Roles of the plasticity regions of Helicobacter pylori in gastroduodenal pathogenesis

Abstract

Putative virulence genes of Helicobacter pylori are generally classified into three categories: strain-specific genes, phase-variable genes and genes with variable structures/genotypes. Among these, there has recently been considerable interest in strain-specific genes found outside of the cag pathogenicity island, especially genes in the plasticity regions. Nearly half of the strain-specific genes of H. pylori are located in the plasticity regions in strains 26695 and J99. Strain HPAG1, however, seems to lack a typical plasticity region; instead it has 43 HPAG1-specific genes which are either undetectable or incompletely represented in the genomes of strains 26695 and J99. Recent studies showed that certain genes or combination of genes in this region may play important roles in the pathogenesis of H. pylori-associated gastroduodenal diseases. Most previous studies have focused on the plasticity region in strain J99 (jhp0914–jhp0961) and the jhp0947 gene and the duodenal ulcer promoting (dupA) gene are good candidate markers for gastroduodenal diseases although there are some paradoxical findings. The jhp0947 gene is reported to be associated with an increased risk of both duodenal ulcers and gastric cancers, whereas the dupA gene, which encompasses jhp0917 and jhp0918, is reported to be associated with an increased risk of duodenal ulcers and protection against gastric cancers. In addition, recent studies showed that approximately 10–30% of clinical isolates possess a 16.3 kb type IV secretion apparatus (tfs3) in the plasticity region. Studies on the plasticity region have only just begun, and further investigation is necessary to elucidate the roles of genes in this region in gastroduodenal pathogenesis.

Background

Helicobacter pylori is a well-recognized pathogen that chronically infects more than 50% of the world’s population. H. pylori plays an important role in the development of peptic ulcers, gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma (MALToma). The infection remains latent in the majority of infected patients, with only approximately 20% of infected individuals developing severe disease. It is unclear what determines the outcome of an infection; however, it is thought to involve an interplay between the virulence of the infecting strain, host genetics and environmental factors. Experience with other bacterial pathogens suggests that H. pylori-specific factors may exist that influence the pathogenicity of H. pylori.

Many putative virulence genes of H. pylori have been reported to determine clinical outcome, and these are generally classified into three categories (Table 1). The first category contains strain-specific genes, which are present in only some H. pylori strains. Among this group, the best studied is the cag pathogenicity island (PAI), which encodes a bacterial type IV secretory apparatus (Censini et al., 1996). The cag PAI contains approximately 30 genes, including the cagA gene in the 3′ end of the island, which injects CagA and possibly other bacterial proteins into host cells (Asahi et al., 2000; Backert et al., 2000; Odenbreit et al., 2000; Segal et al., 1999; Stein et al., 2000; Viala et al., 2004). Strains that possess the cag PAI/cagA are statistically more likely to be associated with peptic ulcer and gastric cancer than are strains lacking the PAI/cagA (Blaser et al., 1995; van Doorn et al., 1998). To date, the complete genomes of three H. pylori strains (26695, J99 and HPAG1) have been sequenced (Alm et al., 1999; Oh et al., 2006; Tomb et al., 1997). These are all cag PAI-positive strains; however, many other strain-specific genes lie outside of the cag PAI (Table 2) and nearly half of the strain-specific genes of H. pylori are located in the plasticity region. Genes in this region are also good candidates for H. pylori virulence factors, as described in detail in this review.

Table 1.

Virulence factors of H. pylori

Category	DNA/protein status	Major virulence factors
Strain-specific genes	Gene positive or negative	cag pathogenicity island (PAI)
		Plasticity regions
Phase-variable genes	Gene positive, but some produce functional proteins   and others not	oipA, sabA, babA (regulated by slipped strand mispairing)
Genes with different   structures/genotypes	Gene positive, but function and/or production levels   of proteins different between strains	vacA genotypes (combination of s1/s2 and m1/m2)
		cagA repeat region (East Asian type and Western type)
		alpAB (East Asian type and Western type)

Table 2.

