Abstract
Background and aims
jhp0562 and β-(1,3)galT (jhp0563) of Helicobacter pylori have been suggested as novel virulent factors; however the clinical associations and functions of these genes remain unclear. We examined the prevalence of jhp0562, β-(1,3)galT, and cagA in the United States (U.S.) and Japanese populations.
Methods
A total of 308 strains (171 from the U.S. and 137 from Japan) were examined for the status of jhp0562, β-(1,3)galT, and cagA by polymerase chain reaction.
Results
There were significant differences in the status of jhp0562, β-(1,3)galT and cagA between the U.S. and Japanese populations (P < 0.001). In the U.S., the prevalence of β-(1,3)galT was significantly lower in strains isolated from patients with duodenal ulcer (DU) or gastric ulcer (GU) than those with gastritis (47.8% and 32.1% vs. 72.0%, P < 0.01), and the absence of β-(1,3)galT was an independent factor discriminating DU and GU from gastritis (adjusted odds ratios, 4.21 and 8.52; 95% confidence intervals, 1.75 to 10.12 and 2.76 to 26.33, respectively). In the U.S., the prevalence of the jhp0562-positive/β-(1,3)galT-negative genotype was significantly higher in strains from DU and GU patients than in those from gastritis patients (50.0%, 67.9%, and 24.4%, P < 0.01) and the cagA status was significantly correlated with that of jhp0562 and inversely correlated with that of β-(1,3)galT. In contrast, the prevalence of these three genes was not significantly different in Japan.
Conclusions
jhp0562 or β-(1,3)galT can be used to discriminate peptic ulcers from gastritis in the U.S., but not in Japan.
Keywords: Helicobacter pylori; jhp0562; β-(1,3)galT; glycosyltransferase
Introduction
Helicobacter pylori (H. pylori) infection is etiologically associated with gastritis, peptic ulcer diseases, gastric atrophy, and gastric cancer (GC)1, although most infected people remain asymptomatic. Factors thought to be associated with clinical gastroduodenal diseases include H. pylori virulence, host genetics, and environmental factors such as diet2. There has been considerable interest in the molecular epidemiology of H. pylori putative virulence factors, especially CagA, VacA, and OipA3, 4. However, no such factors have been exclusively linked to a specific H. pylori-related disease (e.g., GC).
jhp0562, which encodes a glycosyltransferase involved in the synthesis of lipopolysaccharide (LPS), was reported to be associated with peptic ulcer diseases in children, but not in adults, in the Portuguese population5. The sequence of β-(1,3)galT (jhp0563), which is involved in the Lewis (Le) antigen expression of LPS, is highly similar to that of jhp05626, 7. In a subsequent study by the same group in the Portuguese population, the presence of jhp0562 alone (jhp0562-positive/β-(1,3)galT-negative) was associated with peptic ulcers rather than with gastritis, and the presence of β-(1,3)galT alone (jhp0562-negative/β-(1,3)galT-positive) was associated with gastritis rather than with peptic ulcers8.
However, no previous study investigated the association between these two genes and H. pylori-related diseases in other countries. This study investigated whether there was also an association between these genes and clinical outcomes in populations from another Western country (the United States [U.S.]) where there is a low prevalence of H. pylori (e.g., 10–15 % at the aged > 50 years)9 and an Asian country (Japan) where there is a high prevalence of H. pylori (e.g., over 60 % at the aged > 50 years)10.
Methods
Patients and H. pylori
H. pylori strains were obtained from the gastric mucosa of H. pylori-infected patients who underwent endoscopy at Oita University Faculty of Medicine (Oita, Japan) and Michael E. DeBakey Veterans Affairs Medical Center (Houston, TX). Presentations included gastritis, duodenal ulcer (DU), gastric ulcer (GU), and GC. DU, GU, and GC were identified by endoscopy, and GC was further confirmed by histopathology. Gastritis was defined as H. pylori gastritis in the absence of peptic ulcers or gastric malignancy. Patients with a history of partial gastric resection were excluded. Patients who received H. pylori eradication therapy or treatment with antibiotics, bismuth-containing compounds, H2-receptor blockers, or proton pump inhibitors within 4 weeks prior to the study were also excluded. Informed consent was obtained from all participants, and the protocol was approved by local hospital ethics committees.
