Molecular Basis of Pathogenicity in Helicobacter pylori Clinical Isolates

Abstract

This study identified pathogenicity genes in 40 Helicobacter pylori clinical isolates. The cagA, vacA, and iceA genes were detected in 65%, 97.5%, and 97.5% of the isolates, respectively. The cagA, iceA1, and vacAs1a/m1 genes were related to erosive gastritis, whereas the vacAs2/m2 and iceA2 genes were associated with enanthematous gastritis.

Helicobacter pylori is considered the major etiologic agent of chronic active gastritis, an essential catalyst in the emergence of peptic ulcer, and a risk factor for the development of gastric cancer (17). Studies indicate that the evolution of the infection depends in part on the expression of specific bacterial pathogenicity genes, such as cagA (cytotoxin-associated gene A), vacA (vacuolating cytotoxin), and iceA (induced by contact with epithelium) (2).

The cagA gene is considered to be a marker for the presence of a cagA pathogenicity island (8). The cagA-positive H. pylori strains increase interleukin-8 production and gastric inflammation (5). The vacA gene encodes a vacuolating cytotoxin able to induce the formation of cytoplasmic vacuoles in epithelial cells (11). This gene comprises two variable regions: the signal region, with two alleles, s1 (subtypes s1a, s1b, and s1c) and s2, and the middle region, with the alleles m1 and m2 (3, 28). In general, the s1/m1 strains produce large amounts of vacuolating cytotoxin, the s1/m2 strains produce moderate amounts, and the s2/m2 strains produce little or none (3). The iceA gene has two alleles: iceA1 and iceA2. The iceA1 allele is associated with peptic ulcer, and iceA2 is related to asymptomatic gastritis (24, 29).

This study analyzed the presence of cagA, vacA, and iceA genes in clinical isolates and correlated these findings with the endoscopic diagnosis. Forty isolates of H. pylori were obtained from biopsy specimens of the gastric antrum collected from dyspeptic patients admitted to the upper gastrointestinal endoscopic ward in the Hospital of the Federal University of Rio Grande, Rio Grande do Sul, Brazil. This study was approved by the ethics committee of our university. Informed consent was obtained from all patients.

After collection, the biopsy specimens were kept in brain heart infusion broth (Acumedia, United States) with 20% glycerol and refrigerated (4 to 8°C) for a maximum of 4 h (22). This broth was thereafter vortexed, and 200 μl was added to medium Columbia agar (Oxoid, United Kingdom), supplemented with 7% sheep blood and with a selective mixture for Helicobacter species isolation (Cefar, Brazil). The agar plates were incubated under microaerophilic conditions (5 to 15% O₂ and 10% CO₂) at 37°C for 4 to 10 days (14). The identification of H. pylori was performed using catalase, oxidase, and urease tests, microscopy, and ureA gene detection (12, 19).

The DNA extraction was performed after 48 h of bacterial growth. Colonies were collected and resuspended in 500 μl of 1× TE buffer. The suspension was centrifuged at 10,000 × g for 5 min, and the supernatant was thereafter discarded. The DNA from the clinical isolates was then extracted with DNAzol reagent (Invitrogen, United States) by the method of the manufacturer.

The presence of the ureA, cagA, vacA, and iceA genes in the isolates was investigated by PCR using the primers described previously (6, 10, 21, 31). The PCR was performed as described by Rota et al. (for the ureA and cagA genes) and by Benenson et al. (for the alleles of the vacA and iceA genes) (4, 27).

The statistical analysis was performed by using Fisher's exact test, a chi-squared test, and a chi-squared test for linear trend. P values of less than 0.05 were considered statistically significant.

The presence of the pathogenicity genes was studied in 40 clinical isolates of H. pylori. From those, 50% (20 of 40) were obtained from patients with endoscopic diagnosis of enanthematous gastritis and 50% (20 of 40) were obtained from patients with erosive gastritis.

The cagA gene was identified in 65% (26 of 40) of the isolates. This frequency is similar to that found in previous studies of cagA in Brazil (14, 16, 18). The vacA and iceA genes were detected in 97.5% (39 of 40) of the samples. The vacAs1b (43.6%) and vacAm2 (53.9%) alleles were the most frequently detected in the 39 isolates, as well as the iceA2 allele (71.8%). This is an expected result, because these alleles have been reported in other studies (7, 18, 26). Moreover, 12.8% of the isolates verified the presence of the m1 and m2 alleles of the vacA gene, and 5.1% of the isolates had both iceA alleles. The detection of more than one allele of the middle region of vacA, as well as the identification of both iceA alleles in the same isolate, suggests coinfection of two different strains of H. pylori. Cases of patients being infected with multiple strains of H. pylori are not uncommon, being more frequent in areas of high H. pylori prevalence (9, 15, 23).

