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Microbial Communities of the Gut

OVERVIEW

The gastrointestinal tract represents an important and challenging system for exploring how microbial communities become established within their hosts, how their members maintain stable ecological niches, and how these dynamics relate to host health and disease. The complex, dynamic, and spatially diversified microbial community of the human gut is believed to be composed of at least 1013 microorganisms, including more than 800 species of bacteria (most of which have not yet been successfully cultured in the laboratory), numerous viral species including bacteriophages, archaea (e.g., methanogens), and eukaryotes (e.g., helminths and protozoa). The collective genome of the microbiota in the human gut is approximately one hundred-fold larger than that of its host. Therefore, as Bäckhed et al. (2005) state in their contribution to this chapter, “It seems appropriate to view ourselves as a composite of many species and our genetic landscape as an amalgam of genes embedded in our Homo sapiens genome and in the genomes of our affiliated microbial partners.”

The first paper in this chapter, contributed by workshop presenter Karen Guillemin, focuses on the establishment of the gut microbiota (in humans, during the early days of infancy), its influence on host development (e.g., immunity), and the mechanisms by which hosts perceive and respond to the presence of colonizing microbes. Guillemin and coworkers pursue these fundamental questions in germ-free (GF) zebrafish, an experimental system that simplifies analyses of microbial influence on host development, while closely approximating gastrointestinal tract and immune system maturation, as well as gut microbiota



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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary 1 Microbial Communities of the Gut OVERVIEW The gastrointestinal tract represents an important and challenging system for exploring how microbial communities become established within their hosts, how their members maintain stable ecological niches, and how these dynamics relate to host health and disease. The complex, dynamic, and spatially diversified microbial community of the human gut is believed to be composed of at least 1013 microorganisms, including more than 800 species of bacteria (most of which have not yet been successfully cultured in the laboratory), numerous viral species including bacteriophages, archaea (e.g., methanogens), and eukaryotes (e.g., helminths and protozoa). The collective genome of the microbiota in the human gut is approximately one hundred-fold larger than that of its host. Therefore, as Bäckhed et al. (2005) state in their contribution to this chapter, “It seems appropriate to view ourselves as a composite of many species and our genetic landscape as an amalgam of genes embedded in our Homo sapiens genome and in the genomes of our affiliated microbial partners.” The first paper in this chapter, contributed by workshop presenter Karen Guillemin, focuses on the establishment of the gut microbiota (in humans, during the early days of infancy), its influence on host development (e.g., immunity), and the mechanisms by which hosts perceive and respond to the presence of colonizing microbes. Guillemin and coworkers pursue these fundamental questions in germ-free (GF) zebrafish, an experimental system that simplifies analyses of microbial influence on host development, while closely approximating gastrointestinal tract and immune system maturation, as well as gut microbiota

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary diversity, in mammals. This approach has demonstrated the pervasive influence of the microbiota over a variety of events in the maturation of the gastrointestinal tract, but it raises further questions regarding the potential for individual developmental variation arising from differences in microbiota from one member of a species to another. In humans, such variation could accrue among contemporaries who live in different environments or have different diets, as well as over the course of history. Once established, the gut microbiota acts as an exquisitely tuned metabolic “organ” within the host, according to presenter Jeffrey Gordon, senior author of the second paper in this chapter. He and coworkers review current knowledge of the structure and function of the human gut microbiota, as well as recent research that reveals coevolution between humans and gut microbes to their mutual benefit. Over the course of evolution, symbiotic gut bacteria have become, in Gordon’s words, “master physiological chemists,” employing a broad range of strategies to manipulate host genomes. Details of these microbial strategies are revealed in the final contribution to this chapter, in which presenter Abigail Salyers surveys the microbial activities in the human colon that are influenced by diet and that in turn affect human health. These include genetic exchanges among microbes that occur through transformation, phage transduction, and conjugation—interactions that are known to contribute to antibiotic resistance and which may also influence the evolution and virulence of pathogens. Salyers notes that several basic and longstanding questions regarding the composition, function, and evolution of the human intestinal microflora can now be investigated with the advent of molecular technology. THE ROLE OF THE INDIGENOUS MICROBIOTA IN ZEBRAFISH GASTROINTESTINAL TRACT DEVELOPMENT Karen Guillemin, Ph.D. University of Oregon Although the anatomy of the human gastrointestinal (GI) tract has been explored since at least the time of Leonardo da Vinci, who secretly produced detailed drawings of human organs at a time when such studies were considered heretical, our understanding of this organ is still largely incomplete. That is because we know so little about its cellular composition, which is dominated by microbes. The bacterial community of the GI tract contains an enormous wealth of unsequenced genomic information, and it raises important questions as to its function in the normal physiology and development of this organ. My coworkers and I are interested in the role commensal bacteria play in animal development, a phenomenon that has gone largely unexplored by developmental biologists. We are using a model vertebrate, the zebrafish, to study GI tract development in the presence and absence of the microbiota. Here I will