Strain-specific genes

Category	Strain
	26695	J99	HPAG1
R–M system	6	6	5
Transposases	14	1	0
cag genes	0	0	3
Other	22	15	7
Hypothetical	90	37	28
Total	132	59	43

The second group is the phase-variable genes such that the gene status can be changed during growth or in different conditions. Based on the comparison of the three completed genomes of H. pylori, six genes encoding outer-membrane proteins (oipA, sabA, sabB, babB, babC and hopZ) are thought to undergo phase variation (Alm et al., 1999; Oh et al., 2006; Tomb et al., 1997). The functional status is regulated by a slipped strand mispairing mechanism and is mediated by the number of CT dinucleotide repeats in the 5′ region of the genes. Among these, OipA (outer inflammatory protein) and SabA (sialic acid binding adhesin) are reported to be associated with peptic ulcers and gastric cancer (Yamaoka et al., 2002, 2006). Recently, another putative virulence factor, the outer-membrane protein BabA (blood group antigen binding adhesin), was shown to be regulated by the slipped strand mispairing mechanism in some clinical isolates (Colbeck et al., 2006; Hennig et al., 2006) and has been reported to be associated with peptic ulcers and gastric cancer (Fujimoto et al., 2007; Gerhard et al., 1999; Yamaoka et al., 2006).

The last group is the genes with variable structures/genotypes depending on the strain. For example, specific vacA genotypes containing different mosaic combinations of signal regions and middle region allelic types have been associated with different clinical outcomes (Atherton et al., 1995). In addition, the structure of many genes differs between Western strains and East Asian strains, and the structural differences in some genes (e.g. 3′ repeat region of the cagA, alpAB genes) are reported to influence virulence (Lu et al., 2007; Yamaoka et al., 1998b, 1999a).

Among these virulence factors, there has recently been considerable interest in strain-specific genes outside of the cag PAI: the plasticity regions. This review focuses on the plasticity regions and describes the current knowledge of the regions in relation to clinical outcomes.

Plasticity regions of H. pylori

Comparison of the complete genome sequences of two H. pylori strains (26695 and J99) revealed several regions whose G+C content was lower than that of the rest of the H. pylori genome (35% compared with 39 %), suggestive of horizontal DNA transfer from other species (Alm et al., 1999; Tomb et al., 1997). One such region is the cag PAI and the other has been termed the plasticity region based on the variability in gene content between different isolates. Nearly half of the strain-specific genes of H. pylori are located in the plasticity region (Alm & Trust, 1999).

In H. pylori strain J99, the plasticity region has been reported to range from jhp0914 to jhp0961 (Alm et al., 1999). This region is continuous in strain J99; however, only 6 and 10 of the 48 open reading frames (ORFs) present in the plasticity region of strain J99 were found in strains 26695 and HPAG1, respectively. Two DNA microarray studies have analysed the gene content of 15 (Salama et al., 2000) and 56 (Gressmann et al., 2005) isolates of H. pylori. Table 3 shows the prevalence of jhp0914–jhp0961 in 72 strains, including strains 26695, J99 and HPAG1, and confirms that the ORFs in the plasticity region really do display diversity, except for jhp0915, which was present in all strains studied.

Table 3.

