Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, S-750 07 Uppsala, Sweden1
Author for correspondence: Hans Jonsson. Tel: +46 18 673382. Fax: +46 18 673392. e-mail: hans.jonsson{at}mikrob.slu.se
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ABSTRACT |
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Keywords: adhesion, mucin
Abbreviations: CnBP, collagen-binding protein; MBP, maltose-binding protein; NANB, non-A, non-B
The GenBank accession number for the sequence reported in this paper is AF120104.
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INTRODUCTION |
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Lactobacillus reuteri frequently occurs in the gastrointestinal tract of various mammals, including man (Molin et al., 1993 ; Naito et al., 1995
). Strains of this species have been described to possess several properties believed to be important for its capacity to colonize. These are adherence to epithelial cells (Wadström et al., 1987
), binding of fibronectin (Lindgren et al., 1992
), expression of the cell-surface CnBP (Aleljung et al., 1994
), production of an autoaggregation-promoting protein (Roos et al., 1999
) and production of the antimicrobial substance reuterin (Axelsson et al., 1989
). Recently, Jonsson et al. (2001)
reported that many strains of Lb. reuteri adhere to components in mucus. Interestingly, for some of the strains the adhesion to mucus was triggered by addition of mucin to the growth medium.
In this report we have characterized the adhesion of Lb. reuteri strain 1063 to mucus material in vitro. The results presented show that a high-molecular-mass protein, which possesses the typical features of cell-surface proteins from Gram-positive bacteria, adheres to components in mucus.
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METHODS |
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Proteins and reagents.
Lb. reuteri 1063 was grown for 16 h in 500 ml LDM II broth and the cells were harvested by centrifugation at 10000 g. Proteins in the spent culture medium were precipitated by adding (NH4)2SO4 to 40% saturation and pelleted by centrifugation at 15000 g. The proteins were dissolved in 50 mM acetic acid (HAc), dialysed (cut-off 10000 Da) against the same buffer and subsequently lyophilized. The protein material was finally dissolved in 1 ml PBS (g l-1: NaCl, 8·0; KCl, 0·2; Na2HPO4.2H2O, 1·44; KH2PO4, 0·2; pH 7·3) and stored at -20 °C. To remove loosely bound surface material, the pelleted bacteria were washed five times with distilled water (50 ml each time). Thereafter, the wash solution was lyophilized, the material was dissolved in PBS (1 ml) and stored at -20 °C. Antibodies were raised against material from the culture medium and from cell-surface water wash in a rabbit immunized with approximately 100 µg protein and given three booster doses at 2-week intervals. The animal was sacrificed 8 weeks after the first immunization. To raise the specific activity of the antiserum (P108) against the mucus adhesion factor, it was preadsorbed against cells of non-adhering Lb. reuteri 1068. The bacteria were grown in 200 ml MRS for 16 h and washed twice in PBS supplemented with 0·05% Tween 20 (PBST) after which they were suspended in 20 ml PBST. One millilitre of antiserum was mixed with 1 ml bacterial suspension and incubated at room temperature for 2 h. After centrifugation, the adsorbed antiserum was passed through a 0·2 µm filter. The IgG fraction from the adsorbed antiserum was purified on a Protein A-Sepharose CL-4B column (Amersham Pharmacia Biotech), according to the manufacturers instructions. Mucus from pig or hen was prepared from the small intestine of freshly slaughtered animals. The intestine was rinsed with cold PBST after which the mucus was released by gently scraping the mucosa and washing with PBST. Particles were pelleted by centrifugation and the mucus was stored at -20 °C. Urea was added (final concentration 8 M) to some of the mucus material after which it was fractionated on a Superose 6 PC 3.2/30 column in a Smart System (Amersham Pharmacia Biotech) at a flow rate of 50 µl min-1. Fractions (50 µl) were collected and stored at -20 °C. The relative molecular masses of the eluted fractions were estimated by calibration of the column with a Gel Filtration HMW Calibration Kit (Amersham Pharmacia Biotech).
Binding of bacteria to immobilized mucus.
