Mechanisms of disease: Helicobacter pylori virulence factors

Abstract

Helicobacter pylori plays an essential role in the development of various gastroduodenal diseases; however, only a small proportion of people infected with H. pylori develop these diseases. Some populations that have a high prevalence of H. pylori infection also have a high incidence of gastric cancer (for example, in East Asia), whereas others do not (for example, in Africa and South Asia). Even within East Asia, the incidence of gastric cancer varies (decreasing in the south). H. pylori is a highly heterogeneous bacterium and its virulence varies geographically. Geographic differences in the incidence of gastric cancer can be explained, at least in part, by the presence of different types of H. pylori virulence factor, especially CagA, VacA and OipA. However, it is still unclear why the pathogenicity of H. pylori increased as it migrated from Africa to East Asia during the course of evolution. H. pylori infection is also thought to be involved in the development of duodenal ulcer, which is at the opposite end of the disease spectrum to gastric cancer. This discrepancy can be explained in part by the presence of H. pylori virulence factor DupA. Despite advances in our understanding of the development of H. pylori-related diseases, further work is required to clarify the roles of H. pylori virulence factors.

Introduction

Helicobacter pylori is a Gram-negative spiral bacterium whose ecological niche is the human stomach. H. pylori gastritis is etiologically associated with peptic ulcer, primary gastric B-cell lymphoma and gastric carcinoma. Despite a general decline in the incidence of gastric cancer, it remains the fourth most common cancer and second leading cause of cancer-related deaths worldwide.¹ In 2010, it is estimated that there will be 21,000 new gastric cancer cases and 10,570 deaths from gastric cancer in the USA.² In Africa and South Asia, the incidence of gastric cancer is much lower than in East Asian countries, although the prevalence of H. pylori infection is high in each of these regions (Table 1).³ Even within East Asia, the incidence of gastric cancer varies, decreasing at increasingly southerly latitudes. What is the cause of these geographic differences in disease pattern? In addition, H. pylori infection is thought to be involved in the development of both gastric cancer and duodenal ulcer, which are at opposite ends of the disease spectrum. What makes it possible for H. pylori infection to be involved in both of these gastroduodenal diseases?

Table 1.

Frequency of CagA phenotype and incidence of gastric cancer in 2008

Geographic region/country	Number of strains studied	Number of sequences by CagA type		Age-standardized incidence rates per 100,000 population for gastric cancer
		Western	East Asian	Overall incidence	Male	Female
World total	NA	NA	NA	14.1	19.8*	9.1
Asia	NA	NA	NA	18.6*	25.9*	11.7
East Asia	NA	NA	NA	30.0*	42.4*	18.3*
South Korea	175	1	174	41.4*	62.2*	24.6*
Japan	419	21	398	31.1*	46.8*	18.2*
China	65	4	61	29.9*	41.3*	18.5*
Taiwan	34	0	34	10.4	13.5	7.6
West Asia	NA	NA	NA	9.4	12.6	6.7
Southeast Asia	NA	NA	NA	8.6	10.9	6.7
Vietnam	122	4	118	18.9*	24.4*	14.6
Malaysia	3	2	1	8.4	10.7	6.4
Thailand	106	54	52	3.5	4.2	3.0
South-Central Asia	NA	NA	NA	5.3	6.7	3.9
Kazakhstan	4	4	0	20.6*	31.7*	13.7
Iran	91	91	0	15.6*	21.9*	9.0
Turkey	51	51	0	13.5	18.9*	8.8
Pakistan	26	26	0	6.3	8.0	4.5
India	4	4	0	3.8	4.7	2.9
Latin America and Caribbean	NA	NA	NA	11.7	15.7*	8.4
South America	NA	NA	NA	12.4	17.3*	8.4
Colombia	147	147	0	17.4*	23.4*	12.5
Chile	1	1	0	17.9*	27.3*	10.3
Brazil	10	10	0	10.9	16.2*	6.6
Central America	NA	NA	NA	10.9	12.7	9.3
Costa Rica	33	33	0	21.8*	28.5*	15.6*
Caribbean	NA	NA	NA	8.5	11.2	6.1
Europe	NA	NA	NA	10.3	14.7	7.0
Central-East Europe	NA	NA	NA	14.7	22.2*	9.7
West Europe	NA	NA	NA	6.5	9.0	4.4
Italy	57	57	0	10.9	14.8	7.7
Germany	1	1	0	7.7	10.3	5.5
Ireland	3	3	0	7.3	10.1	4.8
Greece	100	100	0	5.7	7.9	3.9
France	63	63	0	4.9	7.5	2.8
Sweden	5	5	0	4.3	5.7	3.1
North Europe	NA	NA	NA	6.2	8.6	4.2
Oceania	NA	NA	NA	5.5	7.5	3.7
Australia	1	1	0	5.2	7.3	3.2
North America	NA	NA	NA	4.2	5.8	2.8
Canada	20	20	0	4.8	6.7	3.1
USA	152	152	0	4.1	5.7	2.8
Africa	NA	NA	NA	4.0	4.7	3.3
South Africa	12	12	0	3.2	4.5	2.3

CagA sequencing data were initially obtained from the NCBI (April 16, 2007).²⁶ Data from previous studies on genotyping of the cagA repeat region were also included for South Korea, Japan, China, Taiwan, Kazakhstan, Pakistan, Italy, France, South Africa, Canada, USA, Colombia and Brazil,²⁰ in addition to Vietnam,^20,24 Thailand,^20,25 Iran²³ and Turkey.²² Data on age-standardized incidence rates (ASRs) per 100,000 population obtained from the GLOBOCAN database, which provides access to the most recent (2008) estimates of the incidence of, and mortality from, 27 major cancers worldwide; organized by the International Agency for Research on Cancer (IARC).¹

^*Regions and countries with an ASR ≥15.0. Abbreviation: NA, not available.