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary describe how we have used this system to explore the establishment and role of the intestinal microbiota in early development and the means by which the host perceives and responds to its microbiota. The Zebrafish Model Zebrafish offer a number of advantages as a model for microbiota development and establishment. The embryos develop ex utero, which facilitates our ability to create GF animals. We harvest embryos and surface-sterilize them with a bleaching procedure that has been shown not to cause developmental defects; the embryos are then grown in sterile tissue culture flasks containing sterile media. The larvae are transparent, allowing us to follow the development of internal organs and monitor the dynamics of bacteria in live animals. GI tract development and physiology in zebrafish closely resemble those of mammals. Zebrafish also possess both adaptive and innate immune systems similar to those in mammals. Finally, zebrafish are readily amenable to genetic analysis. The zebrafish gut is simpler than the human equivalent, but it exhibits regional specialization similar to the human organ: the proximal tract is specialized for lipid absorption, while most protein absorption occurs in the distal tract. This area near the anal vent is thought to be involved in osmoregulation. Figure 1-1 shows some of the key events in gut maturation and immune system development; our focus is on zero through eight day postfertilization (dpf). The embryos hatch out of their eggshells between 2 and 3 dpf, prior to the completion of gut maturation. FIGURE 1-1 Important events in zebrafish development. SOURCE: Guillemin (2005).

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary One of the first questions we addressed was how the gut microbiota is established. We have used scanning electron microscopy to visualize the rich microbial community on the surface of the embryonic chorion. We have also performed fluorescent in situ hybridization to look at the distribution of bacteria during development. Using a combination of three eubacterial probes that recognize sequences of 16S ribosomal RNA genes, we found that there is a dense community of microbes on the surface of the early embryo, but the interior of the embryo appears sterile. Microbes do not begin to accumulate within the embryo’s internal organs, as viewed in transverse sections of the GI tract, until after the embryo hatches. In adult zebrafish, microbes are distributed throughout the lumen of the gut; the fish also appear to have immune cells that sample the microbial contents of the gut, possibly like mammalian dendritic cells. We quantified the bacterial load over the course of zebrafish development using quantitative polymerase chain reaction (PCR), producing results similar to those obtained through in situ hybridization and suggesting that the microbial load of the zebrafish GI tract, relative to its weight, is roughly comparable to that in humans. Enumeration studies of the zebrafish microbiota reveal a preponderance of Aeromonas and Pseudomonas species, so we wanted to examine the distribution of these genera during developmental time. We hybridized the proximal and distal intestine of zebrafish at both 4 and 8 dpf with the eubacterial probes, as well as with specific probes for Aeromonas or Pseudomonas. At 4 dpf, we found a preponderance of Pseudomonas species in the distal intestine. By 8 dpf, these were largely replaced with Aeromonas species, but a large number of Pseudomonas species remained in the proximal intestine of these animals. Acquisition of this microbiota happens concurrently with the later events of gut maturation; a similar process of postnatal gut maturation occurs in mammals as they acquire their microflora. Role of the Microbiota in Gut Development and Function To examine the functional significance of the temporal relationship between microbiota establishment and gut maturation, we turned to gnotobiology, the use of GF animals (see subsequent paper by Bäckhed et al.). We raised GF zebrafish under sterile conditions, as previously described. For both GF and control fish, the egg yolk was the sole source of nutrients during these experiments. We verified the sterility of the GF zebrafish by plating fish homogenates on tryptic soy agar, by PCR with 16S panbacterial primers, and by conventional and scanning electron microscopy. To determine whether adding bacteria to GF zebrafish at a later stage of development would restore them to the same status as control animals, we raised GF animals until 5 dpf, when the conventionally-reared animals’ guts become functional, then exposed the GF fish to a mixture of bacteria found in the water of their conventionally reared clutch mates. We examined a variety of traits to compare the phenotypes of control and

