, and flagellin are strongly immunogenic but give nonspecific cross-reactions with antibodies to antigens from other gram-negative bacteria and, more particularly, Campylobacter jejuni (10, 12, 17, 21, 26). Catalase, groEL, and flagellin will therefore be of poor utility for the envisaged predictive test. Metabolic enzymes, such as identified -ketoacyl-ACP S and PPiase, are considered to be promising candidates for a serological test kit because they have few cross-reactions with NUD sera and are more conserved than virulence factors such as CagA and VacA (14). Reported antibody frequencies to VacA, -ketoacyl-ACP S, and PPiase make it possible to discriminate between the groups of NUD patients and ulcer patients, but it is rather
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unlikely that a single antigen could be sufficient as a highly reliable predictive tool. Neither the frequencies of positive sera for CagA and/or VacA nor the respective performance indices are sufficient as unique predictive markers for ulcer disease. This is due to the well-known fact that there exist H. pylori strains which are phenotypically negative for one or both antigens (5). In this regard, patterns including antibodies to -ketoacyl-ACP S and PPiase could be promising for the prediction of GDU because their performance indices are close to 80%. In conclusion, we identified the primary structure of five H. pylori protein antigens, and two of them–-ketoacyl-ACP S and PPiase–are proposed for the first time as antigens of diagnostic interest. Antibody patterns detected by using these antigens may be useful in developing a diagnostic test designed to predict the clinical evolution of the H. pylori infection toward an ulcer disease within the adult population of France. ACKNOWLEDGMENTS This work was supported by the Hospital and University of Poitiers, Poitiers, France. REFERENCES 1. Alm, R. A., L. S. Ling, D. T. Moir, B. L. King, E. D. Brown, P. C. Doig, D. R. Smith, B. Noonan, B. C. Guild, B.L. deJonge, G. Carmel, P. J. Tummino, A. Caruso, M. Uria-Nickelsen, D. M. Mills, C. Ives, R. Gibson, D. Merberg, S. D. Mills, Q. Jiang, D. E. Taylor, G. F. Vovis, and T. J. Trust. 1999. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176–180. 2. Aucher, P., M. L. Petit, P. R. Mannant, L. Pezennec, P. Babin, and J. L. Fauchère. 1998. Use of an immunoblot assay to define serum antibody patterns associated with Helicobacter pylori infection and with H. pylorirelated ulcers. J. Clin. Microbiol. 36:931–936. 3. Bothmann, H., and A. Plückthun. 2000. The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. I. Increased functional expression of antibody fragments with and without cis-prolines. J. Biol. Chem. 275: 17100–17105. 4. Chatha, L., P. Ray, D. K. Bhasin, D. Panigrahi, T. Khanna, K. Vaiphei, K. Singh, and H. Singh. 1997. Western blot analysis of serological response in Helicobacter pylori in acid peptic diseases. Indian J. Med. Res. 105:170–175. 5. Cover, T. L., Y. Glupczynski, A. P. Lage, A. Burette, M. K. R. Tummuru, G. I. Perez-Perez, and M. J. Blaser. 1995. Serologic detection of infection with CagA⫹ Helicobacter pylori strains. J. Clin. Microbiol. 33:1496–1500. 6. Dunn, B. E., N. B. Vakil, B. G. Schneider, M. M. Miller, J. B. Zitzer, T. Peutz, and S. H. Phadnis. 1997. Localization of Helicobacter pylori urease and heat shock protein in human gastric biopsies. Infect. Immun. 65:1181– 1188. 7. European Helicobacter pylori Group. 1997. Current European concepts in the management of Helicobacter pylori infection. The Maastricht Consensus Report. European Helicobacter pylori Study Group. Gut 41:8–13. 8. Faulde, M., J. Cremer, and L. Zöller. 1993. Humoral immune response against Helicobacter pylori as determined by immunoblot. Electrophoresis 14:945–951. 9. Hazell, S. L., D. J. Evans, and D. Y. Graham. 1991. Helicobacter pylori catalase. J. Gen. Microbiol. 137:57–61. 10. Høiby, N., M. T. Collins, F. Espersen, J. B. Hertz, G. E. Hoff, and P. O. Schiøtz. 1987. Taxonomic application of crossed immunoelectrophoresis. Int. J. Syst. Bacteriol. 37:229–240. 11. Höök-Nikanne, J., G. L. Perez-Perez, and M. J. Blaser. 1997. Antigenic characterization of Helicobacter pylori strains from different parts of the world. Clin. Diagn. Lab. Immunol. 4:592–597. 12. Johansen, H. K., A. N兾orgaard, L. P. Andersen, P. Jensen, H. Nielsen, and N. H兾oiby. 1995. Cross-reactive antigens shared by Pseudomonas aeruginosa, Helicobacter pylori, Campylobacter jejuni, and Haemophilus influenzae may cause false-positive titers of antibody to H. pylori. Clin. Diagn. Lab. Immunol. 2:149–155. 13. Karvar, S., H. Karch, M. Frosch, W. Burghardt, and U. Gross. 1997. Use of serum-specific immunoglobulins A and G for detection of Helicobacter pylori infection in patients with chronic gastritis by immunoblot analysis. J. Clin. Microbiol. 35:3058–3061. 14. Kimmel, B., A. Bosserhoff, R. Frank, R. Gross, W. Goebel, and D. Beier. 2000. Identification of immunodominant antigens from Helicobacter pylori and evaluation of their reactivities with sera from patients with different gastroduodenal pathologies. Infect. Immun. 68:915–920. 15. Lebart, L., A. Morineau, and K. M. Warwick. 1984. Multivariate descriptive
552
16. 17. 18. 19. 20.
