Pan-genomic analyses identify key Helicobacter pylori pathogenic loci modified by carcinogenic host microenvironments

Abstract

Objective: Helicobacter pylori is the strongest risk factor for gastric cancer; however, the majority of infected individuals do not develop disease. Pathological outcomes are mediated by complex interactions among bacterial, host and environmental constituents, and two dietary factors linked with gastric cancer risk are iron deficiency and high salt. We hypothesised that prolonged adaptation of H. pylori to in vivo carcinogenic microenvironments results in genetic modification important for disease.

Design: Whole genome sequencing of genetically related H. pylori strains that differ in virulence and targeted H. pylori sequencing following prolonged exposure of bacteria to in vitro carcinogenic conditions were performed.

Results: A total of 180 unique single nucleotide polymorphisms (SNPs) were identified among the collective genomes when compared with a reference H. pylori genome. Importantly, common SNPs were identified in isolates harvested from iron-depleted and high salt carcinogenic microenvironments, including an SNP within fur (FurR88H). To investigate the direct role of low iron and/or high salt, H. pylori was continuously cultured in vitro under low iron or high salt conditions to assess fur genetic variation. Exposure to low iron or high salt selected for the FurR88H variant after only 5 days. To extend these results, fur was sequenced in 339 clinical H. pylori strains. Among the isolates examined, 17% (40/232) of strains isolated from patients with premalignant lesions harboured the FurR88H variant, compared with only 6% (6/107) of strains from patients with non-atrophic gastritis alone (p=0.0034).

Conclusion: These results indicate that specific genetic variation arises within H. pylori strains during in vivo adaptation to conditions conducive for gastric carcinogenesis.

Keywords: Helicobacter pylori; gastric cancer; high salt; iron deficiency.

Figure 1

Experimental design. Helicobacter pylori strain B128 was originally isolated from a human patient. Strain B128 (green) was used for orogastric challenge of Mongolian gerbils and was passaged three times through gerbils in a previous study. The in vivo-adapted output strain, designated H. pylori strain B8 (orange), was isolated from those gerbils, sequenced and used as the reference strain in this study. In an independent experiment, strain B128 (green) was also used for orogastric challenge of a single Mongolian gerbil, and the in vivo-adapted output strain isolated from this gerbil was designated H. pylori strain 7.13 (orange). Carcinogenic H. pylori strain 7.13 was then used to infect another cohort of Mongolian gerbils maintained on either iron-replete or iron-depleted diets. Single colonies (n=3) from three independent gerbils maintained on iron-replete diet (n=9) and three independent gerbils maintained on iron-depleted diet (n=9) were subjected to whole genome sequence analysis. In vivo-adapted strain from iron-replete (red) and iron-depleted (blue) conditions was also compared with previously described H. pylori single colonies (n=4) harvested from one gerbil maintained on a regular salt (pink) or high salt (light blue) diet.

Figure 2

(A) Phylogenetic analyses of whole genome sequences from Helicobacter pylori strains B128 (green) and 7.13 (orange), and in vivo-adapted iron-replete (red) and iron-depleted (blue) strains. R1-3 and D1-3 designate independent gerbils maintained on either iron-replete (n=3) or iron-depleted (n=3), respectively, while a–c designate different single colonies isolated from each gerbil. H. pylori strains isolated from humans or gerbils are grouped within the grey boxes, respectively. The scale bar represents amino acid changes per site. (B) Venn diagram showing the number of common and unique single nucleotide polymorphisms (SNPs) among human-adapted H. pylori strains B128 (green) and 7.13 (orange), and in vivo-adapted H. pylori strains isolated from gerbils maintained on iron-replete (red) and iron-depleted (blue) diets, all relative to the reference H. pylori strain B8. The number in the parentheses indicates the total number of unique SNPs identified in each strain and present exclusively in that strain, but not any other strain.

Figure 3

Phylogenetic analysis of Fur amino acid sequence. Phylogenetic analysis was performed for full-length Fur from all available Helicobacter pylori sequence data within the National Center for Biotechnology Information NCBI database. H. pylori single colonies from strains B128 (green) and 7.13 (orange), and in vivo-adapted strains isolated from gerbils maintained on iron-replete (red) and iron-depleted (blue) diets as well as in vivo-adapted strains isolated from gerbils maintained on regular salt (pink) and high salt (light blue) diets were included in these analyses. R1-3 and D1-3 designate independent gerbils maintained on either iron-replete (n=3) or iron-depleted (n=3), respectively, while a–c designate different single colonies isolated from each gerbil. HSO and RSO designate independent high salt output and regular salt output H. pylori strains, respectively. REFSEQ (#) represents the number of unique H. pylori sequences from different H. pylori strains that cluster under the same phylogeny. Although all Fur sequences from the NCBI database were used, this phylogenetic analysis shows only the branch to which the newly sequenced strains cluster. The scale bar represents amino acid changes per site.

Figure 4

Analysis of genetic variation within fur following continuous culture in vitro under high salt or low iron conditions. (A) Helicobacter pylori strain 7.13 was grown in normal Brucella broth, modified Brucella broth with iron supplementation (100 µM FeCl₃), iron chelation (100 µM dipyridyl) or high salt (1.1% NaCl) for 20 hours and continuously over a 6-day time course. The growth rate or ratio of the final OD₆₀₀ to the initial starting OD₆₀₀ is represented as mean values with SE from experiments performed on at least six independent occasions. A one-way analysis of variance with multiple comparisons was used to determine statistical significance. ****p<0.0001. (B) H. pylori strain 7.13 was continuously cultured in vitro under these conditions for 6 days to assess the presence (CAC) or absence (CGC) of the SNP in fur (FurR88H). SNP, single nucleotide polymorphism.

Figure 5

Analysis of genetic variation within fur in vivo. The frequency of the FurR88H variant was examined among (A) 224 in vivo-adapted Helicobacter pylori strains isolated from gerbils maintained on iron-replete or iron-depleted diets and (B) 339 clinical H. pylori strains isolated from three independent and well-defined patient cohorts within Colombia and the USA.Fischer’s exact test was used to determine statistical significance. **p<0.005 and ****p<0.0001. DYS, dysplasia; IM, intestinal metaplasia; MAG, multifocal atrophic gastritis; NAG, non-atrophic gastritis alone.