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary experimental animals, including the expression of an enzyme on the brush border of the intestinal epithelium, alkaline phosphatase. This enzyme is known to be induced during the developmental period; by staining for its activity in transverse sections of the gut, we found that by 8 dpf, alkaline phosphatase activity was considerably higher in the control zebrafish than in the GF animals. Alkaline phosphatase activity was restored to control levels in GF animals that were exposed, 5 dpf, to bacteria from their siblings. Another marker of maturation we examined was glycan expression. The glycan landscape of the GI tract is known to be very sensitive to the presence of microbes, so we used a number of different lectins to sensitively detect the expression of a variety of sugar moieties. Using image analysis software to quantify sugar expression, we found that galactoseα1,3galactosyl (Galα1,3Gal) is down-regulated in conventionally reared animals, but persists at high levels in GF zebrafish; exposure to bacteria reduces the expression of this glycan in formerly GF fish. Experiments in GF rats have found that the number of mucus-secreting goblet cells in the GI tract is reduced in the GF animals (Ishikawa et al., 1986; Sharma and Schumacher, 1995). We found that in GF zebrafish the number of goblet cells at 8 dpf is less than in conventionally reared 8 dpf animals and similar to the number found in 5 dpf conventionally reared animals. Exposure to the microbiota again reverses this GF trait to that of a conventional animal of the same age. We are also examining the relationship between microbiota establishment and GI tract function. For example, we have compared the ability of GF and control zebrafish to absorb proteins in their distal intestines. This trait can be evaluated by feeding the fish a high concentration of protein in the form of the enzyme horseradish peroxidase, then assaying the enzyme’s activity after it is absorbed into cells. In these experiments, the GF animals—which we know to be equally proficient at swallowing as the conventionally raised controls—were found to be dramatically defective in protein absorption. This deficiency was reversed in GF animals following exposure to the microbiota. We also visualized GI motility in GF and control animals, which is possible in the transparent zebrafish. The GF animals had markedly shorter and more regular peristaltic waves along their GI tract, a trait that was once again reversed by exposure to the microbiota. Host Perception of and Response to the Microbiota Having accumulated evidence that the microbiota plays important roles in maturation and function of the zebrafish GI tract, we next explored how the host perceives the presence of the microbes, and attempted to identify the types of signals sent by the microbiota to its host that promote gut development. To do this, we created monocolonized animals by inoculating GF zebrafish, at 5 dpf, with pure cultures of either of two major bacterial constituents of the zebrafish GI

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary tract: Aeromonas sobria and Pseudomonas fluorescens. We found that monoassociation with either bacterial strain was sufficient to reverse the previously discussed traits of low alkaline phosphatase activity and high Galα1,3Gal levels in GF zebrafish. We next tested whether live bacteria were required to reverse these phenotypes or whether a heat-killed preparation of the microbiota was sufficient to signal to the host to promote gut maturation. We found that heat-killed microbiota was sufficient to induce alkaline phosphatase activity in GF animals; preliminary studies also indicate that bacterial lipopolysaccharide produces the same effect. By contrast, heat-killed bacteria failed to suppress the expression of Galα1,3Gal in GF animals. Thus, we have found evidence for two different modes of host perception for the presence of microbes: one that uses a generic signal of microbial-associated molecular patterns and another that requires active signaling from constituents of the microbiota. Conclusion Our findings on the role of the microbiota in the development of the GI tract in zebrafish show this to be a promising model for investigating the role of microbial communities in the developmental biology of the host. We have been able to show that the microbiota is required for gut maturation—as indicated by patterns of expression of glycans, by alkaline phosphatase activity, and by goblet cell census—and that the microbiota is important for such functions as protein absorption and GI motility. The possibility that alkaline phosphatase enzymatic activity can be up-regulated by a heat-killed bacterial preparation immediately brings to mind the toll-like receptors (TLRs) of the innate immune system, and their ability to recognize generic microbial-associated molecular patterns. Much attention has been focused on the role of TLRs in protection against infection, but it is also important to consider these molecules in the context of gut development and homeostasis. A recent publication by Rakoff-Nahoum et al. (2004) examines susceptibility to intestinal injury in animals that are deficient for TLR signaling. In this study, wild-type mice survived an intestinal injury, while animals deficient in MyD88, an adaptor molecule essential for TLR-mediated induction of inflammatory cytokines, manifested severe morbidity and mortality in response to the same insult. A similar phenotype was observed in animals in which the ligand for TLRs was depleted by an antibiotic reduction of the microbiota. This trait was reversed, and the animals’ viability restored, by exposing them to lipopolysaccharide and lipoteichoic acid, two conserved molecular products of microorganisms recognized by TLRs. These results indicate that the constituents of the microbiota continually shape GI tract homeostasis. Given such findings, it will now be important to determine the extent to which the microbiota is stereotyped during development. Most of us would agree

