Structural and immunochemical studies of neutral exopolysaccharide produced by Lactobacillus johnsonii 142

Carbohydrate Research 345 (2010) 108–114

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Structural and immunochemical studies of neutral exopolysaccharide produced by Lactobacillus johnsonii 142 Sabina Górska a, Wojciech Jachymek a, Jacek Rybka a, Magdalena Strus b, Piotr B. Heczko b, Andrzej Gamian a,c,* a

Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland Chair of Microbiology, Jagiellonian University Medical College, Czysta 18, 31-121 Kraków, Poland c ´ skiego 10, 50-368 Wrocław, Poland Department of Medical Biochemistry, Wrocław Medical University, Chałubin b

a r t i c l e

i n f o

Article history: Received 16 July 2009 Received in revised form 8 September 2009 Accepted 13 September 2009 Available online 27 September 2009 Keywords: Lactobacillus johnsonii Exopolysaccharide Lactic acid bacteria Crohn disease Inflammatory bowel disease Probiotic

a b s t r a c t This paper describes the structure of neutral exopolysaccharide (EPS) produced by Lactobacillus johnsonii 142, strain of the lactic acid bacteria isolated from the intestine of mice with experimentally induced inflammatory bowel disease (IBD). Sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, NOESY, and 1H,13C HSQC experiments revealed that the repeating unit of the EPS is a pentasaccharide: ?3)-a-D-Galp-(1?3)-b-D-Glcp-(1?5)-b-D-Galf-(1?3)-a-D-Galp-(1?3)-a-D-Galp-(1? The rabbit antiserum raised against whole cells of L. johnsonii 142 reacted with homologous EPS, and cross-reacted with exopolysaccharide from Lactobacillus animalis/murinus 148 isolated also from mice with IBD, but not reacted with EPS of L. johnsonii 151 from healthy mice. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Bacteria of the genus Lactobacillus are Gram-positive microorganisms, belonging to the group of lactic acid bacteria (LAB), natural inhabitants of mammalian gastrointestinal tract. For some LAB strains the probiotic activity has been identified, which means that strains had a beneficial influence on the health of the host.1 Colonization of the intestine by probiotic microorganisms is considered to be an important factor for antagonistic activity against enteropathogens, modulation of the immune system activity of the host, improved healing of damaged gastric and intestinal mucosa, reducing lactose intolerance or hypocholesterolemic action.2,3 Most of these activities are primarily connected with adherence of the microbial cells to the intestinal mucosa. The surface antigens of

Abbreviations: COSY, correlation spectroscopy; 1D and 2D, one- and twodimensional; DEAE, diethylaminoethyl; EPS, exopolysaccharide; GLC–MS, gasliquid chromatography–mass spectrometry; HMBC, 1H-detected heteronuclear multiple-bond correlation spectroscopy; HSQC, 1H-detected heteronuclear single quantum coherence spectroscopy; HSQC-TOCSY, the NMR experiment consisting of a HSQC sequence followed by a TOCSY sequence; IBD, inflammatory bowel disease; LAB, lactic acid bacteria; NA, nucleic acid; NOESY, nuclear Overhauser effect spectroscopy; PBS, phosphate-buffered saline; TA, teichoic acid; TCA, trichloroacetic acid; TFA, trifluoroacetic acid; TOCSY, total correlation spectroscopy. * Corresponding author. Tel.: +48 71 370 99 82; fax: +48 71 370 99 75. E-mail address: [email protected] (A. Gamian). 0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.09.015

bacterial cell, for example, capsular polysaccharides or proteins of the cell wall play the essential role in this process.4 It has been shown that the exopolysaccharides (EPSs) from LAB alter the adhesion of pathogenic bacteria to intestinal mucus.5 There is also a growing number of publications reporting on the use of purified exopolysaccharides as immunostimulatory substances in the experimental therapy.6,7 During the investigations concerning the immunological activity of various polysaccharides from probiotic bacterial strains, the information about the molecular structure of the polysaccharide is essential. While structures of a number of EPSs of LAB are described, they come mainly from the strains of industrial significance.8 In the present work we report the structure of the neutral exopolysaccharide produced by Lactobacillus johnsonii 142, the strain isolated from mice with experimentally induced inflammatory bowel disease (IBD). IBD is a term covering two human diseases with chronic gastrointestinal inflammation: ulcerative colitis and Crohn’s disease, whose pathology is characterized by inflammatory cell infiltration in gut submucosa. The etiology of IBD is largely unknown but it is generally accepted that the chronic inflammation is perpetuated by gut commensal microflora in genetically predisposed hosts with altered immune response to bacterial products.9 Typical IBD changes strongly resembling human disease can be evoked in specific animal models, mostly in knockout mice colonized with specifically altered gut microflora (Altered Schaedler Flora) containing several species