The questions above may be answered by considering various host-related factors, the duration of infection and/or environmental factors. Host genetics play an important role in interactions between the host and H. pylori. For example, polymorphisms in those genes that control the host’s inflammatory response can either accentuate or attenuate the inflammatory response and thus the incidence of adverse outcomes associated with an infection.⁴ The duration of infection and the age at acquisition of an H. pylori infection are also important, as is poor nutrition in childhood (or childhood infections), which lead to reduced acid secretion and increased susceptibility to the development of H. pylori-induced atrophic gastritis and gastric cancer.⁵ Environmental factors known to protect against gastric cancer include storage of food in a refrigerator (that is, a food preservation surrogate and an indication of reduced consumption of a seasonal diet) and the increased consumption of fresh fruit and vegetables; correlates with gastric cancer include a high salt intake and nitrate consumption.⁶ In addition to host-related factors, duration of infection and/or environmental factors, considering the bacterial factors present should provide one of the most important clues needed to answer the questions.

H. pylori has co-evolved with humans at least since they migrated out of Africa approximately 58,000 years ago and probably throughout their evolution (Figure 1); H. pylori is, therefore, a highly heterogeneous bacterium,^7–10 the virulence of which has also changed geographically. In this Review, current knowledge of H. pylori virulence factors and how they contribute to the differing geographic gastric cancer disease patterns and to the development of both gastric cancer and duodenal ulcer is discussed, with a focus on cytotoxin-associated gene A product (CagA), vacuolating cytotoxin (VacA), outer inflammatory protein (OipA) and duodenal ulcer promoting (DupA).

Figure 1.

Distribution of Helicobater pylori genotypes before Columbus found the New World and human migration to America and Oceania began. There are seven modern H. pylori population types—hpEurope, hpEastAsia, hpAfrica1, hpAfrica2, hpAsia2, hpNEAfrica and hpSahul.^7–9 hpEurope includes almost all H. pylori strains isolated from ethnic Europeans, including people from countries colonized by Europeans. Most H. pylori isolates from East Asia belong to hpEastAsia, which includes hspMaori (Polynesians, Melanesians, and native Taiwanese), hspAmerind (American Indians) and hspEAsia (East Asia) subpopulations. hpAsia2 strains are isolated in South, Southeast and Central Asia; hpAfrica1 in West Africa, South Africa and African Americans. hpNEAfrica is predominantly made up of isolates from Northeast Africa. hpAfrica2 is very distinct from any other type and has currently only been isolated in South Africa. hpSahul is a novel group specific to H. pylori strains isolated from Australian Aborigines and Highlanders of New Guinea. H. pylori is predicted to have spread from East Africa over the same time period as anatomically modern humans (~58,000 years ago), and has remained intimately associated with their human hosts ever since. Estimated global patterns of H. pylori migration are indicated by arrows and the numbers show the estimated time since they migrated (years ago). The broken arrow indicates an unconfirmed migration pattern. Detailed analyses of migration patterns have been explained previously.^8,9

CagA

CagA is the most extensively studied H. pylori virulence factor. There are two types of clinical H. pylori isolate: CagA-producing (cagA positive) strains and CagA-nonproducing (cagA negative) strains. Studies of Mongolian gerbils showed that gastric cancer developed in those animals infected with wild-type H. pylori, but not in animals infected with isogenic cagA mutants.^11,12 Since then, another study reported that gastric cancer and other malignant neoplasms occurred in some transgenic mice that had CagA protein artificially introduced.¹³ These data have led to increasing knowledge of CagA as a bacteria-derived carcinogen. In Western countries, it has been reported that individuals infected with cagA-positive strains of H. pylori are at a higher risk of peptic ulcer or gastric cancer than those infected with cagA-negative strains.^14,15 In East Asia, however, most strains of H. pylori have the cagA gene irrespective of the disease; the pathogenic difference in East Asia is, therefore, difficult to explain simply in terms of the presence or absence of the cagA gene alone.¹⁶

Caga type: Western versus East Asian

cagA is a polymorphic gene. In particular, there are different numbers of repeat sequences located in the 3′ region of the cagA gene of different H. pylori strains.^17–19 The repeat regions were initially classified into two types—the first repeat and the second repeat—and the sequence of the second repeat region was found to differ considerably between East Asian strains and Western strains of H. pylori.^17–20

Each repeat region of the CagA protein contains Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs, including a tyrosine phosphorylation site. It has now become more common to name the first repeat region as EPIYA-A and EPIYA-B segments and to name the second repeat region as EPIYA-C or EPIYA-D segments for Western and East Asian strains, respectively.²¹ As such, each CagA is assigned a sequence type consisting of the names of the EPIYA segments in its sequence (that is, ABC, ABCC or ABCCC for Western-type CagA and ABD for East-Asian-type CagA) (Figure 2). Analyses of the repeat regions of CagA has demonstrated that, although rare, some East Asian strains have a Western-type CagA sequence, especially strains isolated from Okinawa, the south islands of Japan (Table 1).^20,22–26 By contrast, none of the Western strains studied have an East-Asian-type CagA sequence.^20,26 A study published in 2009 reported that the Western-type CagA detected in strains from Okinawa formed a different cluster compared with the ordinary Western-type CagA and it was named as the J-Western-type CagA subtype.²⁷ As H. pylori, together with humans, is thought to have migrated out of Africa approximately 58,000 years ago, and reached parts of Europe and finally East Asia via Central Asia (Figure 1), the original H. pylori strain is thought to have a Western-type CagA sequence that possibly evolved to the J-Western-type CagA and then to the East-Asian-type CagA. Further studies are needed to clarify the role of the different Western-type CagA sequences in the pathogenesis of disease.

