Abstract
The gut microflora can be considered a metabolically active organ composed of a vast and complex community of microorganisms that has an important role in the stability and functional activity of the intestinal ecosystem.
Recently, thanks to microarray technology, a global screening of the microflora's regulated genes has allowed the analysis of the complex bacteria–host interplay. In particular, most of our knowledge comes from studies on Bacteroides thetaiotaomicron, a prominent member of the intestinal microflora of mice and humans.
The results of published studies have revealed that Bacteroides thetaiotaomicron modulate the expression of a large quantity of genes implicated in different aspect of host physiology.
This review aims to illustrate the specific contributions of this intestinal microorganism in three important aspects of host physiology: mucosal barrier reinforcement, immune system modulation and nutrients metabolism. In particular, we focus on recent insights about the molecular mechanisms by which Bacteroides thetaiotaomicron help the host in these important functions.
Keywords
1. Introduction
The human intestine is populated by a complex and abundant consortium of microorganisms that, together with the mucosal barrier and the local immune system, are part of the intestinal ecosystem [
[1]
].The total number of bacteria that colonize our body surfaces is estimate to be 10 times greater than the total number of our somatic cells. The impact of these indigenous microbial communities on human physiology is more pronounced in the intestine, since this organ contains most of our bacteria [
[2]
]. It has been shown that gut microflora contains more than 100 times the number of genes in our genome, thus it can be considered as a postnatally acquired organ composed of bacterial cells that can perform different functions [[3]
].Host–microbial interactions seem to be of high complexity since they span a continuum from mutually beneficial to pathogenic in different conditions. Several studies in the last years have emphasized the importance of understanding the contributions of this microflora to human health and disease and some of the factors involved in a particular host–microbial relationship are now being elucidated at the molecular level [
4
, 5
].Much of our understanding of the molecular mechanisms that can explained the host–bacterial mutualism comes from studies of Bacterioides thetaiotaomicron (BT), a prominent member of the distal intestine microflora of mice and humans that modulates a number of essential host functions [
6
, 7
].BT is Gram-negative anaerobe that comprised 6% of all bacteria and 12% of all Bacteroides in the human intestine and represents the most comprehensive 16S rRNA sequence based enumeration of the adult human colonic microflora published to date [
[7]
]. Based on these important features, this genetically manipulatable organism has been used as a model for understanding the impact of constituents of the microflora on gut gene expression.In particular, elegant studies performed on germ-free mice colonized with BT have demonstrated the importance of host–bacteria interactions for the establishment of the intestinal ecosystem [
8
, 9
].The host is protected from the resident intestinal microflora by the physical and chemical barriers formed by the gastrointestinal epithelium [
10
, 11
]. These barriers are reinforced by the acquired mucosal immune response. During the development of the ecosystem, antigen specific B and T cells learn to regulate their responses to resident microorganisms, such that a state of controlled inflammation is established in adult hosts [[12]
]. On the other hand, the gut microflora is a key regulator of the immune system since it is able to induce tolerance to certain microbial epitopes and to potentiate the immune response against others [[13]
]. Moreover, these microorganisms and their genome provide us with important physiological activities that are not encoded in our own human genome, such as the ability to carry out a set of biochemical reactions [14
, 15
, 16
].Since most of BT functions seem to be associated with modulation of gene expression, recent advances in functional genomics and in particular the introduction of microarray technology, are creating new opportunities for better understanding how hosts and their microbes collaborate in many functions of the gut [
8
, 15
, 17
].A comprehensive molecular analysis of the cross-talk between BT and the gut should consider different aspects of their interaction ranging from mucosal barrier function to immure regulation and nutrient processing.
2. BT and mucosal barrier
The intestine has an important function in working as a barrier against the surrounding environment to permit only the favourable substances to be absorbed and the dangerous compounds to be secreted via the faeces.
