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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Genome-wide association studies of IBD: a window into host-symbiotic factors
  5. Symbiotic factors of the microbiota: lessons from successful symbionts
  6. Conclusions and perspectives
  7. References

Humans are colonized by a diverse collection of microbes, the largest numbers of which reside in the distal gut. The vast majority of humans coexist in a beneficial equilibrium with these microbes. However, disruption of this mutualistic relationship can manifest itself in human diseases such as inflammatory bowel disease. Thus the study of inflammatory bowel disease and its genetics can provide insight into host pathways that mediate host–microbiota symbiosis. Bacteria of the human intestinal ecosystem face numerous challenges imposed by human dietary intake, the mucosal immune system, competition from fellow members of the gut microbiota, transient ingested microbes and invading pathogens. Considering features of human resident gut bacteria provides the opportunity to understand how microbes have achieved their symbiont status. While model symbionts have provided perspective into host–microbial homeostasis, high-throughput approaches are becoming increasingly practical for functionally characterizing the gut microbiota as a community.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Genome-wide association studies of IBD: a window into host-symbiotic factors
  5. Symbiotic factors of the microbiota: lessons from successful symbionts
  6. Conclusions and perspectives
  7. References

Humans are colonized by upwards of 1014 bacteria cells, the majority of which reside in the distal gut. Human are outnumbered by their bacterial symbionts by an estimated ratio of 10:1, and we are just beginning to appreciate the diversity and function of this microbiota. A great percentage of the human population lives in a peaceful, albeit dynamic, equilibrium with these microbes. Disruption of this mutualistic relationship manifests in human diseases such as inflammatory bowel disease (IBD).

Increasingly, 16S ribosomal RNA (rRNA) and whole-genome shotgun (WGS) sequencing surveys of the gut microbiota have been performed to link community membership and/or features with human disease states. However, high-level metagenomic analyses of microbial communities employing microbiome quantitative data for tasks like biomarker discovery and functional or phylogenetic subtyping are challenging for a number of reasons. Many diseases represent a spectrum of disorders with diverse pathophysiology. There is tremendous inter-subject variability (even between identical twins) and potentially multiple healthy configurations of a microbial community. Furthermore, when shifts in microbial communities are observed in disease states it is difficult to discern cause from effect. Nevertheless, consideration of the microbiota as a ‘forgotten organ’ functioning in health and disease is a tantalizing prospect.

Herein, we consider host and microbial contributions to symbiosis. IBD represents a disrupted intestinal ecosystem. As such, IBD genome-wide association studies serve as a guide for identifying host genes necessary for coexistence with the gut microbiota. Current understanding of the roles of a few IBD-associated genes in both innate and adaptive immune responses are reviewed. We examine the contribution of members of the microbial world for examples of robust and dynamic systems that contribute to colonization. Specifically, we focus on features of bacterial symbionts from three genera: Bacteroides, Helicobacter and Lactobacillus that contribute to their fitness as colonizers and may provide benefits to the host. We conclude with a brief discussion of microbial community based high-throughput sequencing technologies and their analysis methods.

Genome-wide association studies of IBD: a window into host-symbiotic factors

  1. Top of page
  2. Summary
  3. Introduction
  4. Genome-wide association studies of IBD: a window into host-symbiotic factors
  5. Symbiotic factors of the microbiota: lessons from successful symbionts
  6. Conclusions and perspectives
  7. References

Inflammatory bowel disease is a model disease for investigating the regulation of homeostasis between host and microbiota. IBD-focused genome-wide and targeted mapping association studies afford insight into the genes and regulatory pathways that promote host–microbial symbiosis. Studies of IBD (Barrett et al., 2008) have identified numerous loci and genes, many of which play multifunctional roles in innate and adaptive immunity. Here we focus on four: Nod2 (nucleotide-binding oligomerization domain containing 2), Atg16L1 (autophagy-related gene 16-like 1), IRGM (immunity-related GTPase M) and IL-23R (IL-23 receptor).

