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Journal of Clinical Microbiology, September 1999, p. 2979-2982, Vol. 37, No. 9
Department of Medicine,
Received 30 December 1998/Returned for modification 19 March
1999/Accepted 4 June 1999
Alleles of the vacuolating cytotoxin gene (vacA) of
Helicobacter pylori vary between strains, particularly in
the region encoding the signal sequence (which may be type s1 or s2)
and the midregion (which may be type m1 or m2). Using a PCR-based
typing system developed in the United States, we showed that 36 strains
from Asia and South America were all vacA signal sequence
type s1; 3 were midregion type m1 and 11 were m2, but 22 could not be
typed for the vacA midregion. All strains possessed
cagA (cytotoxin-associated gene A), another virulence
marker. vacA nucleotide sequence analysis showed that
midregion typing failure was due to base substitutions at the primer
annealing sites. Using the new sequence data, we developed two new
PCR-based vacA midregion typing systems, both of which
correctly typed 41 U.S. strains previously typed by the old system and
successfully typed all 36 of the non-U.S. strains. All previously
untypeable strains were vacA m1, other than one m1/m2
hybrid. In summary, we describe and validate a simple PCR-based system
for typing vacuolating cytotoxin (vacA) alleles of H. pylori and show that this system correctly identifies the signal
and midregion types of vacA in 77 strains from Asia and
North and South America.
The two best-established methods for
grading Helicobacter pylori strain virulence are typing of
the allele of the vacuolating cytotoxin gene, vacA, and
determination of the presence or absence of the cytotoxin-associated
gene, cagA (a marker for the cag pathogenicity island) (4). A copy of vacA is present in
essentially all strains of H. pylori, but its nucleotide
sequence varies between strains. This variation is most marked in the
region encoding the signal sequence, which may be type s1 (with
subtypes a, b, or c) or type s2, and the midregion, which may be type
m1 or type m2 (1, 16). vacA alleles with all
possible combinations of signal and midregion types have been found
(1, 9). Among strains from the United States, the
vacA midregion type of a strain correlates with its ability
to induce vacuolation in HeLa cells in vitro and with gastric
epithelial damage in vivo, but not with gastric inflammatory cell
infiltrate in vivo or with peptic ulceration (2). However,
in Taiwan, an association between the m1 type of vacA (the
more cytotoxic type) and peptic ulceration has been described
(17). In contrast, in the United States, the vacA signal region type correlates with the level of gastric inflammatory cell infiltration (2) and in many but not all studies from the United States and Europe, it also correlates with peptic ulceration (2, 5, 12, 14, 15). In the Far East, this association is not
found, as essentially all strains described are vacA signal type s1a or s1c (8, 10, 11, 16, 17). This work on the association of vacA poymorphisms with disease has been
hampered by the finding of several groups, especially in the Far East, that the originally described primers used for PCR typing of the vacA midregion do not satisfactorily categorize all strains
(8, 10, 11, 12, 14, 17). We aimed to assess the extent of this problem, define the reasons for typing failure, and develop a
modified system which would allow simple and accurate vacA
typing of all strains.
H. pylori isolates.
In the first part of the
study, we used 36 previously obtained isolates from subjects outside
the United States: 8 from dyspeptic patients without ulcers from Lima,
Peru; 13 from dyspeptic patients (6 with duodenal ulceration, 5 with
gastric ulceration, 1 with both types of ulceration, and 1 with no
ulcers) from Yamagucchi, Japan; 9 from dyspeptic patients from Bangkok,
Thailand; and 6 from asymptomatic patients from Shandong Province,
China. In the standardization of our new PCR-based
vacA-typing strategies, we used 41 isolates from patients
from Nashville, Tenn., who underwent upper gastrointestinal endoscopy
for a variety of indications. We have previously reported on the
characterization of the cagA and vacA genotypes
of most of these U.S. isolates with our previous systems
(2).
vacA and cagA typing using original
methodology.
