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Previous Article | Next Article 
Infection and Immunity, December 2001, p. 7832-7838, Vol. 69, No. 12
Swedish Institute for Infectious Disease
Control, 171 82 Solna,1 Department of
Medicine,2 Microbiology and Tumor
Biology Center,4 and Department of
Pathology,6 Karolinska Institute, 171 77 Stockholm, and Department of Molecular Biology, AstraZeneca
R&D, 431 83 Mölndal,7 Sweden;
Department of Molecular Biology and Pharmacology, Washington
University School of Medicine, St. Louis, Missouri
631103; and Department of Microbiology
and Immunology, Stanford University School of Medicine, Stanford,
California 943055
Received 4 June 2001/Returned for modification 25 July
2001/Accepted 31 August 2001
Helicobacter pylori has a very plastic genome,
reflecting its high rate of recombination and point mutation. This
plasticity promotes divergence of the population by the development of
subclones and presumably enhances adaptation to host niches. We have
investigated the genotypic and phenotypic characteristics of two such
subclones isolated from one patient as well as the genetic evolution of these isolates during experimental infection. Whole-genome genotyping of the isolates using DNA microarrays revealed that they were more
similar to each other than to a panel of other genotyped strains
recovered from different hosts. Nonetheless, they still showed
significant differences. For example, one isolate (67:21) contained the
entire Cag pathogenicity island (PAI), whereas the other (67:20) had
excised the PAI. Phenotypic studies disclosed that both isolates
expressed adhesins that recognized human histo-blood group
Lewisb glycan receptors produced by gastric pit and surface
mucus cells. In addition, both isolates were able to colonize, to
equivalent density and with similar efficiency, germ-free transgenic
mice genetically engineered to synthesize Lewisb glycans in
their pit cells (12 to 14 mice/isolate). Remarkably, the Cag
PAI-negative isolate was unable to colonize conventionally raised
Lewisb transgenic mice harboring a normal gastric
microflora, whereas the Cag PAI-positive isolate colonized 74% of the
animals (39 to 40 mice/isolate). The genomic evolution of both isolates
during the infection of conventionally raised and germ-free mice was monitored over the course of 3 months. The Cag PAI-positive isolate was
also surveyed after a 10 month colonization of conventionally raised
transgenic animals (n = 9 mice). Microarray analysis
of the Cag PAI and sequence analysis of the cagA,
recA, and 16S rRNA genes disclosed no changes in recovered
isolates. Together, these results reveal that the H. pylori
population infecting one individual can undergo significant divergence,
creating stable subclones with substantial genotypic and phenotypic differences.
Helicobacter pylori
colonizes the stomachs of about half of the world's population. The
infection is lifelong unless treated. A subpopulation of infected
individuals develops severe pathology, including peptic ulcers,
atrophic gastritis, and gastric adenocarcinoma. Unfortunately, it is
not yet possible to predict the outcome of H. pylori
infection in a colonized untreated individual or to identify human
populations at risk for significant pathology. All available data
indicate that the outcome is determined by factors emanating from the
host, the microorganism, and the environment (13, 21, 24).
H. pylori is a species with a plastic, adaptable genome that
presumably helps the bacteria to persist for decades in the
inhospitable niche of the human stomach. Isolates from different
individuals display considerable genomic variation as defined by
fingerprinting methods such as restriction fragment length polymorphism
(RFLP) or arbitrary primed PCR (AP-PCR) (2, 22). Strains
isolated from unrelated individuals rarely have identical fingerprints, in contrast to strains isolated from members of the same family (26). Most of this genomic variation appears to result in
silent, synonymous base substitutions (40) and thus is not
accompanied by great variations in the proteome. Still, it is
conceivable that this variation can give rise to cohabitating organisms
that differ in phenotype. This might be significant for the outcome of
infection. It also poses a technical challenge in evaluating the
virulence potential of a cultured bacterial population that is
recovered from an infected individual at a particular point during the
course of infection. If the net virulence of the population, determined
by the relative ratio of more- and less-virulent subclones, shifts to a
more-virulent composition, the host-microbial relationship could skew
towards pathogenesis.
