Prevalence of tumor necrosis factor alpha inducing protein (tipα) gene of Helicobacter pylori and its association with upper gastrointestinal diseases in India

Helicobacter pylori (H. pylori) is known to cause several gastroduodenal diseases including chronic Gastritis, Peptic Ulcer disease and Gastric Cancer. Virulent genes of H. pylori like cagA, vacA are known to be responsible for the disease pathogenesis. However, these virulence genes are not always found to be associated with disease outcome in all populations around the world. Tumor necrosis factor alpha inducing protein tipα is a newly discovered virulence gene of H. pylori and is an inducer of certain cytokines and chemokines that are responsible for causing stomach cancer. Therefore, we conducted a study, which aims to find the prevalence of tipα gene in the Indian patients with gastroduodenal symptoms, and its association with H. pylori related gastroduodenal diseases. 267 clinical H. pylori isolates are included in our study for finding the prevalence of tipα gene and its association with cagA and vacA gene using PCR assay. The current study shows that the prevalence rate of tipα gene is 59.9%. Our study has found a significant association (p < 0.05) of tipα gene with Non Ulcer Dyspepsia (NUD) and an association of cagA and vacAs1m1 with Gastritis and Duodenal Ulcer. Our study demonstrates for the first time the presence of tipα as virulence factor of H. pylori strain in Indian population isolated from patients suffering from gastroduodenal diseases. Further, tipα is significantly associated with NUD but not with other gastroduodenal diseases in India.


Introduction
Helicobacter pylori (H. pylori) is a Gram negative microaerophilic bacterium that chronically colonized the gastric epithelium of more than half of the world population and plays an important role in the pathogenesis of several gastroduodenal diseases such as chronic Gastritis, Peptic Ulcer Disease (PUD) and Gastric Cancer (Thirumurthi et al. 2012). In India, about 53.4-73.5% of the population is infected by H. pylori (Hooi et al. 2017). There is a wide geographical variation in both H. pylori related gastroduodenal diseases as well as distribution of its virulent genes. The prevalence Shweta Mahant and Shubham Mehra share equal authorship.
* Sudeep Bose sbose1@amity.edu of Gastric Cancer is high in South India as compared to North India while the Eastern and the Northern regions of India have high rates of Duodenal Ulcer (DU) (Misra et al. 2007). Role of various virulence factors of H. pylori, such as cytotoxin-associated gene Pathogenicity Island (cag PAI), cagA, vacuolating cytotoxin A (vacA), and urease have been studied extensively in gastroduodenal diseases in India and other parts of the world (Patel et al. 2017;Jeyamani et al. 2018;Baj et al. 2021). In East-Asian countries, most of the H. pylori strains are cagA-positive, while 20-40% of isolates from Europe and Africa are cagA-negative strains. It is shown that patients infected by cagA-positive H. pylori have a higher risk of developing PUD or Gastric Cancer compared to those infected with their cagA-negative counterparts. However, in East Asia, most strains of H. pylori have the cagA gene irrespective of the disease (Shimoyama et al. 1997). In India, there are wide geographical differences in the prevalence of cagA and vacA. About 90% strains from East India (Patra et al. 2012), 66.6% H. pylori strains of North-East India (Sarma et al. 2017) and whereas only 50% of the strains from North India are cagA positive (our unpublished data). Also about 80-90% of the strains from East India are vacAs1m1 whereas 60.7% of the North-East India are vacAs1m1 (Chattopadhyay et al. 2002;Sarma et al. 2017). Therefore, as in the Indian population the strains with s1m1 are predominant. Since there is wide variation in cagA and vacA prevalence rates in different geographies in India and in different parts of the world, it demonstrates that the gastroduodenal diseases are multifactorial. Hence, there is a need to identify other virulence factors, which may be responsible for disease causation. Tumor necrosis factor alpha inducing protein (Tipα) of H. pylori is a carcinogenic factor that induces tumor promotion in vitro and in vivo (Suganuma et al. 2001(Suganuma et al. , 2005(Suganuma et al. , 2008. In vitro studies also shows that the binding of tipα to surface nucleolin on human Gastric Cancer cell line and that internalization of the tipα and nucleolin complex induces tumor progression and epithelial mesenchymal transition in human Gastric Cancer (Watanabe et al. 2010). Tipα is a new nuclear factor Kappa B cells (NF-κb) activating protein of H. pylori associated with strong induction of Tumor Necrosis Factor alpha (TNF-α) in combination with Ras activation. When H. pylori infection occurs in the stomach epithelium in which Ras protein is activated or overexpressed, Tipα dimer is assumed to play a carcinogenic role leading to Gastric Cancer. However, if H. pylori infection occurs in the stomach epithelium without activated Ras protein, Tipα dimer will probably not produce Gastric Cancer, but only inflammation (Gastritis and Gastric Ulcer) (Suganuma et al. 2008). Tipα protein was found to be significantly higher in gastric cancer patients as compared to chronic gastritis patients, suggesting that tipα induces the expression of a variety of pro-inflammatory cytokines including TNFα and chemokine genes, is overexpressed in gastric mucosa exposed to H. pylori (Suganuma et al. 2008). Although India constitutes around one-sixth of the world population, there are no studies on the prevalence of tipα in H. pylori related gastroduodenal diseases. This prompted us to study the prevalence of the tipα genes from H. pylori strains isolated from various parts of India and its association with H. pylori related gastroduodenal diseases.