Figure 2.

Structural polymorphism in CagA. Western-type CagA contain EPIYA-A, EPIYA-B, and EPIYA-C segments. By contrast, East-Asian-type CagA contain the EPIYA-A, EPIYA-B and EPIYA-D segments, but not the EPIYA-C segment. The EPIYA motif in each segment (shown in green) represents the tyrosine phosphorylation sites of CagA. The sequence flanking the tyrosine phosphorylation site of the EPIYA-D segment (EPIYATIDF), but not the EPIYA-C segment (EPIYATIDD), matches perfectly the consensus high-affinity binding sequence for the SH2 domains of SHP2. In Western countries, the incidence of gastric cancer is significantly higher in patients infected with strains containing multiple EPIYA-C segments than in patients infected with strains containing a single EPIYA-C segment (that is, ABCCC versus ABC). By contrast, almost all East Asian strains contain a single EPIYA-D segment. CagA forms dimers in cells in a phosphorylation-independent manner, and the CagA multimerization (CM) sequence (also named the conserved repeat responsible for phosphorylation-independent activity [CRPIA] or MARK2/PAR1b kinase inhibitor [MKI]) in yellow was identified as the site responsible for dimerization, for inhibition of MARK2/PAR1b kinase and for the interaction of CagA with activated c-Met.

Pathogenicity of multiple EPIYA segments

The first reports to suggest that the number of EPIYA segments in the second repeat region is associated with gastric cancer were published in the late 1990s.^17,18 Subsequent studies confirmed that, in Western countries, the incidence of gastric cancer is notably higher in patients infected with strains containing multiple EPIYA-C segments than in patients infected with strains containing a single EPIYA-C segment (that is, ABCCC versus ABC).^17,18,28,29 However, as almost all East Asian strains contain a single EPIYA-D segment,²⁶ it is difficult to differentiate between simple gastritis and gastric cancer simply by considering the number of repeated sequences in East Asia.

With regard to the function of the repeat regions, initial demonstrations suggest that H. pylori strains that have a larger number of EPIYA segments in their repeat regions are less resistant to gastric acid.¹⁸ This finding seems to indicate that H. pylori strains containing many EPIYA segments can survive only in the presence of advanced atrophic gastritis (such as gastric cancer), in which gastric acid secretion is low. If this is the case, multiple EPIYA segments may have developed after the onset of atrophy and are not the cause, but rather the effect of atrophy.

Tyrosine phosphorylation and pathogenicity

There have been many reports that the site of tyrosine phosphorylation in the EPIYA motif of CagA plays a direct role in the pathogenicity of H. pylori. Following injection of CagA into epithelial cells, the EPIYA motifs are tyrosine phosphorylated by Src and Abl family kinases,^30–34 which results in the impairment of a variety of intracellular signaling systems (Figure 3). Backert et al. reported that there are at least 20 known cellular binding partners of CagA, which is the highest number currently known for any virulence-associated effector protein in the microbial world.³⁵ Of these binding partners, 10 host cell proteins bind CagA in a phosphorylation-dependent manner.^35,36 The number of EPIYA motifs present is related to the level of CagA phosphorylation that occurs in epithelial cells infected by either East Asian³⁷ or Western strains.²⁸

Figure 3.

The pathogenesis of CagA-related signaling. In the early steps of CagA recognition, CagF binds CagA.^117,118 CagL, CagY and probably CagI utilize host integrin β1 as a cell-surface receptor, which triggers delivery of CagA into target cells.^41,119 Injected CagA can then undergo tyrosine phosphorylation by Src and Abl family kinases at the EPIYA motifs located near the C-terminal end (in EPIYA-A [A], EPIYA-B [B], EPIYA-C [C] or EPIYA-D [D] segments). CagA binds to and activates or inactivates multiple signaling proteins in both a phosphorylation-dependent and phosphorylation-independent manner. There are currently 20 known cellular binding partners of CagA, 10 of which form phosphorylation-dependent interactions with CagA.³⁵ CagA itself forms dimers in a phosphorylation-independent manner, via the CagA multimerization (CM) sequence⁴² (also known as MKI or CRPIA). CM sequence is also essential for formation of the CagA–Par1⁴² and CagA–c-Met complexes.⁴⁶ The proposed functions of complexes formed by CagA with Grb7, SHP1, Ras-GAP, PI3K, α-Pix and integrin β1 are unknown. Experimental data obtained via natural infection versus transfection of CagA are conflicting—PI3K, Grb2 and Crk did not interact with CagA in transfection experiments.³⁴

The best studied of the host signaling factors that interact with phosphorylated CagA is the Src homology-2 domain-containing phosphatase 2 (SHP2).³⁸ SHP2 is able to bind EPIYA-B, EPIYA-C and EPIYA-D segments. Importantly, however, CagA with an EPIYA-D segment has a higher binding affinity for SHP2 than CagA with an EPIYA-C segment.²¹ The sequence flanking the tyrosine phosphorylation site of the EPIYA-D segment (EPIYATIDF), but not the EPIYA-C segment (EPIYATIDD), matches perfectly the consensus high-affinity binding sequence for the SH2 domains of SHP2 (Figure 2).