Recent studies have focused on the mechanisms underlying this genomic
diversity (30, 31). The mutation frequency in H. pylori is unusually high (~10 One bacterial factor that has been ascribed particular significance
regarding the outcome of infection is the Cag pathogenicity island
(PAI) (10). The 38.5-kb Cag PAI contains 27 genes
(6). Some of its genes encode a multiprotein type IV
secretion system that translocates microbial compounds to host cells
(6, 33, 36, 38). Inactivation of the type IV secretion
machinery, by disruption of the cagE gene, produced a marked
reduction in pathogenic potential in a Mongolian gerbil model
(34). The cagA gene encodes a protein that,
like the intimin receptor (Tir) of enteropathogenic Escherichia
coli, is transferred and tyrosine phosphorylated in host cells
(29, 33, 36, 38). The CagA protein has been associated
with a more aggressive course of infection (6, 10).
These and other findings imply that bacteria that carry the Cag PAI are
more virulent and more likely to drive the host-microbial relationship
towards pathogenesis (7, 9, 10, 37). Identical 31-bp
sequences located at both ends of the Cag PAI facilitate incorporation
and deletion of this fragment (6). The ratio of Cag
PAI-positive to -negative isolates could change by clonal expansion of
the variant that best fits the requirements for survival in a given
host's niche at a given time during the course of infection (19). This, in turn, could affect the overall virulence of
the colonizing population in a host (7). The 31-bp repeats
can also serve to introduce the Cag PAI into previously Cag
PAI-deficient strains in a multistrain infection with Cag PAI-positive
and -negative bacteria (30).
The emergence of divergent clones within a single individual is
unlikely to be conferred solely by the PAI, but rather it is likely to
reflect general plasticity of the whole genome (35). A
recent case control study provided some support for this hypothesis (17). In an attempt to describe the clonal heterogeneity
in the H. pylori population within an individual, antral
biopsy samples were collected from patients referred for endoscopy for
their upper gastrointestinal tract symptoms (16). Colonies
from primary cultures were analyzed by AP-PCR (17). The
results revealed the existence of subclones within individual patients.
These subclones differed based on the presence or absence of the
cagA gene but had identical genomic fingerprints.
In this study, we have characterized two such H. pylori
subclones obtained from a single patient using DNA microarray-based whole-genome genotyping and an experimental transgenic mouse model. The
results reveal substantial genotypic as well as phenotypic differences.
Bacterial strains and culture conditions.
H.
pylori 67:20 (Cag PAI negative) and 67:21 (Cag PAI positive) were
isolated as single colonies from the primary culture of two pooled
antral biopsy samples obtained from a 90-year-old Swedish female with
gastric ulcer. Hp1 is a previously described clinical isolate obtained
from a Peruvian female with gastritis (25). The cultures
were stored at
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.12.7832-7838.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Comparison of Genetic Divergence and Fitness
between Two Subclones of Helicobacter pylori
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
7) (3a,
43). H. pylori seems to be a panmictic
species, i.e., recombination is frequent, and in addition, selective
sweeps where only a subset (the most-fit variants) of the population
survives are rare. Consequently, H. pylori lacks sequential
bottlenecks that purify the population (1, 23, 32, 39).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
70°C until analyzed.
Defining the genotypes of subclones 67:20 and 67:21. (i) Isolation of genomic DNA. Genomic DNA from single colonies was prepared with the respiratory sample preparation kit (Roche Diagnostic Systems, Branchburg, N.J.) as described in a previous study (17). DNA for sequence and microarray analyses was prepared using the QIAamp tissue kit (Qiagen GmbH, Hilden, Germany).
(ii) Fingerprinting. RFLP analysis of the flaA gene was performed to verify the strain identities of the two primary colonies according to the method of Enroth et al. (15). The amplified flaA fragment was digested with AluI, DpnII and HhaI, and the products were analyzed on a 1% agarose gel.
(iii) PCR. A PCR assay was used to distinguish an intact from an absent Cag PAI. The reaction mixture contained PCR buffer (Boehringer Mannheim GmbH, Mannheim, Germany), 100 µM concentrations of deoxynucleoside triphosphates, 0.5 U of DNA polymerase (Boehringer Mannheim), 7 µM concentrations of primers UpCagF (ACT TTC ACG CCC TTT CCC TCC) and DownCagR (TTG CAT GCG TTA TTA TTT CAC), and H. pylori genomic DNA. The cycling conditions were denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and elongation at 72°C for 1 min for 30 cycles followed by a final elongation step of 7 min at 72°C. This PCR assay generates a 562-bp PCR product if the PAI is absent and no product if the PAI is present.