H. pylori genotyping
Antral biopsy specimens were obtained for the isolation of H. pylori using standard culture methods as previous described11. Chromosomal DNA was extracted from confluent plate cultures expanded from a single colony using a commercially available kit (QIAGEN, Valencia, CA). Two H. pylori strains with fully sequenced genomes deposited in GenBank, strain 26695 (ATCC 700392), which is negative for jhp0562 and positive for β-(1,3)galT, and strain J99 (ATCC 700824), which is positive for both genes, were used as control strains. Polymerase chain reaction (PCR) amplification for jhp0562 and β-(1,3)galT was performed using one primer pair, 5′-TGA AAA GCC CTT TTG ATT TTG-3′ and 5′-GCT GTA GTG GCC ACA TAC ACG-3′, as described by Oleastro et al., who originally reported the importance of the genes5. The PCR conditions were initial denaturation for 5 min at 95°C, 35 amplification steps (95°C for 30 s, 56°C for 30 s, and 72°C for 30 s), and a final extension cycle of 7 min at 72°C, as described by Oleastro et al5. These primers generated two PCR products with 301 and 602 bp in the strain J99, corresponding to jhp0562 and β-(1,3)galT (jhp0563), respectively, and only one PCR product with 558 bp in the strain 26695, corresponding to β-(1,3)galT (HP0619). Two copies of β-(1,3)galT gene fragments of different lengths were found in several cases. For this study, we constructed a new primer pair, 5′-ATG CAA GCC TTA GAA GAT TG-3′ and 5′-ATA TCC ACC GGC TCT ATG AT-3′. These primers generated two PCR products with 404 and 704 bp in the strain J99, corresponding to jhp0562 and β-(1,3)galT, respectively, and only one PCR product with 661 bp in the strain 26695, corresponding to β-(1,3)galT. The PCR products obtained using primers for jhp0562 had the same lengths (301 bp for the first primer and 404 bp for the second primer) in all experiments, but the PCR products obtained using primers for β-(1,3)galT had several lengths (approximately 550–600 bp for the first primer and 660–700 bp for the second primer) with 21 nt-tandem repeat sequences, consistent with a previous finding8. The status of cagA was determined by PCR using primer pair 5′-ACC CTA GTC GGT AAT GGG-3′ and 5′-GCT TTA GCT TCT GAY ACY GC-3′ (Y = C + T) as described previously12. The amplified fragment was detected by a 1.5% agarose gel electrophoresis using an ultraviolet transilluminator.
Statistical analysis
Variables such as gender, mean age, and the status of jhp0562, β-(1,3)galT, and cagA (negative or positive for the three genes) were evaluated. The univariate association between each genotype and the clinical outcomes were quantified by the chi-square test. A multivariate logistic regression model was used to calculate the odds ratios (OR) of the clinical outcomes by including age, sex, and the H. pylori genotypes. All determinants with P values of < 0.10 were entered together in the full model of logistic regression, and the model was reduced by excluding variables with P values of > 0.10. OR and 95% confidence intervals (CIs) were used to estimate the risk. Spearman rank coefficients (r) were also determined to evaluate the association between the different genotypes of the strains. A P value of less than 0.05 was accepted as statistically significant. The SPSS statistical software package version 18.0 (SPSS, Inc., Chicago, IL) was used for all statistical analyses.
