Mutation frequency and biological cost of antibiotic resistance in Helicobacter pylori.

Among the several factors that affect the appearance and spread of acquired antibiotic resistance, the mutation frequency and the biological cost of resistance are of special importance. Measurements of the mutation frequency to rifampicin resistance in Helicobacter pylori strains isolated from dyspeptic patients showed that approximately 1/4 of the isolates had higher mutation frequencies than Enterobacteriaceae mismatch-repair defective mutants. This high mutation frequency could explain why resistance is so frequently acquired during antibiotic treatment of H. pylori infections. Inactivation of the mutS gene had no substantial effect on the mutation frequency, suggesting that MutS-dependent mismatch repair is absent in this bacterium. Furthermore, clarithromycin resistance conferred a biological cost, as measured by a decreased competitive ability of the resistant mutants in mice. In clinical isolates this cost could be reduced, indicating that compensation is a clinically relevant phenomenon that could act to stabilize resistant bacteria in a population.

Genomics and proteomics converge on Helicobacter pylori.

During the past year, a series of studies have provided new perspectives about genetic diversity in Helicobacter pylori. The results illustrate how the current revolution in genomics and proteomics is being used to understand how this organism co-evolves with its host. The approaches should have broad applications to other host-bacterium relationships.

Molecular analysis of commensal host-microbial relationships in the intestine.

Human beings contain complex societies of indigenous microbes, yet little is known about how resident bacteria shape our physiology. We colonized germ-free mice with Bacteroides thetaiotaomicron, a prominent component of the normal mouse and human intestinal microflora. Global intestinal transcriptional responses to colonization were observed with DNA microarrays, and the cellular origins of selected responses were established by laser-capture microdissection. The results reveal that this commensal bacterium modulates expression of genes involved in several important intestinal functions, including nutrient absorption, mucosal barrier fortification, xenobiotic metabolism, angiogenesis, and postnatal intestinal maturation. These findings provide perspectives about the essential nature of the interactions between resident microorganisms and their hosts.

Theoretical and experimental approaches for studying factors defining the Helicobacter pylori-host relationship.

Mathematical modeling has helped develop hypotheses about the role of microbial and host parameters in the initial and subsequent phases of Helicobacter pylori colonization. Transgenic mice have been used to test the hypothesis that the outcome of colonization is influenced by whether bacteria can adhere to available epithelial cell receptors. Complementary use of modeling and experimental approaches should facilitate studies of H. pylori pathogenesis.

Analyzing the molecular foundations of commensalism in the mouse intestine.

We maintain complex societies of nonpathogenic microbes on our mucosal surfaces. Although the stability of this flora is important for human health, very little is known about how its constituents communicate with us to forge stable and mutually advantageous relationships. The vast majority of these indigenous microbes reside in the intestine. Recent studies of a gut commensal, Bacteroides thetaiotaomicron, has revealed a novel signaling pathway that allows the microbe and host to actively collaborate to produce a nutrient foundation that can be used by this bacterium. This pathway illustrates the type of dynamic molecular interactions that help define commensal relationships.

A molecular sensor that allows a gut commensal to control its nutrient foundation in a competitive ecosystem.

Little is known about how members of the indigenous microflora interact with their mammalian hosts to establish mutually beneficial relationships. We have used a gnotobiotic mouse model to show that Bacteroides thetaiotaomicron, a component of the intestinal microflora of mice and humans, uses a repressor, FucR, as a molecular sensor of L-fucose availability. FucR coordinates expression of an operon encoding enzymes in the L-fucose metabolic pathway with expression of another locus that regulates production of fucosylated glycans in intestinal enterocytes. Genetic and biochemical studies indicate that FucR does this by using fucose as an inducer at one locus and as a corepressor at the other locus. Coordinating this commensal's immediate nutritional requirements with production of a host-derived energy source is consistent with its need to enter and persist within a competitive ecosystem.

Helicobacter pylori attaches to NeuAc alpha 2,3Gal beta 1,4 glycoconjugates produced in the stomach of transgenic mice lacking parietal cells.