(iv) Whole-genome genotyping using DNA microarrays. The two primary isolates, 67:20 and 67:21, were genotyped according to the method of Salama et al. (35). Each DNA microarray contained duplicate copies of a set of 1,660 PCR products from the two fully sequenced strains, TIGR 26695 and Astra J99 (3, 42). These PCR products represent 98.9% of the open reading frames (ORFs) in the 26695 and J99 genomes. Ninety-one ORFs were unique to J99 (26695 was arbitrarily set as the reference strain). Hp1, an isolate previously genotyped using these DNA microarrays (35), was used as a reference control.
Genomic DNA (2 µg) from either the 67:20 or 67:21 isolate was labeled with Cy5. Genomic DNA (1 µg) from each of the two control strains (26695, J99) was labeled with Cy3. The Cy3-labeled control DNA was then cohybridized with the Cy5-labeled test strain DNA using previously published protocols (35). All hybridizations were performed in duplicate. Data from duplicate datasets were merged and analyzed according to the method of Salama et al. (35). Previous control experiments using Cy3-26695 or Cy3-J99 DNA alone had established that the hybridization conditions employed for the microarray analysis result in 0% false positives with the 26695 probe and 7% false positives with the J99 probe. The false-negative rates are 1 and 2% for the 26695 and J99 probes, respectively (35). After hybridization and washing, microarrays were scanned on the red and green channels, and the red/green ratio for each gene was normalized as outlined in the information available at http://genome-www4.stanford.edu/Microarray/help/results_normalization.html. If the normalized red/green ratio for a gene was less than 0.5, it was called absent in the 67:20 or 67:21 strain.Studies using gnotobiotic transgenic mice.
FVB/N transgenic
mice, expressing the human 
-1,3/4-fucosyltransferase gene under the
control of transcriptional regulatory elements from a Fabp
gene, have been described in earlier reports (18, 25).
These mice produce the human histo-blood group antigen Lewisb (Leb)
(Fuc1,2Gal
1,3[Fuc1,4]GlcNAc) in their gastric pit and
surface mucus cells. This fucosylated epitope serves as a receptor for H. pylori adhesins (27).
Genotyping colonies recovered from the stomachs of colonized mice. To assess the stability of the genome in vivo, we investigated the original primary isolates from the human host and organisms recovered from infected mice. To verify the cagA status in the reisolated H. pylori isolate 67:21 after experimental infection, DNA was prepared from 1 to 16 primary colonies recovered from each mouse. In total, 332 colonies were analyzed by PCR according to previously described methods (17).
PCR amplifications of the recA (forward primer, 5'-GAA ATT TAT GGG CAG AGT C; reverse primer, 5'-GAT AAA AAT GAG AGT GGT GTT) (41), cagA (forward primer, 5'-TTG GAA ACC ACC TTT TGT ATT AGC; reverse primer, 5'-GTG CCT GCT AGT TTG TCA GCG) (17), and 16S rRNA (forward primer, 5'-TGG CAA TCA GCG TCA GGT AAT G; reverse primer, 5'-GCT AAG AGA TCA GCC TAT GTC C) (14) genes were performed according to standard procedures. Sequence analysis of these genes was also performed on the primary isolates and on 67:21 recovered from conventionally raised mice infected for 3 or 10 months (n = 2 mice/time point and 5 colonies per animal). The same genes were also sequenced from (i) five reisolates of 67:20 (except cagA) from ex-germ-free mice, (ii) five reisolates of 67:21 from ex-germ-free animals, and (iii) the two primary subclones after 50 rounds of in vitro passage. To identify genetic variation in the Cag PAI, we analyzed DNA using a custom microarray containing PCR products from each gene in the Cag PAI (A. Sillén, C. Nilsson, H. Enroth, P. Falk, and L. Engstrand, submitted for publication). These microarrays were used to assay 20 reisolated 67:21 colonies from a conventionally raised mouse infected for 3 months, 20 colonies from an ex-germ-free animal infected for 3 months, and 10 colonies from a conventionally raised animal infected for 10 months.| |
RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Genotyping studies indicate that 67:20 and 67:21 are subclones of
the same strain.
We used a stepwise approach to compare, with
increasing comprehensiveness, the genomes of two isolates recovered
from one H. pylori-infected patient. RFLP analysis of
flaA provides one way of distinguishing distinct H. pylori strains (15, 20). RFLP analysis yielded
identical patterns for the 67:20 and 67:21 isolates (Fig.
