Genomic Changes during Chronic Helicobacter pylori Infection
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细菌学杂志 2006年第1期
Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany,Institute of Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Str. 2, D-97080 Würzburg, Germany,Berna Biotech, Ltd., Berne, Switzerland,Louisiana State University Medical Center, New Orleans, Louisiana,Division of Comparative Medicine, MIT, Cambridge, Massachusetts 02139,Mathematical Genetics Group, Department of Statistics, University of Oxford, Peter Medawar Building for Pathogen Research, South Parks Road, Oxford OX1 3SY, United Kingdom
ABSTRACT
The gastric pathogen Helicobacter pylori shows tremendous genetic variability within human populations, both in gene content and at the sequence level. We investigated how this variability arises by comparing the genome content of 21 closely related pairs of isolates taken from the same patient at different time points. The comparisons were performed by hybridization with whole-genome DNA microarrays. All loci where microarrays indicated a genomic change were sequenced to confirm the events. The number of genomic changes was compared to the number of homologous replacement events without loss or gain of genes that we had previously determined by multilocus sequence analysis and mathematical modeling based on the sequence data. Our analysis showed that the great majority of genetic changes were due to homologous recombination, with 1/650 events leading to a net gain or loss of genes. These results suggest that adaptation of H. pylori to the host individual may principally occur through sequence changes rather than loss or gain of genes.
INTRODUCTION
Helicobacter pylori is thought to be the second-most-prevalent human bacterial pathogen, infecting the stomachs of more than one-half of the human population (20). H. pylori induces chronic gastritis in all infected persons and causes complications such as peptic ulcers, gastric cancer, and gastric lymphoma of the mucosa-associated lymphoid tissue in subgroups of patients. H. pylori shows exceptional genetic variability and intraspecies diversity. Allelic diversity is so high that almost every unrelated isolate of H. pylori has a unique sequence when a fragment of several hundred base pairs is sequenced from housekeeping or virulence genes (7, 12, 19). This allelic diversity is the result of the combination of a high (mutator-type) mutation rate (4), a high frequency of recombination between strains during mixed colonization (8, 13, 19), and the ability of H. pylori to integrate unusually small pieces of exogenous DNA into its chromosome (6, 19).
Much of the current knowledge about genetic diversity of H. pylori has been gained from population genetic analysis of sequence data obtained from H. pylori strains isolated from unrelated persons of diverse geographical origins (1, 17). Since H. pylori infection is chronic and can persist for decades, an alternative approach is the analysis of genetic relationships between H. pylori strains that have been isolated sequentially from the same patient. We have used mathematical modeling on sequence data from 24 pairs of sequential H. pylori isolates from Colombia and the United States to estimate basic parameters of recombination and mutation of H. pylori in vivo. The data showed that the estimated mean size of imported fragments was only 417 bp, the shortest import length that has been reported for a bacterium. Half of the genome is replaced by homologous sequence in a time frame estimated to be from 40 to 2,000 years (6). The wide credibility region of this estimate is principally due to a high degree of uncertainty about the time since the last common ancestor of each pair of strains, which were taken from middle-aged patients at intervals from 3 months to 4 years, but even the higher values in this range represents extremely rapid import of DNA compared with other bacteria (6).
H. pylori strains not only show allelic diversity but also differ in their gene contents. The two completely sequenced genomes of H. pylori strains 26695 and J99 share only 94% of their genes (2), whereas approximately 7% of the genes are unique for each strain, respectively (22). A comparison of 15 strains with DNA microarrays showed that in this set of strains, 81 of 1,643 genes present on the array (combined from 26695 and J99 genomes) were shared by all strains, whereas 22% of genes were only present in some of the strains (15). We have recently further refined the estimate of the number of genes present in all H. pylori strains to 1,111 by comparative genome hybridizations performed with a globally representative collection of 54 H. pylori strains (9). Furthermore, Israel et al. found numerous genomic differences between H. pylori J99, a strain whose genome sequence was published in 1999, and multiple isolates cultured from the same patient at the same time as J99 and 6 years later (10). Similar results were found in another study, where two clones of a patient were taken at the same time and compared to each other (3).
These results suggested that the H. pylori genome has a highly plastic gene content. To quantitate the rate of genomic changes (gene loss or acquisition) during chronic colonization and to compare it to rates of allelic replacement events, we have used whole-genome DNA microarray hybridization to study gene loss and gene acquisition in the same collection of sequential H. pylori isolates previously studied to determine the rates of mutation and recombination in vivo (6). Where the microarray data indicated a possible genomic change, the event was verified by PCR and sequence analysis. Changes of gene content occurred in 5 of the 21 pairs analyzed, indicating significant plasticity of the genome content. However, quantitative analysis shows that genetic events leading to loss or acquisition of genes are far less frequent than events leading to allelic exchanges alone.
