Helicobacter pylori induced gastric immunopathology is associated with distinct microbiota changes in the large intestines of long-term infected Mongolian gerbils

Abstract

Background: Gastrointestinal (GI) inflammation in mice and men are frequently accompanied by distinct changes of the GI microbiota composition at sites of inflammation. Helicobacter (H.) pylori infection results in gastric immunopathology accompanied by colonization of stomachs with bacterial species, which are usually restricted to the lower intestine. Potential microbiota shifts distal to the inflammatory process following long-term H. pylori infection, however, have not been studied so far.

Methodology/principal findings: For the first time, we investigated microbiota changes along the entire GI tract of Mongolian gerbils after 14 months of infection with H. pylori B8 wildtype (WT) or its isogenic ΔcagY mutant (MUT) strain which is defective in the type IV secretion system and thus unable to modulate specific host pathways. Comprehensive cultural analyses revealed that severe gastric diseases such as atrophic pangastritis and precancerous transformations were accompanied by elevated luminal loads of E. coli and enterococci in the caecum and together with Bacteroides/Prevotella spp. in the colon of H. pylori WT, but not MUT infected gerbils as compared to naïve animals. Strikingly, molecular analyses revealed that Akkermansia, an uncultivable species involved in mucus degradation, was exclusively abundant in large intestines of H. pylori WT, but not MUT infected nor naïve gerbils.

Conclusion/significance: Taken together, long-term infection of Mongolian gerbils with a H. pylori WT strain displaying an intact type IV secretion system leads to distinct shifts of the microbiota composition in the distal uninflamed, but not proximal inflamed GI tract. Hence, H. pylori induced immunopathogenesis of the stomach, including hypochlorhydria and hypergastrinemia, might trigger large intestinal microbiota changes whereas the exact underlying mechanisms need to be further unraveled.

Conflict of interest statement

Competing Interests: The authors have the following interests. Co-authors Markus M. Heimesaat and Stefan Bereswill are PLOS ONE Editorial Board members. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. Gastric histopathology in Mongolian gerbils 14 months following H. pylori infection.

Mongolian gerbils were infected with H. pylori wildtype strain B8 (WT; middle panels) or H. pylori mutant strain lacking cagY (Mut; right panels) as described in Methods. Fourteen months following infection, stomach biopsies separated in antrum (A) and corpus (C) mucosa were taken and paraffin sections stained with H&E. Uninfected age-matched animals served as negative controls (Control; left panels). Representative photomicrographs are shown (100 x magnification).

Figure 2. In situ T lymphocyte responses in colonic sections of Mongolian gerbils 14 months following H. pylori infection.

Mongolian gerbils were infected with H. pylori wildtype strain B8 (B8; middle) or H. pylori mutant strain lacking cagY (B8ΔcagY; right). Fourteen months following infection, colonic biopsies were taken and paraffin sections stained for CD3 by immunohistochemistry to visualize T lymphocytes in situ. Uninfected age-matched animals served as negative controls (Naïve; left). Representative photomicrographs of positively stained cells are shown (200 x magnification, scale bar 50 µm).

Figure 3. Stomach microbiota composition in Mongolian gerbils 14 months following H. pylori infection.

Fourteen months following oral infection of Mongolian gerbils with H. pylori wildtype strain B8 (B8; black circles) or H. pylori mutant strain lacking cagY (B8ΔcagY; grey circles), the microbiota composition of luminal stomach contents were quantitatively analyzed by culture as described in Methods. Uninfected age-matched animals served as negative controls (Naïve; white circles). Numbers of live (A) E. coli, (B) Proteus sp., (C) Enterococci, (D) Lactobacilli, (E) Bacteroides/Prevotella spp. as well as the (F) total bacterial load are indicated as colony forming units (CFU) per g luminal content. Numbers of animals harboring the respective bacterial species are given in parentheses. Medians and significance levels (p-values) determined by Mann-Whitney-U test are indicated. Data shown were pooled from three independent experiments.

Figure 4. Duodenal microbiota composition in Mongolian gerbils 14 months following H. pylori infection.

Fourteen months following oral infection of Mongolian gerbils with H. pylori wildtype strain B8 (B8; black circles) or H. pylori mutant strain lacking cagY (B8ΔcagY; grey circles), the microbiota composition of luminal duodenum contents were quantitatively analyzed by culture as described in Methods. Uninfected age-matched animals served as negative controls (Naïve; white circles). Numbers of live (A) E. coli, (B) Proteus sp., (C) Enterococci, (D) Lactobacilli, (E) Bacteroides/Prevotella spp. as well as the (F) total bacterial load are indicated as colony forming units (CFU) per g luminal content. Numbers of animals harboring the respective bacterial species are given in parentheses and medians indicated. Data shown were pooled from three independent experiments.