1). This finding was consistent with the
results of an earlier AP-PCR analysis of these two isolates
(17). In addition, analysis of the PCR fragments,
representing 318 bp of the recA ORF and 396 bp of the 16S
rRNA gene from the primary 67:20 and 67:21 clinical isolates, revealed
that they had identical nucleotide sequences.
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Phenotypic divergence manifested by differences in the ability of the subclones to colonize conventionally raised transgenic mice with an epithelial glycan receptor for H. pylori adhesin(s). To determine if the observed differences in the genotypes of the 67:20 and 67:21 isolates confer differences in their fitness, we investigated their ability to establish a niche in the stomachs of transgenic mice that are engineered to produce a receptor to a known H. pylori adhesin.
The ability of H. pylori to attach to the gastric epithelium represents the coincidence of host and microbial factors: the capacity of a colonizing strain to produce adhesins recognized by gastric epithelial receptors and the ability of the host to express these receptors prior to or during the course of infection. A majority of clinical isolates of H. pylori appear to be able to bind to the human histo-blood group antigen Leb (27). Approximately 70% of humans produce Leb-containing glycans in their gastric epithelia, where expression is restricted to the mucus-producing pit cell lineage (4, 27). Forced expression of the human-1,3/4 fucosyltransferase in the pit cell
lineage of neonatal and adult transgenic mice belonging to the FVB/N
strain allows the production of Leb-containing glycans
(18).
In vitro binding assays (25) disclosed that both 67:20 and
67:21 bound equally well to Leb-expressing pit cells
present in sections of transgenic mouse stomach. No binding was
detectable when the same organisms were incubated with stomach sections
prepared from Leb-negative nontransgenic littermates (data
not shown). These latter results indicate that the observed differences
in the genotypes of the subclones do not correlate with their capacity
to express adhesins that recognize these Leb glycan receptors.
The efficiency of colonization of conventionally raised adult
Leb transgenic mice with the Cag PAI-positive 67:21 isolate
was 74% (29 of 39 animals sacrificed 3 months after inoculation). This efficiency is similar to that observed with the unrelated Hp1 isolate,
containing the first 8 genes of the Cag PAI (HP0520 to HP0527)
(25). Ten months after inoculation, 6 of 9 conventionally raised animals were colonized with 67:21. In contrast, the Cag PAI-negative 67:20 subclone failed to colonize any conventionally raised transgenic mice (0 of 40 animals surveyed 3 months following inoculation).
To define colonization potential in the absence of a competing
indigenous microflora, 67:20 or 67:21 was introduced into the stomachs
of adult germ-free Leb transgenic mice. After 3 months,
100% (12 of 12) of the gnotobiotic transgenic mice were colonized with
the 67:21 isolate while 85% (12 of 14) of mice were colonized with the
Cag PAI-negative 67:20 isolate. There were no statistically significant
differences in the densities of colonization between mice harboring the
different subclones (range = 104 to 106
CFU/stomach).
67:20 and 67:21 subclones: a model strain pair for testing the role of Cag PAI in inducing inflammation. The 67:20 and 67:21 subclones provide an important opportunity to assess the role of the Cag PAI in inducing inflammation given the presence of the PAI in the one clone, its complete absence in the other, and the relatively high degree of genotypic similarities throughout the rest of their genomes. The ability to induce interleukin-8 (IL-8) production in cultured AGS gastric epithelial cells (8) was only seen in the 67:21 isolate (data not shown). This finding was not unexpected given that IL-8 induction in this cell line has been clearly associated with the Cag PAI (8, 9, 37). Surprisingly, a single-blind study failed to reveal any discernible differences in the histopathologic changes produced in the stomachs of ex-germ-free mice after a 3-month colonization with either the Cag PAI-negative 67:20 or Cag PAI-positive 67:21 subclone (n = 26 animals).