MATERIALS AND METHODS
Bacteria. Strains were isolated and single-colony purified from patients in Narino, Colombia (10 pairs; designated the NQ strain collection), and New Orleans, LA (14 pairs; designated the LSU strain collection), as described previously (6, 21). The time interval between the isolations of the first and follow-up strains was 3 to 48 months for the LSU strains and 36 months for the NQ strains. Of the 24 pairs characterized in the previous study (6), 2 were excluded because they were completely unrelated according to their DNA sequences. Two further pairs of strains were identical at 3 and 5 of the 10 sequenced fragments, respectively, making their degree of clonal relatedness ambiguous. These strains were analyzed by microarrays. The former pair was unrelated in gene content according to microarray analysis and was excluded from further analysis, while the latter pair was identical (no single difference) according to microarrays and was included, resulting in 21 pairs of related strains in total.
Whole-genome DNA microarray. The composition of the H. pylori whole-genome microarray used in this study has been previously described (14). It is based on PCR products derived from the two available H. pylori whole-genome sequences of strains 26695 (22) and J99 (2), and contains 1,655 probes representing 96.1% of genes present in H. pylori 26695 or J99. The performance of the array for H. pylori genome comparisons was validated by experiments comparing the two strains, 26695 and J99, whose genomes have been sequenced. These experiments showed that we detected 100% of the known strain-specific genes of J99 and 97.3% of the known strain-specific genes of 26695 as defined by Salama et al. (15). Four strain-specific genes of 26695 (HP0315, HP1001, HP1516, and HP1537) were incorrectly scored as present in J99 when a conservative cutoff of 2 for the ratio was used but scored as absent using a cutoff of 1.5. For these genes, overall low signal intensities were obtained under our hybridization conditions, very likely due to short sequence lengths and/or low G-C content of these genes, making scoring errors more likely.
Hybridization. Fluorescent labeling of DNA and competitive hybridizations were performed as described by Salama et al. (15), with several modifications. Whole-genomic DNA was prepared from blood agar plate-grown H. pylori with QIAGEN Tip-100 columns. Labeling of the DNA with aminoallyl-dUTP was performed with the BioPrime DNA labeling system (Invitrogen) minus the deoxynucleoside triphosphates (dNTP) set. A dNTP set was made using 0.5 mM dGTP, dATP, dCTP, 0.3 mM dTTP (Amersham Pharmacia), and 0.2 mM aminoallyl dUTP (Sigma). One microgram of DNA was diluted to 24 μl in water, 20 μl of random octamer-primer solution was added, and they were heated for 5 min at 99°C. The DNA was then placed directly on ice, 5 μl of dNTPs and 1 μl of high-fidelity Klenow enzyme were added to the reaction mixture, and the reaction was incubated at 37°C for 1 h. The reaction was stopped with 5 μl 0.5 M Na2EDTA, pH 8.0. Purification was done by a modified QIAGEN PCR purification protocol. PE and AE buffers (QIAGEN) were replaced with 5 mM KPO4 (pH 8.0)-80% EtOH and 4 mM KPO4 (pH 8.5), respectively. The Cy3 and Cy5 dyes (Amersham Pharmacia) were diluted using 72 μl of water, and 4.5 μl was aliquoted and dried before storage in the dark at 4°C. Labeling of the probe with the Cy dyes followed the protocol by Salama et al. (15) with the exception that before hybridization the Cy3 and Cy5 probes were combined and 100 μg of yeast tRNA was added. After being dried, the probe was resuspended in 32 μl of hybridization buffer (50% formamide, 6x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.5% sodium dodecyl sulfate, 50 mM Na phosphate [pH 8.0], and 5x Denhardt's solution). The probe was heated for 2 min at 99°C and applied to the slide. Microarray scanning and data processing were preformed as previously described (11, 18).
Empty-site PCR and sequencing. All loci where the microarray analysis indicated that loss or gain of genes had occurred were further characterized by PCR and sequence analysis. The loci were amplified by PCR with primers targeting genes that flank the deletion-acquisition event. PCR products were purified and sequenced as previously described (19).
Southern hybridization. Loss of insertion (IS) element copies was verified by Southern blot hybridization under high-stringency conditions (16). Genomic DNAs of the LSU1062 strains and the pair NQ315-NQ1712 were digested by EcoRI and electrophoretically separated on a 1% agarose gel. A 600-bp fragment of HP0414, which is part of the IS606 element, labeled with digoxigenin-dUTP was used as the probe.
Accession numbers. All new sequences generated in this study have been submitted to the GenBank/EMBL/DDBJ databases (accession numbers AM086402 to AM086418).
