Manuel R. Amieva

Host-bacterial interactions in Helicobacter pylori infection.

Helicobacter pylori are spiral-shaped gram-negative bacteria with polar flagella that live near the surface of the human gastric mucosa. They have evolved intricate mechanisms to avoid the bactericidal acid in the gastric lumen and to survive near, to attach to, and to communicate with the human gastric epithelium and host immune system. This interaction sometimes results in severe gastric pathology. H pylori infection is the strongest known risk factor for the development of gastroduodenal ulcers, with infection being present in 60%-80% of gastric and 95% of duodenal ulcers.(1)H pylori is also the first bacterium to be classified as a definite carcinogen by the World Health Organizations International Agency for Research on Cancer because of its epidemiologic relationship to gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma.(2) In the last 25 years, since H pylori was first described and cultured, a complete paradigm shift has occurred in our clinical approach to these gastric diseases, and more than 20,000 scientific publications have appeared on the subject. From the medical point of view, H pylori is a formidable pathogen responsible for much morbidity and mortality worldwide. However, H pylori infection occurs in approximately half of the world population, with disease being an exception rather than the rule. Understanding how this organism interacts with its host is essential for formulating an intelligent strategy for dealing with its most important clinical consequences. This review offers an insight into H pylori host-bacterial interactions.

The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations

The small intestine epithelium undergoes rapid and continuous regeneration supported by crypt intestinal stem cells (ISCs). Bmi1 and Lgr5 have been independently identified to mark long-lived multipotent ISCs by lineage tracing in mice; however, the functional distinctions between these two populations remain undefined. Here, we demonstrate that Bmi1 and Lgr5 mark two functionally distinct ISCs in vivo. Lgr5 marks mitotically active ISCs that exhibit exquisite sensitivity to canonical Wnt modulation, contribute robustly to homeostatic regeneration, and are quantitatively ablated by irradiation. In contrast, Bmi1 marks quiescent ISCs that are insensitive to Wnt perturbations, contribute weakly to homeostatic regeneration, and are resistant to high-dose radiation injury. After irradiation, however, the normally quiescent Bmi1+ ISCs dramatically proliferate to clonally repopulate multiple contiguous crypts and villi. Clonogenic culture of isolated single Bmi1+ ISCs yields long-lived self-renewing spheroids of intestinal epithelium that produce Lgr5-expressing cells, thereby establishing a lineage relationship between these two populations in vitro. Taken together, these data provide direct evidence that Bmi1 marks quiescent, injury-inducible reserve ISCs that exhibit striking functional distinctions from Lgr5+ ISCs and support a model whereby distinct ISC populations facilitate homeostatic vs. injury-induced regeneration.

Helicobacter pylori enter and survive within multivesicular vacuoles of epithelial cells

Although intracellular Helicobacter pylori have been described in biopsy specimens and in cultured epithelial cells, the fate of these bacteria is unknown. Using differential interference contrast (DIC) video and immunofluorescence microscopy, we document that a proportion of cell‐associated H. pylori enter large cytoplasmic vacuoles, where they remain viable and motile and can survive lethal concentrations of extracellular gentamicin. Entry into vacuoles occurs in multiple epithelial cell lines including AGS gastric adenocarcinoma, Caco‐2 colon adenocarcinoma and MDCK kidney cell line, and depends on the actin cytoskeleton. Time‐lapse microscopy over several hours was used to follow the movement of live H. pylori within vacuoles of a single cell. Pulsed, extracellular gentamicin treatments show that the half‐life of intravacuolar bacteria is on the order of 24 h. Viable H. pylori repopulate the extracellular environment in parallel with the disappearance of intravacuolar bacteria, suggesting release from the intravacuolar niche. Using electron microscopy and live fluorescent staining with endosomal dyes, we observe that H. pylori‐containing vacuoles are similar in morphology to late endosomal multivesicular bodies. VacA is not required for these events, as isogenic vacA– mutants still enter and survive within the intravacuolar niche. The exploitation of an intravacuolar niche is a new aspect of the biological life cycle of H. pylori that could explain the difficulties in eradicating this infection.

Tolerance rather than immunity protects from Helicobacter pylori-induced gastric preneoplasia.