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of Lactobacillus.10 As it has been previously demonstrated by us, lactobacilli can actively modify the inflammatory process in both ways.11 In this paper we present data on the structure of exopolysaccharide produced by bacterial strain Lactobacillus johnsonni 142, which has been isolated from the intestine of mouse with IBD. We show also the results of preliminary immunochemical studies of two other exopolysaccharides, namely of Lactobacillus animalis/murinus 148 isolated also from mice with IBD and of L. johnsonii 151 from healthy mouse. 2. Results Bacterial strains were grown on MRS broth under anaerobic conditions. Electron micrograph of L. johnsonii 142 cells showed symmetrical bacterial rods with EPS accumulated around cells, as revealed by ruthenium red staining specific for polysaccharide (Fig. 1). Exopolysaccharide was obtained by TCA extraction of bacterial mass, then was precipitated from the solution with ethanol (5 vol) and was purified by treatment with DNase, RNase, protease, further by ion-exchange chromatography on DEAE-Sephadex A-25, and finally by gel filtration. Typical chromatographic pattern of ion-exchange chromatography consisted of saccharide-positive fraction of neutral exopolysaccharide (EPS), saccharide- and phosphate-positive fraction typical for teichoic acid (TA) and phosphate-positive fraction of nucleic acid (NA). Average molecular mass estimated from gel filtration chromatography on TSK HW55S column was around 1.0  105 Da. The average yield of EPS preparation from dry bacterial mass was 1.2%. The sugar analysis and absolute configuration determination of the EPS components have shown that the EPS is composed of D-Glc and D-Gal in molar ratio of 1:4. Methylation analysis revealed the presence in EPS of 5-substituted galactofuranose or 4-substituted galactopyranose (the 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylgalactitol-1-d), 3-substituted galactopyranose (1,3,5-tri-O-acetyl-2,4,6tri-O-methylgalactitol-1-d), and 3-substituted glucopyranose (1,3,5tri-O-acetyl-2,4,6-tri-O-methylglucitol-1-d) in molar ratios 1:3:1. 4-Substituted galactopyranose was excluded due to NMR data. The detailed NMR analysis of the L. johnsonii strain 142 polysaccharide showed signals from five anomeric carbons and protons. The sugar residues are indicated by capital letters as shown in the structure given in Figure 2, the numbers denote carbons and protons, and these letters and numbers refer to the relevant sugars throughout the entire text, tables, and figures. Due to overlaps of spin systems HSQC-TOCSY and HMBC connectivities were used for unambiguous assignments of chemical shifts of sugars. By taking into account the previously published NMR data and comparing the chemical shifts for the respective monosaccharides8,12–14

Figure 1. Electron micrograph showing EPS accumulation around Lactobacillus johnsoni 142 cells.

Figure 2. Structure of the L. johnsoni 142 polysaccharide. The capital letters refer to sugar residues.