RESULTS
Comparisons of the genomes of sequentially isolated H. pylori strains by whole-genome DNA microarray hybridization. We analyzed the genomes of 21 pairs of H. pylori strains isolated from predominantly middle-aged, adult patients in Narino, Colombia (NQ strains), and New Orleans (LSU strains). Each pair of isolates was taken from the same patients at two different time points (these time points are referred to below as early and later). These pairs were compared by competitive hybridization with a whole-genome DNA microarray based on the two published genome sequences (14). The strategy used to screen for genomic changes in sequential isolates of H. pylori was a variant of the classical approach to comparative genome hybridization, where a test strain is hybridized against the strain upon whose genome the microarray is based (15). To permit more reliable identification of genomic changes, pairs of sequential isolates were directly hybridized against each other. Thus, genomic DNA of the paired strains was labeled with Cy3 or Cy5 and competitively hybridized to the microarray. The arrays were scanned for the two colors separately, and a signal ratio was calculated.
A signal ratio of 1 indicated the presence of the gene in both strains. Signal ratios of <0.5 or >2 were interpreted as evidence of potential loss or gain of the respective gene. When both channels yielded signal intensities of <350, the gene was excluded from the analysis, the most likely reason being the absence of the gene in both strains. This cutoff for missing genes was determined empirically, with data from comparative genome hybridizations of either strain LSU2003-1 or strain LSU1010-1, with 26695 and J99 on the same microarray (9) being used for calibration.
26695 and J99 have 1,590 and 1,495 predicted genes, respectively, of which 96% were represented on the array. An average of 1,453 spots were detected per tested genome pair. Assuming that they have a genome size similar to those of J99 and 26695, this indicates that the great majority of genes contained in the NQ and LSU strains were assayed by our array system.
In three of seven of NQ strain pairs and 9 out of 14 of LSU strain pairs, microarray hybridizations did not yield evidence of single-genome content difference. These pairs were not investigated further. In the remaining nine pairs, microarray hybridizations indicated potential gene content differences at one or more loci.
To validate the results of microarray hybridizations and to further analyze the events underlying the genomic changes, "empty-site PCRs" were designed for all changes indicated by the microarray hybridizations and sequenced. Primers were designed based on the sequences of genes flanking the predicted genomic events. The validation experiments confirmed the predicted changes in six cases (Table 1; Fig. 1 and 2A). An additional two events indicated by the microarray experiments could be explained by homologous recombination events, which changed the degree of sequence homology to the DNA spotted on the microarray but involved no loss or gain of sequence (Fig. 2B). Analysis by PCR and sequence analysis did not confirm five of the changes that were predicted from the microarray results, because the genes were found either to be absent from both strains or not to show any sequence differences. Only those events validated by sequencing were included in the subsequent calculations.
Changes involving the cag PAI. Two sequentially isolated strain pairs showed differences in the cag pathogenicity island (cag PAI) (5). NQ1712 lacked the entire cag PAI and one of the two imperfect copies of the 31-bp repeat sequence flanking the island in the earlier isolate NQ315 (Fig. 1A). The sequences upstream and downstream of the remaining copy of the repeat were identical to the sequence of NQ315, indicating that a deletion had occurred by intrachromosomal recombination between the two 31-bp repeat sequences and subsequent excision of the PAI, rather than by allelic replacement after import of an empty-site allele from an unrelated strain, as described by Kersulyte et al. (13).
A partial loss of the cag island was observed in a second pair, LSU1062 (Fig. 1B). The earlier strain, LSU1062-1, contained a complete cag PAI that was followed by a complete IS606 element, consisting of tnpA and tnpB homologs. The deletion in LSU1062-3 started in the "middle repeat region" of gene HP0527 (cagY) and comprised all remaining cag island genes (HP0528 to -0548), as well as part of the IS606 element (tnpA and the first 192 nucleotides of tnpB).
Changes involving IS elements and restriction-modification systems. Within the LSU1062 pair, the later strain showed evidence of having lost its only copy of the IS606 element. The 26695-J99 whole-genome microarray contains several spots that hybridize with components of IS elements (e.g., the tnpA and tnpB homologs HP0413/0414, HP1007/1008, and JHP826/827). In the LSU1062 pair, none of these spots showed hybridization with the later strain, in contrast to the earlier strain. To ascertain that no other copies of IS elements were still present in the genome of the later strain, a Southern blot hybridization was performed, which confirmed that the later strain did not contain any copy of these IS elements. Southern blot hybridization also showed that an IS606 element was lost in the strain pair NQ315/1712. The location of this event could not be determined.
The later strain of the LSU1016 pair contained the restriction-modification system HpyAIV (HP1351/1352), of which only small fragments were present in the earlier strain, interrupted by a putative open reading frame of unknown function. In the later strain, this region contained the complete restriction-modification system (Fig. 1C).
Changes involving the plasticity region. In addition to the cag PAI, a region termed the "plasticity zone" has been described as harboring the majority of strain specific genes (2). Microarray hybridization indicated that 14 plasticity region genes (HP0990 to -0995 and JHP929 to -936) were present in the later strain of the LSU1014 pair that were absent from the earlier strain. The event underlying this change could not be further characterized by empty-site PCR, because the region flanking the acquired genes was too different from strains 26695 and J99. The microarray results were therefore verified by single-gene PCRs for three selected genes (HP0995, JHP933, and JHP934), which were positive only in the later strain and therefore confirmed the microarray results in all cases.
