Exported proteins in probiotic bacteria: adhesion to intestinal surfaces, host immunomodulation and molecular cross-talking with the host (original) (raw)

Abstract

The group of exported proteins of a bacterium are those proteins that are sorted from the cytoplasm to the bacterial surface or to the surroundings of the microorganism. In probiotic bacteria, these proteins are of special relevance because they might determine important traits such as adhesion to intestinal surfaces and molecular cross-talking with the host. Current knowledge about the presence and biological relevance of exported proteins produced by the main genera of probiotic bacteria in the gastrointestinal environment is reviewed in this minireview. As will be seen, some of these proteins are involved in host adhesion or are able to modify certain signalization pathways within host cells, whereas others are important for the physiology of probiotic bacteria in the gastrointestinal tract.

Introduction

Probiotics are a group of intestinal microorganisms that, when consumed alive, enhance gastrointestinal homeostasis by positively affecting the intestinal microbiota balance. Members of the genera Bifidobacterium and Lactobacillus are the most commonly used probiotics in human nutrition (Araya et al., 2002). Some probiotic strains have been shown to reduce intestinal colonization by pathogens through direct competition for attachment sites, the production of antimicrobials, modulation of the host-acquired immune response, improvement of lactose intolerance and reduction of the serum cholesterol levels (Salminen et al., 2005). In addition, scientific evidence suggests that probiotic bacteria exert some kind of beneficial influence over epithelial cells that regulate their development and function (Ljungh & Wadstrom, 2006; Sonnenburg et al., 2006).

Exported proteins present on the surface or secreted by probiotic bacteria might be responsible for some of those probiotic traits. Their identification and characterization could be relevant for a better understanding of the interaction of probiotic bacteria with the gastrointestinal environment.

The term ‘exported protein’ refers to those bacterial proteins that contain within their sequences export signals and surface-retention domains. These special motifs allow exported proteins to be secreted into the bacterial surroundings or to become associated with the cytoplasmic membrane/cell wall either by covalent or noncovalent interactions. In addition, some cytoplasmic proteins lacking such domains are also found to be associated with the bacterial surface or released into the extracellular environment, being normally referred to as anchorless multifunctional proteins or moonlighting proteins.

Many exported proteins produced by probiotic microorganisms have been described and characterized during recent years, mainly in members of the genus Lactobacillus (for further reading, see Velez et al., 2007); it has been shown that some are able to bind to epithelial components like mucus or extracellular matrix (ECM) proteins (Navarre & Schneewind, 1999). However, we have to keep in mind that other cell wall components can affect the interaction of a probiotic bacterium with host cells. One example is lipoteichoic acid purified from Lactobacillus johnsonii, which can inhibit the adhesion of several enteropathogens, maybe as a result of a direct competition for the adhesion sites (Granato et al., 1999).

The aim of this minireview is to compile the current knowledge about exported proteins produced by probiotic bacteria, as well as to examine their potential roles in the interaction with the host, such as adhesion, bacteria–host molecular cross-talking and immunomodulation.

Exported proteins and intestinal ecology and physiology

In the gastrointestinal tract (GIT) of a healthy adult, both commensal and potentially pathogenic microorganism populations cohabit in permanent contact between them and with mucosal cells. Continuous exchange of information flows among such different cell lineages, which, in a normal situation, is properly conducted by the intestinal epithelium and the gut-associated lymphoid tissue (GALT), resulting in the internal stability or homeostasis of the GIT. Exported proteins might be responsible for some of the mechanisms by which probiotic bacteria contribute to the maintenance of such homeostasis, due to their privileged situation on the bacterial surface that placed them in direct contact with the bacterial environment (Fig. 1). Several proteins have been shown to be present on the surface or secreted by probiotic bacteria, mainly in bifidobacteria and lactobacilli, but also in the strain Escherichia coli Nissle 1917. In addition, several surface-associated proteins have been identified by bioinformatic analysis, although no defined and precise function has been described (Boekhorst et al., 2006).

Figure 1

Exported proteins produced by probiotic bacteria can cooperate in the maintenance of gastrointestinal homeostasis through several mechanisms. (a) Some exported proteins are responsible for bacterial adhesion to intestinal surfaces, such as mucin, epithelial cells and components of the ECM; (b) others can modulate the function of both epithelial and immune cells (represented here by an M-cell with a dendritic cell and several leucocytes in its inner cavity); and (c) certain can induce specific changes in gene expression, being responsible for the molecular cross-talking between probiotic bacteria and the host cells. By means of these mechanisms, probiotics can exert some beneficial effects over the host. For example modulation in gene expression caused by exported proteins can enhance the epithelial barrier reinforcing cell junctions, therefore diminishing the risk of pathogen invasion.

The molecular mechanisms by which exported proteins produced by probiotic bacteria could participate in GIT homeostasis are still unclear. Exported proteins are frequently found to mediate adhesion to intestinal components like mucus or ECM components like collagen, fibronectin or laminin (Flock, 1999). In addition, a high proportion of intestinal bacteria are present in the luminal content if we compare with those that remain attached to the mucosa, and so we can suppose that secreted proteins might play important roles in the molecular intercommunication between probiotic bacteria and the host epithelium.

Although the precise bacterial molecules underlying the process of immunomodulation in certain probiotic bacteria have not yet been identified, it is known that cell wall preparations of probiotic bacteria can act either as adjuvants of the immune response (Sekine et al., 1994) or change the cytokine secretion profiles of human cells (Ruiz et al., 2005; O'Hara et al., 2006; O'Mahony et al., 2006). The sum of all these individual responses to each microbe leads normally to a low immune responsiveness of the intestinal mucosa toward the intestinal microbiota, mainly due to a predominance of the anti-inflammatory response to commensal bacteria (Cario, 2005; Cario & Podolsky, 2006). This response is promoted by probiotic bacteria (Ma et al., 2004), and, when this equilibrium is affected, often ends in the appearance of inflammatory intestinal disorders (Jones & Foxx-Orenstein, 2007).

