Identification of markers for Helicobacter pylori strains isolated from children with peptic ulcer disease by suppressive subtractive hybridization

Abstract

Peptic ulcer disease (PUD) occurs after a long-term Helicobacter pylori infection. However, the disease can develop earlier, and rare cases have been observed in children, suggesting that these H. pylori strains may be more virulent. We used suppressive subtractive hybridization for comparative genomics between H. pylori strains isolated from a 5-year-old child with duodenal ulcer and from a sex- and age-matched child with gastritis only. The prevalence of the 30 tester-specific subtracted sequences was determined on a collection of H. pylori strains from children (15 ulcers and 30 gastritis) and from adults (46 ulcers and 44 gastritis). Two of these sequences, jhp0562 (80.0% versus 33.3%, P = 0.008) and jhp0870 (80.0% versus 36.7%, P = 0.015), were highly associated with PUD in children and a third sequence, jhp0828, was less associated (40.0% versus 10.0%, P = 0.048). Among adult strains, none of the 30 sequences was associated with PUD. However, both jhp0562 and jhp0870 were less prevalent in adenocarcinoma strains than in PUD strains from children and adults, the difference being statistically significant for jhp0870. In conclusion, two H. pylori genes were identified as being strongly associated with PUD in children, and their putative roles as an outer membrane protein for jhp0870 and in lipopolysaccharide biosynthesis for jhp0562, suggest that they may be novel virulence factors of H. pylori.

FIG. 1.

Schematic representation of the PCR assay for the detection of the jhp0562, jhp0563(HP0619)-like genes in H. pylori strains (A) and analysis of their detection by PCR in an ethidium bromide-stained 1.5% agarose gel (B). (A) The black arrows represent the forward F1-jhp0562/jhp0563, labeled F, and the reverse R1-jhp0562/jhp0563, labeled R, conserved primers between H. pylori strains, respectively. These F and R primers hybridize both on jhp0562 and on jhp0563-like genes. Genes that belong to the same family are represented in an identical way. (B) MW, molecular weight marker ΦX174 RF DNA/HaeIII fragments (72-1353 pb; Invitrogen).

FIG. 2.

Genomic organization of the jhp0870 (A) and jhp0649/HP0710 (B) loci in J99 and 26695 H. pylori strains. The jhp0870 gene is specific for the J99 strain. No homologue of this gene exists in the 26695 strain (5). Instead, an intergenic space is observed, flanked by genes homologous to those flanking jhp0870 in the J99 strain (52). Genes which belong to the same family are represented in an identical way. The boxes represent the 27-, 2-, and 4-bp additional sequences present in jhp0870 but absent in jhp0649/HP0710.

FIG. 3.

Schematic representation of the PCR assay for the detection of the jhp0870 and jhp0649-like genes in H. pylori strains (A) and analysis of their detection by PCR in ethidium bromide-stained 2,5% agarose gel (B). (A) The black arrows represent the forward F1-jhp0870/jhp0649, labeled F, and reverse F1-jhp0870/jhp0649, labeled R, conserved primers between the jhp0870 and jhp0649-like genes in H. pylori strains, respectively. These F and R primers hybridize both on jhp0649 and jhp0870-like genes. The three internal boxes in the jhp0870 scheme represent the 27-bp, 2-bp and 4-bp additional sequences present in jhp0870 but absent in jhp0649. (B) MW, molecular weight marker ΦX174 RF DNA/HaeIII fragments (72-1353 pb; Invitrogen).

FIG. 4.

Distribution of jhp0562 and jhp0870 genes among H. pylori strains isolated from subjects with PUD, gastritis, gastric adenocarcinoma, and gastric MALT lymphoma, including both children only and children and adults together (all strains). P values, determined by the Fisher exact test, are shown for the significant associations only.