1 Laboratory of Enteric Pathogens, Specialist and Reference Microbiology Division, Health Protection Agency, London NW9 5HT, UK
2 Department of Endoscopy and Gastrointestinal Endoscopy, National Cancer Centre Hospital, Tokyo 104-0045, Japan
3 The Centre for Digestive Diseases, The General Infirmary at Leeds, Leeds LS1 3EX, UK
4 Health Protection Agency Laboratory, York Road, Leeds LS15 7TR, UK
Correspondence
Robert Owen
robert.owen{at}hpa.org.uk
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ABSTRACT |
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INTRODUCTION |
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Isolates of H. pylori, irrespective of geographical location, have identical phenotypes as defined by conventional bacteriological tests (Owen et al., 2001b) although there is evidence that strains can differ in some expressed features such as vacuolating cytotoxin activity (Forsyth et al., 1998
), lectin reaction patterns (Hynes et al., 2002
) and interleukin-8 induction in gastric epithelial cells (Owen et al., 2003
). In contrast, strains of H. pylori from unrelated individuals exhibit an unusually high level of genomic diversity as indicated by various fingerprinting techniques (Owen et al., 2001b
). Multilocus sequence analyses indicate that the species has a panmictic (non-clonal) population structure due to frequent horizontal transfer and free recombination of metabolic enzyme genes, and of pathogenicity-associated genes such as cagA and vacA (Suerbaum et al., 1998
; Achtman et al., 1999
). These genetic events are thought to occur in individual patients during mixed gastric colonization by unrelated strains over several years (Falush et al., 2001
). Nevertheless, analysis of sequence variability also provides evidence of two weakly clonal groupings (Asian, and African/Afro-American group) in strains from different parts of the world despite extensive inter-strain recombination masking evidence of phylogenetic relatedness (Achtman et al., 1999
). Sequence analysis of housekeeping and pathogenicity-associated genes, mainly on isolates from Switzerland but with representatives from China and South Africa, also demonstrated that the species existed as a recombinant population, but no evolutionary relationships between strains could be discerned in that strain set (Solca et al., 2001
). More recently, evidence that H. pylori can be divided into seven populations and subpopulations with distinct geographical distributions was established from a more extensive analysis of multilocus data implementing a Bayesian approach for deducing population structure (Falush et al., 2003
). The possibility that geographical separation of H. pylori may have resulted in distinct populations evolving on different continents is also suggested by analyses of the virulence-associated cagA and vacA genes, where differences were reported between isolates originating from China and Japan compared to those from the United States and Europe (van der Ende et al., 1998
; Ito et al., 1997
; Pan et al., 1998
; van Doorn et al., 1998
; Yamaoka et al., 1998
). Geographically associated characters have also been demonstrated for urease subunit genes (Campbell et al., 1997
), and for an outer-membrane protein gene (HP0638) (Ando et al., 2002
).
H. pylori from different parts of the world, nevertheless, remain difficult to stratify in relation to disease severity. Our study focuses on patient isolates from Japan to define East Asia population-specific markers by comparison with isolates from subSaharan Africa, represented by Nigeria and South Africa, and from the United Kingdom, represented by England and Northern Ireland. Sequence diversity was examined in core fragments of two established housekeeping genes (ureI and atpA), and at a previously uninvestigated locus (ahpC), in H. pylori from unrelated individuals who were selected wherever possible as members of the local indigenous population. We aimed also to determine the frequencies of the specific East Asian markers in the other strain subpopulations.
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METHODS |
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Nine geographically diverse but internationally recognized reference strains of H. pylori were also examined. Seven cultures were obtained (in lyophilized form) from the National Collection of Type Cultures (NCTC): NCTC 11637 (type strain, Australia); NCTC 11638 (Australia); NCTC 13081 (strain Tx30a, USA); NCTC 13206 (CCUG 38770, antibiotic susceptibility control strain, Belgium) (Glupczynski et al., 2001); NCTC 13207 (CCUG 38772, antibiotic susceptibility control strain, France) (Glupczynski et al., 2001
); NCTC 12455 (strain 26695, genome-sequenced strain, England) (Tomb et al., 1997
); and NCTC 12491 (formerly type strain of Helicobacter nemestrinae, USA) (Suerbaum et al., 2002
). Cultures of the genome-sequenced strain J99 (USA) (Alm et al., 1999
) and of strain SS1 (Australia) (Lee et al., 1997
) were kindly provided by Professor D. Taylor (University of Alberta, Canada) and Dr S. Rypkema (National Institute for Biological Standards and Control, Potters Bar, UK), respectively.