Results
Prevalence of candidate genes in the U.S. and Japan
A total of 308 patients were included in this study: 171 from the U.S. (82 with gastritis, 46 with DU, 28 with GU, and 15 with GC) and 137 from Japan (37 with gastritis, 45 with DU, 39 with GU, and 16 with GC). The distribution of the status of cagA, jhp0562, and β-(1,3)galT in the two countries are shown in Table 1. There were significant differences in the cagA, jhp0562, and β-(1,3)galT status between strains isolated from the U.S. and Japanese populations (P < 0.001, 0.001, and 0.001, respectively). Regarding jhp0562 and β-(1,3)galT, four different combinations of the two genes were observed (Figure 1): single fragment of jhp0562 (jhp0562-positive/β-(1,3)galT-negative) (profile 1), single fragment of β-(1,3)galT (jhp0562-negative/β-(1,3)galT-positive) (profile 2), double positive for jhp0562 and β-(1,3)galT (profile 3), and two β-(1,3)galT fragments with different lengths (profile 4). The prevalence of the jhp0562-positive/β-(1,3)galT-negative genotype was significantly higher in strains from Japan than in strains from the U.S (81.0% vs. 39.3%, P < 0.001). The jhp0562-negative/β-(1,3)galT-positive genotype, and two fragments of β-(1,3)galT were found only in strains isolated from the U.S. The prevalence of double positive genotype was significantly higher in strains from the U.S than in strains from Japan (19.0% vs. 41.0%, P = 0.006).
Table 1.
Prevalence of jhp0562 and β-(1,3)galT
| Japan | U.S. | ||||
|---|---|---|---|---|---|
| n | 137 | 171 | P value | ||
| mean age | 60.1 ± 12.3 | 50.8 ± 14.5 | |||
| male | 76 | 55.5% | 137 | 79.2% | |
| cagA positive | 132 | 96.4% | 144 | 83.2% | <0.001 |
| jhp0562 positive | 137 | 100.0% | 140 | 80.9% | <0.0001 |
| β-(1,3)galT positive | 26 | 19.0% | 99 | 57.2% | <0.0001 |
| jhp0562-positive/β-(1,3)galT-negative | 111 | 81.0% | 68 | 39.3% | <0.0001 |
| jhp0562-negative/β-(1,3)galT-positive | 0 | 0.0% | 28 | 16.2% | <0.0001 |
| double positive | 26 | 19.0% | 71 | 41.0% | 0.006 |
| Two β-(1,3)galT with different lengths | 0 | 0.0% | 4 | 2.3% | 0.07 |
Figure 1.
jhp0562 and β-(1,3)galT profiles observed in Helicobacter pylori strains. Four different combinations of the two genes were observed: single fragment of jhp0562 (jhp0562-positive/β-(1,3)galT-negative) (profile 1), single fragment of β-(1,3)galT (jhp0562-negative/β-(1,3)galT-positive) (profile 2), double positive for jhp0562 and β-(1,3)galT (profile 3), and two β-(1,3)galT fragments of different lengths (profile 4).
MW, molecular weight marker
The association between candidate genes and clinical outcomes
The prevalence of cagA was significantly higher in strains from the U.S. patients with DU, GU, and GC than in those with gastritis (89.1%, 92.9%, and 100% vs. 75.6%, P < 0.05) (Table 2). In contrast, that of β-(1,3)galT was significantly lower in strains from the U.S. patients with DU and GU than in those with gastritis (47.8% and 32.1% vs. 72.0%, P < 0.01). The prevalence of jhp0562 or β-(1,3)galT were not different between gastritis and GC. Considering the ethnicity in U.S., the same tendency was observed although the differences were not statistically significant probably due to the lack of ethnic data from approximately 30% of the U.S. patients (data not shown). Table 3 shows the association between clinical outcomes and the presence of cagA, jhp0562, and β-(1,3)galT in multivariate analysis in the U.S. population. Interestingly, both age and the absence of β-(1,3)galT were independent factors for discriminating DU from gastritis (adjusted OR = 4.21; 95% CI = 1.75–10.12). Likewise, the absence of β-(1,3)galT was an independent factor discriminating GU from gastritis (adjusted OR = 8.52; 95% CI =2.76–26.33). On the other hand, the prevalence of cagA, jhp0562, and β-(1,3)galT was not significantly different between patients with these two diseases in Japan.
Table 2.
