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Proc Natl Acad Sci U S A. Oct 6, 2009; 106(40): 17193–17198.
Published online Sep 17, 2009. doi:  10.1073/pnas.0908876106
PMCID: PMC2746127
Microbiology

Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human- mucus binding protein

Abstract

To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins (spaCBA) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues.

Keywords: genome, probiotics, adhesion, pilus, lactic acid bacteria

The Gram-positive lactobacilli are commensal inhabitants of the gastrointestinal (GI) tract that also play important roles in the production and preservation of food. Based on their health-promoting effects, these bacteria are commonly marketed as probiotics (13). One postulated feature considered indispensable for some probiotic lactobacilli is adherence to human intestinal tissues, which may promote a variety of specific interactions with the host (46). Despite many studies demonstrating the adherent properties of lactobacilli, the molecular mechanisms governing these host–microbe interactions are only beginning to emerge as comparative and functional analyses of their genome sequences progress (7, 8). Cell surface components that promote the adherence of lactobacilli include exopolysaccharides (EPSs); teichoic acids (TAs); and surface-exposed proteins, such as the S-layer and LPXTG-like proteins (4). Several of these components also function as modulators of the immune response, such as in the Lactobacillus plantarum D-alanine–depleted TA activation of the Toll-like receptor 2 (9) and the Lactobacillus acidophilus S-layer stimulation of the DC-SIGN receptor on dendritic cells (10). Recently, it was reported that specific immune responses were elicited in human subjects by L. plantarum and Lactobacillus rhamnosus, although the molecular mechanisms of these interactions await further characterization (11, 12).

A possible mechanism for adherence and colonization that has not yet been identified in lactobacilli (4) but is well established in many Gram-positive pathogens involves proteinaceous surface-exposed polymeric structures known as pili (13, 14). These Gram-positive pili have a narrow diameter (1–10 nm) but can project outwardly from the cell surface with lengths of 1 μm or more. Unlike the Gram-negative pili, each Gram-positive pilus is an assembly of multiple pilin subunits coupled to each other covalently by the transpeptidase activity of the pilin-specific sortase (1315). Recent studies have established that the resulting isopeptide bonds are formed between the threonine of an LPXTG-like motif and the lysine of an YPKN pilin motif in the different pilin subunits (15, 16). Another membrane-bound transpeptidase, the housekeeping sortase, also recognizing the LPXTG-like motif on target pilins is responsible for attaching the base of the elongated pilus covalently to the peptidoglycan in the cell wall (1315). Typically, the pilus is a heterotrimer composed of a major pilin forming the pilus shaft, a minor pilin decorating the pilus backbone, and another minor pilin with adhesive properties often situated at the pilus tip (13, 14). The genes coding for the 3 pilin subunits and the pilin-specific sortase are usually localized at the same locus as a gene cluster and are frequently flanked on both ends by transposable elements, suggesting an origin by horizontal gene transfer (13).

L. rhamnosus GG, originally cultured from a healthy human intestinal source, has been thoroughly studied and used safely as a probiotic strain in a variety of functional foods for nearly 20 years (2, 3, 1720). In our efforts to decipher the molecular mechanisms involved in the interaction between strain GG and the human host, we determined the complete genome sequences for L. rhamnosus GG and L. rhamnosus LC705, an industrial strain used routinely as an adjunct starter culture in dairy products (21). Several studies have compared the adhesion properties of these two strains and concluded that GG is considerably more adherent than LC705 to human tissue (22, 23). In this study, the presence of a cluster of pilus-encoding genes (spaCBA) associated with the genome of strain GG is reported, and the expression of a pilin subunit (SpaC) was confirmed by immunogold electron microscopy. Employing anti-SpaC immune serum and an L. rhamnosus GG mutant carrying an inactivated spaC gene, it was demonstrated that SpaC is a key factor for adhesion between strain GG and human intestinal mucus. These findings and the comparative genomic analysis with strain LC705 provide an important framework for understanding a molecular mechanism for the interaction of L. rhamnosus GG with human host tissues.

Results

Comparative Genomics of the L. rhamnosus GG and LC705 Genomes.

