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THE HUMAN INTESTINAL MICROBIOTA

Extracellular proteins secreted by probiotic bacteria as mediators of effects that promote mucosa–bacteria interactions

Borja Sánchez, María C. Urdaci, Abelardo Margolles

  • 1Instituto de Productos Lácteos de Asturias, Consejo Superior de Investigaciones Científicas (IPLA-CSIC), Ctra. Infiesto s/n, 33300 Villaviciosa, Asturias, Spain
  • 2Université de Bordeaux, UMR 5248 CNRS, UBX1-ENITAB, ENITAB, 1 cours du Général de Gaulle, 33175 Gradignan Cedex, France
  • Correspondence
    Borja Sánchez
    borja{at}ipla.csic.es

    • Microbiology 2010; 156(11):3232–3242 · https://doi.org/10.1099/mic.0.044057-0

    View at publisher PubMed

    Abstract

    During the last few years, a substantial body of scientific evidence has accumulated suggesting that certain surface-associated and extracellular components produced by probiotic bacteria could be responsible for some of their mechanisms of action. These bacterial components would be able to directly interact with the host mucosal cells; they include exopolysaccharides, bacteriocins, lipoteichoic acids and surface-associated and extracellular proteins. Extracellular proteins include proteins that are actively transported to the bacterial surroundings through the cytoplasmic membrane, as well as those that are simply shed from the bacterial surface. Compared to the other bacterial components, the interactive ability of extracellular proteins/peptides has been less extensively studied. In this review, current findings supporting an interaction between extracellular proteins/peptides produced by probiotic bacteria (strains of the genera Bifidobacterium, Lactobacillus and Escherichia) and host mucosal cells are discussed. Research needs and future trends are also considered.

    Proteins secreted by probiotic bacteria

    The human gastrointestinal tract (GIT) is a vast surface inhabited by a complex and diverse community of micro-organisms. After thousands of years of co-evolution, the human body is completely tolerant to most of them, especially to bacteria (Gill et al., 2006; Stringer, 2003; Turnbaugh et al., 2007). However, some of these micro-organisms possess pathogenic potential and take advantage of certain gastrointestinal targets, colonizing the GIT and causing different intestinal disorders. Indeed, the commensal microbiota is quantitatively and qualitatively unbalanced in a wide range of intestinal and autoimmune disorders (Willing et al., 2009). In this regard, probiotic bacteria could positively affect the balance between pathogenic and non-pathogenic bacteria (Araya et al., 2002; Borody, 2000). Probiotics are defined as live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host (Araya et al., 2002). Probiotics exert several beneficial effects on human health, including interaction with the immune system, production of antimicrobial substances, enhancement of the mucosal barrier function and competition with enteropathogens for adhesion sites (Boesten & de Vos, 2008).

    Nowadays, the unravelling of the molecular mechanisms underlying these beneficial effects is an attractive field for gut microbiologists. Among the different extracellular compounds responsible for these processes, proteins secreted and released into the environment by probiotic bacteria (extracellular proteins) might mediate certain interactions, since they would be able to interact directly with mucosal cells, such as epithelial and immune cells (Sánchez et al., 2008a). Evidence of a direct interaction between bacterial extracellular proteins and the human immune system is mainly available for commensal and pathogenic species. In this context, the existence of antibodies directed against certain proteins secreted by the commensal microbiota in the framework of certain autoimmune diseases is known. Some examples of these proteins are porin OmpC from Escherichia coli, porin OmpW from Bacteroides caccae, protein I2 from Pseudomonas fluorescens and flagellin from Clostridium coccoides (Adams et al., 2008; Furrie et al., 2004; Iltanen et al., 2006; Müller et al., 2008). Theoretically, it is possible that certain extracellular proteins secreted by probiotic bacteria might also reach the gut mucosa, acting as molecular effectors responsible for downstream responses in mucosal cells.

    Extracellular proteins can be divided into two groups. The first group is composed of proteins that contain a signal peptide, which is located in the N-terminal part of the sequence to direct the protein to the protein export machinery. The second group includes surface-associated proteins that are simply shed from the bacteria due to the normal turnover of the cell wall (Anokhina et al., 2006b; Turner et al., 2004; Driessen & Nouwen, 2008; Sánchez et al., 2009b). Secretion systems are highly conserved among eubacteria, given the high degree of homology among the genes coding for their monomeric components (van Wely et al., 2001). These systems are particularly well known in Gram-negative bacteria, in which at least seven different secretion systems have been described, including those corresponding to macromolecules (Types I–VI and the twin-arginine translocation system) (Natale et al., 2008; Sibbald & van Dijl, 2009). In probiotic bacteria, most of which are Gram-positive, the majority of proteins are secreted through two molecular systems: the general secretory pathway and the specific protein export systems (Natale et al., 2008; van Wely et al., 2001). The first is characterized by the presence of a signal peptide carrying a particular sortase cleavage motif, the best known being the Ala-X-Ala (signal peptide type I) and the Leu-Ala-Gly-Cys (signal peptide type II or lipobox sequence) (von Heijne & Abrahmsen, 1989; Willing et al., 2009). In this system, proteins are synthesized as pre-proteins and transported in an unfolded state through a hydrophilic channel formed by the Sec proteins (von Heijne, 1989). The second group gathers specific export systems, in which proteins are secreted in a folded/oligomeric state, including ABC transporters, and the twin-arginine translocation system (Berks, 1996; Binet et al., 1997; Jongbloed et al., 2006). Finally, several glycolytic, housekeeping and ribosomal proteins are often found on the surface of bacteria (Jeffery, 2003). It is still unknown how these intracellular proteins can span the cytoplasmic membrane and reach the cell surface, although it is assumed that, once surface-exposed, these proteins could develop other functions, serving, for example, as adhesins (Candela et al., 2009; Castaldo et al., 2009; Hurmalainen et al., 2007; Kinoshita et al., 2008a, b; Ramiah et al., 2008). Because of their property of having different functions, depending on the subcellular localization, these proteins are termed ‘moonlighting’ proteins (Jeffery, 2003). To date, putative extracellular proteins of probiotic bacteria have mainly been identified by bioinformatic means (usually through the identification of certain domains, such as signal peptide or cell wall anchoring motifs, by sequence homology) (Barinov et al., 2009), and only a few of them have been experimentally identified and partially characterized (Sánchez et al., 2008b; van Pijkeren et al., 2006).