Prevalence of strain-specific genes in the plasticity region of strain J99: analyses for 72 strains

Gene number	Strains hybridized/positive (%)
jhp0914	17
jhp0915	100
jhp0916	43
jhp0917	39
jhp0918	36
jhp0919	36
jhp0920	38
jhp0921	36
jhp0922	42
jhp0923	40
jhp0924	39
jhp0925	19
jhp0926	19
jhp0927	61
jhp0928	58
jhp0929	50
jhp0930	24
jhp0931	47
jhp0932	47
jhp0933	51
jhp0934	47
jhp0935	49
jhp0936	46
jhp0937	22
jhp0938	85
jhp0939	85
jhp0940	22
jhp0941	89
jhp0942	86
jhp0943	42
jhp0944	21
jhp0945	51
jhp0946	49
jhp0947	26
jhp0948	31
jhp0949	24
jhp0950	60
jhp0951	44
jhp0952	88
jhp0953	39
jhp0954	63
jhp0955	94
jhp0956	86
jhp0957	94
jhp0958	72
jhp0959	13
jhp0960	38
jhp0961	44

Although the ORFs of the major part of the plasticity region encode putative proteins with unknown functions, some have been found to share similarity with genes encoding functional proteins. For example, in the plasticity region of strain J99, jhp0917 and jhp0918 are homologous to vir factors; jhp0919, jhp0920 and jhp0931 are homologous to DNA topoisomerase I (topA) involved in DNA replication; jhp0921, jhp0922 and jhp0923 are homologous to DNA transformation competence ComB8 (jhp0921 and jhp0922) or ComB9 (jhp0923) homologues; jhp0928 is homologous to a methylase gene; jhp0935 is homologous to partitioning protein A; and jhp0941 and jhp0951 are homologous to integrase/recombinase genes (xerCD family) involved in DNA restriction, modification, recombination and repair systems.

Strain HPAG1 seems to lack a typical plasticity region; instead it has 43 HPAG1-specific genes which are either undetectable or incompletely represented in the genomes of strains 26695 and J99 and are scattered throughout the genome evenly without indication of islands (Table 2). Accordingly, other H. pylori strains would be expected to contain their own set of strain-specific genes, and in fact novel candidates for virulence factors have been found experimentally in the plasticity regions of additional H. pylori strains (for example, a novel 16.3 kb segment, tfs3; see below) (Kersulyte et al., 2003).

Plasticity regions and clinical outcomes

The strain-specific genes are likely to play a similar role to the cag PAI in the pathogenesis of H. pylori infection. The highly variable gene content in the plasticity regions may play an important role in this regard, and some loci within the plasticity regions have previously been proposed to serve as markers of clinical outcomes. Most previous studies have focused on the plasticity region in strain J99, probably due to the fact that strain J99 was isolated from a duodenal ulcer patient whereas the other fully sequenced strains, HPAG1 and 26695, were from gastritis patients (Alm et al., 1999; Oh et al., 2006; Tomb et al., 1997).

In an initial systematic study, Occhialini et al. (2000) examined the prevalence of 21 ORFs inside the plasticity region of strain J99 (jhp0914–jhp0961) in a small group of 17 gastric cancer and 26 gastritis patients from Costa Rica and found an increased prevalence of the jhp0940 and jhp0947 genes in H. pylori from gastric cancer patients. A follow-up study to further examine this relationship was performed with 200 strains of H. pylori from Brazil, including strains from duodenal ulcer, gastric cancer and H. pylori gastritis patients (Santos et al., 2003). The original hypothesis was only partially confirmed as the new study demonstrated an association between jhp0947 (but not jhp0940) and both duodenal ulcer and gastric cancer. de Jonge et al. (2004) also reported that disruption of the jhp0945–0947–0949 locus in H. pylori strain 1061 significantly decreased its ability to induce interleukin (IL)-12 production in monocyte THP-1 cells, and the requirement for the jhp0945–0947–0949 locus for IL-12 induction was subsequently confirmed using four wild-type H. pylori strains. These authors also reported that the presence of the jhp0947 and jhp0949 genes, but not jhp0945, was significantly associated with duodenal ulcer when compared with gastritis in a small group of 26 duodenal ulcer and 19 gastritis patients from the Dutch population. Recently, Rizwan et al. (2008) reported that recombinant JHP0940 protein elicited strong and significant levels of TNF-alpha and IL-8 in human macrophages and enhanced translocation of nuclear factor (NF)-κB in cultured macrophages. However, they could not find a relationship between the presence of jhp0940 and clinical outcomes or cag PAI status in seven countries studied (India, South Africa, Japan, Costa Rica, Peru, France and Spain). In India, South Africa and France, most strains possessed the jhp0940 gene, whereas in Spain fewer than 10% of strains possessed the gene irrespective of disease status. As a preliminary study, Salih et al. (2007) recently examined the prevalence of jhp0926, jhp0931, jhp0933, jhp0944 and jhp0945 in a small group containing 21 gastritis and 14 peptic ulcer patients from Turkey and found that the jhp0931 gene was more likely to be found in H. pylori from patients with peptic ulcer disease than in those with gastritis (14.2% vs 0 %). However, the jhp0931 gene was not related to clinical outcomes in the original study in the Costa Rican population (Occhialini et al., 2000).

