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.

Helicobacter pylori is associated with development of gastritis, gastric ulcers, and adenocarcinomas in humans. The Lewis(b) (Le(b)) blood group antigen mediates H. pylori attachment to human gastric mucosa. Soluble glycoproteins presenting the Leb antigen or antibodies to the Leb antigen inhibited bacterial binding. Gastric tissue lacking Leb expression did not bind H. pylori. Bacteria did not bind to Leb antigen substituted with a terminal GalNAc alpha 1-3 residue (blood group A determinant), suggesting that the availability of H. pylori receptors might be reduced in individuals of blood group A and B phenotypes, as compared with blood group O individuals.

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.

No abstract

A molecular sensor that allows a gut commensal to control its nutrient foundation in a competitive ecosystem Communicated by Stuart A. Kornfeld, Washington University School of Medicine, St. Louis, MO, June 21, 1999 (received for review April 21, 1999 ABSTRACTLittle 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.Humans must adapt to life in a microbial world. As adults, the number of microbes associated with our mucosal surfaces exceeds our total number of somatic and germ cells by more than an order of magnitude (1). The gastrointestinal tract is home to our most complex and populous society of microbes. The composition of the microflora varies along the length of the gut and during the life of the host. The microflora provides a functional barrier to colonization by pathogens (2, 3), plays an important role in normal nutrition and metabolism, and is thought to help shape development of the intestine's mucosal immune system (4). Despite its importance, almost nothing is known about the molecular mechanisms that allow components of the microflora to interact with their hosts so as to establish relationships that are advantageous to both. Understanding such relationships is important in considering the origins of opportunistic infections, various immunopathologic states, and the propagation of antibiotic-resistant organisms (2-8).Assembly of the gut microflora commences at birth. When space and nutrients are not limiting, commensals with high division rates predominate. As the population increases and nutrients are depleted, niches become occupied with more specialized species (4). One conceptualization of how this process may be orchestrated is that the distribution of early-colonizing gut commensals is defined by a preformed nutrient foundation that has been laid down by the host. The ability of other commensals to enter occupied habitats would depend on their ability to utilize these nutrient substrates more efficiently and/or to engineer alterations in the nutrient reservoir to better suit their own metabolic capacities. In a mutualistic relationship, coordination of microbial nutrient utilization and host nutrient production should be achieved at a minimal energetic cost to mi...

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 Ϸ1͞4 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.

SUMMARY 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.

Paneth cells represent one of the four major epithellal llneages In the mouse small Intestine. It is the only llneage that migrates downward from the stem-cell zone located in the lower portion of the crypt of Lieberkuhn to the crypt base. Mature Paneth cells release growth factors, digestive enzymes, and antimicrobial peptides from their apical secretor granules. Some of these factors may affect the crypt stem cell, its transit-cell descendants, differentiating villusassociated epithelal lages, and/or the gut microflora. We used single and multllabel immunocytochemical methods to study Paneth cell days (2) before being removed by phagocytosis (3). A variety offunctions have been attributed to Paneth cells. These functions include modulation of the intestinal microflora and maintenance of mucosal defense barriers through production of antimicrobial peptides (cryptdins, lysozyme). The location of Paneth cells at the crypt base, combined with their production of growth factors and other regulatory molecules (4-6), suggests that they may also contribute to the stem-cell niche through short-circuit paracrine loops and/or regulate the proliferation and differentiation programs of other cell lineages.One way of examining Paneth cell function is to study the differentiation program of this lineage during gut development. Morphogenesis of the mouse small intestinal epithelium is not completed until the end of the third postnatal week. A pseudostratified endoderm undergoes conversion to a monolayer overlying nascent villi in a morphologic "wave" of cytodifferentiation. This wave moves from the duodenum to the ileum from embryonic day 15 (E15) through E19. Crypts form from an intervillus epithelium during the first two postnatal weeks (7). Crypt number increases rapidly between the second and third postnatal week through crypt fission (7). Analyses of mouse aggregation chimeras indicate that the perinatal mouse gut contains a polyclonal intervillus epithelium, supplied by stem cells with multiple genotypes (8). A process of cell selection occurs during crypt morphogenesis, yielding monoclonal crypts by postnatal day 14 (P14). An adult mouse crypt appears to be supplied either by a single slowly dividing master stem cell and its more rapidly cycling transit-cell descendants (the stem-cell pedigree concept) or by several equivalent stem cells with similar cycling times and probabilities for self-maintenance (9).We have examined the Paneth cell lineage from E15 to P42 in germ-free, ex-germ-free, and conventional mice and in intestinal isografts. The results of these studies form a basis for interpreting the significance of the pattern of expression of a mouse cryptdin 2/human growth hormone (hGH) transgene in several pedigrees of mice.MATERIALS AND METHODS Animals. FVB/N mice were caged in microisolators and given autoclaved chow (Ralston Purina) ad libitum. Germfree NMRI mice (10) were maintained in gnotobiotic isolator cages. Conventional NMRI animals were housed in a nonsterile but pathogen-free environment. Ex-...