Culture conditions.
All strains of H. pylori from biopsy specimens and allied species were cultured for 23 days on Columbia agar base (Oxoid) containing 10 % (v/v) defibrinated horse blood, and incubated at 37 °C under microaerobic conditions (4 % oxygen, 5 % hydrogen, 5 % carbon dioxide and 86 % nitrogen) in a variable atmosphere incubator (Don Whitley Scientific). Identity of H. pylori was confirmed by Gram staining, and catalase, cytochrome oxidase and urease tests. Stock cultures were preserved on glass beads at -80 °C (Microbank system; Pro-Lab Diagnostics) or over liquid nitrogen.
DNA preparation, PCR and sequencing.
Genomic DNA was extracted from sweep cultures by the method of Wilson (1987). Diluted DNA (100 ng) was used to amplify an internal fragment from three genes (ureI, atpA and ahpC) which were selected because of their potential roles in core cytoplasmic metabolic housekeeping activities and not in encoding outer-membrane proteins or secreted proteins that might be under greater selection pressure in a stressful environment such as the gut mucosal surface. The ureI locus, which was included previously in a panel of seven housekeeping genes used for a population genetic study (Achtman et al., 1999
), is part of the H. pylori urease gene cluster and encodes a urea accessory (transporter) protein thought to be an integral cytoplasmic membrane protein forming a proton-gated urea-channel-regulating cytoplasmic urease (Weeks et al., 2000
; Mobley, 2001
). The following primers were designed for amplification and sequencing of a 585 bp fragment within the ureI sequence (HP0071) of H. pylori strain 26695: forward primer (ureIS4) 5'-GGAAGGAAAAGGCAATGC-3' and reverse primer (ureIAS2) 5'-CTAAACGCTCTATGATCA-3'. These primers were used as alternatives to those described by Achtman et al. (1999)
, which were initially tested but did not amplify ureI from all our isolates. The target region for these primers corresponded closely to those described by Achtman et al. (1999)
.
The atpA gene encodes an ATP synthase F1 alpha homologue forming the catalytic portion of the multisubunit enzyme found in the cytoplasmic membrane. The enzyme has a key function in the synthesis of ATP and may have a role in adaptation by H. pylori to acid stress by proton translocation (McGowan et al., 1997). Primers for amplification and sequencing of a 627 bp region within the atpA (HP1134) sequence were as described by Achtman et al. (1999)
.
The ahpC gene encodes the AhpC subunit of an alkylhydroperoxide reductase a cysteine-based peroxidase homologue involved in the defence against oxidative stress. It is present in H. pylori strain 26695 (HP1563) as well as in at least eight other species of Helicobacter (Lundstrom et al., 2001), and although not used in previous sequence typing studies, it was evaluated in the present study as a potential novel locus to provide population-specific markers. The forward primer (HP26:F) 5'-TTAGTTACAAAACTTGCCCC-3', and reverse primer (HP26:R) 5'-GCTTTCATCCCTTTATCGCC-3') were used to amplify and to sequence a 542 bp fragment as described by Lundstrom et al. (2001)
.
PCR and nucleotide sequencing.
The three sets of primers were synthesized commercially (MWG BIOTECH) and PCR amplification reactions were performed in a total volume of 50 µl containing 100 ng of diluted template DNA, 1·5 mM MgCl2, 0·05 mM each deoxynucleotide (dATP, dCTP, dGTP and dTTP), 0·4 mM each oligonucleotide primer, 0·2 µl (1 U) Taq polymerase (Invitrogen) and 5 µl of 10x buffer provided by the manufacturer. Automated sequencing from both strands of PCR products of the internal fragments was then performed by standard protocols either in house using a CQ2000 sequencer (Coulter Beckman) or commercially (MWG BIOTECH). Use of these six primer sets enabled complete double-stranded sequences to be obtained from a single sequencing run in each direction. Twenty-one sequences representing the range of diversity of loci within the East Asian (four strains) and Nigerian (two strains) population sets have been deposited in EMBL with accession numbers AJ583137AJ583157.