    The issue of surface-associated proteins, among other protein groups supporting probiotic action in probiotic bacteria, has recently been extensively reviewed by Lebeer and co-workers (Lebeer et al., 2008, 2010), by Kleerebezem et al. (2010) and by Buist et al. (2006). Another recent report has focused on the adhesins present in one of the major probiotic groups, the genus Lactobacillus (Vélez et al., 2007). Often, surface-associated proteins are identified by bioinformatic means (Båth et al., 2005), with a few exceptions (Beck et al., 2009). Furthermore, the presence of a specific genomic island coding for a pilus in Lactobacillus rhamnosus GG, in which a mucin-binding protein has been identified, has recently been reported (Kankainen et al., 2009). There are clearly considerable difficulties in studying the specific effects of extracellular proteins in human models (i.e. clinical studies).

    The aim of this review is to gather together the current information on the interaction between extracellular proteins produced by probiotic bacteria and the human gut mucosa. This group of proteins may trigger downstream responses in the host mucosa, potentially explaining some of the molecular mechanisms of action of probiotics. Current knowledge about the signalling mechanisms and the genetic/physiological changes induced by secreted proteins in mucosal cells and animal models is discussed. We will not deal with other cell-surface molecules with potential roles in the interaction with the human host, such as exopolysaccharides (Lebeer et al., 2007a), indoles (Bansal et al., 2010) or lipoteichoic acids (Anokhina et al., 2006a).

    Why study extracellular proteins secreted by probiotic bacteria?

    Extracellular proteins from probiotic bacteria could diffuse through the mucus layer that covers the intestinal mucosa, enabling interaction with epithelial and immune cells. In some cases, the signal is transmitted to the nucleus via different pathways, which generally involve the consecutive action of several kinases, such as mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI-3K) and glycogen synthase kinase-3 (GSK-3) (Hoarau et al., 2008). The activation of these cascades finally triggers changes in genetic expression and produces physiological changes in the cell. As we will discuss in this review, probiotic extracellular proteins regulate certain signalling pathways and cellular responses, including secretion of different effector molecules such as chemokines, cytokines or antibacterial peptides (defensins), mucus secretion, production of pseudopods, induction of changes in the surface properties, rearrangement of the tight junctions and modulation of the immune function and the response of the gut-associated lymphoid tissue (GALT) cells. These proteins are potential candidates for understanding the interaction between probiotic bacteria and the human host. The study of extracellular proteins may provide novel strategies for the clinical application of probiotic bacteria and may allow understanding of their mechanism of action. As previously stated, current knowledge about the interaction between extracellular proteins and the GALT mainly derives from data involving commensal bacterial groups. In this context, it has been observed that some proteins secreted by the commensal microbiota might be responsible for the anomalous immune response observed in the framework of inflammatory bowel disease (Adams et al., 2008; Ivison & Steiner, 2008; Schoepfer et al., 2008).

    There is a considerable amount of scientific evidence relating peptides secreted by probiotic bacteria to host-health promoting effects (Table 1⇓). However, we currently lack molecular information about the identity of such peptides. Further research is needed to identify these peptides and to establish their precise molecular mechanism of action.

    Table 1.

    Probiotic extracellular proteins/peptides with a known role in the interaction of potential probiotic strains with mucosal cells

    Receptors involved in the recognition of extracellular proteins

    Molecules naturally present on the mucosal surfaces of the gut can be targets for extracellular proteins. For instance, it is known that S-layer protein from Lactobacillus crispatus is able to interact, directly, with the collagen molecules on the surface of epithelial cells (Antikainen et al., 2002). This ability could be responsible for the competitive exclusion of enteropathogens, including E. coli O157 : H7 (Chen et al., 2007). Some studies have revealed that the interaction of the commensal microbiota and the host cells drives the correct development of the naïve intestinal mucosa, for example triggering and modulating the intestinal angiogenesis (Stappenbeck et al., 2002). In fact, it is known that the commensal microbiota and the host cells have a continuous exchange of molecular information, called cross-talk (Hooper & Gordon, 2001; Hooper et al., 2001). Very little is known about the cellular receptors responsible for the recognition of extracellular proteins secreted by probiotic bacteria, in contrast to receptors recognizing other bacterial products (Asong et al., 2009). One example is some bacterial flagellins, recognized by Toll-like receptor 5 (TLR-5) and by the ICE protease-activating factor (IPAF) (Gewirtz, 2006; Ren et al., 2006). Interestingly, recent data support the involvement of IPAF in the recognition of other bacterial molecules (Abdelaziz et al., 2010). Recently, it has been suggested that the C-type lectin receptor (CLR) DC-specific ICAM-3-grabbing non-integrin (DC-SIGN) may be involved in the recognition of certain extracellular components of probiotic bacteria (Konstantinov et al., 2008). CLRs are normally present on the surface of immune cells, such as dendritic cells (DCs) and macrophages, and recognize carbohydrate patterns that could be present, for instance, in extracellular glycoproteins (Benz & Schmidt, 2002). Also, the existence of an intestinal receptor for glycolipids of lactobacilli has been reported (Iwamori et al., 2009). Finally, it has been suggested that some TLRs may recognize more than one type of molecule; for example TLR-2 is able to recognize different glycolipids and lipoproteins (Yan et al., 2007).

    Future studies should shed light on the involvement of other receptors, which could be specific for certain extracellular proteins.

    Extracellular proteins secreted by probiotic bifidobacteria: enhancement of the mucosal barrier and immunomodulation

    Members of the genus Bifidobacterium are among the first colonizers of the human GIT (Favier et al., 2003). The beneficial effects of bifidobacteria on human health have largely been assessed during the last few years; moreover, decreases in the number of bifidobacteria in the colon have been associated with several diseases, including inflammatory bowel disease, irritable bowel syndrome, autoimmune disorders such as rheumatoid arthritis or lupus, and infections by enteropathogens (Gueimonde et al., 2007; López et al., 2010; Salminen et al., 2005, 2009; Turroni et al., 2009). Bifidobacterial strains are included in the formulation of functional foods for human nutrition, notably in dairy products and infant milks. Fig. 1⇓ shows the extracellular proteins/peptides with a known interaction with mucosal cells.

    Figure image not available in archive
    Fig. 1.

