Fig 1.
Bacterial populations within individual glands are heterogeneous.
(A) Recovered CFU per gram of stomach at 2 weeks post-infection with Hp GFP or Hp tdT (eight mice per group). Data represent two independent experiments. Gray dotted line, limit of detection; black bars, median. Relevant data values are included in S1 Data. (B) Left, cartoon schematic of the gastric epithelium. The gastric epithelium consists of a single layer of columnar epithelial cells that is organized into repeated, invaginated units called glands. The epithelium is covered with a protective mucus layer (gray) that shields the underlying cells from the acidic lumen. H. pylori (green) reside within the surface mucus in a free-swimming state, or directly attached to the surface epithelium and the epithelium deep in the gastric glands. The gland-associated bacteria are concentrated in the mid-glandular region, which contains gastric epithelial precursor cells. The mid-glandular region is indicated by the inset, which zooms into a 3D confocal image of H. pylori within a gland. Middle, 3D confocal images of gland-associated bacteria in Hp GFP–or Hp tdT–infected mice from panel A. Nuclei (blue), F-actin (white), GFP (green), tdT (red); scale bar, 10 μm. Right, 113 total colonized glands from three mice per group analyzed to determine the number of bacteria per gland. Black bars, median; error bars, interquartile range. Relevant data values are included in S1 Data. (C) Total CFU/g recovered from mice co-infected with Hp GFP and Hp tdT at 2 weeks post-infection (12 mice). Black dashed lines connect Hp GFP and Hp tdT counts recovered from the same mouse. Data represent three independent experiments. Gray dotted line, limit of detection. Relevant data values are included in S1 Data. (D) Three-dimensional confocal images of individual gastric glands from co-infected mice from panel C. Nuclei (blue), F-actin (white), GFP (green), tdT (red); scale bar, 10 μm. (E) Percent Hp GFP bacteria per gland (1,050 colonized glands analyzed from seven co-infected mice). Each dot represents a colonized gland. (F) Data from panel E plotted as a histogram. The x-axis represents percentage of Hp GFP bacteria per colonized gland, separated into 12 bins; the y-axis represents percentage of analyzed glands that are present in each bin. Relevant data values are included in S1 Data. Statistics: p-values obtained using Mann–Whitney test (panels A,B) or Wilcoxon signed-rank test (panel C). CFU, colony-forming unit; GFP, green fluorescent protein; NS, no significance; tdT, tdTomato; 3D, three-dimensional.
Fig 2.
H. pylori gland populations are organized into patches within the mucosa.
(A) Schematic of stomach sectioning. Mouse stomachs were opened along the lesser curvature and flattened. The corpus, transition zone (tz, solid black line), and antrum and the lesser (magenta line) and greater (blue line) curvatures are identified. Longitudinal sections were taken along the greater curvature and include all gastric regions. (B) Image of longitudinal section from 2-week co-infected mouse. x = 0 is the junction between the antrum and transition zone. For clarity, top image shows bacterial signal without nuclei and F-actin overlaid. Yellow dots mark the top and base of each gland (scale bar, 150 μm). Boxes i, ii, and iii are magnified in the upper right corner of the figure (scale bar, 50 μm). (C) Gland height (black line) and bacteria per gland (green and red bars) are mapped according to their location within the longitudinal section in panel B. Boxes i, ii, and iii correspond to boxed regions in panel B. Total CFU/g of each strain recovered from this mouse indicated. Relevant data values included in S1 Data. CFU, colony-forming unit; GFP, green fluorescent protein; tdT, tdTomato; tz, transition zone.
Fig 3.
PACT reveals that gland-associated H. pylori form stable population islands.
(A) Mouse stomach processed through PACT. Before hydrogel embedding and clearing via SDS, the stomach is opaque. After, it is optically transparent. Scale box, 4.9 × 5.3 mm. (B, C) Images of glands from mice co-infected with Hp GFP and Hp tdT, shown from a top-down view. Images from mouse infected as adult at 2 weeks post-infection (B) or infected as neonate at 1 month post-infection (C). Scale bar, 1 mm. (D) Percentage Hp GFP bacteria per gland in co-infected mice at 1 month post-infection (300 colonized glands analyzed from three mice, includes one infected as adult and two infected as neonates). Relevant data values are included in S1 Data. (E) Representative examples of computer simulations of gland colonization and spread. Ten randomly selected glands were colonized by Hp GFP and another set of ten by Hp tdT in a field of 20,000 available glands, and bacteria were allowed to spread over time. Green, Hp GFP–occupied glands; red, Hp tdT–occupied glands; yellow, 50:50 co-occupied glands; black, unoccupied. Three different colonization scenarios were simulated, including or excluding parameters of adjacent spread or gland resistance. GFP, green fluorescent protein; PACT, passive CLARITY technique; SDS, sodium dodecyl sulfate; tdT, tdTomato; TZ, transition zone.
Fig 4.