Changes involving housekeeping genes. Microarray hybridization indicated a genomic difference affecting the ceuE genes (HP1561 and HP1562) in pair NQ315/1712. PCR analyses showed that, in fact, the earlier strain NQ315 contained a single copy of ceuE, while NQ1712 contained two paralogous copies arranged in tandem, which is similar to the situation in the sequenced strains 26695 and J99, where the two gene copies have >90% nucleotide identity (Fig. 1D). Mosaic sequences were identified upstream and downstream of the ceuE genes, suggesting that the fragment containing one ceuE copy present in the earlier strain had been replaced by a fragment containing two copies in the later strain or vice versa.
The microarray data indicated absence of a cluster of pseudogenes (HP0903/0904) that contains truncated homologs of an acetate kinase gene (ackA; HP0903) and a phosphotransacetylase gene (pta; HP0904) in the later strain of pair LSU1040. Empty-site sequence analysis showed that HP0903 was truncated in both paired strains (Fig. 1E). In the later strain, a recombination event had caused an even larger truncation of HP0903 and also the partial deletion of gene HP0904. The total size of the deletion was approximately 1 kb. A mosaic sequence was only present downstream of the deletion.
DISCUSSION
A whole-genome microarray was used to compare the genome content of paired sequential isolates of H. pylori. We detected only six genomic changes in 21 pairs, despite the fact that we sampled the great majority of the genome. The strains are undoubtedly closely related to each other, because they were isolated from the same patient at a short time interval. The strains come from middle-aged individuals and thus are likely to represent well-established infections, so we may not have captured changes that happened during the initial period of adaptation to the host. Despite the high degree of conservation of gene content, our previous analysis of sequence variations in 10 gene fragments showed that a large number of recombination events had taken place in the same strains (6). In that study, we estimated that across the genome an average of 114 recombination events had occurred per pair. However, this estimate excluded the pair of strains with the highest number of differences (5 of 10 gene fragments), which we have now shown to be clonally related and thus should be included in the analysis. Inclusion of this strain pair raises the estimated average total number of homologous recombination events to 194 per pair or 4,000 for all 21 strain pairs. Thus, genetic changes associated with gain or loss of sequence were 650 times rarer than simple homologous replacement events.
In three of six cases, the later strain had fewer genes than the earlier strain; in the other three cases, it had more. However, we cannot be certain whether each of these events were a deletion or an acquisition, because the event may have occurred before the isolation of the first strain, with both variants cocolonizing the stomach, when the first sample was taken. Indeed, samples were taken at intermediate time points for a subgroup of pairs (Table 2), which in one case (LSU1014) showed both strains coexisting during the observation period.
With one exception, all genomic changes occurred among the 7 of 21 pairs in which recombination had been detected within the 10 sequenced gene fragments (Table 1). Indeed, four of the six events required homologous recombination with DNA from another H. pylori genome, the two exceptions being deletions of the cag PAI. This emphasizes the importance of transient or persistent cocolonization by two or more unrelated H. pylori strains for generating genomic changes in H. pylori.
The observed deletions could have been produced by intrachromosomal events or by import of empty-site alleles from unrelated strains. In the latter case, the import should have resulted in mosaic sequences flanking the deletion. Surprisingly, in most cases, mosaic sequences were only identified on one side of the deletion (Fig. 1 and 2). The mechanism responsible for this phenomenon is not known. No mosaic sequences were found in the pair that deleted the cag PAI, indicating that recombination between the imperfect copies of the 31-bp repeat were sufficient to delete the island, presumably by a RecA-dependent mechanism, similar to an event described by Bjrkholm et al. (3).
It is widely assumed that H. pylori uses genetic variation to adapt to individual hosts and specific niches within a host. While some variation can be generated in the absence of mixed colonization (e.g., by point mutations, slipped-strand mispairing, or intrachromosomal recombination), import of DNA fragments from an exogenous H. pylori strain appears to be the principal source of allelic variation, as well as genomic changes in H. pylori. Most of these events involve no loss or gain of sequence and are likely to have phenotypic and fitness effects that are subtle and difficult to detect.
The effectiveness of recombination as an adaptive strategy depends critically on the prevalence of H. pylori in a population. H. pylori is likely to have evolved in a situation when infection was quasi-universal, permitting the panmictic exchange of genes and alleles. With declining prevalence of H. pylori, coinfections are likely to become much rarer, making genetic exchange ineffective as a means of genetic adaptation, which in turn may further accelerate the disappearance of H. pylori from certain populations.
ACKNOWLEDGMENTS
We thank Matthias Frosch, Mark Achtman, and Thomas F. Meyer for encouragement and support.