    Some of the effects mediated by extracellular proteins secreted by different strains of the genus Bifidobacterium. The proteins are represented by blue dots. (a) The pentapeptide CHWPR is able to increase c-myc and il-6 expression through a mechanism involving the nuclear receptor RORγ. (b) Proteinaceous compounds can bind to TLR-2 present on the surface of dendritic cell pseudopods, increasing their survival and the production of interleukins 10 and 12, as well as affecting their maturation. This is done by a mechanism involving several kinases, including ERK, p38, PI-3K or GSK-3. (c) Proteinaceous compounds are able to increase the production of TJPs, therefore increasing transepithelial resistance (TER), through a signalling pathway involving ERK. (d) An extracellular serin protease inhibitor (serpin) produced by B. dentium, B. breve and B. longum subsp. longum (and probably other bifidobacterial species) is a suicide inhibitor of neutrophil elastase, which is involved in acute inflammatory episodes within the gut.

    Enhancement of the mucosal barrier

    Some extracellular proteins produced by bifidobacteria are known to be responsible for the enhancement of the mucosal barrier and to modulate the GALT immune function in animal and cellular models. Among the extracellular proteins secreted by bifidobacteria, serine protease inhibitor (serpin) from Bifidobacterium longum subsp. longum NCC2705 was the first extracellular protein shown to interact directly with the host factors (Ivanov et al., 2006). Extracellular serpin is also produced by other bifidobacterial species, including Bifidobacterium breve, Bifidobacterium dentium and B. longum subsp. infantis. It has been shown that serpin efficiently inhibits pancreatic and neutrophil elastases (Ivanov et al., 2006). Neutrophils are recruited in the intestinal mucosa from the blood vessels by means of the secretion of inflammatory cytokines, and therefore are involved in inflammatory episodes. Bifidobacterial serpin, acting on enzymes directly involved in the inflammatory response, might thus mediate some of the anti-inflammatory effects of bifidobacteria (Ivanov et al., 2006).

    Another well-known probiotic strain, Bifidobacterium animalis subsp. lactis BB-12, produces a pentapeptide (CHWPR) in the stationary phase of growth; CHWPR has been shown to upregulate the c-myc and il-6 genes in the cell line HL-60 (Mitsuma et al., 2008). This pentapeptide crosses the cytoplasmic membrane and binds to the nuclear receptor ROR-γ. The complex binds to the promoter region of the genes and activates their transcription. The effect of CHWPR on intestinal cells might have a great impact on GIT physiology, since IL-6 is a dual anti- and pro-inflammatory cytokine, and since c-myc deregulation is observed in several human cancers.

    Tight junctions in epithelial cells are key components for the regulation of the mucosal barrier function, selectively affecting the movement of solutes and water through the epithelium. In a recent study, uncharacterized extracellular proteins secreted by B. longum subsp. infantis, isolated from a known probiotic cocktail, increased the production of zonula occludens-1 and occludin, two tight-junction proteins (TJPs), in epithelial cells. In addition, these increased transepithelial resistance (TER). Both events produced a reduction of colon permeability in a murine model, thus helping to maintain the mucosal barrier function (Ewaschuk et al., 2008).

    Immunomodulation

    Extracellular proteins secreted by B. longum subsp. infantis attenuated both inflammation and colonic permeability in IL-10-deficient mice, which lack the ability to produce sufficient quantities of the anti-inflammatory cytokine IL-10. The mechanism of action of these extracellular proteins is mediated by the modulation of two cytoplasmic mitogen-activated protein kinases (MAPKs): an increase in the levels of extracellular regulated kinase (ERK) together with a decrease in p38 MAPK (p38) (Ewaschuk et al., 2008).

    Finally, extracellular proteins secreted by B. breve C50 were shown to interact with the TLR-2 present on the surface of immature human DCs, inducing different functional and physiological changes through different pathways. Prolonged DC survival was mediated by the PI-3K pathway, DC maturation by the p38 and PI-3K pathways, increase in IL-10 production by the MAPK (p38, ERK) and PI-3K pathways, and finally an increase in IL-12 production was mediated by means of the p38 and GSK-3 pathways (Hoarau et al., 2008). Further research is needed to elucidate the precise role of those proteins.

    Cellular responses to extracellular proteins produced by probiotic lactobacilli

    Lactobacilli are important commensal bacteria in the human GIT. Several Lactobacillus species are used in the food industry for the production of a panoply of fermented products. Again, certain strains are considered as probiotics. L. rhamnosus GG (ATCC 53103) is one of the probiotic strains that has been most closely studied, and in addition has one of the most extensive safety assessment records (Lebeer et al., 2007b). Fig. 2⇓ shows the main cellular pathways modulated in response to extracellular proteins secreted by probiotic lactobacilli. The action of certain extracellular proteins might explain some of the beneficial effects exerted by certain probiotic lactobacilli.

    Figure image not available in archive
    Fig. 2.

    Schematic representation of the molecular mechanisms modulated by extracellular proteins produced by lactobacilli. The proteins are represented by brown dots. Specific details of the signal transduction mechanisms are given in the main text. (a) Proteinaceous compounds increase muc-2 expression in epithelial cells, resulting in an increase of mucin secretion. (b) S-layer protein A (SlpA) from L. acidophilus NCFM binds DC-SIGN, whose signalling increases IL-10 production by epithelial cells, whereas S-layer protein B (SlpB) increases the production of IL-12p70, TNF-α and IL-1β. (c) Extracellular proteins increase the production of HSP25 and HSP72, and affect electrolyte transport through PI-3K signalling (d). (e) Proteins p40 and p75, secreted by L. rhamnosus GG, promote enterocyte growth, decrease the incidence of apoptotic processes, and enhance transepithelial resistance through the increase of TJP synthesis. (f) Proteins secreted by lactobacilli increase hBD2 and IL-8 production.