On the other hand, some exported proteins have been shown to act over the internal host cell structure, inducing rearrangement of the cytoskeleton and cell junctions and thus a strengthening of epithelial barrier function through heat shock protein induction (Tao et al., 2006); this enhancement could also be achieved by the induction of mucin secretion (Mack et al., 2003). Recently, a small secreted peptide produced by Bifidobacterium animalis ssp. lactis BB-12 has been shown to be able to induce changes in the expression of certain regulatory genes within the host cells (Mitsuma et al., 2008).

Types of exported proteins

Owing to its physical extension and situation, the cell wall is of great importance because it constitutes, with the cytoplasmic membrane, the interface between the extracellular environment and the cytoplasm of the bacteria. Basically, the cell wall of probiotic bacterium is composed of a group of accessory molecules (such as proteins, teichoic and teichuronic acids, lipoglycans, polyphosphates or carbohydrates) that are embedded in a peptidoglycan macromolecule (Hancock, 1997). Its main function is to act as an exoskeleton, conferring protection from both osmotic and mechanical stresses, but it is also the physical support of a variety of molecules, including surface-associated proteins. The cell wall contains different attachment sites for certain exported proteins, which, once surface exposed, would be able to interact with the bacterial environment.

The surface of most probiotic bacteria is formed by the cytoplasmic membrane and a thick peptidoglycan layer (Bifidobacterium and Lactobacillus genera, for example), whereas in others it consists of a thin peptidoglycan layer and an outer membrane containing lipopolysaccharide in addition to the cytoplasmic membrane (E. coli Nissle 1917), which corresponds to the former classification of gram-positive and gram-negative bacteria, respectively. Moreover, some strains synthesize a polysaccharide layer, often referred to as exopolysaccharide (EPS) (Ruas-Madiedo et al., 2006), and others can also form a crystalline layer (S-layer) by combining a special class of surface-associated proteins (Ävall-Jääskeläinen & Palva, 2005).

Generally, exported proteins contain a signal peptide, which direct them to the protein export machinery and allow their migration to the bacterial surface. In addition, some proteins contain one or more supplementary domains, which allow their anchoring to the cell wall or to the cytoplasmic membrane, preventing their secretion into the external medium. Logically, proteins containing a signal peptide and lacking surface-retention domains will be secreted into the external medium. Therefore, exported proteins can be surface associated (to both the cytoplasmic membrane and the cell wall) or secreted into the bacterial surroundings. All the different types of exported proteins are represented in Fig. 2a.

Figure 2

Different types of exported proteins identified in probiotic bacteria. (a) 1: LPXTG proteins, 2: lipoproteins, 3: proteins with N-terminal or C-terminal transmembrane helices, 4: LysM proteins, 5: GW proteins, 6: proteins with choline-binding domains, 7: WXL proteins, 8: S-layer proteins, 9: anchorless proteins and 10: secreted proteins. (b) Schematic representation of the main types of protein secretion. Proteins can be exported via the ATP-dependent GSP, which implies protein linearization and subsequent surface refolding 1 or, in contrast, they can be transported in a folded state by specific systems like the Tat pathway or ABC transporters 2. Finally, it remains unknown how anchorless proteins can cross the cytoplasmic membrane 3. N: N-terminal ending, C: C-terminal ending, +: positively charged residues.

Surface-associated proteins

Within this class we can make a distinction between covalent (LPXTG and lipoproteins) and noncovalent surface-associated proteins. Probably the best-known surface-associated proteins are those that possess a C-terminal LPXTG motif in their amino acidic sequence. This motif is recognized by the enzyme sortase, a surface peptidase that catalyses a cleavage between threonine and glycine and the subsequent covalent binding of the protein to the peptidoglycan network (Navarre & Schneewind, 1999). Although this motif is widely conserved among bacteria, other amino acidic patterns like NPQTN, NAKTN or QVPTGV can be recognized by other sortases (Boekhorst et al., 2005). Another type of covalently surface-associated proteins are lipoproteins, which harbour an N-terminal lipobox sequence (LXXC) that allows their binding to a lipid of the cytoplasmic membrane (Sutcliffe & Harrington, 2002). Lipoproteins are usually the binding subunits of nutrient transport systems and are thought not to be normally surface exposed, although there are several exceptions such as the laminin-binding protein of Streptococcus agalactiae (Spellerberg et al., 1999).

While LPXTG and lipoproteins are covalently linked to the cell surface, other proteins become attached by noncovalent associations. This includes proteins with N-terminal or C-terminal transmembrane helices, which are domains composed by hydrophobic amino acids (followed or preceded by positively charged residues) by means of which they become literally anchored in the cytoplasmic membrane (Bath et al., 2005); proteins containing LysM-type repeats, which have been proposed as the attachment mechanism of the autolysin AcmA of Lactococcus lactis (Steen et al., 2003); proteins with glycine-tryptophan (GW) motifs that have been shown to mediate the binding of several listerial and streptococcal surface proteins (Yother & White, 1994; Cabanes et al., 2002); proteins harbouring choline-binding domains, detected in the muramidase of Streptococcus pneumonie (Garcia et al., 1988); and finally the presence in the amino acidic sequence of C-terminal WXL motifs, which have been suggested as a possible binding domain of a subset of Lactobacillus plantarum proteins (Kleerebezem et al., 2003).