Relationship between cagA,jhp0562, and β-(1,3)galT and clinical outcomes in the U.S. (a) and Japan (b)
| (a) US | ||||||||
|---|---|---|---|---|---|---|---|---|
| gastritis | DU | GU | GC | |||||
| n | 82 | 46 | 28 | 15 | ||||
| mean age | 43.8 ± 13.0 | 54.5 ± 12.6 | 56.1 ± 12.3 | 68.4 ± 6.9 | ||||
| male | 60 | 73.2% | 39 | 84.8% | 27 | 96.4% | 12 | 80.0% |
| cagA positive | 62 | 75.6% | 41 | 89.1%* | 26 | 92.9%* | 15 | 100.0%* |
| jhp0562 positive | 65 | 79.3% | 40 | 87.0% | 22 | 78.6% | 13 | 86.7% |
| β-(1,3)galT positive | 59 | 72.0% | 22 | 47.8%* | 9 | 32.1%* | 9 | 60.0% |
| jhp0562-positive/β-(1,3)galT-negative | 20 | 24.4% | 23 | 50.0%* | 19 | 67.9%* | 6 | 40.0% |
| jhp0562-negative/β-(1,3)galT-positive | 15 | 18.3% | 5 | 10.9% | 6 | 21.4% | 2 | 13.3% |
| double positive | 44 | 53.7% | 17 | 37.0% | 3 | 10.7%* | 7 | 46.7% |
| Two β-(1,3)galT with different lengths | 3 | 3.7% | 1 | 2.2% | 0 | 0.0% | 0 | 0.0% |
| (b) Japan | ||||||||
|---|---|---|---|---|---|---|---|---|
| gastritis | DU | GU | GC | |||||
| n | 37 | 45 | 39 | 16 | ||||
| mean age | 60.4 ± 12.6 | 50.3 ± 13.0 | 62.5 ±11.5 | 64.7 ± 9.3 | ||||
| male | 22 | 59.5% | 25 | 55.6% | 20 | 51.3% | 9 | 56.3% |
| cagA positive | 34 | 91.9% | 45 | 100.0% | 37 | 94.9% | 16 | 100.0% |
| jhp0562 positive | 37 | 100.0% | 45 | 100.0% | 39 | 100.0% | 16 | 100.0% |
| β-(1,3)galT positive | 5 | 13.5% | 11 | 24.4% | 7 | 17.9% | 3 | 18.8% |
| jhp0562-positive/β-(1,3)galT-negative | 32 | 86.5% | 34 | 75.6% | 32 | 82.1% | 13 | 81.3% |
| jhp0562-negative/β-(1,3)galT-positive | 0 | 0.0% | 0 | 0.0% | 0 | 0.0% | 0 | 0.0% |
| double positive | 5 | 13.5% | 11 | 24.4% | 7 | 17.9% | 3 | 18.8% |
| Two β-(1,3)galT with different lengths | 0 | 0.0% | 0 | 0.0% | 0 | 0.0% | 0 | 0.0% |
DU; duodenal ulcer, GU; gastric ulcer, GC; gastric cancer
P < 0.05 compared with gastritis
Table 3.
Multivariate analyses of the risk for DU (a) and GU (b) by age, gender, cagA status, and β-(1,3)galT status in the U.S. population
| (a) DU | |||
|---|---|---|---|
| Adjusted OR | 95% CI | P value | |
| Age (per 1 year) | 1.07 | 1.03–1.10 | <0.001 |
| Gender (female) | 0.54 | 0.18–1.57 | 0.26 |
| cagA positive | 1.83 | 0.51–6.54 | 0.35 |
| β-(1,3)galT negative | 4.21 | 1.75–10.12 | 0.001 |
| (b) GU | |||
|---|---|---|---|
| Adjusted OR | 95% CI | P value | |
| Age (per 1 year) | 1.08 | 1.03–1.12 | <0.001 |
| Gender (female) | 0.10 | 0.01–1.01 | 0.05 |
| cagA positive | 1.54 | 0.26–9.06 | 0.63 |
| β-(1,3)galT negative | 8.52 | 2.76–26.33 | <0.001 |
The association between the combination of the two genes and clinical outcomes in the U.S.
