Neutrophil-activating Protein Polymorphism of Helicobacter pylori Determines the Host Risk of Dyspepsia.

Helicobacter pylori infection is an accepted cause of dyspepsia, according to the Rome IV criteria, because its eradication significantly resolves dyspepsia; however, no H pylori virulence factor that can cause dyspepsia has been identified. Here, we identified a polymorphism in H pylori neutrophil-activating protein A (NapA) closely associated with dyspepsia occurrence.

There is evidence that chronic gastric inflammation caused by reactive oxygen species (ROS) is associated with dyspepsia severity in individuals with H pylori infection. ROS production is characteristic during innate immune responses to H pylori, and H pylori uses various enzymes to counteract ROS, which facilitate the establishment of persistent infection.3, 4 We investigated the sequences of genes encoding 2 antioxidant proteins, NapA and alkyl hydroperoxide reductase (AhpC), in patient-derived H pylori strains.

Patients with and without dyspepsia (n = 33 and 88, respectively) were enrolled (Supplementary Methods), and H pylori strains were isolated from their stomachs (Supplementary Figure 1A). The baseline characteristics of participants are presented in Supplementary Table 1. H pylori sequencing analysis revealed 2 polymorphisms in each of napA (encoding amino acids 70 and 73) and ahpC (encoding amino acids 126 and 140). H pylori with NapA including serine at amino acid 70 (Ser 70-NapA) was isolated from dyspeptic patients more frequently than H pylori carrying threonine at the same position (Thr 70-NapA) (age-adjusted odds ratio, 2.88; 95% confidence interval, 1.19–6.94; P = 0.019) (Figure 1). The prevalence of Ser 70-NapA H pylori infection did not differ between patients with meal-related and meal-unrelated dyspepsia (Supplementary Figure 1B and C).

Supplementary Figure 1

( (B and C) Prevalence of H pylori NapA polymorphisms in patients without dyspepsia and with each subgroup of dyspepsia.

Supplementary Table 1

Participant Characteristics

	Dyspepsia (–) (n = 88)	Dyspepsia (+) (n = 33)	P value
Age (y), mean ± SD	54.8 ± 13.2	47.1 ± 13.3	.005a
Sex
Male	37 (42.0%)	12 (24.5%)	.68b
Female	51 (58.0%)	21 (63.6%)
BMI (kg/m2), mean ± SD	22.0 ± 3.2	22.0 ± 4.1	.97c
Frequency of alcohol consumption
Social drinker (<5 days/wk)	43 (48.9%)	19 (57.6%)	.11c
Frequent drinker (5–7 days/wk)	15 (17.0%)	1 (3.0%)
Smoking habit
Ex-smoker	21 (23.9%)	7 (21.2%)	.20c
Current smoker	6 (6.8%)	6 (18.2%)
Medication
Proton pump inhibitor	10 (11.4%)	9 (27.3%)	.052b
Histamine 2 receptor antagonist	12 (13.6%)	5 (15.2%)	1.00b
Dyspeptic symptom categories			NA
Postprandial fullness	0 (0%)	18 (54.5%)
Early satiation	0 (0%)	17 (51.5%)
Epigastric pain	0 (0%)	20 (60.6%)
Epigastric burning	0 (0%)	12 (36.4%)

NOTE. Bold values indicate significant differences.

BMI, body mass index; NA, not applicable; SD, standard deviation.

Student t test.

Fisher exact test.

Pearson χ2 test.

Figure 1

Associations between dyspepsia and polymorphisms in Error bars indicate 95% confidence intervals. *P < .05 by logistic regression analysis.

According to the homo-12-mer 3D structure of NapA (RCSB Protein Data Bank ID: 1JI4, http://www.rcsb.org), amino acid 70 lies close to the monomer junction and is exposed on the dodecameric complex surface (Supplementary Figure 2A); therefore, it likely contributes to the stability of 12-mer formation and NapA neutrophil activation function, which may influence dyspepsia. NapA must form a dodecameric complex to sequester and store iron, which are functions associated with the antioxidant abilities of H pylori. NapA has dual roles, enhancing bacterial antioxidant ability and stimulating ROS production by neutrophil recruitment. To examine phenotypic differences in H pylori with polymorphisms of NapA amino acid 70, we cultured five Ser 70-NapA H pylori strains isolated from dyspeptic patients and five Thr 70-NapA H pylori strains from non-dyspeptic patients (Supplementary Figure 2B). Investigation of susceptibility to H2O2 and t-BuOOH demonstrated that Ser 70-NapA H pylori strains had significantly smaller inhibition zones (P = .021 and .047, respectively), indicating that Ser 70-NapA H pylori strains are better adapted to ROS exposure than those with Thr 70-NapA (Supplementary Figure 2C). When we exposed mouse neutrophils to H pylori culture supernatants, intracellular ROS production was marginally higher in those exposed to Ser 70-NapA H pylori than Thr 70-NapA H pylori supernatants (Pfor trend = .06) (Supplementary Figure 2D), although migration activity did not differ (Pfor trend = .20) (Supplementary Figure 2E).

