Clinical relevance and distribution of Helicobacter pylori virulence factors in isolates from Chinese patients

Introduction

Helicobacter pylori (H. pylori) infections can cause severe gastroduodenal diseases, including chronic gastritis (CG), peptic ulcer disease (PUD), gastric carcinoma (GC), and mucosa-associated lymphoid tissue lymphoma (1). It is reported that majority of H. pylori-infected individuals remain asymptomatic throughout life and some individuals have the risk of developing severe disease, such as GC. The bacterial factors have been implicated in various clinical symptoms, especially numerous virulence factors with heterogeneity across strains (2,3). Various virulence factors enable the survival of H. pylori in the acidic environment, successful colonization of the gastric mucosa, and persistent infection, causing chronic inflammation and tissue damage (3). The most well-studied factor is cytotoxin-associated gene A (cagA), which has been considered an important carcinogen (4). In addition, several other virulence factors may also play an important role in the pathogenicity of H. pylori (5).

CagE, which is the second most studied gene of the cag pathogenicity island (cagPAI), is essential for CagA translocation and phosphorylation (6). Studies have revealed that it is a better marker for cagPAI presence than cagA and is considered an important risk factor for duodenal ulcer (DU) development (7,8). VacA is present in almost all H. pylori strains, and the different combinations of s and m regions determine the cytotoxic activity of different strains (9). Previous studies showed that the genotype s1m1 is highly related with PUD or GC (10,11). Jhp0562, which is involved in the synthesis of lipopolysaccharide, is located upstream of the jhp0563 gene (12). The jhp0562 and jhp0563 genes are highly similar (>80%) and are highly associated with PUD (13-15). Many studies have focused on the homA and homB genes, which are 90% identical (16,17). Previous studies showed that the presence of homB was associated with PUD and development of GC (18,19). HopQ belongs to the largest outer membrane protein family in H. pylori, and two families of hopQ alleles have been described, namely, hopQI and hopQII (20,21). The hopQI genotype has been commonly detected in both Western and East Asian strains, whereas the hopQII genotype is prevalent in Western strains (22). Additionally, a previous report revealed a correlation between the hopQI gene and PUD (23). In numerous Asian and Western strains, a novel hrgA gene has been identified upstream of hpyIIIM in the hpyIII R-M system of H. pylori (24,25). Previous studies suggested that the hrgA gene frequency increased among GC patients and indicated that hrgA might be a marker for GC development among East Asian patients (26,27).

Due to geographical variations, the distribution and roles of these virulence factors in gastric diseases are still under investigation. Furthermore, there is a paucity of data regarded the distribution of cagE, jhp0562, jhp0563, and hrgA in China, which has a higher H. pylori infection incidence and GC occurrence than Western countries. Heilongjiang (HLJ) and Jiangxi (JX), with significant geographic variations, are high-risk areas for the development of GC. Therefore, we investigated the distribution of the cagA, cagE, vacA, jhp0562, jhp0563, homA, homB, hopQI, hopQII, hrgA, and hpyIIIR genes in clinical strains isolated from patients with gastroduodenal diseases in HLJ and JX to assess the association between diseases and virulence factors. We present the following article in accordance with the MDAR reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-23-1404/rc).

Methods

Study subjects

A total of 160 patients were involved in this study, including 59 patients from the Affiliated Hospital of Harbin Medical University (Heilongjiang Province, China) and 101 patients from the First Affiliated Hospital of Nanchang University (Jiangxi Province, China). The gastrointestinal disease type was determined by gastrointestinal endoscopy and histopathological tests. The gastric mucosal biopsies were taken from the greater curvature of the gastric antrum during upper gastrointestinal endoscopy. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of the National Institute for Communicable Disease Control and Prevention (Approval No. ICDC-2013001) and written informed consent was obtained from all patients.