Nod-2: intracellular sensor and multifunctional effector in symbiosis

Nod2, a member of the Nod-like receptor family, is an intracellularly localized microbial-associated pattern recognition receptor. Nod2 consists of three main functional motifs: a leucine-rich repeat domain, a nucleotide-binding oligomerization domain and two caspase recruitment domains (Proell et al., 2008). Biochemical and functional data suggest that Nod2 senses muramyl dipeptide (MDP) (Girardin et al., 2003), a constituent of peptidoglycan and results in NF-κB pathway activation (Kelsall, 2005). Nod2's recognition of MDP also engages MAP kinase pathways and induces activator protein 1 transcription factors. Recent studies suggest how Nod2 functions in Crohn's disease and thus how Nod2 is an important mediator of host–microbiota symbiosis.

Nod2 is expressed by intestinal epithelial cells and antigen presenting cells. Loss-of-function mutations of Crohn's disease-associated alleles reveal Nod's multiple roles. Paneth cells, a subset of epithelial cells in the small intestine, secrete antimicrobial peptides and proteins. Paneth cell production of α-defensins is decreased in Crohn's disease patients, with greater deficits observed in patients with Nod2 mutations (Wehkamp et al., 2004). A similar decrease of α-defensins, specifically defensin-related cryptdin (Defcr) 4 and Defcr-related sequence 10, was observed in Nod2−/− mice (Kobayashi et al., 2005). Nod2−/− mice also have increased bacterial colonization in their terminal ilea, suggesting that Nod2-dependent defensin production regulates size and membership of regional mucosal bacterial communities (Petnicki-Ocwieja et al., 2009). Commensal bacteria also modulate Nod2 expression, as germ-free mice showed reduced ileal Nod2 levels while colonization with Lactobacillus plantarum and Escherichia coli strain Nissle 1917 restored Nod2 expression levels (Petnicki-Ocwieja et al., 2009). Feedback mechanisms with microbes inducing the expression of host genes that regulate bacterial numbers or membership is a common theme of this symbiosis.

Unravelling how Crohn's disease-related Nod2 mutations drive pro-inflammatory responses in macrophages and dendritic cells has revealed that MDP sensing by Nod2 may cross-regulate other PRR pathways. Nod2−/− mice have increased susceptibility to colitis induced by the mucosal disruptant dextran sulfate sodium and mice bearing a mutation similar to one observed in Crohn's disease that truncates Nod2 have elevated levels of NF-κB activation and IL-1β in macrophages (Maeda et al., 2005). As this mutation truncated Nod2 and abrogated MDP binding (as do many other Crohn's disease-associated mutations), how was NF-κB activation occurring? Microbes express multiple pattern recognition receptor ligands. Studies of Nod2−/− mice suggest that Nod2 signalling may inhibit Toll-like receptor (TLR) 2-induced NF-κB activation, resulting in the pro-inflammatory cytokine pattern observed in Crohn's disease (elevated IL-12, IFN-γ) (Watanabe et al., 2004; 2006). Nod2 sensing of MDP may down-modulate subsequent TLR2, 3, 4, 5 and 9 activation. Furthermore, the cytokine reduction observed upon MDP pre-treatment followed by TLR-ligand exposure was dependent upon IFN regulatory factor 4 (Watanabe et al., 2008). While the MDP–Nod2 pathway acts in host defence against pathogens, Nod2 may also function as a damper of pro-inflammatory cytokine responses to the commensal microbiota.

MDP stimulation of Nod2 has recently been shown to impact a dendritic cell's intracellular handling of bacteria. These observations connect Nod2 to the autophagy pathway and explicate links between Nod2 and Crohn's disease-risk alleles in the autophagy genes, Atg16L1 (Homer et al., 2010) and IRGM. In monocyte-derived dendritic cells, MDP engagement of Nod2 results in autophagosomes that contain core components of the antigen processing machinery and classical autophagy effectors (Cooney et al., 2010). Nod1 and Nod2 recruit Atg16L1 to the plasma membrane where bacterial internalization is occurring and cells expressing a Crohn's disease risk-associated Nod2 allele or its murine orthologue cannot recruit Atg16L1 (Travassos et al., 2010). Dendritic cells isolated from Crohn's disease patients bearing the prevalent Nod2-susceptibility alleles or the Atg16L1 risk allele T300A showed deficient formation of these MDP-induced autophagosomes. In addition, when challenged with Salmonella enterica or adherent and invasive E. coli (AIEC), there was reduced localization of these bacteria to autophagosomes (Cooney et al., 2010). Nod2's contribution to symbiosis encompasses regulating α-defensin production in Paneth cells, dampening TLR ligand driven inflammation, and extends to autophagy-mediated clearance of bacteria (Netea and Joosten, 2010). As such, impaired Nod2 function can explain both the hyper-immunity and the immunodeficiency reflected in Crohn's disease (see Fig. 1 for a depiction of Nod2's multiple roles in intestinal homeostasis).