Helicobacter pylori infection of the human stomach is associated with altered acid secretion, loss of acid-producing parietal cells, and, in some hosts, adenocarcinoma. We have used a transgenic mouse model to study the effects of parietal cell ablation on H. pylori pathogenesis. Ablation results in amplification of the presumptive gastric epithelial stem cell and its immediate committed daughters. The amplified cells produce sialylated oncofetal carbohydrate antigens that function as receptors for H. pylori adhesins. Attachment results in enhanced cellular and humoral immune responses. NeuAc alpha 2,3Gal beta 1,4 glycoconjugates may not only facilitate persistent H. pylori infection in a changing gastric ecosystem, but by promoting interactions with lineage progenitors and/or initiated cells contribute to tumorigenesis in patients with chronic atrophic gastritis.

Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from gnotobiology.

Studying the cross talk between nonpathogenic organisms and their mammalian hosts represents an experimental challenge because these interactions are typically subtle and the microbial societies that associate with mammalian hosts are very complex and dynamic. A large, functionally stable, climax community of microbes is maintained in the murine and human gastrointestinal tracts. This open ecosystem exhibits not only regional differences in the composition of its microbiota but also regional differences in the differentiation programs of its epithelial cells and in the spatial distribution of its component immune cells. A key experimental strategy for determining whether "nonpathogenic" microorganisms actively create their own regional habitats in this ecosystem is to define cellular function in germ-free animals and then evaluate the effects of adding single or several microbial species. This review focuses on how gnotobiotics-the study of germ-free animals-has been and needs to be used to examine how the gastrointestinal ecosystem is created and maintained. Areas discussed include the generation of simplified ecosystems by using genetically manipulatable microbes and hosts to determine whether components of the microbiota actively regulate epithelial differentiation to create niches for themselves and for other organisms; the ways in which gnotobiology can help reveal collaborative interactions among the microbiota, epithelium, and mucosal immune system; and the ways in which gnotobiology is and will be useful for identifying host and microbial factors that define the continuum between nonpathogenic and pathogenic. A series of tests of microbial contributions to several pathologic states, using germ-free and ex-germ-free mice, are proposed.

Host-microbial symbiosis in the mammalian intestine: exploring an internal ecosystem.

The mammalian intestine contains a complex, dynamic, and spatially diversified society of nonpathogenic bacteria. Very little is known about the factors that help establish host-microbial symbiosis in this open ecosystem. By introducing single genetically manipulatable components of the microflora into germfree mice, simplified model systems have been created that will allow conversations between host and microbe to be heard and understood. Other paradigms of host-microbial symbiosis suggest that these interactions will involve an exchange of biochemical signals between host and symbionts as well as among the bacteria themselves. The integration of molecular microbiology, cell biology, and gnotobiology should provide new insights about how we adapt to a microbial world and reveal the roles played by our indigenous, 'nonpathogenic' flora.

Epithelial attachment alters the outcome of Helicobacter pylori infection.

Genetically defined in vivo models are needed to assess the importance of target cell attachment in bacterial pathogenesis. Gastric colonization by Helicobacter pylori in human populations is common and persistent, and has various outcomes including peptic ulcers and cancer. The impact of attachment on the course of infection was examined in transgenic mice expressing a human receptor for H. pylori in their gastric epithelium. Persistent infection by a clinical isolate occurred at comparable microbial densities in transgenic and nontransgenic littermates. However, microbial attachment in transgenic mice resulted in production of autoantibodies to Lewisx carbohydrate epitopes shared by bacteria and acid-secreting parietal cells, chronic gastritis, and parietal cell loss. This model should help identify bacterial and host genes that produce attachment-related pathology.

Methods for the Identification of H. pylori Host Receptors.

Bacterial attachment to host receptors is a prerequisite for colonization of epithelial cell surfaces, in particular, continuously renewing mucosal surfaces, such as the gastrointestinal tract. Microbes express adhesion molecules for interactions with eukaryotic cell surface proteins or glycoconjugates, such as glycoproteins and glycolipids (1). The combination of high receptor specificity (2) and restricted receptor distribution will target bacteria to specific tissues, i.e., cell populations. This is referred to as tissue tropism and partly determines the niche a bacterium is able to occupy. In addition, competition between bacterial species for space and nutrients selects for bacteria able to colonize specific niches. Bacteria unable to adhere to the epithelial cells and mucus lining will be exposed to the local nonspecific host defense mechanisms (such as peristalsis and turnover of the epithelial cell populations and the mucus layer) and eventually removed. The biological relevance of adherence as an initial step in the infectious process has focused interest to the structures involved in these processes. Bacterial adhesins and host receptors are both potential targets for novel antimicrobial drug design (3). Antimicrobial agents could be chemically coupled to soluble high-affinity receptor analogs and kill pathogens, such as H. pylori, once they are targeted by the complex. Soluble receptor analogs would competitively interfere with bacterial attachment, utilizing the same mechanism as naturally occurring scavenger molecules in human secretions, such as milk and saliva. Receptor analogs could be developed for high-affinity interactions and would thereby be efficient inhibitors at low concentrations. Both drug targeting and competitive adhesion inhibition receptor analogs could exhibit a higher specificity for the pathogenic microbes, circumventing the negative effects of broad spectrum antibiotics.

