©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mosaicism in Vacuolating Cytotoxin Alleles of Helicobacter pylori
ASSOCIATION OF SPECIFIC vacA TYPES WITH CYTOTOXIN PRODUCTION AND PEPTIC ULCERATION (*)

(Received for publication, March 14, 1995; and in revised form, May 15, 1995)

John C. Atherton (1) Ping Cao (1) Richard M. Peek , Jr. (2) Murali K. R. Tummuru (1) Martin J. Blaser (1) (3) Timothy L. Cover (1) (3)(§)

From the  (1)Divisions of Infectious Disease and (2)Gastroenterology, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2605 and the (3)Department of Veterans Affairs Medical Center, Nashville, Tennessee 37232-2637

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Approximately 50% of Helicobacter pylori strains produce a cytotoxin, encoded by vacA, that induces vacuolation of eukaryotic cells. Analysis of a clinically isolated tox strain (Tx30a) indicated secretion of a 93-kDa product from a 3933-base pair vacA open reading frame. Characterization of 59 different H. pylori isolates indicated the existence of three different families of vacA signal sequences (s1a, s1b, and s2) and two different families of middle-region alleles (m1 and m2). All possible combinations of these vacA regions were identified, with the exception of s2/m1 (p < 0.001); this mosaic organization implies that recombination has occurred in vivo between vacA alleles. Type s1/m1 strains produced a higher level of cytotoxin activity in vitro than type s1/m2 strains; none of 19 type s2/m2 strains produced detectable cytotoxin activity. The presence of cagA (cytotoxin-associated gene A) was closely associated with the presence of vacA signal sequence type s1 (p < 0.001). Among patients with past or present peptic ulceration, 21 (91%) of 23 harbored type s1 strains compared with 16 (48%) of 33 patients without peptic ulcers; only 2 (10%) of 19 subjects harboring type s2 strains had past or present peptic ulcers (p < 0.005). Thus, specific vacA genotypes of H. pylori strains are associated with the level of in vitro cytotoxin activity as well as clinical consequences.


INTRODUCTION

Helicobacter pylori is the causative agent of chronic superficial gastritis in humans, and infection with this organism is a significant risk factor for the development of peptic ulcer disease and gastric cancer(1, 2, 3, 4, 5) . An important virulence determinant of H. pylori is the vacuolating cytotoxin(6, 7, 8) . The H. pylori cytotoxin induces cytoplasmic vacuolation in a variety of mammalian cell lines in vitro(9) , and produces epithelial cell damage and mucosal ulceration when administered intragastrically to mice(10) . Encoded by vacA, the cytotoxin is translated as a 1287-1296-amino acid precursor, which then undergoes both N-terminal and C-terminal processing to yield a mature 87-kDa secreted product(6, 8, 10, 11, 12) . Although only about 50% of H. pylori strains induce vacuolation of epithelial cells in vitro(9, 13) , nearly all strains hybridize with vacA probes(8, 10, 11, 12) . A second putative virulence determinant is the high molecular weight protein encoded by the cytotoxin-associated gene, cagA. About 60% of strains possess cagA, and nearly all of these express the cagA gene product(14, 15) . Production of vacuolating cytotoxin activity in vitro and the presence of cagA are closely associated characteristics (14, 15) . However, insertional mutagenesis of cagA fails to ablate cytotoxin activity(16) .

The genetic basis for the absence of detectable cytotoxin activity in supernatants from 50% of H. pylori strains is not yet well understood, but in a previous study we demonstrated that several tox and tox strains differed substantially within the middle region of vacA(8) . Therefore, the objectives of this study were (i) to characterize further the vacA alleles that are present in different strains of H. pylori, (ii) to correlate vacA genotypic differences with levels of cytotoxin production in vitro, (iii) to determine whether there is a correlation between particular vacA genotypes and presence of cagA, and (iv) to determine whether the vacA genotype of infecting H. pylori strains is related to the occurrence of peptic ulceration. We describe here the molecular cloning of a 3933-bp()vacA open reading frame from a toxH. pylori strain and show that clinical H. pylori isolates contain one of five different mosaic vacA structures. In addition, we show that the vacA genotype of a strain is strongly associated with its cytotoxin phenotype and its capacity to induce peptic ulceration.