BACKGROUND & AIMS Chronic infection with the bacterial pathogen Helicobacter pylori causes gastric disorders, ranging from chronic gastritis to gastric adenocarcinoma. Only a subset of infected persons will develop overt disease; most remains asymptomatic despite lifelong colonization. This study aims to elucidate the differential susceptibility to H pylori that is found both across and within populations. METHODS We have established a C57BL/6 mouse model of H pylori infection with a strain that is capable of delivering the virulence factor cytotoxin-associated gene A (CagA) into host cells through the activity of a Cag-pathogenicity island-encoded type IV secretion system. RESULTS Mice infected at 5-6 weeks of age with CagA(+)H pylori rapidly develop gastritis, gastric atrophy, epithelial hyperplasia, and metaplasia in a type IV secretion system-dependent manner. In contrast, mice infected during the neonatal period with the same strain are protected from preneoplastic lesions. Their protection results from the development of H pylori-specific peripheral immunologic tolerance, which requires transforming growth factor-β signaling and is mediated by long-lived, inducible regulatory T cells, and which controls the local CD4(+) T-cell responses that trigger premalignant transformation. Tolerance to H pylori develops in the neonatal period because of a biased ratio of T-regulatory to T-effector cells and is favored by prolonged low-dose exposure to antigen. CONCLUSIONS Using a novel CagA(+)H pylori infection model, we report here that the development of tolerance to H pylori protects from gastric cancer precursor lesions. The age at initial infection may thus account for the differential susceptibility of infected persons to H pylori-associated disease manifestations.

Bone morphogenetic protein 2 induces pulmonary angiogenesis via Wnt–β-catenin and Wnt–RhoA–Rac1 pathways

Mutations in bone morphogenetic protein (BMP) receptor II (BMPRII) are associated with pulmonary artery endothelial cell (PAEC) apoptosis and the loss of small vessels seen in idiopathic pulmonary arterial hypertension. Given the low penetrance of BMPRII mutations, abnormalities in other converging signaling pathways may be necessary for disease development. We hypothesized that BMPRII supports normal PAEC function by recruiting Wingless (Wnt) signaling pathways to promote proliferation, survival, and motility. In this study, we report that BMP-2, via BMPRII-mediated inhibition of GSK3-β, induces β-catenin (β-C) accumulation and transcriptional activity necessary for PAEC survival and proliferation. At the same time, BMP-2 mediates phosphorylated Smad1 (pSmad1) or, with loss of BMPRII, pSmad3-dependent recruitment of Disheveled (Dvl) to promote RhoA–Rac1 signaling necessary for motility. Finally, using an angiogenesis assay in severe combined immunodeficient mice, we demonstrate that both β-C– and Dvl-mediated RhoA–Rac1 activation are necessary for vascular growth in vivo. These findings suggest that the recruitment of both canonical and noncanonical Wnt pathways is required in BMP-2–mediated angiogenesis.

Listeria monocytogenes invades the epithelial junctions at sites of cell extrusion.

Listeria monocytogenes causes invasive disease by crossing the intestinal epithelial barrier. This process depends on the interaction between the bacterial surface protein Internalin A and the host protein E-cadherin, located below the epithelial tight junctions at the lateral cell-to-cell contacts. We used polarized MDCK cells as a model epithelium to determine how L. monocytogenes breaches the tight junctions to gain access to this basolateral receptor protein. We determined that L. monocytogenes does not actively disrupt the tight junctions, but finds E-cadherin at a morphologically distinct subset of intercellular junctions. We identified these sites as naturally occurring regions where single senescent cells are expelled and detached from the epithelium by extrusion. The surrounding cells reorganize to form a multicellular junction that maintains epithelial continuity. We found that E-cadherin is transiently exposed to the lumenal surface at multicellular junctions during and after cell extrusion, and that L. monocytogenes takes advantage of junctional remodeling to adhere to and subsequently invade the epithelium. In intact epithelial monolayers, an anti-E-cadherin antibody specifically decorates multicellular junctions and blocks L. monocytogenes adhesion. Furthermore, an L. monocytogenes mutant in the Internalin A gene is completely deficient in attachment to the epithelial apical surface and is unable to invade. We hypothesized that L. monocytogenes utilizes analogous extrusion sites for epithelial invasion in vivo. By infecting rabbit ileal loops, we found that the junctions at the cell extrusion zone of villus tips are the specific target for L. monocytogenes adhesion and invasion. Thus, L. monocytogenes exploits the dynamic nature of epithelial renewal and junctional remodeling to breach the intestinal barrier.