    Enhancement of the mucosal barrier and maintenance of GIT homeostasis

    Extracellular proteins secreted by probiotic lactobacilli have been shown to help maintain the mucosal barrier, mainly through MAPK-dependent mechanisms (Schlee et al., 2008). The signalling mechanisms of the proteins are better characterized in lactobacilli than in bifidobacteria. Uncharacterized extracellular proteinaceous compounds secreted by Lactobacillus acidophilus PZ 1138, Lactobacillus fermentum PZ 1162 and Lactobacillus paracasei subsp. paracasei LMG P-17806 have been shown to induce production of the antimicrobial peptide human β-defensin 2 (hBD2) in epithelial cells. The signal of these extracellular proteins was shown to be transduced to the nucleus through the MAPKs ERK, p38 and c-Jun terminal kinase (JNK), where hBD2 synthesis was increased through the modulation of nuclear factor κB (NF-κB) and activator protein 1 (AP-1), ending finally in an increase of IL-8 production (Schlee et al., 2008). In addition, two peptides present in L. rhamnosus GG conditioned media, NPSRQERR and PDENK, were shown to possess antimicrobial activity against E. coli EAEC 042, Salmonella enterica serovar Typhimurium and Staphylococcus aureus (Lu et al., 2009).

    Extracellular proteinaceous compounds secreted by the lactobacilli contained in a probiotic formula (L. plantarum, L. acidophilus, L. casei and L. delbrueckii subsp. bulgaricus) induced mucin secretion through muc2 gene expression in murine colonic epithelial cells, although the possible signal transduction pathways involved are not yet known (Caballero-Franco et al., 2007). Further, extracellular proteins present in growth media conditioned by L. rhamnosus GG increased the production of the heat-shock proteins HSP25 and HSP72 in young adult mouse colon (YAMC) cells (Tao et al., 2006). On the one hand, HSP overproduction was due to the transcriptional regulation mediated by heat-shock factor 1 (HSF-1); on the other hand, the MAPKs p38 and JNK were also shown to be involved in the signalling pathway leading to HSP increase.

    Extracellular proteins p40 and p75, both secreted by L. rhamnosus GG, are perhaps the best studied extracellular proteins. p40 is homologous to an uncharacterized surface antigen of Lactobacillus casei ATCC 334 (gi|116493594), whereas p75 is homologous to a cell wall-associated hydrolase of the same bacterium (gi|116493849) (Yan et al., 2007). Yan and co-workers have shown that both p40 and p75 are efficient growth promoters, as they induced the proliferation of YAMC cells by activating protein kinase B (AKT) through a PI-3K-dependent pathway. In the same study, these two proteins were also able to reduce the colon injuries induced by tumour necrosis factor alpha (TNF-α) in murine colon tissue explants (Yan et al., 2007). Moreover, the two proteins completely inhibited TNF-α-induced apoptosis in the KSRI−/− MCE cell line, which undergoes apoptosis following TNF-α treatment (Yan et al., 2007). Finally p40 and p75 were capable of attenuating the TER decrease induced by hydrogen peroxide, also preventing the rearrangement of several TJPs (occludin, zonula occludens-1, E-cadherin and β-cathetin) through a protein kinase C- and MAPK-dependent signalling pathway (Seth et al., 2008). p40 and p75 thus appear to be important secreted proteins for GIT homeostasis, involved in both cell proliferation and apoptosis, and in the maintenance of the mucosal barrier.

    To sum up, extracellular proteins secreted by probiotic lactobacilli have been shown to promote the stabilization and enhancement of the mucosal barrier function by increasing the production of human defensins, the secretion of mucus and the concentration of HSPs in epithelial cells, and by stabilizing the arrangement and concentration of TJPs.

    Electrolyte absorption

    Certain studies have shown that probiotic lactobacilli are promising agents for the treatment of diarrhoea (Arvola et al., 1999; Pant et al.,1996). In this context, it has recently been shown that unidentified extracellular proteinaceous compounds secreted by L. acidophilus and L. rhamnosus increased the chloride/hydroxyl exchange activity (Cl−/OH−) in Caco-2 cells through the PI-3K signal transduction pathway. This triggered an increase in Cl− absorption, which was also related to an overproduction of the apical anion-exchange transporter SLC26A3 on the cell surface (Borthakur et al., 2007). As suggested by the authors, this increased electrolyte absorption in epithelial cells might be an alternative treatment for people suffering from diarrhoea or other intestinal disorders in which electrolyte absorption is perturbed.

    Immunomodulation and modulation of cytokine production

    Extracellular proteins secreted by probiotic lactobacilli can modulate the activity of immune cells. S-layer protein A (SlpA) released from L. acidophilus NCFM cells has been shown to induce IL-10 production in DCs (Konstantinov et al., 2008). DCs matured in the presence of purified SlpA expressed lower levels of the maturation marker CD86 with respect to controls, and did not induce T-cell proliferation, suggesting that this protein might somehow induce changes in the immune functions of DCs. The effects of SlpA on DCs were shown to be produced through a direct interaction of SlpA with the surface lectin DC-SIGN (Konstantinov et al., 2008). This result suggests a direct role for SlpA as an anti-inflammatory protein, given that a SlpA knockout strain, which overproduced another S-layer protein (SlpB), preferentially induced a pro-inflammatory response through an increase in production of IL-12p70, TNF-α and IL-1β. Unfortunately, no information about the components involved in the downstream signal transduction pathway was reported.

    Flagellin shed from E. coli Nissle 1917 cells

    E. coli is an abundant and important micro-organism within the human intestinal microbiota, and is well adapted to this environment. Non-pathogenic and pathogenic E. coli strains differ in the presence of virulence factors, normally acquired by horizontal gene transfer, and encoded on mobile genetic elements or in genome islands (Grozdanov et al., 2004). E. coli strain Nissle 1917 (O6 : K5 : H1) is a probiotic strain isolated from a soldier who survived a severe outbreak of diarrhoea during World War I. It has been successfully used for the treatment of various intestinal disorders (Grozdanov et al., 2004). The main molecular mechanisms modulated in the host by extracellular proteins of E. coli Nissle 1917 are summarized in Fig. 3⇓.

    Figure image not available in archive
    Fig. 3.

    Flagellin shed from E. coli Nissle 1917 cells induces hBD2 and IL-8 production. The molecular mechanism of action involves signalling through the kinases ERK, JNK and p38, and the nuclear effectors NF-κB and AP-1.

    Enhancement of the mucosal barrier

    E. coli Nissle 1917 secretes proteinaceous molecules capable of blocking the adhesion and colonization of the intestinal cell line INT407 by Salmonella sp., Yersinia enterocolitica, Shigella flexneri, Legionella pneumophila and Listeria monocytogenes (Altenhoefer et al., 2004; Lasaro et al., 2009). Flagellin secreted by this strain promotes hBD2 secretion in Caco-2 cells. This is achieved through a direct interaction with TLR-5, which activates several MAPKs (ERK1/2, JNK and p38) responsible for the signal transduction to the nucleus. The increase in hBD2 was abolished by mutation of the NF-κB and AP-1 binding sites of the hBD2-encoding gene (Schlee et al., 2007).