The association between the cagA and vacA genes is described in Table 1. All cagA-positive isolates confirmed the presence of vacA. The combinations vacAs1a/m1, vacAs1b/m1, vacAs1a/m2, vacAs1b/m2, and vacAs1b/m1m2 were present mainly in cagA-positive samples. A statistically significant association was observed between cagA and vacA (P < 0.001).

TABLE 1.

Association between the cagA gene and the allelic combinations of the vacA gene in isolates of H. pylori

Genotypea	% with or without cagA gene (no. with gene status/total no. of samples)
	cagA positive	cagA negative
vacAs1a/m1	100.0 (5/5)
vacAs1b/m1	87.5 (7/8)	12.5 (1/8)
vacAs1a/m2	100.0 (5/5)
vacAs1b/m2	66.7 (4/6)	33.3 (2/6)
vacAs2/m2	10.0 (1/10)	90.0 (9/10)
vacAs1b/m1m2	100.0 (3/3)
vacAs2/m1m2	50.0 (1/2)	50.0 (1/2)
vacA negative		100.0 (1/1)

^aP < 0.001.

The relationship of pathogenicity genes with gastric disorders is described in Table 2. The cagA gene and the combination vacAs1a/m1 were frequently detected in isolates from patients with erosive gastritis. Similar findings were reported by other authors (14, 20). These genes are directly related to the infiltration of polymorphonuclear cells, which causes severe epithelial damage. Already, the combination vacAs2/m2 was frequently observed in isolates from patients with enanthematous gastritis, a finding that suggests that such alleles are related to minor damage in gastric mucosa (1). However, a statistically significant difference was not found in the association between either cagA or vacA and the clinical manifestations. The iceA1 allele was detected in 66.7% of isolates from patients with erosive gastritis, while iceA2 was identified in 57.1% of isolates from patients with enanthematous gastritis. The iceA1 allele may be associated with a more severe form of gastritis because iceA1-positive strains produce more inflammation-inducing cytokines, such as interleukin-8, which are potent chemotactic factors that activate polymorphonuclear leukocytes that contribute to enhanced inflammatory responses (13, 30). This finding agrees with those of previous studies (24, 25). In this work, a statistically significant association was observed between iceA and the endoscopic diagnosis (P = 0.047).

TABLE 2.

Distribution of the cagA gene and of the vacA and iceA alleles in isolates of H. pylori deriving from patients with different clinical manifestations

Genotype	% with clinical manifestation (no. affected/total no. of samples)
	Enanthematous gastritis	Erosive gastritis
cagA genesa
cagA positive	42.3 (11/26)	57.7 (15/26)
cagA negative	64.3 (9/14)	35.7 (5/14)
vacA genesb
vacAs1a/m1	20.0 (1/5)	80.0 (4/5)
vacAs1b/m1	50.0 (4/8)	50.0 (4/8)
vacAs1a/m2	40.0 (2/5)	60.0 (3/5)
vacAs1b/m2	33.3 (2/6)	66.7 (4/6)
vacAs2/m2	80.0 (8/10)	20.0 (2/10)
vacAs1b/m1m2	66.7 (2/3)	33.3 (1/3)
vacAs2/m1m2	50.0 (1/2)	50.0 (1/2)
vacA negative		100.0 (1/1)
iceA genesc
iceA1 + iceA2		100.0 (2/2)
iceA1	33.3 (3/9)	66.7 (6/9)
iceA2	57.1 (16/28)	42.9 (12/28)
iceA negative	100.0 (1/1)

^aP = 0.185.

^bP = 0.350.

^cP = 0.047.

Based on the data presented above, we conclude that the detection of cagA, vacA, and iceA genes allows an improved evaluation of the pathogenic potential from clinical isolates. In this study, the cagA gene, the combination vacAs1a/m1, and the iceA1 allele were related to erosive gastritis; similarly, the combination vacAs2/m2 and the iceA2 allele were related to an attenuated form of gastritis. Therefore, the genotyping of the microorganism appears to be a clinically relevant procedure and can contribute to the prognosis of H. pylori infection.