Proc Natl Acad Sci U S A 102(14): 5186-5191

Intracellular <em>Helicobacter pylori</em> in gastric epithelial progenitors

^{Center for Genome Sciences and Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63108; and}^{Department of Anatomy, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain 17666, United Arab Emirates}

^{To whom correspondence should be addressed. E-mail:}ude.ltsuw.loocelom@nodrogj.

Edited by Stanley Falkow, Stanford University, Stanford, CA, and approved February 28, 2005

Received 2004 Oct 14

Freely available online through the PNAS open access option.

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 NeuAcα2,3Galβ1,4-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.

Keywords: adult mammalian epithelial progenitors, bacterial pathogenesis, intracellular bacterial communities, gnotobiotic mice, chronic atrophic gastritis

Abstract

The stomachs of more than half of all humans are colonized by Helicobacter pylori. Typically acquired during childhood, this Gram-negative bacterium can persist in the gastric ecosystem throughout the life span of untreated hosts (1). H. pylori is found mainly in the mucous layer of the stomach; at any given moment, only a small fraction appears to adhere to the gastric epithelium (2). H. pylori expresses a number of adhesins that mediate attachment to gastric epithelial glycan receptors, including SabA, which binds to NeuAcα2,3Galβ1,4-containing glycans such as sialyl-Lewis⁽3). These glycans are prominently represented in the stomachs of H. pylori-infected individuals who have developed chronic atrophic gastritis (4), a preneoplastic condition characterized by loss of acid-producing parietal cells (5).

H. pylori has long been viewed as an extracellular bacterium, although the ineffectiveness of non-cell-penetrating antibiotics in eradicating infection in a subset of individuals, and the predominance of a T helper 1 adaptive immune response characteristic of invasive pathogens, suggest the existence of an intracellular population (6, 7). Several electron microscopic studies of gastric biopsy samples from infected individuals have reported intact and degraded forms of the bacterium within epithelial cells (8). Additionally, time-lapse microscopic analyses have documented H. pylori invasion of a gastric adenocarcinoma-derived epithelial cell line (9, 10).

Recently, uropathogenic strains of Escherichia coli, which cause acute and recurrent urinary tract infections, were found to spend part of their life cycle as intracellular bacterial communities within bladder urothelial cells (11). These communities progress through distinct developmental stages, characterized by changes in the growth rate, morphology, and motility of its members. Deconstruction occurs as community members exit the host cell and disperse to other urothelial cells. This mechanism allows a reservoir of quiescent bacteria to be established that helps support persistent infection (12). This discovery raises the question of whether other bacterial pathogens form communities within differentiated host epithelial cells and/or epithelial progenitors.

In this current study, we address this issue using a gnotobiotic transgenic mouse model of persistent H. pylori infection in humans with chronic atrophic gastritis. Colonizing germ-free animals with clinical isolates greatly simplifies analysis of bacterial tropism, because mice normally contain a complex gastric microbiota due to their coprophagy. Our transgenic mice have an engineered ablation of parietal cells, achieved by expressing an attenuated diphtheria toxin A fragment (tox176) under the control of a lineage-specific promoter (Atp4b; ref. 13). Parietal cells were selected for ablation because they function as a guardian of the stem cell niche: they are the only major lineal descendant of the multipotent gastric stem cell that completes its differentiation within the niche; their acid impedes colonization of the niche; and their presence affects the proliferative activity of multi- and oligopotential progenitors (ablation stimulates proliferation and produces a progressive amplification of normally rare NeuAcα2,3Galβ1,4 glycan-positive GEPs; ref. 14 and Fig. 1 A and B). The loss of parietal cells and the increased representation of NeuAcα2,3Galβ1,4 glycans are features that tox176 mice share with humans with chronic atrophic gastritis (14).

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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.)

Below, we report how scanning confocal microscopy, combined with multilabel immunohistochemistry plus transmission EM (TEM), has revealed that a subset of dividing and nondividing gastric epithelial progenitors (GEPs) provides a milieu that supports formation of intracellular collections of H. pylori strains recovered from patients with or without chronic atrophic gastritis. The development of intracellular bacterial collections (IBCs) in adult mammalian epithelial progenitors provides a previously unappreciated view of how H. pylori may persist in some of its hosts, as well as an opportunity to consider how the biological features of these progenitors, revealed from ongoing functional genomics studies, may not only support but also be influenced by IBCs.

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Acknowledgments

We thank Maria Karlsson and David O'Donnell for their assistance with rearing and analyzing gnotobiotic mice, Janaki Guruge for culture-based assays, Jaime Dant for help preparing samples for TEM studies, and Sabrina Wagoner for superb technical assistance. This work was supported by grants from the National Institutes of Health (DK58529 and DK63483).

Acknowledgments

Notes

Author contributions: J.D.O. and J.I.G. designed research; J.D.O. and S.M.K. performed research; J.D.O., S.M.K., and J.I.G. analyzed data; and J.D.O. and J.I.G. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: IBC, intracellular bacterial collection; GEP, gastric epithelial progenitor; MAA, Maackia amurensis agglutinin; TEM, transmission EM; FS/Z, forestomach/zymogenic transition.

Notes

Author contributions: J.D.O. and J.I.G. designed research; J.D.O. and S.M.K. performed research; J.D.O., S.M.K., and J.I.G. analyzed data; and J.D.O. and J.I.G. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: IBC, intracellular bacterial collection; GEP, gastric epithelial progenitor; MAA, Maackia amurensis agglutinin; TEM, transmission EM; FS/Z, forestomach/zymogenic transition.

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