Culture and extraction of genomic DNA

Gastric biopsy specimens were homogenized thoroughly and streaked onto Karmali agar plates (Oxoid) supplemented with 5% fresh defibrinated sheep blood and cultured under microaerophilic conditions (5% O₂, 10% CO₂, and 85% N₂) at 37 ℃ for 48 hours. H. pylori colonies were identified by typical morphology, Gram staining, and urease, oxidase, and catalase activity tests. The bacterial cells were washed twice with phosphate buffered saline (PBS) and centrifuged at 8,000 rpm for 3 minutes. The genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions.

Polymerase chain reaction (PCR) amplification

The PCR reaction was performed in a volume of 25 µL, containing forward and reverse primers (1 µM). The amplification was as follows: 94 ℃ for 5 minutes, followed by 35 cycles of 94 ℃ for 45 seconds, 54 ℃ for 45 seconds, and 72 ℃ for 45 seconds, and finally 72 ℃ for 5 minutes. The primers used to amplify the targeted genes are summarized in Table 1. The H. pylori strain 26695 and strain J99 were used as controls. The PCR products were analyzed on a 1% agarose gel containing 1 X TAE (Tris, acetic acid, EDTA) buffer.

Statistical analysis

The relationship between each genotype and different regions, as well as clinical outcomes, was quantified using the chi-squared test. A P value (bilateral test) less than 0.05 was considered statistically significant. The logistic models were used to explore the relationship between diseases and the candidate genes. The odds ratio and 95% confidence interval (CI) were obtained for multivariate analysis. When one condition (e.g., GC) was studied, the other conditions were used as reference (e.g., CG and PUD). Initially, the full model was used, which included ‘age’, ‘sex’, and all candidate genes. Thereafter, the model was subjected to backward stepwise selection for candidate risk factors. The R function ‘glm ()’ was used to build the full model and the ‘MASS: stepAIC ()’ function was used to perform model selection. The R function ‘rcorr ()’ was used to calculate the Spearman correlation coefficient between studied genes.

Results

A total of 160 H. pylori strains were successfully isolated from gastric biopsy specimens, including 59 from HLJ (39 with CG, 9 with PUD, and 11 with GC) and 101 from JX (38 with CG, 36 with PUD, and 27 with GC). There was no significant difference in the sex nor age of the patients between the two regions. The average age of the examined patients was 47.5±15.2 years, and the average age in patients with GC was significantly higher than that in CG and PUD patients. The percentage of male patients with GC was significantly higher than that of CG patients (P<0.05; Table 2).

The presence of virulence factors in all 160 strains was determined by PCR and the results are summarized in Table 3.

cagA, cagE, and vacA status

All strains tested were successfully detected for the cagA and cagE genes. The most common vacA s genotype was s1 (158/98.8%), while the vacA s2 subtype was detected in only two (1.2%) strains in our study (Table 3, Figure 1). For the vacA m region, the m2 subtype was present in 109 (68.1%) strains, and the m1 subtype was present in 51 (31.9%) strains. The frequencies of the m1 subtype strains were 30.5% and 32.7% in HLJ and JX, respectively. Conversely, the m2 subtype was present in 69.5% and 67.3% of strains from HLJ and JX, respectively (Table 3, Figure 1A,1B). There were no significant differences in the m subtypes between different geographic regions (P>0.05). The dominant vacA combination was s1m2 (67.5%), which was significantly higher than s1m1 (31.3%, P<0.001), but no statistical significance was obtained between the different regions (P>0.05; Table 3).

Figure 1 The distribution of vacAs and m region genotypes of H. pylori strains from different regions and diseases. (A) The prevalence of each genotype in JX. (B) The prevalence of each genotype in HLJ. (C) The prevalence of each genotype in strains from CG patients. (D) The prevalence of each genotype in strains from PUD patients. (E) The prevalence of each genotype in strains from GC patients. JX, Jiangxi; HLJ, Heilongjiang; CG, chronic gastritis; PUD, peptic ulcer disease; GC, gastric carcinoma.