EXPERIMENTAL PROCEDURES

Cloning of vacA from H. pylori Tx30a

Chromosomal DNA from toxH. pylori strain Tx30a, which fails to produce detectable cytotoxin activity in vitro(8, 9) and which lacks cagA(14) , was partially digested with SauIIIa and ligated to BamHI-digested GEM11 arms (Promega). After packaging (Promega), the library was titered and screened in E. coli ER1793 cells, using a P-labeled 0.7-kb XbaI fragment of vacA from strain 60190 as a probe(8) . Bacteriophage DNA was isolated from a purified, reactive clone (Tx30a-10), and vacA-containing restriction fragments were identified by Southern hybridization. A 2.0-kb HindIII fragment was subcloned into pBluescript (Stratagene, La Jolla, CA) to create pA153, and a 2.3-kb SacI fragment was subcloned to create pA145 (which contained nucleotides 1-762 and 2285-4350, respectively, of GenBank sequence U29401). A 1.5-kb vacA fragment bridging pA145 and pA153 was PCR-amplified from Tx30a chromosomal DNA, using primers (5` GATGGAGGTTGGGATTGG 3`) and (5` TAGGGTTAGGTTATTAAACATAAG 3`) (nucleotides 699-717 and 2317-2340), and subcloned into pT7Blue (Novagen) to create pA147. Nucleotide sequences were determined on both strands by primer walking, using the dideoxy chain termination procedure. The 1.5-kb vacA fragment contained in pA147 was PCR-amplified from Tx30a chromosomal DNA on two different occasions, and sequencing of the two products yielded identical results. A search of data bases for homologous proteins was accomplished using the BLAST network service of the National Center for Biotechnology Information, FastA, and Wordsearch programs.

Colony Hybridization of H. pylori Isolates

Colony hybridizations were performed as described previously(8) , except that washes were with 0.1 SSC at 68 °C. pCTB4 is a 458-bp probe, derived from the middle region of vacA in tox strain 60190(8) . A second probe (VA6) was PCR-amplified from the corresponding vacA region of toxH. pylori strain 87-203(8) , using primers (5`GCAATATTTATCTGGGAAAATC 3`) and (5` GCTATATCCCGTTTGCAAAC 3`). The cagA probe was a 2334-bp BamHI/EcoRI restriction fragment of pMC3 (14) .

PCR Amplification and Sequencing of vacA Fragments

vacA fragments 1.5 or 1.3 kb in size were PCR-amplified from the middle region of vacA from tox strain 84-183 and tox strain 86-313, using primers derived from the vacA sequence of H. pylori 60190(8) . Primers (5` ATGGAAATACAACAAACACAC 3`) and (5`GAGCTTGTTGATATTGAC 3`) were used to amplify a 1.5-kb fragment from the 5` end of vacA from strain 93-68. Primers (5` ATTTTACCTTTTTACACATTCTAGCC 3`) and (5` AGAAGCCCTGAGACCG 3`) were used to amplify 0.5-kb fragments encoding the vacA signal sequences of multiple H. pylori strains. PCR products to be sequenced were subcloned into the pT7 Blue vector (Novagen), and nucleotide sequences of plasmid DNA then were determined on both strands by the dideoxy chain termination procedure.

PCR-based Methodology for Typing vacA Homologs and Detecting cagA

Primers for PCR-based typing of vacA homologs are shown in Table 1. Thermal cycling for each set of primers (0.5 µM each) was at 95 °C for 1 min, 52 °C for 1 min, and 72 °C for 1 min, for a total of 35 cycles. PCR amplification of cagA used previously described primers F1 (5` GATAACAGGCAAGCTTTTGAGG 3`) and B1 (5` CTGCAAAAGATTGTTTGGCAGA 3`), with annealing at 55 °C, to amplify a 349-bp product from the middle of cagA(14) .



Detection of Vacuolating Cytotoxin Activity by HeLa Cell Assay

H. pylori cells were grown for 64 h in brucella broth (17) containing 5% fetal bovine serum at 37 °C in 6% carbon dioxide, and culture supernatants were concentrated 40-fold by ultrafiltration(13) . Concentrated culture supernatants were tested for vacuolating cytotoxin activity using a HeLa cell assay, as described previously(13) . For the final 29 consecutive isolates, the concentrated culture supernatants were serially diluted, using doubling dilutions, such that the final concentration applied to cells ranged from 8-fold concentrated to 4-fold diluted (arbitrarily assigned dilution units of 1, 2, 4, 8, 16, and 32). All supernatants were tested on at least two occasions, and cytotoxin activity is expressed as the mean maximum dilution unit score yielding >80% HeLa cell vacuolation. We define high grade cytotoxin activity as a dilution unit score 8, low grade cytotoxin activity as a score between 1 and 8, and tox as a score <1.

Detection of Vacuolating Cytotoxin by Antigen Detection Enzyme-linked Immunosorbent Assay (ELISA)

Separate aliquots of the same 29 supernatants used in the quantitative HeLa cell culture assay were tested for reactivity with rabbit antiserum to the purified cytotoxin of H. pylori strain 60190, as described previously (6) . ELISA values were calculated as log (optical density 100).

Immunoblotting and RNA Dot Blot Analysis

Western blotting of H. pylori culture supernatants with antiserum prepared against the purified cytotoxin of strain 60190 was accomplished as described previously(6) . For RNA dot blot analysis, RNA was isolated from H. pylori strains and transferred to nylon membranes (18) . The RNA then was hybridized with a P-labeled 0.7-kb XbaI fragment of vacA(8) at 42 °C in the presence of 50% formamide(18) .