A model of host-microbial interactions in an open mammalian ecosystem.

The maintenance and significance of the complex populations of microbes present in the mammalian intestine are poorly understood. Comparison of conventionally housed and germ-free NMRI mice revealed that production of fucosylated glycoconjugates and an alpha1, 2-fucosyltransferase messenger RNA in the small-intestinal epithelium requires the normal microflora. Colonization of germ-free mice with Bacteroides thetaiotaomicron, a component of this flora, restored the fucosylation program, whereas an isogenic strain carrying a transposon insertion that disrupts its ability to use L-fucose as a carbon source did not. Simplified models such as this should aid the study of open microbial ecosystems.

Genetic engineering of carbohydrate biosynthetic pathways in transgenic mice demonstrates cell cycle-associated regulation of glycoconjugate production in small intestinal epithelial cells.

Proliferation, migration-associated differentiation, and cell death occur continuously and in a spatially well-organized fashion along the crypt-villus axis of the mouse small intestine, making it an attractive system for studying how these processes are regulated and interrelated. A pathway for producing glycoconjugates was engineered in adult FVB/N transgenic mice by expressing a human alpha 1,3/4-fucosyltransferase (alpha 1,3/4-FT; EC 2.4.1.65) along the length of this crypt-villus axis. The alpha 1,3/4-FT can use lacto-N-tetraose or lacto-neo-N-tetraose core chains to generate Lewis (Le) blood group antigens Le(a) or Le(x), respectively, and H type 1 or H type 2 core chains to produce Leb and Le(y). Single- and multilabel immunohistochemical studies revealed that expression of the alpha 1,3/4-FT results in production of Le(a) and Leb antigens in both undifferentiated proliferated crypt cells and in differentiated postmitotic villus-associated epithelial cells. In contrast, Le(x) antigens were restricted to crypt cells. Villus enterocytes can be induced to reenter the cell cycle by expression of simian virus 40 tumor antigen under the control of a promoter that only functions in differentiated members of this lineage. Bitransgenic animals, generated from a cross of FVB/N alpha 1,3/4-FT with FVB/N simian virus 40 tumor antigen mice, expand the range of Le(x) expression to include villus-associated enterocytes that have reentered the cell cycle. Thus, the fucosylations unveil a proliferation-dependent switch in oligosaccharide production, as defined by a monoclonal antibody specific for the Le(x) epitope. These findings show that genetic engineering of oligosaccharide biosynthetic pathways can be used to define markers for entry into, or progression through, the cell cycle and to identify changes in endogenous carbohydrate metabolism that occur when proliferative status is altered in a manner that is not deleterious to the system under study.

Expression of a human alpha-1,3/4-fucosyltransferase in the pit cell lineage of FVB/N mouse stomach results in production of Leb-containing glycoconjugates: a potential transgenic mouse model for studying Helicobacter pylori infection.

Helicobacter pylori is a human pathogen associated with the development of gastric and duodenal ulcers and gastric adenocarcinoma. To test the hypothesis that the human Lewis(b) blood group antigen (Le(b)) functions as a receptor for the bacteria's adhesins and mediates its attachment to gastric pit and surface mucous cells, a human alpha-1,3/4-fucosyltransferase was expressed in these cell lineages in FVB/N transgenic mice. The fucosyltransferase directed production of the Leb epitope without any apparent effect on the proliferation and differentiation programs of this lineage. Moreover, clinical isolates of H. pylori bound to these cells in transgenic mice but not in their normal littermates. Binding was blocked by pretreatment of the bacteria with soluble Le(b). This mouse model could be useful for examining the molecular pathogenesis of diseases caused by H. pylori infection. Creating novel pathways for production of specific oligosaccharides in selected cell lineages of transgenic animals represents an approach for examining the role of complex carbohydrates in regulating cellular differentiation and host-microbe interactions.