The interplay between the microflora and the mucosal barrier is undoubtedly dynamic, reciprocal and very intricate [
[18]
]. First, an intact mucosal barrier is critical to avoid resident intestinal microbes invasion since barrier disruption can induce immune responses that cause pathology such as inflammatory bowel disease [[19]
]. Second, several evidences suggest a role of gut microflora in epithelial barrier development and reinforcement.It has been shown that the absence of indigenous microflora results in altered intestinal morphology and function in germ-free animals. Most reports indicate a prolonged epithelial cell cycle time in germ-free rodents that may be responsible for aberrant mucosal morphologies such as shorter villi, longer microvilli and decreased surface area of mucosa [
[5]
]. On the other hand, the presence of microflora has been associated with a higher intestinal epithelial turnover [[20]
] and with the stimulation of IgA response that is an important aspect of the mucosal barrier function [[21]
].The epithelial surface of the intestinal tract comprises a mucosal layer, in addition to various cell populations, and plays a major role as the first line of protection in the intestine. Susceptibility and resistance to intestinal infections is often associated with the presence or absence of receptors appropriately glycosylated. A recent study has shown that the glycosylation pattern of mice intestine is modified by BT or soluble factors produced by this microorganism in a very specific manner by changing either the level of expression or the topology of the glycans. This process seems to be crucial in signalling and mediating host mucosal immune response [
[22]
].The ability of microflora to modify mucosal responses to pathogens is not confined to physiologic properties such as transport and barrier function, but also extends to the program of gene expression evoked by BT colonization. In particular, the IgA production is associated with increased expression of the polymeric immunoglobulin receptor (pIgR) that transports IgA across the epithelium. A second important response to BT colonization is the upregulation of the gene encoding the small proline-rich protein-2 (sprr2a), a member of the sprr family that contributes to the barrier functions of squamous epithelia, both as a component of the cell envelope and as cross-bridging proteins linked to desmosomal desmoplakin [
[15]
]. BT colonization of germ-free mice has been also associated with an increased expression of the CRP-ductin gene encoding a mucus layer component (MUCLIN) and of the decayaccelerating factor (DAF), an apical epithelial inhibitor of complement-mediated cytolysis [[15]
].Finally, BT has been shown to stimulate matrilysin, an important enzyme involved in the activation of prodefensins, produced in intestinal Paneth cells [
[23]
]. The upregulation of these genes participates in fortifying the intestinal mucosal barrier against pathogens and avoiding mucosal damage from activation of complement components in intestinal secretions.Overall, these reports suggest that a complex network of mechanisms is involved in mucosal barrier reinforcement by intestinal microflora. An altered host–commensal relationships may thus affect microbial contributions to mucosal barrier integrity, leading to enteric infections and contributing to the pathogenesis of disorders such as inflammatory bowel diseases.
3. BT and immune system
It is well established that the intestinal microflora is a key regulator of the immune system.
The colonizing microflora not only provides colonization resistance to potentially pathogenic bacteria but has also a major role in the development of the intestinal immune system, both in terms of gut-associated lymphoid tissue (GALT) and mucosal immunity, and the induction of oral tolerance [
[24]
].Some authors have proposed that the immune system does not react against the commensal microflora [
[25]
]. However, it has been reported that commensal bacteria can elicit systemic antibody responses also in normal conditions even if this immune response does not lead to tissue damage or bacteria elimination from the intestine [[26]
]. In particular, it has been shown that in germ-free mice, BT alone can induce a humoral response characterized by IgA and IgG production that is downregulated in the presence of Bifidobacteria species, thus suggesting that the association of different microbial species can be an important mechanism of tolerance [[27]
].Evidences concerning the specific role of BT in immune system modulation are still limited and most of our knowledge is derived from studies performed in germ-free animals, thus referring to the gut microflora in general and not to this specific strain.
These studies have shown that the innate, as well as the acquired, immune system is affected by the lack of resident bacteria. For example, macrophage chemotaxis and phagocytic activity are inhibited in these animals, and spleen-derived macrophage precursors are fewer in number and express decreased levels of MHC class II [
[28]
].High density oligonucleotide-based microarrays performed to define the transcriptional responses of the distal small intestine to the colonization with BT have shown a 11-fold increase in the transcript of Angiogenin 4 (Ang4), a novel bactericidal gut protein involved in systemic innate immunity and expressed especially by Paneth cells [
15
, 16
]. By inducing the expression of mediators of the innate defence, such as Ang4, BT seems to play a key role in innate immunity, helping to ensure rapid and efficient secretion of these mediators against entheropathogens.Even greater differences are observed in the acquired immune system. Lymphoid constituents of the mucosal immune system are drastically underdeveloped in germ-free animals: the numbers of lymphocytes in the lamina propria are decreased and intestinal lymphoid aggregates, such as the Peyer's patches and mesenteric lymph nodes, are smaller [
[29]
]. Extraintestinal lymphoid organs, such as the spleen and thymus, are also underdeveloped in germ-free mice, providing additional evidence of the role of the resident microflora in the development of the immune system. The immunoglobulin class profile is greatly altered in germ-free animal: IgG concentrations are much lower than in conventional raised animals, with little or no production of IgA. On the other hand, BT colonization is able to induce a systemic as well as local immune response as documented by increased serum levels of IgA and IgG and by the already described stimulation of mucosal IgA production, an important aspect of mucosal barrier function.The human gut microflora is also important in regulating host inflammatory responses. The cellular and molecular bases of these actions are unknown but it has been shown that BT selectively antagonizes transcription factor NF-kappaB, enhancing the nuclear export and nucleocytoplasmic redistribution of the NF-kappaB subunit RelA, through a mechanism dependent on peroxisome proliferator activated receptor-gamma (PPAR-gamma). This PPAR-gamma-dependent anti-inflammatory mechanism defines new cellular targets for therapeutic drug design and interventions for the treatment of chronic inflammation [
[30]
].Finally, BT is able to reverse epithelial damage produced by pro-inflammatory cytokines such as TNF-α- and IFN-γ, modifying both transepithelial resistance and permeability. This effect seems to be related to the activation of the PI3K pathway by BT and may support the role of PI3K, a downstream effector of Toll-like receptors, in the regulation mucosal responses to the intestinal microflora [
[31]
].Based on these evidences commensal bacteria in general and BT in particular may exert a dual function, the stimulation of mucosal mechanism of defence on one side and the maintenance of the homeostasis of the immune response on the other.