    Immunomodulation

    Flagellin released from E. coli Nissle 1917 increased the production of the pro-inflammatory cytokine IL-8 in Caco-2 cells (Schlee et al., 2007). The response to E. coli Nissle 1917 flagellin is particularly atypical, since flagellated micro-organisms are often enteropathogens, such as Salmonella sp. It has been suggested that, in the case of this particular E. coli probiotic strain, the stimulation induced by its flagellin would not reach the threshold levels necessary for the induction of a pro-inflammatory response, producing instead an activation of the immune system through the production of IL-8 and human defensins (Kruis et al., 2004).

    Future perspectives

    In the last few years, various papers have reported on the role of extracellular proteins secreted by probiotic bacteria (Sánchez et al., 2008a, b, 2009a, c, d; Turner et al., 1997, 2004). As has been shown in this review, probiotic extracellular proteins could be linked to some of the beneficial effects ascribed to the corresponding strains, although current information is now restricted to in vitro and animal studies. To date, our knowledge of the identity of these proteins is very limited; although several studies have reported the interaction between extracellular proteinaceous compounds and human cells, few have been identified and characterized so far.

    Further research is needed to elucidate the precise molecular mechanism of action of each of these proteins in both epithelial and immune cells, notably in DCs. This will contribute to the understanding of how probiotics exert beneficial effects on the human host. This knowledge may lead to treatments to reverse some of the processes involved in the initiation, or perpetuation, of various gastrointestinal disorders, such as inflammatory bowel diseases, allergies and autoimmune diseases.

    Acknowledgments

    B. S. was the recipient of a Juan de la Cierva postdoctoral contract from the Spanish Ministerio de Ciencia e Innovación. Research in our group is supported by grant AGL2007-61805.