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary that the human microbiota has probably changed very much since the time of Leonardo da Vinci, due to the onset of antimicrobial therapies and modern sanitation. In that regard, it may be somewhat comforting to imagine that certain aspects of the microbial-directed maturation of the GI tract require generic signals that many different possible constituents could supply. However, it is also clear that certain complex and specific signaling events influence GI tract maturation and function. The gnotobiotic zebrafish model will help elucidate the various signaling mechanisms between animals and their resident microbes. HOST-BACTERIAL MUTUALISM IN THE HUMAN INTESTINE Fredrik Bäckhed, Ruth E. Ley, Justin L. Sonnenburg, Daniel A. Peterson, Jeffrey I. Gordon1 Reprinted with permission from Science (Bäckhed et al. 2005). Copyright 2005 AAAS. The distal human intestine represents an anaerobic bioreactor programmed with an enormous population of bacteria, dominated by relatively few divisions that are highly diverse at the strain/subspecies level. This microbiota and its collective genomes (microbiome) provide us with genetic and metabolic attributes we have not been required to evolve on our own, including the ability to harvest otherwise inaccessible nutrients. New studies are revealing how the gut microbiota has coevolved with us and how it manipulates and complements our biology in ways that are mutually beneficial. We are also starting to understand how certain keystone members of the microbiota operate to maintain the stability and functional adaptability of this microbial organ. The adult human intestine is home to an almost inconceivable number of microorganisms. The size of the population—up to 100 trillion—far exceeds that of all other microbial communities associated with the body’s surfaces and is ~10 times greater than the total number of our somatic and germ cells (Savage, 1977). Thus, it seems appropriate to view ourselves as a composite of many species and our genetic landscape as an amalgam of genes embedded in our Homo sapiens genome and in the genomes of our affiliated microbial partners (the microbiome). Our gut microbiota can be pictured as a microbial organ placed within a host organ: It is composed of different cell lineages with a capacity to communicate with one another and the host; it consumes, stores, and redistributes energy; it mediates physiologically important chemical transformations; and it can maintain and repair itself through self replication. The gut microbiome, which may 1   Materials and methods are available as supporting material on Science Online. We thank L. Angenent for many helpful discussions. Work cited from the authors’ lab is supported by the NIH and NSF. F.B. and J.L.S. are supported by postdoctoral fellowships from the Wenner-Gren and W. M. Keck Foundations, respectively. Supporting online material: www.sciencemag.org/cgi/content/full/307/5717/1915/DC1MaterialsandMethodsTablesS1toS3References.10.1126/science.1104816.

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary contain > 100 times the number of genes in our genome, endows us with functional features that we have not had to evolve ourselves. Our relationship with components of this microbiota is often described as commensal (one partner benefits and the other is apparently unaffected) as opposed to mutualistic (both partners experience increased fitness). However, use of the term commensal generally reflects our lack of knowledge, or at least an agnostic (noncommittal) attitude about the contributions of most citizens of this microbial society to our own fitness or the fitness of other community members. The guts of ruminants and termites are well-studied examples of bioreactors “programmed” with anaerobic bacteria charged with the task of breaking down ingested polysaccharides, the most abundant biological polymer on our planet, and fermenting the resulting monosaccharide soup to short-chain fatty acids. In these mutualistic relationships, the hosts gain carbon and energy, and their microbes are provided with a rich buffet of glycans and a protected anoxic environment (Brune and Friedrich, 2000). Our distal intestine is also an anaerobic bioreactor that harbors the majority of our gut microorganisms; they degrade a varied menu of otherwise indigestible polysaccharides, including plant-derived pectin, cellulose, hemicellulose, and resistant starches. Microbiologists from Louis Pasteur and Ilya Mechnikov to present-day scientists have emphasized the importance of understanding the contributions of this microbiota to human health (and disease). Experimental and computational tools are now in hand to comprehensively characterize the nature of microbial diversity in the gut, the genomic features of its keystone members, the operating principles that underlie the nutrient foraging and sharing behaviors of these organisms, the mechanisms that ensure the adaptability and robustness of this system, and the physiological benefits we accrue from this mutualistic relationship. This review aims to illustrate these points and highlight some future challenges for the field. Microbial Diversity in the Human Gut Bioreactor The adult human gastrointestinal tract contains all three domains of life—bacteria, archaea, and eukarya. Bacteria living in the human gut achieve the highest cell densities recorded for any ecosystem (Whitman et al., 1998). Nonetheless, diversity at the division level (superkingdom or deep evolutionary lineage) is among the lowest (Hugenholtz et al., 1998); only 8 of the 55 known bacterial divisions have been identified to date (Figure 1-2A), and of these, 5 are rare. The divisions that dominate—the Cytophaga-Flavobacterium-Bacteroides (CFB) (e.g., the genus Bacteroides) and the Firmicutes (e.g., the genera Clostridium and Eubacterium)—each comprise ~30 percent of bacteria in feces and the mucus overlying the intestinal epithelium. Proteobacteria are common but usually not dominant (Seksik et al., 2003). In comparison, soil (the terrestrial biosphere’s GI tract, where degradation of organic matter occurs) can contain 20 or more bacterial divisions (Dunbar et al., 2002).

