The impacts of H. pylori virulence factors on the development of gastroduodenal diseases

Although most H. pylori infectors are asymptomatic, some may develop serious disease, such as gastric adenocarcinoma, gastric high-grade B cell lymphoma and peptic ulcer disease. Epidemiological and basic studies have provided evidence that infection with H. pylori carrying specific virulence factors can lead to more severe outcome. The virulence factors that are associated with gastric adenocarcinoma development include the presence, expression intensity and types of cytotoxin-associated gene A (CagA, especially EPIYA-D type and multiple copies of EPIYA-C) and type IV secretion system (CagL polymorphism) responsible for its translocation into the host cells, the genotypes of vacuolating cytotoxin A (vacA, s1/i1/m1 type), and expression intensity of blood group antigen binding adhesin (BabA, low-producer or chimeric with BabB). The presence of CagA is also related to gastric high-grade B cell lymphoma occurrence. Peptic ulcer disease is closely associated with cagA-genopositive, vacA s1/m1 genotype, babA2-genopositive (encodes BabA protein), presence of duodenal ulcer promoting gene cluster (dupA cluster) and induced by contact with epithelium gene A1 (iceA1), and expression status of outer inflammatory protein (OipA). The prevalence of these virulence factors is diverse among H. pylori isolated from different geographic areas and ethnic groups, which may explain the differences in disease incidences. For example, in East Asia where gastric cancer incidence is highest worldwide, almost all H. pylori isolates were cagA genopositive, vacA s1/i1/m1 and BabA-expressing. Therefore, selection of appropriate virulence markers and testing methods are important when using them to determine risk of diseases. This review summarizes the evidences of H. pylori virulence factors in relation with gastroduodenal diseases and discusses the geographic differences and appropriate methods of analyzing these virulence markers.


Background
H. pylori infection is highly prevalent affecting half of the world's population. Once infected, H. pylori can be a lifelong infection in the host unless eradicated. Nevertheless, about 85% of the infected patients only have mild asymptomatic gastritis, while 15% of patients can develop to have peptic ulcer disease (PUD), and less than 1% can develop to have gastric cancer [1].
The diverse clinical presentation of H. pylori infection is a result of interaction between bacterial virulence (e.g. CagA, VacA, BabA), host genetic (e.g. IL-1β, IL-10, TNF-α), and environmental factors (e.g. diet, smoke). The virulence factors of H. pylori can be categorized to be related with 3 major pathogenic processes, including colonization, immune escape and disease induction ( Table 1). The virulence factors responsible for establishing colonization include urease, flagella, chemotaxis system, and adhesins [2,3]. Knocking out the urease, flagella or chemotaxis genes all leads to a failure of H. pylori to establish colonization [3]. With increasing antibiotic resistance, these virulence factors provide alternative drug or vaccine targets for H. pylori eradication and prevention [4]. The virulence factors responsible for immune escape help H. pylori escape from host immune clearance and allow its persistence in the human stomach [5]. This review focused on the virulence factors causing more serious clinical outcomes.

