Intracellular Helicobacter pylori in gastric epithelial progenitors

Abstract

Helicobacter pylori is generally viewed as an extracellular pathogen. We have analyzed the tropism of H. pylori clinical isolates in a gnotobiotic transgenic mouse model of human chronic atrophic gastritis, a preneoplastic condition. These mice lack acid-producing parietal cells and have an amplified population of dividing gastric epithelial progenitors (GEPs) that express NeuAc alpha 2,3Gal beta 1,4-glycans recognized by H. pylori adhesins. Scanning confocal and transmission electron microscopic studies of stomachs that had been colonized for 1 month or 1 year revealed intracellular bacterial collections (IBCs) in a small subset of multi- and oligopotential epithelial progenitors. Transmission electron microscopic and multilabel immunohistochemical analyses disclosed bacteria with several morphotypes, including spiral-shaped, in the cytoplasm and endosomes. Several stages in IBC evolution were documented, from a few solitary bacteria to consolidated populations in dividing and nondividing GEPs, to microorganisms traversing breaches in the GEP plasma cell membrane. IBC formation was not a unique feature of H. pylori strains isolated from patients with chronic atrophic gastritis. The notion that adult mammalian epithelial progenitors can function as a repository for H. pylori broadens the view of host habitats available to this and perhaps other pathogens.

Fig. 1.

Distribution of H. pylori in gnotobiotic tox176 mice. (A and B) Multilabel immunohistochemical study of stomachs from 7-week-old germ-free normal (A) and tox176 (B) mice showing that tox176-mediated parietal cell ablation results in amplification of mitotically active GEPs. Animals were treated with BrdUrd (green) 90 min before death to label cells in S-phase. Dolichos biflorus agglutinin (red) is used to mark parietal cells. Pit cells are tagged with Alexa Fluor 350-conjugated Anguilla anguilla agglutinin (blue), and neck cells are tagged with Alexa Fluor 350- and Alexa Fluor 647-tagged Griffonia simplicifolia II lectin (purple). GEPs produce NeuAcα2,3Galβ1,4 glycans (white after staining with biotinylated MAA and Cy3-labeled streptavidin). (C) Attachment of CAG7:8 (red) to NeuAcα2,3Galβ1,4-positive GEPs (marked green with MAA) in a stomach from a 15-week-old gnotobiotic tox176 mouse killed after 4 weeks of infection. Nuclei are stained blue with bis-benzimide. (D) Frame from a 3D confocal microscopic projection of a focal area of infection in a tox176 stomach showing intracellular bacteria (red, arrows). Cell borders are delineated by using an antibody to E-cadherin (green after treatment with Alexa Fluor 488-tagged donkey anti-rat Ig). To view the 3D projection in its entirety, see Movie 1, which is published as supporting information on the PNAS web site. (Bars, 10 μm.)

Fig. 2.

Confocal microscopic study of CAG7:8 IBCs. Sixteen-week-old gnotobiotic tox176 mice were killed after a 4-week infection with CAG7:8. Sections were processed exactly as in Fig. 1D.(A) A spherical IBC (red). (Insets) A single section stained with antibodies H. pylori (red) and the late endosomal marker, Lamp1 (blue). The results reveal bacterial cells residing within this endosomal compartment. (B) IBC with a dispersed phenotype in a host cell that has developed punctate disruptions of its plasma membrane (the affected cell is also shown in the Inset). (C) CAG7:8 is seen traversing a breach in its host epithelial cell plasma membrane (arrow). 3D reconstructions of serial confocal scans of the sections presented can be found in Movies 2–4, which are published as supporting information on the PNAS web site. [Bars, 10 μm (5 μm in B Inset).]

Fig. 3.

Intracellular collections of H. pylori form in actively dividing GEPs. Six- to 10-week-old tox176 mice were infected for 4 weeks with CAG7:8 and given an i.p. injection of BrdUrd (blue) 1.5 h before death. Cryosections from the zymogenic region of their glandular epithelium were stained with antibodies to H. pylori (red) and E-cadherin (green) and then serially scanned with a confocal microscope at an optical plane thickness of 0.4–0.6 μm. (A and B) S-phase epithelial cells containing small IBCs. Images were taken from 3D reconstructions of serial confocal scans (see Movies 5 and 6, which are published as supporting information on the PNAS web site). A z axis scan of the IBC portrayed in B can be found in Movie 7, which is published as supporting information on the PNAS web site. (C) Conventional photomicrograph of a section containing BrdUrd-positive (blue) and MAA-positive (magenta) GEPs with intracellular CAG7:8 (red). GEP membrane-associated β-catenin appears green. (D) Confocal microscopic image of a MAA-positive (blue) GEP (membranes appear green after staining with antibodies to β-catenin), harboring H. pylori (red). See Movie 8, which is published as supporting information on the PNAS web site, for the corresponding 3D reconstruction. (Bars, 10 μm.)

Fig. 4.

TEM study showing H. pylori strain CAG7:8 within GEPs. (A) Extracellular CAG7:8 in the vicinity of an oligopotential precursor to the pit cell lineage. (B) A spiral-shaped bacterium (closed arrowhead) near a late endosome (open arrowhead) in a preneck cell progenitor. (C) Cross-sectional view of CAG7:8 in the cytoplasm of a preneck cell progenitor surrounded by a clear halo (arrow). (D) Two intracellular H. pylori (arrows) enclosed within a late endosome of a preneck cell. [Bars, 800 nm (A) and 500 nm (B–D).]