Phylogenetic and polymorphism analyses.
The ureI, ahpC and atpA sequences of 76 isolates and seven reference strains of H. pylori as well as additional H. pylori sequences deposited in GenBank for the two genome-sequenced strains (26695 and J99) and nine other miscellaneous strains (Japan, 2 strains; China, 2 strains; Thailand, 2 strains; Gambia, 2 strains; and South Africa, 1 strain) were aligned using CLUSTAL W as implemented in BioEdit (v 5.0.9) (Hall, 1999). Sequences of the three loci for Helicobacter mustelae NCTC 12198 were included in the analyses to provide an outlier to root trees. The sets of aligned sequences for each gene were analysed by the neighbour-joining method of Saitou & Nei (1987)
using the nucleotide substitution model of Dukes & Cantor (1969
), and trees were produced in TREECON (van de Peer & De Wachter, 1994
). Bootstrap sampling analyses were performed with 100 resampled datasets to define confidence limits in the estimated phylogenies. Phylogenetic analyses were also performed using a dataset in which the three sequences (1754 bp) were concatenated in the order ureI/atpA/ahpC for each strain. Further analysis of nucleotide sequences was performed with DnaSP v 3.51 (Rozas & Rozas, 1995
) to determine base composition, the number of polymorphic sites including the number of parsimony-informative sites (PI), and deduced amino acid sequences. Also calculated were the proportions of synonymous substitutions (KS), defined as the percentage of mean differences between pairs of strains at synonymous sites (mutations not resulting in amino acid substitutions) and the proportion of non-synonymous substitutions (KA) defined as the percentage of mean differences between pairs of strains at non-synonymous sites (mutations resulting in amino acid substitutions). Low KS/KA ratios (<1·0) indicated frequent non-synonymous site variation. Sequences were screened to identify single and multiple nucleotide polymorphisms (NPs) with population-specific associations.
Assessment of H. pylori cagA and vacA genotype status.
The primers and PCR conditions for the two assays for cagA (a marker for the 3' end of the cag pathogenicity island and for the cagI region) were as described previously using the F1/B1 primers and the D008/R008 primer sets (Slater et al., 1999; Owen et al., 2001a
). Vacuolating cytotoxin (vacA) genotyping based on signal and mid-region alleles was performed as previously described (Atherton et al., 1999
; Owen et al., 2002
).
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RESULTS |
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The three phylogenetic trees obtained from separate analyses of the ureI, atpA and ahpC loci confirmed the distinctiveness of the East Asian and the Nigerian isolates. By contrast, the detailed structures and internal order of strains within the resultant trees, especially for those of the UK and South Africa isolates, were not completely concordant with each other or with the composite tree (results not presented). For the ureI and atpA analyses, additional sequences from GenBank showed that one Nigerian isolate (H2495) from clade B was closely linked to strain 5596/Gambia, which was a representative of clone 2 (Achtman et al., 1999). Furthermore, three strains (R29/Japan, 97-42/China and 88-8/Thailand), part of the Asian clone (Achtman et al., 1999
), were closely related to clade A. Two other East Asian isolates (F32/Japan and 88-39/Thailand) fell outside clade A and were excluded from further analyses as ethnic details of patients could not be confirmed.