A study that used stable isotope labeling with amino acids in cell culture combined with mass spectrometry of bound protein (SILAC/MS) demonstrated that C-terminal Src tyrosine kinase (Csk) prefers to bind EPIYA-A and EPIYA-B segments, whereas Ras-GAP and Grb7 prefer to bind EPIYA-C segments.³⁶ Malignant neoplasms have been shown to occur with greater frequency in transgenic mice in which East-Asian-type CagA had been introduced compared with transgenic mice in which Western-type CagA had been introduced.³⁹ The discovery that, in vivo, East-Asian-type CagA is more carcinogenic than Western-type CagA is important; however, in this transgenic mouse model, the gastric cancer developed without inflammation and malignancy also occurred in nongastric organs; which raises questions about the applicability of this model to human disease.

Many previous in vitro studies of CagA involved transfection of a CagA expression vector instead of natural injection of CagA via the cag pathogenicity island (PAI) type IV secretion system (T4SS). However, transfection experiments have potentially confounded our understanding of the role of CagA. For example, Grb2 reportedly interacts with phosphorylated CagA during H. pylori infection,³⁶ but such an interaction was not found in studies using transfected CagA.^34,40 Importantly, transfection of CagA does not closely mimic natural injection of CagA by the T4SS. T4SS-injected CagA primarily localizes to focal adhesions,⁴¹ whereas vector-expressed CagA localizes to the entire host membrane.³⁸ Most importantly, transfection prevents assessment of critical bacterial–host cell interactions, such as the activation of cellular signaling associated with VacA and OipA. Therefore, information gained from transfection experiments should be interpreted with caution and the findings confirmed by experiments using natural injection.

Phosphorylation-independent signaling

Nonphosphorylated CagA also impairs intracellular signaling systems. Currently, there are at least 10 known phosphorylation-independent CagA host interaction partners (Figure 3).³⁵ In addition, CagA itself forms dimers in cells in a phosphorylation-independent manner, and the CagA multimerization (CM) sequence (FPLxRxxxVxDLSKVG) was identified by Ren et al. as the site responsible for dimerization.⁴² The CM sequence is located within the EPIYA-C segment, but is just downstream of the EPIYA-D segment (Figure 2).

The CM sequence is also essential for the formation of the CagA–PAR1 (MARK) complex—bound nonphosphorylated CagA inhibits the kinase activity of PAR1 to promote the loss of cell polarity^42,43 and a loss of the elongation phenotype of host cells.⁴⁴ The CM sequence has also been named by Nesic et al. as the MARK2/PAR1b kinase inhibitor (MKI).⁴⁵ In addition, the same region was reported to be responsible for the interaction of CagA with activated c-Met and named as the conserved repeat responsible for phosphorylation-independent activity (CRPIA) by Suzuki and collegues.⁴⁶ The interaction of CRPIA with c-Met subsequently led to the upregulation of β-catenin and nuclear factor κB (NFκB) transcriptional activities, which promoted proliferation and inflammation, respectively. Therefore, there are currently three acronyms that essentially correspond to the same sequence in CagA. Functional differences between the CM/MKI/CRPI sequence in Western-type CagA (FPLKRHDKVDDLSKVG) and East-Asian-type CagA (FPLRRSAAVNDLSKVG) have not been detected^42,43 and have yet to be identified.⁴⁵

Interleukin 8 production

Infection of the gastric mucosa by H. pylori is characterized by the production of proinflammatory cytokines, especially interleukin (IL)-8, a potent neutrophil-activating chemokine.^47–50 The cag PAI, in which the cagA gene is localized at one end, is involved in the induction of gastric IL-8 production; whereas most reports have demonstrated that the CagA protein is not involved in IL-8 induction.^51–53 However, one study has demonstrated that CagA is involved in IL-8 induction in a strain-dependent and time-dependent manner.⁵⁴ IL-8 induction has typically been assessed 12–24 h after infection in vitro using gastric epithelial cells; however, the potentiating effects of CagA only become evident 36–48 h after infection.⁵⁴ Interestingly, findings from transfection experiments suggest that the IL-8 levels induced by East-Asian-type CagA are higher than those induced by Western-type CagA.⁵⁵

Clinical importance of CagA phenotype

As described above, there has been an increasing tendency in the past 10 years to explain the higher incidence of gastric cancer in East Asia using the concept of East-Asian-type CagA and Western-type CagA. The incidence of gastric cancer is clearly higher in East Asian countries than in any other countries when age-standardized rates (ASRs) are considered (Table 1). However, it should be noted that the incidence of gastric cancer is also high in some regions where Western-type CagA strains are reported to account for the majority of H. pylori strains (for example, in Peru and Columbia [ASR per 100,000 population 21.2 and 17.4, respectively]).^20,56 In addition, in Africa the rate of H. pylori infection is high (for example, 70–97% of patients with dyspepsia are infected with H. pylori, as are 80% of asymptomatic volunteers⁵⁷) but gastric cancer is generally uncommon; this seemingly contradictory situation is known as the “African Enigma”.⁵⁷ The incidence of gastric cancer is, however, extremely high in Mali, West Africa (ASR per 100,000 population 20.3) and the frequency of gastric cancer among women in this country is higher than it is among women in Japan (ASR per 100,000 population 19.3 versus 18.2) (Table 1). These facts cannot be explained by the presence of East-Asian-type CagA versus Western-type CagA alone.

In an attempt to explain the geographic difference in the incidence of gastric cancer, my group compared the cagA gene repeat sequences found in Columbia (ASR per 100,000 population 17.4) with those found in the USA (ASR per 100,000 population 4.1). 100 H. pylori isolates from patients with simple gastritis (30 from Columbia and 70 from the USA) were analyzed—57% of the isolates from Columbia had two EPIYA-C segments, whereas only 4% of the isolates from the USA had two EPIYA-C segments (Y. Yamaoka, unpublished data). Overall, the number of EPIYA-C segments may explain, to some extent, the geographic difference in the incidence of gastric cancer in Western countries.