as well as considering the 3JH,H-values estimated from the crosspeaks in the two-dimensional spectra for the coupling between ring protons, each sugar residue could be identified and their anomeric configurations determined. Starting with the anomeric proton H-1, the COSY spectrum determined the H2 signal and the TOCSY spectra, with different mixing times, the H-3 to H-4 and H-6 for galactose and glucose residues, respectively (Fig. 3). To assign all other (i.e., H-5 and H-6) galactose signals as well as for the assignment of overlapping C and D monosaccharide spin systems the HSQC-TOCSY and HMBC connectivities had to be used. From the assigned 1H signals and the one-bond C–H connectivities, the carbon signals were assigned in the HSQC-DEPT spectrum (Fig. 4) in which CH2 moieties of all the sugars were readily identified as negative cross-peaks. By these procedures all the spin systems were determined (Table 1). The substitution positions of the respective monosaccharides were then identified on the basis of the relative high chemical shift values of the signals of the substituted carbons, when compared to values for the unsubstituted monosaccharides.12 The anomeric configurations of the residues were determined from 1H and 13C chemical shifts and the values of 3JH1,H2 and 1JC1,H1. The 1H and HSQC-DEPT NMR spectra of the L. johnsonii strain 142 polysaccharide contained signals for five anomeric protons and carbons (Fig. 4A and B). Residues A with H1/C1 signals at 5.44/99.2 3JH1,H2 3.3 Hz, 1JC1,H1 175 Hz, B with H1/C1 signals at 5.23/95.2 3JH1,H2 3.4 Hz, 1JC1,H1 170 Hz, and C with H1/C1 signals at 5.19/95.6 3JH1,H2  3 Hz, 1JC1,H1 171 Hz were identified as 3-substituted a-D-galactoses due to the large coupling between H-1, H-2, and H-3 and the small vicinal coupling between H-3, H-4, and H-5 and chemical shifts similar to those of a-D-Galp. The 3JH1,H2 and 1JC1,H1 values confirmed pyranosidic configuration of these moieties. Residue D 5.20/109.4 3JH1,H2 <2 Hz, 1JC1,H1 175 Hz was identified as 5-substituted b-D-galactofuranose due to characteristic deshielded anomeric carbon signal at 109.4 as well as characteristic high chemical shifts of C-2, C-3, C-4, and C-5 and the similarities of chemical shifts to the published values.12 Residue E with the H1/C1 signals at 4.66/102.1 3JH1,H2 8 Hz, 1JC1,H1 158 Hz was identified as the 3-substituted b-D-glucopyranose due to the observed large vicinal coupling between all the protons as well as the relative low C-3 carbon shift (82.2 ppm). The 3JH1,H2 1JC1,H1 values confirmed the b pyranosidic configuration of this sugar. Each disaccharide element in the polysaccharide was identified by HMBC and NOESY experiments that showed inter-residue connectivities between adjacent sugar residues and thus provided the sequence of monosaccharides in the polysaccharide. Inter-residue NOEs were found between H-1 of A and H-3 of E, H-1 of E and H-5 of D, H-1 of D and H-3 of C, H-1 of C and H-3 of B, H-1 of B and H-3 of A (Table 2). The HMBC spectra showed cross-peaks between the anomeric proton and the carbon at the linkage position and between anomeric carbon and the proton at the linkage position (Table 3, Fig. 4). Thus the combined results show the pentasaccharide repeating unit of the L. johnsonii strain 142 polysaccharide as shown in Figure 2.

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Figure 3. Part of the 100 ms TOCSY spectrum of L. johnsoni 142 polysaccharide. The spectrum was recorded for 2H2O solution in 25 °C. The cross-peaks are labeled as explained in the legend to Figure 4.

Immunochemical studies of exopolysaccharides have been performed using the rabbit serum obtained after immunization of animals with whole cells of L. johnsonii 142. The titer of anti-EPS serum measured by the passive hemagglutination test was 1/ 2048. This serum showed reactivity in double immunodiffusion test with pure exopolysaccharide of L. johnsonii 142 but not with teichoic acid (TA) from this strain (Table 4). Control sera from healthy non-immunized animals were negative. Biopolymers such as EPS extracted from bacterial mass (crude EPS), purified EPS, TA, and fraction with nucleic acid (NA) from L. johnsonii 151 (isolated from healthy mice) and L. animalis/murinus 148 (isolated from mice with IBD) have also been tested. Pure EPS from L. animalis/murinus 148 reacted with serum anti-L. johnsonii 142, while neither crude EPS nor pure EPS from L. johnsonii 151 did not. The immunoprecipitation test has shown that EPSs from L. animalis/murinus 148 and L. johnsonii 142, both isolated from mice with IBD, gave almost identical patterns, whereas EPS isolated from L. johnsonii 151 revealed much weaker reactivity with this antiserum (Fig. 5). The differences in immunoreactivity may indicate the structural differences of these EPSs. 3. Discussion In the present work we show the structure of the exopolysaccharide from L. johnsonii 142 isolated from mice with experimentally induced inflammatory bowel disease. The molecular mass of EPS has been estimated for 105 Da, as found from gel exclusion chromatography, which explains a weak solubility of the preparation in DMSO before methylation analysis. The exopolysaccharide is a linear polymer with the repeating unit consisting of five monosaccharides residues, namely one 5-substituted galactofuranose, three 3-substituted galactopyranose residues, and 3-substituted

