Host–microbe interactions that facilitate gut colonization by commensal bifidobacteria

Review

Host–microbe interactions that facilitate gut colonization by commensal bifidobacteria Marco Ventura1, Francesca Turroni2, Mary O’Connell Motherway2, John MacSharry2 and Douwe van Sinderen2 1

Laboratory of Probiogenomics, Department of Genetics, Biology of Microorganisms, Anthropology and Evolution, University of Parma, Italy 2 Alimentary Pharmabiotic Centre and Department of Microbiology, Biosciences Institute, University College Cork, Western Road, Cork, Ireland

Microorganisms live in a myriad of ecological niches. The human intestine is among the most densely populated environments; here, a multitude of bacteria appear to have co-evolved to impact beneficially upon the health of their human host. The precise molecular mechanisms and signaling pathways employed by commensal bacteria, including those that facilitate colonization and persistence, remain largely unknown despite the perceived positive effects of such host–microbe interactions. In this review we discuss several fascinating relationships between the gastrointestinal tract and commensal bacteria, with particular emphasis on bifidobacteria as a prototypical group of human enteric microorganisms. Microbial colonization of the infant gut Humans are colonized by a diverse, complex, and dynamic collection of bacteria that outnumber the somatic and germ cells of an individual by about tenfold, and that collectively represent 100-fold more genes than those contained in their host genome [1]. The assembly of microorganisms in the gastrointestinal tract (GIT), also known as the gut microbiota, is on average estimated to consist of eight major bacterial phyla [2–4]. The intestinal microbiota is one of the most densely populated microbial communities and impacts upon different human functions such as intestinal cell proliferation and differentiation, intestinal pH, the development of the immune system, and the innate and acquired immune response to pathogens [5]. The symbiotic interactions between the various bacteria and their human host can be categorized as a continuum ranging from mutualism and commensalism through to pathogenesis. In the human gut environment, co-evolution of bacteria and their host may lead to the development of commensal relationships, where neither partner is disadvantaged, or to symbiotic relationships where unique metabolic activities or other benefits are provided to both partners. It is now well established that the gut microbiota composition is highly variable between individuals, and is also subject to considerable change throughout the lifetime of Corresponding author: van Sinderen, D. ([email protected]). Keywords: gut microbiota; bifidobacteria; host–microbe crosstalk; genomics.

an individual [2,4,6–8]. Development of the human gut microbiota commences at birth with colonization by bacteria from the vaginal and fecal microbiota of the mother, as well as other environmental microbes [9]. Maternal milk may also serve as an inoculum for breast-feeding infants, and is presumed to play a crucial role in the establishment of a bifidobacteria-abundant infant gut microbiota before the weaning period [10]. Thus, initial bacterial colonization is dependent on the microbial communities to which newborns are exposed. In vaginal deliveries, facultative anaerobes including members of the Streptococcus and Escherichia genera dominate during the first days of postnatal life [11]. Following sufficient reduction of oxygen levels in relevant parts of the GIT, obligate anaerobic bacteria, in particular bifidobacteria and clostridia, are established in the infant intestine [12]. In addition, bacterial colonization of the neonatal gut is influenced by many factors, including type of feeding (human milk vs formula milk), mode of delivery (vaginal delivery vs caesarean delivery), and antibiotic treatment [8]. One of the most important effects of the (specific composition of the) early gut microbiota is on the functionality of the host immune system. The early gut microbiota of infants delivered by caesarean section is dominated by Klebsiella, Enterobacter, and Clostridium, in contrast to vaginally delivered infants, which possess a microbiota with a high proportion of bifidobacteria [13]. These differences in the early gut colonization have been associated with immune-linked disorders such as asthma [14], atopy, and allergy [15]. Recent studies have shown that the gut microbiota induces the development of peripheral colonic regulatory T cells (Treg) cells [16] which are commensal-specific, and that microbial exposure early in life is required to prevent the accumulation of colonic and lung invariant natural killer T (iNKT) cells [17]. iNKT cells are associated with mucosal tissue damage and inflammation, and their presence has been associated with the occurrence of inflammatory bowel disease and asthma [17]. An important parameter that influences initial gut colonization and thus microbiota composition is diet. A consistent correlation has been observed between gut microbiota composition and type of feeding (i.e., human