S-layer proteins

S-layer proteins (SLPs) are a special class of noncovalently attached proteins that auto assemble, forming a protective layer on the surface of certain strains. In probiotic bacteria, these proteins present one or various N-terminal S-layer domains (SL), which are responsible for their binding to accessory molecules (such as teichoic acids, lipoteichoic acids and neutral polysaccharides) embedded in the peptidoglycan matrix (Mesnage et al., 2000). In the cases where no SLs are detected within the amino acidic sequence of an SLP, some N-terminal or C-terminal regions have been postulated as cell wall-binding domains (Ävall-Jääskeläinen & Palva, 2005). A direct anchoring of an SLP to the peptidoglycan has been reported at least once (Olabarria et al., 1996).

Moonlighting proteins

Recently, several cytoplasmic proteins with essential roles in bacterial growth and metabolism have been found on the bacterial surface and/or in the extracellular proteome. When fixed on the surface of some probiotic strains, such proteins have been shown to mediate adhesion to the host cells by interacting with the host plasminogen system (Granato et al., 2004). These proteins, also called anchorless proteins, do not possess any export motifs or surface-attachment domains. To date, there is no evidence on what motifs could be responsible for their localization on the surface of bacteria or the hypothetical translocation mechanism across the cytoplasmic membrane.

Secreted proteins

This group is formed by those proteins that are directed to the cell surface by means of a signal peptide but contain no surface-retention domains, therefore being secreted into the external medium of the bacterium. In this group, we can also include the small proportion of surface-associated proteins that are released into the external medium due to the physiological renewal of the cell wall, and that can sometimes be found in the supernatant (Turner et al., 2004). Although cell surface organization differs between bacteria, it is known that all microorganisms secrete their proteins in a similar way, as long as their genomes contain homologous genes that code for the main components of the secretion systems (van Wely et al., 2001).

Two main types of secretion systems are known in probiotic bacteria (Fig. 2b): (1) the so-called, ATP-dependent general secretory pathway (GSP) and (2) the specific protein export pathways. Firstly, GSP is an energy-dependent system in which proteins are synthesized as preproteins, with the presence of a signal peptide in the N-terminal part of the sequence. At least two types of signal peptides are known: the general signal peptide (type I), which carries an Ala-X-Ala cleavage motif, and the lipoprotein signal peptide, which contains a consensus lipoprotein box sequence Leu-Ala-Gly-Cys (von Heijne, 1989; von Heijne & Abrahmsen, 1989). Preproteins are translocated in an unfolded state through a hydrophilic channel formed by the products of the sec genes, after which the signal peptide is proteolytically removed and the protein is refolded with the help of molecular chaperones (for extended reviews on the subject, see Tjalsma et al., 2000; van Wely et al., 2001).

In addition to GSP, different specific protein export pathways exist in bacteria. Some proteins are secreted via the Tat pathway, characterized by the presence of two consecutive arginines in the signal peptide and that are usually formed by ABC or AC systems (Berks, 1996; Jongbloed et al., 2006). Proteins secreted by type I secretion pathways lack signal peptides and are directly secreted by specific ABC transporters (Binet et al., 1997). Finally, secretion of flagellin in the probiotic strain E. coli Nissle 1917 is achieved by a specific mechanism homologous to the type III secretion system present in several pathogenic bacteria (Hueck, 1998). These systems present the advantages of export proteins in a completely folded or oligomeric state, which can be very useful for industrial purposes as efficient protein production as shown recently (Meissner et al., 2007).

Genus Lactobacillus

Members of the genus Lactobacillus belong to the Firmicutes class, characterized by a low GC content in their genomes, and are usually associated with the human GIT. Exported proteins identified in this genus as well as their possible physiological roles are listed in Table 1.

Table 1

Extracellular proteins identified in the genus Lactobacillus with roles in adhesion or interaction with the host

Proteins identified by N-terminal sequencing.

Table 1

Extracellular proteins identified in the genus Lactobacillus with roles in adhesion or interaction with the host

Proteins identified by N-terminal sequencing.

Adhesion to mucus

Supplementation of growth medium with mucin has been shown to induce mucus adhesion in Lactobacillus reuteri; this property is abolished after protease treatment of the bacteria, a fact that points to an overproduction of a hypothetical mucus-binding protein (Jonsson et al., 2001). Conversely, adhesion of probiotic bacteria to host cells could be a mechanism for the induction of mucin secretion through the action of certain bacterial surface proteins. In this way, it has been demonstrated that the coculture of L. plantarum 299v or Lactobacillus rhamnosus GG with HT29-MTX cells induces the secretion of MUC3 mucin, whereas a spontaneous mutant of L. plantarum 299v with reduced adhesion capabilities to such a cell line was unable to induce mucin secretion (Mack et al., 2003). MUC3 mucin can inhibit the adhesion of the enteropathogenic E. coli (EPEC) E2348/69, and so L. plantarum 299v could inhibit EPEC adhesion. These results suggest that the protein or proteins not present in the mutant strain could be in part responsible for the mechanisms by which the probiotic bacteria L. plantarum 299v would inhibit EPEC adhesion to epithelial cells by increasing their MUC3 mucin production.

The proteins involved in adhesion to intestinal surfaces have been reviewed recently by Velez (2007). Firstly, proteins that have already been experimentally shown to be implicated in mucus adhesion are the mucus-binding protein (Mub) of L. reuteri 1063 (Roos & Jonsson, 2002), the Mub of Lactobacillus acidophilus NCFM (Buck et al., 2005), the mannose lectin (Msa) of L. plantarum WCFS1 (Pretzer et al., 2005), the Lactobacillus surface protein A (LspA) of Lactobacillus salivarius UCC118 (van Pijkeren et al., 2006) and the mucin adhesion-promoting protein (MapA) of Lactobacillus fermentum 104R, recently reclassified as L. reuteri 104R (Rojas et al., 2002; Miyoshi et al., 2006).