The disease may result from the breakdown of this global immune regulation at the critical interphase of the host with the external environment.
4. BT and nutrients metabolism
A central aspect of the host–bacterial mutualism is the opportunity to use metabolic capabilities that allow both partners to benefit from an otherwise poorly utilized nutrient substrate. It has been shown that conventionally raised animals require 30% less caloric intake to maintain their body weight than their germ-free counterparts [
[32]
] demonstrating that the microbiota has an important role in extracting maximum nutritional value from the diet. In particular, two complementary mechanisms seem to work at the same time. First, microbial metabolism is responsible for the conversion of many dietary substances into molecules that can be absorbed and utilized by the host. Second, the presence of microbes can alter the intrinsic metabolic apparatus of host cells, resulting in more efficient nutrient uptake and utilization [7
, 14
].As concerning the first aspect important is the contribution of BT to carbohydrate metabolism. Mammals are limited in their capacity to hydrolyse and utilize most polysaccharides ingested with diet. By recruiting a microflora that can hydrolyse these carbohydrates, mammals do not need to evolve the complex repertoire of glycosylhydrolases that would be required to metabolize the wide variety of dietary polysaccharides.
The starch utilization system of BT is the best-understood example of polysaccharide utilization. Functional genomic studies have shown that the genome of BT contains not only genes involved in starch metabolism (susA–susG) but also a transcriptional factor that responds to the presence of glucose oligomers by increasing transcription of metabolic genes [
33
, 34
]. Thus, the bacteria expend energy to express genes involved in nutrient metabolism only when the particular nutrient is available in the gut ecosystem [[35]
].BT has also developed the capacity to degrade a variety of host-derived glycoconjugates (glycans) elaborated on the surfaces of intestinal epithelial cells including chondroitin sulfate, mucin, hyaluronate and heparin [
15
, 36
]. Like components of the starch utilization system, this hydrolases are inducible when the particular molecule is available in the gut ecosystem [35
, 37
].Host glycans are a critical nutrient source for BT and other bacterial species. Several advantages can derive from the ability to utilize these glycans as nutrient source: they are constantly available due to epithelial cells turnover; once degraded they are promptly fermented to obtain carbon and energy; and competition for these glycans is limited since they are degraded only from some bacterial species [
[38]
]. Recent studies performed on animal models have indicated that the regional specificity in glycan production may be functionally linked to the spatial and temporal complexity of the intestinal microflora [[39]
].On the other hand, it has been shown that BT is able to modify host glycoconjugate production as demonstrated by the fucose utilization pathway [
8
, 19
]. The fucose gene cluster in this organism consists of five genes: four genes encode proteins directly involved in fucose uptake and metabolism; one gene is a transcriptional repressor (FucR) that acts regulating transcription of the fucose pathway enzymes and transcriptional repression of a second genetic locus that drive glycan synthesis by the host. Thus, FucR acts as a molecular sensor of environmental fucose availability and matches bacterial demands for fucose with host supply. By using this strategy, the host has to synthesize only has much glycan as necessary to support the nutritional needs of its commensals [[40]
].The host is able to derive nutritional benefit from the products of bacterial breakdown since mammals have developed mechanisms for absorbing and utilizing products of bacterial fermentation. The predominant end products of bacterial fermentation in the gut are short-chain fatty acids that are absorbed in the caecum and the colon by passive diffusion across the epithelium and are metabolized by different organs [
[14]
]. In addition to their nutritional value, SCFA have important effects on other aspects of gut physiology. For example, SCFA have been implicated in water absorption, intestinal blood flow stimulation [[41]
] and epithelial proliferation and differentiation [[42]
].The gastrointestinal microflora can contribute to amino acids homeostasis [
[43]
] and vitamins production in the host. Vitamin synthesis by gut bacteria has been recognized for many years and it has been shown that germ-free rodents require higher amounts of certain B vitamins (e.g. B12, biotin, folic acid and pantothenate) and vitamin K in their diets than their conventionally raised counterparts [[44]