    References

    1. Abdelaziz, D. H., Amr, K. & Am, A. O. (2010). Nlrc4/Ipaf/CLAN/CARD12: more than a flagellin sensor. Int J Biochem Cell Biol 42, 789–791.
    2. Adams, R. J., Heazlewood, S. P., Gilshenan, K. S., O'Brien, M., McGuckin, M. A. & Florin, T. H. (2008). IgG antibodies against common gut bacteria are more diagnostic for Crohn's disease than IgG against mannan or flagellin. Am J Gastroenterol 103, 386–396.
    3. Altenhoefer, A., Oswald, S., Sonnenborn, U., Enders, C., Schulze, J., Hacker, J. & Oelschlaeger, T. A. (2004). The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens. FEMS Immunol Med Microbiol 40, 223–229.
    4. Anokhina, I. V., Kravtsov, E. G., Yashina, N. V., Ermolaev, A. V., Chesnokova, V. L. & Dalin, M. V. (2006a). Characterization of surface adhesins of lactobacilli used in production of probiotic preparations. Bull Exp Biol Med 141, 716–719.
    5. Anokhina, I. V., Kravtsov, E. G., Yashina, N. V., Ermolaev, A. V., Chesnokova, V. L. & Dalin, M. V. (2006b). Surface adhesins of lactobacilli loosely connected to the cell wall and eliminated into the environment during culturing in liquid nutrient media. Bull Exp Biol Med 142, 609–613.
    6. Antikainen, J., Anton, L., Sillanpaa, J. & Korhönen, T. K. (2002). Domains in the S-layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self-assembly. Mol Microbiol 46, 381–394.
    7. Araya, M., Morelli, L., Reid, G., Sanders, M. E. & Stanton, C. (2002). Guidelines for the evaluation of probiotics in foods. FAO/WHO report. .
    8. Arvola, T., Laiho, K., Torkkeli, S., Mykkanen, H., Salminen, S., Maunula, L. & Isolauri, E. (1999). Prophylactic Lactobacillus GG reduces antibiotic-associated diarrhea in children with respiratory infections: a randomized study. Pediatrics 104, e64.
    9. Asong, J., Wolfert, M. A., Maiti, K. K., Miller, D. & Boons, G. J. (2009). Binding and cellular activation studies reveal that Toll-like receptor 2 can differentially recognize peptidoglycan from Gram-positive and Gram-negative bacteria. J Biol Chem 284, 8643–8653.
    10. Bansal, T., Alaniz, R. C., Wood, T. K. & Jayaraman, A. (2010). The bacterial signal indole increases epithelial-cell tight-junction resistance and attenuates indicators of inflammation. Proc Natl Acad Sci U S A 107, 228–233.
    11. Barinov, A., Loux, V., Hammani, A., Nicolas, P., Langella, P., Ehrlich, D., Maguin, E. & van de Gutche, M. (2009). Prediction of surface exposed proteins in Streptococcus pyogenes, with a potential application to other Gram-positive bacteria. Proteomics 9, 61–73.
    12. Båth, K., Roos, S., Wall, T. & Jonsson, H. (2005). The cell surface of Lactobacillus reuteri ATCC 55730 highlighted by identification of 126 extracellular proteins from the genome sequence. FEMS Microbiol Lett 253, 75–82.
    13. Beck, H. C., Madsen, S. M., Glenting, J., Petersen, J., Israelsen, H., Norrelykke, M. R., Antonsson, M. & Hansen, A. M. (2009). Proteomic analysis of cell surface-associated proteins from probiotic Lactobacillus plantarum. FEMS Microbiol Lett 297, 61–66.
    14. Benz, I. & Schmidt, M. A. (2002). Never say never again: protein glycosylation in pathogenic bacteria. Mol Microbiol 45, 267–276.
    15. Berks, B. C. (1996). A common export pathway for proteins binding complex redox cofactors? Mol Microbiol 22, 393–404.
    16. Binet, R., Letoffe, S., Ghigo, J. M., Delepelaire, P. & Wandersman, C. (1997). Protein secretion by Gram-negative bacterial ABC exporters – a review. Gene 192, 7–11.
    17. Boesten, R. J. & de Vos, W. M. (2008). Interactomics in the human intestine: lactobacilli and bifidobacteria make a difference. J Clin Gastroenterol 42, S163–S167.
    18. Borody, T. J. (2000). “Flora power” – fecal bacteria cure chronic C. difficile diarrhea. Am J Gastroenterol 95, 3028–3029.
    19. Borthakur, A., Gill, R. K., Tyagi, S., Koutsouris, A., Alrefai, W. A., Hecht, G. A., Ramaswamy, K. & Dudeja, P. K. (2007). Lactobacillus acidophilus secreted effector molecule(s) increase Cl−/OH− exchange activity in Caco-2 cells via PI-3 kinase mediated pathway. J Nutr 138, 1355–1359.
    20. Buist, G., Ridder, A. N., Kok, J. & Kuipers, O. P. (2006). Different subcellular locations of secretome components of Gram-positive bacteria. Microbiology 152, 2867–2874.
    21. Caballero-Franco, C., Keller, K., De Simone, C. & Chadee, K. (2007). The VSL#3 probiotic formula induces mucin gene expression and secretion in colonic epithelial cells. Am J Physiol Gastrointest Liver Physiol 292, G315–G322.
    22. Candela, M., Biagi, E., Centanni, M., Turroni, S., Vici, M., Musiani, F., Vitali, B., Bergmann, S., Hammerschmidt, S. & Brigidi, P. (2009). Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host. Microbiology 155, 3294–3303.
    23. Castaldo, C., Vastano, V., Siciliano, R. A., Candela, M., Vici, M., Muscariello, L., Marasco, R. & Sacco, M. (2009). Surface displaced alfa-enolase of Lactobacillus plantarum is a fibronectin binding protein. Microb Cell Fact 8, 14.
    24. Chen, X., Xu, J., Shuai, J., Chen, J., Zhang, Z. & Fang, W. (2007). The S-layer proteins of Lactobacillus crispatus strain ZJ001 is responsible for competitive exclusion against Escherichia coli O157:H7 and Salmonella typhimurium. Int J Food Microbiol 115, 307–312.
    25. Choi, C. H., Kim, T. I., Lee, S. K., Yang, K. M. & Kim, W. H. (2008). Effect of Lactobacillus GG and conditioned media on IL-1beta-induced IL-8 production in Caco-2 cells. Scand J Gastroenterol 43, 938–947.
    26. Driessen, A. J. & Nouwen, N. (2008). Protein translocation across the bacterial cytoplasmic membrane. Annu Rev Biochem 77, 643–667.
    27. Ewaschuk, J. B., Díaz, H., Meddings, L., Diederichs, B., Dmytrash, A., Backer, J., Looijer-van Langen, M. & Madsen, K. L. (2008). Secreted bioactive factors from Bifidobacterium infantis enhance epithelial cell barrier function. Am J Physiol Gastrointest Liver Physiol 295, G1025–G1034.
    28. Favier, C. F., de Vos, W. M. & Akkermans, A. D. (2003). Development of bacterial and bifidobacterial communities in feces of newborn babies. Anaerobe 9, 219–229.
    29. Furrie, E., Macfarlane, S., Cummings, J. H. & Macfarlane, G. T. (2004). Systemic antibodies towards mucosal bacteria in ulcerative colitis and Crohn's disease differentially activate the innate immune response. Gut 53, 91–98.
    30. Gewirtz, A. T. (2006). Flag in the crossroads: flagellin modulates innate and adaptive immunity. Curr Opin Gastroenterol 22, 8–12.