Proteins adhering to mucus share common characteristics such as the presence of a signal peptide, a C-terminal cell wall-anchoring motif normally LPXTG-like, several repeated domains with a putative adhesion function and zones with unknown functions (with the exception of MapA). The presence of these proteins on the surface of probiotic bacteria could be important for the persistence of these bacteria in the gut epithelium. This point has been suggested in the strain L. salivarius UCC118, in which an isogenic sortase mutant does not display LPXTG proteins on the surface and shows lower adhesion capabilities either to Caco-2 or to HT29 human cell lines (van Pijkeren et al., 2006).

Adhesion to ECM proteins and epithelial cell lines

A majority of the known ECM-binding proteins in probiotic bacteria have been identified as Lactobacillus SLPs, the monomeric constituents of S-layers. Briefly, S-layer proteins of Lactobacillus species are highly basic proteins (with computed isoelectric point values ranging from 9.4 to 10.4) with a molecular mass comprising between 25 and 71 kDa, and its only known functional role is adhesion to host tissues (for further information, see Ävall-Jääskeläinen & Palva, 2005). Most Lactobacillus species harbour two or more S-layer genes, with some exceptions like Lactobacillus brevis ATCC 8287.

SLPs appear to be directly implied in bacterial adhesion to fibronectin and laminin, but not to type IV collagen (Jakava-Vijanen & Palva, 2007), and it has been suggested that the putative receptors for S-layer proteins could be gut specific (de Leeuw et al., 2006). Direct experimental evidence of binding of SLPs to ECM proteins and to epithelial cell lines has already been obtained for the protein SlpA of L. acidophilus NCFM (protein that additionally carries two mucine-binding domains) (Buck et al., 2005), SlpA of L. brevis ATCC 8287 (de Leeuw et al., 2006), CbsA of Lactobacillus crispatus JCM 5810 (Antikainen et al., 2002), SlpB of L. crispatus ZJ001 (Chen et al., 2007) and the SlpA of Lactobacillus helveticus R0052 (Johnson-Henry et al., 2007). Cell treatments with high concentrations of a chaotropic agent like LiCl or with proteolytic enzymes such as trypsin have been shown to decrease the adhesion to avian and porcine intestinal cells in L. acidophilus ATCC 4356 and L. acidophilus M92 (Sillanpaa et al., 2000; Kos et al., 2003) and to porcine gastric mucin, ECM proteins and Caco-2 cells in the species L. plantarum (Tallon et al., 2007). It is known that SLPs can be extracted easily from the bacterial surface after such a treatment, but it still remains unclear whether this treatment could remove other surface proteins also important for the adhesion (Ävall-Jääskeläinen & Palva, 2005; Jakava-Vijanen & Palva, 2007).

In terms of which part of the S-layer sequence is responsible for the adhesion to ECM components, it is known that the collagen- and laminin-binding domain of the CbsA protein of L. crispatus JCM 5810 would be located at the N-terminal part of the protein, comprising two-thirds of the molecule (Antikainen et al., 2002). To date, the only fully characterized functional binding domain of an S-layer has been described for the SlpA protein of L. brevis ATCC 8287, in which the domain responsible for the adherence to human epithelial cells is comprised between residues 96 and 176, whereas the domain formed by residues 96–245 is responsible for the adhesion to fibronectin (de Leeuw et al., 2006). Another function of SLPs could also be the adhesion to plasma components because it has been found that a 48 kDa SLP of L. brevis OLL2772, with a high homology to SlpA of L. brevis ATCC 8287, is able to specifically bind human blood type-A antigen, which is also present in the intestinal mucosa (Uchida et al., 2006).

However, SLPs are not the sole known proteins able to bind to ECM components. Other examples are the fibronectin-binding protein (FbpA) of L. acidophilus NCFM (Buck et al., 2005), the collagen-binding protein from L. reuteri NCIB 11951 (Roos et al., 1996) and its homologous p29 of L. fermentum RC-14, which possess, in addition, the capacity of inhibit the adhesion of the pathogen Enterococcus faecalis (Heinemann et al., 2000). In addition, at least two small surface-associated proteins (with a molecular mass <3 kDa) have been shown to be responsible for the adhesion of L. fermentum to Caco-2 cells (Baccigalupi et al., 2005).

From a physiological point of view, it is known that growth conditions can affect the adherence of some Lactobacillus strains to laminin and fibronectin. In particular, growth in MRS agar under anaerobic conditions increased the adherence of eight Lactobacillus gasseri and one L. johnsonii strain to laminin, and also improved the adhesion of four L. gasseri strains to fibronectin when compared with the adhesion of the same strains grown in the same broth but under aerobic conditions (Horie et al., 2005). It seems that adhesion to intestinal host surfaces is promoted when experimental conditions mimic the human GIT environment.

Cell-bound proteases and other proteins

Surface-associated proteases present in dairy lactic acid bacteria, usually referred to as cell envelope proteinases (CEPs), are enzymes involved in the breakdown of casein, which supply bacteria with oligopeptides that can be transported and metabolized in the cytoplasm by various peptidases with different specificities. On the other hand, it is known that some peptides, such as those released from milk proteins, can have some kind of beneficial bioactivity over the host, as shown in Fig. 3 (Hebert et al., 2008). To date, four different CEPs types have been identified in several lactic acid bacteria: L. acidophilus, Lactobacillus bulgaricus, L. lactis, Lactobacillus casei, L. helveticus and Streptococcus thermophilus (Germond et al., 2003; Scolari et al., 2006; Hebert et al., 2008). CEPs harbour a C-terminal LPXTG, by means of which they can be covalently linked to peptidoglycan. A known exception to this rule is the protein PrtB of L. bulgaricus, in which a degenerated LPKKT motif is followed and preceded by two imperfect repeats of 59 amino acids (Germond et al., 2003). The mechanism of PtrB cell binding seems to be similar to SLPs, that is, electrostatic interactions of the positive-charged tail of the protein with negative-charged accessory cell wall molecules, but it is also possible that the LPKKT motif could be recognized by the L. bulgaricus sortase. Finally, a solute-binding protein of a cystine uptake system [identified previously as BspA and suggested to bind to some ECM components (Turner et al., 1997)] was extracted by treating L. fermentum BR11 whole cells with a glycine hydrochloride buffer adjusted to a low pH (Turner et al., 2003). These proteins are usually membrane associated by means of a lipoprotein consensus motif (Sutcliffe & Harrington, 2002) but BspA does not appear to harbour such a motif within its sequence. It has been suggested that BspA could be associated with the bacterial surface by means of electrostatic interactions.