Human Subjects and Bacterial Strains

H. pylori isolates were obtained from 59 U.S. subjects, median age 58 (range 23-80), who underwent routine upper gastrointestinal endoscopy for a variety of indications. Of the 59 subjects studied, endoscopic information was available for 56, of whom 23 (41%) had past or present peptic ulcers (20 duodenal, two gastric, and one both). Of these, 12 had at least one ulcer present at the time of the study endoscopy, four had duodenal erosions only (all these had past duodenal ulceration), and seven had no ulcer or duodenal erosions but had active ulceration diagnosed at a previous endoscopy. An active peptic ulcer was defined as a circumscribed break in the mucosa, with apparent depth, measuring more than 1 cm in any dimension; an erosion was defined as a definite circumscribed break in the mucosa not fulfilling the criteria for an active ulcer; and a previous ulcer was defined as an unequivocal diagnosis of peptic ulceration in a previous endoscopy or upper GI series report.

Statistical Methods

The test with Yates' continuity correction or Fisher's exact test was used for analysis of categorical data. The continuous data obtained from the ELISA results were compared across groups using Student's t test. Data on cytotoxin activity were compared using Wilcoxon's rank sum test.


RESULTS

Characterization of the vacA Homolog from toxH. pylori Strain Tx30a

As described under ``Experimental Procedures,'' the entire vacA homolog from tox strain Tx30a was cloned and sequenced. A 3933-bp open reading frame was present, which encoded 1310 amino acids. A potential ribosomal binding site (AGGAA) and an inverted repeat sequence capable of forming a stem-loop structure in the mRNA (G = -11.7 kcal) were each identified. A second open reading frame, homologous to the cysteinyl tRNA synthetase gene of E. coli, was identified upstream from vacA, as has been shown previously in tox strains(8, 12) .

vacA from strain Tx30a encoded a deduced polypeptide with a calculated molecular mass of 141,902 daltons, which is similar to the size (139-140 kDa) of vacA products encoded by toxH. pylori strains(8, 10, 11, 12) . The experimentally determined N-terminal amino acid sequence of the secreted cytotoxin from a tox strain (6) was identified within the deduced VacA sequence from strain Tx30a (95.7% identity) (Fig. 1). In addition, vacA from strain Tx30a encoded a potential signal sequence that was highly homologous within the first 25 residues to the signal sequences of several different tox strains(8, 10, 11, 12) . However, the remainder of the vacA signal sequence encoded by strain Tx30a was markedly divergent from the corresponding region in tox strains (Fig. 1). In addition, compared with the vacA product from strain 60190, VacA from strain Tx30a contained a 20-amino acid deletion (corresponding to residues 342-361 of the H. pylori 60190 vacA product, Fig. 1). The deletion corresponds to a hydrophilic, repeat-containing region in which the vacA product of tox strain CCUG 17874 undergoes proteolytic cleavage(10) . VacA from strain Tx30a also contained a 23-amino acid insertion (Tx30a residues 501-523, Fig. 1) that was absent from the vacA product of strain 60190. The nucleotide sequence encoding most of the insertion (bp 1846-1905) is an imperfect repeat of the immediately preceding sequence (bp 1783-1842) (81.7% identity). The insertion encoded a potential ATP/GTP binding site motif (GNIYLGKS, residues 500-507, Fig. 1), corresponding to a Walker A consensus sequence or phosphate-binding loop(19, 20, 21) . No such potential ATP/GTP binding sites were predicted by translation of vacA sequences from toxH. pylori strains(8, 10, 11, 12) .


Figure 1: Alignment between the deduced VacA amino acid sequence of tox strain Tx30a with that of tox strain 60190. Verticallines denote identity, and colons indicate conservative substitutions.



A comparison of the entire vacA sequence from strain Tx30a with that of tox strain 60190 indicated that there was 86.6% nucleotide identity between the two homologs. A comparison of the two deduced VacA amino acid sequences indicated 82.2% identity and 88.8% similarity. However, the level of relatedness between these proteins varied markedly in different regions. The C-terminal cleaved portion of the VacA protoxin was highly conserved, whereas there was only 59.0% amino acid identity within a 244-amino acid region in the middle of VacA (strain 60190, residues 509-752), and 70-90% amino acid identity within N-terminal regions ( Fig. 1and Fig. 2). A search of protein data bases indicated that the Tx30a vacA product, as well as the vacA products of tox strains, possessed low level homology with the major ring-forming surface protein (Hsr) of Helicobacter mustelae(22) and rickettsial surface layer proteins(23, 24, 25, 26) . For example, there was 25.0% amino acid identity (37.9% similarity) between Tx30a vacA residues 809-932 and the corresponding portion of H. mustelae Hsr (alignment with one gap).


Figure 2: Comparison of the deduced VacA amino acid (aa) sequence from tox strain Tx30a with that of tox strain 60190. The highest level of relatedness was in the C-terminal cleaved portion of the VacA protoxin, and the greatest diversity was in the middle region of the gene. The locations of a 20-amino acid insertion in strain 60190 (), a 23-amino-acid insertion in strain Tx30a (), and paired cysteine residues (CC) are indicated. Levels of homology (percentage of amino acid identity) are denoted as follows: open bar, >90%; barwithhorizontalline, 80-90%; barwithverticallines, 70-80%; barwithdiagonalstripes, <70%.