The complete genomes of the L. rhamnosus GG and LC705 strains were sequenced and annotated. Both genomes are among the largest sized of the lactobacilli genomes, which typically average ≈2 Mbp (7, 8). The genome of strain GG contains a single circular chromosome 3.01 Mbp in size, whereas strain LC705 contains a slightly smaller chromosome (2.97 Mbp) and a 64.5-kb circular plasmid, pLC1 [Table 1 and supporting information (SI) Fig. S1]. Between the two L. rhamnosus strains, ≈3,000 predicted proteins are conserved with a high average amino-acid identity (98%) (Table S1). However, a detailed comparison of the predicted protein sequences equal to or longer than 100 aa revealed that strain GG contains 331 strain-specific proteins, somewhat less than the 383 proteins for LC705.

Table 1.
General genomic features of L. rhamnosus GG and LC705 and selected Lactobacillus spp.

The genomes of strains GG and LC705 exhibit a high degree of synteny that is unexpectedly interrupted by several genomic islands (Fig. S1 and Fig. S2A). Five genomic islands in strain GG (designated GGISL1-5) are predicted to encode 80 unique proteins with lengths of 100 residues or more, whereas in strain LC705, 4 islands (designated LCISL1-4) encode 72 unique proteins. All the genomic islands were identified as those DNA sequences deviating in codon usage, base composition, and dinucleotide frequency from the rest of the genome (24). Moreover, these genomic islands were not conserved between the closely related strains (Fig. S2A), suggesting that they had originated by horizontal gene transfer. In general, horizontally transferred gene islands display special biological functions, and this appeared to be the case for the genomic islands found in strains GG and LC705. Genomic islands with the capacity to transport or metabolize sugars (GGISL1, LCISL1, LCISL2, and LCISL4) and produce specific EPSs (GGISL5 and LCISL3) were observed, as were 2 islands in strain GG enriched with phage-associated genes (GGISL3 and GGISL4). One of the prophages (GGISL3) extending from LGG_01086 to LGG_01143 resembles the Lactobacillus casei American Type Culture Collection (ATCC) 334 prophage (8, 25, 26) to some extent. Remarkably, the GGISL2 island appears to encode a set of genes for 3 pilin proteins (SpaCBA) and a sortase, an obligatory requirement for the assembly of pilus structures, which are considered important for colonization and host interaction in some Gram-positive bacteria (13, 14).

L. rhamnosus belongs to a taxonomic group (L. casei, Lactobacillus paracasei, and Lactobacillus zeae) that is evolutionary distant from other lactobacilli (1). Genomic comparisons with other lactic acid bacteria (LAB) confirmed this taxonomic grouping, and our analysis showed that L. casei ATCC334 (8) is the nearest relative of L. rhamnosus GG and LC705. The majority of the deduced proteomes (68–72%) for the two L. rhamnosus strains and L. casei are conserved and display a high degree of average amino-acid identity (≈85%) (Table S1). Moreover, overall genomic comparisons showed that the colinearity between each of the strains was interrupted by the genomic islands described previously (Fig. S2A). Additional comparisons between the predicted proteomes of different Lactobacillus spp. indicated that, apart from the L. casei strain, strains GG and LC705 were distinctly different from other lactobacilli, showing an average amino-acid identity of only 52–59% (Table S1 and Fig. S2B). In total, 143 and 176 proteins were identified in strains GG and LC705, respectively, without equivalent proteins in other lactobacilli. Many of these predicted proteins (24 in GG and 22 in LC705) were related to carbohydrate transport and metabolism (Fig. S3).

Carbohydrate utilization assays showed that strains GG and LC705 both use a variety of mono- and disaccharide substrates (Table S2), a metabolic activity considered advantageous for bacteria residing in the carbohydrate-rich proximal region of the small intestine (4). Both genomes encode a ubiquitous set of phosphotransferase system (PTS) transporters (Table S3) as well as many carbohydrate-metabolizing enzymes depicted in a metabolic network reconstruction (Fig. S4A). An important metabolic feature commonly exploited in industrial applications is the ability to use lactose, which is lost in strain GG because of frameshifts in the antiterminator (lacT) and 6-phospho-β-galactosidase (lacG) genes (Fig. S4B). Other experimentally verified metabolic differences, including the inability of strain GG to use rhamnose, ribose, and maltose, were explained by genetic variations in enzymes or transporters (Fig. S4A). We also identified 40 and 49 genes predicted to encode potential glycosidases in the genomes of strains GG and LC705, respectively. Because of their annotation and predicted cellular location, several of these (10 in GG and 9 in LC705) may participate in peptidoglycan hydrolysis and conversion of complex polysaccharides and prebiotics to simple carbohydrates.