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Review milk vs formula milk), with a higher abundance of bifidobacteria in breastfed infants as compared to those that are bottle-fed [18]. Genomic analyses of bifidobacterial species that are typically associated with the infant gut microbiota (e.g., Bifidobacterium longum subsp. infantis) has revealed the existence of a gene set that encodes enzymes dedicated to the metabolism of human milk oligosaccharides (HMOs) [19,20], which highlights the genetic specialization and presumed co-evolution of these bacteria to the infant gut and associated diet. Breastfeeding provides many immune-linked benefits such as protection against gastrointestinal and respiratory illnesses, and otitis media [21]. Different components of human milk, such as immunoglobulins, antimicrobial peptides, growth factors, lysozyme, lactoferrin, and HMOs, provide passive immunity, stimulate intestinal epithelial development, and are responsible for at least some of the beneficial effects associated with breastfeeding [22]. However, such compounds are also claimed to shape the infant gut microbiota, and may thus indirectly modulate the host immune system through various different microbial products (see below). For example, recent work has shown that HMO-grown B. longum subsp. infantis stimulates intestinal epithelia inducing the anti-inflammatory cytokine interleukin (IL)-10 and junctional adhesion molecules [23]. Here we will discuss how key members of the human gut microbiota, in particular bifidobacteria, interact with the immune system of the host through the action of a variety of molecules that may also play a pivotal role in gut colonization and persistence. Intestinal immunity and gut microbiota The human intestine is the organ with the highest level of immune activity in the body [24]. It is charged with mounting appropriate responses to harmful bacteria and antigens, and at the same time with developing and maintaining tolerance to other substances (e.g., diet components) and gut commensals, including health-promoting microorganisms [25]. The intestinal mucosa plays a key role as a physical barrier to allow only specific molecules to cross it. However, the contents of the gut lumen are constantly sampled by the gut-associated lymphoid tissue (GALT), such as the Peyer’s patch of the small intestine. Overlying the Peyer’s patch are specialized microfold cells (M cells), which transcytose luminal antigens to underlying antigen-presenting cells (APCs) [26], such as macrophages and dendritic cells (Figure 1). The epithelia, together with the APCs, can distinguish pathogenic from commensal microbes by the expression of pattern recognition receptors (PRRs), which recognize particular microbial components. Although some PRRs may be specific for pathogen signals, many also recognize signals that are common to both pathogens and commensals [lipopolysaccharide (LPS), teichoic acid, CpG motifs, and flagella]. Thus, other factors such as the location where the surveillance takes place, based on the notion that commensals do not invade the mucosa as deeply as pathogens, may play an important role in making this distinction. PRRs include the transmembrane Toll-like receptors (TLRs), C-type lectin receptors (CLRs), cytoplasmic NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs) [27,28]. These 468