This work was supported by grants PTJ-BIO 031U213B from the Bundesministerium für Bildung und Forschung competence center PathoGenoMik and SFB479/A5 from the Deutsche Forschungsgemeinschaft to S.S. and by DFG grant Jo 344/2-1 to C.J.
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ABSTRACT
The gastric pathogen Helicobacter pylori shows tremendous genetic variability within human populations, both in gene content and at the sequence level. We investigated how this variability arises by comparing the genome content of 21 closely related pairs of isolates taken from the same patient at different time points. The comparisons were performed by hybridization with whole-genome DNA microarrays. All loci where microarrays indicated a genomic change were sequenced to confirm the events. The number of genomic changes was compared to the number of homologous replacement events without loss or gain of genes that we had previously determined by multilocus sequence analysis and mathematical modeling based on the sequence data. Our analysis showed that the great majority of genetic changes were due to homologous recombination, with 1/650 events leading to a net gain or loss of genes. These results suggest that adaptation of H. pylori to the host individual may principally occur through sequence changes rather than loss or gain of genes.
INTRODUCTION
Helicobacter pylori is thought to be the second-most-prevalent human bacterial pathogen, infecting the stomachs of more than one-half of the human population (20). H. pylori induces chronic gastritis in all infected persons and causes complications such as peptic ulcers, gastric cancer, and gastric lymphoma of the mucosa-associated lymphoid tissue in subgroups of patients. H. pylori shows exceptional genetic variability and intraspecies diversity. Allelic diversity is so high that almost every unrelated isolate of H. pylori has a unique sequence when a fragment of several hundred base pairs is sequenced from housekeeping or virulence genes (7, 12, 19). This allelic diversity is the result of the combination of a high (mutator-type) mutation rate (4), a high frequency of recombination between strains during mixed colonization (8, 13, 19), and the ability of H. pylori to integrate unusually small pieces of exogenous DNA into its chromosome (6, 19).
Much of the current knowledge about genetic diversity of H. pylori has been gained from population genetic analysis of sequence data obtained from H. pylori strains isolated from unrelated persons of diverse geographical origins (1, 17). Since H. pylori infection is chronic and can persist for decades, an alternative approach is the analysis of genetic relationships between H. pylori strains that have been isolated sequentially from the same patient. We have used mathematical modeling on sequence data from 24 pairs of sequential H. pylori isolates from Colombia and the United States to estimate basic parameters of recombination and mutation of H. pylori in vivo. The data showed that the estimated mean size of imported fragments was only 417 bp, the shortest import length that has been reported for a bacterium. Half of the genome is replaced by homologous sequence in a time frame estimated to be from 40 to 2,000 years (6). The wide credibility region of this estimate is principally due to a high degree of uncertainty about the time since the last common ancestor of each pair of strains, which were taken from middle-aged patients at intervals from 3 months to 4 years, but even the higher values in this range represents extremely rapid import of DNA compared with other bacteria (6).
H. pylori strains not only show allelic diversity but also differ in their gene contents. The two completely sequenced genomes of H. pylori strains 26695 and J99 share only 94% of their genes (2), whereas approximately 7% of the genes are unique for each strain, respectively (22). A comparison of 15 strains with DNA microarrays showed that in this set of strains, 81 of 1,643 genes present on the array (combined from 26695 and J99 genomes) were shared by all strains, whereas 22% of genes were only present in some of the strains (15). We have recently further refined the estimate of the number of genes present in all H. pylori strains to 1,111 by comparative genome hybridizations performed with a globally representative collection of 54 H. pylori strains (9). Furthermore, Israel et al. found numerous genomic differences between H. pylori J99, a strain whose genome sequence was published in 1999, and multiple isolates cultured from the same patient at the same time as J99 and 6 years later (10). Similar results were found in another study, where two clones of a patient were taken at the same time and compared to each other (3).
These results suggested that the H. pylori genome has a highly plastic gene content. To quantitate the rate of genomic changes (gene loss or acquisition) during chronic colonization and to compare it to rates of allelic replacement events, we have used whole-genome DNA microarray hybridization to study gene loss and gene acquisition in the same collection of sequential H. pylori isolates previously studied to determine the rates of mutation and recombination in vivo (6). Where the microarray data indicated a possible genomic change, the event was verified by PCR and sequence analysis. Changes of gene content occurred in 5 of the 21 pairs analyzed, indicating significant plasticity of the genome content. However, quantitative analysis shows that genetic events leading to loss or acquisition of genes are far less frequent than events leading to allelic exchanges alone.