Unlike L. rhamnosus LC705, strain GG is unable to catabolize lactose or casein, the major nutritional components of milk. Given that nitrogen utilization would be important for growth of L. rhamnosus within the host GI tract (4), we compared the predicted protein metabolism of both strains. Biosynthesis and transport of amino acids and peptides and the proteolytic machineries in strains GG and LC705 are similar but differ in one major aspect (Fig. S4C and Table S3). Only strain LC705 harbors a plasmid (pLC1) carrying the cysE and cysK genes, which permits the biosynthetic conversion of serine to cysteine (Fig. S4C). No obvious differences were observed in the predicted enzymatic machinery for casein breakdown between the two L. rhamnosus strains, despite the observation that only strain LC705 can degrade this type of milk protein (Fig. S5). Both strains encode a cell envelope serine protease (PrtP), maturation protein (PrtM), and proteinase (PrtR), in addition to a similar set of 25 peptidases. However, within the LC705 genome, a gene for an additional secreted subtilisin-like serine protease (LC705_02680) was predicted, which may be involved in casein degradation.

Molecules Involved in Host–Microbe Interaction and Adhesion.

Specific secreted and small-sized soluble proteins produced by L. rhamnosus GG have been reported to modulate epithelial cell growth and immune responses (27, 28). We predicted that secreted or cell surface-exposed proteins (Table S3) encompass close to 7% of the deduced proteomes in both L. rhamnosus strains and that 85 proteins associated with each genome are soluble and may possibly contribute to bacterium–host interactions.

Other cell surface components putatively involved in host–cell interactions (29, 30) and potentially implicated in promoting beneficial health effects are the cell envelope-bound or -secreted EPSs. The EPS biosynthetic gene cluster (LCILS3) in the genome of strain LC705 (Fig. S1B) was nearly identical to the clusters present in 4 other L. rhamnosus strains (ATCC9595, R, RW-9595M, and RW-6541M) producing rhamnose-rich EPSs (29). However, these other clustered genes are all genetically different from the EPS gene cluster in L. rhamnosus GG (GGISL5), which has recently been shown to produce a long galactose-rich EPS that reportedly modulates biofilm formation (30).

Bacteriocins are small antimicrobial peptides secreted by many Gram-positive bacteria (31). Although some studies have reported the detection of a bacteriocin-like activity in L. rhamnosus GG, it is still unclear whether this strain actually produces an active bacteriocin (4, 23). The genome of strain GG contains an 8.7-kb putative type IIb bacteriocin operon (LGG_02385–LGG_02392) that encodes the various components required for bacteriocin synthesis, including the export protein, ABC/C39-type peptidase, 2-component signal transduction system, immunity protein, and bacteriocin. The predicted bacteriocin from strain GG consists of 2 short peptides, both containing the bacteriocin type II leader motif required for C39 peptidase-mediated recognition. An identically organized operon (LC705_02382–LC705_02390) was also detected in the genome of strain LC705, although the export protein and the histidine protein kinase both possess a single frameshift mutation, which likely results in the translation of truncated and nonfunctional proteins.

L. rhamnosus GG adheres to mucus and epithelial cell lines about 10-fold more efficiently than strain LC705 (22, 23). A human intervention study showed that strain GG persisted at higher levels than LC705 throughout the study and for 7 days longer in the GI tract of healthy volunteers (Fig. 1). Because the mucus-binding capacity of strain GG appears to be sensitive to protease (22), we speculated that a protein-mediated strain-specific adhesion was responsible for the high binding properties of strain GG. The comparative genomic analysis identified multiple types of proteins (31 in GG and 37 in LC705) with domains related to adhesion and host colonization (Fig. S6). Several of these proteins contained fibronectin-binding domains, but, surprisingly, only a single protein in both genomes (LGG_02337 in GG and LC705_02328 in LC705) contained mucus-binding domains (Table S3). It was noteworthy that 8 proteins in strain GG, including the proteins encoded by the spaCBA gene cluster (see below), and 14 proteins in strain LC705 were strain specific.