Expression of vacA in H. pylori Tx30a

To determine whether a vacA product was expressed by tox strain Tx30a, broth culture supernatant from this strain was immunoblotted with antiserum to the purified H. pylori cytotoxin from tox strain 60190(6) , and an immunoreactive protein of approximately 93 kDa was recognized (Fig. 3). To determine the site of signal peptide cleavage, the immunoreactive protein was purified from Tx30a broth culture supernatant, using previously described methods(6) , and the N-terminal amino acid sequence was determined. The N-terminal sequence XTPXD corresponded to residues 31-35 (Fig. 1), which suggested that a signal sequence was cleaved at an Ala-Asn site. The putative signal sequence (residues 1-30) contained four positive N-terminal charges, a central hydrophobic region, and a six-amino acid cleavage region corresponding to residues 25-30(27, 28) .


Figure 3: Detection of vacA products in H. pylori culture supernatants. H. pylori strains were cultured in Brucella broth containing 0.5% charcoal, and supernatant proteins were concentrated by precipitation with a 50% saturated solution of ammonium sulfate. Concentrated supernatants (4 µg of protein/lane) were immunoblotted with a 1:20,000 dilution of antiserum to the purified cytotoxin of H. pylori 60190(6) . Lanea, strain 60190; laneb, strain Tx30a. Immunoreactive bands were present in supernatants from both strains.



Western blotting of culture supernatants from 13 other tox strains, using antiserum prepared against the purified cytotoxin of strain 60190(6) , indicated that immunoreactive 87-94-kDa bands were clearly detectable in seven, and faint bands were visualized in three of the strains. However, in a dot blot assay, a 0.7-kb vacA probe from strain 60190 (derived from a region with 91% nucleotide identity to vacA from strain Tx30a) hybridized with RNA from all 13 tox strains but not Campylobacter fetus 23D or Escherichia coli DH5 (not shown). Thus, vacA transcription occurred in each of the tox strains tested.

Analysis of the Middle Region of vacA from Multiple H. pylori Strains

To study further the highly divergent middle region of vacA, PCR products spanning this region were amplified from an additional tox and tox strain (strains 84-183 and 86-313, respectively)(8) , as described under ``Experimental Procedures.'' Sequence analysis indicated that each of these 1.5-kb PCR products encoded a partial vacA open reading frame. We then examined a 0.73-kb region in which there was maximum divergence (corresponding to amino acid residues 509-752 of the 60190 vacA product, Fig. 1), by aligning and comparing the vacA sequences of strains 84-183, 86-313, Tx30a, and four previously published vacA sequences. For all four tox strains, the vacA sequences were closely related to each other in this region; similarly, the vacA sequences of all 3 tox strains were closely related to each other (Table 2). However, this region was markedly different between tox strains and tox strains (70.4% mean nucleotide identity and 58.7% mean amino acid identity). Thus, two different families of vacA alleles could be differentiated at this locus. We hereafter designate the family of vacA middle region sequences exemplified by H. pylori strain 60190 as type m1 alleles and the vacA middle region sequences exemplified by strain Tx30a as type m2 alleles. All three of the type m2 vacA alleles encoded an identical 23-amino acid insertion containing a potential ATP/GTP binding site motif (GXXXXGKS), whereas this insertion was absent from all four of the type m1 vacA alleles; whether this site is functionally relevant is not known. Similarly, the previously described 20-amino acid region, present in strain 60190 but absent from strain Tx30a, was deleted from each of the three type m2 alleles but was present in each of the type m1 vacA alleles.



vacA Typing Based on Mid-region Nucleotide Sequences

We next sought to determine the distribution of vacA m1 and m2 allelic types within a collection of 59 H. pylori clinical isolates. Colony hybridizations of these strains were performed with probes pCTB4 (8) and VA6, derived from the vacA mid-regions of tox and toxH. pylori strains, respectively, as described under ``Experimental Procedures.'' Twenty-two (37%) of the strains hybridized selectively with pCTB4 (indicating a type m1 genotype), and the remaining 37 (63%) hybridized selectively with VA6 (indicating a type m2 genotype). Thus, each strain hybridized selectively with only one of the two probes. To type vacA mid-regions by an alternate approach, PCR primers (VA3-F, VA3-R, VA4-F, and VA4-R) were designed to amplify specifically either type m1 or type m2 vacA sequences (Table 1). Of the 59 H. pylori strains tested, all had DNA amplified by one of the two primer sets, and none had DNA amplified by both. PCR typing and colony hybridizations produced identical results for each of the 59 strains tested. Thus, there were two different families of vacA alleles (m1 and m2) that could be differentiated at the mid-region locus.

Characterization of vacA Signal Sequences

To study the vacA signal sequences in H. pylori isolates, a 0.5-kb fragment encoding this region was PCR-amplified from eight additional H. pylori strains, as described under ``Experimental Procedures,'' and the relevant regions were sequenced. Two basic families of putative signal sequences were identified (Fig. 4): 33-amino acid signal sequences closely related to those present in previously characterized tox strains (designated type s1), and sequences closely related to the 30-amino acid signal sequence of strain Tx30a (designated type s2). In comparison with the signal sequences described previously in tox strains(8, 10, 11, 12) , (which we designate as type s1a), four strains possessed variant signal sequences (designated type s1b) containing 12 consistent base pair differences, encoding six consistent amino acid substitutions (Fig. 4).


