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Campylobacter and Helicobacter Research/Reference Unit, Laboratory of Enteric Pathogens, Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London NW9 5HT, UK
Correspondence
Robert Owen
robert.owen{at}hpa.org.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AMO88759–AMO88775.
Accession numbers, source strains and geographical origins of the other GenBank sequences analysed are available as supplementary data with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Gebert et al., 2004), which infects the gastric mucosa of about half of the human population worldwide (Pounder & Ng, 1995). Most infected individuals remain asymptomatic even though the presence of H. pylori typically induces chronic gastritis. In some infected individuals (<20 %), H. pylori is an important causal factor in the development of peptic ulcer disease (gastric and duodenal ulcers) and gastric low-grade mucosal-associated lymphoid tissue (MALT) lymphoma as well as being a key risk factor in the development of gastric cancer (Suerbaum & Michetti, 2002). The presence of H. pylori DNA has been associated with other chronic diseases such as cardiovascular and hepatobiliary disease but viable bacteria have not been isolated from such conditions and direct causality remains unproven (Aceti et al., 2004; Huang et al., 2004; Apostolov et al., 2005).
VacA is a cytotoxin of the gastric epithelial cell layer and a potent immunoregulatory toxin inducing apoptosis in epithelial cells (Boquet et al., 2003; Gebert et al., 2004). The precise mechanisms by which VacA contributes to the onset of chronic disease are unclear but its incidence appears to involve a complex interaction of aetiological factors that include dietary and genetic differences of the host, and bacterial virulence cofactors (Höcker & Hohenberger, 2003). The vacA gene, present as a single copy in the genomes of all H. pylori tested, is highly polymorphic particularly in the signal and mid-regions (Owen et al., 2002; Blaser & Atherton, 2004). Its mosaic structure is the result of frequent recombination (Atherton et al., 1995; Falush et al., 2001). Strains from many parts of the world have been characterized by the sizes of the regions encoding the N-terminal signal (s) peptide, which determines cytotoxic activity (Letley & Atherton 2000; McClain et al., 2001), and the mid (m) 58 kDa toxin subunit, which includes the cell-binding domain determining target cell specificity (Pagliaccia et al., 1998; Atherton et al., 1999a). Common vacA allelic forms (genotypes) identified worldwide are s1/m1 (vacuolating), s1/m2 (selectively vacuolating) and s2/m2 (non-vacuolating), although the latter form is rare in East Asian strains and the s2/m1 form is extremely rare in all populations (Letley et al., 1999). The signal region allelic families can be further divided into s1a, s1b and s1c subfamilies and likewise the mid-region is subdivided into m2a and m2b subfamilies (van Doorn et al., 1998). The vacA genotype is of clinical interest as some alleles have been correlated with the level of vacuolating cytotoxin activity in HeLa cells, with the s1/m1 genotype being more toxigenic than the s1/m2 genotype, whereas toxin activity is blocked in the s2/m2 genotype (Letley & Atherton, 2000). The s1 form is often linked to more severe gastric inflammation; even so, the presence of H. pylori with a particular vacA allelic variant does not necessarily provide a reliable predictor of pathogenic potential (Blaser & Atherton, 2004).
Campbell et al. (1997) presented evidence from an investigation of ethnic groups in New Zealand that genetically distinct H. pylori lineages may have evolved in parallel with race-specific specialization. Subsequent population genetic studies including multilocus sequence typing (MLST) showed that H. pylori from different parts of the world represent seven distinct populations and subpopulations, with modern geographical distributions apparently linked to historical global human migrations (Suerbaum et al., 1998; Achtman et al., 1999; Covacci et al., 1999, Ghose et al., 2002; Falush et al., 2003, Wirth et al., 2004; Kauser et al., 2005). Such genetic analyses have highlighted the panmictic (non-clonal) nature of the species structure and, most significantly, have demonstrated the presence of conserved residual genomic features that can be linked to co-evolution with man. The East Asian (Eastern-type) isolates, particularly those of Japanese and Chinese origin, appear to represent a distinct modern subpopulation. Furthermore, Japanese-specific polymorphisms have been identified within several housekeeping genes (Owen et al., 2004). Geographical separation of H. pylori in distinct separately evolving populations is supported by analyses of the gene encoding CagA, a marker of the cag pathogenicity island, as well as by vacA, in which characteristic differences were identified between isolates originating from China and Japan (East Asian type) compared to those from the USA and Europe (Western type) (van der Ende et al., 1998; Ito et al., 1997; Pan et al., 1998; van Doorn et al., 1998; Yamaoka et al., 1998; van Doorn et al., 1999; Kersulyte et al., 2000). East Asian strains typically have the vacA subtype s1c allele, which is rare in other parts of the world (van Doorn et al., 1998). In Europe, a distribution gradient was observed, with most strains in Northern Europe being s1a whereas in Spain and Portugal most strains were s1b. It remains unclear, however, to what extent H. pylori strain type might contribute to the four times higher rates of gastric cancer observed in Japanese and other East Asians compared to the United Kingdom population (Neugut et al., 1996; Lambert et al., 2002).