The overall conclusion from the series of studies examining ORFs within the plasticity region of strain J99 is that the jhp0947 gene currently appears to be the best candidate for a disease marker. The jhp0947 gene is homologous to jhp0938 (hp0990) and jhp253 (hp1333); however, its function is unknown. The 5′ region of jhp0947 is also homologous to that of jhp0477 (hp0528), which is part of the cag PAI (virB9 homologue) and has been identified as an important structural component of the type IV secretion system (Tanaka et al., 2003). Interestingly, the presence of jhp0947 was associated with the presence of cag PAI in Brazilian strains (Santos et al., 2003), whereas this relationship was not observed in Dutch strains (de Jonge et al., 2004). There are no studies on the jhp0947 gene in East Asian countries, where the strains are considerably different from those in Western countries. Further studies are necessary to confirm the roles of the jhp0947 gene product in gastroduodenal pathogenesis.

Lehours et al. (2004) reported that the presence of jhp0950, which was not included in the 21 ORFs selected by Occhialini et al. (2000), is a candidate marker for gastric extranodal marginal zone B cell lymphoma of the mucosa-associated lymphoid tissue (MALT)-type (MZBL) in the French population. Importantly, jhp0950 seems to be a disease-specific marker for MZBL since the prevalence of jhp0950 (74 %) was significantly higher in MZBL than in all other diseases examined (49% for gastritis, 49% for duodenal ulcer and 39% for gastric adenocarcinoma). The function of jhp0950 is unknown; however, a recent study suggests that jhp0942–jhp0944–jhp0945–jhp0947–jhp0949 may be expressed as an operon, since these genes are consecutive and oriented in the same direction (Occhialini et al., 2000). The jhp0950 gene is also arranged in the same orientation as the jhp0949 gene and may also form part of the operon that includes the jhp0949 gene.

Novel type IV secretion system in the plasticity region

Type IV secretion systems are widely distributed in prokaryotes and are structurally complex molecular machines, typically composed of a cell envelope-spanning translocation channel, cytoplasmic ATPases and a pilus. The most studied type IV secretion systems of H. pylori are the secretion system for effector proteins such as CagA (the cag PAI system), and the DNA import system via natural transformation (the ComB system), which is found in all H. pylori strains (Hofreuter et al., 1998, 2001; Smeets & Kusters, 2002; Censini et al., 1996). The Agrobacterium tumefaciens VirB/D4 type IV secretion system serves as a prototype system for which detailed structural and functional data are available. The VirB/D4 system consists of 11 VirB proteins (VirB1–VirB11) and the substrate recognition factor VirD4, which are assembled into the transport apparatus necessary for the delivery of proteins or nucleoprotein complexes into target cells. In H. pylori, the cag PAI contains seven homologues of the VirB/D4 type IV secretion system, virB4, virB7, virB8, virB9, virB10, virB11 and virD4 (Censini et al., 1996; Selbach et al., 2002; Covacci et al., 1999; Buhrdorf et al., 2003), and the ComB system contains homologues of all vir genes, except virB1, virB5, virB11 and virD4 (Hofreuter et al., 2001). Mutation studies suggest that VirB homologues in the cag PAI, but not the ComB, system are involved in the induction of proinflammatory cytokine IL-8 and activation of transcription factors such as NF-κB in gastric epithelial cells (Censini et al., 1996; Fischer et al., 2001; Selbach et al., 2002; Glocker et al., 1998).