Mucus material, fractionated mucus material, pig gastric mucin (Sigma; M1778) and BSA were dissolved and diluted in 50 mM Na2CO3 buffer, pH 9·7, and immobilized in microtitre wells (Greiner) by incubation of 150 µl solution overnight at 4 °C with slow rotation. The final concentrations or amounts used were an OD280 of 0·1 for the mucus material, 20x dilution for the fractionated mucus and 100 µg ml-1 for mucin and BSA. The wells were blocked with 0·2 ml PBS supplemented with 1% Tween 20 for 1 h and then washed with PBST. Lb. reuteri 1063 was grown in MRS broth for 16 h at 37 °C, washed once in PBST and diluted to an OD600 of 0·5 in the same buffer. A portion of the bacterial suspension was treated with proteinase K (100 µg ml-1) for 1 h at 37 °C and PMSF was added to a final concentration of 1 mM. Another portion was incubated with P108 antiserum IgG (30 µg ml-1), Mub1 (30 µg ml-1),
Mub2 (30 µg ml-1) (
Mub1 and
Mub2 are described below) or IgG from preimmune serum (30 µg ml-1) for 1 h at 37 °C. The bacteria from the different treatments and untreated bacteria were washed four times with PBST and diluted to an OD600 of 0·5 in the same buffer. Bacterial suspensions (0·15 ml) were added to each well and incubated for 1 h at room temperature. The wells were washed with PBST and the degree of binding was examined with an inverted microscope. The buffer was poured off and, after the wells had dried, OD403 was measured in an ELISA plate reader. All measurements were done in triplicate.
Construction and screening of a library from Lb. reuteri 1063.
Lb. reuteri 1063 was grown in 100 ml MRS broth and DNA was extracted according to Axelsson & Lindgren (1987) . The DNA was partially digested with Sau3AI and ligated into
EMBL3 BamHI arms. Packaging into phage particles was performed according to the manufacturers instructions (Promega). After infection of E. coli LE392, the resulting plaques were screened with the adsorbed IgG-fraction from P108, according to the procedure described previously (Roos et al., 1996
).
Subcloning and isolation of positive clones.
DNA from the clones 108:21 and 108:34 was isolated according to Sambrook et al. (1989)
and cleaved in separate reactions with EcoRI, HindIII, PstI, SalI, ScaI (complete cleavages) or Sau3AI (partial cleavage). The material from the five first cleavages was pooled, treated with T4 DNA polymerase to generate blunt ends and then ligated into a SmaI-cleaved pUC18 vector. The Sau3AI-cleaved material was ligated into a BamHI-cleaved pUC18 vector. Ligation mixes were electrotransformed into E. coli TG1 cells and the resulting clones were selected on LB agar plates containing ampicillin, followed by screening with the IgG fraction of the P108 antiserum as described previously (Roos et al., 1996
). Plasmid DNA from positive clones was purified with the Wizard Minipreps DNA purification system (Promega) and characterized by restriction enzyme analyses and sequencing. Deletion clones of 108.21:3 were constructed by using the EraseABase kit (Promega), according to instructions of the manufacturer, and subsequently used in the sequencing work.
DNA sequencing and analysis of the DNA and deduced protein sequences.
The clones and different plasmid subclones were used to determine the nucleotide sequence of the gene. Sequencing was performed by using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) and commercial standard and customized sequencing primers. The sequencing samples were analysed on an automatic sequencing machine (ABI 377; Perkin Elmer). The PC/GENE DNA and protein data-handling package was used for analysis of the DNA and deduced protein sequence. The SignalP server (http://www.cds.dtu.dk/services/SignalP) was used for prediction of signal peptides. NCBIs search tool BLAST (http://www.ncbi.nlm.nih.gov/blast/) was used for similarity searches. Alignments of protein sequences were done with the CLUSTAL W program (Thompson et al., 1994
) and phylogenetic analyses with programs of the PHYLIP package (Felsenstein, 1993
).
Production of fusion proteins and affinity-purified antibodies.