The dominant m2 subtype was detected in 71.1%, 73.7%, and 63.6% of strains isolated from patients with PUD, GC, and CG, respectively. However, the difference was not significant (P>0.05). In the present study, the vacA s2 subtype was present in only two strains isolated from CG patients, and there were no patients with PUD and GC infected with s2 subtype strains (Table 4, Figure 1C-1E).

jhp0562 and jhp0563 status

In this study, three different combinations of jhp0562 and jhp0563 were observed: a single fragment of jhp0562 (profile 1) or jhp0563 (profile 2), and a double positive for two genes (profile 3) with different lengths of jhp0563 (Figure 2A).

Figure 2 The jhp0562/jhp0563 and homA/homB gene profiles observed in H. pylori strains. (A) jhp0562/jhp0563 gene profile. (B) homA/homB gene profile. The gene profiles were determined by PCR performed on 160 H. pylori strains. 1, strain 26695; 2, strain J99; MW, molecular weight marker. The primers generated two PCR products with 301 bp and 602 bp in strain J99, corresponding to jhp0562 and jhp0563, respectively, and only one PCR product with 558 bp in strain 26695 corresponding to jhp0563. Strain 26695 was used as a positive control for homA, and strain J99 was used as a positive control for homA and homB. PCR, polymerase chain reaction.

Overall, 99.4% of the strains carried the jhp0562 gene in our study, which was much higher than those carrying jhp0563 (32.5%, P<0.001). The dominant jhp0562-single positive genotype was detected in 108 (67.5%) strains, whereas the jhp0563-single positive genotype was observed in only 1 strain. There was no significant difference in the prevalence of any of the genotypes between HLJ and JX (Table 3).

Jhp0563 was present in 33.8% of isolates from CG patients and 37.8% from PUD patients, which was higher than that from GC patients (23.7%), however, the difference was not statistically significant (P>0.05). No correlation between jhp0562 single-positive genotype and clinical outcome was observed (P>0.05). The prevalence of double positive strains in patients with CG, PUD, and GC was 33.8%, 37.8%, and 21.1%, respectively. There was also no significant difference between the mixed genotype and various diseases (P>0.05; Table 4).

homA and homB status

The clinical strains can have a single homA gene (profile 1), a single homB gene (profile 2), or both homA and homB genes (profile 3) (Figure 2B).

The homA gene was detected in 53 (33.1%) strains, whereas the homB gene (71.3%) was more frequently detected than homA (P<0.001). Moreover, double positivity for homA and homB strains was rare (4.4%). The homB-single positive genotype was dominant, accounting for 66.9% of strains, whereas the homA-single positive genotype was present in 28.8% of the strains. We found no significant difference in the prevalence of homA or homB between the two geographical regions (Table 3).

A total of 71.4% of CG patients and 75.6% of PUD patients were infected with homB-positive strains, which were higher than the percentage of GC patients (65.8%). However, the difference was not statistically significant (P>0.05). homB single-positive strains were present in 64.9% of CG patients, 71.1% of PUD patients, and 65.8% of GC patients, whereas strains with homA single-positive genotypes accounted for 28.6%, 24.4%, and 34.2%, respectively. In this study, there was no association between the homA or homB gene and clinical outcomes (Table 4).

hopQI and hopQII status

The hopQI gene was present in all strains examined, whereas only 6.9% of the strains carried the hopQII gene. The hopQII-positive strains were significantly more common in HLJ (13.6%) than in JX (2.9%) (P<0.001; Table 3). There was no hopQII-single positive strain in our study. Moreover, the frequency of the hopQI-single positive genotype was significantly higher in JX (97%) than in HLJ (86.4%, P<0.05; Table 3).