Figure 4: Nucleotide sequences encoding putative vacA signal sequences for 12 H. pylori strains (A) and deduced amino acid sequences for three representative vacA types (B). Dots indicate nucleotide or amino acid identity compared with the sequence listed above. Stars indicate nucleotide differences between type s1a and s1b signal sequences. Positions of primers used for subsequent PCR typing of strains are underlined. Arrows denote the experimentally determined sites of putative signal peptide cleavage for strains 60190 and Tx30a. vacA sequence data have been reported previously in (6) and (8) (1), (10) and (12) (2), and (11) (3).



vacA Typing Based on Signal Sequences

We next designed PCR primers (VA1-F and VA1-R) to amplify and differentiate type s1 and s2 vacA signal sequences ( Table 1and Fig. 5). Using these primers, it was predicted that 259- and 286-bp products would be PCR-amplified from type s1 and type s2 strains, respectively. Forty (68%) of 59 strains yielded products of the former size and 19 (32%) yielded products of the latter size, as assessed on a 2% agarose gel (Fig. 5). All 59 strains yielded a PCR product of one of the two sizes; none gave a product of any other size. To differentiate type s1a and s1b signal sequences and to confirm the presence of type s2 sequences, new forward primers (SS1-F, SS2-F, and SS3-F) were designed based on the region encoding the variable second half of the signal sequence (Table 1, Fig. 4), and these were used for separate PCR amplifications with the conserved reverse primer (VA1-R). The designation of all 19 type s2 strains was confirmed by this approach. Of the 40 type s1 strains, 20 were classified as type s1a (DNA amplified by primers SS1-F and VA1-R) and 20 as type s1b (DNA amplified by primers SS3-F and VA1-R). For each of the 59 strains tested, DNA was amplified by only one of the three primer sets, indicating the specificity of this method.


Figure 5: PCR typing of vacA from three H. pylori strains. a, using primers VA3-F and VA3-R; b, using primers VA4-F and VA4-R; c, using primers SS1-F and VA1-R; d, using primers SS3-F and VA1-R; e, using primers SS2-F and VA1-R; f, using primers VA1-F and VA1-R; g, strains typed as s1 or s2 on the basis of the size of the PCR product.



Naturally Occurring Chimeric vacA Gene Structures

Among the 59 strains studied, vacA homologs containing five of the six possible combinations of signal sequence and mid-region types (s1a/m1, s1a/m2, s1b/m1, s1b/m2, and s2/m2) were found, but the s2/m1 combination was not, a highly significant finding (p < 0.001) (Table 3). To further validate the unexpected combination of a type s1 signal sequence with a type m2 mid-region, a 1.5-kb vacA fragment, extending from the ATG start codon to the mid-region of the gene, was PCR-amplified from strain 93-68, as described under ``Experimental Procedures.'' Sequence analysis confirmed the existence of a type s1a/m2 vacA homolog.



Relationship between cagA Genotype of H. pylori Strains and vacA Subtypes

To determine whether the presence of cagA was associated with particular vacA genotypes, the presence of cagA was determined for the 59 H. pylori isolates; 35 (59%) hybridized with a cagA probe. When the vacA signal sequence type was compared with cagA status, 35 (87.5%) of 40 type s1 strains were cagA (17 of 20 s1a and 18 of 20 s1b) compared with none of 19 type s2 strains (p < 0.001) (Table 4). A significant association also was found between vacA mid-region typing and cagA status (p < 0.001) (Table 4), but subgroup analysis showed that only the association between signal sequence type and cagA status was independently significant. Thus, there was a highly significant association between the presence of cagA and the presence of a type s1 vacA signal sequence.



Production of Vacuolating Cytotoxin Activity in Vitro and Relationship to vacA Genotype

Of the 59 H. pylori strains tested, supernatants from 25 (42%) induced detectable vacuolation of HeLa cells (tox). Of 22 strains with the type s1/m1 vacA genotype, 19 (86%) were tox, significantly more than six (33%) of 18 type s1/m2 strains (p < 0.002), which were in turn more likely to be tox than type s2/m2 strains (0 of 19; p < 0.02). In an analysis of the strains from the final 29 consecutive subjects, the vacA genotype also was highly associated with the level of cytotoxin activity. Among tox s1/m1 strains the median cytotoxin activity was 16 units (range 8-32), compared with a median cytotoxin activity of 4 units (range 2-8) for tox s1/m2 strains (p < 0.01). Thus, all tox s1/m1 strains were high grade cytotoxin producers, and all s1/m2 strains producing measurable activity were low grade producers (Table 5). To assess in vitro cytotoxin production by a second method, the same 29 concentrated broth culture supernatants were analyzed by an antigen detection cytotoxin ELISA. ELISA values (mean ± S.E.) for s1/m1 strains (1.93 ± 0.10) were higher than those for s1/m2 strains (1.28 ± 0.09) (p < 0.001, Student's t test), which in turn were higher than those for s2/m2 strains (0.91 ± 0.10) (p < 0.02). These data suggest that there may be different levels of vacA products expressed or secreted by different vacA genotypes. Thus, the vacA genotype of a strain was a strong predictor of the level of vacuolating cytotoxin activity produced by the strain in vitro.