In Thailand, the incidence of gastric cancer is extremely low compared with that in the surrounding countries (ASR per 100,000 population 3.5) (Table 1). A previous study demonstrated that gastric cancer was common among ethnic Chinese and rare among the Thai indigenous population.²⁵ All ethnic Chinese patients who had gastric cancer or peptic ulcers were infected with H. pylori with East-Asian-type CagA; whereas East-Asian-type CagA was found in only 40% of ethnic Chinese patients who had gastritis. These data suggest that East-Asian-type CagA may have increased virulence compared with Western-type CagA. However, interpretation of the relationship between the CagA type and outcome in Thailand is complicated by the fact that the majority of gastric cancers (82%) occurred among ethnic Chinese or Thai–Chinese individuals.²⁵ Taken together, although there is increasing evidence from in vitro studies and animal models that East-Asian-type CagA is more virulent than Western-type CagA, the clinical concept that East-Asian-type CagA versus Western-type CagA explains the geographic difference in the incidence of gastric cancer is still not accepted.

In East Asia, the incidence of gastric cancer decreases at increasingly southerly latitudes. Indeed, the incidence of gastric cancer in Vietnam is half that in South Korea, although most Vietnamese strains of H. pylori (97%) have been reported to contain East-Asian-type CagA (Table 1). In addition, most of the East Asian strains of H. pylori have only one EPIYA-D segment.²⁶ Therefore, in areas where infection with East-Asian-type CagA H. pylori strains is predominant, it is not possible to explain the difference in the incidence of gastric cancer by CagA alone. The structure of the East-Asian-type cagA gene in Vietnamese strains of H. pylori is, however, slightly different to that of strains from other East Asian countries.²⁴ An 18 bp deletion unique to Vietnamese strains was identified in the region located slightly upstream of the EPIYA-A segment, whereas a 39 bp deletion was identified in strains common in East Asian countries such as Japan, and no deletion was identified in Western strains.²⁴ Further research is required to determine whether these subtypes are involved in the pathogenesis of gastric cancer.

VacA

VacA is the second most extensively studied H. pylori virulence factor. In addition to inducing vacuolation, VacA can induce multiple cellular activities, including membrane-channel formation, cytochrome c release from mitochondria leading to apoptosis, and binding to cell-membrane receptors followed by initiation of a proinflammatory response.^58–60 VacA can also specifically inhibit T-cell activation and proliferation.^61–63 Studies indicate that VacA and CagA can even inhibit at least some of each other’s signaling pathways.^64–67 For example, CagA has been shown to promote the expression of the apoptotic suppressor Mcl1, and inhibit epithelial cell apoptosis caused by VacA.^64,65 These data again emphasize the importance of in vitro infection experiments in which interaction among H. pylori virulence factors can be taken into account.

Clinical importance of vacA genotype

Virtually all H. pylori strains have a functional vacA gene. There is variation in the vacuolating activity of different H. pylori strains,^68,69 primarily due to differences in the vacA gene structure at the signal (s) region (s1 and s2) and the middle (m) region (m1 and m2).⁷⁰ In vitro experiments demonstrated that s1/m1 strains are the most cytotoxic, followed by s1/m2 strains, whereas s2/m2 strains have no cytotoxic activity and s2/m1 strains are rare.^70,71 In agreement with in vitro data, in Western countries, including Latin America, the Middle East and Africa, there have been many reports that individuals infected with s1 or m1 H. pylori strains have an increased risk of peptic ulcer or gastric cancer compared with individuals infected with s2 or m2 strains.^14,70,72,73 In East Asia, however, most H. pylori strains have an s1-type s region; therefore the pathogenic difference cannot be explained by the type of s region present.^16,20 Moreover, it is interesting to note that almost all cagA-positive strains are classified as an s1 strain, whereas almost all cagA-negative strains are classified as an s2/m2 strain.⁷⁰

With respect to the m region, however, there is variation within East Asia. Although m1 strains are common in parts of north East Asia, such as Japan and South Korea, m2 strains are predominant in parts of south East Asia, such as Taiwan and Vietnam (Table 2).^20,24 As the incidence of gastric cancer is higher in the north than in the south of East Asia, the m region may play a role in the regional difference in disease pattern. Even within Vietnam, the incidence of gastric cancer is approximately 1.5 times higher in Hanoi in the north than in Ho Chi Minh in the south of the country (Table 2). Research has shown that almost all H. pylori strains are classified as cagA-positive and vacA s1 strains in these two cities, but that the frequency of the vacA m1 strain is considerably higher in Hanoi.²⁴

Table 2.

vacAm region genotype and incidence of gastric cancer in 2008

Country/city	Number of strains studied	vacA genotypes (%)		Age-standardized incidence rates per 100,000 population for gastric cancer
		m1	m2	Overall incidence	Male	Female
South Korea	87	83 (95.4)	4 (4.6)	41.4	62.2	24.6
Japan	83	79 (95.2)	4 (4.8)	31.1	46.8	18.2
China	27	18 (66.7)	9 (33.3)	29.9	41.3	18.5
Vietnam*	78	29 (37.1)	45 (57.9)	18.9	24.4	14.6
Hanoi*‡	38	19 (50.0)	17 (44.7)	NA	27	13.2
Ho Chi Minh*‡	40	10 (25.0)	28 (70.0)	NA	18.7	8.1
Taiwan	28	9 (32.1)	19 (67.9)	10.4	13.5	7.6

Data for vacA genotypes were obtained from previous studies by Yamaoka et al. (for South Korea, Japan, China and Taiwan)²⁰ and by Uchida et al. (for Vietnam).²⁴ To exclude the possible bias among countries, only simple gastritis cases were extracted from the published data. Data for gastric cancer incidence by country were obtained from the GLOBOCAN database, which provides access to the most recent estimates (for 2008) of the incidence of, and mortality from, 27 major cancers worldwide; organized by the International Agency for Research on Cancer (IARC).¹

^*For four strains from Vietnam (two from Hanoi and two from Ho Chi Min) neither m1 nor m2 could be detected; therefore, the sum of m1 and m2 does not match the number of strains studied.