In the present study, no significant difference in hopQI nor hopQII distribution was observed between various diseases.

hrgA and hpyIIIR status

The dominant hpyIIIR-positive genotype was detected in 83.1% of the strains, which was significantly more prevalent than the hrgA-positive genotype (56.9%, P<0.001). We found that 41.9% of the strains were hpyIIIR-single positive genotypes, and 15.6% of the strains had hrgA in place of hpyIIIR. The mixed genotype for hrgA and hpyIIIR was common and accounted for 41.3% (Table 3).

The hpyIIIR gene was present in 88 (87.1%) strains from JX, which was more than that from HLJ (76.3%). In contrast, a significant difference was observed in the frequency of hrgA single-positive genotype between HLJ and JX (23.7% vs. 10.9%, P<0.001). The strains with mixed genotypes were significantly more common in JX (48.5%) than in HLJ (28.8%, P<0.001).

The hrgA-positive strains were more common in GC patients (71.1%) than in CG patients (50.7%), and the difference was statistically significant (P<0.05). In addition, 55.56% of PUD patients were infected with hrgA-positive strains; however, the difference was not statistically significant (P>0.05). The hpyIIIR-positive genotype was dominant, and the frequency was similar between various diseases. However, hypIIIR was detected in 92.6% of strains isolated from GC patients in JX, whereas only 63.6% of strains from HLJ were infected with hpyIIIR-positive strains. The difference was statistically significant (P<0.05). The hrgA single-positive genotype was present in 19.5% and 15.8% of strains from CG and GC patients, respectively, whereas only 8.9% of strains were detected in PUD patients. Moreover, the frequency of this genotype examined in GC strains was significantly different between HLJ and JX patients (36.4% and 7.4%, P<0.05). The frequency of the hpyIIIR single-positive genotype in strains isolated from CG patients (49.4%) was significantly higher than that in GC patients (28.9%, P<0.05; Table 4).

In our cohort, the mixed genotype was common and accounted for 55.3% and 31.2% of strains from GC and CG patients, respectively. The difference was statistically significant (P<0.05). Furthermore, the frequency of mixed genotypes in the CG strains (28.9%) isolated from JX was significantly lower than that in the PUD strains (52.8%, P<0.05) and GC strains (70.4%, P<0.001) (Table 4).

The relationship between different virulence factors

The association between different virulence factors was examined. homA and homB exhibited significant negative associations with each other (P<0.001), as did hrgA and hpyIIIR (P<0.001). A significant correlation between the presence of vacA m and hpyIIIR was observed (P<0.05).

When the virulence factors were combined, we found the hrgA/homB/vacA m2 and hrgA/homB/hpyIIIR genotypes were observed in 39.5% and 36.8% of the strains from GC patients, whereas only 3 strains were infected with the hrgA/homB/vacA m1 or hrgA/homB/jhp0563 genotype. The difference was statistically significant (P<0.001).

The relationship between gene status and clinical outcomes

Univariate analysis showed that only the hrgA gene was associated with GC. There was no significant association between the other genes and different diseases. The presence of the hrgA gene showed a significant association with GC and the risk of GC was 2.224-fold higher in patients infected by strains harboring the hrgA gene (95% CI: 1.014 to 4.882, P<0.05; Table 5).

A multivariate analysis, including age, sex, and the status of different genes, was performed to determine the factors related to clinical outcomes. The results demonstrated that male patients were at significantly increased risk of GC compared to female patients [odds ratio (OR) =2.577, 95% CI: 1.028 to 6.865, P<0.05] and elderly patients were at a higher risk of GC development (OR =1.116, 95% CI: 1.075 to 1.168, P<0.05). Moreover, the hrgA genotype exhibited a positive correlation with GC and increased the risk of GC (OR =3.606, P<0.05; Table 6).