Collection of H. pylori specimen
A total of 267 H. pylori positive isolates across different parts of India were included in the study-North India (n = 65), South India (n = 10), East India (n = 27) and North-East India (n = 165). Three gastric biopsy samples were collected from patients suffering from various GI symptoms as per the inclusion criteria and Exclusion criteria. Inclusion criteria includes: age 18-80 years, consent for upper gastrointestinal endoscopy, symptoms suggestive of H. pylori related gastroduodenal disease. Exclusion criteria were: use of antibiotics, antihistamines, and proton pump inhibitors 3 months prior to the study. The biopsies were taken from gastric antrum, around 2-4 cm from the pylorus. Two biopsies were taken for isolation of H. pylori and one for Rapid urease test. The presence of H. pylori was confirmed by culture, Rapid urease Test and genotyping.
The H. pylori strains included in our study were isolated from patients suffering from various gastroduodenal tract diseases (n = 247) and controls (n = 20). The control group consisted of subjects who underwent upper GI endoscopy due to reasons other than H. pylori related gastroduodenal diseases e.g. chronic diarrhea, chronic liver disease (CLD) and underwent endoscopy as a part of standard treatment protocol. The purpose of including the control group is to compare the prevalence of Tipα inducing protein gene and other H. pylori virulence genes in gastroduodenal diseases (diseased group) to controls (Non H. pylori related Diseased group).

Culturing of H. pylori and DNA extraction
Brucella broth containing the biopsy samples were vortexed in laboratory for 2 min and 200 μl of the mixture was streaked onto on brain heart infusion agar plates with charcoal (BHIA; Becton Dickinson, Sparks, MD, USA) which was supplemented with 5% Horse Serum; 0.4% Iso-vitaleX (Becton Dickinson, Sparks, MD, USA) and antibiotics such as Amphotericin B (8 μg/ml), Trimethoprim (5 μg/ml) and Vancomycin (6 μg/ml) and incubated under microaerophilic condition (5% O 2 ; 10% CO 2 ; 85% N 2 ) in a double gas incubator (Heracell 150i) at 37 °C. H. pylori were identified based on their typical water droplet like morphology and urease, oxidase and catalase test result. The genomic DNA was extracted using standard protocol of CTAB method (Ausubel et al. 1993) with phenol/chloroform and isopropanol precipitation as described elsewhere as well as kit based method (QIAamp DNA Mini Kit) as per the manufacturer protocol and was stored at − 20 °C at Amity University, Noida and NICED, Kolkata.