    31. Gill, S. R., Pop, M., Deboy, R. T., Eckburg, P. B., Turnbaugh, P. J., Samuel, B. S., Gordon, J. I., Relman, D. A., Fraser-Liggett, C. M. & Nelson, K. E. (2006). Metagenomic analysis of the human distal gut microbiome. Science 312, 1355–1359.
    32. Grozdanov, L., Raasch, C., Schulze, J., Sonnenborn, U., Gottschalk, G., Hacker, J. & Dobrindt, U. (2004). Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain Nissle 1917. J Bacteriol 186, 5432–5441.
    33. Gueimonde, M., Ouwehand, A., Huhtinen, H., Salminen, E. & Salminen, S. (2007). Qualitative and quantitative analyses of the bifidobacterial microbiota in the colonic mucosa of patients with colorectal cancer, diverticulitis and inflammatory bowel disease. World J Gastroenterol 13, 3985–3989.
    34. Hoarau, C., Martin, L., Faugaret, D., Baron, C., Dauba, A., Aubert-Jacquin, C., Velge-Roussel, F. & Lebranchu, Y. (2008). Supernatant from Bifidobacterium differentially modulates transduction signaling pathways for biological functions of human dendritic cells. PLoS ONE 3, e2753.
    35. Hooper, L. V. & Gordon, J. I. (2001). Commensal host-bacterial relationships in the gut. Science 292, 1115–1118.
    36. Hooper, L. V., Wong, M. H., Thelin, A., Hansson, L., Falk, P. G. & Gordon, J. I. (2001). Molecular analysis of commensal host-microbial relationships in the intestine. Science 291, 881–884.
    37. Hurmalainen, V., Edelman, S., Antikainen, J., Baumann, M., Lahteenmaki, K. & Korhonen, T. K. (2007). Extracellular proteins of Lactobacillus crispatus enhance activation of human plasminogen. Microbiology 153, 1112–1122.
    38. Iltanen, S., Tervo, L., Halttunen, T., Wei, B., Braun, J., Rantala, I., Honkanen, T., Kronenberg, M., Cheroutre, H. & other authors (2006). Elevated serum anti-I2 and anti-OmpW antibody levels in children with IBD. Inflamm Bowel Dis 12, 389–394.
    39. Ivanov, D., Emonet, C., Foata, F., Affolter, M., Delley, M., Fisseha, M., Blum-Sperisen, S., Kochhar, S. & Arigoni, F. (2006). A serpin from the gut bacterium Bifidobacterium longum inhibits eukaryotic elastase-like serine proteases. J Biol Chem 281, 17246–17252.
    40. Ivison, S. M. & Steiner, T. S. (2008). Anti-flagellin antibodies in irritable bowel syndrome: another attack on our commensals? Neurogastroenterol Motil 20, 1081–1085.
    41. Iwamori, M., Shibagaki, T., Nakata, Y., Adachi, S. & Nomura, T. (2009). Distribution of receptor glycolipids for lactobacilli in murine digestive tract and production of antibodies crossreactive with them by immunization of rabbits with lactobacilli. J Biochem 146, 185–191.
    42. Jeffery, C. J. (2003). Moonlighting proteins: old proteins learning new tricks. Trends Genet 19, 415–417.
    43. Jongbloed, J. D., van der Ploeg, R. & van Dijl, J. M. (2006). Bifunctional TatA subunits in minimal Tat protein translocases. Trends Microbiol 14, 2–4.
    44. Kankainen, M., Paulin, L., Tynkkynen, S., von Ossowski, I., Reunanen, J., Partanen, P., Satokari, R., Vesterlund, S., Hendrickx, A. P. & other authors (2009). Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human- mucus binding protein. Proc Natl Acad Sci U S A 106, 17193–17198.
    45. Kinoshita, H., Uchida, H., Kawai, Y., Kawasaki, T., Wakahara, N., Matsuo, H., Watanabe, M., Kitazawa, H., Ohnuma, S. & other authors (2008a). Cell surface Lactobacillus plantarum LA 318 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) adheres to human colonic mucin. J Appl Microbiol 104, 1667–1674.
    46. Kinoshita, H., Wakahara, N., Watanabe, M., Kawasaki, T., Matsuo, H., Kawai, Y., Kitazawa, H., Ohnuma, S., Miura, K. & other authors (2008b). Cell surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Lactobacillus plantarum LA 318 recognizes human A and B blood group antigens. Res Microbiol 159, 685–691.
    47. Kleerebezem, M., Hols, P., Bernard, E., Rolain, T., Zhou, M., Siezen, R. J. & Bron, P. A. (2010). The extracellular biology of the lactobacilli. FEMS Microbiol Rev 34, 199–230.
    48. Konstantinov, S. R., Smidt, H., de Vos, W. M., Bruijns, S. C., Singh, S. K., Valence, F., Molle, D., Lortal, S., Altermann, E. & other authors (2008). S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions. Proc Natl Acad Sci U S A 105, 19474–19479.
    49. Kruis, W., Fric, P., Pokrotnieks, J., Lukas, M., Fixa, B., Kascak, M., Kamm, M. A., Weismueller, J., Beglinger, C. & other authors (2004). Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut 53, 1617–1623.
    50. Lasaro, M. A., Salinger, N., Zhang, J., Wang, Y., Zhong, Z., Goulian, M. & Zhu, J. (2009). F1C fimbriae play an important role in biofilm formation and intestinal colonization by the Escherichia coli commensal strain Nissle 1917. Appl Environ Microbiol 75, 246–251.
    51. Lebeer, S., Ceuppens, J., Geboes, K., Van Assche, G., Rutgeerts, P., Vanderleyden, J. & De Keersmaecker, S. C. (2007a). Mechanisms of probiotic-host interaction with IBD as a case study: a role for exopolysaccharides? Commun Agric Appl Biol Sci 72, 41–45.
    52. Lebeer, S., Verhoeven, T. L., Perea, V. M., Vanderleyden, J. & De Keersmaecker, S. C. (2007b). Impact of environmental and genetic factors on biofilm formation by the probiotic strain Lactobacillus rhamnosus GG. Appl Environ Microbiol 73, 6768–6775.
    53. Lebeer, S., Vanderleyden, J. & De Keersmaecker, S. C. (2008). Genes and molecules of lactobacilli supporting probiotic action. Microbiol Mol Biol Rev 72, 728–764.
    54. Lebeer, S., Vanderleyden, J. & De Keersmaecker, S. C. (2010). Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens. Nat Rev Microbiol 8, 171–184.
    55. López, P., Gueimonde, M., Margolles, A. & Suarez, A. (2010). Distinct Bifidobacterium strains drive different immune responses in vitro. Int J Food Microbiol 138, 157–165.
    56. Lu, R., Fasano, S., Madayiputhiya, N., Morin, N. P., Nataro, J. & Fasano, A. (2009). Isolation, identification, and characterization of small bioactive peptides from Lactobacillus GG conditional media that exert both anti-Gram-negative and Gram-positive bactericidal activity. J Pediatr Gastroenterol Nutr 49, 23–30.
    57. Mitsuma, T., Odajima, H., Momiyama, Z., Watanabe, K., Masuguchi, M., Sekine, T., Shidara, S. & Hirano, S. (2008). Enhancement of gene expression by a peptide p(CHWPR) produced by Bifidobacterium lactis BB-12. Microbiol Immunol 52, 144–155.
    58. Müller, S., Schaffer, T., Schoepfer, A. M., Hilty, A., Bodmer, T. & Seibold, F. (2008). Partial overlap of anti-mycobacterial, and anti-Saccharomyces cerevisiae mannan antibodies in Crohn's disease. World J Gastroenterol 14, 3650–3661.
    59. Natale, P., Bruser, T. & Driessen, A. J. (2008). Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane – distinct translocases and mechanisms. Biochim Biophys Acta 1778, 1735–1756.