Stock cultures of H. pylori stored at
Nucleotide sequencing of vacA regions of selected
strains for which PCR typing was unsuccessful.
For selected
strains from which vacA midregions could not be typed by our
original PCR-based methods, 1.7-kb vacA fragments (coordinates 726 to 2445 bp in the published vacA sequence
of strain Tx30a [1]) were amplified by PCR with
primers VA10-F (5'-CGCTGAAATCTCTCTTTATG) and VA10-R
(5'-CATGCTTTGATTGCCGATAGC). PCR products were subcloned into
pT7Blue (Novagen, Madison, Wis.), and then both strands were sequenced
with a minimum of twofold redundancy with vector primers and then by
primer walking. For selected strains whose vacA signal
sequence regions could not be subtyped, we determined the nucleotide
sequence of this region by sequencing directly PCR amplicons from
primers A1043 (5'-ATTTTACCTTTTTACACATTCTAGCC) and B771
(5'-AGAAGCCCTGAGACCG). Nucleotide sequencing was performed with an automated ABI sequencer.
PCR-based vacA genotyping using new strategies.
Two new strategies were used in this study to classify the midregion
types of the vacA alleles. New strategy 1 used primers VAG-F
(5'-CAATCTGTCCAATCAAGCGAG) and VAG-R
(5'-GCGTCAAAATAATTCCAAGG) (Fig. 1). The PCR protocol was
similar to that described previously (1, 3), except that the
reaction mixtures were heated initially to 95°C for 90s and then
underwent 35 cycles of 30 s at 95°C, 60 s at 56°C, and
90 s at 72°C. After cycling, the products were extended for a
further 5 min at 72°C. New strategy 2 used four primers together in
one reaction tube: VA7-F (5'-GTAATGGTGGTTTCAACACC) and VA7-R
(5'-TAATGAGATCTTGAGCGCT) and the previously described primers VA4-F and VA4-R (1) (Fig. 1). The thermocycling
conditions were as described for new strategy 1.
vacA and cagA typing using original
methodology.
The vacA signal regions of all 36 non-U.S.
isolates were categorized as type s1 by PCR with conserved primers
VA1-F and VA1-R (1, 2). Further PCR subtyping with allelic
subtype-specific primers was successful for all strains other than two
from Peru (Table 1). vacA
midregion typing with (separately) m1-specific primers (VA3-F and
VA3-R) and m2-specific primers (VA4-F and VA4-R) (Fig. 1) had
previously successfully amplified vacA fragments from all 70 U.S. strains tested (1, 2). However, for the strains in the
current study, the typing method failed to amplify DNA from 21 of 28 Asian strains, including all 13 Japanese strains tested, and DNA from 1 of 8 Peruvian strains (Table 1). The strains from which DNA was not
amplified hybridized weakly with the m1-specific probe (pCTB4) but did
not hybridize detectably with the m2-specific probe (VA6) (Fig. 1). All
28 Asian and 6 of 7 Peruvian strains were cagA positive.
Thus, vacA alleles from most Asian strains were sufficiently
different from those of U.S. strains to prevent effective midregion
typing, and alleles from two Peruvian vacA s1 strains were
sufficiently different to prevent vacA signal region
subtyping.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Simple and Accurate PCR-Based System for Typing Vacuolating
Cytotoxin Alleles of Helicobacter pylori
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C were regrown under microaerobic conditions (Campypak plus;
Becton Dickinson, Cockeysville, Md.), and chromosomal DNA was extracted
by lysis with GES (guanadinium thiocyanate, EDTA, Sarkosyl), chloroform
extraction, and isopropanol precipitation as described previously
(1, 3). The vacA signal and midregion were typed
by both PCR and high-stringency colony hybridization with previously
described primers and probes (those for the vacA midregion
are shown schematically in Fig. 1).
vacA signal sequence typing was by PCR with primers VA1-F
and VA1-R to categorize this region as type s1 or s2 and the reverse
primer VA1-R together with allelic subtype-specific forward primers to subclassify type s1 alleles (the primers used were primer SS1-F for
subtype s1a or s1c, whose predicted peptide signal sequences differ by
only two amino acids [15], and primer SS3-F for
subtype s1b [1, 3]). vacA midregion typing
was performed by two separate PCRs with m1-specific primers (primers
VA3-F and VA3-R) in the first PCR and m2-specific primers (primers
VA4-F and VA4-R) in the second PCR (1, 3) (Fig. 1). The
presence of cagA was determined by colony hybridization with
a 2,334-bp probe, as described previously (1, 3).