Figure 5. Jejunal microbiota composition in Mongolian gerbils 14 months following H. pylori infection.

Fourteen months following oral infection of Mongolian gerbils with H. pylori wildtype strain B8 (B8; black circles) or H. pylori mutant strain lacking cagY (B8ΔcagY; grey circles), the microbiota composition of luminal jejunum contents were quantitatively analyzed by culture as described in Methods. Uninfected age-matched animals served as negative controls (Naïve; white circles). Numbers of live (A) E. coli, (B) Proteus sp., (C) Enterococci, (D) Lactobacilli, (E) Bacteroides/Prevotella spp. as well as the (F) total bacterial load are indicated as colony forming units (CFU) per g luminal content. Numbers of animals harboring the respective bacterial species are given in parentheses and medians indicated. Data shown were pooled from three independent experiments.

Figure 6. Ileal microbiota composition in Mongolian gerbils 14 months following H. pylori infection.

Fourteen months following oral infection of Mongolian gerbils with H. pylori wildtype strain B8 (B8; black circles) or H. pylori mutant strain lacking cagY (B8ΔcagY; grey circles), the microbiota composition of luminal ileum contents were quantitatively analyzed by culture as described in Methods. Uninfected age-matched animals served as negative controls (Naïve; white circles). Numbers of live (A) E. coli, (B) Proteus sp., (C) Enterococci, (D) Lactobacilli, (E) Bacteroides/Prevotella spp. as well as the (F) total bacterial load are indicated as colony forming units (CFU) per g luminal content. Numbers of animals harboring the respective bacterial species are given in parentheses. Medians and significance levels (p-values) determined by Mann-Whitney-U test are indicated. Data shown were pooled from three independent experiments.

Figure 7. Caecal microbiota composition in Mongolian gerbils 14 months following H. pylori infection.

Fourteen months following oral infection of Mongolian Gerbils with H. pylori wildtype strain B8 (B8; black circles) or H. pylori mutant strain lacking cagY (B8ΔcagY; grey circles), the microbiota composition of luminal caecum contents were quantitatively analyzed by culture as described in Methods. Uninfected age-matched animals served as negative controls (Naïve; white circles). Numbers of live (A) E. coli, (B) Proteus sp., (C) Enterococci, (D) Lactobacilli, (E) Bacteroides/Prevotella spp. as well as the (F) total bacterial load are indicated as colony forming units (CFU) per g luminal content. Numbers of animals harboring the respective bacterial species are given in parentheses. Medians and significance levels (p-values) determined by Mann-Whitney-U test are indicated. Data shown were pooled from three independent experiments.

Figure 8. Colonic microbiota composition in Mongolian gerbils 14 months following H. pylori infection by culture.

Fourteen months following oral infection of Mongolian gerbils with H. pylori wildtype strain B8 (B8; black circles) or H. pylori mutant strain lacking cagY (B8ΔcagY; grey circles), the microbiota composition of luminal colon contents were quantitatively analyzed by culture as described in Methods. Uninfected age-matched animals served as negative controls (Naïve; white circles). Numbers of live (A) E. coli, (B) Proteus sp., (C) Enterococci, (D) Lactobacilli, (E) Bacteroides/Prevotella spp. as well as the (F) total bacterial load are indicated as colony forming units (CFU) per g luminal content. Numbers of animals harboring the respective bacterial species are given in parentheses. Medians and significance levels (p-values) determined by Mann-Whitney-U test are indicated. Data shown were pooled from three independent experiments.

Figure 9. Molecular caecum and colon microbiota analysis in Mongolian gerbils 14 months following H. pylori infection.

Fourteen months following oral infection of Mongolian gerbils with H. pylori wildtype strain B8 (Hp B8; right lanes within each panel), the microbiota composition of luminal caecum (left panel) and colon (right panel) contents were subjected to PCR-DGGE analysis of PCR-amplified total bacterial 16S rRNA gene fragments as described in Methods, and compared to uninfected age-matched controls (Naïve; left lanes within each panel). Sequence analysis revealed that indicated DNA bands appearing 14 months following H. pylori B8 infection refer to enterobacteria and Akkermansia spp. Shown DGGE profiles of the bacterial microbiota are representative for three independent experiments.