Kersulyte et al. (2003) identified a novel 16.3 kb type IV secretion apparatus (tfs3) in the plasticity region of a strain from a Peruvian patient with gastric cancer (PeCan18B), seven of the 16 ORFs of which were homologues of the virB/D operon of A. tumefaciens, the third putative type IV secretion system present in H. pylori. The clustering of DNA with a lower G+C content in tfs3 is suggestive of horizontal DNA transfer from other species. Four of these genes are transmembrane pore genes (virB7, virB8, virB9 and virB10), and three encode cytoplasmic membrane-associated ATPases that move their cognate macromolecule substrates VirB4, VirB11 and VirD4 to and through the pore. Full-length and partial tfs3 elements were each found in approximately one-fifth of clinical H. pylori strains from Spain, Peru, India and Japan. Our subsequent study confirmed that 8% (Japan), 13% (Korea) and 33% (Colombia) of clinical isolates possess complete tfs3 (Lu et al., 2005a). Strain 26695 contains a truncated tfs3 element that corresponds to 7369 bp of the 5′ region of full-length tfs3 from strain PeCan18B (18 144 bp). The function of the tfs3 elements is still unknown, and there is no evidence to support a critical role in transformability, general bacterial conjugation, intracellular entry, survival or mouse colonization (Kersulyte et al., 2003). In addition, there was no correlation between the presence of complete or partial tfs3 and clinical outcomes (Kersulyte et al., 2003), and the presence of complete tfs3 was not linked to other vir homologue genes (Lu et al., 2005a). Although other type IV secretion systems of H. pylori show homology to components of the tfs3 system, it is not known whether they work independently and are unable to complement each other functionally or not, as shown between the cag PAI and ComB system (Hofreuter et al., 2001). Further studies are necessary to elucidate the function of this third type IV secretion system in H. pylori and to determine the interplay among the three type IV secretion systems.

Identification of the duodenal ulcer promoting gene (dupA) as another virB4 homologue in the plasticity region

The plasticity region of strain J99 contains two virB4 homologues (jhp0917 and jhp0918). The jhp0917 gene Y. Yamaoka 548 Journal of Medical Microbiology 57 encodes a protein of 475 amino acids, but lacks a region homologous to the C-terminus of VirB4, while the jhp0918 gene encodes a product of 140 amino acids that is homologous to the missing VirB4 region. Lu et al. (2005a) recently found that all of the clinical isolates that they studied (eight each from Japan, Korea and Colombia) contained a 1 bp insertion (C or T) in the 3′ region of the jhp0917 gene (after position 1385 in strain C142; GenBank accession no. AB196363), resulting in a frameshift leading to a continuous gene. Subsequent studies in Brazilian strains reported that 86 out of 89 (97 %) isolates contained a 1 bp insertion after position 1385 (Gomes et al., 2008), confirming that jhp0917–0918 usually form one continuous gene homologous to the intact virB4 gene, with strain J99 being a rare exception.