Figure 3

Some examples of the role of surface-associated proteins identified in the genus Lactobacillus. Left, cell-bound proteases are capable of producing bioactive peptides from proteins present in the outer milieu. Centre, moonlighting proteins develop roles that might be important for GIT physiology, such as pathogen aggregation and binding of plasminogen. Right, the bioactivity of extracellular nuclease produced by several Lactobacillus species can give rise to oligonucleotides with immunomodulatory activity.

Moonlighting proteins I: glycolytic enzymes

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase are important glycolytic enzymes that catalyse, respectively, the interconversion of glyceraldehyde-3-P to 1,3-bis-phosphoglycerate and dehydration of 2-phosphoglycerate to phosphoenolpyruvate. Both enzymes have been detected on the surface of some lactic acid bacteria species: L. acidophilus, Lactobacillus amylovorus, L. crispatus, Lactobacillus gallinarum, L. gasseri, L. johnsonii, Lactobacillus paracasei, L. rhamnosus and L. lactis (Hurmalainen et al., 2007), as well as in other commensal bacteria (Pancholi & Chhatwal, 2003). The molecular mass of surface-associated L. crispatus ST1 enolase was estimated to be around 370 kDa, not very different from the theoretical mass of the predicted octameric multimer (374 kDa). This suggests that enolase, as well as other moonlighting proteins, could be translocated in a folded/oligomeric state from the bacterial cytoplasm (Antikainen et al., 2007a).

Cell wall association of enolase and GAPDH in probiotic and pathogenic bacteria has been shown to be reversible and pH dependent (Fig. 4); these enzymes are attached to the cell surface at pH 5 but released into the medium at a neutral or a slightly alkaline pH (Nelson et al., 2001; Antikainen et al., 2007b). Both enzymes are positively charged at acidic pH values below their isoelectric point and thus they could bind to negatively charged cell surface components like lipoteichoic acids, in the same way as SLPs do (Antikainen et al., 2007b). This pH dependency for the attachment/release of surface-associated enolase and GAPDH is likely to affect probiotic/host interactions. The acidic pH of the intestinal mucosa will favour their attachment to the bacterial surface, whereas a rapid detachment will occur at the neutral or slightly alkaline pH of the intestinal content. This could be one of the mechanisms by which probiotic bacteria respond to the physicochemical changes of the gastrointestinal environment.

Figure 4

Effect of pH on the binding of moonlighting proteins to the bacterial cell wall. Left, proteins are positively charged at acidic pH values below their isoelectric points, and remain associated with the bacterial surface by means of electrostatic interactions with negatively charged components of the cell wall. Right, proteins lose their positive charge when the pH is above their isoelectric points, which leads to their release in to the bacterial surroundings. Symbol + denotes positive net charge.

Not much data is available in scientific databases regarding the involvement of anchorless proteins in the adhesion to ECM components. It has been shown recently that GAPDH from L. plantarum LA 318 is able to bind to human colonic mucin (Kinoshita et al., 2008). Antikainen (2007a) have shown that enolase of L. crispatus is the only protein among those present in its surface-associated proteome able to bind to laminin and collagen. However, surface-associated forms of bacterial GAPDH and enolase have been shown to be involved in plasminogen (Plg)/plasmin binding and activation (Pancholi & Fischetti, 1992, 1998; Pancholi, 2001; Pancholi & Chhatwal, 2003; Seifert et al., 2003; Bergmann et al., 2004). The interaction of probiotic bifidobacteria and lactobacilli with the human plasminogen system has been reported recently (Antikainen et al., 2007a; Candela et al., 2007; Hurmalainen et al., 2007).

Plasminogen activation in probiotic bacteria is mainly dependent on the gradual release of surface-associated enolase and GAPDH, in contrast to pathogens where plasminogen activation passes by the immobilization of the plasminogen/plasmin system on their surface (Fig. 3) (Lähteenmaki et al., 2005). Moreover, no homologues of any of the known plasminogen activators have been found in the genomes of the sequenced lactobacilli species (Khil et al., 2003), suggesting that probiotic lactobacilli, and probably other probiotic bacteria, lack the endogenous potential for plasminogen activation present in several enteropathogens. For this reason, plasminogen activation by ‘probiotic’ enolase and GAPDH might interfere in the interaction between plasminogen and gastrointestinal pathogens such as Helicobacter pylori and Salmonella sp., as has been suggested (Jönsson et al., 2004; Lähteenmaki et al., 2005; Hurmalainen et al., 2007).