MATERIALS AND METHODS
Bacteria. Strains were isolated and single-colony purified from patients in Narino, Colombia (10 pairs; designated the NQ strain collection), and New Orleans, LA (14 pairs; designated the LSU strain collection), as described previously (6, 21). The time interval between the isolations of the first and follow-up strains was 3 to 48 months for the LSU strains and 36 months for the NQ strains. Of the 24 pairs characterized in the previous study (6), 2 were excluded because they were completely unrelated according to their DNA sequences. Two further pairs of strains were identical at 3 and 5 of the 10 sequenced fragments, respectively, making their degree of clonal relatedness ambiguous. These strains were analyzed by microarrays. The former pair was unrelated in gene content according to microarray analysis and was excluded from further analysis, while the latter pair was identical (no single difference) according to microarrays and was included, resulting in 21 pairs of related strains in total.
Whole-genome DNA microarray. The composition of the H. pylori whole-genome microarray used in this study has been previously described (14). It is based on PCR products derived from the two available H. pylori whole-genome sequences of strains 26695 (22) and J99 (2), and contains 1,655 probes representing 96.1% of genes present in H. pylori 26695 or J99. The performance of the array for H. pylori genome comparisons was validated by experiments comparing the two strains, 26695 and J99, whose genomes have been sequenced. These experiments showed that we detected 100% of the known strain-specific genes of J99 and 97.3% of the known strain-specific genes of 26695 as defined by Salama et al. (15). Four strain-specific genes of 26695 (HP0315, HP1001, HP1516, and HP1537) were incorrectly scored as present in J99 when a conservative cutoff of 2 for the ratio was used but scored as absent using a cutoff of 1.5. For these genes, overall low signal intensities were obtained under our hybridization conditions, very likely due to short sequence lengths and/or low G-C content of these genes, making scoring errors more likely.
Hybridization. Fluorescent labeling of DNA and competitive hybridizations were performed as described by Salama et al. (15), with several modifications. Whole-genomic DNA was prepared from blood agar plate-grown H. pylori with QIAGEN Tip-100 columns. Labeling of the DNA with aminoallyl-dUTP was performed with the BioPrime DNA labeling system (Invitrogen) minus the deoxynucleoside triphosphates (dNTP) set. A dNTP set was made using 0.5 mM dGTP, dATP, dCTP, 0.3 mM dTTP (Amersham Pharmacia), and 0.2 mM aminoallyl dUTP (Sigma). One microgram of DNA was diluted to 24 μl in water, 20 μl of random octamer-primer solution was added, and they were heated for 5 min at 99°C. The DNA was then placed directly on ice, 5 μl of dNTPs and 1 μl of high-fidelity Klenow enzyme were added to the reaction mixture, and the reaction was incubated at 37°C for 1 h. The reaction was stopped with 5 μl 0.5 M Na2EDTA, pH 8.0. Purification was done by a modified QIAGEN PCR purification protocol. PE and AE buffers (QIAGEN) were replaced with 5 mM KPO4 (pH 8.0)-80% EtOH and 4 mM KPO4 (pH 8.5), respectively. The Cy3 and Cy5 dyes (Amersham Pharmacia) were diluted using 72 μl of water, and 4.5 μl was aliquoted and dried before storage in the dark at 4°C. Labeling of the probe with the Cy dyes followed the protocol by Salama et al. (15) with the exception that before hybridization the Cy3 and Cy5 probes were combined and 100 μg of yeast tRNA was added. After being dried, the probe was resuspended in 32 μl of hybridization buffer (50% formamide, 6x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.5% sodium dodecyl sulfate, 50 mM Na phosphate [pH 8.0], and 5x Denhardt's solution). The probe was heated for 2 min at 99°C and applied to the slide. Microarray scanning and data processing were preformed as previously described (11, 18).
Empty-site PCR and sequencing. All loci where the microarray analysis indicated that loss or gain of genes had occurred were further characterized by PCR and sequence analysis. The loci were amplified by PCR with primers targeting genes that flank the deletion-acquisition event. PCR products were purified and sequenced as previously described (19).
Southern hybridization. Loss of insertion (IS) element copies was verified by Southern blot hybridization under high-stringency conditions (16). Genomic DNAs of the LSU1062 strains and the pair NQ315-NQ1712 were digested by EcoRI and electrophoretically separated on a 1% agarose gel. A 600-bp fragment of HP0414, which is part of the IS606 element, labeled with digoxigenin-dUTP was used as the probe.
Accession numbers. All new sequences generated in this study have been submitted to the GenBank/EMBL/DDBJ databases (accession numbers AM086402 to AM086418).
RESULTS
Comparisons of the genomes of sequentially isolated H. pylori strains by whole-genome DNA microarray hybridization. We analyzed the genomes of 21 pairs of H. pylori strains isolated from predominantly middle-aged, adult patients in Narino, Colombia (NQ strains), and New Orleans (LSU strains). Each pair of isolates was taken from the same patients at two different time points (these time points are referred to below as early and later). These pairs were compared by competitive hybridization with a whole-genome DNA microarray based on the two published genome sequences (14). The strategy used to screen for genomic changes in sequential isolates of H. pylori was a variant of the classical approach to comparative genome hybridization, where a test strain is hybridized against the strain upon whose genome the microarray is based (15). To permit more reliable identification of genomic changes, pairs of sequential isolates were directly hybridized against each other. Thus, genomic DNA of the paired strains was labeled with Cy3 or Cy5 and competitively hybridized to the microarray. The arrays were scanned for the two colors separately, and a signal ratio was calculated.