^‡Data for gastric cancer incidence in two cities in Vietnam were obtained from the CI5 (Cancer Incidence in Five Continents) database, which provides access to detailed information on the incidence of cancer recorded by cancer registries (regional or national) worldwide; organized by the IARC (http://ci5.iarc.fr/). Abbreviations: m, middle; NA, not analyzed.

Novel genotypes

In 2007, a third disease-related region of vacA was identified between the s region and the m region; it was named the intermediate (i) region.⁷⁴ According to the authors of the study, all s1/m1 strains were classified as type i1, and all s2/m2 strains were classified as type i2, but s1/m2 strains were classified as either type i1 or i2, and i1 strains were shown to be more pathogenic. In the same study, typing of the i region was also reported to be more effective for determining the risk of gastric cancer in Iranian patients than typing of the s region or m region.⁷⁴ An additional study conducted by the same group showed that the i region plays a role in the development of peptic ulcer in people in Iraq and Italy.^75,76 However, in a study of patients from East and Southeast Asia, there was no association between the i region and disease.⁷⁷ More recently, a fourth disease-related region—the deletion (d) region—was identified between the i region and the m region.⁷⁸ The d region is divided into d1 (no deletion) and d2 (a 69–81 bp deletion). The study of Western strains demonstrated that d1 was a risk factor for gastric mucosal atrophy; however, almost all East Asian strains are classified as s1/i1/d1.⁷⁸ At present, therefore, typing of the m region may be the best overall marker for gastric cancer, especially in East Asia.

OipA

Approximately 4% of the H. pylori genome is predicted to encode outer membrane proteins (OMPs), some of which may function as adhesins.^79–85 One such OMP is OipA, which was identified in 2000.⁸⁶ The functional status of OipA is regulated by slipped strand mispairing that is determined by the number of CT dinucleotide repeats in the 5′ region of the gene (switch ‘on’ and OipA is functional; switch ‘off’ and OipA is nonfunctional).⁸⁶

Interleukin 8 production

OipA was initially identified as a proinflammatory-response-inducing protein based, in part, on the fact that oipA isogenic mutants reduced the production of IL-8 from gastric epithelial cell lines.⁸⁶ In vivo experiments using human gastric biopsy specimens also confirmed that a functional oipA was independently and significantly associated with high mucosal IL-8 levels.¹⁵ Some subsequent conflicting reports regarding IL-8 induction in vitro^87–90 might be at least partially explained by the fact that multiple in vitro passages can allow factors other than OipA to enhance IL-8 induction.⁹¹ Binding sites for the transcription factors NFκB, activator protein 1 (AP1), and interferon-stimulated responsive element (ISRE)-like element within the IL-8 promoter are involved in regulating IL-8 gene transcription in H. pylori-infected gastric epithelial cells in both an OipA-dependent and cag PAI-dependent manner.^92–94 However, data from my group demonstrated that the activation of NFκB is strain dependent and this might also be a reason for the conflicting reports (Figure 4).⁸³

Figure 4.

The pathogenesis of OipA-related signaling. Phosphorylation by OipA reportedly regulates various epithelial cell signaling pathways, most of which are also important for cag PAI-related signaling. p38/STAT1 pathways are regulated independently of the cag PAI. OipA and the cag PAI are both necessary for full activation of the IL-8 promoter, but act via different pathways that diverge upstream of IRF1: only OipA is involved in the STAT1/IRF1/ISRE pathway.⁹² OipA is believed to function as an adhesin; but its target receptors have not been identified. OipA phosphorylation sites are shown for all molecules. EGFR contains more than 10 phosphorylation sites but only the two investigated as part of OipA-related signaling pathways^98,99 are shown. OipA is reportedly involved in phosphorylation of FAK at tyrosine (Y) 397, Y576, Y577, Y861 and Y925, but not Y407; whereas the cag PAI is involved in phosphorylation of FAK at Y407 only.⁹⁸ However, another study reported that FAK phosphorylation at Y397 was cag PAI dependent.⁴¹ Broken arrows indicate unconfirmed interactions. Abbreviations: S, serine; T, threonine.

Gradually it has become clear that one of the functions of OipA is to induce inflammation and actin dynamics via phosphorylation of multiple signaling pathways (Figure 4).^92,95–99 However, most of these signaling pathways, with the exception of p38-related pathways, are also involved in cag PAI-related pathways. Therefore, there might have been some interaction between OipA and cag PAI and/or CagA; however, evidence of such an interaction has not been reported and further studies will be necessary to clarify the specific roles of OipA on these cag PAI-related pathways. One study has demonstrated that OipA in addition to CagA is involved in β-catenin signaling, leading to the opening of cell–cell junctions and proliferation.¹²

Gastric colonization

OipA functions as an adhesin and is reported to be involved in the attachment of H. pylori to gastric epithelial cells in vitro.^89,92,100 In addition, animal studies have revealed that OipA plays a role in the bacterial colonization of the gastric mucosa. In one study of mice infected with either wild-type H. pylori strain CPY2052 or its isogenic oipA mutant, the bacterial density was considerably decreased in the mice infected with the mutant strain compared with the mice infected with the wild-type strain.¹⁰⁰ In a study of Mongolian gerbils, the isogenic oipA mutant of the TN2 wild-type H. pylori strain did not cause infection.⁹⁰ By contrast, the isogenic oipA mutant of wild-type H. pylori strain 7.13 has been shown to infect animals; however, no animals infected with the oipA mutant developed gastric cancer, whereas 27% of those infected with the wild-type strain developed gastric cancer.¹² These animal studies demonstrate that OipA alone plays a role in the development of gastric cancer.