H. pylori exerts intraspecies colonization resistance early in infection.
(A) Sequential infection schematic. Mice were infected with the initial strain for 1 week, challenged with the opposite-colored isogenic strain, and stomachs were harvested after 1 additional week for CFU enumeration of both initial and challenge strains and visualization of gland colonization by microscopy. As an example, Hp GFP was depicted as the initial strain and Hp tdT as the challenge strain in this diagram. However, the reverse experiment, in which Hp tdT was the initial strain and Hp GFP was the challenge strain, was also conducted. (B) Total CFU/g recovered from sequentially infected mice as depicted in panel A. Both iterations in which Hp GFP was either the initial strain (left) or challenge strain (right) are shown (five mice per group). Black dashed lines connect Hp GFP and Hp tdT counts recovered from the same mouse at 2 weeks post-inoculation of the initial strain. Gray dotted line, limit of detection. Relevant data values are included in S1 Data. (C) Three-dimensional confocal images of bacteria in glands from sequentially infected mice in panel B. GFP (green), tdT (red); scale bar, 30 μm. (D) Total CFU/g recovered from mice pre-colonized with Hp GFP for different time points (1, 3, 5, or 7 days) prior to challenge with Hp tdT (5–6 mice per group). Stomachs were harvested at 2 weeks post-inoculation of the initial strain for CFU enumeration of both initial and challenge strains. Data represent two independent experiments. Black dashed lines connect Hp GFP and Hp tdT counts recovered from the same mouse. Gray dotted line, limit of detection. Relevant data values are included in S1 Data. (E) Three-dimensional confocal images of bacteria in glands from sequentially infected mice in panel D, 2 weeks post-inoculation of the initial strain. GFP (green), tdT (red); scale bar, 30 μm. CFU, colony-forming unit; GFP, green fluorescent protein; tdT, tdTomato.
Fig 5.
Knockdown of pre-established gland population with antibiotics abolishes colonization resistance.
(A) Left, total CFU/g recovered from mice colonized with Hp GFP for 1 week (pre-amoxicillin), and then treated with amoxicillin for 3 days (post-amoxicillin) (11 mice per group). Gray dotted line, limit of detection; black bars, median. Data represent three independent experiments. Statistics: p-value obtained using Mann–Whitney test. ****p < 0.0001. Right, magnified images of regions indicated by insets from panel B. Scale bar, 50 μm. Relevant data values are included in S1 Data. (B) Images of longitudinal stomach sections from mice in panel A. Regions indicated by insets are magnified above. Percentage of antral and transition zone glands colonized indicated for each individual mouse. Scale bar, 75 μm. (C) Total CFU/g recovered from mice pre-colonized with Hp GFP for 1 week, given water or water infused with amoxicillin for 3 days, and challenged with Hp tdT. After 1 additional week, stomachs were harvested for CFU enumeration of the initial and challenge strains (10 mice per group). Black dashed lines connect Hp GFP and Hp tdT counts recovered from the same mouse. Gray dotted line, limit of detection. Data represent two independent experiments. Relevant data values are included in S1 Data. (D) Three-dimensional confocal images of bacteria in glands from sequentially infected mice in panel C. GFP (green), tdT (red). Scale bar, 30 μm. CFU, colony-forming unit; GFP, green fluorescent protein; tdT, tdTomato.
Fig 6.
A bacterial mutant that does not colonize the antral glands cannot exert colonization resistance.
(A) Left, total CFU/g recovered from mice infected with Hp GFP ΔchePep for 1 week (three mice). Gray dotted line, limit of detection; black bars, geometric mean. Right, Three-dimensional confocal images of antral or transition zone glands from infected mice. The transition zone is characterized by the presence of parietal cells in the bottom third of gastric glands (arrows). Nuclei (blue), F-actin (white), H. pylori (green); scale bar, 30 μm. Relevant data values are included in S1 Data. (B) Total CFU/g recovered from mice pre-colonized with Hp GFP ΔchePep for 1 week and then challenged with Hp tdT. Stomachs were harvested at 2 weeks post-inoculation of the initial strain for CFU enumeration of initial and challenge strains (six mice). Black dashed lines connect Hp GFP ΔchePep and Hp tdT counts recovered from the same mouse. Gray dotted line, limit of detection. Data represent two independent experiments. Relevant data values are included in S1 Data. (C) Mapping of location and number of gland-associated bacteria across a longitudinal stomach section of a mouse from panel B. Gland height (black line) and bacteria per gland (green or red bars) are plotted. x = 0 is the junction between the antrum and transition zone. Total CFU/g for each strain recovered from this mouse: Hp GFP ΔchePep = 5.36 × 105 CFU/g, Hp tdT = 3.87 × 106 CFU/g. Relevant data values are included in S1 Data. (D) Three-dimensional confocal images of antral or transition zone glands from the longitudinal section analyzed in panel C. GFP (green), tdT (red); scale bar, 30 μm. CFU, colony-forming unit; GFP, green fluorescent protein; tdT, tdTomato.
Fig 7.
Host T cells impact the density of gland-associated bacteria.
(A) Total CFU/g recovered from WT or TCRβ/δ−/− (TCR KO) mice infected with H. pylori PMSS1 WT for 8 weeks (8–22 mice per group). Gray dotted line, limit of detection; red bars, median. Data represent two independent experiments. Relevant data values are included in S1 Data. (B) Bacteria per gland in antrum and transition zone of WT or TCR KO mice at 8 weeks post-infection. A total of 200–300 glands were analyzed per mouse for both WT and TCR KO groups (five mice per group). Red bars, median. Relevant data values are included in S1 Data. (C) Percentage of colonized glands in the antrum and transition zone of WT or TCR KO mice at 8 weeks post-infection. Bars, median; error bars, interquartile range. Relevant data values are included in S1 Data. (D) Images of antrum of WT or TCR KO mice at 8 weeks post-infection. Scale bar, 80 μm. Statistics: p-values obtained using Mann–Whitney test (panels A–C). **p < 0.01, ***p < 0.001, ****p < 0.0001. CFU, colony-forming unit; KO, knockout; TCR, T-cell receptor; WT, wild-type.