A signal ratio of 1 indicated the presence of the gene in both strains. Signal ratios of <0.5 or >2 were interpreted as evidence of potential loss or gain of the respective gene. When both channels yielded signal intensities of <350, the gene was excluded from the analysis, the most likely reason being the absence of the gene in both strains. This cutoff for missing genes was determined empirically, with data from comparative genome hybridizations of either strain LSU2003-1 or strain LSU1010-1, with 26695 and J99 on the same microarray (9) being used for calibration.
26695 and J99 have 1,590 and 1,495 predicted genes, respectively, of which 96% were represented on the array. An average of 1,453 spots were detected per tested genome pair. Assuming that they have a genome size similar to those of J99 and 26695, this indicates that the great majority of genes contained in the NQ and LSU strains were assayed by our array system.
In three of seven of NQ strain pairs and 9 out of 14 of LSU strain pairs, microarray hybridizations did not yield evidence of single-genome content difference. These pairs were not investigated further. In the remaining nine pairs, microarray hybridizations indicated potential gene content differences at one or more loci.
To validate the results of microarray hybridizations and to further analyze the events underlying the genomic changes, "empty-site PCRs" were designed for all changes indicated by the microarray hybridizations and sequenced. Primers were designed based on the sequences of genes flanking the predicted genomic events. The validation experiments confirmed the predicted changes in six cases (Table 1; Fig. 1 and 2A). An additional two events indicated by the microarray experiments could be explained by homologous recombination events, which changed the degree of sequence homology to the DNA spotted on the microarray but involved no loss or gain of sequence (Fig. 2B). Analysis by PCR and sequence analysis did not confirm five of the changes that were predicted from the microarray results, because the genes were found either to be absent from both strains or not to show any sequence differences. Only those events validated by sequencing were included in the subsequent calculations.
Changes involving the cag PAI. Two sequentially isolated strain pairs showed differences in the cag pathogenicity island (cag PAI) (5). NQ1712 lacked the entire cag PAI and one of the two imperfect copies of the 31-bp repeat sequence flanking the island in the earlier isolate NQ315 (Fig. 1A). The sequences upstream and downstream of the remaining copy of the repeat were identical to the sequence of NQ315, indicating that a deletion had occurred by intrachromosomal recombination between the two 31-bp repeat sequences and subsequent excision of the PAI, rather than by allelic replacement after import of an empty-site allele from an unrelated strain, as described by Kersulyte et al. (13).
A partial loss of the cag island was observed in a second pair, LSU1062 (Fig. 1B). The earlier strain, LSU1062-1, contained a complete cag PAI that was followed by a complete IS606 element, consisting of tnpA and tnpB homologs. The deletion in LSU1062-3 started in the "middle repeat region" of gene HP0527 (cagY) and comprised all remaining cag island genes (HP0528 to -0548), as well as part of the IS606 element (tnpA and the first 192 nucleotides of tnpB).
Changes involving IS elements and restriction-modification systems. Within the LSU1062 pair, the later strain showed evidence of having lost its only copy of the IS606 element. The 26695-J99 whole-genome microarray contains several spots that hybridize with components of IS elements (e.g., the tnpA and tnpB homologs HP0413/0414, HP1007/1008, and JHP826/827). In the LSU1062 pair, none of these spots showed hybridization with the later strain, in contrast to the earlier strain. To ascertain that no other copies of IS elements were still present in the genome of the later strain, a Southern blot hybridization was performed, which confirmed that the later strain did not contain any copy of these IS elements. Southern blot hybridization also showed that an IS606 element was lost in the strain pair NQ315/1712. The location of this event could not be determined.
The later strain of the LSU1016 pair contained the restriction-modification system HpyAIV (HP1351/1352), of which only small fragments were present in the earlier strain, interrupted by a putative open reading frame of unknown function. In the later strain, this region contained the complete restriction-modification system (Fig. 1C).
Changes involving the plasticity region. In addition to the cag PAI, a region termed the "plasticity zone" has been described as harboring the majority of strain specific genes (2). Microarray hybridization indicated that 14 plasticity region genes (HP0990 to -0995 and JHP929 to -936) were present in the later strain of the LSU1014 pair that were absent from the earlier strain. The event underlying this change could not be further characterized by empty-site PCR, because the region flanking the acquired genes was too different from strains 26695 and J99. The microarray results were therefore verified by single-gene PCRs for three selected genes (HP0995, JHP933, and JHP934), which were positive only in the later strain and therefore confirmed the microarray results in all cases.