Clinical importance of OipA

Interestingly, the oipA ‘on’ status and cagA positivity are closely linked with each other (correlation coefficient = 0.82).¹⁵ As described above, the cagA status is also linked to the vacA s region type and it is further closely linked to the presence of the babA gene; the BabA protein is another virulence factor that is also an OMP.⁸⁰ The links among these factors should have a certain biological significance and they may somehow interact with each other. It might be more relevant to hypothesize that these factors interact synergistically with each other and induce serious diseases, rather than to discuss which factor is the most virulent. According to one investigation so far, it is interesting to note that all East Asian strains are classified as oipA status ‘on’ and as functional OipA-protein-producing strains.⁸⁶ Half-collapsed CT repeat sequences in the s region of the oipA gene (for example, Japanese-derived strain JK51 contains a CTGCCTTTCT repeat sequence and its status is ‘on’) suggest that this sequence may result from an intentional change in the status in the course of the evolution of the bacteria to prevent the switch from being turned ‘off’ easily.

DupA

The production of CagA, VacA and OipA is linked and the majority of H. pylori strains produce either all of these proteins or none of them. Almost all East Asian strains of H. pylori are classified as CagA-producing, VacA-producing (vacA s1), and OipA-producing (oipA status ‘on’) strains and are highly pathogenic. In addition, CagA, VacA and OipA are all thought to be involved in the development of both gastric cancer and duodenal ulcer, which are at the opposite ends of the disease spectrum. In 2005, the first disease-specific H. pylori virulence factor that induced duodenal ulcer and had a suppressive action on gastric cancer was identified, and was named duodenal ulcer promoting gene A (DupA).¹⁰¹

Cytokine induction

Initial in vitro experiments using dupA-deleted and dupA-complemented mutants showed that the presence of dupA was associated with increased IL-8 production from both the antral gastric mucosa in vivo and gastric epithelial cells.¹⁰¹ The relationship between dupA and IL-8 induction from gastric epithelial cells in vitro is still controversial;^102,103 however, one study supports the original data that the presence of the dupA gene is associated with increased IL-8 production from gastric mucosa in vivo.¹⁰² Importantly, in vitro experiments using dupA mutants showed that dupA substantially increased H. pylori-induced IL-12p40 and IL-12p70 production by CD14⁺ mononuclear cells.¹⁰² The production of other T-helper-1-associated cytokines and IL-8 were also modestly induced by the cells. These data suggest that virulent H. pylori strains cause inflammation by stimulating epithelial cells via CagA, OipA and/or VacA-related proteins and mononuclear inflammatory cells through DupA.

Clinical outcomes

In an initial study of a total of 500 H. pylori isolates, including 160 from Japan, 175 from South Korea and 165 from Colombia, the positive rate for the dupA gene was high in patients with duodenal ulcer and low in patients with gastric cancer, regardless of a patients’ nationality (42% versus 9% on average).¹⁰¹ Since this report was published, studies have been conducted all over the world.¹⁰⁴ Reports from India,¹⁰⁵ China¹⁰⁶ and Iraq⁷⁵ support the initial data, whereas reports from Iran,^75,107 Brazil,^108,109 Singapore,¹⁰³ Malaysia¹⁰³ and Japan¹¹⁰ failed to demonstrate a correlation between the presence of the dupA gene and disease.

A pooled analysis of data from three Western countries (the USA, Belgium and South Africa) showed that the presence of the dupA gene was correlated with peptic ulcer as well as with gastric cancer.¹¹¹ One report from Iran, which was not supportive of the hypothesis that dupA contributes to the development of duodenal ulcer but not gastric cancer, showed that the incidence of dysplasia—a premalignant condition—was notably lower in dupA-positive patients,¹⁰⁷ partially supporting the hypothesis. One report from Brazil¹⁰⁸ was also unsupportive of the hypothesis, but showed that the rate of infection with dupA-positive strains in pediatric patients was 100% and higher than that in adult patients. This finding indicates that the number of dupA-positive strains decreases as gastric mucosal atrophy gradually progresses with aging, which also partially supports the hypothesis.¹⁰⁸ In a review of previous reports on dupA that included a total of 2,358 cases, the positive rate for dupA was 48% and confirmed that dupA was a risk factor for duodenal ulcer.¹¹² However, a large regional difference was also reported: dupA might, therefore, be a risk factor for both duodenal ulcer and gastric cancer in some regions.

Of note, an academic report on Brazilian strains that was presented at the Annual Meeting of the American Gastroenterological Association in 2008, showed that a dupA gene mutation (deletion or insertion) was found in 50% of patients with gastric cancer, whereas it was found in only approximately 20% of patients with duodenal ulcer.¹¹³ As a result, the positive rate for the functional dupA gene was considerably higher in patients with duodenal ulcer than in patients with gastric cancer, and this finding supported the original hypothesis. That such mutations occur at an unexpectedly high frequency should not be ignored. However, it is impossible to detect dupA mutations by using a simple PCR method; therefore, detection of functional dupA by measuring intact DupA protein using immunoblotting techniques, which has not been reported, should be attempted.