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Figure 1. Nod2: hub of intestinal homeostasis. Nod2 is an intracellular sensor which influences intestinal homeostasis via multiple pathways. Connections to ATG16L1 and IRGM influence autophagy. Nod2 also impacts inflammasome activation. By regulating α-defensin production, Nod2 modulates mucosal-associated bacterial colonization level in the gut. Nod2 also functions as an inflammatory damper of Toll-like receptors (TLR) responses.

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Autophagy genes and gut homeostasis

Autophagy is a basic physiologic function that sequesters cellular and cytoplasmic matter for lysosomal degradation (Mizushima et al., 2008). This core process is essential for survival in response to nutrition deprivation, hypoxia, and other cellular and organismal stressors. The autophagy pathway has been implicated in numerous disease processes: neurodegenerative diseases, cancer and inflammatory disorders, including IBD (Heath and Xavier, 2009). Autophagy plays critical roles in the immune system: clearance of intracellular microbes, antigen presentation, type 1 interferon production, lymphocyte development and regulation of cytokine signalling; and in turn, autophagy is regulated by the immune system via TLR, cytokine and Nod2 signalling pathways (Virgin and Levine, 2009). Upwards of 50 studies have validated the involvement of two autophagy genes, ATG16L1 and IRGM, in Crohn's disease, and ongoing research is elucidating the role of these genes in brokering peace between host and microbe.

Atg16L1

Coding variants in Atg16L1 have been strongly associated with Crohn's disease (Hampe et al., 2007; Rioux et al., 2007) and Atg16L1 is essential for autophagosome generation in response to bacterial invasion and serum starvation (Xavier et al., 2008). In addition, Atg16L1 plays a critical role in exocytosis of antimicrobial containing granules from Paneth cells (Cadwell et al., 2008). Thus Atg16L1 may function in host–microbiota symbiosis by limiting mucosal bacterial populations.

Like many IBD-associated susceptibility alleles, homozygous expression of Atg16L1T300A does not confer 100% susceptibility to Crohn's disease. A recent study using mouse models implicates an enteric virus in addition to the endogenous microbiota to explain how environment and genetics contribute to Crohn's disease. Murine norovirus CR6 (MNV CR6) was required for the Paneth cell abnormalities observed in mice expressing hypomorphic levels of Atg16L1 (Atg16L1HM). Further, MNV CR6 infected Atg16L1HM mice exposed to dextran sulfate sodium displayed an intestinal inflammatory response that was striking for both its resemblance to Crohn's disease and its antibiotic sensitivity (Cadwell et al., 2010). MNV infection of Atg16L1HM mice resulted in altered gene expression of Paneth cell amino acid and carbohydrate metabolic pathways that may reveal additional functions for Atg16L1 in symbiosis.

The autophagy pathway provides a mechanism for the host to degrade intracellular bacteria. Knock-down of Atg16L1 expression impairs autophagy of Salmonella typhimurium and in cells from donors expressing Atg16L1T300A, the capture of S. typhimurium within autophagosomes was markedly reduced (Kuballa et al., 2008; Travassos et al., 2010). Crohn's disease-associated strains of AIEC appear to have a selective advantage in Atg16L1-deficient cells as demonstrated by enhanced replication compared with other commensal and pathogenic E. coli strains (Lapaquette et al., 2010). These findings suggest that an inability to mobilize effective autophagy may compromise clearance of intracellular infections creating niches permissive for bacteria contributing to chronic inflammation.

IRGM

Genetic variants within and upstream of IRGM alter its expression and contribute to Crohn's disease susceptibility (Parkes et al., 2007; McCarroll et al., 2008). The immunity-related GTPase (IRG) family (p47 GTPases), including IRGM, consists of proteins that play important roles against intracellular pathogens (Howard, 2008). IRGM is necessary for autophagy-mediated clearance of Mycobacterium tuberculosis and S. typhimurium (Singh et al., 2006; Howard, 2008). Unlike Atg16L1, the mechanism by which IRGM promotes autophagy is unknown (Delgado et al., 2009); however, expression levels of IRGM play a critical role in regulating autophagosome encapsulation of bacteria (McCarroll et al., 2008). As with Atg16L1T300A, cells expressing Crohn's disease-genetic variants of IRGM offer a selective advantage for the replication of AIEC.