More importantly, Lu et al. (2005a) reported that the jhp0917–0918 gene is a marker for development of duodenal ulcer disease and for protection against gastric adenocarcinoma. They also found that the gene is a marker for a protective effect against atrophy and intestinal metaplasia. Consequently, they have designated the jhp0917–jhp0918 gene the duodenal ulcer promoting (dupA) gene. They examined 500 H. pylori isolates, 160 from Japan, 175 from Korea and 165 from Colombia, and found that the prevalence of the dupA gene was significantly greater among strains isolated from duodenal ulcer patients (42 %) than from patients with H. pylori gastritis (21 %), with gastric ulcer (27 %) or with gastric cancer (9%) (P <0.001 for duodenal ulcer vs gastritis or cancer). Importantly, these associations were consistently observed in isolates from both Asian and Western countries. Although a number of putative H. pylori virulence genes have been associated with increased risks of a clinical outcome such as peptic ulcer or gastric cancer, none have clearly been linked to one specific H. pylorirelated disease such as duodenal ulcer, thus dupA is the first disease-specific virulence marker in H. pylori.

As expected, the prevalence of the jhp0917 and jhp0918 genes was closely linked: only 10 of 500 (2 %) strains were jhp0917-negative/jhp0918-positive while the status of the two genes in the remaining strains was either both positive or both negative (Lu et al., 2005a). Subsequent studies confirmed that most isolates were both positive or both negative: for example, 474 of 482 (98%) in Brazilian strains (Gomes et al., 2008) and 149 of 157 (95 %) in Iranian strains (Douraghi et al., 2008). This relationship was also observed in North Indian strains; however, in this case only 75% (122/166) of the strains were either both positive or both negative, with 15 (9 %) jhp0917-positive/jhp0918-negative and 27 (16 %) jhp0917-negative/jhp0918-positive (Arachchi et al., 2007). The absence of the jhp0917 and/or jhp0918 genes (especially where one is present and the other is absent) is probably due to primer mismatches rather than to gene truncation or genetic decay. We could categorize strains as intact dupA-positive only if positive for both jhp0917 and jhp0918 genes with a 1 bp insertion after position 1385 corresponding to strain C142.

Possible functions of DupA

The function of DupA is still not fully understood. The dupA gene encodes homologues of VirB4 ATPase, which is thought to be involved in DNA uptake/DNA transfer and protein transfer. Bioinformatic analyses show that the N-terminus of DupA (encoded by jhp0917; position 3–201) has homology to members of the FtsK/SpoIIIE family, whereas the central region (encoded by jhp0917 3′– jhp0918; position 203–610) shares homology with the TraG/TraD family (Lu et al., 2005a). The FtsK/SpoIIIE domain contains a putative ATP-binding P-loop motif and is involved in cell division and peptidoglycan synthesis or modification, and has been implicated in intercellular chromosomal DNA transfer. Members of the TraG/TraD family are potential NTP hydrolases that are essential for DNA transfer in bacterial conjugation and are thought to mediate interactions between DNA processing and mating pair formation systems.

In vitro experiments using dupA-deleted and -complemented mutants showed that the absence of the dupA gene was associated with increased susceptibility to low pH (Lu et al., 2005a). In addition, the presence of the dupA gene was associated with increased IL-8 production from the antral gastric mucosa in vivo as well as from the gastric epithelial cells in vitro (Lu et al., 2005a). The authors further confirmed that the dupA gene is involved in the activation of transcription factors that bind to the IL-8 promoter, such as NF-κB and AP-1. These findings are in agreement with in vivo results showing higher neutrophil infiltration and IL-8 production in the antral gastric mucosa of duodenal ulcer patients compared with that in other diseases (Yamaoka et al., 1998a, 1999b). It is possible that the dupA gene acts in combination with other vir homologues in the plasticity region to form a type IV secretion system similar to the cag PAI, which is also involved in IL-8 secretion; however, a search for genes in the plasticity region of strain J99 did not find potential partner genes for dupA (jhp0917–jhp0918) that were components of a type IV secretion system. Recent preliminary data from my laboratory showed that IL-8 induction from gastric epithelial cells was decreased in dupA mutants of some strains compared with their parental strains, but was not reduced in dupA mutants of other strains (Y. Yamaoka, unpublished data). Therefore, it is possible that some strains form a novel type IV secretion system involving the dupA gene that may have a role in IL-8 induction, whereas others do not. The dupA gene might be an ancestral remnant of a type IV secretion system in some strains. In other words, only strains that are both dupA-positive and form a novel type IV secretion system might be involved in gastroduodenal diseases. Further studies are required to elucidate the roles of genes surrounding the dupA gene.