The application of gene sequence analysis to improve the understanding of population structures of bacterial pathogens (Cooper & Feil, 2004) has resulted in a rapid expansion in the availability of high-quality DNA sequences. As various complete and partial H. pylori vacA sequences are now available from public databases, the aim of our study was to utilize that unique information in a comprehensive comparative genomic analysis to define more precisely the global nature of variation of insert sequences within the signal and mid-region allelic families of vacA present in the modern H. pylori gene pool.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Naylor et al., 2006). Nine reference cultures of H. pylori were obtained in lyophilized form from the National Collection of Type Cultures (NCTC, London): NCTC 11637 (type strain, Australia); NCTC 11638 (Australia); NCTC 13081 (strain Tx30a, USA); NCTC 13082 (Canada), NCTC 13085 (strain 60190, England), NCTC 13086 (Canada), NCTC 11916 (England), NCTC 13207 (CCUG 38772), NCTC 12455 (strain 26695, genome sequenced strain, England; Tomb et al., 1997). 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, England), respectively.
Bacterial culture conditions.
All strains of H. pylori were recovered from the archived stock of cultures preserved on glass beads at –80 °C (Microbank system, Pro-Lab Diagnostics). Strains were subcultured for 2–3 days on Columbia agar base (Oxoid) containing 10 % (v/v) defibrinated horse blood and incubated at 37 °C under microaerophilic conditions (4 % oxygen, 5 % hydrogen, 5 % carbon dioxide and 86 % nitrogen) in a MACS-VA500 microaerophilic workstation (Don Whitley Scientific). Identity of H. pylori cultures was confirmed by Gram staining and by tests for catalase, cytochrome oxidase and urease activity.
PCR and sequencing.
Genomic DNA was extracted from sweep cultures by the method of Wilson (1987). Diluted DNA (100 ng) was used to amplify internal fragments from vacA signal- and mid-regions as previously described (Atherton et al., 1999b; Owen et al., 2002). Briefly the primers used for amplification and sequencing were as follows. For the signal region 259 bp fragment, the primer pair used was forward primer VAIF (5'-ATGGAAATACAACAAACACAC-3') and reverse primer VAIR (5'-CTGCTTGAATGCGCCAAAC-3'). For the mid-region 566 bp fragment, the primer pair used was: forward primer VAGF (5'-CAATCTGTCCAATCAAGCGAG-3') and reverse primer VAGR (5'-GCGTCAAAATAATTCCAAGG-3'). The primers were synthesized commercially (MWG Biotech) and PCR amplification reactions were performed in a total volume of 50 µl containing 100 ng diluted template DNA, 1.5 mM MgCl2, 0.05 mM of each deoxynucleotide (dATP, dCTP, dGTP and dTTP), 0.4 mM of each oligonucleotide primer, 0.2 µl (1 U) Taq polymerase (Invitrogen) and 5 µl 10x buffer, provided by the manufacturer. Automated nucleotide sequencing, from both strands of PCR products, of the internal fragments of vacA was then performed using the CEQ dye terminator cycle sequencing quick start kit run on a Beckman CEQ 8000 (Beckman Coulter). The primer sets used enabled complete double-stranded nucleotide sequences to be obtained from a single sequencing run in each direction.