Moonlighting proteins II: housekeeping proteins

Translation elongation factor Tu (EF1A or EF-Tu) is a G-protein responsible for the selection and binding of the cognate aminoacyl-tRNA to the acceptor site of the ribosome during protein synthesis (Nilsson & Nissen, 2005). Despite being a cytoplasmic protein, EF-Tu has been described as a common antigen for many lactobacilli (Nakamura et al., 1997). Granato and co-workers demonstrated the presence of EF-Tu on the surface of the probiotic strain L. johnsonii NCC 533 (La1) in an attempt to identify molecules mediating attachment to intestinal epithelial cells and mucin. Furthermore, recombinant La1 EF-Tu (rEF-Tu) was also able to bind to intestinal epithelial cells lines (Caco-2 and HT29) as well as to human gut mucin (Granato et al., 2004). Adhesion of EF-Tu to epithelial cells or mucus has been found to be pH dependent, as has been observed for otherMub mucus-binding proteins of other probiotic strains (Fig. 4) (Green & Klaenhammer, 1994; Blum et al., 2000; Roos & Jonsson, 2002; Granato et al., 2004). By contrast, rEF-Tu has been shown to stimulate IL-8 release from HT29 cells in the presence of soluble CD14, suggesting that EF-Tu could participate in gut homeostasis through its binding to the intestinal mucosa (Granato et al., 2004).

Heat shock proteins of the GroEL class, also designated chaperones of the Hsp60 class, are a highly conserved group of essential proteins required for the proper folding of many proteins in all living organisms (Gupta, 1995). Similar to other moonlighting proteins, no motifs have been found in its sequence supporting GroEL translocation across the cytoplasmic membrane. As with EF-Tu, GroEL has been detected at the surface of La1 by a whole-cell enzyme-linked immunoassay and is also detectable in the spent culture medium throughout the logarithmic growth phase (Bergonzelli et al., 2006). La1 recombinant GroEL (rGroEL) expressed in E. coli was shown to be capable of attaching to the mucus as well as to the HT29 cell line, suggesting a substantial contribution of GroEL to the adhesion abilities of La1 strain (Bergonzelli et al., 2006). As for La1 EF-Tu, GroEL binding to mucus or human cells was more efficient at acidic pH, also being able to stimulate IL-8 secretion by HT29 cells in the presence of soluble CD 14 (Bergonzelli et al., 2006).

The immunomodulatory capacity of La1 rGroEL tested on macrophages was found to be similar to rGroEL of other bacteria (L. helveticus, Bacillus subtilis and L. lactis) (Bergonzelli et al., 2006). It has also been shown that La1 rGroEL induces a strong aggregation of the gastric pathogen H. pylori, suggesting that it might contribute to a decrease of the bacterial load by facilitating clearance of the aggregated pathogen with the mucus (Fig. 3) (Bergonzelli et al., 2006).

Secreted proteins

One well-known protein secreted by members of the genus Lactobacillus is nuclease. After abundant evidence of extracellular DNAse activity, nucleases from several Lactobacillus species have been identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, coupled to in-gel protein renaturalization and nuclease assay (Peant & LaPointe, 2004). Nuclease activity over the DNA present in the luminal content can lead to the formation of a set of diverse oligonucleotides, some of them with immunomodulatory properties (Fig. 3) (Takahashi et al., 2006a, b; Iliev et al., 2008).

Several researchers have reported on the interaction between small proteinaceous compounds secreted by some probiotic strains and pathogenic bacteria, although, to our knowledge, most of them have not yet been identified. These small proteins, sometimes smaller than 5 kDa, have been shown to exert in vitro certain probiotic effects that are also observed for the whole bacteria. Several examples are available in the scientific databases; soluble proteinaceous compounds secreted by several Bifidobacterium and Lactobacillus probiotic strains have been shown to protect C2Bbe1 monolayers against infection by Listeria monocytogenes; these substances are only active at acid pH in the case of Lactobacillus sp. (Corr et al., 2007). Proteinaceous factors secreted by the probiotic strain L. acidophilus La-5 have been shown to downregulate the locus of enterocyte effacement (LEE) and autoinducer-2 production (AI-2) in E. coli O157:H7, suggesting that they can somehow interfere with the quorum sensing of this enteropathogen (Medellin-Pena et al., 2007). Acid-stable peptides secreted by the probiotic bacterium L. rhamnosus GG caused heat shock protein upregulation (Hsp25 and Hsp72) through the activation of certain Mitogen-activated protein kinase (MAPK) signalization pathways (Tao et al., 2006). Finally, bacteria contained in the VSL#3 probiotic formula (manufactured by Seaford Pharmaceuticals) secrete soluble compounds that are able to induce mucin secretion and muc2 gene expression in murine colonic epithelial cells (Caballero-Franco et al., 2007). All of these mechanisms are summarized in Fig. 5.

Figure 5

Small proteinaceous factors produced by Lactobacillus strains (small dots) can interfere with the pathogenicity of Escherichia coli O157:H7 by downregulating the locus of LEE and the autoinducer-2 production (AI-2). Epithelial cells can reply to these compounds, increasing the production of heat shock proteins and mucin secretion through the modification of MAPK signalization pathways. As a result, the epithelial barrier becomes reinforced and thus infections by invasive microorganisms are markedly reduced.

Similar results showed that a 47 kDa surface protein of L. reuteri JCM1081 and L. reuteri TM105 was able to adhere to the human glycolipids gangliotetraosylceramide and sulphatide (Mukai et al., 2002). Mukai and co-workers postulated this protein to be partly responsible for the inhibitory effect of the surface protein extract over H. pylori adhesion to epithelial cells, probably by means of a direct competition for the binding sites. Finally, the Sep protein of L. fermentum BR11, similar to the L. gasseri and L. johnsonii aggregation-promoting factor (Ventura et al., 2002), has been shown to be secreted by such bacteria and has been used satisfactorily to construct fusion proteins (Turner et al., 2004).

Genus Bifidobacterium

Bifidobacteria are formed by microorganisms of the class Actinomycetes and, in contrast to lactobacilli, are distinguished by the high GC content of their genomes. As for the genus Lactobacillus, a list of the exported proteins identified in this genus is presented in Table 2.