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary Our knowledge of the composition of the adult gut microbiota stems from culture-based studies (Moore and Holdeman, 1974), and more recently from culture-independent molecular phylogenetic approaches based on sequencing bacterial ribosomal RNA (16S rRNA) genes. Of the > 200,000 rRNA gene sequences currently in GenBank, only 1,822 are annotated as being derived from the human gut; 1,689 represent uncultured bacteria. rRNA sequences can be clustered into relatedness groups based on their percent sequence identity. Cutoffs of 95 and 98 percent identity are used commonly to delimit genera and species, respectively. Although these values are somewhat arbitrary and the terms “genus” and “species” are not precisely defined for microbes, we use them here to frame a view of human gut microbial ecology. When the sequences (n = 495 greater than 900 base pairs) are clustered into species, and a diversity estimate model is applied, a value of ~800 species is obtained (Figure 1-3). If the analysis is adjusted to estimate strain number (unique sequence types), a value > 7,000 is obtained (Figure 1-3). Thus, the gut microbiota, which appears to be tremendously diverse at the strain and subspecies level, can be visualized as a grove of eight palm trees (divisions) with deeply divergent lineages represented by the fan(s) of closely related bacteria at the very top of each tree trunk. Diversity present in the GI tract appears to be the result of strong host selection and coevolution. For example, members of the CFB division that are predominantly associated with mammals appear to be the most derived (i.e., farthest away from the common ancestor of the division), indicating that they underwent accelerated evolution once they adopted a mutualistic lifestyle. Moreover, a survey of GenBank reveals that several subgroups in CFB are distributed among different mammalian species (Figure 1-2B), suggesting that the CFB-mammal symbiosis is ancient and that distinct subgroups coevolved with their hosts. The structure and composition of the gut microbiota reflect natural selection at two levels: at the microbial level, where lifestyle strategies (e.g., growth rate and substrate utilization patterns) affect the fitness of individual bacteria in a competitive ensemble, and at the host level, where suboptimal functionality of the microbial ensemble can reduce host fitness. Microbial consortia whose integrated activities result in a cost to the host will result in fewer hosts, thereby causing loss of their own habitat. Conversely, microbial consortia that promote host fitness will create more habitats. Thus, the diversity found within the human GI tract, namely, a few divisions represented by very tight clusters of related bacteria, may reflect strong host selection for specific bacteria whose emergent collective behavior is beneficial to the host. This hypothesis has two important implications: (1) A mechanism exists to promote cooperation, and (2) the structure promotes functional stability of the gut ecosystem. To benefit the host, bacteria must be organized in a trophic structure (food web) that aids in breaking down nutrients and provides the host with energetic substrates. Cooperative behavior that imposes a cost to the individual while benefiting the community can emerge within groups of bacteria (Rainey and Rainey,