glucose residue. Galactofuranose has already been found together with galactopyranosyl residue in some EPSs produced by Lactobacillus genus like Lactobacillus rhamnosus C83,15 L. rhamnosus KL37C,16 L. rhamnosus GG,17 Lactobacillus helveticus NCDO 766,18 L. helveticus TN-4,14 and L. helveticus Lh 59.19 However, none of the structures of EPSs produced by L. johnsonii as reported so far, contain galactofuranosyl residue. In immunochemical assays we have compared the serological properties of EPS from L. johnsonii 142 (colitis +), L. animalis/murinus 148 (colitis +), and L. johnsonii 151 (colitis ). Exopolysaccharides from L. johnsonii 142 and L. animalis/murinus 148 (both colitis +) cross-reacted in hemagglutination assay with studied serum, whereas EPS from L. johnsonii 151 (colitis ) was not reactive. This was confirmed by the immunoprecipitation test, where EPS from strains isolated from mice with IBD reacted better than L. johnsonii 151 from healthy mice (Fig. 5). These results may indicate structural similarity between L. johnsonii 142 and L. animalis/ murinus 148. Therefore the EPS of L. johnsonii 151, isolated from healthy mice, and not reacting with this serum, must be structurally different from the former two EPSs. It is quite tempting to speculate a relation between inflammatory process and particular bacterial strains or production of EPS with a particular structure by these bacteria multiplying in the intestine. It is suggested that commensal intestinal bacterial flora is an important factor in inflammatory process in IBD.20–22 All three Lactobacillus strains used in this study are constantly present in the colony of the Ga1,2 mice used by us11 but their proportions and numbers differ depending on inflammatory process. The question arises whether these resident strains could, upon potential of gut inflammation, switch to the production of EPSs with specific motifs in their structure, absent in EPSs of lactobacilli present on normal gut mucosa. Another explanation of these results could be a preferential

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Figure 4. (A) 1H NMR spectrum of the L. johnsoni 142 polysaccharide. (B) Selected 1JC,H connectivities in HSQC-DEPT spectrum. (C and D) Selected 3JC,H connectivities in HMBC spectrum of the EPS L. johnsoni 142. The spectra were obtained for 2H2O solution at 600 MHz and 25 °C. The capital letters refer to sugar residues as in Figure 2 and the numbers refer to protons in the respective monosaccharide. Underlined characters and dotted lines show HMBC connectivities through glycosidic bonds.

Table 1 1 H and 13C NMR chemical shifts of Lactobacillus johnsonii strain 142 exopolysaccharide Residue

A ?3)-a-D-Galp(1? B ?3)-a-D-Galp(1? C ?3)-a-D-Galp(1? D ?5)-b-D-Galf(1? E ?3)-b-D-Glcp(1?

Chemical shift (ppm)

Table 2 Selected inter-residue and intra-residue NOESY connectivities from the anomeric atoms of the isolated exopolysaccharide of Lactobacillus johnsonii strain 142 Residue

H1 C1

H2 C2

H3 C3

H4 C4

H5 C5

H6 C6

H60

5.44 99.2 5.23 95.2 5.19 95.6 5.20 109.4 4.66 102.1

3.98 67.6 4.04 67.1 3.99 67.57 4.20 81.9 3.43 72.5

4.06 74.11 4.11 74.6 4.02 77.7 4.27 76.6 3.69 82.2

4.29 65.68 4.27 66.1 4.16 69.7 4.18 81.8 3.65 70.2

4.27 71.0 4.21 71.25 4.22 71.1 4.07 77.8 3.47 75.6

3.81a 61.4 3.74a 61.4 3.82a 61.4 3.77 61.8 3.89 61.1

—

H1 dH Connectiv- Intra- Inter-residue (ppm) ities to dH residue atom/residue (ppm) atom

A ?3)-a-D-Galp-(1? 5.44

— —

B ?3)-a-D-Galp-(1? 5.23

—

C ?3)-a-D-Galp-(1? 5.19 D ?5)-b-D-Galf-(1? 5.20

3.75

Spectra were obtained for 2H2O solutions at 25 °C. Acetone (dH 2.225, dC 31.05) was used as an internal reference. a Assignment of resonance can be interchanged.

E ?3)-b-D-Glcp-(1? 4.66

w—Weak signal.

3.69 3.43w 3.98 4.06w 4.06 4.29 4.11 4.02 4.27 4.07 3.69 3.47

H3 of E ?3)-b-D-Glcp-(1? H2 of E ?3)-b-D-Glcp-(1? H2 H3 H3 H4 H3 H3

of of of of

A ?3)-a-D-Galp-(1? A ?3)-a-D-Galp-(1? B ?3)-a-D-Galp-(1? C ?3)-a-D-Galp-(1?

H3 H5 of D ?5)-b-D-Galf-(1? H3 H5

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Table 3 Selected intra-residue and inter-residue 3JH,C connectivities from the anomeric atoms of the isolated exopolysaccharide of Lactobacillus johnsonii strain 142 Residue

H1 C1

A ?3)-a-D-Galp-(1?

5.44 99.2

Connectivities to (ppm) dC

Intra-residue atom

dH 3.69 4.27

H5 C3 of E ?3)-b-D-Glcp-(1?