Relationship of vacA Subtype to Occurrence of Peptic Ulceration

Finally, we sought to determine whether particular vacA genotypes were associated with the occurrence of peptic ulceration. Infection with a type s1 strain was found in 21 (91%) of the 23 subjects with past or present peptic ulceration compared with 16 (48%) of the 33 subjects with no documented ulcers (p < 0.005). Three of the patients with peptic ulcer disease were infected with type s1/cagA strains. Thus, only two (11%) of 19 patients harboring type s2 strains had past or present peptic ulcers. Of these, one was a 69-year-old male smoker taking enteric coated aspirin (325 mg/day), and the other was a 32-year-old male nonsmoker with no other significant medical problems, who did not report taking aspirin or other nonsteroidal anti-inflammatory agents. Twelve (63%) of 19 subjects infected with strains possessing a type m1 vacA mid-region had peptic ulcer disease compared with 11 (30%) of 37 subjects infected with type m2 strains (<0.05). However, subgroup analysis showed that the vacA mid-region type was not independently associated with occurrence of peptic ulcer disease. Twelve (67%) of the 18 persons infected with type s1a strains had peptic ulcer disease compared with nine (47%) of 19 infected with type s1b strains, a nonsignificant difference (p = 0.3).

As expected(29, 30, 31, 32) , H. pylori isolates from patients with ulcers produced cytotoxin activity in vitro more frequently than isolates from patients without ulcers; 14 (61%) of 23 patients with ulcers harbored tox strains compared with nine (27%) of 33 patients without ulcers (p < 0.05). Subgroup analysis of the 33 tox strains (nine from ulcer patients and 24 from non-ulcer patients) indicated that seven (50%) of 14 type s1 strains were associated with ulcers compared with two (11%) of 19 type s2 strains (p < 0.05). Thus, the vacA signal sequence type was associated with occurrence of ulceration independent of the in vitro cytotoxin phenotype.


DISCUSSION

As described in this study, the vacA homolog in H. pylori strain Tx30a encodes a deduced protein of 142 kDa, which is similar to the size of vacA products from three previously characterized toxH. pylori strains (8, 10, 11, 12) . The presence of an immunoreactive 93-kDa protein in culture supernatant from strain Tx30a suggests that this vacA product undergoes C-terminal cleavage and secretion through the outer membrane, probably via a mechanism analogous to that described for tox strains(8, 10, 11) . Despite these similarities, vacA from strain Tx30a differed markedly from previously characterized vacA homologs in two regions: the mid-region of the gene and the signal sequence. That a large proportion of H. pylori strains contain vacA alleles resembling that of strain Tx30a suggests that this type of vacA product may have functional properties, probably independent of the capacity to induce cell vacuolation.

Based upon divergence among H. pylori strains within the vacA signal sequence (s1a, s1b, and s2) and mid-region (m1 and m2), we have developed methodology for typing vacA genes; five of the six possible signal sequence/mid-region combinations were found among the strains examined. Several bacterial genes with similar mosaic structures have been described, including IgA proteases from Neisseria gonorrhoeae and Haemophilus influenzae and penicillin binding proteins from Neisseria meningitidis and Streptococcus pneumoniae(33, 34, 35, 36, 37) . These genes are characterized by regions that are highly conserved interspersed with regions that are markedly divergent, entirely analogous to the structure of vacA in H. pylori. In N. meningitidis, highly divergent regions within the penicillin binding protein genes of different strains appear to have arisen by horizontal transfer of DNA from the corresponding genes of Neisseria flavescens or Neisseria cinerea(35) . Most bacterial species in which mosaic genes have been described are competent for natural transformation, a property shared by H. pylori(38, 39) . We speculate that DNA transfer from a non-H. pylori species originally may have given rise to divergence within H. pylori vacA genes. Simultaneous infection of humans with multiple H. pylori strains occurs (40, 41) , and therefore the opportunity for subsequent DNA transfer between H. pylori strains exists. That this occurs is suggested by the existence of four different gene structures in strains possessing a type s1 signal sequence (s1a/m1, s1b/m1, s1a/m2, and s1b/m2). A striking finding of this study was that the vacA genotype s2/m1 was not identified. A possible explanation is that the recombinant event required to produce this genotype simply never occurred, but we consider it more likely either that strains with the s2/m1 genotype are non-viable, or that this genotype confers a selective disadvantage.

Several previous studies have demonstrated an association between expression of CagA and production of vacuolating cytotoxin activity in vitro by H. pylori strains(14, 15) . This study demonstrates a strong genetic association between the presence of cagA and vacA signal sequence type s1. Why two genetic elements without any physical linkage on the H. pylori chromosome (42) should be so closely associated is not clear. One hypothesis is that there are two clonal H. pylori populations (vacA s1/cagA and vacA s2/cagA), but evidence from analysis of other H. pylori genes fails to support this (43) . Another possibility is that there may be a functional linkage, whereby a selective advantage conferred by each gene product is manifested only in the presence of the other.