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary FIGURE 1-2 Representation of the diversity of bacteria in the human intestine. (A) Phylogenetic tree of the domain bacteria based on 8,903 representative 16S rRNA gene sequences. Wedges represent divisions (superkingdoms): Those numerically abundant in the human gut are red, rare divisions are green, and undetected are black (for colors please refer to the original article). Wedge length is a measure of evolutionary distance from the common ancestor. (B) Phylogenetic tree of the CFB division based on 1561 sequences from GenBank (> 900 nucleotides) and their ecological context. Wedges are major subgroups of CFB; symbols are sources of the sequences [Earth, environmental; cow, ruminants; rodent, rat and/or mouse; person, human GI tract; others are termite, cockroach, worm (including hydrothermal), and pig]. Ratios are the number of sequences represented in the human gut relative to the total number in the subgroup; red, yellow, and black indicate majority, minority, and absence of sequences represented in human GI tract, respectively. (C) Phylogenetic (parsimony) tree of Bacteroides. Strains classified as B. thetaiotaomicron based on phenotype are in red; 16S rRNA analysis did not confirm this classification for all strains. Bacteroides spp. with sequenced genomes are in bold. Black circles indicate nodes with high (> 70 percent) bootstrap support. Scale bars indicate the degree of diversity (evolutionary distance) within a division or subgroup ([A] and [B]), respectively] in terms of the fraction of the 16S rRNA nucleotides that differ between member sequences; in (C), the evolutionary distance between organisms is read along branch lengths, where scale indicates number of changes in 16S rRNA nucleotide composition. SOURCE: Bäckhed et al. (2005).

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary 2003) and can be maintained by group selection as long as consortia are isolated and new consortia form periodically (e.g., new GI tracts). Furthermore, selection must act simultaneously at multiple levels of biological organization (Travisano and Velicer, 2004). These criteria are met in the human GI tract where new acts of colonization occur at birth, with a small founding population of noncheaters from the mother, and selection occurs both at the microbial and host level. Diversity is generally thought to be desirable for ecosystem stability (McCann, 2000). One important way diversity can confer resilience is through a wide repertoire of responses to stress (referred to as the insurance hypothesis [Yachi and Loreau, 1999]). In man-made anaerobic bioreactors used to treat wastewater (a system analogous to the gut but where no host selection occurs), rates of substrate degradation can remain constant, whereas bacterial populations fluctuate chaotically as a result of blooms of subpopulations (Fernandez et al., 2000). Functional redundancy in the microbial community ensures that key processes are unaffected by such changes in diversity (Goebel and Stackebrandt, 1994). By contrast, in the human gut, populations are remarkably stable within individuals (Zoetendal et al., 1998), implying that mechanisms exist to suppress blooms of subpopulations and/or to promote the abundance of desirable bacteria. A study of adult monozygotic twins living apart and their marital partners has emphasized the potential dominance of host genotype over diet in determining microbial composition of the gut bioreactor (Zoetendal et al., 2001). The role of the immune system in defining diversity and suppressing subpopulation blooms remains to be defined. One likely mediator of bacterial selection is secretory immunoglobulin A (Suzuki et al., 2004). The human gut is faced with a paradox: How can functional redundancy be maintained in a system with low diversity (few divisions of bacteria), and how

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary TABLE 1-2 Effects of the Microbiota on Host Biology, Defined by Comparing Germ-Free (GF) and Conventionally-Raised (CONV-R) Rodents Phenotype Comments References Gut structure and function GF mice and rats have enlarged ceca Likely a consequence of accumulation of undegraded host and dietary polysaccharides that bind water (Gordon et al., 1966a; Wostmann and Bruckner-Kardoss, 1959) GF mice villi are thinner Mesenchymal core is less cellular; reduced immune population (see below) (Banasaz et al., 2002) GF mice have slower epithelial renewal rates Evolutionary conserved response, also seen in germ-free zebrafish; mechanisms to be defined (e.g., what is contribution of undeveloped mucosal immune system). Epithelial regeneration markedly reduced in GF mice with dextran sodium sulfate-induced colitis: effect requires Myd88-dependent signaling through mesenchymal cells and recruitment of pericryptal macrophages. (Banasaz et al., 2002; Pull et al., 2005; Rawls et al., 2004; Savage et al., 1981) GF mice have slower gut motility Migrating motor complexes move more slowly with more restricted spatial distribution; detailed comparisons of the enteric nervous system in GF versus CONV-R animals are needed; potential implications concerning the pathogenesis of irritable bowel syndrome in humans (Abrams and Bishop, 1967) GF rats have reduced bile acid deconjugation Impaired excretion of bile acids (Gustafsson et al., 1966) GF rats produce more bile acids This effect, together with deconjugation defect, results in increased bile acid pools; impact on cholesterol homeostasis (Wostmann, 1973) GF rats have fewer enteroendocrine cells There are multiple subpopulations on enteroendocrine cells, defined by their peptide hormone products. The effects of microbiota on these subpopulations has not been well-defined. (Uribe et al., 1994) GF rats have mesenteric microvaculture that is hyporesponsive to norepinephrine and pitressin Kallikrein-like molecules produced by GF cecum may contribute to phenotype (Baez and Gordon, 1971; Gordon, 1964)

