Background
Helicobacter pylori is a Gram-negative, genetically diverse spiral bacteria that infects more than half of the population worldwide [
1]. Infection with
H. pylori is associated with duodenal ulcer (DU) or gastric ulcer, gastritis, and gastric adenocarcinoma [
2]. About 65–70 % of the Indian population is infected with
H. pylori [
3,
4]. 15–20 % of overall infected population develop gastric or duodenal ulcer and less than 1 % develop gastric adenocarcinoma [
5].
The genome of various
H. pylori strains demonstrates significant genetic diversity. Genetic variation in specific virulence genes of
H. pylori may participate in the pathogenic process of
H. pylori infection in the stomach, thereby contributing to the variable risk of diverse clinical outcomes. So, in addition to the host immunological factors and environmental factors, another important reason for the diverse clinical outcomes is the differences in virulence factors among
H. pylori strains [
6].
Cytotoxin-associated gene (
cagA) was the first reported gene that varies in
H. pylori strains and is considered as a marker for the presence of the
cag pathogenicity island (
cag PAI), which includes a number of other genes associated with increased virulence [
7‐
9]. About one-half to two-thirds of US and European strains carry the
cag PAI and is associated with overt disease. In contrast,
cag PAI is distributed among most of the Asian strains, irrespective of disease status. In India the asymptomatic individuals also carry the
cag PAI [
10]. It was found that most of the
H. pylori strains in Indian subcontinent have
cagA typeA and any particular type of
cagA is not associated with disease outcome [
11].
Some
H. pylori strains can produce vacuoles in epithelial cells by the action of VacA protein which consists of a signal region (
s1 or
s2) and a middle region (
m1 or
m2). Strains harboring the
s1m1 mosaic combination are said to be more cytotoxically potent than the strains with
s1m2 genotype, while
s2m2 strains do not secrete vacuolating cytotoxin [
12]. In Western countries,
H. pylori strains with
s1m1 genotype usually also carry the
cag pathogenicity island (
cag PAI) and are more significantly associated with the disease than those strains which don’t have this
cag PAI [
7]. In Indian population, the strains with
s1m1 genotype are predominant [
13]. It concludes that the diseases are multi-factorial and thereby we need to identify other bacterial virulence factors which play important role in diseases manifestation.
It is generally known that bacterial adherence to the gastric epithelium is the first critical stage of colonization by
H. pylori in the human stomach [
14]. The blood group antigen binding adhesin (BabA) is one of the major outer membrane proteins of
H. pylori that binds to the fucosylated Lewis
b blood group antigens on the gastric epithelium and plays a key role in facilitating bacterial colonization to the stomach [
15,
16]. It has been hypothesized that the adherent bacteria may be more successfully able to transfer their products to the host cells. Thus, a higher bacterial density coupled with more capable delivery of bacterial products to the host cells may provoke a stronger inflammatory response. It has been already shown that
H. pylori is able to commence nonspecific immune responses [
17].
H. pylori has various adhesins, including blood group antigen binding adhesin (BabA), which binds to the Lewis B (Le
b) antigen [
15,
18].
H. pylori adhesin-mediated colonization is multi-factorial and no single adhesin may be essential, although adherence of this bacteria to the gastric epithelium is a compulsory early step in colonization and renders
H. pylori 100–1000 times more resistant to antibiotics than the non-adherent ones [
19]. Blood group antigen binding adhesin is encoded by a polymorphic gene named
babA2, while the
babA1 allele is non-functional. Gerhard et al. [
20] in 1999 first demonstrated a positive association between a
babA2 carrying strain and duodenal ulcer (DU) and gastric cancer (GC). Subsequently, a series of studies of the association between
babA2 gene and peptic ulcer diseases and GC have been performed, but with inconsistent or conflicting conclusions [
21‐
24]. However, in Asian countries, most of the strains are
babA2 positive irrespective of disease status [
25,
26]. These data clearly indicates that the frequency of
H. pylori
babA2 gene may vary geographically and their associations with disease outcome also vary accordingly.
