Integrative and Conjugative Elements of Helicobacter pylori Are Hypothetical Virulence Factors Associated With Gastric Cancer

Abstract

Helicobacter pylori is a bacteria with high genome plasticity that has been associated with diverse gastric pathologies. The genetic diversity of this bacteria has limited the characterization of virulence factors associated with gastric cancer (GC). To identify potentially helpful disease biomarkers, we compared 38 complete genomes and 108 draft genomes of H. pylori isolated worldwide from patients with diverse gastric pathologies and 53 draft genomes of H. pylori isolated from Mexican patients with GC, intestinal metaplasia, gastritis, peptic ulcer, and dyspepsia. H. pylori strains isolated from GC were 3-11 times more likely to harbor any of seven genes encoded within an integrative and conjugative element (ICE) than H. pylori isolated from subjects with other gastric pathologies. We tested the cytopathic effects on AGS cells of selected H. pylori strains with known cytotoxin-associated gene pathogenicity island (cag-PAI) and ICE status (H. pylori strains 29CaP, 29CaCe, 62A9, 7C, 8822, and 26695) and the histopathological damage of H. pylori 29CaP and 62A9 in a mouse model. H. pylori 29CaP, which harbors a complete ICEHptfs3 but lacks cag-PAI, elicited distinctive morphology changes and higher histopathological scores compared with other H. pylori strains carrying cag-PAI and hybrid ICE with incomplete TFSS. The presence of intact segments of ICE regions might be a risk factor to develop GC that needs to be addressed in future studies.

Keywords: Helicobacter pylori; comparative genomics; gastric cancer; integrative and conjugative elements (ICE); virulence factor.

Figure 1

Comparison of protein orthologous groups (POGs) predicted with three H. pylori genome datasets. (A) Number of genomes included in each genome set and the number of gastric cancer-associated POGs detected. (B) Number of POGs detected in the three datasets.

Figure 2

Gastric cancer-associated protein orthologous groups (POGs) mapped on H. pylori 29CaP genome. tblastx comparisons of the four ICEs described, and KHP30 and phiHP33 bacteriophage genomes with H. pylori 29CaP genome to show the regions where POGs are encoded. Alignments and circular map were obtained with BRIG v0.95.

Figure 3

Distribution of integrase and ICE coverage in 38 H. pylori complete genomes. Genomes selected were clustered using ANI-BLAST to match their phylogeographic origin. Presence of integrase gene in genomes is marked as a blue star. Genomes with ICE coverage >50% are marked as an orange square. There is no association between phylogeographic origin and presence of elements mentioned, only a high frequency of these elements in genomes of H. pylori isolated from gastric cancer.

Figure 4

Comparison of H. pylori 29CaP and 29CaCe genomes. Linear maps of H. pylori genomes are connected by vectors matching the similar regions between them. Red vectors represent sequences with the same orientation, whereas blue vectors represent regions with inverted sequences. cag-PAI, ICEs, and prophages are signaled in the genomes. Images were generated using Artemis Comparison Tool v 18.1.0 (Sanger Institute) with blastn alignments.

Figure 5

Evaluation of the functional effects of the complete ICEHptfs3. (A) In silico evaluation of TFSS completeness in H. pylori strains used for infection in AGS cells and mouse model. We show that H. pylori 29CaP has a complete TFSS located in the ICE element. (B) Cytopathic effects on AGS cells using strains with different cag-PAI and ICE genotypes. (C) Histopathological damage induced by H. pylori 29CaP and 62A9 in mice. Stomachs from infected mice were evaluated by hematoxylin and eosin staining. Scores were normalized, dividing the individual score of each infected mouse by the average score of their respective control group.