Table 2

Extracellular proteins/peptides identified in the genus Bifidobacterium with roles in the adhesion to intestinal surfaces or in bacterial–host interaction

Proteins identified by N-terminal sequencing.

Not yet identified.

Table 2

Extracellular proteins/peptides identified in the genus Bifidobacterium with roles in the adhesion to intestinal surfaces or in bacterial–host interaction

Proteins identified by N-terminal sequencing.

Not yet identified.

Surface-associated proteins

It has been shown recently that certain Bifidobacterium surface proteins are able to interact with the human plasminogen system. By means of such proteins, bifidobacteria are able to retain the plasminogen at the bacterial surface in the form of aggregates (Candela et al., 2007). Remarkably, all those proteins were identified as moonlighting proteins (DnaK, glutamine synthetase, enolase, bile salt hydrolase, and phosphoglycerate mutase).

With regard to SLPs, no reports have been published in the genus Bifidobacterium although a search in the Conserved Domain Database (CDD; http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) has revealed the presence of one gene harbouring one S-layer domain in the two sequenced bifidobacterial species [GenBank, accession numbers 696305 (Bifidobacterium longum NCC2705), ZP_00120975 (B. longum DJO10A), YP_909374 (Bifidobacterium adolescentis ATCC 15703) and ZP_02028389 (B. adolescentis L2-32)]. Further work is needed to decide to whether or not such a protein is really an SLP.

Another surface-associated protein identified in the genus Bifidobacterium is the CEP of B. adolescentis 94-BIM. It is postulated that this cell-bound protease is the precursor of the enzyme secreted into the surrounding medium (Samartsev et al., 2000). As discussed earlier, CEP bioactivity in the GIT could produce bioactive peptides with immunological and antihypertensive properties from different substrates (Yamamoto et al., 1998; Seppo et al., 2003) and, in addition, CEPs could be a way of transient binding to the intestinal epithelium, as by definition proteases catalyse the hydrolytic breakdown of proteins into peptides or amino acids, and so they can bind proteins temporarily.

Secreted proteins

Recently, we have identified fourteen proteins in the culture supernatant of the probiotic strain B. longum NCIMB 8809. Three were identified as the solute-binding proteins for peptides/phosphate. Five corresponded to homologues of TraG-like proteins, four were identified as cell wall hydrolases, probable homologues of the listerial cell wall-associated hydrolase p60 and two were identified as peptidoglycan synthetase and the moonlighting protein transaldolase (Sánchez et al., 2008). These proteins or products derived from their bioactivity over substrates present at the GIT could play important roles in the physiology of these bacteria and in their interaction with the host cells, although this point requires further research.

Another example of a protein secreted by bifidobacteria is the amylase produced by B. adolescentis Int-57, which is involved in starch degradation (Lee et al., 1997). Starch is a complex carbohydrate of vegetal origin that reaches the human colon in large proportions (Cummings & Macfarlane, 1991). Simple sugars and short-chain fatty acids (SCFA) resulting from the glycolytic activity of probiotics (mainly bifidobacteria and lactobacilli) over nondigestible polysaccharides like resistant starch (Fig. 6) are fermentable substrates for other members of the Firmicutes division (such as Eubacterium sp. and Anaerostipes sp.), which produce other SCFAs such as butyric and propionic acids (Belenguer et al., 2006). SCFAs are quickly absorbed in the intestinal epithelium, where they are supposed to protect the mucosa by decreasing the formation of secondary bile acids (van Munster et al., 1994); moreover, butyrate is used as an energy source by colonocites and is postulated to regulate their growth and differentiation (Bugaut & Bentejac, 1993). All these reasons suggest the important role of secreted amylases in the health and ecology of the large intestine, and how they can indirectly affect the normal development and physiology of host intestinal cells.

Figure 6

Secreted proteins identified in probiotic bifidobacteria. (a) Serpin (serine protease inhibitor) can inactivate the neutrophil elastase, which is involved in inflammatory events. Serpin could thus be an important immunomodulatory protein. (b) Secreted amylase catalyses the breakdown of starch into oligosaccharides and simple sugars, which can be used as energy sources by other intestinal bacteria. (c) Small peptides can produce changes of gene expression in human cells. It has been shown that the peptide CHWPR can increase the expression of the protooncogen c-myc, the IL-6 and the oxalyl-CoA decarboxylase (oxd).

Recently, a serine protease inhibitor, denominated serpin, has been characterized in the probiotic strain B. longum NCC2705 and has been shown to inhibit proteases from the elastase family, in particular, pancreatic and neutrophil elastases (Fig. 6) (Ivanov et al., 2006). This last protease is important from an immune point of view, because neutrophils are recruited and activated in the gut epithelium following inflammatory episodes (Burg & Pillinger, 2001). Because neutrophil elastase is released during the course of an inflammatory response, B. longum serpin could be responsible for a part of the observed immunomodulatory properties of bifidobacteria (Reeves et al., 2002).

Regarding the possible receptors of these peptides on the epithelial cells, Fujiwara and co-workers observed that extracellular protein extracts of B. longum SBT2928 were able to inhibit the adhesion of the enterotoxigenic E. coli Pb176 to the intestinal epithelial cell line HCT-8. Further experiments suggested that unidentified components of that extract could be interfering with the interaction between the colonization factor antigen of the enteropathogen and the glycolipid-binding receptor gangliotetraosylceramide present on the surface of the intestinal cells (Fujiwara et al., 2001).