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary Phenotype Comments References GF mice have reduced capillary network complexity in their villus mesenchymal cores Potential influence on nutrient absorption and mucosal barrier functions (Stappenbeck et al., 2002) Cardiac function GF mice and rats have lower cardiac weight Detailed morphometric studies lacking, mechanism not described (Bruckner-Kardoss and Wostmann, 1974; Gordon et al., 1966a, 1966b; Wostmann et al., 1982) GF rats have lower cardiac output Physiological mechanisms, myocardial energetics not defined (Gordon et al., 1963; Wostmann et al., 1968) Endocrine system GF mice are more insulin sensitive than CONV-R animals Could reflect, at least in part, reduced fat stores; other similarities to mice that are subjected to chronic caloric restriction need to be delineated (Bäckhed et al., 2004) GF mice have lower circulating levels of leptin and adiponectin Associated with reduced fat stores (Bäckhed et al., 2004; Bäckhed and Gordon, unpublished observation) Nutrition/metabolism GF mice are vulnerable to vitamin deficiencies The gut microbiota produces vitamin K, B6, B12, biotin, folic acid, and pantothenate (Gustafsson et al., 1962; Sumi et al., 1977; Wostmann et al., 1963) GF rats do not extract as much energy from their diet See text for details (Wostmann et al., 1983; Yamanaka et al., 1977)

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary Phenotype Comments References GF mice and rats have lower metabolism (VO2) Hexose monophosphate shunt and TCA cycle activity reduced. Mechanisms remain ill-defined; possible role for leptin (Bruckner-Kardoss and Wostmann, 1978; Levenson et al., 1969; Wostmann et al., 1982, 1968) GF mice absorb more cholesterol GF mice have large bile acid pools (Gustaffsson et al., 1975) Immune system development B-cells and immunoglobulin secretion GF mice have 40–1000 fold less serum IgM/IgG and intestinal IgA (Horsfall et al., 1978) Natural immunoglobulin GF mice have normal levels of natural Ig and B1 B-cells. However the majority of IgA that reacts with components of the microbiota originates from B2 B-cells. While class switching can occur in a T-cell independent pathway, most reactive antibodies are generated in a classical T-cell dependent manner. (Bos et al., 2001; Macpherson and Uhr, 2004) Ochsenbein et al., 1999; Thurnheer et al., 2003) Anti-‘commnesal’ IgA The segmented filamentous bacteria (SBF; Clostridia) are stronger inducers of intestinal IgA than other bacteria (including Bacteroidetes). Interestingly, SBF found to be enriched in the intestines of mice lacking secretory IgA. (Fagarasan et al., 2002; Suzuki et al., 2004) Intraepithelial lymphocytes Both γδ and αβ T-cells are significantly decreased in the intraepithelial lymphocyte population of GF mice: γδ>αβ (Bandeira et al., 1990) Cytotoxic T lymphocytes Size and composition of CD8 repertoire to non-gut related antigen is unaffected by presence of absence of the microbiota. (Bousso et al., 2000) T-cells (αβ) General T-cell function and number is not reduced in GF rats (Nielsen, 1972) T-cells (γδ) Decreased number of mesenteric lymph node γδ T-cells in GF mice (Yoshikai et al., 1988) Mucosal-associated invariant T-cells Invariant Vα19-Jα33 TCR+ cells located in lamina propria, fail to develop in GF mice (Treiner et al., 2003) CD4+CD25+ T-cells Levels and function are the same in GF and CONV-R mice (Gad et al., 2004)

