Introduction

The incidence of Helicobacter pylori (H. pylori) infection continues to be higher in developing countries and is estimated at 3–10 % per year in contrast to approximately 1 % per year in developed countries [1]. Many virulence factors have been identified in H. pylori: urease, the blood group antigen-binding adhesin (babA), the cag pathogenicity island, the vacuolating cytotoxin (vacA), and H. pylori neutrophil-activating protein (HP-NAP) are the important ones [2].

A 40-kb cag pathogenicity island (cag PAI) encodes T4SS, which injects the CagA oncoprotein as well as peptidoglycan into host cells, resulting in induction of potent proinflammatory chemokines such as interleukin (IL)-8 [3]. Translocated CagA undergoes tyrosine phosphorylation by Src, leading to actin cytokine rearrangements, scattering, and elongation of infected host cells.

Previously we reported a high prevalence of cagA in our study populations [80 % in gastric cancer (GC), 83.3 % in peptic ulcer disease (PUD), and 76.7 % in non-ulcer dyspepsia (NUD)] and also found no correlation of cagA with GC and PUD [4]. Therefore, it is likely that cagA cannot be considered as the sole virulence marker for the determination of disease outcome, at least in India. Hence, it is possible that some other genes of cagA-PAI are responsible for pathogenicity and disease outcome. Genotyping of cagL would be the better molecular tool to understand this problem. CagL is a pilus protein that interacts with host cellular α5β1 integrins through its arginine-glycine-aspartate (RGD) motif, guiding proper positioning of the T4SS and translocation of CagA [5]. Consequent activation of host signaling pathways stimulates the transcription factor NF-κB and upregulates inflammation-associated genes such as IL-8.

Therefore, in the current study our aim was to determine the prevalence of cagL genopositivity and its amino acid polymorphisms in patients with GC, PUD, and NUD.

Materials and methods

Patient population

We enrolled 200 adult patients (NUD 120, PUD 30, GC 50) undergoing upper gastrointestinal endoscopies at Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India, between September 2007 and October 2010. The diagnosis of gastroduodenal diseases was based on clinical, endoscopic, and histopathological examination. Patients with NUD were considered as controls in our study. The ethics committee of the institute granted approval for the study, and all the patients gave their consent to participate. Subjects who had received antimicrobial therapy, H2-receptor blockers, proton-pump inhibitors, and non-steroidal antiinflammatory drugs in the 30 days before the endoscopies were excluded from the study.

Detection of H. pylori infection

During each endoscopic examination, antral biopsies were obtained and subjected to the following tests: rapid urease test (RUT), culture, histopathology, and H. pylori-specific ureA polymerase chain reaction (PCR) following the standard protocol as described elsewhere [6]. H. pylori infection was diagnosed if culture was positive or any two of the remaining three tests were found to be positive.

H. pylori genotyping

DNA was isolated from the bacterial growth and H. pylori-positive gastric tissues using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) as per the manufacturer’s instructions and subjected to PCR for the presence of cagA and cagL genotypes of H. pylori, as described previously [7, 8].

CagL-genosequencing for translating into amino acid sequences

The DNA fragments amplified by the cagL region were purified by the Qiagen quick PCR purification kit (Qiagen) and sequenced using both the forward and reverse primers to verify that they truly represented the H. pylori cytotoxin-associated gene L. Sequencing was performed by Eurofins, MWG operon (Bangalore, India), and analyzed by the BLAST program at the National Center for Biotechnology Information. Furthermore, amino acid sequences of cagL were predicted by the standard code, and aligned by the Expasy online tool for translation (http://www.expasy.org).

Statistical analysis

Data were analyzed using SPSS software (version 12.0; SPSS, Chicago, IL, USA). H. pylori genotypes and cagL amino acid polymorphism status in relationship to gastroduodenal diseases were assessed by Chi square test. All P values were two sided and considered significant when <0.05.

Results

Patient characteristics

A total of 200 patients (mean age, 46.34 ± 14.71 years; 123 males) were enrolled and their distributions were as follows: GC 50 (mean age, 52.12 ± 12.41 years; 37 males), PUD 30 (mean age, 47.10 ± 14.71 years; 18 males), and NUD 120 (mean age, 43.77 ± 14.99; 68 males).

Detection of H. pylori infection

The prevalence of H. pylori infection in our study population was 52.5 %. H. pylori infection was significantly higher in patients with PUD than with GC (70 vs. 48 %, P = 0.045) and NUD (70 vs. 50 %, P = 0.038).