Changes involving housekeeping genes. Microarray hybridization indicated a genomic difference affecting the ceuE genes (HP1561 and HP1562) in pair NQ315/1712. PCR analyses showed that, in fact, the earlier strain NQ315 contained a single copy of ceuE, while NQ1712 contained two paralogous copies arranged in tandem, which is similar to the situation in the sequenced strains 26695 and J99, where the two gene copies have >90% nucleotide identity (Fig. 1D). Mosaic sequences were identified upstream and downstream of the ceuE genes, suggesting that the fragment containing one ceuE copy present in the earlier strain had been replaced by a fragment containing two copies in the later strain or vice versa.
The microarray data indicated absence of a cluster of pseudogenes (HP0903/0904) that contains truncated homologs of an acetate kinase gene (ackA; HP0903) and a phosphotransacetylase gene (pta; HP0904) in the later strain of pair LSU1040. Empty-site sequence analysis showed that HP0903 was truncated in both paired strains (Fig. 1E). In the later strain, a recombination event had caused an even larger truncation of HP0903 and also the partial deletion of gene HP0904. The total size of the deletion was approximately 1 kb. A mosaic sequence was only present downstream of the deletion.
DISCUSSION
A whole-genome microarray was used to compare the genome content of paired sequential isolates of H. pylori. We detected only six genomic changes in 21 pairs, despite the fact that we sampled the great majority of the genome. The strains are undoubtedly closely related to each other, because they were isolated from the same patient at a short time interval. The strains come from middle-aged individuals and thus are likely to represent well-established infections, so we may not have captured changes that happened during the initial period of adaptation to the host. Despite the high degree of conservation of gene content, our previous analysis of sequence variations in 10 gene fragments showed that a large number of recombination events had taken place in the same strains (6). In that study, we estimated that across the genome an average of 114 recombination events had occurred per pair. However, this estimate excluded the pair of strains with the highest number of differences (5 of 10 gene fragments), which we have now shown to be clonally related and thus should be included in the analysis. Inclusion of this strain pair raises the estimated average total number of homologous recombination events to 194 per pair or 4,000 for all 21 strain pairs. Thus, genetic changes associated with gain or loss of sequence were 650 times rarer than simple homologous replacement events.
In three of six cases, the later strain had fewer genes than the earlier strain; in the other three cases, it had more. However, we cannot be certain whether each of these events were a deletion or an acquisition, because the event may have occurred before the isolation of the first strain, with both variants cocolonizing the stomach, when the first sample was taken. Indeed, samples were taken at intermediate time points for a subgroup of pairs (Table 2), which in one case (LSU1014) showed both strains coexisting during the observation period.
With one exception, all genomic changes occurred among the 7 of 21 pairs in which recombination had been detected within the 10 sequenced gene fragments (Table 1). Indeed, four of the six events required homologous recombination with DNA from another H. pylori genome, the two exceptions being deletions of the cag PAI. This emphasizes the importance of transient or persistent cocolonization by two or more unrelated H. pylori strains for generating genomic changes in H. pylori.
The observed deletions could have been produced by intrachromosomal events or by import of empty-site alleles from unrelated strains. In the latter case, the import should have resulted in mosaic sequences flanking the deletion. Surprisingly, in most cases, mosaic sequences were only identified on one side of the deletion (Fig. 1 and 2). The mechanism responsible for this phenomenon is not known. No mosaic sequences were found in the pair that deleted the cag PAI, indicating that recombination between the imperfect copies of the 31-bp repeat were sufficient to delete the island, presumably by a RecA-dependent mechanism, similar to an event described by Bjrkholm et al. (3).
It is widely assumed that H. pylori uses genetic variation to adapt to individual hosts and specific niches within a host. While some variation can be generated in the absence of mixed colonization (e.g., by point mutations, slipped-strand mispairing, or intrachromosomal recombination), import of DNA fragments from an exogenous H. pylori strain appears to be the principal source of allelic variation, as well as genomic changes in H. pylori. Most of these events involve no loss or gain of sequence and are likely to have phenotypic and fitness effects that are subtle and difficult to detect.
The effectiveness of recombination as an adaptive strategy depends critically on the prevalence of H. pylori in a population. H. pylori is likely to have evolved in a situation when infection was quasi-universal, permitting the panmictic exchange of genes and alleles. With declining prevalence of H. pylori, coinfections are likely to become much rarer, making genetic exchange ineffective as a means of genetic adaptation, which in turn may further accelerate the disappearance of H. pylori from certain populations.
ACKNOWLEDGMENTS
We thank Matthias Frosch, Mark Achtman, and Thomas F. Meyer for encouragement and support.
This work was supported by grants PTJ-BIO 031U213B from the Bundesministerium für Bildung und Forschung competence center PathoGenoMik and SFB479/A5 from the Deutsche Forschungsgemeinschaft to S.S. and by DFG grant Jo 344/2-1 to C.J.
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