Nucleotide sequences used in comparative genomic analysis.
The dataset compiled for this study comprised 550 complete and three partial sequences of vacA for isolates of H. pylori originating from patients in 32 countries in widely separated geographical regions. The set included a total of 484 vacA signal region sequences representing isolates from 30 countries (Table 1). For the signal region s1 allelic family, 60 sequences were determined in-house and 223 were obtained from GenBank. For the signal region s2 allelic family, 159 sequences were determined in-house and 42 were obtained from GenBank. The subset of vacA mid-region sequences represented a total of 411 isolates of H. pylori from 27 countries (Table 1). For the mid-region m1 allelic family, 43 sequences were determined in-house and 113 were obtained from GenBank. For the mid-region m2 allelic family, sequences of 179 isolates were determined in-house and 76 were obtained from GenBank. Seventeen partial vacA (mid-region) sequences determined in this study have been deposited in the EMBL nucleotide sequence database with the accession numbers AMO88759–AMO88775. Accession numbers of the other GenBank sequences used are provided in supplementary Table S1, available with the online version of this paper.
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). Translated and predicted amino acid sequences were analysed visually for marker motifs. Nucleotide and amino acid sequence motifs were compared to public databases using BLAST analysis to identify possible phylogenetic origins. The sets of aligned sequences for signal and mid-regions of vacA were analysed by the neighbour-joining method (Saitou & Nei, 1987) using the nucleotide substitution model of Jukes & Cantor (1969). Phylogenetic trees from nucleotide and predicted amino acid sequences rooted with reference to either NCTC 12455 (genome sequenced strain, vacA s1/m1 form) or Tx30a (vacA s2/m2 form), as well as unrooted trees, were generated in TREECON (van de Peer & de Wachter, 1994).
Statistical analysis.
The datasets were analysed using GraphPad InStat version 3.05. Differences between groups were compared using Fisher's exact one-tailed test. Results were considered statistically significant at a P value of <0.05.
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RESULTS |
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For the signal region inserts, 98 % had G+C contents of 
52 mol% and nucleotide sequences in which polymorphisms were limited to single synonymous substitutions despite diversity in strain geographical origin, encompassing 17 widely separated countries (Table 1
). The nucleotide sequences of each of these inserts translated to give a common predicted nine amino acid sequence NDPIHSESR (SRI type A, Table 2). Inserts with a lower G+C content of 48 mol% were identified in three isolates originating from Wales (one patient) and from South Africa (two patients); these had a single S to N substitution at amino acid position 8 (SRI type B). The insert sequence of a strain from a patient in England (London) had a markedly higher G+C content of 56 mol% and contained a single E to Q substitution at amino acid position 7 (SRI type C). No homologies between any of these insert sequences and other known nucleotide or amino acid sequences were identified in a BLAST search.
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Overall diversity of s2 alleles.
The 286 bp amplicons from 201 isolates of the s2 allelic subfamily had conserved G+C contents (47–51 mol%, mean 50 mol%) and, like the s1 allele family sequences, were heterogeneous at the nucleotide level, with 108 nucleotide sequence types (42 amino acid sequence types). Nearest neighbour analysis of these sequences (nucleotide and predicted amino acid) indicated a high degree of overall relatedness (>75 %) between sequences with no significant branches or subgroups irrespective of the geographical origin of the isolates. Because of the rarity of the s2 allele family in the East Asian populations, no Japanese or Chinese sequences were available for inclusion in the analyses.
Features of pre-insert site sequences.
Alignments of s-region sequences showed a conserved amino acid motif within the signal region immediately adjacent to the 5'-end of the insert. Most (87 %) s2 sequences contained a signal region pre-insert motif (SRP type I) with ten amino acids giving the sequence MGTELGANTP immediately before the insert site (Fig. 1a). BLAST homology searches confirmed that the SRP motif was not present elsewhere in the H. pylori genome or in any other bacterial sequences in GenBank. Twenty-five other s2 strains were represented by five other SRP types (II to VI) defined on single or double substitutions within the motif. For instance, a further 10 % of isolates had either M(1) to I or G(2) to S substitutions. By contrast, when the insert was not present in vacA, 68 % of s1 allele strains had the completely different VSITPQQSHA (35 %) or VSITPQKSHA (33 %) sequence at the SRP motif location, with 88 other strains represented by 11 variants containing minor amino acid substitutions.