Plasmid DNA from 108.34:1 (encoding Mub2 repeats) was cleaved at SmaI and PstI sites in the multilinker of the vector and 108.34:3 (encoding Mub1 repeats) with the NaeI site in the gene and the PstI site in the multilinker (Fig. 1). The first cleavage resulted in a 2031 bp fragment and the second in a 1247 bp fragment that were purified and ligated into an XmnI/PstI-cleaved pMAL-c2 vector (encoding the maltose-binding protein, MBP; New England Biolabs). The constructs were electrotransformed into TG1 cells and transformants were selected on LB plates with 50 µg ampicillin ml-1. A number of clones were analysed by restriction enzyme digestion and DNA sequence determination. Production of fusion protein was verified by SDS-PAGE and Western blot analysis. Fusion proteins (MBP-Mub1 and MBP-Mub2) were produced and affinity-purified according to New England Biolabs instructions. The concentrations of the proteins were estimated by measuring A280 and the purity by SDS-PAGE analyses. The fusion proteins were coupled to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) and used for affinity purification of the P108 antiserum mentioned above. The purified antibodies (
Mub1 and
Mub2) were transferred to PBS buffer supplemented with 1% BSA and 0·05% NaN3 and stored at 4 °C. Antibodies against MBP (
MBP) were purchased from New England Biolabs.
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SDS-PAGE and Western blotting of Mub.
The protein preparations from the spent growth medium and the cell surface of strain 1063, Mub purified from strain 1063 and the two affinity-purified fusion proteins were mixed 1:1 with sample buffer containing SDS and 2-mercaptoethanol and separated by SDS-PAGE. Electrophoresis was performed on PhastGel Gradient 825% gels with the PhastSystem (Amersham Pharmacia Biotech), according to the manufacturers instructions. The proteins were blotted to a Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech) by diffusion at 65 °C for 45 min (PhastSystem Development Technique File No. 220). The membrane was blocked in PBST for 1 h at 37 °C followed by an incubation overnight at 4 °C with the P108 antiserum (diluted 1/1000) or affinity-purified antibodies Mub1 and
Mub2 (both diluted 1/1000). After washing with PBST, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (Bio-Rad), diluted 1/1000, at 37 °C for 1 h. After washing, the membranes were developed with 4-chloro-1-naphthol as substrate. The gels were stained with Coomassie blue after the blotting procedure.
Immunofluorescence detection of Mub on the bacterial surface.
Strains 1063 and 1068 were grown overnight in MRS broth and washed once in PBST. The bacteria were attached onto Poly Prep slides (Sigma) by drying 10 µl bacterial suspension (OD600 of 1·0) onto the slide. The slides were washed with PBST and blocked with PBST containing 5% goat serum for 1 h at room temperature with slow rotation in a humidity chamber. After washing with PBST, the slides were incubated with a mixture of the affinity-purified antibodies Mub1 and
Mub2, both diluted 1/30 in PBST. The slides were washed and incubated with FITC-labelled goat anti-rabbit F(ab')2 fragments (Sigma), diluted 1/40 in PBST, for 1 h in a humidity chamber. After washing, the presence of Mub was visualized by fluorescence microscopy. Controls where the primary antibodies were excluded were done in parallel.
Adhesion of Mub to immobilized mucus.
Mucus material, mucus fractions, mucin or BSA were immobilized in microtitre wells as described above. ELISA was performed by incubation of 150 µl lysate (produced according to Sambrook et al., 1989
), diluted to an A280 of 0·2, 150 µl purified Mub, diluted to an A280 of 4 x 10-4, or 150 µl fusion protein or MBP diluted to an A280 of 10-3, per microtitre well for 1 h at room temperature. Normally the proteins were diluted in PBST, but 10 mM citrate buffers, pH 36, with 0·15 M NaCl or 10 mM phosphate buffers, pH 67·4, with 0·15 M NaCl were used in some experiments. The wells were washed and incubated with the
Mub1,
Mub2 or
MBP antibodies, diluted 1/2000 in PBST, for 1 h at room temperature. After washing, the wells were incubated with an anti-rabbit IgG peroxidase conjugate (Bio-Rad), diluted 1/5000 in PBST, for 1 h at room temperature, washed and developed with 3,3',5,5'-tetramethylbenzidine as substrate, according to Bos et al. (1981)
. A450 was measured in an ELISA plate reader. All assays were performed in triplicate. Inhibition experiments were done by addition of different components to the microtitre wells together with the fusion proteins MBP-Mub1 and MBP-Mub2. The different components were mucin, fetuin, asialofetuin, BSA (each 1 mg ml-1) or maltose, mannose, glucose, fucose, raffinose, N-acetylgalactosamine, N-acetylglucosamine and sialic acid (each 10 mg ml-1).