The convergence of Crohn's disease-associated genes on the autophagy pathway suggests that autophagosome-mediated processing and presentation of intracellular microbes is necessary for intestinal host–microbiota symbiosis. Additional links exist between autophagy and inflammation. Macrophages with Atg16L1 deficiency demonstrate an elevated level of TRIF-dependent caspase 1 activation following TLR4 stimulation or after co-culture with E. coli, resulting in high levels of IL-1β and IL-18 (Saitoh et al., 2008). Numerous cytokines have been implicated in IBD pathogenesis and while connections between these cytokines and autophagy are unknown; their role in IBD pathogenesis is significant. Crohn's disease has been viewed as a canonical T helper (Th) type 1 immune (IFN-γ and IL-12)-driven disease. Recently, the pendulum has swung towards a Th IL-17 (Th17) centric view. However, IBD likely reflects Th1 and Th17 perturbations (Strober et al., 2010).

IL-23, IL-23R and IL-17

Disease-associated IL23R (interleukin 23-receptor) polymorphisms have been observed in IBD (Abraham and Cho, 2009). In contrast, genetic polymorphisms have also been identified that appear to afford protection against developing Crohn's disease (Dubinsky et al., 2007; Li et al., 2010). IL-23R is a heterodimer expressed by dendritic cells, macrophages, neutrophils and T cells. IL-23 is also a heterodimer (composed of a p19 subunit and a p40 subunit shared with IL-12) and is expressed by dendritic cells, macrophages and epithelial cells. The IL-23–IL-23R pathway is an important mediator of inflammatory responses necessary for host defence. Mouse models of innate-driven and T cell-mediated colitis have been instrumental in understanding how IL-23 functions in IBD pathogenesis (Ahern et al., 2008). IL-23-driven mucosal inflammation is mediated by innate and adaptive cellular effectors with IFN-γ and IL-17 functioning as key molecular effectors. IL-23 may also function in the differentiation of a subset of CD4+ T-helper cells that secrete IL-17 (Th17) and aid in the expansion and stabilization of Th17 populations (O'Quinn et al., 2008).

Th17 cells produce a variety of cytokines including: IL-17, IL-21, IL-22, IL-26 and mediate host defence against both extracellular and intracellular bacteria and fungi (Peck and Mellins, 2010). IL-17 exerts pleiotropic inflammatory effects via inducing cytokines, chemokines and prostaglandins. Recently, an innate lymphoid cell subset that produces IFN-γ and IL-17 has been identified in inflamed colons and its absence ameliorates colitis (Buonocore et al., 2010). Notably, absence of IL-17 or IL-17R in donor T cells in the T-cell transfer model of colitis exacerbated disease with an increase in T helper type 1 cytokines (e.g. IFN-γ) suggesting that IL-17's role in the pathophysiology of IBD is controversial (O'Connor et al., 2009).

IL-22 may participate in gut homeostasis via its induction of β-defensins. Th17 cells demonstrate a striking dependence on the intestinal microbiota. Segmented, filamentous bacteria (SFB) residing in the terminal ileum appear to direct the development of mouse gut Th17 cells (Gaboriau-Routhiau et al., 2009; Ivanov et al., 2009). The immunological abnormalities of germ-free animals have established the dependence of the immune system on the commensal microbiota. However, the connection linking SFBs to Th17 development raises questions regarding what features of SFBs are responsible and what other members of the gut microbiota influence immune cell development.

Symbiotic factors of the microbiota: lessons from successful symbionts

  1. Top of page
  2. Summary
  3. Introduction
  4. Genome-wide association studies of IBD: a window into host-symbiotic factors
  5. Symbiotic factors of the microbiota: lessons from successful symbionts
  6. Conclusions and perspectives
  7. References

Bacteria of the human intestinal ecosystem face numerous challenges imposed by vicissitudes of human dietary intake, the mucosal immune system, competition from fellow members of the gut microbiota, transient ingested microbes and invading pathogens. 16S rRNA surveys and WGS sequencing of microbial communities and their members provide data sets that are informing the understanding of taxa and genomic potential of the human intestinal microbiota (Nelson et al., 2010). However, there is still much to be learned about the requirements for being a successful gut symbiont. (See Table 1 for a summary of gut symbiont factors, traits and features promoting symbiosis.)