Finally, the identification of a pentapeptide (CHWPR) produced by the probiotic strain B. animalis ssp. lactis BB-12 has been reported (Mitsuma et al., 2008). This pentapeptide was able to increase the gene expression of oxalyl-CoA decarboxylase and, moreover, the gene expression of c-myc and IL-6 in the cell line HL-60 (Fig. 6). Oxalyl-CoA decarboxylase has been shown to be involved in the response to bile of the strains of B. animalis ssp. lactis IPLA 4549 and 4549dOx, whereas IL-6 is a dual anti- and proinflammatory cytokine and c-myc is an important protooncogene whose misregulation can lead to the development of several human cancers (Schorl & Sedivy, 2003; Sánchez et al., 2007). The genes whose expression is regulated by the CHWPR therefore appear to have an important role in the GIT physiology.

With regard to the mechanism of action, it has been shown that the pentapeptide can cross the cytoplasmic membrane binding to the nuclear receptor ROR-γ, the ensemble binding to the promoter region of the c-myc gene. To our knowledge, this is the first time that the molecular mechanism of a peptide produced by a probiotic bacterium is reported.

Genus Escherichia

In spite of not belonging to the best-known probiotic genera, one of the best-studied probiotic strains is E. coli Nissle 1917, commercialized as Mutaflor®. This strain, which can efficiently colonize the GIT, was isolated from a soldier who survived a severe outbreak of diarrhoea during World War I (Stentebjerg-Olesen et al., 1999), and is capable of diminishing the effect of several gastrointestinal disorders (Schulze & Sonnenborn, 1995; Kruis et al., 1997). Other E. coli strains have also been used as probiotics, such as E. coli PZ720, which, when administered to newborns, can reduce the incidence of allergies and gastrointestinal infections (Lodinova-Zadnikova & Sonnenborn, 1997). It has been shown recently that flagellin, a protein present at the surface of E. coli Nissle 1917, can mediate an increase in the production of β-defensin 2 (hBD-2), a human antimicrobial peptide (Schlee et al., 2007).

Flagellins are the major constituents of bacterial flagella (98% of the final flagellar mass) and, when organized on the surface, give a characteristic helicoidal shape to the flagellar filament, which, in addition, comprise a basal body and a hook (Kuwajima et al., 1986; LaVallie & Stahl, 1989; Nuijten et al., 1990). Both the basal body and the hook form a type III-like secretion system, by which flagellin monomers are specifically exported to the bacterial surface (Hueck, 1998). Flagellin is formed by four domains, two of them highly conserved among species (the N-terminal and C-terminal domains or D0 and D1 domains, respectively) that are buried into the flagellar filament, and two other variable globular domains (D2 and D3 domains) that can show differences of 1000 residues, depending on the microorganism (Beatson et al., 2006). D2 and D3 domains are surface exposed and represent the targets for antibody responses.

As mentioned earlier, monomeric flagellin has been identified as a soluble factor secreted by the probiotic strain E. coli Nissle 1917 that induces the production of hBD-2 in Caco-2 cells, resulting in a reduction of pathogen adhesion and invasion. This induction has been shown to be dependent on the activation of certain MAP kinase-signalling pathways (ERK1/2, JNK, and p38) and on an increase of the inflammatory cytokine IL-8 (Fig. 7) (Schlee et al., 2007). The signal could be transmitted by the human innate immune system through toll-like receptor 5 (TLR-5) and ICE protease-activating factor (IPAF) because D0 and D1 domains, as highly conserved zones, represent special molecular patterns that can be recognized easily (Gewirtz, 2006; Zamboni et al., 2006). While TLR-5 responds to extracellular flagellin, IPAF detects cytosolic flagellin, due to their differential subcellular location in human epithelial cells (Miao et al., 2007). In addition, it has been proposed that neural apoptosis inhibitory protein 5 (Naip5) could also be involved in flagellin recognition (Wright et al., 2003).

Monomeric flagellin secreted by the probiotic bacterium Escherichia coli Nissle 1917 is recognized by TLR-5 and IPAF, receptors of the innate immune system, whose signalization alters genetic expression via MAPK pathways. This results in an increase of the production of human β-defensin, an antimicrobial peptide, and IL-8, a proinflammatory cytokine.

Figure 7

Monomeric flagellin secreted by the probiotic bacterium Escherichia coli Nissle 1917 is recognized by TLR-5 and IPAF, receptors of the innate immune system, whose signalization alters genetic expression via MAPK pathways. This results in an increase of the production of human β-defensin, an antimicrobial peptide, and IL-8, a proinflammatory cytokine.

Concluding remarks

Recent evidence suggests that a continuous exchange of communication between enteric bacteria and host cells is essential for the correct development and establishment of commensal relationships at the GIT. Some probiotic strains can improve GIT health and, for example, prevent enteropathogen colonization and induce host immunomodulation.

The results discussed in this review demonstrate the potential importance of exported proteins in GIT physiology and ecology, notably in the adhesion to intestinal surfaces and in the cross-talking between probiotic bacteria and the host. The main challenges for the near future are elucidation of the mechanism of anchorless-protein translocation and the identification and characterization of the small proteins secreted by probiotic bacteria involved in bacteria–host interaction. This will help to understand how probiotic bacteria are able to exert their positive effects on the host. Another challenge is to establish how moonlighting proteins (notably GADPH and EF-Tu) are able to cross the cytoplasmic membrane and arrive at the cell wall. Bioinformatics analysis should help produce new insights into the selection of putative surface proteins with a possible role in host interaction.

Identification of exported proteins, together with their mechanism of action over the host, will help elucidate the biological relevance and functions of probiotic bacteria in the GIT.

Acknowledgements

B.S. was the recipient of a Clarín postdoctoral contract from the Gobierno del Principado de Asturias funded by the Plan de Ciencia, Tecnología e Innovación (PCTI) de Asturias 2006–2009.

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Author notes

Editor: Willem van Leeuwen