Although Indian subcontinent constitutes about the 1/5th of the world population, but there was not much studies about the role of babA2 gene in disease outcome from this subcontinent. The contradictory conclusions from previous studies and the inadequate information from India provided the impetus to study (1) the clinical relevance of the babA2 gene by examining its association with H. pylori virulence-associated genes and with clinical outcome as well as (2) the comparative analysis of IL-8 production and apoptosis by co-culturing the AGS cell line with Indian H. pylori strains with variant genetic makeup.
Discussion
Various studies have indicated that the incidence and/or severity of gastroduodenal pathologies related to
H. pylori may vary according to geographical regions [
27]. There is also evidence for the existence of different strains of
H.
pylori with different degrees of virulence indicating the variation in the distribution of the different virulence attributes of
H. pylori in different populations [
29‐
31]. The
cagA gene was found at a frequency of 86 % among the 114 tested strains of this region and this virulence marker was found at almost equal frequencies in strains from DU patients (90.6 %) and NUD patients (82 %), indicating that the prevalence of the
cagA gene cannot be considered as a key virulence marker for determination of the clinical status of the host, as has been reported in other Indian studies [
13,
32]. In our study most of the strains have type A
cagA without any association in disease outcome. All the strains used in sequence analysis consist of Western-CagA-specific sequence and it is in agreement with our previous report [
11]. We also found that highly virulent
s1m1 allele of
vacA gene was present among 71.9 % of Indian
H. pylori strains and presence of this allele did not show any correlation with the disease outcome (DU = 73.6 %, NUD = 70.5 %) of the host. This data is in accord with the previous report having 70 % presence of
s1m1 allele in Indian
H. pylori isolates [
5].
Here we found that the Indian
H. pylori strains exhibited 67.5 % prevalence of
babA2 gene and the
babA2 gene was uniformly distributed (DU = 69.8 %, NUD = 65.6 %) irrespective of disease status. These results indicate that, unlike in the Western countries [
33], presence of
babA2 is not associated with clinical outcome in India. In our study among 84 double positive strains, 61 were found as triple positive. These 61 triple positive strains also showed an almost equal distribution in DU (29/53, 54.7 %) patients and NUDs (32/61, 52.4 %), which is inconsistent with the report from few Western countries [
34].
More recent analyses of
babA2 as a virulence marker have produced conflicting data on the usefulness of
babA2 expression in predicting clinical outcome, which is most likely dependent on the geographic origin of the
H. pylori strains. In Portuguese and Thai populations,
babA2 is not a biomarker for peptic ulcer disease or gastric cancer [
35,
36]. However, for strains isolated from Germany, Turkey, or northern Portugal,
babA2 expression is associated with the severity of gastric disease [
18,
37,
38]. This inconsistency may be due to the specific geographic variation of the circulating bacterial lineages. Another explanation for this lack of association may be due to the allelic variation of the
babA2 gene which was studied by Pride et al. [
39].
An inflammatory response is one of the main pathophysiological events in
H. pylori mediated infection. It has been shown that epithelial cells secrete IL-8 as a result of
H. pylori infection [
40‐
42]. IL-8 acts as a potent chemo-attractant for neutrophils and is thought to play a crucial role in
H. pylori induced tissue damage [
43]. Several studies have shown that induction of IL-8 secretion from host cells is dependent on the presence of CagA, VacA, OMPs (OipA) and LPS [
43‐
46]. In order to see the effect of
babA2 gene on the IL-8 induction, triple positive (
cagA
−/
vacA
−/
babA2
+) strains and triple negative strains were cultured with AGS cells. The triple positive strains induced the highest amount of IL-8 secretion from AGS cells, and the double negative strains but with
babA2 gene caused significantly higher amount of IL-8 secretion than the triple negative ones (Fig.
2,
P < 0.05). Therefore, it may be emphasized that the triple positive strains and also the presence of
babA2 somehow enhances the ability of
H. pylori to induce IL-8 in the gastric mucosa.
IL-8 secretion and inflammation due to
H. pylori infection lead to epithelial cell damage or apoptosis. Apoptosis is a genetically programmed form of cell death which is mainly characterized by some distinct morphological and molecular features. Microbial pathogens or their products can directly activate the apoptotic pathway which plays a role in pathogenesis [
47]. It has been reported that
cagA, and
vacA have an effect on
H. pylori mediated apoptosis [
48,
49]. In order to elucidate the role of
babA2 in apoptosis, the triple positive,
cagA
+/
vacA
+/
babA2
−,
cagA
−/
vacA
−/
babA2
+ and triple negative strains were used independently to infect the AGS cells. It was observed that the triple positive strains caused more epithelial cell death than the other strains, whereas the
cagA
−/
vacA
−/
babA2
+ strains caused significantly higher level of apoptosis than the triple negative strains (
P < 0.05).