Virulence factors associated with gastric adenocarcinoma
Gastric adenocarcinoma is the most deadly disease cause by H. pylori. The virulence factors that are associated with development of gastric adenocarcinoma are summarized in Table 2.
Cytotoxin-associated gene a (CagA) & type IV secretion system (T4SS) CagA is a well-recognized oncoprotein which is injected into host cells via a pilus structure called type IV secretion system (T4SS) [6]. Successful pilus formation and CagA translocation relies on the binding between CagL on the tip of T4SS and integrin α5β1 receptor on the host cell [7]. The gene locus that encodes CagA and the T4SS is called cag pathogenicity island (cag PAI). After being injected into host cells, CagA alters intracellular signal transduction pathways that facilitates malignant transformation of gastric epithelial cells or activates Lgr5-positive stem cells [8,9]. Importantly, transgenic mice overexpressing phosphorylation-competent CagA developed gastrointestinal adenocarcinoma, myeloid leukemia and B cell lymphoma, while phosphorylation-resistant CagA could not confer the same pathological changes [10]. These data provided direct evidence that CagA is an oncoprotein and the need of phosphorylation in conferring oncogenesis. In addition to the above cellular changes, CagA and T4SS also increase gastric inflammation via NFκB signaling and increased IL-8 secretion [11,12], which predispose to genetic instability and carcinogenesis. CagA can also cause epigenetic changes, such as DNA promoter or histone hypermethylation, which in turn leads to downregulation of tumor suppressor genes (e.g. MGMT) or microRNAs (e.g. let-7) [13,14]. Alternatively, CagA can attenuate tumor suppressing micro-RNA expression (e.g. miR-320a, miR-4496) via unknown mechanisms [15,16]. Therefore, CagA and T4SS can contribute to gastric carcinogenesis via multiple mechanisms.
In concordance with the above-mentioned in vitro and in vivo evidences, several epidemiologic studies also support infection with CagA-positive H. pylori can increase the gastric cancer risk, especially for the non-cardiac location. Most of these studies just tested the serum antibodies against CagA protein to define the status of a CagA-positive H. pylori infection [17][18][19]. Meta-analyses of case-control studies showed CagA-seropositive is associated with 2-fold risk of distal gastric cancer among H. pylori infectors [19], while cagA-genopositive is associated with 2.1-2.4 fold risk of gastric cancer [20]. A cohort study with long-term follow-up also demonstrated that infection with cagA-genopositive strain was associated with greater risk of progression of gastric precancerous lesion (OR = 2.28). However, in East Asia, where nearly all H. pylori strains possess cagA gene [21], presence of serum anti-CagA antibody or cagA gene may not be sensitive enough [22], and CagA subtyping is suggested to identify high risk infectors (Table 2).
Accordingly, the risk of gastric cancer in CagA-positive H. pylori infector can be further stratified by CagA expression level [23,24], the amount of translocation into host cell [25,26] and its biological activity [27][28][29]. CagA expression level is higher with the presence of genetic AATAAGATA motif upstream of the translation-starting site, which was associated with greater risk of advanced gastric precancerous lesion [23,24]. The amount of CagA translocation is greater in strains harboring an amino acid sequence polymorphism (Y58E59) in the CagL of T4SS, which increases its binding affinity with integrin receptor α5β1 on the gastric epithelial cell [26]. Accordingly, gastric cancer risk was increased by 4.6-fold in patients infected by CagL-Y58E59 strain compared with those infected by non-Y58E59 strain [25].
The biological activity of CagA protein is determined by the types and numbers of the EPIYA motifs on its C-terminal region. There are four types of EPIYA motifs based on their distinct conserved flanking sequences, namely EPIYA-A, -B, -C and -D. H. pylori isolates from East Asia where gastric cancer incidence is highest often contains EPIYA A-B-D motif, whereas isolates from Western countries often contains EPIYA A-B-C motif. oipA "on" (vs. oipA "off") 2.4 DNA sequencing or immunoblot [74] PCR Polymerase chain reaction The A-B-D motif has stronger Src homology 2 phosphatase (SHP-2) binding affinity than the A-B-C motif [27]. A meta-analysis showed 1 EPIYA-D motif was associated with 1.91-fold gastric cancer risk in Asia, compared with 1 EPIYA-C motif [30]. In Western countries, strains harboring multiple EPIYA-C motifs (A-B-C-C or A-B-C-C-C) have higher phosphorylation capacity, SHP-2 binding affinity, and confer higher gastric cancer risk (OR = 3.28) compared with only 1 EPIYA-C motif [30]. Notably, a higher CagA phosphorylation ability was associated with increased risk of gastric precancerous lesions in Taiwan [31]. In addition, amino acid sequence polymorphism within the Western EPIYA-B motif also influences CagA activity, as strains harboring EPIYT-B motif have attenuated ability of inducing hummingbird phenotype and IL-8 in gastric epithelial cells and confer less gastric cancer risk than strains harboring EPIYA-B motifs [29]. Interestingly, EPIYT-B motif was associated with higher duodenal ulcer risk [29].