SDS-PAGE and Western blotting of mucus material.
The mucus material was mixed with sample buffer, separated by SDS-PAGE and blotted as described above. After blocking in PBST for 1 h at 37 °C the membrane was incubated overnight at 4 °C with MBP-Mub2, MBP-Mub1 fusion proteins or MBP (diluted to an A280 of 5 x 10-3 in PBST) followed by incubation for 1 h at 37 °C with the affinity-purified antibodies Mub2 or
MBP (diluted 1/1000 in PBST). The incubation with the secondary antibody and development was performed as above.
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RESULTS |
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The best match in a database search using the BLAST search engine was an unknown protein from Lactococcus lactis subsp. lactis encoded by the gene ywfG (GenBank accession no. AAK06278). The protein has previously not been assigned any features, but it is possible to predict an N-terminal signal sequence and a C-terminal cell-wall anchoring motif (LPXTG), putative membrane-spanning region and a cell-membrane anchor. The protein consists of 926 aa and has four repeats of approximately 175 aa each. In a phylogenetic analysis, the repeats of this protein were shown to be more related to the Mub1 repeats (37% sequence identity) than the Mub2 repeats are. The Mub repeats also showed similarities to a non-A, non-B (NANB) hepatitis virus antigen (Reyes et al., 1990 ) and the human ocular component hr44 (Braun et al., 1995
). Some less pronounced similarities were also found with a family of high-molecular-mass adhesion proteins from Haemophilus influenzae (Barenkamp & St Geme, 1996
).
Production and purification of the MBP-Mub fusion protein
To produce and purify recombinant Mub, two constructs encoding fusions of the MBP with Mub1 and Mub2 repeats, respectively, were constructed in the protein expression vector pMAL-c2. MBP-Mub1 contains approximately two repeats whereas MBP-Mub2 contains almost four repeats and the molecular masses of the proteins are 94·4 and 123·2 kDa, respectively (Fig. 1). The purified fusion proteins were immobilized on Sepharose beads and used for affinity purification of the P108 antiserum. The antibodies obtained,
Mub1 and
Mub2, were used in Western immunoblot analyses and shown to react specifically with MBP-Mub1 and MBP-Mub2, respectively (data not shown). Both fusion proteins were subjected to degradation as shown on immunoblots (Fig. 2
, lanes 4 and 5).
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The N terminus of the purified protein was sequenced. The result showed a heterogeneous population of N-terminal sequences. However, by also looking at the second and third choice in some of the positions, the sequence of the predicted mature protein, ATTESNASAK, could be identified. This indicates that the predicted cleavage site is correct, but also that the protein is easily degraded.
By using affinity-purified antibodies against recombinant Mub in an SDS-PAGE analysis (Fig. 2), it was evident that the large protein purified from strain 1063 was recognized by the
Mub1 and
Mub2 antibodies and most probably represents the full-length protein encoded by mub. By using the antibodies in an immunofluorescence microscopy experiment, Mub could also be detected on the cell surface of strain 1063, but not on strain 1068 (Fig. 3
).
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Adhesion of the MBP-Mub2 (Fig. 4) and MBP-Mub1 (data not shown) fusion proteins to mucus material was also demonstrated in Western blot analyses. Both fusion proteins showed a similar adhesion pattern, while the fusion partner MBP itself did not adhere. The fusion proteins were shown to adhere to more than 10 different components of pig mucus with molecular masses between 15 and 45 kDa (Fig. 4b
), but also to components with very high molecular mass that could not enter the gel. Interestingly, they seemed to adhere only to a high-molecular-mass component in the hen mucus.