H. pylori genotyping

In the present study, 86.6 % (91/105) of the H. pylori isolates were positive for the cagL genotype by PCR. The prevalence of cagL-positive H. pylori infection was uniformly distributed in patients with GC (75 %; 18/24), NUD (86.6 %; 52/60), and PUD (100 %; 21/21). The cagA genotype was present in 86 % (82/105) of our H. pylori isolates. Presence of cagA in GC, PUD, and NUD was 66.6 % (16/24), 86 % (18/21), and 88.3 % (53/60), respectively. The majority of our H. pylori isolates, i.e., 78 (74.2 %), were positive for both cagA and cagL genotypes and were equally distributed in cases of GC (12, 50 %), PUD (18, 86 %), and NUD (48, 80 %).

cagL amino acid sequence polymorphisms in disease population

The amplified products (354 bp) of the cagL gene from all clinical isolates were sequenced. After matching to the sequences available in the GenBank database, the nucleotide sequence of our isolates had uniform identities (>95 % homology) to the cagL gene of the standard J99 strain, H. pylori strain 26695, and NCTC 11637. With the help of the expasy online translation tool, the amino acid sequences of cagL were translated for the selected isolates. Based on these predicted amino acid sequences, our all H. pylori isolates expressed the RGD motif (Fig. 1). There were no differences of the cagL amino acid sequence polymorphisms between patients with PUD and NUD (P > 0.05). However, the isolates from GC patients had significantly higher rates of D58 amino acid sequence polymorphism than those from PUD (P = 0.025) and NUD patients (P = 0.046). The presence of K59 was also found to be significantly higher in GC when compared to PUD (P = 0.011) and NUD (P = 0.010) (Table 1). Moreover, the presence of K59 usually accompanied D58 (Fig. 1), and these combined D58K59 polymorphisms had significantly higher rates of GC [P = 0.029, odds ratio (OR) (95 % confidence interval, CI) = 3.82 (1.18–12.37)] (Table 2).

Fig. 1
figure 1

The comparison of cagL amino acid sequences between J99 Helicobacter pylori strains and nine clinical isolates submitted in the NCBI gene bank. The indicates the RGD motif, and the asterisks indicate sequences with variations at the 35, 58, 59, 60, 62, and 122 residues. The nucleotides marked “·” indicate identical sequence regions to the J99 sequence, and nucleotides marked “–” are donated as an unamplified region

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Table 1 Differences in polymorphisms of cagL amino acid sequences of Helicobacter pylori isolates from patients with gastric cancer (GC), peptic ulcer disease (PUD), and non-ulcer dyspepsia (NUD)
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Table 2 Association between amino acid polymorphism with gastric cancer (GC) and peptic ulcer disease (PUD)
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Accession numbers

Based on the combined 58 and 59 positions of cagL amino acid sequences, all cagL-positive isolates were divided into nine groups (DT, DK, NE, NK, YE,YK, MK, ME, and DE) and one representative sequence each from all groups was deposited in Gen-Bank with accession no. JN087496, JN022446, JN564705, JQ417888, JQ417890, JQ417889, JQ417891, JQ436696, and JQ417887, respectively (Supplementary Table SI).

Discussion

The prevalence of H. pylori in patients with GC in our study was low and had no association. Other tissue-based studies from India also failed to show an association between H. pylori infection and GC [4, 9, 10]. The chances of finding H. pylori in the biopsy specimens become less when the changes of chronic atrophic gastritis and intestinal metaplasia are set in the stomach. These changes lead to absence or decrease in the H. pylori load in the stomach, probably because of lack of nutrients for this organism [11]. So, these may be the probable reasons for the low prevalence of H. pylori infection when a tissue-based diagnosis is made.

No correlation of cagA could be established with GC and PUD, although cagA had been identified as a virulence marker and associated with increased severity of disease in some geographic regions [12]. The high prevalence of cagA (80–90 %) independent of disease status had been reported in other Indian studies as well [13, 14]. Therefore, we targeted the cagL genotype, which is responsible for the translocation of cagA into the gastric epithelium. The present study indicated 86.6 % of our isolates were cagL positive and all carried the RGD motif in their amino acid sequences. Studies from Malaysia, Singapore, and Taiwan reported >85 % prevalence of cagL in their H. pylori isolates, and the results are concordant with the current finding [15, 16]. Moreover, the presence of RGD motif in all the current isolates suggests that the CagA translocation is mediated through an RGD-dependent pathway. Therefore, it is rational to check whether the cagL amino acid sequence polymorphisms correlate with clinical disease. Previously, a study from Taiwan reported a higher rate of the amino acid sequence polymorphisms Y58 and E59 in GC patients (P < 0.05) and concluded that cagL-Y58E59 isolates possibly exert stronger acid suppression during chronic infection [16]. On the other hand, the results of Saha et al. [17] help in part to explain that CagL may dissociate ADAM17 of integrin a5b1 to contribute to hypochlorhydria during H. pylori infection. In the present study, different amino acid polymorphisms, D58 and K59, were significantly associated with GC. However, an experimental approach that probes mechanistically the observed associations between D58K59 cagL amino acid polymorphisms and GC is required to reach any conclusion.

Conclusion

In summary, the majority of Indian H. pylori isolates were cagL positive and all such strains carried the RGD motif. Patients infected with cagL-genopositive H. pylori strains having cagL-D58K59 amino acid polymorphisms had a 3.8-fold increased risk for development of gastric cancer. However, further studies are required to know the exact role of particular cagL amino acid polymorphisms in the pathogenicity of H. pylori-associated gastroduodenal diseases.