Identification of H. pylori nucleotide and amino acid sequence types
The numbers of H. pylori representing each geographical group and results on nucleotide sequence type (NST) and amino acid sequence type (AST) derived for the three genes (ureI, atpA and ahpC) are listed in Table 1. There was a high degree of sequence diversity amongst alleles from all isolates. Every patient was infected by a strain with a unique NST except for the paired antrum/corpus isolates from eleven Japanese and from five UK patients, where paired strains had an identical ST. In the UK set, there were 17 NSTs for both ureI and atpA compared to 18 for ahpC the difference was attributed to two isolates being from members of the same family and they had identical alleles at the former two loci. Nucleotide sequences were translated into amino acid sequences and amino acid sequence types were defined (Table 1
). The precise significance of these differences is difficult to assess fully because of the small number of strain sequences analysed and their relatively short lengths (<200 codons); nevertheless, the results suggested that AtpA was relatively more highly conserved in the Japanese and UK population sets whereas AhpC was more conserved in the Nigerian strains. For instance, in the UK (non-Asian) subset, there were just five AtpA ASTs for the 23 isolates compared to 17 NSTs for that locus. Furthermore, AtpA AST-1 was a feature of most (12/16) isolates in the UK set and of most (13/16) isolates in the Japanese set. By contrast, all except one isolate (7/8) in the Nigerian set were AhpC AST-11, which was a type rare in the other geographical groups.
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The cagA and vacA genotype status of geographical strain sets
Overall, the cagA gene was present in 86 % of all the strains of H. pylori and for individual population sets the highest frequency of 100 % was in the Nigerian isolates (8/8) (Table 1). The commonest overall vacA genotype was s1m1 (67 %) and that type was a feature of all Japanese isolates, although the Chinese and Thai isolates were typically s1m2 (4/5) (Table 1
). The m2 subtype was more common in the UK isolates (14/24, 58 %) compared to the other sets (East Asian, 4/22 patients; Nigeria, 1/8 patients; and South Africa, 2/7 patients). The s2m2 type was less common (11 %) present in the UK set (six patients), in the South African set (one patient), and in the reference set (three patients; SS1, Tx30a and CCUG 38772), whereas the combination s2m1 was not found in any of the strains.
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DISCUSSION |
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Additional evidence in support of distinctions between the East Asian isolates and those from other geographical regions was apparent from our analysis of other parameters of genetic variability, in particular the proportion of synonymous to non-synonymous substitutions in the target sequences. Synonymous substitutions, particularly at the third codon position, are generally more frequent and so contribute most to divergence between strains. However, they are biologically neutral whereas an increased proportion of non-synonymous mutations indicates increased organismhost interaction and the selection of protein variants for instance a low KS/KA ratio of about 4 for cagA could be interpreted as an indicator of antigenic diversity (van der Ende et al., 1998). In H. pylori, synonymous mutations are estimated to be four times more frequent than non-synonymous mutations (Falush et al., 2001
), so marked deviations in frequencies may provide valuable population markers. Interestingly, we found several such differences in the KS/KA ratios when a mean value of 24 for bacterial genes was used as a comparative reference (van der Ende et al., 1998
). The Japanese isolates had similar ratios (about 14·5) for both ureI and ahpC whereas the ratio for atpA of 56·6 was higher (fourfold) and indicated positive selection pressure to conserve the catalytic site of the AtpA protein in that population. Most striking was the sixfold difference between the ahpC mean KS/KA ratio of 90·6 for the Nigerian isolates compared to the ratio of 14·6 for the Japanese isolates. Most Nigerian strains had the same AhpC amino acid type, indicating that the protein was highly conserved in that population. By contrast, the South African and UK strain sets had ahpC and atpA KS/KA ratios of 38·165·4, which indicated some variability, with a general trend of decreased variability compared to the Japanese but increased variability compared to the Nigerian set. By contrast, the ureI ratios were of the same order (11·216·3) irrespective of geographical origin, but with a bias compared to the other two gene products towards increased non-synonymous substitution and a higher number of UreI amino acid types. The mean ureI ratio of 14·1 for all strain sets in this study closely matched the mean value of 16·2 reported previously by Achtman et al. (1999)
. As the number of isolates tested was small, the statistical significance of these observations needs to be validated on larger strain sets and their biological significance established.