Other groups have reported that cagE inactivation of an already gerbil-passaged strain had a minor effect on the colonization of Mongolian gerbils, although IL-8 production and inflammation were diminished to the level obtained with Cag PAI-deficient strains (28, 34). However, when using a piglet-adapted 26695 strain in gnotobiotic piglets, or the well-established mouse-adapted strain SS1 in C57BL/6 mice, Eaton and coworkers noted that there was no effect of inactivation of the Cag PAI on the ability to colonize or to induce inflammation (12). Our findings in gnotobiotic Leb transgenic mice are consistent with these latter findings. In addition, studies in conventionally raised Leb transgenic mice support the notion that elements of the Cag PAI may help H. pylori to compete with other microbial species in patients that harbor a complex indigenous gastric flora (e.g., as in chronic atrophic gastritis).Gnotobiotic transgenic mice reveal that the subclones exhibit a high degree of genetic stability. The rate at which subclones emerge during the course of infection with H. pylori is not known. Studies of humans suggest that genetic drift can occur over the course of several years (31). To examine the time frame during which changes may occur in an individual subclone (67:21), we examined single colonies recovered after a 3- or 10-month infection of Leb transgenic mice (n = 140 colonies from conventionally raised animals infected for 3 months, n = 152 colonies from conventionally raised animals infected for 10 months, and n = 40 colonies from ex-germ-free animals mono-associated for 3 months). PCR studies revealed that all reisolates remained positive for cagA.
Since partial deletions in the Cag PAI would not be detected by the cagA PCR assay, we proceeded to analyze 67:21 colonies reisolated from one ex-germ-free mouse infected for 3 months, one conventionally raised mouse infected for 3 months, and one conventionally raised mouse infected for 10 months. DNA microarrays containing all of the Cag PAI genes were employed for this survey of 20 colonies from the first two experimental conditions and 10 colonies from the long-term infection. All reisolates contained all the genes of the Cag PAI, just like the primary 67:21 clinical isolate. PCR and DNA microarray analyses can detect major genomic deletions. A large portion of the genetic diversity of H. pylori arises from single-base mutations (43). Therefore, we compared the nucleotide sequences of three genes in five reisolates per experimental condition. After passage in conventionally raised and ex-germ-free mice for 3 months or in conventionally raised mice for 10 months, no nucleotide substitutions were detected in cagA (only 67:21), recA and 16S rRNA genes in any of the reisolates. The same was true when the 67:21 isolate was passaged 50 times in vitro. (See Materials and Methods for a list of the primers used to generate the 322-bp cagA, 318-bp recA, and 396-bp 16S rRNA gene fragments used for this sequence analysis.) It is possible that there is less selective pressure exerted on H. pylori for genetic divergence in the environment of the germ-free or conventionally raised mouse stomach than in the human stomach. Alternatively, the 67:20 and 67:21 subclones may be derived from a parent whose genotype (e.g., repertoire of restriction modification and DNA repair enzymes) favors a relatively slow rate of genetic evolution compared to other strains. It has been postulated that in humans, there may be clonal expansion of Cag PAI-positive and -negative subclones from the same colonizing strain within the same host (7, 17). This presumably occurs in an ecosystem with few competing microbial species as long as acid production is maintained. Therefore, it is interesting to note that in conventionally raised Leb transgenic mice with dense indigenous gastric microbial populations, there were no detectable Cag PAI-negative clones after 10 months of infection with the Cag PAI-positive isolate. This might indicate selection of Cag PAI-positive clones in an environment where H. pylori benefits from a competitive edge against other microorganisms, or that the 3- to 10-month time frame of the colonization experiments is simply too short for any discernible genetic changes to occur.Prospectus. We have determined that two H. pylori subclones, recovered from a 90-year-old human with gastric ulcer disease, have undergone divergent genetic and phenotypic evolution since the time of their separation. Our findings lend support to the concept that clonal expansion of more-fit variants may provide a means for the bacterial population to coevolve with its host and adapt to changes in the gastric niche. These studies emphasize the importance of initiating longitudinal studies of H. pylori-infected humans over an extended time scale (decades) so that subspecies development can be examined using tools, such as DNA microarrays, that are emerging from the current revolution in genomics and proteomics.
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ACKNOWLEDGMENTS |
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We thank Margareta Rodensjö, Gunilla Bergo, Lena Ericsson, and Ewa Österlund for technical assistance. We are grateful to Stanley Falkow and Tore Midtvedt for allowing us to conduct parts of this study in their labs.
This work was supported by grants from the Swedish Cancer Society, the Swedish Research Council, the Swedish Foundation for Strategic Research, and the National Institutes of Health.
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FOOTNOTES |
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* Corresponding author. Mailing address: Swedish Institute for Infectious Disease Control, 171 82 Solna, Sweden. Phone: 46-8-457 24 15. Fax: 46-8-30 17 97. E-mail: lars.engstrand{at}smi.ki.se.
Editor: V. J. DiRita
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