DupA and clinical outcomes: discrepancies in recent studies

In late 2007 and early 2008, four studies evaluating the relationship between the presence of dupA and clinical outcomes in different geographical areas were published (Arachchi et al., 2007; Argent et al., 2007; Douraghi et al., 2008; Gomes et al., 2008). Detailed distribution of the dupA gene in relation to different disease states is summarized well by Douraghi et al. (2008). One study examining North Indian strains supported the original findings and reported that the dupA gene was more common in strains from patients with duodenal ulcer (38 %; 36/96) than in those from patients with functional dyspepsia (23 %; 16/70) (P <0.05); this study did not examine isolates from patients with gastric cancer (Arachchi et al., 2007). In contrast, a study on Iranian strains reported that the presence of the dupA gene was independent of the clinical outcome, including duodenal ulcer, gastric ulcer and gastric cancer; however, histological analyses confirmed that the presence of the dupA gene was inversely associated with the presence of the precancerous lesion gastric dyspepsia and with the presence of lymphoid follicles, which represent a relatively common histological feature of chronic gastritis (Douraghi et al., 2008). Therefore, this study provided further support for dupA as a protective marker against gastric cancer, in agreement with the original study.

In a study examining strains from Brazilian children and adults, the prevalence of the dupA gene was extremely high (92 %; 445/482) irrespective of the nature of the gastro-duodenal diseases, gastritis, duodenal ulcer and gastric cancer (Gomes et al., 2008). Interestingly, the frequency of the dupA gene was significantly higher in strains from children than in those from adults (P=0.01). H. pylori infection is typically acquired in childhood and persists throughout life unless treated with a combination of anti-acid and antimicrobial therapy, so it is speculated that the dupA gene might be lost during long-term infections in which the gastric mucosa gradually develop chronic atrophic gastritis and gastric cancer. Therefore, their results might partially support the original hypothesis that dupA is a marker associated with reduced gastric damage that leads to the development of gastric cancer. Familial studies or longitudinal studies will be necessary to confirm the possible loss of dupA over time.

Another study examining strains from four countries (Belgium, South Africa, China and the United States) reported no significant association between the presence of the dupA gene and peptic ulcer or gastric cancer in any individual population, although the prevalence of these diseases in each population may have been too small to reveal a true effect; for example, there was only one case of gastric cancer in the Chinese population studied (Argent et al., 2007). When the authors combined Belgian and South African populations, the presence of the dupA gene was significantly associated with the presence of gastric cancer [70% (26/37) in gastric cancer vs 44% (40/92) in gastritis] (P <.01). There was also a non-significant trend towards an association between the dupA gene and duodenal ulcer [60% (32/53)] (P=0.057) in the combined Belgian and South African population. However, it is not relevant to combine random populations since it is unclear whether the South African strains were taken from patients of European descent and recent studies confirmed that the genomic structures of some South African strains (e.g. HpAfrica2 type) were relatively different from those of strains from the European population (i.e. HpEurope type) (Falush et al., 2003; Linz et al., 2007).

The relationship between the presence of the dupA gene and the cag PAI/cagA gene is also somewhat controversial. One study involving Indian patients with gastritis or duodenal ulcer reported that the presence of the dupA gene was significantly associated with the cagA-positive genotype (Arachchi et al., 2007). Interestingly, two studies reported that the presence of dupA was associated with the cagA-positive genotype in strains isolated from adults with duodenal ulcer, but not in those from patients with other diseases or from children (Douraghi et al., 2008; Gomes et al., 2008); however, these relationships were not observed in the remaining two studies (Argent et al., 2007; Lu et al., 2005a).