Fig. 1.
The colonization properties of L. rhamnosus GG and LC705 in the human GI tract. A short-term intervention study involving 12 healthy adults assessed the colonization properties of two L. rhamnosus strains in the GI tract. Fecal samples were collected ...

Demonstration of Mucus-Binding Pili in L. rhamnosus GG.

Pili are cell surface-localized protrusions that have been well characterized in several Gram-positive pathogens. In general, two or three subunit genes encoding a Gram-positive pilus are organized into an operon, along with at least one sortase gene (13, 14). Genes encoding pilin subunits and pilin-specific sortases were identified in the genomes of strains GG and LC705, an observation that has not been documented previously in the genomes of other Lactobacillus species. Specifically, GG harbors 2 separate pilus loci in its genome: the spaCBA (GGISL2) and spaFED gene clusters (Fig. S1A). In contrast, only 1 pilus locus identical to the spaFED gene cluster in strain GG was detected in the genome of strain LC705 (Fig. 2Aand Fig. S1B). Aside from the closely related Lactobacillus strains, a homology search did not yield matches to other bacterial pilins that exhibited high amino-acid identities. However, a low degree of amino-acid sequence identity (≈30–40%) was observed for pilins found in related Gram-positive species, such as Enterococcus faecalis and Enterococcus faecium (32). Based on these analyses, SpaA and SpaD were predicted to be the major pilin subunits forming the pilus shaft or backbone, SpaB and SpaE the ancillary minor pilin subunits, and SpaC and SpaF the large-sized minor pilin subunits likely functioning as adhesins. Moreover, several motifs found typically in the primary structure of pilin were identified (Fig. 2A). Each pilin subunit contains a Sec-dependent secretion signal; an LPXTG-like motif; and, excluding SpaE, an E box. Moreover, the YPKN pilin-like motif (13, 14), which contains an essential lysine residue whose side-chain amino group has been implicated in isopeptide bonding, was also detected in the SpaA, SpaB, and SpaD pilins (Fig. 2A and SI Text).

Fig. 2.
The unique spaCBA pili cluster of L. rhamnosus GG and its expression. (A) Schematic illustration of the spaCBA and spaFED gene clusters. Depicted are the sortase (dark gray arrows), pilin subunits (white arrows), Cna protein B-type domain (gray), E box ...

To confirm the expression of pili on the cell surface, cell wall protein extracts from strains GG and LC705 were immunoblotted using polyclonal antibodies specific for Escherichia coli-expressed SpaC pilin (Fig. 2B). In addition to the 90-kDa monomeric SpaC subunit, a ladder of high molecular weight (HMW) protein complexes (lane 3) representing the various extended lengths of pili was detected in the cell wall-associated protein fraction from strain GG, as typically observed for piliated Gram-positive pathogens (14). Cell wall protein extracts from cells of strain LC705 (lane 4) served as a negative control, because the spaCBA cluster is not present in the LC705 genome. To establish whether the SpaC pilin is a component of the pilus structure, the spaC gene was inactivated using genetic techniques described previously (30) and the resulting insertional mutant strain (GG-ΩspaC) was then analyzed by immunoblotting. We observed that bands for the HMW protein complex were not present in the mutant strain (lane 2), suggesting that SpaC had not polymerized into the pilus structure. However, we did detect a protein band that corresponds to the predicted size (56 kDa) for a C-terminal truncated SpaC protein produced because of the insertional inactivation.

Subsequently, direct localization of pili on the cell surface of L. rhamnosus GG was demonstrated by immunogold transmission electron microscopy (Fig. 3). Briefly, strain GG cells cultivated to stationary phase were treated with anti-SpaC polyclonal antibodies, labeled with protein A-conjugated gold particles (10 nm) and then negatively stained. As shown in the electron micrograph (Fig. 3), multiple pili, averaging 10 to 50 per cell and with lengths of up to 1 μm, extend outwardly from the surface of the cells, with the majority situated predominantly near the cell poles (Fig. 3B). Significantly, the gold particles are not merely confined to the pilus tip but are also found throughout the length of the pilus, indicating that multiple copies of SpaC can be incorporated within the pilus structure.