    60. Pant, A. R., Graham, S. M., Allen, S. J., Harikul, S., Sabchareon, A., Cuevas, L. & Hart, C. A. (1996). Lactobacillus GG and acute diarrhoea in young children in the tropics. J Trop Pediatr 42, 162–165.
    61. Ramiah, K., van Reenen, C. A. & Dicks, L. M. (2008). Surface-bound proteins of Lactobacillus plantarum 423 that contribute to adhesion of Caco-2 cells and their role in competitive exclusion and displacement of Clostridium sporogenes and Enterococcus faecalis. Res Microbiol 159, 470–475.
    62. Ren, T., Zamboni, D. S., Roy, C. R., Dietrich, W. F. & Vance, R. E. (2006). Flagellin-deficient Legionella mutants evade caspase-1- and Naip5-mediated macrophage immunity. PLoS Pathog 2, e18.
    63. Salminen, S. J., Gueimonde, M. & Isolauri, E. (2005). Probiotics that modify disease risk. J Nutr 135, 1294–1298.
    64. Salminen, S., Collado, M. C., Isolauri, E. & Gueimonde, M. (2009). Microbial-host interactions: selecting the right probiotics and prebiotics for infants. Nestle Nutr Workshop Ser Pediatr Program 64, 201–213.
    65. Sánchez, B., Bressollier, P. & Urdaci, M. C. (2008a). Exported proteins in probiotic bacteria: adhesion to intestinal surfaces, host immunomodulation and molecular cross-talking with the host. FEMS Immunol Med Microbiol 54, 1–17.
    66. Sánchez, B., Champomier-Vergès, M. C., Anglade, P., Baraige, F., de los Reyes-Gavilan, C. G., Margolles, A. & Zagorec, M. (2008b). A preliminary analysis of Bifidobacterium longum exported proteins by two-dimensional electrophoresis. J Mol Microbiol Biotechnol 14, 74–79.
    67. Sánchez, B., Arias, S., Chaignepain, S., Denayrolles, M., Schmitter, J. M., Bressollier, P. & Urdaci, M. C. (2009a). Identification of surface proteins involved in the adhesion of a probiotic Bacillus cereus strain to mucin and fibronectin. Microbiology 155, 1708–1716.
    68. Sánchez, B., Chaignepain, S., Schmitter, J. M. & Urdaci, M. C. (2009b). A method for the identification of proteins secreted by lactic acid bacteria grown in complex media. FEMS Microbiol Lett 295, 226–229.
    69. Sánchez, B., Schmitter, J. M. & Urdaci, M. C. (2009c). Identification of novel proteins secreted by Lactobacillus plantarum that bind to mucin and fibronectin. J Mol Microbiol Biotechnol 17, 158–162.
    70. Sánchez, B., Schmitter, J. M. & Urdaci, M. C. (2009d). Identification of novel proteins secreted by Lactobacillus rhamnosus GG grown in de Man-Rogosa-Sharpe broth. Lett Appl Microbiol 48, 618–622.
    71. Schlee, M., Wehkamp, J., Altenhoefer, A., Oelschlaeger, T. A., Stange, E. F. & Fellermann, K. (2007). Induction of human beta-defensin 2 by the probiotic Escherichia coli Nissle 1917 is mediated through flagellin. Infect Immun 75, 2399–2407.
    72. Schlee, M., Harder, J., Köten, B., Stange, E. F., Wehkamp, J. & Fellermann, K. (2008). Probiotic lactobacilli and VSL#3 induce enterocyte beta-defensin 2. Clin Exp Immunol 151, 528–535.
    73. Schoepfer, A. M., Schaffer, T., Seibold-Schmid, B., Muller, S. & Seibold, F. (2008). Antibodies to flagellin indicate reactivity to bacterial antigens in IBS patients. Neurogastroenterol Motil 20, 1110–1118.
    74. Seth, A., Yan, F., Polk, D. B. & Rao, R. K. (2008). Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism. Am J Physiol Gastrointest Liver Physiol 294, G1060–G1069.
    75. Sibbald, M. J. J. B. & van Dijl, J. M. (2009). Bacterial Secreted Proteins: Secretory Mechanisms and Role in Pathogenesis. Edited by Wooldridge, K.. Norwich, UK. : Caister Academic Press. Horizon Scientific Press.
    76. Stappenbeck, T. S., Hooper, L. V. & Gordon, J. I. (2002). Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proc Natl Acad Sci U S A 99, 15451–15455.
    77. Stringer, C. (2003). Human evolution: out of Ethiopia. Nature 423, 692–693.
    78. Tao, Y., Drabik, K. A., Waypa, T. S., Musch, M. W., Alverdy, J. C., Schneewind, O., Chang, E. B. & Petrof, E. O. (2006). Soluble factors from Lactobacillus GG activate MAPKs and induce cytoprotective heat shock proteins in intestinal epithelial cells. Am J Physiol Cell Physiol 290, C1018–C1030.
    79. Turnbaugh, P. J., Ley, R. E., Hamady, M., Fraser-Liggett, C. M., Knight, R. & Gordon, J. I. (2007). The human microbiome project. Nature 449, 804–810.
    80. Turner, M. S., Timms, P., Hafner, L. M. & Giffard, P. M. (1997). Identification and characterization of a basic cell surface-located protein from Lactobacillus fermentum BR11. J Bacteriol 179, 3310–3316.
    81. Turner, M. S., Hafner, L. M., Walsh, T. & Giffard, P. M. (2004). Identification and characterization of the novel LysM domain-containing surface protein Sep from Lactobacillus fermentum BR11 and its use as a peptide fusion partner in Lactobacillus and Lactococcus. Appl Environ Microbiol 70, 3673–3680.
    82. Turroni, F., Marchesi, J. R., Foroni, E., Gueimonde, M., Shanahan, F., Margolles, A., van Sinderen, D. & Ventura, M. (2009). Microbiomic analysis of the bifidobacterial population in the human distal gut. ISME J 3, 745–751.
    83. van Pijkeren, J. P., Canchaya, C., Ryan, K. A., Li, Y., Claesson, M. J., Sheil, B., Steidler, L., O'Mahony, L., Fitzgerald, G. F. & other authors (2006). Comparative and functional analysis of sortase-dependent proteins in the predicted secretome of Lactobacillus salivarius UCC118. Appl Environ Microbiol 72, 4143–4153.
    84. van Wely, K. H. M., Swaving, J., Freudl, R. & Driessen, A. J. M. (2001). Translocation of proteins across the cell envelope of Gram-positive bacteria. FEMS Microbiol Rev 25, 437–454.
    85. Vélez, M. P., De Keersmaecker, S. C. & Vanderleyden, J. (2007). Adherence factors of Lactobacillus in the human gastrointestinal tract. FEMS Microbiol Lett 276, 140–148.
    86. von Heijne, G. (1989). The structure of signal peptides from bacterial lipoproteins. Protein Eng 2, 531–534.
    87. von Heijne, G. & Abrahmsen, L. (1989). Species-specific variation in signal peptide design. Implications for protein secretion in foreign hosts. FEBS Lett 244, 439–446.
    88. Willing, B., Halfvarson, J., Dicksved, J., Rosenquist, M., Järnerot, G., Engstrand, L., Tysk, C. & Jansson, J. K. (2009). Twin studies reveal specific imbalances in the mucosa-associated microbiota of patients with ileal Crohn's disease. Inflamm Bowel Dis 15, 653–660.
    89. Yan, F., Cao, H., Cover, T. L., Whitehead, R., Washington, M. K. & Polk, D. B. (2007). Soluble proteins produced by probiotic bacteria regulate intestinal epithelial cell survival and growth. Gastroenterology 132, 562–575.