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show that the signal region pre-insert motif (SRP) type I, present in strains such as Tx30a, was more commonly associated with the presence of signal region insert (SRI) type A than with other insert types (82 % vs 18 %). Thus the two groups of strains defined on the basis of presence or absence of insert type A differed significantly (P=0.001) even though the combination of signal region insert type A with pre-insert motif type I was a feature of geographically diverse strains.
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Diversity amongst the mid-region insert peptide sequences was evident when they were aligned with the insert sequence of strain Tx30a, the well-characterized non-toxigenic strain (McClain et al., 2001) used here as the reference for the vacA m2-allelic subfamily. The Tx30a mid-region insert with the amino acid motif LRVNGHTAHFKNIDATKSDNGLNTS was designated the reference for MRI type 1. A total of 23 amino acid variants (arbitrarily numbered 1 to 23) were defined with each differing by multiple amino acid substitutions when compared with the MRI type 1 sequence. The frequencies of the predominant MRI sequence types (all other types were represented by just one or two sequences) are given in Table 4 with the amino acid substitutions used for defining each type. The commonest form was MRI type 4, which was a feature of 158 (62 %) geographically diverse strains of the m2 allelic subfamily, and was defined by the 25 amino acid sequence LRVNGHSAHFKNIDASKSDNGLNTS. The second most common variant was MRI type 2 with the 25 amino acid sequence LRVNGHSAHFKNIDATKSDNGLNTS, which was a feature of 37 (14 %) geographically diverse strains. The type 4 and type 2 forms differed only in a single S to T substitution at amino acid 16 and together represented 76 % of all available MR insert sequences (Table 4). Five other less common (up to 6 %) MRI forms were also characterized by single amino acid substitutions when compared to the type 1 sequence. Most MR inserts characteristically contained a short internal amino acid SDNGLN motif (positions 18 to 23) except for inserts from 15 MRI type 22 sequences (from China and Peru) that were mainly of Chinese origin. These all contained a unique GRNGID sequence that was also found in MRIs from several other miscellaneous Chinese sequences that were not assigned an MRI type.
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Overall diversity of vacA m2 alleles.
The 255 vacA sequences of 641 bp (213 amino acids) of the m2-allelic subfamily strains likewise all had highly conserved G+C contents (40–42 mol%) and were also heterogeneous at the nucleotide level, with 179 nucleotide sequence variants (155 deduced amino acid sequence variants). Nearest neighbour analysis of these mid-region m2 allele sequences (nucleotide and predicted amino acid sequences) indicated a high degree of overall sequence relatedness (>75 %), irrespective of geographical origin. As with the m1 sequences, there were no clear-cut distinctions between European/North American/African sequences. However, a subset of 14 Chinese sequences formed a distinct separate lineage that included three sequences (HK-41, HK-46 and CH-4), which was consistent with the previously reported subdivision of m2 alleles into m2a and m2b genotypes (van Doorn et al., 1998).
Features of pre-insert site sequences.
Multiple alignments of the 255 complete vacA sequences of the mid-region m2 genotype showed a short conserved motif that was located immediately upstream of the insert and was designated the mid-region pre-insert site (MRP) motif. That 11 amino acid motif (MRP type 1) had the sequence NGNIYLGKSTN (Fig. 1b) and was a feature of 215 sequences (84 %) representing the m2 alleles including strainTx30a (m2a genotype). A variant (MRP type 2) with an I to V substitution at motif position 4 was found in 23 strains (9 %), while another variant (MRP type 3) with four amino acid substitutions (positions 4, 8, 9 and 11) was a feature of 16 Chinese sequences including the genotype m2b sequences described by van Doorn et al. (1998). One additional sequence (MRP type 4) from a Peruvian strain resembled the latter type except for an M to L substitution at position 4.