Considering the four different combinations of the two genes, the prevalence of the jhp0562-positive/β-(1,3)galT-negative genotype was significantly higher in strains isolated from the U.S. patients with DU and GU than those with gastritis (50.0% and 67.9% vs. 24.4%, P < 0.01). Even in the case of three combination of cagA, jhp0562 and β-(1,3)galT, the tendency was not changed because all of jhp0562-positive strains possess cagA. Double positivity for jhp0562 and β-(1,3)galT was significantly less frequent in strains from GU patients than in those from gastritis patients (10.7% vs. 53.7%, P < 0.001). Although statistical significance was not observed, double positivity for jhp0562 and β-(1,3)galT tended to be less prevalent in strains from DU patients than in those from gastritis patients (37.0% vs. 53.7%, P = 0.05). The prevalence of the jhp0562-negative/β-(1,3)galT-positive genotype was neither related with DU nor GU. These combinations of the two genes did not associate with GC compared with gastritis.
Correlations between cagA and jhp0562 or β-(1,3)galT
In the U.S., there was a significant positive correlation between the presence of cagA and jhp0562 (r = 0.58, P < 0.001). In contrast, there was a negative correlation between the presence of cagA and β-(1,3)galT (r = −0.25, P < 0.001), and the prevalence of jhp0562 was inversely correlated with that of β-(1,3)galT (r = −0.23; P = 0.001) in the U.S. In Japan, the prevalence of jhp0562 and β-(1,3)galT in the cagA-positive strains was 100% and 19.6%, respectively.
Discussion
Our study revealed that the absence of β-(1,3)galT might be an independent discriminating factor for distinguishing DU and GU from gastritis in the U.S. population. Interestingly, multivariate analysis showed that the absence of β-(1,3)galT, but not the presence of cagA, was an independent factor discriminating DU and GU from gastritis. Regarding the expression patterns of these two genes, the jhp0562-positive/β-(1,3)galT-negative genotype was significantly associated with peptic ulcer, consistent with previous findings8. In addition, we found that the prevalence of double positivity for jhp0562 and β-(1,3)galT was lower in strains from patients with peptic ulcer in the U.S., although the previous report in Portugal did not elucidate a difference between gastritis and peptic ulcer. The prevalence of the jhp0562-positive/β-(1,3)galT-negative genotype was higher in strains from patients peptic ulcer in the U.S. than that reported previously (56.7% vs. 33.3%)8.
Oleastro et al. originally reported that jhp0562 was significantly more prevalent in peptic ulcer than in gastritis in children but not adults5. They did not examine the prevalence of β-(1,3)galT in adults8. We revealed jhp0562 and β-(1,3)galT as candidate markers for peptic ulcers, even in adults in the U.S. Although the presence of β-(1,3)galT alone (jhp0562-negative/β-(1,3)galT-positive) was associated with gastritis in their report, we did not find any association between the jhp0562-negative/β-(1,3)galT-positive genotype and clinical outcomes. The prevalence of the jhp0562-negative/β-(1,3)galT-positive genotype in the U.S. in our study was lower than that in their report (16.2 vs. 37.6%). The difference in subjects (children or adults) between that report and our study may have contributed to this discrepancy. Oleastro et al. reported that the prevalence of jhp0562 was 48.8% (22/45) in children and 66.6% (60/90) in adults. There are several reports indicating that the prevalence of strains harboring more virulent genotypes such as cagA and vacA s1 alleles was higher in older patients than in younger patients13, 14. Oleastro et al. gave the following hypothesis: after infection in childhood, a specific strain will undergo changes during the course of infection in response to (i) changes occurring in the gastric mucosa and (ii) the evolution of the associated pathology14. On the other hand, the most common H. pylori genotypic pattern among children was similar to the pattern in adults in the U.S.15. These differences may once more reflect geographical variations in the strain reservoirs and host immune response. It is necessary to examine the prevalence of jhp0562 and β-(1,3)galT in children in the U.S.