In contrast, the presence of hrgA exhibited a negative correlation with CG and represented a protective factor for CG (OR =0.499, P<0.05). In addition, sex and age also significantly decreased the risk of CG (OR =0.387, 95% CI: 0.191 to 0.765; and OR =0.960, 95% CI: 0.935 to 0.984, respectively; P<0.05). In patients with PUD, only age was observed to be associated with the disease, and the risk of PUD was 0.965-fold lower in elderly patients (OR =0.965, 95% CI: 0.939 to 0.991, P<0.05; Table 6).

Discussion

This current study demonstrated the distribution of distinct virulence genes in strains isolated from patients with various diseases and revealed a possible relationship with gastric diseases.

It is accepted that cagA is the most important virulence factor, and the prevalence of cagA ranges from 50% to 70% in European and American countries (32), whereas the prevalence is above 90% in some Asian countries (28,33). In addition, the prevalence of cagE ranges from 30 to 90% according to geographic regions (8,34,35). A recent study suggested that cagE combined with cagA can be used as marker of DU (8), however, their role in gastric pathogenesis remains unclear. In the present study, all strains tested were cagA and cagE positive, which may contribute to the higher incidence rate of GC in China compared to Western countries.

The vacA s1m1 genotype strains produce higher levels of toxins and are more highly associated with PUD and GC than the s2m2 genotype strains, which produce little or no toxins (11,36). After examining the vacA subtypes, we found that 98.7% of strains were of the s1 subtype, and strains containing m2 (68.1%) were more common, which is in contrast to previous studies (32,37), suggesting a difference between Chinese and foreign strains. The cagA and vacA s1 genotypes have been identified as markers of strains associated with DU or GC among Western populations (5). However, such a pattern is not observed in East Asia, where nearly all isolates have cagA and vacA s1 genotypes (28,38,39).

Almost all strains in our study cohort were positive for jhp0562, consistent with a study in Japan which reported the prevalence of jhp0562 was 100% and higher than that in the USA (12). Jhp0562 contributes to the synthesis of both type 1 and 2 Le antigens, which are expressed by East Asian strains, whereas the type 1 Le antigen is expressed by Western strains (40-42), and this may explain the geographical variations of jhp0562. In addition, two jhp0563 gene fragments of different lengths, which were observed in Western strains, were absent in our study cohort (profile 3 in Figure 2A). The length of jhp0563 varies due to the number of 21-nt tandem repeats, which is used as a gene switching mechanism (43,44). Studies have shown that jhp0562 is significantly associated with PUD and jhp0563 is associated with gastritis in some Western and American countries (12,13). Oleastro et al. reported a positive association between jhp0562 with cagA, likely leading to the strong link between jhp0562 with PUD (13). Moreover, jhp0562 and jhp0563 were found to describe the different incidences of GC (14). However, the present study did not reveal any association between the jhp0562 nor jhp0563 gene and clinical outcomes, consistent with studies in other Asian countries (12,14).

The homA and homB gene profiles vary greatly based on the geographic origin of the strains (45,46). Previous studies have shown that homB is found more frequently than homA in East Asian strains (19,30). In the present study, we observed that the positive rate of homB (71.3%) was significantly higher than that of homA (33.1%). The homB can increase secretion of proinflammatory cytokine interleukin (IL)-8 and increased inflammation is associated with more severe diseases, such as PUD and GC (47). The presence of homB is associated with PUD in Western strains, whereas homA is associated with non-ulcer diseases (48). However, we did not identify a relationship between homA nor homB and any disease type.

The homA and homB can be found interchangeably at two genomic loci, and various combinations of the genes can be found, including a single homA or homB gene, two copies of homA or homB, a single copy of each gene, or neither of these genes (18,49). Studies revealed that two copies-genotype of homB was strongly associated with PUD, therefore, the association between homB and disease is probably a result of the variations in genotype, geographic location, genomic loci and gene copy number in various strains (18,19,30). Further investigation is warranted to determine the copy number of these genes, as well as obtaining samples from other regions in China.