By contrast, when the insert was absent, 91 sequences representing the m1 genotype including those of NCTC 12445, NCTC 11637 and J99 had the KGIDTGNGGFN sequence at that position. A similar sequence was present in 59 Asian m1 strains but there was a G to D substitution at position 2. Likewise, minor amino acid variations were present in five other m1 genotype strain sequences. Different conserved motifs identified at additional locations within the m-region (see unlabelled boxes in Fig. 1) will be described elsewhere.
Results summarized in Table 3 show that the two most common inserts MRI types (2 and 4) and the respective mid-region pre-insert motif (MRP type 1) were significantly associated (P=0.0001). These types represented isolates from many countries so the associations were independent of geographical origin. By contrast, a third insert type, MRI type 22, which was a feature of strains of Chinese origin, was significantly associated with pre-insert motif sequence MRP type 3 (P=0.004). Overall, the analysis showed that 119 strains, representing 71 % of the available complete s2/m2 allele sequences, had the insert/motif combination SRI type A/MRI-type 4/MRP type 1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Cover & Blanke, 2005). The aim of the present study was to analyse diversity amongst the short vacA insert sequences using new sequences and all currently available online database sequences. Although the presence or absence of vacA inserts provides the basis of a widely used scheme for H. pylori strain genotyping (Atherton et al., 1999b), the clinical significance of a particular genotype remains unclear, and consequently vacA typing is of limited value in predicting severity of infection and possible clinical outcome (Go et al., 1998; Blaser, 1999; Höcker & Hohenberger, 2003). Analyses of the 892 sequences of the vacA s and m alleles in the dataset compiled for this study demonstrated that insert sequences were highly conserved according to a number of criteria, notably their location within vacA, overall nucleotide length, G+C content and similarities within allelic families in nucleotide and hence in predicted amino acid sequences. Extensive recombination is a recognized feature of the H. pylori genome within both housekeeping and pathogenicity-associated genes (Suerbaum et al., 1998). Furthermore, there is evidence of gain and loss of multiple genes during evolution of the species (Gressmann et al., 2005). The globally conserved nature of these short vacA inserts evident from our analysis was therefore unexpected. Despite the presence of numerous nucleotide polymorphisms in the sets of signal- and mid-region alleles, we found that almost all (98 %) of the signal region inserts were defined by the unique NDPIHSESR amino acid sequence. Likewise, 76 % of the mid-region inserts in the sequence dataset also were highly conserved at the predicted amino acid level, as most nucleotide mutations were synonymous, irrespective of geographical origin of the H. pylori isolates. The predominant m-insert sequences were represented by just two peptide motifs (types 2 and 4), which differed only by a single serine to threonine polymorphism.
The mid-region and signal-region peptide sequences were unrelated, and the insert sequences shared no homologies with either of the two sequenced genomes of H. pylori (Tomb et al., 1997; Alm et al., 1999), with the Helicobacter hepaticus genome (Suerbaum et al., 2003), or with genomes of Campylobacter jejuni and other allied enteric bacterial species deposited in GenBank. The possible origins of the phylogenetically unrelated inserts in H. pylori vacA therefore remain unclear. Their sequence and size uniqueness, and global distribution combined with a high degree of conservation suggest they may have ancestral origins rather than representing recent acquisitions by the H. pylori genome. Although we have referred to the target sequences as ‘inserts' for the purpose of this study, they could possibly be viewed as ancestral remnants of regions that have undergone selective deletion from vacA leading to the emergence of more pathogenic forms of H. pylori during adaptation to colonize the human gastric mucosa in different geographical environments. During human migrations over millennia, there could have been selective loss of forms containing the insert, resulting in the eventual emergence of the more virulent vacA s1 form as the predominant component of local species gene pools. Interestingly, there is evidence suggesting a link between the presence of vacA s2 alleles and the absence of cagA (Andreson et al., 2002) and by inference possibly the complete cag pathogenicity island. Such an association could be interpreted as an indicator of the primitive origins of the s-insert, although it can only be speculated that ancestral forms of H. pylori may have lacked the cag pathogenicity island (Gressmann et al., 2005).