DupA as part of a novel T4SS

DupA is found in an area called the plasticity zone, in which many genes thought to be associated with pathogenicity can be found (Box 1).^101,104 DupA has a high homology with the VirB4 ATPase, but there are vir genes before and after the region of the dupA locus. In the strain Shi470, for example, virB2, virB3, virB4 (dupA), virB8, virB9, virB10, virB11, virD4 and virD2 can all be found (Figure 5). These vir genes before and after the region of the dupA locus are structurally similar to the T4SS called cag PAI and ComB, and are thought to be the third T4SS. Kersulyte et al. named the third T4SS in the plasticity zone tfs3 (type IV secretion 3).¹¹⁴ The T4SS containing dupA has since been named as tfs3a, and T4SS containing the virB4 sequence but not dupA was named as tfs3b.¹¹⁵ The strain G27 that contains tfs3a is unlikely to be functional because dupA has a frame-shift mutation. In addition, the strain J99 has a mutation in dupA and contains only a part of tfs3a. These observations suggest that only strains with functional dupA and complete tfs3a may be considered as pathogenic strains that have the same action as T4SS, such as cag PAI and ComB. The study of the plasticity zone is only at its beginning and may be the most attractive area for future investigation.

Box 1. Helicobacter pylori plasticity zones.

• There are several regions of the H. pylori genome in which the G+C content is lower than in the rest of the genome (33–34% vs 39%)

• One region of such low G+C content is the cag PAI—the others have been named collectively as plasticity zones on the basis of the variability in gene content among different H. pylori isolates

• Nearly half of the strain-specific genes of H. pylori are located in plasticity zones

Figure 5.

Type IV secretion system in the Helicobacter pylori plasticity zone. Gene arrangement in the plasticity region of H. pylori strain Shi470, which has a complete dupA cluster (named tfs3a [type IV secretion 3a]) and functional dupA gene sequence, in comparison with corresponding regions of genome sequences of other H. pylori strains (G27, J99, P12 and 26695). Genes encoding type IV secretion system (T4SS) components are represented by arrows, with frameshift mutations indicated by asterisks. Genes encoding proteins that have 90–95% sequence similarity to the Shi470 proteins are shown in green and genes encoding proteins that have 50–75% sequence similarity are shown in pink. It is possible that only those H. pylori strains that have both a functional dupA gene sequence and a complete tfs3a are pathogenic and have the same action as the cag PAI and ComB T4SS. The numbers shown represent the gene number of each strain deposited in GenBank (GenBank accession numbers for Shi470, G27, J99, P12 and 26695 are CP001072, CP001173, AE001439, CP001217 and AE000511, respectively).

Conclusions

So, can H. pylori factors explain both the geographic differences in gastric cancer disease pattern and the role of H. pylori infection in gastric cancer and duodenal ulcer? CagA, VacA and probably OipA (that is, different CT repeat sequence patterns) are key factors needed to answer the first question and DupA is a key factor needed to answer the second question. However, although these four virulence factors are obviously important they can provide only partial answers to the questions. First, since H. pylori consist of approximately 1,600 genes, there remains the possibility that additional important pathogenic genes will be identified. Second, it is also true that gastric cancer incidence has changed remarkably with environmental factors such as diet (for example, salt intake) or with migration. Host factors (for example, IL-1 polymorphisms) and duration of infection (for example, early infection with duodenal ulcer and late infection with gastric cancer) should also be taken into account. These various factors are thought to interact with each other in a complex manner in the actual development of disease.

In the past 10 years, tremendous progress has been made in understanding the mechanisms by which H. pylori virulence factors induce gastroduodenal damage. However, controversial data are also increasing as research moves forward. Transfection of CagA versus natural infection via T4SS gives us different results. The relationship between induction of IL-8 and CagA or OipA is also controversial, and ‘time-dependent’ and ‘strain-dependent’ phenomena have proved confusing. The terminology used in the field is also confusing, with CM, MKI and CRPIA all being used to describe the same sequence. In addition, tfs3a and tfs3b in strain P12 were both named tfs4.¹¹⁶ We should, therefore, take the time to understand and interpret each report carefully, with methodology and terminology used taken into account.

There also remains a considerable knowledge gap between the detailed dissection of events observed in vitro or in animal models and the clinical and/or epidemiological confirmation of their role in the pathogenesis of human disease. One reason for this discrepancy is that H. pylori is surprisingly heterogenic and we use only limited H. pylori strains in vitro or in animal models. By detailed typing of H. pylori in addition to searching for interactions between bacterial factors and other factors such as environmental factors and host factors, we will gradually understand the mechanisms by which H. pylori induces gastric injury, leading to gastric cancer.

Key points.

• Experimental evidence suggests that East-Asian-type CagA is more virulent than Western-type CagA, but the concept that this explains geographic differences in the incidence of gastric cancer is still controversial

• The number of CagA Glu-Pro-Ile-Tyr-Ala (EPIYA)-C segments (with multiple repeats increasing the virulence) may explain, to some extent, geographic differences in the incidence of gastric cancer in Western countries

• The genotype of the vacA middle region (m1 versus m2; m1 being more virulent) may, in part, explain geographic differences in the incidence of gastric cancer in East Asian countries

• CagA, OipA and VacA are thought to interact synergistically with each other to induce serious disease

• The presence of functional DupA with an intact, full size dupA cluster (tfs3a) is associated with duodenal ulcers

• Bacterial factors, environmental factors and host factors are thought to form complex interactions with each other in the actual development of disease

Review criteria.

The references cited in this review were identified by searching the MEDLINE database. No date restrictions were applied to the search, which was limited to articles published in the English language. The search terms used were “Helicobacter pylori”, “CagA”, “cag pathogenicity island”, “VacA”, “OipA”, “DupA”, and “plasticity”. The reference lists of the articles identified were also searched to identify additional relevant references.