Fig. 3.
Identification of pili in L. rhamnosus GG by immunogold electron microscopy. L. rhamnosus GG was grown to stationary phase, treated with anti-SpaC serum, labeled with protein A-conjugated gold particles (10 nm), negatively stained, and examined by transmission ...

To assess the receptor specificity of SpaC-containing pili, we examined the adhesion properties between E. coli-expressed SpaC protein and human intestinal mucus. Binding of radiolabeled (125I) SpaC to mucus was observed and was approximately 6-fold more than the background level of mucus binding by radiolabeled ovalbumin (Fig. 4). Competition with unlabeled SpaC protein showed that the mucus binding was inhibited in a dose-related manner (P = 2.0 × 10−7), suggesting that the SpaC pilin may recognize mucosa-related components. Moreover, binding of L. rhamnosus GG cells to mucus has been determined previously (6, 22), but we observed a 10-fold reduction in mucus binding when cells were pretreated with SpaC antiserum (Fig. 5). To rule out steric hindrance by the antibodies bound to the pilus structure as the reason for the reduced mucus adherence, we tested the mucus-binding properties of an insertional mutant strain (GG-ΩspaC). Our results showed that insertional inactivation of the spaC gene caused mucus binding to be nearly eliminated (Fig. 5), providing strong evidence for a role of the SpaC pilin in mucin binding and likely contributing to retention of strain GG during transit through the GI tract.

Fig. 4.
In vitro competitive binding of L. rhamnosus GG SpaC pilin to human intestinal mucus. Recombinant SpaC pilin was radiolabeled (125I), and the binding to human intestinal mucus was competitively inhibited by 2-, 10-, and 100-fold increases in unlabeled ...
Fig. 5.
Adhesion of L. rhamnosus GG and GG-ΩspaC to human intestinal mucus. Radiolabeled (3H) cells of the L. rhamnosus GG strain (with or without SpaC antiserum pretreatment) and the GG-ΩspaC mutant were tested for binding to human intestinal ...

Discussion

We determined the complete genome sequences of probiotic strain L. rhamnosus GG and adjunct starter culture strain LC705 and performed an in-depth comparative genomic survey complemented by functional analyses. Our comparative analysis of the two genomes, each ≈3.0 Mbp in size, revealed a high level of sequence homology and genomic synteny. However, several differences were observed in the number of strain-specific genes and the genomic islands encoding proteins for EPS biosynthesis (29, 30), specific sugar utilization (Table S2), and bacteriophage production. Considering that only a few studies have addressed bacteriophages in L. rhamnosus strains (26), it is noteworthy that an isometric bacteriophage was observed in the electron micrographs of GG cells, possibly indicating that the phage components encoded by one of the genome islands are functional (Fig. 3A).

An unexpected observation in the GG and LC705 genomes was the presence of genes for 3 canonical pilus subunits and a dedicated sortase typically required for assembling pili, proteinaceous appendages characterized previously in Gram-positive pathogens for facilitating host–cell attachment (13, 14). Two pilus gene clusters (spaCBA and spaFED) were identified within the GG genome, whereas only the spaFED cluster was predicted in strain LC705. To characterize the unique spaCBA cluster, we established SpaC pilin expression and verified the presence of multiple SpaCBA pili on the surface of GG cells. Moreover, using a mutant with an insertionally inactivated spaC gene and by treating GG cells with SpaC antiserum, it was shown that SpaCBA pili mediate strain GG adherence to human intestinal mucus. Because the spaC insertional mutant used here had secreted a C-terminal truncated SpaC protein, the most likely explanation for a reduction in mucosal adhesion is that the truncated form was missing the consensus sequences (E box, YPKN pilin-like motif, and sortase recognition site) considered important for pilus assembly, and thus could not be coupled within the polymerizing pilus structure. Alternate explanations for the reduced mucus adherence may include a polar effect of the ΩspaC mutation, the disruption of the pilus assembly process by incorporating a truncated SpaC, or the assembly of a truncated SpaC with an impaired mucus-binding site. However, the latter explanation is less likely, because the most probable adhesion domain for SpaC is located within the translated N-terminal region of the truncated protein. Interestingly, a stretch of SpaC sequence (residue 137–262) is similar to the type A domain of the von Willebrand factor (vWFA), which is considered a prerequisite for the adherence of Streptococcus agalactiae pili to epithelial cells (33). Despite interacting primarily with human extracellular matrix proteins (34), a region of the vWFA domain (residue 201–299) demonstrates weak homology with a fucose-binding lectin domain, suggesting that the vWFA-like domain in SpaC may bind in a lectin-type manner, a distinct plausibility considering that heavily glycosylated mucin glycoproteins are the main component of mucus (35). Because the L. rhamnosus GG and LC705 genomes only encode a limited set of strain-specific adhesins, it is reasonable to speculate that the prolonged intestinal persistence of strain GG found during an intervention study (Fig. 1) may be attributed to the mucus-binding capacity of SpaCBA pili.