Molecular characterization of genotypic status by PCR amplification
Genotyping of urease gene was done to confirm the presence of H. pylori using the primer listed in Table 1 (Roth et al. 2001). 267 H. pylori isolates included in our study were further amplified for cagA and vacA gene by multiplex PCR assay using 2.5 pmol of primers VAG-F and VAG-R, 25 pmol of primers VAI-F and VAI-R, 10 pmol of primers cag5c-F and cag3c-R as shown in Table 1 (Chattopadhyay et al. 2004).
Genotyping of tipα gene was carried out using simplex PCR (Table 1). The PCR was performed with the final volume of 20 µl holding 10 ng of bacterial genomic DNA, 20 pmol of each primer, 0.25 mM of each dNTPs (Bangalore Genei), 1U of Taq DNA polymerase (Bangalore Genei) in standard PCR buffer (Bangalore Genei) containing 1.5 mM MgCl 2 and were amplified according to cycling conditions: 35 cycles at 94 °C for 1 min, 60 °C for 1 min and 72 °C for 1 min with final extension of 10 min at 72 °C in Eppendorf (Vapo-protect). Amplified products of multiplex and simplex PCR were then analyzed using 2% and 1% agarose gel, respectively, in 1X TAE buffer containing 0.05% µg/ml EtBr followed by screening running under UV trans-illuminator (Tarsons).

Statistical analysis
Fisher exact test or chi square was used to calculate the p value which was defined as statistically significant with p value < 0.05 and also the 95% confidence interval (CI) was calculated using SPSS V.18.0 software for Windows (SPSS Inc., Chicago, IL, USA). Microsoft Word and Excel have been used to generate graphs, tables.
The genotyping of tipα was done using primer as shown in Table 1, which gave an amplicon size of 168 bp (Fig. 1).

Association of tipα with cagA and vacA gene
In our study we found 56.8% (121/213) cagA positive and 72.2% (39/54) cagA negative showed presence of tipα gene but the association was found to be not significant (Table 4). We found that 95.2% of vacAs1m2 also shows the presence of tipα and the association is found to be significant (p < 0.05). 71.1% of vacAs2m2 and 53.7% of vacAs1m1 showed the presence of tipα but the association was not significant.