Vacuolating cytotoxin a (VacA)
VacA was named for its ability to induce vacuole formation in eukaryotic cells. The difference in vacuolating abilities are determined by the variations in the three regions of the vacA genethe signal (s1 and s2), intermediate (i1 and i2) and middle regions (m1 and m2). A combination of different sequences in the 3 regions leads to multiple alleles and determines the vacuolating activity. Vacuolating activity is high in s1/m1 genotypes, intermediate in s1/m2 genotypes, and absent in s2/m2 genotypes [32]. In clinical isolates, only s1/m2 strain varies in i-type; s1/m1 and s2/m2 strains are exclusively i1 and i2, respectively [33]. The i-type determines vacuolating activity among the s1/m2 strains [33]. Even though the physiologic role of vacuolation is unclear, higher vacuolation activity was linked with more severe clinical outcomes of the infection. Meta-analysis showed individuals infected with H. pylori harboring vacA s1 or m1 has an increased risk of gastric cancer in Western populations (OR = 2.50-5.32, Table 2) [20]. Moreover, vacA i1 type H. pylori infection is associated with higher gastric cancer risk in the Middle Asia and Middle East area (OR = 10.9-15.0) [34]. However, similar to CagA, the high prevalence of vacA s1/i1/m1 genotype in the East Asian and Southeast Asian population dampens its application as a disease determinant in these areas [35].
Interestingly, the s1/i1/m1 type of vacA is often linked with genopositive cagA [36]. Therefore, neither of the virulence markers can be considered an independent factor for disease outcome [37]. In fact, when multiple virulence factors are present, the risk of severe clinical outcome is greater. For example, in a long-term follow-up cohort, infection with strains that are simultaneously cagA-genopositive and vacA s1/m1 was associated 4.8-fold risk of progression of gastric precancerous lesions compared to those infected with cagA-negative/vacA s2/m2 strains, which was higher than each of the virulence factors considered alone (OR = 2.28-3.38) [38].

Blood group antigen binding adhesin (BabA)
BabA encoded by babA2 gene is a major adhesin on the outer membrane of H. pylori, which recognizes Lewis b (Le b ) blood group antigens on the host cells and determines H. pylori colonization density [39,40]. Two other paralogs of BabA had been foundthe BabB and BabC, encoded by babB and babC gene, respectively. The sequence of the 3 bab genes was similar in the 5′ and 3′ region particularly between babA and babB, but the middle region was diverse and likely mediates the binding function. Thus, only BabA has Le b antigen binding ability [41,42]. The BabA protein expression is mainly regulated by the recombination between babA and babB gene, which forms chimeric proteins (BabA/B or BabB/A) [41,43,44]. For example, intra-genomic recombination with babB brings variable number of cytosine-thiamidine (CT) dinucleotide to the 5′-region of the babA sequence, leading to phase variation and affects the expression of BabA [43]. Other mechanisms that regulate BabA expression includes mutation in the coding region of the babA2 gene, or the presence of 4 additional adenines (poly[A] nucleotides) within the − 10 to − 35 region of the babA2 promoter, which diminishes the strength of the promoter [43].
Therefore, using a single pair of PCR primers to determine babA2 genopositivity may not reflect the actual expression status of BabA. This may explain the conflicting results of studies exploring the correlation between babA2 genopositivity and gastric cancer [45]. Fujimoto et al. determined BabA expression level by immunoblotting and classified H. pylori into BabA high producers (BabA-H) with Le b binding activity, BabA low producers (BabA-L) without Le b binding activity, and BabA-negative strain (babA2-genonegative) [46]. Notably, infection with BabA-L strains was associated with highest risk of gastric cancer, followed by infection with BabA-H and BabA-negative strains. In Western countries, infection with BabA-L and BabA-H strain are associated with 54.8-fold and 19.8-fold risk of gastric cancer compared to BabA-negative infectors. Moreover, BabA-L strain infectors had highest gastric H. pylori colonization density, neutrophil infiltration, and mucosal atrophy. However, since all H. pylori isolates from East Asia are either BabA-H or BabA-L, the categorization is not sensitive enough to risk stratify infectors in this area.
In Taiwan, we explored babA and babB recombination using multiple pairs of PCR primers. Four types of babA and babB recombination can be foundthe A B genotype without recombination, AB B with babB inserted into babA, A BA with babA inserted into babB, and AB BA with both of the recombination [44]. The isolates from gastric cancer patients had a higher rate of AB BA genotype than those from non-cancer patients (40.0% vs. 9.7%, OR = 6.2, p < 0.05). Interestingly, isolates with AB BA genotype had lower BabA expression level than isolates with A B genotype [44]. Therefore, although babA2-genonegative strain was associated with lowest gastric cancer risk [46], in babA2-genopositive strain, a lower BabA expression level seemed to be associated higher gastric cancer risk [44,46]. These data suggest multiple pairs of PCR primers should be used to reflect actual BabA status and determine the risk of gastric cancer, especially in East Asia where nearly 100% H. pylori are babA2-genopositive [40,46].
Notably, H. pylori that simultaneously expresses multiple virulence factors is associated with an even higher risk of severe clinical outcomes. A case-control study showed Infection with strains "triple-positive" for cagA, vacAs1 and babA2 genes significantly correlates to the development of peptic ulcer (p < 0.0001) and adenocarcinoma (p = 0.014) and discriminated adverse disease outcome better than did the dual-positive (cagA and vacA1) classification [47].