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receptors recognize and bind ligands present in the gut lumen, and initiate maturation of APCs and subsequent immune responses, thus playing a pivotal role in the interaction between intestinal microorganisms and host immune defense, immune-cell recruitment, IgA production, and mucosal homeostasis [28]. Furthermore, TLR recognition of commensal bacteria by intestinal epithelia and subsequent MYD88-dependent signaling has been shown to be indispensable for the maintenance of gut homeostasis, which reinforces the hypothesis that specific microorganisms and the human host have a symbiotic relationship [29,30]. However, although intestinal APCs monitor and are relatively unresponsive to gut commensal bacteria, a recent study has shown that they can discriminate between pathogenic and commensal bacteria via the intracellular NLRC4 inflammasome which mediates clearance of the pathogenic bacteria from the gut [31,32]. In addition, specific interaction of gut bacteria-derived molecules with intestinal immune cells affects the development and maturation of the innate and adaptive immune system. For example, the exopolysaccharide (EPS) Polysaccharide A (PSA), produced by the common gut bacterium Bacteroides fragilis, exerts immunomodulatory activities, including the correction of systemic T cell deficiencies and Th1/Th2 imbalance, while also playing a role in immune maturation by directing lymphoid organogenesis [33]. Bacteroides thetaiotaomicron also induces secretion of the species-specific bactericidal protein, angiogenin 4, by gut Paneth cells [34]. It is now generally accepted that gut commensal bacteria promote the induction of Treg to sustain the Th1/Th2 balance [35]. Indeed, Bifidobacterium bifidum-derived membrane vesicles have recently been shown to induce dendritic cell-mediated Treg differentiation [36]. Considering the importance of the gut microbiota in driving the host immune system, it is important to understand how gut commensals colonize and establish themselves within their host. Models of gut commensalism Three representative groups of human gut commensals, in other words Bacteroides, bifidobacteria, and lactobacilli, are currently used as model bacteria to investigate host– microbe interactions, as well as crosstalk and other types of molecular interplay between members of the autochthonous gut microbiota [37,38]. Bifidobacteria were originally isolated from the feces of a breastfed infant, and to date 38 different species are recognized, all of which have been isolated from the GIT contents of mammals, birds, or insects [39,40]. Furthermore, the genus Bifidobacterium includes three subspecies divisions: B. animalis subsp. animalis and B. animalis subsp. lactis; B. pseudolongum subsp. globosum and B. pseudolongum subsp. pseudolongum; and B. longum subsp. infantis, B. longum subsp. longum and B. longum subsp. suis. Bifidobacteria and lactobacilli that have been isolated from the human gut, or that are used for human consumption, are the subject of scientific research in view of their commercial application as health-promoting probiotic cultures. In recent years, genome sequencing and subsequent functional analysis of enteric bifidobacteria and lactobacilli has seen major progress, generating insights into the diversity and evolution of

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Villus Epithelial adherence, ght juncon and cytokine inducon. Mucin producon, mucus adherence and degradaon.

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Figure 1. Commensal interaction with the intestinal immune system. The gut lumen is separated from the lamina propria by a single layer of epithelium overlying intestinal villi and lymphoid aggregates such as the Peyer’s patch. Intestinal villi are folded to increase surface area, thus allowing increased nutrient absorption while lymphoid aggregates continuously monitor the microbial contents of the lumen. The intestinal epithelial cells differentiate from epithelial stem cells in the crypts, at the base of villi, to specific functions depending on gut location. Specialized enterocytes absorb nutrients (small intestine) and water (colon), and have apical microvilli which again increase the surface area and absorptive capacity. The epithelial layer is coated in a layer of mucus, secreted by goblet cells, which is adhered to, metabolized, degraded, and induced by commensal bacteria, as is bactericidal protein production by Paneth cells at the crypt base. Commensal bacteria also adhere tightly to the apical face of the enterocytes via specialized pili, and induce host cytokine signaling and tight-junction maintenance. The gut luminal environment also induces commensal capsular polysaccharide production and microbial competitive factors such as bacterocins. Specialized epithelial M (microfold) cells in the follicle-associated epithelium overlying the secondary lymphoid tissue, such as the Peyer’s patch, sample (transcytose) the luminal contents and initiate processing and antigen presentation by dendritic cells and macrophages – which induce the adaptive T cell and B cell responses. The specialized polysaccharide coat of commensal engages pattern recognition receptors (PRRs), which lead to a homeostatic immune response by Treg cell induction and IgA-producing plasma cell differentiation in the mesenteric lymph nodes. These home to the intestinal villi as do dendritic and T cell subsets (intraepithelial lymphocytes). The IgA-producing plasma cells secrete the dimeric antibody, sIgA, into the lumen – this subsequently binds antigens and commensal bacteria and leads to further intestinal immune sampling. This immune sampling cascade helps to maintain gut homeostasis between the host and the commensal microflora.

commensal bacteria, as well as into the molecular basis for health-promoting host–microbe interactions. Furthermore, integration of comparative and functional genomic information with gene expression data of the human gut has expanded our understanding regarding the roles of gut commensals in both microbe–microbe and host–microbe interactions, and has revealed several key molecules, produced by particular Bacteroides, Bifidobacterium, and Lactobacillus species, that promote the establishment of these microorganisms in the human intestine. Bifidobacterial adaptation to the human gut Genomic analyses of representative members of the gut microbiota suggest that the genetic capacity to metabolize complex, host-indigestible carbohydrates can significantly impact upon the composition of the gut microbiome [39]. This genomic adaptation is obvious in many bifidobacterial genomes where over 8% of annotated genes encode enzymes involved in carbohydrate metabolism [41]. The presence of a diverse repertoire of CAZymes in bifidobacterial genomes suggests that they target a diverse array of different complex carbohydrates, but the precise targets of