].The ability of gut microflora to maximize the nutritional value of the diet derives from direct microbial degradation of dietary macromolecules but also from microbial induction of intestinal genes that facilitate recovery of nutrients [
15
, 38
, 45
].Functional genomic approaches showed that BT colonization of germ-free mice produced changes in expression of a number of host genes involved in the processing and absorption of carbohydrates, as well as the breakdown and absorption of complex dietary lipids [
[46]
]. Bacterial colonization led to increased ileal expression of the NaC/glucose co-transporter, the principal mediator of the epithelial glucose uptake [[47]
]. There was, also, an upregulation of different genes involved in lipid absorption and metabolism: pancreatic lipase-related protein 2, which breaks down triacylglycerols; co-lipase, which stimulates pancreatic lipase-related protein 2 activity; L-FABP, a fatty acid-binding protein involved in intracellular trafficking of fatty acids; and apolipoprotein A-IV, which mediates export of triacylglycerols from the enterocytes [[46]
]. In addition, colonization of germ-free mice with BT resulted in a decrease in expression of fasting induced adipocyte factor (FIAF) [[48]
].BT also affected genes involved in regulating dietary metal ions absorption and drugs detoxification [
[15]
]. In particular, it has been reported an upregulation of the epithelial high affinity copper transporter 1 (CRT1), as well as a downregulation of glutathione S transferase, which conjugates glutathione to a variety of electrophiles, and of multidrug resistance protein-1a (Mdr1a), which exports glutathione-conjugated compounds from the epithelium [[49]
].Overall, these evidences suggest that the role of BT in nutrients metabolism is complex and dynamic, involving not only direct participations in metabolic activities but also an influence on gut gene expression. Both these aspects are fundamental for host–bacterial relationship allowing the host to gain some control on the composition of the gut microflora on one hand, and to improve its own metabolism on the other.
5. Conclusions
The adult gastrointestinal ecosystem is based on a stable alliance among the resident microflora, immune mediators and the mucosal barrier. All three components are essential for complete functional and developmental maturity of the gastrointestinal ecosystem. Gut microflora participates in the development and functional activity of the other two members and impart stability to the gut ecosystem by having the capacity to conduct a multitude of biochemical reactions involved in host and microbial metabolism.
The studies described above provide a broad-based characterization of functional and molecular responses to colonization with a prototypic gut commensal, BT. In particular, functional genomics studies reveal that this microorganism is able to modulate expression of wide variety of host genes that participate in different and important physiological functions.
These observations emphasize the need to understand more about the roles played by other components of the intestinal microflora in host biology. The capacity to enumerate these microorganisms represents the first step in understanding molecular contributions of specific microbial species to human physiology. The discover of a species selectivity for some of the colonization-associated changes in gene expression could help to better understand how our physiology can be affected by changes in the composition of indigenous microflora. By identifying these host genes and the microbial effectors of their expression, we should be able to identify new molecular targets and new chemical strategies for influencing different aspects of host physiology.
Practice points
- •BT colonization leads to important functional and molecular responses of the gut ecosystem.
- •The BT-host interplay results in the upregulation of different genes involved in mucosal barrier reinforcement against pathogens.
- •BT acts as a regulator of the intestinal immune system.
- •BT has a central role in nutrients metabolism, involving not only a direct participation in gut metabolic activities but also an influence on gut gene expression.
Research agenda
- •The analysis of molecular contributions of different microbial species to gut physiology.
- •The identification of species selectivity for some changes in gene expression associated with bacterial colonization.
- •The identification of molecular targets for influencing different aspects of host physiology.
Conflict of interest statement
None declared.
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Article info
Publication history
Accepted:
April 16,
2007
Received:
December 15,
2006
Identification
Copyright
© 2007 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Inc. All rights reserved.
