Topographical localisation of cagA positive and cagA negative Helicobacter pylori strains in the gastric mucosa; an in situ hybridisation study

Abstract

Background: The cagA gene is a marker for the presence of the cag pathogenicity island, and the presence of cagA positive strains of Helicobacter pylori can identify individuals with a higher risk of developing gastrointestinal diseases.

Aims: To study the interaction between H. pylori cagA(+) and cagA(-) strains and the gastric mucosa.

Methods: Patients with H. pylori associated gastritis and peptic ulcers were studied. Biopsies were obtained from the antrum, corpus, fundus, and incisura for H pylori culture, and for in situ hybridisation studies. From each biopsy, multiple single H. pylori colonies were isolated and propagated for DNA isolation, and cagA was detected by the polymerase chain reaction (PCR). For in situ detection of H. pylori an oligonucleotide specific for an H. pylori common antigen and an oligonucleotide specific for cagA were used as probes. Biotinylated probes were incubated with biopsy sections, developed with streptavidin-horseradish peroxidase, and amplified with the tyramide system.

Results: PCR results for cagA in isolated colonies confirmed the in situ hydridisation studies. In situ hybridisation identified cagA(+) bacteria in patients with cagA(+) isolates; cagA(-) bacteria in patients with cagA(-) isolates, and cagA(+) and cagA (-) bacteria in patients with both cagA(+) and cagA(-) isolates. CagA(-) bacteria usually colonised the mucous gel or the apical epithelial surface, whereas cagA(+) bacteria colonised the immediate vicinity of epithelial cells or the intercellular spaces.

Conclusions: These results document a different in vivo interaction between H. pylori cagA(+) or cagA(-) strains and the gastric mucosa.

Figure 1

In situ hybridisation for the detection of cagA(+) and cagA(−) strains in a mixture of Helicobacter pylori ATCC 49503 (cagA(+)) and Tx30a (cagA(−)) strains fixed on glass slides. The DNA probe for the common antigen was developed with fluorescein isothiocyanate (green) and the probe for cagA was developed with Cy3 (red). (A) green filter; (B) red filter; and (C) triple filter, yellow areas indicate overlapping of the green common antigen and the red cagA, thereby demonstrating the presence of cagA(+) strains.

Figure 2

In situ hybridisation for the detection of Helicobacter pylori cagA(+) and cagA(−) strains in gastric biopsies of patients colonised with H pylori. The DNA probe for the common antigen was developed with fluorescein isothiocyanate (FITC; green) and the probe for cagA was developed with Cy3 (red). (A) The gastric biopsy of a patient colonised with both cagA(+) and cagA(−) strains; a cagA(−) bacterium was scanned to show the presence of FITC and the absence of Cy3 fluorescence; the arrowhead points to cagA(−) and the arrow to cagA(+) bacteria. (B) The gastric biopsy of a patient colonised exclusively with cagA(+) strains; a bacterium was scanned to show the presence of both FITC and Cy3 fluorescence.

Figure 3

In situ hybridisation for the detection of Helicobacter pylori cagA(+) and cagA(−) strains in gastric biopsies of patients colonised with H pylori. The DNA probe for the common antigen was developed with fluorescein isothiocyanate (FITC; green) and the probe for cagA was developed with Cy3 (red). (A, D) Green filter; (B, E) red filter; and (C, F) triple filter, the yellow areas indicate overlapping of the green common antigen and the red cagA, thereby demonstrating the presence of cagA(+) strains. Patient 256 was colonised with cagA(+) strains, and colonisation is seen close to the epithelium; patient 254 was colonised with cagA(−) strains, and colonisation is seen mainly in the mucous.

Figure 4

In situ hybridisation for the detection of Helicobacter pylori cagA(+) and cagA(−) strains in the gastric biopsy of a patient (259) colonised with both cagA(+) and cagA(−) strains. The DNA probe for the common antigen was developed with fluorescein isothiocyanate (FITC; green) and the probe for cagA was developed with Cy3 (red). (A) green filter; (B) red filter; (C) triple filter, yellow areas indicate overlapping of the green common antigen and red cagA, thereby demonstrating the presence of cagA(+) strains; and (D) triple filter in confocal microscopy. The arrowhead points to cag(−) and the arrow to cagA(+) bacteria. cagA(−) strains are seen colonising mainly the mucous, whereas cagA(+) strains are in intimate contact with epithelial cells.

Figure 5

In situ hybridisation for the detection of Helicobacter pylori cagA(+) and cagA(−) strains in gastric biopsies of patients colonised with H pylori. The DNA probe for the common antigen was developed with fluorescein isothiocyanate (FITC; green) and the probe for cagA was developed with Cy3 (red). All preparations were observed with the triple filter. Patients 251 and 252 were colonised with cagA(−) strains, and colonisation is seen mainly in the mucous or in the proximity of the epithelial cells. Patients 256 and 261 were colonised with cagA(+) strains, and colonisation is seen in the interepithelial spaces and in intimate contact with the epithelium. Patients 285 and 247 were colonised with both, cagA(−) and cagA(+) strains. The arrowheads point to cagA(−) and the arrows to cagA(+) bacteria; in patient 285 some cagA(+) bacteria are seen in intimate contact with the epithelial cells; whereas in patient 247 a few cagA(−) bacteria are seen in the mucous.

Figure 6

In situ hybridisation for the detection of Helicobacter pylori cagA(+) and cagA(−) strains in gastric biopsies of patient 285. The DNA probe for the common antigen was developed with fluorescein isothiocyanate (FITC; green) and the probe for cagA was developed with Cy3 (red). (A, D) green filter; (B, E) red filter; (C, F) triple filter, the yellow areas indicate the overlapping of the green common antigen and the red cagA, thereby demonstrating the presence of cagA(+) strains. Two different regions of the epithelium are presented to show colonisation in the intercellular epithelial space.