A key feature of the global epidemiology of vacA is the non-random nature of the frequencies of the different allelic forms. Surveillance data on naturally occurring H. pylori strain populations show consistent differences in the relative frequencies of the two allelic families; for instance, in England and Wales up to 65 % of m alleles, depending on the demographics of patients sampled, had an insert whereas 11 % or fewer have s alleles containing an insert (Owen et al., 2002; Elviss et al., 2004, 2005). Likewise, similar frequencies of the s2 and m2 genotypes have been reported in many other parts of the world (van Doorn et al., 1999). Overall, these cumulative data highlight the fact that the s1 allelic family is the predominant vacA signal form of strains infecting humans globally with suballelic forms showing some geographical associations; for instance, allele s1c was described as the major subtype in East Asian strains whereas allele s1a was more typical of Western, in particular Northern European, strains (van Doorn et al., 1999; Yakoob et al., 2002; Wang et al., 2003; Zhou et al., 2004a, b). It should be noted that these subtypes were determined by differences in the main signal region rather than within the insert. No subtypes within s2 forms have been defined, a fact that reflects the rarity and conserved nature of such inserts in present H. pylori populations globally. For the vacA m alleles, the m1 and m2a forms generally have equal frequencies, except in Spain and Central and South America, where m1 is more prevalent, and in China, where the m2a form predominates (van Doorn et al., 1999; Wang et al., 2003).
The relative frequencies of the combined s/m allelic forms are also highly conserved but some differences according to geographical location have been noted; for instance, subtype m2 was found exclusively amongst East Asian s1c strains (van Doorn et al., 1999). The results on isolates from English and Welsh dyspeptics (Owen et al., 2002; Elviss et al., 2004, 2005) were consistent with data from other parts of the world in showing that the vacA s1/m1 and s1/m2 genotypes occur at similar frequencies and together constitute up to 90 % of local H. pylori gene pools. The frequency of forms with inserts in both the signal and mid-regions (s2/m2 form), by contrast, was lower at about 15 % or less, while isolates with an insert in the signal but not the mid-region (s2/m1 form) were rare and only found in South Africa (Letley et al., 1999). The s2 alleles occur only in combination with m2 alleles, which suggests that the s2/m1 combination results in a disabled non-functional form of VacA toxin, and consequently that such strains would be unable to colonize and/or persist on gastric epithelia. Data on human populations in European, African and North American countries as well as in China show that the frequency of the s2/m2 allelic family is low (generally around 10 %). In Japanese human populations, the s2/m2 form appears even rarer: for example, such forms were found in only 4 % of H. pylori infecting patients in Okinawa (Zhou et al., 2004b) and were completely absent from isolates infecting adult gastritis patients in Tokyo (Owen et al., 2004). In Japanese children the predominant genotype reported was s1c/m1b (Azuma et al., 2004). There may well be differences in allelic frequencies within the numerically vast and geographically diverse East Asian population. The s1/m2 form is prevalent in China although there are indications of regional differences (Pan et al., 1998; Yakoob et al. 2002; Zhou et al., 2004a, b; Gong et al., 2005). For instance, in the Shaanxi Province (Central China) the relative frequencies for the s1/m1, s1/m2 and s2/m2 allele combinations were 52 %, 39 % and 9 % respectively (Qiao et al., 2003), whereas in two areas of Northern China, the predominant genotypes were s1/m1b and s1/m2 (no s2 forms were reported), in individuals with multiple strain infections (Gong et al., 2005). Overall these results highlight the complexity of H. pylori strain populations and the difficulty of drawing general conclusions.
It has been proposed that VacA may contribute to colonization and persistence in vivo at different stages of the infection process as part of its multifunctional role (Cover & Blanke, 2005). The fact our results indicate a high degree of conservation within the vacA inserts suggests that other factors may also contribute to overall VacA functionality. The simple presence or absence of the insert appears to be the key functional marker in the case of the signal region. Here the direct effect of the presence of the insert is to cause an amino acid terminal (hydrophilic) extension of VacA that in turn either causes blocking or markedly alters the functional properties of the toxin, leading to loss of toxigenic activity (Letley & Atherton, 2000; McClain et al., 2001). The VacA proteins encoded by the s2/m2 forms nevertheless apparently retain an important biological function in some individuals (McClain et al., 2001); for instance, s2/m2 allele strains from cases of chronic gastric disease in England have proved difficult to eradicate despite repeated antibiotic therapy. Contributing factors may be intrinsic antibiotic resistance as well as lower metabolic activity and low colonization levels of s2 strains. As a consequence they may be more difficult to detect with diagnostic tests such as the urea breath test. By contrast, the mid-region insert appears to have no role in determining target cell specificity, with differences between the m1 and m2 forms of the protein being determined by a short region of amino acids from the beginning of the mid-region to the m1–m2 junction (Ji et al., 2000).