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary Phenotype Comments References Modulation of Immunity and Disease GF mice are resistant to developing inflammatory bowel disease GF mice with aberrations in T-cell development and function (IL2–/–, IL10–/–, TCRα–/–) do not develop spontaneous colitis, unlike their CONV-R counterparts (Dianda et al., 1997; (Mizoguchi et al., 2000; Sadlack et al., 1993; Sellon et al., 1998) GF rodents have reduced susceptibility to arthritis CONV-R β2-microglobulin-HLA-B27 transgenic rats develop spontaneous colitis and arthritis; B10.BR mice develop enthesopathy; GF counterparts do not (Rath et al., 1996; Rehakova et al., 2000) GF NOD mice have higher rate of autoimmune diabetes No primary papers; phenotype mentioned in reviews (Bach, 1994a,1994b; Wicker et al., 1987) Xenobiotic metabolism The gut microbiota metabolizes dietary oxalates 50 percent of GF rats have kidney stones versus 0 percent for CONV-R. Oxalate-degrading Oxalobacter formigenes reduces kidney stone formation in a rat model; currently in clinical trials. (Allison et al., 1985; (Gustaffson and Norman, 1962; Sidhu et al., 2001) The gut microbiota is required for nitroreduction of xenobiotics See http://umbbd.ahc.umn.edu/ for a database of microbial biocatalytic/biodegradation reactions involving xenobiotics and various chemical compound classes. Consider microbiota when defining factors that influence bioavailability of orally administered drugs. (Larsen et al., 1998) General health and disease GF rats live longer than their CONV-R counterparts Mechanism to be determined; possible similarities to insulin-sensitive, calorie-restricted animals (Gordon et al., 1966b; Pollard and Wostmann, 1985) Removing the microbiota decreases incidence of intestinal neoplasia in some mouse models GF IL-10–/– mice and Tgfβ-1–/–, Rag2–/–; Tcrb–/–, p53–/–; and Gpx1–/–,Gpx2–/– compound homozygous knockout mice have reduced inflammation and tumor formation. Microbial communities associated with pre-neoplastic and neoplastic lesions in mouse models and humans have yet to be enumerated. (Balish and Warner, 2002; Chu et al., 2004; Engle et al., 2002; Kado et al., 2001) GF mice are more radioresistant Mechanism to be defined; potential implications for ameliorating GI syndrome in patients receiving radiotherapy for abdominal/pelvic malignancies (Matsuzawa, 1965)   SOURCE: Bäckhed et al. (2005).

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Ending the War Metaphor: The Changing Agenda for Unraveling the Host-Microbe Relationship - Workshop Summary TABLE 1-3 Six Ways Environmental Engineers Improve Bioreactor Efficiency Approach Method Reference Engineering Altering reactor configuration: e.g., changing to a plug-flow reactor with higher substrate levels at the entrance to the reactor increases conversion rates according to Monod kinetics and promotes staging of the reactor by compartmentalization. This results in differing local environmental conditions that are optimal for sequential functional subpopulations. (Angenent et al., 2002; Rittmann and McCarty, 2001) Engineering Removal of product. Even if the product is not inhibiting, thermodynamics dictate that the energy for the conversion will be higher if the concentration of products are maintained at a very low level. (Stams, 1994; Thauer et al., 1977) Operational Elongation of the sludge retention time (mean residence time) by adding an attachment matrix to the reactor. (Zaiat et al., 2001) Microbial Ecology Selection of a different community: e.g., sulfate reducers (SRBs) are not wanted in anaerobic methanogenic bioreactors because they produce a toxic product—H2S. Preventing SRBs improves reactor efficiency. (Elferink et al., 1994) Microbial Ecology Bioaugmentation: addition of a functional microbe that can remove a specific substrate at higher rates. (Abeysinghe et al., 2002; Patureau et al., 2001) Microbial Ecology Bacteriophage addition: bacteriophage modulate bacterial population dynamics in bioreactors and, therefore, can be used to optimize reactor efficiency (e.g., as a biocontrol agent to prevent foam formation by microbes). (Hantula et al., 1991; Lu et al., 2003; Thomas et al., 2002)   SOURCE: Bäckhed et al. (2005). REFERENCES Abeysinghe DH, De Silva DG, Stahl DA, Rittmann BE. 2002. The effectiveness of bioaugmentation in nitrifying systems stressed by a washout condition and cold temperature. Water and Environmental Research 74(2):187–199. Abrams GD, Bishop JE. 1967. Effect of the normal microbial flora on gastrointestinal motility. Proceedings for the Society of Experimental Biology and Medicine 26(1):301–304. Allison MJ, Dawson KA, Mayberry WR, Foss JG. 1985. Oxalobacter formigenes gen. nov., sp. nov.: Oxalate-degrading anaerobes that inhabit the gastrointestinal tract. Archives of Microbiology 141(1):1–7. Angenent LT, Zheng D, Sung S, Raskin L. 2002. Microbial community structure and activity in a compartmentalized, anaerobic bioreactor. Water and Environmental Research 74(5):450–461. Bach JF. 1994a. Insulin-dependent diabetes mellitus as an autoimmune disease. Endocrine Reviews 15(4):516–542.

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