View larger version (18K):
[in a new window]
FIG. 1.
Schematic of vacA midregions for the m1 and
m2 allelic types, showing the original midregion allelic type-specific
typing system (1) and the new PCR strategies. The diagram is
drawn to scale except for the sizes of the arrows representing the PCR
primers. The boxed area is 910 bp for strain 60190 (coordinates 1395 to
2304 numbered from the adenine of the start codon [8])
and 1,000 bp for strain Tx30a (coordinates 1371 to 2370 [1]). X represents an insertion of 75 bp, and Y
represents an insertion of 15 bp; both are present in m2 alleles but
not in m1 alleles. The arrows labeled VA3-F and VA3-R and those labeled
VA4-F and VA4-R are the allelic type-specific primers used in our
original PCR-based system (1). The solid lines labeled pCTB4
and VA6 are the allelic type-specific probes used for hybridization.
VAG-F and VAG-R are the primers used in new strategy 1, and they anneal
to regions of vacA conserved between m1 and m2 alleles; the
types are then differentiated on the basis of PCR product size. Primers
VA7-F and VA7-R and primers VA4-F and VA4-R are allelic type-specific
primers used together in new strategy 2. Product size depends on which
specific primers anneal, and the expected annealing primers are shown
below the m1 and m2 regions.
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
cagA status and vacA genotypes of
strains used in this study as assessed by PCR typing
Determination by nucleotide sequencing of reasons for vacA midregion typing failure. To define why PCR-based typing of the vacA midregion was unsuccessful for some strains, we sequenced a 1.7-kb region of vacA containing the primer and probe annealing sites from five of the strains that had not been successfully typed: 90-40 (Peru), 88-29 (Thailand), 88-32 (Thailand), HPK3 (Japan), and Ch2 (China). At the m1 forward primer (VA3-F) annealing site, from 0 to 3 of 19 bases were noncomplementary, whereas at the reverse primer (VA3-R) annealing site, 6 to 7 of 20 bases were noncomplementary. In the 439-bp annealing region for the m1 probe (pCTB4), 9% or fewer of the bases were noncomplementary. Comparative analysis with previously obtained nucleotide sequences from other type m1 vacA alleles showed that these newly sequenced alleles did not form a specific subgrouping within the m1 family, in that they were not related more closely to each other than to other type m1 alleles (7, 13). The annealing region for the 308-bp m2 probe (VA6) included a 75-bp insertion not found in type m1 vacA alleles (Fig. 1). Of the remaining 234 bp to which the m2 probe could potentially anneal, more than 25% of bases were noncomplementary in all cases. These findings explain the annealing of the m1 probe and the absence of annealing of the m2 probe under high-stringency conditions. The final strain, strain Ch2, had a vacA midregion with an m1-like sequence at the forward primer annealing site and an m2-like sequence at the reverse primer annealing site and appeared to be a naturally occurring m1/m2 hybrid. Thus, overall, poor annealing of primers or probes due to sequence divergence between vacA alleles explained PCR and hybridization failures for all but one strain (vacA m1/m2 hybrid strain Ch2).