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DISCUSSION |
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The fact that strain 1063 adhered efficiently to both pig and hen mucus indicates that the bacterium has little or no host specificity regarding adhesion to mucus. No inhibition of adhesion of strain 1063 cells could be seen when Mub1 or
Mub2 antibodies were used separately. However, the significant inhibition observed when using the antibodies together suggests that both types of repeats are important for binding of the bacteria to mucus components. Moreover, since the P108 antibodies, raised against a mix of cell-surface and secreted proteins from strain 1063, blocked binding more efficiently than the mixture of
Mub1 and
Mub2, other proteins may also be involved in the binding.
The presence of two types of amino acid residue repeats and a >500 aa N-terminal region makes it likely that Mub is a multifunctional protein, in common with many described adhesins from Gram-positive organisms (Fischetti, 2000 ). The two types of repeats both adhere to mucus, mucus fractions and to mucin (Table 2
). Western blot experiments showed that the Mub1 and Mub2 repeats adhere to the same components in pig intestinal mucus and to the same components in hen mucus. Furthermore, the inhibition of binding by the glycoproteins fetuin and asialofetuin suggests that Mub adheres to carbohydrate structures in mucus. However, the binding of MBP-Mub1 was less sensitive to the addition of glycoproteins, indicating that it also interacts with other components in mucus. Of the simple carbohydrates tested, only fucose gave a reduction in binding of MBP-Mub1 and MBP-Mub2, indicating that it might form part of a more complex carbohydrate receptor structure. There was also a pH dependence in the binding with maximum binding below neutral pH. Interestingly, MBP-Mub1 and MBP-Mub2 had different pH optima for binding, which could reflect an adaptation to the interaction with different niches in the gastrointestinal tract. Mukai et al. (1998)
showed that three Lb. reuteri strains adhered to different sugar residues of glycoconjugates. Specificity of binding was determined for only one of the strains that, in contrast to Mub, exhibited binding that was sensitive to addition of N-acetylgalactosamine.
The type 1 and 2 repeats of Mub display extensive sequence similarities with each other and also to the repeats of the Lc. lactis protein YwfG. Lactococci are not usually considered as members of the normal intestinal microflora, but in studies during the 1970s Lc. lactis was commonly isolated from faeces from many different groups of humans (Finegold et al., 1983 ). The presence of a protein with strong similarities to Mub suggests that Lc. lactis actually is adapted to the gastrointestinal tract environment. The Mub repeats also share similarities with an NANB hepatitis virus antigen (Reyes et al., 1990
) and the human ocular component hr44 (Braun et al., 1995
). The hepatitis peptide is described as a peptide that is recognized by antiserum from a human infected with the virus and hr44 is a protein located on the cell surface of epithelial cells of the eye. The functions of these proteins are not known, but both might be present at surfaces that are in contact with a mucus layer. The finding that proteins from such distant organisms appear related is intriguing. It could be speculated that the similarities between the proteins reflect some common mechanism for interaction with mucus components.
In most cases, the initial step in infection by any micro-organism is adhesion or association with host mucosal surfaces. Therefore, blocking of this step is a potential point of disease control. In several reports, probiotic lactobacilli have been shown to inhibit the in vitro attachment of enterovirulent bacteria to mucus and enterocytes and also to prevent invasion of the pathogen (Craven & Williams 1998 ; Tuomola et al., 1999
). Whether the mechanism of inhibition is competition for the same receptor structures or an effect of steric hindrance by Lactobacillus cells is not known. Lb. reuteri has been tested for its probiotic activities in different animal systems and has been found to have protecting effects against pathogenic micro-organisms such as Salmonella (Edens et al., 1997
) and Cryptosporidium (Alak et al., 1997
). Furthermore, children suffering from rotavirus diarrhoea were found to have a significantly reduced duration of the disease when treated with Lb. reuteri (Shornikova et al., 1997
). The importance of the mucus-binding properties of lactobacilli in this context has not yet been evaluated. Preliminary results show that mub is not present in a majority of other Lb. reuteri strains tested. However, tested strains of the species Lactobacillus mucosae harboured it (Roos et al., 2000
). Construction of a mub mutant is planned in the near future. This is the key for a thorough evaluation of the role of Mub in colonization and in the control of pathogenic micro-organisms.
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ACKNOWLEDGEMENTS |
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Received 17 April 2001;
revised 24 September 2001;
accepted 10 October 2001.