In our study, the presence of cagE, jhp0562, homB, and hopQI was prevalent, which probably attributed to the fact that the presence of these genes was common in cagA and vacA s1 strains (21,30,40,41,50). The differences in these gene profiles may contribute to the diversity of virulence between Chinese and Western strains and emphasize the characteristics of strains in higher-risk areas of GC, such as HLJ and JX. The strains harboring the cagA and vacA s1 genotypes could be considered highly virulent (49). Although we did not identify any relationship between the studied factors and different diseases in China, we also assumed that the presence of these genes could increase the virulence of strains in China. These factors can interact synergistically in some fashion and contribute to high levels of gastric mucosal inflammation, which is associated with the precancerous process (51,52).

In the present study, the frequency of hrgA (56.9%) in our strains was higher than that of other East Asian strains (e.g., 18% in Vietnam, 36% in Hong Kong, 31% in Korea), and similar to that of Western strains (e.g., 46% in Colombia, 55% in Canada) (27). It is worth noting that the mixed genotype for hpyIIIR and hrgA was observed in 41.25% of our strains, which was rare in other studies (25,27). Moreover, there was a significant difference in hrgA single-positive or mixed genotype between HLJ and JX strains, suggesting the obvious geographic diversity of this gene.

The frequency of hrgA-positive or mixed genotypes was significantly higher in GC strains than in CG strains, indicating that there was a strong correlation between these genotypes and GC. The significant difference was still apparent after multivariate analysis that adjusted for age, sex, and other factors. The multivariate analysis showed that the hrgA gene was independently associated with the development of GC and an increased risk of developing GC was associated with strains containing hrgA.

A previous study showed that there was no difference in the induction of IL-8 release nor apoptosis in gastric epithelial cells between hrgA and hpyIIIR strains (25). Therefore, whether the acquisition of hrgA is selected by GC pressure or is a causative factor was difficult to determine. We observed that the hpyIIIR single-positive genotype was more common in CG strains; moreover, the hrgA gene showed a negative association with CG. It is accepted that the development of GC begins with H. pylori-induced chronic superficial gastritis (53,54). Therefore, we speculated that the CG individuals with hrgA gene potentially develop GC due to interactions with host factors (e.g., single nucleotide polymorphisms of inflammatory cytokines) and environmental factors (38,55,56).

There were still some limitations in our research. First, the sample size of GC patients was small. Second, there were only two regions of China selected in our study. The distribution of virulence factors varied greatly in different regions of China. Although the two regions are representative of regional disparity, the 160 strains in our study couldn’t completely stand for the strains from other regions. Third, the function of HrgA protein and its role in GC progression is unclear. Further study is necessary to make adequate comparisons involving more GC patients from more regions of China to confirm the correlation between hrgA and GC, as well as to examine the function of HrgA. Once confirmed, hrgA may be used to identify individuals at higher GC risk in China.

Conclusions

In China, where there is a high prevalence of H. pylori infection and GC occurrence, the universal presence of studied virulence factors makes it impossible to examine disease-specific associations with any of these virulence factors. However, the distribution pattern of these factors suggested the characteristics of Chinese strains, which showed higher virulence and, ultimately, resulted in severe diseases. We found a strong association between hrgA status and progression to GC. We speculated that hrgA may serve as a promising candidate factor for severe disease development in China, and further study is warranted to confirm our speculation.

Acknowledgments

We would like to thank all the participants from the Department of Diagnosis for Communicable Diseases, the National Institute for Communicable Disease Control and Prevention, and the Chinese Center for Disease Control and Prevention.

Funding: This research was supported by the Research on Equipment for Rapid Diagnosis and Treatment of Marine “Superbug” Infection project (No. HJ20172B07021).

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-23-1404/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-23-1404/dss

Peer Review File: Available at https://atm.amegroups.com/article/view/10.21037/atm-23-1404/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-23-1404/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Ethics Committee of the National Institute for Communicable Disease Control and Prevention (No. ICDC-2013001) and informed consent was obtained from all patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Teoh)