Design and testing of two new PCR strategies for vacA midregion typing. Next, so that the vacA alleles of all strains could be typed successfully, we sought to develop new PCR strategies to distinguish between m1 and m2 alleles. We used these strategies to type the 36 non-U.S. strains described above and also (to ensure that our new strategies were valid) 41 strains from the United States previously successfully typed by our previous strategy. For new strategy 1, taking advantage of the conserved m2-specific 75-bp insertion, we designed primers VAG-F and VAG-R to span this region and to anneal to sites conserved among all presently known m1 and m2 vacA alleles (Fig. 1). This permitted sorting of PCR products by size (predicted size of 567 bp for type m1, coordinates 1278 to 1844 from beginning of the open reading frame in strain 60190 [6], and predicted size of 642 bp for type m2, coordinates 1254 to 1895 in strain Tx30a [1]). By use of these primers, PCR amplification of vacA alleles gave, for every strain, a product of one of the two predicted sizes, and for all 58 strains for which previous PCR typing had been successful, the result was the same. All 22 previously untypeable vacA alleles were now typed as m1 with the new primers, including the m1/m2 allele from strain Ch2 (Fig. 2B).
|
Determination by nucleotide sequence analysis of the reasons for vacA s1 signal sequence subtyping failure in two Peruvian strains. To deduce why vacA s1 alleles from two Peruvian strains (strains 90-16 and 90-22) could not be subtyped as s1a or s1b, we determined the nucleotide sequences of their vacA signal regions. The vacA alleles from these strains were more similar to s1b alleles than to s1a alleles (data not shown). Because of the difficulty subtyping the s1 vacA alleles of these two Peruvian strains, we similarly obtained the nucleotide sequences of two Peruvian strains classified as s1a by the PCR-based system and the single strain classified as s1b. Nucleotide sequence analysis of the two s1a strains (strains 90-14 and 90-40) revealed that although they had s1a-like sequences at the primer annealing site, 5' to this their sequences were closer to s1b sequences (data not shown). Nucleotide analysis of the Peruvian s1b strain showed a typical s1b allele. Thus, in summary, all 57 U.S. and Asian strains could be successfully typed as s1a, s1b, or s2. Peruvian strains were successfully typed as s1, but since they were often intermediate between s1a and s1b, this subtyping system was not appropriate for these strains.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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With the modifications to the vacA midregion typing system described in this paper, the vacA alleles of all strains that we have examined can be typed by just two PCRs: one for typing of the midregion as m1 or m2 and one for typing of the signal sequence as s1 or s2. For midregion typing, we recommend new strategy 1 (Fig. 1 and 2A) as the most direct strategy. For signal sequence typing, we recommend the use of primers VA1-F and VA1-R (1, 3). vacA s1 alleles can be further subtyped by PCR, although this will need validation in individual countries and may not be suitable for all locales (as was found for Peru in this study). Other groups have also improved PCR typing of vacA alleles on the basis of studies with European (12, 14) and international (15, 16) strain collections. The latter strategy uses a PCR approach similar to the one that we have described but, additionally, uses reverse hybridization with oligonucleotide probes immobilized on a nitrocellulose strip. While this confirms specificity, it is unlikely to be convenient for most researchers, unless premade nitrocellulose strips with immobilized oligonucleotides are inexpensively available commercially. The improved typing system described in our present study is easy to use and should facilitate studies to assess which groups of vacA alleles are prevalent in H. pylori strains colonizing different populations around the world. Studies can then be designed for appropriate populations to assess whether H. pylori strains with specific vacA alleles are differently associated with particular diseases.
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ACKNOWLEDGMENTS |
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This work was supported by grants AI 39657 and DK 5707, by a center grant from the National Cancer Institute (CA 68485), and by the Medical Research Service of the U.S. Department of Veterans Affairs. John Atherton is funded by a Clinician Scientist Fellowship from the Medical Research Council (United Kingdom).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medicine, Division of Gastroenterology, University Hospital, Nottingham NG7 2UH, United Kingdom. Phone: 44 115 9249924. Fax: 44 115 9422232. E-mail: John.Atherton{at}nottingham.ac.uk.
Present address: Facultad de Medicina, Universidad Nacional
Autonoma de Mexico, Mexico City, Mexico.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, September 1999, p. 2979-2982, Vol. 37, No. 9
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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