Overall, there are distinct geographical variations in the prevalence of the dupA gene, and there appears to be an association between dupA and duodenal ulcers in some populations but not in others. As Argent et al. (2007) reported, the association of dupA with duodenal ulcer in only some populations could reflect differences in the definition or diagnosis of ulcers or in the use of drugs that either cause or heal ulcers in these populations. In addition, the discrepancy could be related to the limitation of PCR techniques for detecting the intact dupA gene. In some studies, only one set of primer pairs for jhp0917 and jhp0918 was used (Douraghi et al., 2008; Lu et al., 2005a): use of multiple primer pairs is recommended for detection of the dupA gene in future studies. None of the previous reports considered the 1 bp insertion after position 1385 as a criterion for the presence of the dupA gene, although the insertion is very common (97–100 %) (Gomes et al., 2008; Lu et al., 2005a). More importantly, Gomes et al. (2008) reported frameshift mutations in 14/86 (16%) dupA-positive sequenced samples; a single adenine insertion after position 1426 of dupA or at position 2998 of the jhp0917–jhp0918 gene of the J99 strain that created a premature stop codon and may have considerable effects on protein expression or function. In their study, they counted the truncated samples as dupA-positive; however, it is clear that these mutated sequences would not produce intact DupA protein. It is definitely unacceptable to ignore such mutations that occur at an unexpectedly high frequency; however, it is impossible to detect them using a simple PCR method. It might be better to detect intact dupA by measuring intact DupA protein using immunoblotting techniques, which has not been reported previously. In addition, as mentioned above, only strains that are dupA-positive and form a novel type IV secretion system might be involved in gastroduodenal diseases. If this is true, examining the presence of DupA/dupA alone might not be sufficient. Studies on DupA and the role of the plasticity region are still in their early stages, and great progress is expected in the near future.

Conclusions

There are few studies investigating the roles of genes in the plasticity regions in H. pylori; however, as described in this review, there are several good candidate markers for gastroduodenal diseases, including the jhp0947 gene and the dupA gene. It has been proposed that a H. pylori virulence factor should (1) have a disease or other in vivo correlation, (2) be epidemiologically consistent across populations and regions and (3) be biologically plausible and the effect should be reduced or eliminated by gene deletion and be restored by complementation (Lu et al., 2005b). Only criterion (1) is cleared for the jhp0947 gene, and the studies have been limited to strains from non-Asian countries. Further studies in the Asian population will be necessary to confirm the importance of the jhp0947 gene for gastroduodenal diseases. Although there are some paradoxical findings, studies to date suggest that DupA clears all three criteria and might be the first disease-specific marker for increased risk of duodenal ulcers and protection from gastric malignancies. In contrast, although a number of putative virulence factors of H. pylori have been reported, including the jhp0947 gene, their presence has typically been associated with an increased risk of both gastric cancer and peptic ulcers. However, there are currently no established methods to detect the intact dupA gene or DupA protein and further studies are necessary, first to establish such detection procedures and subsequently to elucidate the role of DupA, the interaction of the dupA gene with surrounding genes, and possible formation of a novel type IV secretion system. In addition, most previous studies have focused on the plasticity region in strain J99; however, there are many strain-specific genes in strains 26695 and HPAG1, not only in the plasticity regions but also outside of these regions, that may be involved in gastritis. Novel strain-specific genes are expected to be present in other clinical isolates, and we expect that identification of the roles of such strain-specific genes will give further insight into the pathogenesis of H. pylori-induced gastroduodenal diseases. Recently, the high parallel Genome Sequencer 20 System (GS-20) (454 Life Sciences) was introduced and successfully used to sequence H. pylori genomes (Oh et al., 2006). Such instrumentation provides an opportunity to rapidly sequence multiple individual H. pylori isolates, and will allow more efficient identification of novel strain-specific genes that are related to specific diseases.