Table 1.  Select factors, traits and features promoting symbiosis.
OrganismTrait/factorRole in mutualism
Lactobacillus  
 L. plantarumF1F0-ATPaseAids in acid tolerance
Sodium-proton antiportersAids cells in maintaining an internal pH
Encodes genes for multiple potential adhesionsAdhesion to the epithelium
 L. reuteriUreaseLocalized decrease in pH
Production of antimicrobial compound reuterinProvides a competitive advantage in gut
 L. rhamnosus GGPili with subunit SpaC (functions as an adhesin)Adhesion to the epithelium
Helicobacter  
 H. pyloriUreaseLocalized decrease in pH
Adhesins (BabA)Adhesion to the epithelium
Altered flagellin FlaAPrevents TLR5 binding while remaining functional
Vacuolating cytotoxinDecreases proliferation of human (VacA)
 CD4+ and CD8+ T cells and B cells
 H. hepaticusType VI secretion systemRegulates colonization
 Modulates host pro-inflammatory responses
Bacteroides  
 B. thetaiotaomicronPolysaccharide utilization loci (PULs)Allows for the utilization of diverse polysaccharides
Hybrid two-component sensor regulator systems (HCTS)Facilitates a quick response to nutrient flux in the gut allowing for rapid PUL regulation
 B. fragilisPolysaccharide AImmunomodulatory factor influencing T-regulatory cells

Bacteroides

Two Bacteroides spp., Bacteroides thetaiotaomicron and fragilis are highly adept symbionts of the human gut and both exhibit unique features that facilitate their commensalism. B. thetaiotaomicron is a saccharolytic gut bacterium that devotes 18% of its genome to polysaccharide utilization (Sonnenburg et al., 2005). This fit glycan forager ferments indigestible polysaccharides, providing the host with short chain fatty acids. B. thetaiotaomicron also degrades host mucins via gene clusters dubbed polysaccharide utilization loci (Martens et al., 2008). In B. thetaiotaomicron, a vast number of polysaccharide utilization loci are linked with hybrid two-component sensor regulator systems (HCTS) (Sonnenburg et al., 2006). By investigating bacteroides proliferation in response to fructans and unravelling how a fructose-binding HCTS regulates the fructan utilization structurally and functionally, Sonnenburg et al. provide insight into bacteroides' nimble responses to gut nutrient fluxes (Sonnenburg et al., 2010). Other studies illustrate B. thetaiotaomicron's reaction to the mucosal immune system and how the adaptive immune system regulates host–microbiota symbiosis. Using mice with adaptive immunity restricted to a single IgA directed against B. thetaiotaomicron's capsular polysaccharide and with a microbiota limited to B. thetaiotaomicron, Peterson et al. demonstrated the importance of IgA as a host symbiotic factor and how B. thetaiotaomicron dynamically adapts to the immune system by modulating its own epitope expression (Peterson et al., 2007). A recent study has elegantly leveraged massive parallel sequencing, transposon mutagenesis (termed insertion-sequencing), and gnotobiotic mouse models to develop a robust platform to reveal the microbial genes necessary for human gut symbiosis and identified many genomic features of B. thetaiotaomicron that influences its in vivo fitness (Goodman et al., 2009). Interestingly, the use of immunodeficient hosts (MyD88−/− and Rag1−/−) in this system did not reveal B. thetaiotaomicron genes related to immuno-adaptation, suggesting potential functional genomic redundancy in such pathways. This powerful forward genetics platform has the potential to illuminate symbiotic factors in many microbes beyond B. thetaiotaomicron and provide a wealth of knowledge about the genetics of symbiosis both within microbial communities and with their hosts.