Discussion
There are several studies, which have shown that the incidence and/or severity of gastroduodenal diseases related to H. pylori may vary geographically (Smith et al. 2019). Further, there are several reports for the presence of different strains of H. pylori with different degrees of virulence indicating variation in the distribution of different virulence genes of H. pylori in different populations (Blaser et al. 1995;Queiroz et al. 2000;Yamaoka et al. 2008). Several Indian studies show that cagA gene was found at a high frequency of 86% of the H. pylori strains 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 (Chattopadhyay et al. 2002;Saxena et al. 2011). This is very similar to the results in our present study, where we found the prevalence rate of 79.8% for cagA positivity among H. pylori related gastroduodenal diseases. We found large geographical differences in the prevalence rates in different parts of India. We found that the prevalence of cagA in North India, South India, North-East India were 44.6%, 90% and 93.9%, respectively. cagA was significantly associated (p < 0.05) with Gastritis and DU, where its prevalence was 92.5% and 88.3%, respectively, compared to prevalence rate of 65% (13/20) among controls. On the other hand, the prevalence of cagA gene among GERD, NUD and Gastric Ulcer were 50%, 72.5% and 71.4%, respectively, but was not significant. Our results are in line with the findings and observations of other studies from India, which show a high cagA prevalence among DU and Gastritis patients. Prevalence was also high among NUD patients but they did not reach significance. The prevalence of cagA phenotype was also lower in patients with GERD as has been the observation of other studies as well (Shavalipour et al. 2017). We also found the overall prevalence rate of 75.2% for the vacAs1m1 allele was highest followed by vacAs2m2 (16.9%) and vacAs1m2 (7.9%). This data is in accordance to previous studies having about 70% s1m1 allele in Indian H. pylori isolates (Chattopadhyay et al. 2002). We found significant association (p < 0.05) of the virulent allele s1m1 with Gastritis and Duodenal Ulcer but not with others viz. GERD, DU and Gastric Ulcer. Our present observations are in line with the other studies from India stating higher prevalence of virulent genes in PUD (Saxena et al. 2011). Saxena et al. reported a high occurrence of cagA in H. pylori isolates; 80% in GC, 83.3% in PUD 76.7% in NUD. Although there is geographical variation in the prevalence of vacAs1m1 with 91.5% in North-East India, 90% in South India, 74% in East India. In North India, the prevalence of s1m1 is 40% whereas 43% is s2m2, which is similar to reports from Kauser et al. (2005) from Ladakh, India where they found that 60% of the strains have s2 genotype (Kauser et al. 2005). This study shows the prevalence of tipα gene among the Indian H. pylori strains and its association with virulent genes cagA, vacA gene in various gastroduodenal diseases. Our study shows that the overall prevalence of tipα, among the Indian H. pylori isolates is 59.9%. NUD has been significantly associated with tipα with a prevalence rate of 86.3% compared to the prevalence of 60% among controls. The prevalence rate was similar to NUD (86.3%) and GERD while the prevalence rates were much lower in Gastritis (41.3%), DU (57.1%) and Gastric Ulcer (42.9%). The highest prevalence rate of tipα gene was found in 95.6% (22/23) vacAs1m2 positive H. pylori strains indicating a strong correlation between them. Another study has also reported increased mucosal IL-18 mRNA expression in vacAs1m2 allele causing increase in the risk of gastroduodenal disease although no significance was found (Bagheri et al. 2013). It has been found that Gastric Cancer patients infected with tipα positive strains of H. pylori produce significantly higher amounts of TNF-α than patients with chronic Gastritis and that TNF-α induced inflammatory response plays a significant role in the development of Gastritis and Gastric Cancer associated with H. pylori infection (Suganuma et al. 2008).
Our study also demonstrates the significant association of the tipα gene with Non Ulcer Dyspepsia (NUD). Non Ulcer Dyspepsia refers to heterogeneous and broad range of chronic upper abdominal symptoms, which are widely shared with different gastroduodenal disorders. Therefore, NUD is generally diagnosed when other frequent gastroduodenal diseases are excluded, and upper endoscopy ruled out macroscopic lesions-i.e., Gastritis or Peptic ulcer. Such a condition is frequently encountered in clinical practice, its prevalence being close to 20%-30% in the general population and probably predates the occurrence of Gastritis and PUD. To date, no conclusive data have been reported on the role of H. pylori infection on these dysfunctions. A Cochrane Meta-analysis of 17 trials with 3,566 patients showed that there was a 10% (95% CI 6-14) relative risk reduction of dyspepsia following H. pylori eradication as compared to placebo, with a NNT of 14 (95% CI 10-25). H. pylori infection may cause dyspeptic symptoms in NUD through other mechanisms such as: (1) alterations of gastric acid secretion; (2) persistent and active inflammation of gastric mucosa; and (3) post-infective changes in gastroduodenal mucosa. Our study raises a possibility of tipα being the link between H. pylori and NUD. This merits further larger studies to comprehensively understand this possibility (Mosso et al. 2020).
In summary, our results revealed the prevalence rate of tipα gene is 59.9% (160 out of 267) among the Indian patients with gastroduodenal symptoms. There is a highly significant association between virulent gene cagA and vacAs1m1 alleles with Duodenal Ulcer and Gastritis. We found a significant association of the tipα gene with Non Ulcer Dyspepsia (NUD). Limitation of this study is detailed demographics like alcohol, smoking, dietary habits, Weight, Height was not available for all samples. In addition, the sample size of the study is limited.

Conclusion
The present study concludes for the first time that the Indian patients with gastroduodenal symptoms has 59.9% prevalence rate of tipα gene. Our study also shows that there is an association of tipα with vacAs1m1 and cagA gene with duodenal ulcer and tipα gene with non-ulcer dyspepsia (NUD) disease.