The multiple alignments we performed of complete vacA sequences showed two important features in relation to geographical diversity of alleles. Firstly, there were high levels of homology within geographically diverse sequences defined as belonging to either the signal or mid-region families of alleles. Our phylogenetic analysis confirmed the close relatedness reported previously amongst the m1 allelic family (Ito et al., 1997) and the more marked distinction between the m1 and m2 forms (Pagliaccia et al., 1998; Atherton et al., 1999a; Ji et al., 2002). Our analysis also confirmed the findings of van Doorn et al. (1998) of divergence within the m2 allelic family with two distinct subfamilies of sequences, one of which was predominantly of Chinese origin (designated here as m2 B). Although the level of similarity between these two m2 subgroups is unclear, the uniqueness of the m2 B subgroup was attributable to polymorphisms in the m-insert, all of which had closely related forms (MRI types 21 and 22), as well as at other locations within the m-region (Fig. 1c). These results further support the existence of two possible vacA m2 sequence sublineages within the Chinese H. pylori gene pool. Sequences of vacA appear to highlight finer differences in population structure than discernible from MLST analyses based on housekeeping genes, which grouped Chinese and Japanese isolates in one East Asian subpopulation designated hspEAsia (Suerbaum & Achtman, 2004). The distinctiveness of the Chinese H. pylori gene pool is also evident from analyses of cagA, which suggest a more active East Asian form (Zhou et al., 2004b; Zhang et al., 2005), and from analysis of hopQ alleles (Cao et al., 2005).
The second interesting finding in our analysis was the presence of several conserved short sequences in core vacA regions that provided markers for a specific location directly associated with the gaining of an insert. The SRP motif identified upstream of the signal-region insert provided a flanking marker in 92 % of strains for the location of the signal region insert. Likewise the MRP motif identified upstream of the mid-region insert provided a flanking marker in 84 % of strains for the location of the 75 bp mid-region insert. The locations of these motifs fell within the region identified as determining the phenotypic differences between the m1 and m2 forms of the proteins in target cell specificity (Ji et al., 2000). The characterization of SRP and MRP motifs provides the first evidence of site-specific markers within vacA for the insert sequences that might explain the constancy of their locations, despite an enormous geographical diversity of isolate origin. This has a functional relevance as the SRP motif contains the signal-region sequence processing site, which differs in the s1 and s2 forms, and as a consequence, the extension of the mature N-terminus is three amino acids less as a result of the insert. In view of the role of this extension in blocking vacuolating activity (Letley & Atherton 2000; Letley et al., 2003; McClain et al., 2001), it is an important finding that this processing site is well conserved amongst s2 strains as it suggests similar signal processing and, together with the insertion, a conserved N-terminal extension to the s2 VacA form.
In conclusion, our comprehensive analysis of geographically diverse vacA sequences demonstrated that the signal and mid-region inserts providing the basis for vacA allelic typing have several unique conserved features. We speculate that internal insert variation appears unlikely to contribute markedly to differences in H. pylori strain virulence but nevertheless the findings support the continued use of the insertion-typing primers for determining vacA type. Future work should determine how geographical variation at other locations within vacA might determine variation in toxin structure and function as these could provide more specific markers for use as predictors of severity of H. pylori–host interactions. The identification of a H. pylori subtype found only in Chinese patients highlights the need to extend investigations into geographical diversity, particularly in regions where there is a high incidence of gastric cancer and host factors may favour colonization by particular strain pathotypes.
Edited by: G. E. Duhamel
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Received 29 September 2006;
accepted 4 January 2007.
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