Following the initial reported health benefit of L. rhamnosus GG in 1987 (36), strain GG has become one of the most comprehensively studied probiotic cultures in use today (2, 3). However, a detailed understanding of the molecular mechanisms governing the interplay between the human host and strain GG has been hindered and largely undiscovered because of insufficient genomic information on L. rhamnosus GG. By determining the complete genome sequence of strain GG and by comparing it with the closely related strain LC705, the molecular framework has been expanded for the discovery of cell surface components and other factors involved in host–microbe interactions. Our findings represent the previously unreported observation of pili in probiotic LAB, indicating how the probiotic strain GG may persist in the host and possibly compete with pathogens for residence sites in the human intestinal tract (6). As well, because pili from Gram-positive pathogens have established immunostimulatory effects (37), it is tempting to speculate that the observed pilus structures could function as immune stimuli that may support some health-promoting properties of L. rhamnosus GG (1719).

Materials and Methods

L. rhamnosus GG and LC705 strains were manipulated, sequenced, and analyzed as described in SI Text. The genomic sequences of strains GG and LC705 and the plasmid pLC1 have been deposited in the European Molecular Biology Laboratory (EMBL) database under accession numbers FM179322, FM179323, and FM179324, respectively.

Detailed descriptions of methods for cloning, immunoblotting, adhesion assays, insertional mutant construction, intervention trials, and immunogold electron microscopy are provided in SI Text. The GI colonization study was approved by the Coordinating Ethics Committee for the Hospital District of Helsinki and the Uusimaa region. Resected human intestinal tissue was the source of mucus, and its recovery and use were approved by the Ethics Committee for the Hospital District of Southwest Finland and with the fully informed written consent of the patients.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Docent Ilkka Palva for helpful discussions on pilus-related topics. We are grateful to the technician team at the Institute of Biotechnology and thank Juha Laukonmaa and Tuula Vähäsöyrinki at Valio Ltd., Tine Verhoeven and Lindsey Broos at Katholieke Universiteit Leuven, and Katariina Kojo and Marko Sutinen at the University of Helsinki for their skillful technical assistance. Heikki Huhtinen, MD, PhD, from the Department of Surgery, Turku University Central Hospital, is acknowledged for collecting human intestinal tissue samples. The Finnish Funding Agency for Technology and Innovation (Grants 40274/06 and 259/05) and Academy of Finland (Grants 118165 and 118602), including the funding for the Center of Excellence in Microbial Food Safety Research, are acknowledged for supporting this work.

Footnotes

Conflict of interest statement: S.T., R.K., T.M.-S., K.H., K.T.K., H.K., M.S., K.L., A.S., and T.S. are employed by Valio Ltd, which produces and markets the L. rhamnosus GG and LC705 strains. M.K., L.P., P.A., S. Lebeer, S.C.J.D.K, J.V., and A.P. have received research funds from Valio Ltd.

Data deposition: The sequences reported in this paper have been deposited in the European Molecular Biology Laboratory Nucleotide Sequence Database, www.ebi.ac.uk/embl (accession nos. FM179322, FM179323, and FM179324).

This article contains supporting information online at www.pnas.org/cgi/content/full/0908876106/DCSupplemental.

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