these enzymes remain to be identified ([42] for review). In addition, glycans and glycoproteins produced by the host represent an important carbon source for bacteria, in particular in the distal colon where the availability and accessibility of simple carbohydrates is limited. Mucins are such host-produced glycoproteins secreted by goblet cells in the intestine, and form the main glycoprotein component of the mucus layer covering the intestinal mucosa. The main monosaccharide constituents in mucin-derived glycoproteins are N-acetylglucosamine, N-acetylgalactosamine, fucose, and galactose, and these glycoproteins are sometimes decorated with sialic acid and sulfate groups [43]. The infant isolate B. bifidum PRL2010 was shown to degrade mucin [44], and in silico analyses of its chromosome together with functional genome approaches revealed the existence of a gene set involved in mucin metabolism (Figure 2). Recently, another human gut microbiota member, Akkermansia muciniphila, was identified as an important mucin degrader [45–50], and this has expanded the known repertoire of enteric bacteria that degrade mucin beyond Bacteroides spp. [51] and Ruminococcus spp. [52,53]. 469

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Figure 2. Comparative analysis of Bifidobacterium genomes (modified from [53]). Circular genome atlas of Bifidobacterium bifidum PRL2010 with mapped orthologs (defined as reciprocal best FastA hits with more than 30% identity over at least 80% of both protein lengths) (from the exterior, circle 1) in six publicly available Bifidobacterium genomes: circle 2 B. bifidum S17; circle 3 Bifidobacterium adolescentis ATCC 15703, circle 4 Bifidobacterium dentium Bd1; circle 5 Bifidobacterium animalis subsp. lactis DSM 10140; circle 6 Bifidobacterium longum subsp. longum NCC2705; circle 7 B. longum subsp. infantis ATCC 15697. Circle 8 illustrates B. bifidum PRL2010 G+C% deviation, followed by circle 9 which highlights B. bifidum PRL2010 GC skew (G-C/G+C). The lighter versus the darker color in circles 8 and 9 represent the region with higher and lower GC content respect to the genome average. In addition, the outer arrowheads indicate the main genetic loci encoding enzymes involved in the mucin metabolism, which are mapped on the circular genome atlas of B. bifidum PRL2010. White regions represent non-coding sequences and rRNA loci. The genetic loci encoding for different mucin-breakdown enzymes identified in the genome of B. bifidum PRL2010 are shown in the panel on the right. Their position in the genome sequences of PRL2010 are marked on the atlas map with red arrowheads. Abbreviation: Hyp, hypothetical.

Another intriguing example of host-produced glycans that are fermented by particular elements of the gut microbiota are HMOs, which are present in human milk but which are not utilized by the host. The genome sequence of B. longum subsp. infantis ATCC 15697, a common infant gut inhabitant, contains a gene cluster that encodes glycosidases and carbohydrate transporters necessary for importing and metabolizing HMOs [19]. This 43 kb gene cluster encodes a variety of predicted or proven catabolic enzymes, such as fucosidases, sialidases, a bhexosaminidase, and b-galactosidases, as well as extracellular solute-binding proteins and permeases that are devoted to HMO metabolism [19,20,54,55]. Furthermore, the genome of this organism contains a complete urease operon predicted to be involved in the utilization of urea which represents an important nitrogen source in milk [19]. Nevertheless, given that HMOs and mucus O-linked glycans are so similar in structure it is difficult to predict if 470

enzymes are specific for one or the other, or target both. Recently, Marcobal et al. [56] demonstrated that B. longum subsp. infantis ATCC 15697 cannot utilize purified mucus O-glycans, whereas other enteric bacteria such as Bacteroides can. By contrast, B. longum subsp. infantis ATCC 15697 was optimized for HMOs and could outcompete Bacteroides that use these substrates somewhat poorly through mucus O-glycan-degrading pathways. Further evidence of commensal adaptation to the human gut is demonstrated by the ability of these microorganisms to survive the stressful conditions that are encountered in the intestine. GIT stresses encountered include exposure to bile salts, osmotic stress resulting from diet variation, and acidic stress during passage through the stomach. Bifidobacteria counteract these stressful conditions through a repertoire of molecular chaperones, bile efflux transporters, bile salt hydrolases, and ATPases [57]. In addition, bifidobacteria, similarly to