Found in upwards of 70% of humans, Bacteroides fragilis represents another highly successful member of the human gut. Similar to B. thetaiotaomicron, glycans are at the centre of the mutualism between B. fragilis and its hosts. However, it is B. fragilis' glycans that have shaped the understanding of its symbiotic adaptations (Comstock, 2009). Protein glycosylation is core to B. fragilis' fitness and colonization capability; and the O-glycosylation systems of B. fragilis, common across Bacteroides species, may explain the prominence of this genus in the human gut (Fletcher et al., 2009). B. fragilis generates eight polysaccharide complexes (PSA–H), two of which are zwitterionic (PSA, PSB). Of these, PSA is unique in its immunomodulatory potential (Troy and Kasper, 2010). Using germ-free mice, B. fragilis has been shown to restore the physiological balance of Th1 and Th2 subsets in the gut and restore lymphoid organogenesis in a PSA-dependent fashion (Mazmanian et al., 2005). B. fragilis and specifically PSA also protect mice from Helicobacter hepaticus-induced colitis via T cell- and IL-10-dependent effects (Mazmanian et al., 2008). A recent study explains PSA's effect on IL-10 production – PSA, via a TLR2-dependent mechanism, induces FOXP3 transcription and directs the development of a specific gene expression profile for inducible Foxp3+ T-regs with increased T effector cell suppressive abilities (Round and Mazmanian, 2010). The recognition of PSA as an immunomodulatory factor and elucidation of its function in T-regs will hopefully spur the search for additional microbial symbiotic molecules that influence immune cell fate/function to maintain intestinal homeostasis.

Helicobacter

For several bacterial colonizers of the mammalian gut, their symbiotic status is not fixed and many maintain a potential to induce pathogenesis even within the gastrointestinal tract. The Helicobacter genus has several members which display this property: H. bilis, cinaedi, pylori and hepaticus (Fox, 2007). About 50% of thepopulation is colonized with H. pylori (Torres et al., 2007) and over 80% of those are asymptomatic (Amieva and El-Omar, 2008) illustrating this microbe's success as a symbiont. H. pylori is acid resistant and accesses the stomach epithelium by burrowing through the gastric mucus. Adhesins, including BabA, promote its epithelial adherence and urease decreases the local stomach pH, both of which exemplify its adaptation to the gastric niche. However, Warren and Marshall (1983) have established that H. pylori can cause gastritis and gastric and duodenal ulcers, as well as a subclinical gastritis which can progress in some individuals to cancer – inflammatory conditions at odds with its success as a symbiont. Significant emphasis has been placed on identifying and characterizing virulence factors, e.g. its type IV secretion system and CagA gene; however, much remains to be learned about its symbiotic persona – how H. pylori colonizes so many individuals without driving inflammation or epithelial injury.

Helicobacter pylori's lipotrichous flagella, consisting of FlaA and B, and H. pylori's close association with epithelial cells might provide an inflammatory stimulus as flagellins are ligands for TLR5 driven pro-inflammatory NF-κB responses (Tallant et al., 2004). However, H. pylori has adapted to this host detection system. Alterations in the N-terminal D1 domain of FlaA abolish TLR5 binding (Andersen-Nissen et al., 2005). Additionally, H. pylori's vacuolating cytotoxin (VacA), which affects the in vitro proliferation of CD4+ and CD8+ T cells and B cells, suggests that H. pylori has evolved mechanisms to blunt adaptive immune cell responses (Torres et al., 2007). H. pylori's success as a colonizer is reflected in its niche specialization, evasion of immune detection and immune suppression. However, changes in human ecology – antibiotics use, access to clean water and widespread use of acid blockade medications – may be altering the prevalence of H. pylori with unknown consequences for the health of human hosts and for their microbiota (Blaser and Falkow, 2009).

Helicobacter hepaticus is a spiral Gram-negative microaerophilic bacterium that was isolated from the livers and intestinal mucosal scrapings of mice with hepatitis (Fox et al., 1994). H. hepaticus colonizes the gastrointestinal tracts of WT mice (usually without incident) and is pervasive in many mouse colonies, attesting to its fitness as a commensal (Chow and Mazmanian, 2010). Mice with defects in adaptive and/or innate immunity, such IL-10−/− or RAG2−/− mice, develop spontaneous colitis upon colonization with H. hepaticus. This H. hepaticus-driven colitis has proven useful to dissect the contribution of the innate and adaptive immune systems, to intestinal homeostasis (Coombes et al., 2005; Fox et al., 2010). The availability of whole-genome sequence will provide additional insight into the symbiotic and dysbiotic relationships H. hepaticus can have with its murine hosts (Suerbaum et al., 2003).