High temperature requirement a (HtrA)
H. pylori can secrete proteases as well as induce the expression of host proteases to cleave extracellular matrix and intercellular junctional proteins. Disruption of junctional protein is particularly important for H. pylori to exploit the host receptors located on the basolateral side of the cell membrane, such as integrin [48]. The serine protease and chaperone HtrA is most studied protease expressed by H. pylori. Intracellular HtrA acts as chaperone that refold and degrade misfolded proteins. Thus, HtrA is important for bacterial survival under stressful conditions, such as extreme temperature, pH or salt concentration [49]. All clinical H. pylori isolates possesses htrA gene and suppression of HtrA proteolytic activity is sufficient to kill H. pylori [50]. Therefore, HtrA is a promising target for anti-H. pylori therapy. In addition to the essential role in H. pylori survival, secreted HtrA can cleave E-cadherin and fibronectin [51]. E-cadherin cleavage disrupts cell junctions which exposes basolateral integrin receptors for binding by the H. pylori T4SS, as well as induces epithelial-mesenchymal transition. Since fibronectin has integrin binding motif -RGD, its proteolysis may release integrin receptors on the gastric epithelial cells to interact with H. pylori T4SS, and subsequently facilitate the translocation of CagA [7]. It is novel to assess whether htrA genetic polymorphism is associated with gastric cancer risk, especially in the high gastric cancer incidence area.
In vitro study showed CagA is able to be translocated into human B lymphocytes via T4SS [54]. Once in the cytoplasm, it binds to SHP-2, which stimulates B lymphocyte proliferation and inhibits apoptosis via regulation of intracellular pathways, including activation of endoplasmic reticulum kinases 1 and 2 (ERK 1 and ERK 2) and p38 MAP kinase (MAPK) and increased expression of Bcl-2 and Bcl-xL [54]. Clinical study also showed positive correlations between the expression of CagA and phospho-SHP-2 (p-SHP-2), p-ERK, p-p38 MAPK, Bcl-2 and Bcl-xL in gastric MALToma tissue [55]. Therefore, CagA may promote gastric low-grade MAL-Toma transformation to high-grade B cell lymphoma via the above pathways.
Recently, the genomes of three H. pylori strains isolated from MALToma patients were sequenced. Nine genes were identified to be shared by 3 MALToma strains and absent in the reported 5 gastritis/ulcer strains [56]. Further investigations are needed to clarify the impact of these genes in gastric lymphomagenesis.

Virulence factors associated with peptic ulcer disease (PUD)
The virulence factors that are associated with development of PUD are summarized in Table 3.

Cytotoxin-associated gene a (CagA)
In a large meta-analysis including 44 studies and 17,374 patients from both Eastern and Western regions, CagA-seropositive was associated with a 1.69-fold risk of PUD, which was lower than its association with gastric cancer (OR = 2.44) [20]. However, due to diverse cagA genoprevalence in the various geographic areas, the methods used to identify high risk population for PUD should be different. In Western and Southeast Asian population, where cagA-genopositive rate is lower, cagA-genopositive is sensitive enough to identify high risk infector for PUD [57,58]. In East Asia, where nearly all H. pylori strains possess cagA gene [21], CagA subtyping is suggested to identify high risk infectors. Accordingly, a meta-analysis showed multiple EPIYA-C motifs is associated with 2.3-fold risk of DU compared with 1 EPIYA-C motif in Asian population [30].

Vacuolating cytotoxin a (VacA)
As mentioned above, the higher vacuolation activity of strains carrying vacA s1, i1 or m1 genotypes were linked with more severe clinical outcomes than the s2, i2 or m2 genotypes [20,32,33,36,58,59]. However, similar to cagA genoprevalence, diversity in the prevalence of vacA risky genotypes (s1, i1 and m1) was noted in different geographic regions. Therefore, the use of vacA genotypes to determine PUD risk depends on the prevalence of risky genotypes in each geographic region. In America, Europe, Africa and Middle East where the prevalence of vacA risky genotypes (s1/m1) is lower, individuals infected with vacA s1 or m1 H. pylori strains have an increased risk of PUD compared with those with s2 or m2 strains [20,32,36,59]. In Southeast Asia, vacA m1 is associated with increased risk of PUD [58]. In East Asia, where most strains are vacA s1/i1/s1 genotype, vacA genotypes cannot differentiate high risk infectors, and other virulence markers should be used [35]. The vacA i1 genotype is not associated with risk of PUD in meta-analysis [34].