Review other gut commensals, have evolved sensing systems and defenses against stress which allow them to withstand harsh conditions and sudden environmental changes. In this context, microbial stress responses rely on coordinated gene expression to alter various cellular processes and structures (e.g., DNA metabolism, housekeeping genes, and membrane composition) which act in concert to improve bacterial stress tolerance [57]. The integration of these stress responses is accomplished by regulatory networks that allow the cell to react rapidly to various and sometimes complex environmental changes [58]. Microbial molecules mediating host interaction The cell envelope of gut commensals represents a pivotal structural interface between bacteria and the environment, and harbors proteins and carbohydrates that facilitate bacterial attachment and colonization. Most current knowledge on the mechanisms of these latter processes derives from studies involving pathogens [59]. These previously acquired mechanistic insights offer an important basis to unravel the molecular strategies used by commensals for intestinal colonization and establishment. Capsular polysaccharides Genomic analyses of genomic sequences of human-derived species of the genus Bacteroides (i.e., B. fragilis, B. vulgatus, B. thetaiotaomicron, and Parabacteroides distasonis) highlight that these bacteria encode an extensive repertoire of glycosyl hydrolases directed to the metabolism of complex carbohydrates [60]. Among the members of this bacterial group, B. thetaiotaomicron modulates carbohydrate decoration of the intestinal mucosa [61], and B. fragilis was the first within the genus Bacteroides to be found to produce multiple capsular polysaccharides [62]. This latter genetic feature has also been identified in other gut Bacteroidetes [63]; B. fragilis can synthesize an immunomodulatory polysaccharide, designated PSA, which adjusts particular aspects of the host immune system [33]. Consequently, these findings catalyzed research in capsular or exopolysaccharides produced by gut commensals as important mediators of gut microbiota colonization, host–microbe crosstalk, and/or immune modulation. A significant body of experimental data exists on the role of pathogen-encoded surface polysaccharides in pathogenesis, such as biofilm formation, tissue adherence, and anti-phagocytic activity during immune evasion [64]. In addition, capsular polysaccharide possesses a clearly demonstrated key role as a virulence factor in animal models, and this has prompted their exploitation as components of vaccines to prevent bacterial infections [65,66]. In gut commensals, such as various Bacteroides species, capsular polysaccharides are predicted to alter the physical properties of cell surfaces and exert an important role in host–bacterial commensalism [67]. Several studies have reported that B. fragilis expresses multiple capsular polysaccharides; attempts to eliminate capsular-mediated protection against the host immune system result in competitive defects of acapsular mutants, with subsequent spontaneous phenotypic reversion [62,67–69]. This reversion is due to the presence of an alternative pathway by which B. fragilis acapsular mutants are capable of re-establishing capsule production.