The Complete Genome Sequence of Helicobacter pylori Strain G27

Helicobacter pylori is a gram-negative pathogen that colonizes the stomachs of over half the worlds population and causes a spectrum of gastric diseases including gastritis, ulcers, and gastric carcinoma. The H. pylori species exhibits unusually high levels of genetic variation between strains. Here we announce the complete genome sequence of H. pylori strain G27, which has been used extensively in H. pylori research.

Hypoxia increases human keratinocyte motility on connective tissue.

Re-epithelialization of skin wounds depends upon the migration of keratinocytes from the cut margins of the wound and is enhanced when human keratinocytes are covered with occlusive dressings that induce hypoxia. In this study, two independent migration assays were used to compare cellular motility on connective tissue components under normoxic or hypoxic conditions. Human keratinocytes apposed to collagens or fibronectin exhibited increased motility when subjected to hypoxic (0.2 or 2% oxygen) conditions compared with normoxic (9 or 20% oxygen) conditions. When compared with normoxic cells, hypoxic keratinocytes exhibited increased expression and redistribution of the lamellipodia-associated proteins (ezrin, radixin, and moesin). Furthermore, hypoxic keratinocytes demonstrated decreased secretion of laminin-5, a laminin isoform known to inhibit keratinocyte motility. Hypoxia did not alter the number of integrin receptors on the cell surface, but did induce enhanced secretion of the 92-kD type IV collagenase. These data demonstrate that hypoxia promotes human keratinocyte motility on connective tissue. Hypoxia-driven motility is associated with increased expression of lamellipodia proteins, increased expression of collagenase and decreased expression of laminin-5, the locomotion brake for keratinocytes.

Pathobiology of Helicobacter pylori–Induced Gastric Cancer

Colonization of the human stomach by Helicobacter pylori and its role in causing gastric cancer is one of the richest examples of a complex relationship among human cells, microbes, and their environment. It is also a puzzle of enormous medical importance given the incidence and lethality of gastric cancer worldwide. We review recent findings that have changed how we view these relationships and affected the direction of gastric cancer research. For example, recent data have indicated that subtle mismatches between host and microbe genetic traits greatly affect the risk of gastric cancer. The ability of H pylori and its oncoprotein CagA to reprogram epithelial cells and activate properties of stemness show the sophisticated relationship between H pylori and progenitor cells in the gastric mucosa. The observation that cell-associated H pylori can colonize the gastric glands and directly affect precursor and stem cells supports these observations. The ability to mimic these interactions in human gastric organoid cultures as well as animal models will allow investigators to more fully unravel the extent of H pylori control on the renewing gastric epithelium. Finally, our realization that external environmental factors, such as dietary components and essential micronutrients, as well as the gastrointestinal microbiota, can change the balance between H pyloris activity as a commensal or a pathogen has provided direction to studies aimed at defining the full carcinogenic potential of this organism.

Quantitative Imaging of Gut Microbiota Spatial Organization

Genomic technologies have significantly advanced our understanding of the composition and diversity of host-associated microbial populations. However, their spatial organization and functional interactions relative to the host have been more challenging to study. Here we present a pipeline for the assessment of intestinal microbiota localization within immunofluorescence images of fixed gut cross-sections that includes a flexible software package, BacSpace, for high-throughput quantification of microbial organization. Applying this pipeline to gnotobiotic and human microbiota-colonized mice, we demonstrate that elimination of microbiota-accessible carbohydrates (MACs) from the diet results in thinner mucus in the distal colon, increased proximity of microbes to the epithelium, and heightened expression of the inflammatory marker REG3β. Measurements of microbe-microbe proximity reveal that a MAC-deficient diet alters monophyletic spatial clustering. Furthermore, we quantify the invasion of Helicobacter pylori into the glands of the mouse stomach relative to host mitotic progenitor cells, illustrating the generalizability of this approach.