One of the striking findings of this study was that strains containing a type s2 vacA signal sequence consistently failed to produce detectable vacuolating cytotoxin activity in vitro; only strains with type s1 vacA produced such activity. In addition, the type of vacA middle region was independently associated with the level of cytotoxin activity produced by strains. The divergent middle region of vacA comprises a sizable portion of the gene, and thus structural differences between type m1 and type m2 gene products could easily give rise to differences in cytotoxin phenotypes. However, the basis for the highly significant differences in phenotype between strains with type s1 and type s2 signal sequences is less clear. One hypothesis is that strains with type s2 signal sequences export the VacA protoxin less efficiently across the cytoplasmic membrane. Alternatively, differences in the N-terminal residues of the mature secreted vacA products, arising from different signal sequence cleavage sites in type s1 and type s2 VacA proteins, may account for differences in protein function. Finally, it may be that vacA signal sequence types are markers for other unidentified structural, regulatory, or secretory elements that influence cytotoxin activity.

A potentially important aspect of the vacA typing system presented here is its clinical relevance. In this and several previous studies(29, 30, 31, 32) , H. pylori isolates from patients with peptic ulcer disease expressed cytotoxin activity in vitro more commonly than isolates from patients with gastritis alone. The present investigation demonstrates an even stronger link between the clinical status of patients and the vacA genotype of infecting strains. Why might the vacA genotype be a better predictor of a strain's ulcerogenic properties than direct testing of cytotoxin production in vitro? One possible explanation is that cytotoxin expression may occur at significantly higher levels in vivo than in vitro. Alternatively, induction of vacuolation in transformed cell lines by the cytotoxin may be only an imperfect marker for production of epithelial damage in vivo. The strong association between peptic ulcer disease and vacA type s1 strains is complemented by the equally important finding that vacA type s2 strains are rarely associated with peptic ulceration. The identification of H. pylori strains associated with different risks of ulcerogenicity may have clinical implications.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants R29 DK45293 and R01 CA58834, the Medical Research Service of the Department of Veterans Affairs, and a Pfizer Scholars Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Infectious Diseases, Medical Center North A3310, Vanderbilt University School of Medicine, Nashville, TN 37232-2605. Tel.: 615-322-2035; Fax: 615-343-6160.

The abbreviations used are: bp, base pair(s); PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; kb, kilobase pair(s).