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The establishment of the expression of other capsular polysaccharides restores the reduced fitness of acapsular mutants in the gut. As mentioned above, bifidobacteria represent one of the dominant bacterial groups of the infant gut microbiota. Recently, the genome sequence of Bifidobacterium breve UCC2003 has been decoded [70] and was shown to contain a gene cluster predicted to be involved in the production of two different cell surface-associated EPSs. Notably, the alternative biosynthesis of each of these surface EPS is directed by either half of this bidirectional gene cluster, and their expression appears to be subject to phase variation by means of an apparent promoter-inversion mechanism [71]. Surface EPS produced by UCC2003 facilitates increased stress tolerance to both bile and low pH and influences gut persistence, but not initial colonization of UCC2003 in the murine gut, at least under the specific conditions tested. Surface EPS-producing B. breve UCC2003 cells elicit only a weak adaptive immune response as compared to isogenic mutants lacking this cell envelope-associated structure. Furthermore, colonization by B. breve UCC2003 expressing surface EPS in a murine model, as compared to an isogenic EPS-negative derivative, reduced infection levels of the murine pathogen Citrobacter rodentium. These data implicate bifidobacterial surface EPS in modulating host–microbe crosstalk, resulting in host-mediated immune tolerance of the commensal, while providing protection against a pathogen in an as yet unknown manner [71]. Pili and gut bacteria Most bacterial pathogens possess long filamentous structures, known as pili or fimbria, which protrude from their cellular surface. These structures are often required for adhesion of the bacterium to host tissues during colonization [72]. However, little is known about the presence and function of such structures in human gut commensals. Adherence and persistence of the human gut commensal Lactobacillus rhamnosus GG to human cells has been shown to be mediated by two sortase-dependent pili, SpaCBA and SpaEF [73]. The SpaCBA pilus was demonstrated to be pivotal for efficient adherence to the Caco-2 intestinal epithelial cell line as well as for biofilm formation [74]. In addition, a derivative L. rhamnosus GG spaCBA mutant promoted elevated mRNA levels of the chemokine IL-8 in vitro compared to the wild-type, possibly involving an interaction between lipoteichoic acid and TLR2 [74]. These studies represent clear molecular indications of a health-promoting bacterium eliciting immunemodulatory activities upon interaction with its human host [75,76]. Pili are known to adhere to carbohydrate moieties that are present in glycoprotein or glycolipid receptors [77], and it has been postulated that pili form the first opportunity for microorganisms to attach to their host, which is followed by a much more closer and tighter association between the bacterial cell and the host cell surface ([72] for review). Recently, a member of the so-called type IVb or Tad (tight-adherence) pilus family was shown to be specifically expressed by B. breve UCC2003 under in vivo conditions in a murine gut [70]. Mutational analysis of the corresponding tad locus of UCC2003 was shown to be 471

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Figure 3. Tad fimbria and bifidobacteria. (a) Schematic comparative representation of tad loci of various bifidobacterial strains. Each arrow indicates an open reading frame (ORF), the size of which is proportional to the length of the arrow. Coloring of the arrows represents the different function of the gene as indicated above each arrow. The amino acid identity of the relevant encoded proteins is indicated in percentages. (b) Morphology of Tad fimbria of Bifidobacterium longum subsp. infantis ATCC15697. The genome of B. longum subsp. infantis ATCC15697 encodes only a Tad fimbria and does not encompasses any sortase-dependent pilus-encoding genes [78]. Scale bar, 0.5 mm.

essential for efficient in vivo murine gut colonization, whereas immunogold transmission electron microscopy of UCC2003 cells retrieved from the murine gut demonstrated the presence of Tad pili at the poles of the cells. Notably, the tad locus of UCC2003 is highly conserved among all sequenced bifidobacterial strains (Figure 3), and this supports the notion of a ubiquitous pilus-mediated host-colonization and persistence mechanism for intestinal bifidobacteria. Atomic force microscopy (AFM) of various human intestinal bifidobacterial strains belonging to B. bifidum, B. longum subsp. longum, and B. adolescentis revealed the presence of cell surface-located pilus-like appendages (Figure 4) [78]. The genomes of the corresponding microorganisms revealed the presence of one to three predicted sortase-dependent pilus gene clusters, and each is predicted to encode one major pilin subunit plus one or two minor pilin subunits, as well as a so-called sortase required for covalent assembly of these pilin subunits. Quantitative reverse transcription (qRT)-PCR analysis revealed that the genes encompassing the major and minor pilin subunits of each of these sortase-dependent pilus gene clusters are transcribed as a polycistronic mRNA, and that these genes are differentially expressed depending on 472

the available carbohydrate in the growth medium [78]. This suggests that, in addition to Tad pili, other types of pili may play a role in bifidobacterial colonization, and/or other host interactions, in a fashion similar to that described for L. rhamnosus GG [73]. Other bioactive molecules produced by gut bacteria involved in host–microbe interactions Among bacterial proteins that are believed to drive host– microbe interactions and result in host benefit are the eukaryotic-type serine protease inhibitors (serpins), which are synthesized by particular members of the gut microbiota. Members of the serpin family regulate a wide range of signaling pathways in eukaryotes, and some are recognized for their ability to suppress inflammatory responses by inhibiting elastase activity [79]. Analyses of bifidobacterial genome sequences have highlighted the presence of genes encoding serpin-like proteins among enteric bifidobacteria including B. breve, B. longum subsp. longum, and B. longum subsp. infantis [80]. Furthermore, the bifidobacterial serpin-like protein performs an immune-modulatory role by reducing intestinal inflammation in a murine colitis model [81]. In addition, an outer-surface lipoprotein encoded by B. bifidum MIMBb75, named BopA, modulates