As the triple positive strains exhibit highest level of IL-8 induction as well as apoptosis that is why we made isogenic
babA2 mutants of these strains [I-121 and 135(1) respectively] to reconfirm the role of
babA2 in
H. pylori virulence. The IL-8 induction assay and cell cycle analysis by flow cytometry were performed with these wild type and mutant strains as described in methods section. In agreement, it was found that the wild type strains were significantly (
P <
0.05) more capable of IL-8 induction as well as mediating the apoptotic cell death in AGS cells (Figs.
3,
5). These data more strongly establish the virulence potential of the strains with
babA2 positive genotype.
It has been already reported that ex vivo IL-8 production requires the adherence of viable
H. pylori to the AGS cells [
50], and as the induction of apoptosis and IL-8 secretion are often linked [
51], so it can be said that the apoptosis in AGS cells also require the adherence of
H. pylori. In this study, it was found that the adherence of
babA2 positive and
babA2 negative strains were comparable in AGS cells. In agreement, it was also found that the extent of adherence to the AGS cells were comparable between the wild type strains and isogenic
babA2 mutants. This report suggests that although adherence is crucial, the
babA2 gene products may regulate the IL-8 secretion and apoptosis by an adherence independent manner.
H. pylori has at least five different adhesin factors which use different receptors for adhering on gastric epithelial cells [
43], suggesting that presence or absence of only one of these genes may not affect its adherence to gastric cells. Further studies will be required to explain what other factors may impact the extent of adherence.
In this report, although it was observed that Indian
H. pylori strains harbored
babA2 gene independent of disease status and also the triple positive strains showed no association with disease status, but on the other hand, the triple positive strains and even only the
babA2 positive strains showed more virulence as they induced more IL-8 secretion and as well as caused more apoptosis in AGS cell line than the triple negative strains. In accordance, the wild type strains also exhibited more capability to induce IL-8 secretion as well as apoptosis than their isogenic
babA2 mutants in the cell culture study. Apparently, it seems that these two statements are contradictory. What could be the probable reasons?
H. pylori is one of the genetically diverse bacterial species with regard to genotyping [
52‐
54]. Previous reports also suggested that
H. pylori genotype varies geographically [
2,
54]. The polymorphisms of some cytokine genes have been found to be associated with
H. pylori mediated clinical consequences, probably because they modulate the amount of cytokine production in response to
H. pylori infection [
55]. It was already been established that dietary salt intake can play an important role in enhancing the likelihood of severe clinical consequences of
H. pylori mediated infection [
56]. Environmental iron level also brings about the changes in the composition of the
H. pylori outer membrane which ultimately influences the pattern of clinical outcomes [
57]. These above mentioned information clearly establish the role of host factors and environmental factors in the disease outcomes of
H. pylori mediated infection. Importantly,
H. pylori toxin gene expression is inconsistent with the general phenomenon observed for other pathogenic bacteria, that is, that expression of the important toxin genes are strongly associated with diseases [
58]. The expressions of toxin genes by
H.
pylori are not solely dependent on the disease status. So, this result can not lead to the underestimation of the role of
babA2 gene as a toxin gene. The genetic constitution of host may play a critical role in the successful colonization and ultimately in the disease consequences of
H. pylori infection. We can say that the virulent effect of
babA2 gene of
H. pylori may come into action only after interplay with certain host factors. Our results also showed that
babA2 gene in combination with the presence of
cagA and
vacA s1m1 allele become much more toxic as the triple positive strains showed highest degree of IL-8 induction as well as apoptotic cell death in cell culture study, although the PCR-based genotyping results showed a uniform distribution of triple positive strains independent of disease status. These discrepancies may be due to the fact that at the time of
H. pylori isolation, the NUD people harboring the triple positive strains may be at the risk of developing an ulcer disease in future which cannot be come into our count.