Blood group antigen binding adhesin (BabA)
Both animal and human studies showed that infection by BabA-expressing strains is associated with higher bacterial density and more severe injury in the gastric mucosa [46,60]. A meta-analysis of case-control studies showed that babA2 genopositive is associated with an increased risk of PUD (OR = 2.07) in Western countries, but not in Asian countries [45]. As mentioned above, Fujimoto et al. determined BabA expression level by immunoblotting [46]. BabA-L (BabA low producers) and BabA-H (BabA high producers) strains were also associated with higher risk of duodenal ulcer than BabA-negative strains in Western countries (OR = 33.9 and 18.2, respectively) [46]. However, the underlying mechanisms remained to be elucidated. Despite the positive findings of babA2 genopositive and BabA expression intensity in determining peptic ulcer risk in Western countries, these methods are not sensitive enough to differentiate high risk infector in East Asia. Further studies using multiple sets of babA and babB PCR primers [44] are warranted to determine whether babA/B recombination can determine ulcer risk.

Duodenal ulcer promoting gene (DupA)
DupA was initially identified in 2005 and named for its role to increase risk of DU (i.e. duodenal ulcer promoting). The original data showed the presence of dupA gene was associated with increased risk of DU, as well as neutrophil infiltration and IL-8 expression in the antrum [61]. In contrast, its presence was also associated with decreased risk of gastric atrophy, intestinal metaplasia, and gastric cancer [61]. The data are compatible with the findings that antral predominant gastritis often leads to higher gastric acid secretion and duodenal ulcer formation. Nevertheless, although two meta-analyses found a small increase in DU risk (OR = 1.4) by dupA-genopositive strain [62,63], conflicting results were found [64,65]. In addition, the oipA "on" (vs. oipA "off") 4.0 DNA sequencing or immunoblot [74] PCR Polymerase chain reaction association was only found in Asian countries, but not in Western countries [63]. It has been reported that dupA forms T4SS with vir genes around it (called dupA cluster).
H. pylori with complete dupA cluster was associated with 2.1-fold risk of DU than that with incomplete dupA cluster or negative dupA [66]. Therefore, merely testing the presence/absence of dupA gene may not reflect the competent function of DupA and analysis of whole dupA cluster may be more accurate to determine DU risk, especially in Western countries [67].
Induced by contact with epithelium gene a (IceA) The iceA gene was originally identified in 1998 when investigating genes "induced by contact of H. pylori with epithelium" [68]. Two families of iceA have been found, iceA1 and iceA2. Infection with iceA1-genopositive strain is associated with PUD and increased mucosal levels of IL-8 [57,68,69]. Meta-analysis showed the presence of iceA1 gene was associated with a small increase of peptic ulcer risk (OR = 1.28) in Western countries [70].

Outer inflammatory protein (OipA)
OipA is an outer membrane protein that functions in adhesion and IL-8 induction. Its functional status (on or off ) is regulated by slipped-strand mispairing based on the number of CT dinucleotide repeats in the 5′ region of oipA gene [71]. Infection with oipA "on" strain has been linked with higher H. pylori colonization density, neutrophil infiltration and IL-8 levels in the human stomach [72]. However, the corresponding receptor for OipA has not been identified. Previous study showed oipA "on" status was closely linked to cagA-positive, vacA s1/m1, and babA2-positive genotypes [73]. Moreover, oipA "on" status was associated with increased risk of DU independent of the other virulence factors [72]. A meta-analysis also reported that the oipA "on", but not "off", status is significantly associated with an increased risk of PUD (OR = 3.97) and gastric cancer (OR = 2.43), especially in the Western countries [74]. Importantly, merely investigating the presence/absence of oipA gene would overlook its functional on/off status and may be unreliable to predict risks of PUD or GC [74].

Conclusions
Epidemiological studies had provide evidence that infection with H. pylori carrying specific virulence factors is associated with increased risk of serious clinical outcomes. To identify infectors who are at high risk of serious clinical outcomes, one should select appropriate virulence factors and testing methods according to the epidemiological data of each geographic area and ethnic group. Ethics approval and consent to participate Not applicable.

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