21. 22.
23.
ATANASSOV ET AL. analysis and related techniques for large matrices. Wiley Interscience, New York, N.Y. Moche, M., G. Schneider, P. Edwards, K. Dehesh, and Y. Lindqvist. 1999. Structure of the complex between the antibiotic cerulenin and its target, beta-ketoacyl-acyl carrier protein synthase. J. Biol. Chem. 274:6031–6034. Newell, D. J. 1987. Identification of the outer membrane proteins of Campylobacter pyloridis and antigenic cross-reactivity between C. pyloridis and C. jejuni. J. Gen. Microbiol. 133:163–170. Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10:1–6. Nilsson, I., A. Ljungh, P. Aleljung, and T. Wadstrom. 1997. Immunoblot assay for serodiagnosis of Helicobacter pylori infections. J. Clin. Microbiol. 35:427–432. Opazo, P., I. Muller, A. Rollan, P. Valenzuela, A. Yudelevich, R. Garcia-de la Guarda, S. Urra, and A. Venegas. 1999. Serological response to Helicobacter pylori recombinant antigens in Chilean infected patients with duodenal ulcer, non-ulcer dyspepsia and gastric cancer. APMIS 107:1069–1078. Perez-Perez, G. L., and M. J. Blaser. 1987. Conservation and diversity of Campylobacter pyloridis major antigens. Infect. Immun. 55:1256–1263. Phadnis, S. H., M. H. Parlow, M. Levy, D. Ilver, C. M. Caulkins, J. B. Connors, and B. E. Dunn. 1996. Surface localization of Helicobacter pylori urease and a heat shock protein homolog requires bacterial autolysis. Infect. Immun. 64:905–912. Qiu, X., C. A. Janson, A. K. Konstantinidis, S. Nwagwu, C. Silverman, W. W. Smith, S. Khandekar, J. Lonsdale, and S. S. Abdel-Meguid. 1999. Crystal structure of beta-ketoacyl-acyl carrier protein synthase III. A key condensing enzyme in bacterial fatty acid biosynthesis. J. Biol. Chem. 274:36465–36471.
J. CLIN. MICROBIOL. 24. Ramm, K., and A. Plückthun. 2000. The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. II. Isomerase-independent chaperone activity in vitro. J. Biol. Chem. 275:17106–17113. 25. Rocha, G. A., A. M. R. Oliveira, D. M. M. Queiroz, A. S. T. Carvalho, and A. M. M. F. Nogueira. 2000. Immunoblot analysis of humoral immune response to Helicobacter pylori in children with and without duodenal ulcer. J. Clin. Microbiol. 38:1777–1781. 26. Sipos, A., M. Klocke, and M. Frosch. 1991. Cloning and sequencing of the genes coding for the 10- and 60-kDa heat shock proteins from Pseudomonas aeruginosa and mapping of a species-specific epitope. Infect. Immun. 59: 3219–3226. 27. Tomb, J., O. White, A. R. Kerlavage, R. A. Clayton, G. G. Sutton, R. D. Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H. G. Khalak, A. Glodek, K. McKenney, L. M. Fitzegerald, N. Lee, M. D. Adams, E. K. Hickey, D. E. Berg, J. D. Gocayne, T. R. Utterback, J. D. Peterson, J. M. Kelley, M. D. Cotton, J. M. Weidman, C. Fujii, C. Bowman, L. Watthey, E. Wallin, W. S. Hayes, M. Borodovsky, P. D. Karp, H. O. Smith, C. M. Fraser, and J. C. Venter. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388: 539–547. 28. Vanet, A., and A. Labigne. 1998. Evidence for specific secretion rather than autolysis in the release of some Helicobacter pylori proteins. Infect. Immun. 66:1023–1027. 29. Widmer, M., J. D. de Korwin, P. Aucher, J. M. Thiberge, S. Suerbaum, A. Labigne, and J. L. Fauchère. 1999. Performance of native and recombinant antigens for diagnosis of Helicobacter pylori infection. Eur. J. Clin. Microbiol. Infect. Dis. 18:823–826.