A recent study suggests that H. hepaticus may maintain its symbiotic status in the gut of WT mice through its type VI secretion system (T6SS) (Chow and Mazmanian, 2010). T6SS secretion systems were originally discovered in pathogens and conceptualized as virulence factors (Cascales, 2008). By employing T6SS mutant strains (ΔIcmF and ΔHcp), Chow and Mazmanian (2010) demonstrated that H. hepaticus regulates both its colonization of the gut and limits its access to the cytoplasm of epithelial cells when internalized, via its T6SS. Internalization of WT H. hepaticus by epithelial cells elicits lower levels of inflammatory cytokines and mediators (TNFα, IL-6, iNOS and IL-1β) as compared with the T6SS mutants. Exposure of mice to the mutant T6SS strains resulted in heightened Th17 responses in vitro. While the factor(s) secreted by the T6SS mediating these effects remain(s) to be identified, this study establishes a novel perspective on T6SS as a host-symbiotic factor. Furthermore, recent data suggest that T6SS may play an important role in mediating communication between bacteria as well between eukaryotic hosts and their microbiota (Jani and Cotter, 2010).

Lactobacilli

Lactobacilli are common symbionts in the gut exhibiting numerous traits that facilitate their colonization of the gastrointestinal tract, some of which may confer benefits to the host. Whole-genome sequencing of human gut-derived Lactobacillus strains is providing insight into factors that contribute to the success of members of this genus. The L. plantarum genome possesses a variety of genes that confer an ability to adapt to the fluctuating pH environments of the gut such as an F1F0-ATPase and 10 sodium-proton antiporters (Kleerebezem et al., 2003). Lactobacillus reuteri also has evolved mechanisms for surviving the extreme pH shifts of the gastrointestinal tract. Like H. pylori, L. reuteri produces urease, which likely plays a role in its acid resistance (Walter et al., 2010). Binding to the intestinal epithelium confers a fitness advantage and may promote more permanent states of colonization. The L. plantarum genome encodes several potential adhesion molecules with a high degree of homology to other, previously classified adhesions (Kleerebezem et al., 2003). Many Gram-negative organisms display pili on their outer surface that allow for adhesion to mucosal surfaces; however, pili are less commonly found in Gram-positive bacteria (Telford et al., 2006). In contrast to the streptococcal pathogens in which pili have been identified, recent data suggest that Lactobacillus rhamnosus GG, a widely studied beneficial microbe originally isolated from the intestinal tract of a healthy human, possesses pili as well (Kankainen et al., 2009). Both spaCBA and spaFED encode subunits for these pili and SpaC appears to function as an adhesin as strains bearing non-functional SpaC showed drastically reduced mucus adherence (Kankainen et al., 2009). Similar to H. pylori, several lactobacilli possess traits (pH resistance and adhesion mechanisms) that promote their colonization along the gastrointestinal tract.

Certain lactobacilli may confer benefits on the host. Maintenance of the intestinal barrier and minimizing bacterial numbers adjacent to this border is essential for gut homeostasis. L. plantarum stimulates the expression of the tight junction-associated proteins, zonula occludens-1 and occludin, and thus the presence of L. plantarum along the mucosa may contribute to barrier integrity (Karczewski et al., 2010). L. reuteri produces several antimicrobials, one of which is reuterin, which may regulate bacterial colonization density along the mucosa. Reuterin (3-hydroxyprionaldehyde) exerts it antimicrobial activity by inducing oxidative stress in other microbes (Schaefer et al., 2010) and may contribute to colonization resistance against pathogens and eliminate niche competition for L. reuteri (Spinler et al., 2008). Numerous studies (> 1000) have examined the potential mucosal immunomodulatory effects of Lactobacillus strains isolated from the human gut. Whole-genome sequencing, transcriptomic analysis and functional metagenomic screens are beginning to offer an opportunity for a well-nuanced understanding of how this genus brokers its symbiotic relationship with its hosts.