REFERENCES

  1. Blaser, M. J. (1992)Clin. Infect. Dis. 15, 386-393 [Medline] [Order article via Infotrieve]
  2. Hentschel, I., Brandstatter, G., Dragosics, B., Hirschl, A. M., Nemec, H., Schutze, K., Taufer, M., and Wurzer, H.(1993)N. Engl. J. Med. 328, 308-312 [Abstract/Free Full Text]
  3. Nomura, A., Stemmermann, G. N., Chyou, P.-H., Kato, I., Perez-Perez, G. I., and Blaser, M. J. (1991)N. Engl. J. Med. 325, 1132-1136 [Abstract]
  4. Parsonnet, J., Friedman, G. D., Vandersteen, D. P., Chang, Y., Vogelman, J. H., Orentreich, N., and Sibley, R. K.(1991)N. Engl. J. Med. 325, 1127-1131 [Abstract]
  5. Cover, T. L., and Blaser, M. J.(1995)Adv. Intern. Med.41,in press
  6. Cover, T. L., and Blaser, M. J.(1992)J. Biol. Chem. 267, 10570-10575 [Abstract/Free Full Text]
  7. Cover, T. L., Cao, P., Murthy, U. K., Sipple, M. S., and Blaser, M. J.(1992) J. Clin. Invest. 90, 913-918 [Medline] [Order article via Infotrieve]
  8. Cover, T. L., Tummuru, M. K. R., Cao, P., Thompson, S. A., and Blaser, M. J.(1994) J. Biol. Chem. 269, 10566-10573 [Abstract/Free Full Text]
  9. Leunk, R. D., Johnson, P. T., David, B. C., Kraft, W. G., and Morgan, D. R.(1988) J. Med. Microbiol. 26, 93-99 [Abstract]
  10. Telford, J. L., Ghiara, P., Dell'Orco, M., Comanducci, M., Burroni, D., Bugnoli, M., Tecce, M. F., Censini, S., Covacci, A., Xiang, Z., Papini, E., Montecucco, C., Parente, L., and Rappuoli, R.(1994) J. Exp. Med. 179, 1653-1658 [Abstract]
  11. Schmitt, W., and Haas, R.(1994)Mol. Microbiol 12, 307-319 [Medline] [Order article via Infotrieve]
  12. Phadnis, S. H., Ilver, D., Janzon, L., Normark, S., and Westblom, T. U.(1994) Infect. Immun. 62, 1557-1565 [Abstract]
  13. Cover, T. L., Cao, P., Lind, C. D., Tham, K. T., and Blaser, M. J.(1993) Infect. Immun. 61, 5008-5012 [Abstract]
  14. Tummuru, M. K. R., Cover, T. L., and Blaser, M. J.(1993)Infect. Immun. 61, 1799-1809 [Abstract]
  15. Covacci, A., Censini, S., Bugnoli, M., Petracca, R., Burroni, D., Macchia, G., Massone, A., Papini, E., Xiang, Z., Figura, N., and Rappuoli, R.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5791-5795 [Abstract]
  16. Tummuru, M. K. R., Cover, T. L., and Blaser, M. J.(1994)Infect. Immun. 62, 2609-2613 [Abstract]
  17. Hawrylik, S. J., Wasilko, D. J., Haskell, S. L., Gootz, T. D., and Lee, S. E. (1994)J. Clin. Microbiol. 32, 790-792 [Abstract]
  18. Tummuru, M. K. R., and Blaser, M. J.(1992)J. Bacteriol. 174, 5916-5922 [Abstract]
  19. Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J.(1982)EMBO J. 8, 945-951
  20. Hodgman, T. C. (1988)Nature333,22-23 [Medline] [Order article via Infotrieve]
  21. Saraste, M., Sibbald, P. R., and Wittinghofer, A.(1990)Trends Biochem. Sci. 15, 430-434 [CrossRef][Medline] [Order article via Infotrieve]
  22. O'Toole, P. W., Austin, J. W., and Trust, T. J.(1994)Mol. Microbiol. 11, 349-361 [Medline] [Order article via Infotrieve]
  23. Gilmore, R. D., Jr., Cieplak, W., Jr., Policastro, P. R., and Hackstadt, T.(1991) Mol. Microbiol. 5, 2361-2370 [Medline] [Order article via Infotrieve]
  24. Carl, M., Dobson, M. E., Ching, W.-M., and Dasch, G. A.(1990)Proc. Natl. Acad. Sci. U. S. A. 87, 8237-8241 [Abstract]
  25. Anderson, B. E., McDonald, G. A., Jones, D. C., and Regnery, R. L.(1990) Infect. Immun. 58, 2760-2769 [Medline] [Order article via Infotrieve]
  26. Hahn, M.-J., Kim, K.-K., Kim, I., and Chang, W.-H.(1993)Gene (Amst.)133,129-133 [Medline] [Order article via Infotrieve]
  27. Pugsley, A. P. (1993)Microbiol. Rev. 57, 50-108 [Abstract]
  28. Izard, J. W., and Kendall, D. A.(1994)Mol. Microbiol. 13, 765-773 [Medline] [Order article via Infotrieve]
  29. Figura, N., Guglielmetti, P., Rossolini, A., Barberi, A., Cusi, G., Musmanno, R. A., Russi, M., and Quaranta, S.(1989)J. Clin. Microbiol. 27, 225-226 [Medline] [Order article via Infotrieve]
  30. Tee, W., Lambert, J. R., and Dwyer, B.(1995)J. Clin. Microbiol. 33, 1203-1205 [Abstract]
  31. Rautelein, H., Blomberg, B., Jarnerot, G., and Danielsson, D.(1994)Scand. J. Gastroenterol. 29, 128-132 [Medline] [Order article via Infotrieve]
  32. Goossens, H., Glupczynski, Y., Burette, A., Lambert, J.-P., Vlaes, L., and Butzler, J.-P.(1992)Med. Microbiol. Lett. 1, 153-159
  33. Smith, J. M., Dowson, C. G., and Spratt, B. G.(1991)Nature 349, 29-31 [CrossRef][Medline] [Order article via Infotrieve]
  34. Halter, R., Pohlner, J., and Meyer, T. F.(1989)EMBO J. 8, 2737-2744 [Abstract]
  35. Spratt, B. G., Zhang, Q.-Y., Jones, D. M., Hutchison, A., Brannigan, J. A., and Dowson, C. G. (1989)Proc. Natl. Acad. Sci. U. S. A. 86, 8988-8992 [Abstract]
  36. Dowson, C. G., Hutchison, A., Brannigan, J. A., George, R. C., Hansman, D., Linares, J., Tomasz, A., Smith, J. M., and Spratt, B. G.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8842-8846 [Abstract]
  37. Poulsen, K., Reinholdt, J., and Kilian, M.(1992)J. Bacteriol. 174, 2913-2921 [Abstract]
  38. Wang, Y., Roos, K. P., and Taylor, D. E.(1993)J. Gen. Microbiol. 139, 2485-2493 [Medline] [Order article via Infotrieve]
  39. Ferrero, R. L., Cussac, V., Courcoux, P., and Labigne, A.(1992)J. Bacteriol. 174, 4212-4217 [Abstract]
  40. Prewett, E. J., Bickley, J., Owen, R. J., and Pounder, R. E.(1992) Gastroenterology 102, 829-933 [Medline] [Order article via Infotrieve]
  41. Fujimoto, S., Marshall, B., and Blaser, M. J.(1994)J. Clin. Microbiol. 32, 331-334 [Abstract]
  42. Bukanov, N. O., and Berg, D. E.(1994)Mol. Microbiol.11,509-523 [Medline] [Order article via Infotrieve]
  43. Garner, J. A., and Cover, T. L. (1995) J. Infect. Dis.172, in press

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