Supplementary Figure 2

(Red and blue spheres indicate locations of amino acids 70 and 73, respectively. (B) NapA amino acid sequences. ATCC700392 and J99 are genome-sequenced references. (C) Inhibition zone assay data were collected by 5 strains from each group in 1 independent experiment. P values, Student t test. Data of neutrophil ROS induced by H pylori culture supernatants (D) and neutrophil migration (E) were collected by 3 independent experiments for five Thr70-NapA and five Ser70-NapA strains. P values for trend, Pearson correlation analysis.

We also compared changes in Mongolian gerbils infected with Ser 70-NapA and Thr 70-NapA H pylori. Four months after infection, gastric emptying was significantly delayed in gerbils infected with Ser 70-NapA H pylori relative to those with Thr 70-NapA H pylori or uninfected controls (Pfor trend = .004) (Supplementary Figure 3A). Seven months after infection, hematoxylin-eosin staining of the gastric antrum revealed more severe infiltration of inflammatory cells in gastric mucosa infected with Ser 70-NapA H pylori, and more severe inflammatory cell infiltrations around the myenteric plexus (MP) were detected after Ser 70-NapA H pylori infection, compared with Thr 70-NapA H pylori or uninfected controls (Figure 2). The activity of myeloperoxidase (MPO), a hemoprotein secreted during inflammatory cell activation, was higher in gastric mucosa infected with Ser 70-NapA H pylori strains (Pfor trend = .0003), resulting in a tendency toward increased levels of thiobarbituric acid reactive substances (TBARS), an indicator of free radical–mediated lipid peroxidation injury (Pfor trend = .08) (Supplementary Figure 3B). These results suggest that Ser 70-NapA H pylori infection delays gastric emptying through induction of ROS by enhancing inflammatory cell recruitment.

Supplementary Figure 3

(. (B) MPO activity (left panel) and TBARS levels (right panel) in gastric mucosa from Mongolian gerbils (n = 15 for each). P values for trend, Pearson correlation analysis. (C) More than 1 year after successful eradication therapy for 33 dyspeptic patients, a questionnaire was posted to them about the presence/absence of dyspepsia. Among the 18 analyzed patients, 10 reported the resolution of dyspepsia after eradication therapy. (D) Symptom relief rates were 40.0% (2/5) and 61.5% (8/13) after eradication of Thr70-NapA and Ser70-NapA H pylori, respectively.

Figure 2

Light microscopy images of hematoxylin-eosin–stained normal gastric mucosa and Thr 70- or Ser 70-NapA MP, myenteric plexus. Scale bars in upper and lower panels indicate 1 mm and 50 μm, respectively.

Finally, we used a conventional mail survey to compare symptom relief rates of patients after eradication of Ser 70-NapA or Thr 70-NapA H pylori (Supplementary Figure 3C). More than 1 year after successful eradication therapy, we sent 33 dyspeptic patients a questionnaire regarding the presence/absence of dyspepsia, and 18 responses were received. Symptom improvement rates were 26.6% greater after eradication of Ser 70-NapA H pylori than Thr 70-NapA H pylori (61.5% [8/13] vs 40.0% [2/5]), although the difference was not statistically significant because of the small sample size (Supplementary Figure 3D). Treatment response in dyspeptic patients with Thr 70-NapA H pylori (40.0%) was comparable to a previously reported placebo effect. To prove this effect, a double-blind study comparing eradication and non-eradication groups would provide more conclusive results; however, because of the cancer-preventive effect of eradication therapy, it would be ethically inappropriate to include a non-eradication group.

Notably, 2 previous reports support a potential key role for polymorphism of amino acid 70 in H pylori NapA.7, 10 We did not perform whole genome sequencing of isolated H pylori strains; therefore, experiments using genetically modified H pylori strains expressing Thr 70-NapA or Ser 70-NapA on the same genetic background could confirm our hypothesis. Thus, although there is a possibility that differences at other H pylori loci contribute to the development of dyspepsia, our data represent the first molecular-level evidence of the mechanism underlying H pylori–associated dyspepsia. These findings could assist in efficient diagnosis and prediction of treatment responses in patients with H pylori–associated dyspepsia.