The microbiota as a metagenomic community

In considering microbial symbiosis, we have so far focused on model symbionts and mentioned genomic methods only briefly. High-throughput metagenomic approaches are also becoming increasingly practical, and the first questions typically asked in such investigations is which microorganisms are present in a community, and how abundant are they? Sequencing technologies have been developed to determine the taxonomic composition of the gut microbiota from faecal samples by detecting organism-specific variants of highly conserved genes. The 16S rRNA gene is the most popular phylogenetic marker as it is ubiquitous, contains conserved regions appropriate for PCR amplification and possesses highly variable regions (Tringe and Hugenholtz, 2008). The nucleotide sequences within these regions reflect evolutionary divergence and can be matched with specific microorganisms or grouped into highly similar operational taxonomic units (OTUs) (Schloss, 2010). This process of mapping the short high-throughput reads to taxa requires computational approaches to handle sequencing errors, chimeric reads and uncertainty in matching 16S databases (Wooley and Ye, 2009). These data are typically analysed as relative abundances – that is, the fraction of the total microorganismal population comprised by each specific organism or OTU. However, 16S studies can be prone to biological and technical variability arising from: DNA extraction from a mixed microbial population, sequencing and PCR amplification bias, the multicopy and non-unique nature of the 16S gene, and subject-to-subject biological variation in the composition of the gut microbiota. Thus, care must be taken to quality-check phylometagenomic data, and careful normalization is required for comparison within and across studies (Hamady and Knight, 2009).

Conversely, functional characterization of a microbial community quantifies the microorganismal genes in the community, referred to as the metagenome. This is typically obtained using WGS sequencing, in which short reads from all DNA in the community are sequenced. Unlike WGS sequencing of individual organisms, which typically produce a fully assembled genome, metagenomes have been analysed using a range of techniques. Simple communities have been near-fully assembled (Tyson et al., 2004), but this is impractical in complex gut microbiota. Instead, open reading frames (ORFs) can be assembled in a targeted manner (Rho et al., 2010) and assigned putative function, much as would ORFs in a newly sequenced bacterial isolate. Alternatively, short reads produced as primary data can be treated as taxonomic markers or as gene fragments and analysed directly (Turnbaugh et al., 2009). Any of these approaches result in functional, rather than taxonomic, data describing a community: the abundances of metabolic enzymes, the presence or absence of biosynthetic gene clusters or the abundances of functional modules and pathways.

The microbiota represent a key component of future personalized medicine. Even without explicit causative or modulatory roles, there is tremendous potential in the ability to use the taxonomic or metagenomic composition of a subject's gut or oral flora as a diagnostic or prognostic biomarker. The microbiota are amazingly plastic; changing metagenomically within hours and metatranscriptomically within minutes in response to perturbations ranging from antibiotics to caloric intake (Dethlefsen and Relman, 2010). For any phenotype to which they are causally linked, this opens the possibility of pharmaceutical, prebiotic or probiotic treatments. However, as has been shown in recent years for polygenic genetic disorders, a combination of many environmental and microorganismal factors will undoubtedly interact in tandem to influence health and disease status.

Conclusions and perspectives

  1. Top of page
  2. Summary
  3. Introduction
  4. Genome-wide association studies of IBD: a window into host-symbiotic factors
  5. Symbiotic factors of the microbiota: lessons from successful symbionts
  6. Conclusions and perspectives
  7. References

Several diseases with an inflammatory component to their pathophysiology are associated with alterations in the gut microbiota: obesity, the metabolic syndrome, type 1 and 2 diabetes, asthma, irritable bowel syndrome and IBD (Blaser and Falkow, 2009). As well, acute diarrhoeal infections with intestinal pathogens, salmonella and campylobacter, are associated with IBD (Jess et al., 2010). A causality dilemma has remained intrinsic to considerations of the role of the microbiota in these diseases. The experimental and computational tools are evolving to explicate these associations. Animal models, which bear host mutations similar to those in human diseases and where microbial communities can be manipulated as in gnotobiotic animals, are vital experimental tools for unravelling the molecular mechanisms behind the associations observed between the microbiota and disease. Novel methods, such as insertion sequencing which pairs transposon mutagenesis with massive parallel sequencing, will facilitate the forward genetic screens of defined community members to unravel their symbiotic features. Overall, for gaining insight into the metagenomic aspects of host and microbiota symbiosis, it is increasingly crucial to have proper computational and statistical tools to support these investigations.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Genome-wide association studies of IBD: a window into host-symbiotic factors
  5. Symbiotic factors of the microbiota: lessons from successful symbionts
  6. Conclusions and perspectives
  7. References
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