These associations between markers in H. pylori housekeeping gene sequences and geographical origin were in agreement with other reports that East Asian isolates had independent virulence-associated genotypic features for instance, the type II cag right-junction motif was present in 95 % of East Asian strains compared to 1 % or less of strains from Africa, North America and South Asia (Kersulyte et al., 2000). Moreover, independent cag pathogenicity-island-related evidence that East Asian strains may constitute a genetically distinct population was provided by analysis of the 5' region of cag A (Yamaoka et al., 1998
), of other regions of cagA (van der Ende et al., 1998
) and of the right end of the cag pathogenicity island (Kersulyte et al., 2000
), as well as by presence of the vacA s1c subtype allele as an East Asian strain marker (van Doorn et al., 1998
). Recently, analysis of polymorphism motifs in the ORF (HP0638) encoding the OipA protein segregated East Asian isolates from isolates from the Indian subcontinent and Western countries including African-American isolates (Ando et al., 2002
). Our findings support the concept of a nonrandom geographical distribution of certain polymorphism motifs in unrelated genes in the H. pylori genome. No geographical segregation was observed in phylogenetic analysis based either on atpD, scoB, glnA and recA sequences (Solca et al., 2001
) or on glmM sequences (van der Ende et al., 1998
), which could indicate that some genes contain only limited or no geographical characteristics although the strain selection in those studies may have influenced interpretation. Sequence disruptions due to frequent recombination and mutation events mask evidence of clonality in all isolates except for those from more recent infections amongst family members (Suerbaum et al., 1998
; Owen & Xerry, 2003
), and multiple or sequential isolates from the same individual (Falush et al., 2001
), so evidence from several unrelated loci supports the relative stability of the geographical motifs. The latter point was confirmed in the present study, as all marker polymorphisms were identical in paired isolates from different gastric sites in 11 of the Japanese and in five of the UK patients and in some family isolates. Overall, the Japanese polymorphisms were relatively rare in H. pylori infecting the UK and South African patients, although data on a larger number of isolates are necessary for rigorous statistical analysis.
Covacci et al. (1999) suggested from the global geographical distribution of H. pylori that the species might have co-evolved with man over prolonged periods of some 50 000 years of human migration leading to genetically distinct human and H. pylori populations. The deduced population structure from multilocus data of the four modern populations of H. pylori suggests that their gene pools were derived from ancestral populations that arose in Africa, Central Asia and East Asia (Falush et al., 2003
). However, attempts to define subpopulations within European isolates (the hpEurope population) of H. pylori are confounded by the complex history of human migrations (Falush et al., 2003
). Analysis of the UK isolates including the Indian subgroup in the present study showed that while they were distinct from the African and East Asian variants, their gene sequences contained a higher proportion of alleles of African origin. They were also more diverse than the East Asian and Nigerian strains with respect to cagA presence and vacA alleles. Our overall findings are consistent with a humanbacterial comigration hypothesis (Falush et al., 2003
) although it is essential for interpretation of population markers to take into account the more recent history of human hosts, as suggested by similarities between Spanish and Latin American strains (Kersulyte et al., 2000
). Our investigation of sequences from strains from several neighbouring countries in the East Asian region (Japan, China and Thailand) confirmed the presence of common marker polymorphisms for local geographical variants of H. pylori, and gives support to the concept of a distinct Asian clone (Achtman et al., 1999
), subsequently designated as the hpEastAsia subpopulation (Falush et al., 2003
). The ureI and atpA sequences for Asian clone strains 88-28, R29 and 97-42 included in the present analyses segregated in clade A with our Japanese (Tokyo) isolates, and had the typical East Asian marker polymorphisms. The associations of our European isolates with the hpEurope population represented by strain 26695 await further clarification, as do the associations of the Nigerian strains with the two modern Africa populations (hpAfrica1 and hpAfrica2).
In conclusion, the nucleotide polymorphism analysis described here provides new information on the biogeographical diversity of H. pylori, and in particular identifies specific polymorphisms that could be used to define the Japanese subpopulation, which we propose should be designated H. pylori geovar orientalis' according to the general criteria of Staley (1999). Application of this biogeographical approach to other population groups could provide a more precise framework for investigating the subtleties of H. pylorihost interactions and disease associations.
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ACKNOWLEDGEMENTS |
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Received 8 May 2003;
revised 18 September 2003;
accepted 3 October 2003.
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