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B. adolescens ATCC 15703

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Figure 4. Morphology of various bifidobacterial species with sortase-dependent pili. Samples were viewed by atomic force microscopy. Scale bar, 0.5 mm.

adhesion and also affects the production of IL-8 by Caco-2 epithelial cells [82]. Further investigations of bacterial surface and secreted proteins are likely to reveal additional molecular mechanisms by which commensals influence and modulate human health. Metabolic end-products, such as short-chain fatty acids (SCFAs), vitamins, and polyunsaturated fatty acids such as conjugated linoleic acid, should also be mentioned in the context of gut commensal-produced molecules that mediate host–microbe interactions. SCFAs are the end-products of bacterial fermentation of complex carbohydrates in the GIT, and are highly important for human metabolism because they increase the amount of energy intake, stimulate water and sodium absorption, lower luminal pH, and lower the bioavailability of toxic amines. Recently, it was shown that acetate produced by bifidobacteria enhances intestinal defense mediated by epithelial cells, thereby protecting the host against infection by enterohemorrhagic bacteria [83]. An integrated ‘omics’ approach revealed that genes encoding an ATP-binding-cassette-type carbohydrate transporter present in an autochthonous member of the gut microbiota, B. longum subsp. longum, contributes to the protection of mice against an otherwise lethal infection by Escherichia coli O157:H7 [83]. In addition, although bifidobacteria do not produce butyrate as an end-product of fermentation,

de Vuyst and Leroy demonstrated the importance of acetate, which is produced by bifidobacteria, as a substrate for the production of butyrate by butyrate-producing bacteria (e.g., clostridia) in the gut [84]. Butyrate is the primary energy source for colonocytes and has attracted much research interest due to its potential use for the prevention of colon cancer [85]. Bacteriocins are ribosomally-synthesized, extracellularly-released peptides that have bactericidal or bacteriostatic effect on other (usually related) species [86]. Production of bacteriocins is claimed to be an important characteristic of probiotics, promoting colonization of the GIT by the producer strain, and exerting antagonistic activity against pathogens [87]. In addition, bacteriocins may function as signaling peptides, signaling to other bacteria through quorum sensing and crosstalk within microbial communities, while also signaling to cells of the host immune system. Many bacteriocins produced by lactic acid bacteria have been shown to be active against several Gram-positive food spoilage and/or pathogenic bacteria such as Listeria monocytogenes. This was elegantly demonstrated by Corr et al. [88], who observed that the human gut isolate Lactobacillus salivarius UCC118 exhibited bacteriocinmediated anti-infective activity against L. monocytogenes EGDe in a murine model. More recently the human isolate 473