There were significant differences in the jhp0562 and β-(1,3)galT status between strains isolated from the U.S. and Japanese populations. These findings were consistent with other virulent factors such as cagA and vacA; the prevalence of the virulent genotypes (i.e., East Asian type cagA and vacA s1 type) were extremely high in Japan regardless of clinical outcomes16. High prevalence of jhp0562 and low prevalence of β-(1,3)galT also supports that most H. pylori strains in Japan show high virulence compared with these in the U.S. In this study, the prevalence of jhp0562 was strongly associated with that of cagA in the U.S. population. Consistent with our result, Oleastro et al. reported a positive association of jhp0562 with the cag pathogenicity island (PAI)8. Although the correlation could not be calculated because of the high prevalence of cagA in Japan, all cagA-positive strains were also positive for jhp0562. These patterns were consistent with the relationship between cagA and other virulent factors in Japan. The cagA status is also linked to the vacA s region type, and it is further closely linked to the presence of babA and oipA “on” status, which are other virulence factors coding outer membrane proteins4, 17, 18. As a result, almost all H. pylori strains circulating in Japan are extremely virulent, harboring cagA, vacA s1 genotype, oipA “on” status, and babA irrespective of the clinical outcomes4, 19, 20. Thus, it is likely that the phenotype resulting from the expression of cagA, vacA s1, and jhp0562 confers a biological advantage to the strain, with the cumulative action of each factor contributing simultaneously to the fitness of the strains in vivo and a more pronounced pro-inflammatory response. It might be better to hypothesize that these factors interact synergistically with each other and induce serious diseases than to discuss which of these factors is the most virulent. In addition, we found that the prevalence of β-(1,3)galT was inversely associated with that of cagA, although the inverse correlation was not strong. Multivariate analysis showed that the absence of β-(1,3)galT but not the presence of cagA was an independent factor for discriminating DU and GU from gastritis in the U.S. This means that the absence of β-(1,3)galT may be the true candidate marker for peptic ulcer in the U.S. When we compared the sequence of jhp0562 and β-(1,3)galT by using Asian strains (full-sequenced strains 51 and 52) and Western strains deposited by Oleastro in Genbank, we could not find the genetic polymorphism among these two area (data not shown). This suggests that the prevalence of these genes but not polymorphism associated with the clinical outcomes.
jhp0562 encodes a glycosyltransferase involved in the synthesis of the chemical structure of LPS and is located immediately upstream of β-(1,3)galT, which in turn encodes a β-(1,3) galactosyltransferase involved in type I Le antigen synthesis (Lea and Leb synthesis of the LPS)21, 22. Although these Le antigenic structures were reported to be important for bacterial colonization, adhesion, and evasion of host immune response23–25, the role of these in H. pylori infection has not been elucidated. Therefore, additional in vitro and in vivo studies are necessary to elucidate the causal relationship between jhp0562 and β-(1,3)galT and to investigate the mechanisms by which these gene products correlate with clinical outcomes. Because these two genes were inversely correlated, the products of the two genes may have the same cell function, thus producing functional redundancy.
In conclusion, the absence of β-(1,3)galT was independent factor discriminating DU and GU from gastritis in the U.S., but not in Japan. The jhp0562-positive/β-(1,3)galT-negative genotype was significantly associated with the presence of peptic ulcer in the U.S. jhp0562 and β-(1,3)galT were correlated with cagA similarly as other virulence factors, and there was a difference in the presence of jhp0562 or β-(1,3)galT among strains isolated from the U.S and Japan. In the U.S., the status of jhp0562 or β-(1,3)galT can predict the future development of peptic ulcer for the patients with gastritis. Prospective study is necessary to elucidate it. Moreover, the study of the distribution of these genes in other populations would also be interesting to further elucidate the associations found in the present study and the possible virulent role of these factors in H. pylori infection.
Acknowledgements
This report is based on work supported in part by grants from the National Institutes of Health (DK62813), grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (22390085 and 22659087), and Special Coordination Funds for Promoting Science and Technology from MEXT of Japan. We thank Ms. Ayaka Takahashi for excellent technical assistance.
Footnotes
Potential competing interests: None
References
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