Review Lactobacillus johnsonii La1, which is commercially used as a probiotic strain, has been shown to inhibit Helicobacter pylori both in vitro and in clinical trials [89]. Bacteriocin production by bifidobacteria appears to be rare, but bifidocin B and bifidin I are proposed to be class IIa bacteriocins that are produced by B. bifidum NCFB1454 and B. longum subsp. infantis, respectively. However, since their identification there has been no further work on their characterization [90,91]. More recently a B. thermophilum strain, RBL67, was reported to produce a bacteriocin-like inhibitory substance or ‘lantibiotic’ [92]. Finally, the genome sequence of B. longum subsp. longum DJ010A revealed a complete lantibiotic gene cluster, although production of this lantibiotic could only be detected on agar, and this has hindered efforts to purify and characterize this bacteriocin further. This bacteriocin exhibited a broad spectrum of activity against Gram-positive and some Gram-negative bacteria [93]. Interestingly, remnants of this lantibiotic cluster can be found in the genomes of B. longum subsp. infantis ATCC15697 and B. angulatum DSM 20098, suggesting that bacteriocin production may be important for particular bifidobacteria in the GIT. Taken together, the data mentioned above suggest that gut bacteria have evolved various genetic strategies through the biosynthesis of molecules (e.g., serpin and bacteriocin) or extracellular structures (e.g., EPS and pili) that allow the gut microbiota to cope with the harsh conditions in the human gut as well as to adhere to the intestinal mucosa and to protect themselves against the immune system of the host. Molecular basis of the interaction with other commensal bacteria Approaches based on global genome transcription profiles have greatly assisted expression studies and have been successful in determining how individual organisms in bacterial communities affect the transcriptome of each other. Transcriptome investigations on mice that were mono-associated with B. thetaiotaomicron (one of the dominant components of the human gut microbiota) and subsequently colonized by B. longum subsp. longum NCC2705, showed that the presence of NCC2705 increased the diversity of polysaccharides targeted for breakdown by B. thetaiotaomicron, including mannose and containing glycans [37]. The modifications in the transcriptional profiles of polysaccharide utilization-related genes by B. longum subsp. longum and B. thetaiotaomicron may imply a symbiosis between these microbes, where each species possesses a complementary set of glycosyl hydrolase activities, which when combined allow both to participate in a synergistic harvest of xylose and mannose-containing sugars. This event has already been noted in other microbial communities, for example those involved in the breakdown of complex carbohydrates such as cellulose [94]. Similar data have been described for a combination of lactobacilli, and involving L. reuteri and L. johnsonii, for which it has been shown that a specific nutritional adaptation can assist cohabitation by these two strains through resource partitioning in the mouse fore-stomach [95]. The molecular impact of members of the human microbiota on a murine host was analyzed by studying the host 474

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epithelium mRNA response to co-colonization by B. longum subsp. longum NCC2705 and B. thetaiotaomicron [37]. Notably, the host response to these two bacterial species was different. In fact, the host response to B. thetaiotaomicron enhanced tumor necrosis factor a (TNFa)-centered signaling, whereas NCC2705 induced cytokine interferon-g-mediated responses and reduced production of host antibacterial proteins such as Reg3g (regenerating islet-derived-3g) and Pap (pancreatitis-associated protein). Although one must be very careful in interpreting such information from a simplified gut model, these results suggest that the host response to enteric bifidobacteria might not only promote their own survival in the human intestine but may also affect the composition of the overall human gut microbiota. Concluding remarks For millennia mammals have evolved with their commensal partners, and adaptive co-evolution has formed an inextricable bond between the gut microbiota and their host [67]. Imbalances in the gut microbiota may contribute to some human intestinal diseases. Furthermore, it is generally accepted that gut bacteria influence the regulatory network of the immune system, and this compounds the intricate connection between gut microbiota composition and host health. Thus, if specific bacterial groups have evolved to maintain or even promote intestinal health, diseases and malfunctions may consequently be the result of the absence of such microorganisms and/or their metabolites. We have provided here several examples of key molecules, such as pili and capsular or surface polysaccharides, that mediate host–microbe interaction in specific human gut commensal bacteria, emphasizing bifidobacteria in particular. However, in the process of understanding the molecular basis underlying host–microbe interactions, the exploration of the molecular composition of the gut commensal cell envelope represents an important target. In fact, these surfaces can be viewed as the structural interface between microorganisms and their environment, where essential functions and activities required for bacterial attachment and colonization occur. The first decade of genomic exploration of the biology of gut commensals, such as bifidobacteria, has afforded unprecedented insights into the genetic adaptation of these microorganisms to the human gut through the decoding of their genome sequences (probiogenomics) [39]. The next decade holds the promise of being even more rewarding as new discoveries on the molecular mechanisms underpinning host–microbe interactions are generated by means of functional genomics efforts. Acknowledgments This work was financially supported by the Cariparma Bank Foundation to M.V., by a Federation of European Microbiological Societies (FEMS) Advanced Fellowship 2011 and an Irish Research Council for Science, Engineering, and Technology (IRCSET) Embark postdoctoral fellowship to F.T., and a H.R.B. postdoctoral fellowship (Grant no. PDTM/20011/9) awarded to M.O.C.M.. D.v.S. is a member of The Alimentary Pharmabiotic Centre and the Alimentary Glycoscience Research Cluster, both funded by Science Foundation Ireland (SFI) through the Irish Government National Development Plan (Grant numbers 07/CE/ B1368 and 08/SRC/B1393, respectively). We thank also Elena Foroni for the contribution of AFM images.

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