82.2 74.1 71.0 B ?3)-a-D-Galp-(1?

5.23 95.2

C ?3)-a-D-Galp-(1?

Inter-residue atom/residue H3 of E ?3)-b-D-Glcp-(1?

C3 C5 H3 of A ?3)-a-D-Galp-(1?

4.06 74.6 71.25

5.19 95.6

C3 C5 H3 of B ?3)-a-D-Galp-(1?

4.11 4.22

H5 C3 of B ?3)-a-D-Galp-(1?

74.6 D ?5)-b-D-Galf-(1?

5.20 109.4

H3 of C ?3)-a-D-Galp-(1?

4.02 4.20

H2 C3 of C ?3)-a-D-Galp-(1?

77.7 81.9 E ?3)-b-D-Glcp-(1?

4.66 102.1

C2 4.07

H5 of D ?5)-b-D-Galf-(1? C5 of D ?5)-b-D-Galf-(1?

77.8

Table 4 Reactivity in double immunodiffusion test of extracellular antigens from L. johnsonii 142 and L. animalis/murinus 148 (from mice with IBD) and L. johnsonii 151 (from healthy mice) with serum anti-L. johnsonii 142 Antigens

Serum anti-L. johnsonii 142

L. johnsonii 142a

EPS TA NA EPS TA NA EPS TA NA

+  /+ +  +   

L. animalis/murinus 148a

L. johnsonii 151b

EPS—exopolysaccharide, TA—teichoic acid, NA—nucleic acid fraction, a—isolated from mice with IBD, b—isolated from healthy mice.

160

Precipitated protein [µg]

Strains

180

140 151

120

142

100

148

80 60 40 20 0

multiplication of only some bacteria with particular exopolysaccharide structure on inflamed mucosa; it is known that patients with IBD have higher numbers of some bacteria attached to their colon mucosa than it is found for healthy persons.23 It is generally accepted that the unrestrained activation of the intestinal immune system by elements of the flora is probably a key event in the pathophysiology of IBD. The mechanism of this phenomenon has been studied extensively,24–26 but further research is needed. The comparative studies of the EPS isolated from Lactobacillus strains isolated from mice with IBD may shed the light on the mechanisms of the pathogenesis of this disease. 4. Experimental 4.1. Bacterial strains and isolation of exopolysaccharide Strains of L. johnsonii 142 and L. animalis/murinus 148 were isolated from an intestinal tract of mice with experimentally induced inflammatory bowel disease (IBD), whereas L. johnsonii 151 was isolated from that of healthy mouse of the same colony. We used CD4+CD45RB high T cell transfer SCID mice with colitis housed under SPF conditions as an animal model of IBD.11 The bacteria were identified by cell morphology and phenotypic identification was performed using commercial identification systems (API 20E, API20A, APIStaph, APIStrep, API50CHL (bioMerieux and BBL Crystal ID System, BD, USA). The species of the Lactobacillus genus were

100

50

25

12,5

6,25

Antigen [µg] Figure 5. Quantitative immunoprecipitation of serum against whole cells of L. johnsonii 142 with EPS from L. johnsonii 151 ( ), L. johnsonii 142 ( ), and ). L. animalis/murinus 148 (

designated using either PCR with primers for 16S–23S rRNA27 or rep-PCR using GTG5 primers.28 The strains were stored at 75 °C in MRS broth supplemented with 20% glycerol. Bacteria were cultivated in MRS liquid broth (Biocorp) under anaerobic conditions at 37 °C for 48 h. Cells were harvested by centrifugation at 6600g (4 °C, 20 min) and washed twice with PBS and once with MiliQ water. Freeze-dried bacterial mass was extracted with 10% TCA (25 °C, 2.5 h) and then centrifuged at 14,500g for 20 min. The pellet was discarded and the EPS from the supernatant was precipitated with 5 vol of cold 96% ethanol (4 °C, 16 h) and collected by centrifugation at 23,500g (4 °C, 50 min). The pellet was suspended in water, dialyzed for 48 h against water and then lyophilized. 4.2. Purification of the exopolysaccharide The freeze-dried preparation of crude EPS was dissolved in buffer (50 mM Tris–HCl pH 7.5, 10 mM MgCl2) and treated with DNase

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and RNase (37 °C, 6 h) and with protease (37 °C, 16 h), then dialyzed against water at 4 °C for 24 h. The resuspended polysaccharide was purified by ion-exchange chromatography on a DEAESephadex A-25 column (1.6  20 cm). The neutral EPS was eluted with 20 mM Tris buffer, pH 8.2 whereas TA and NA were obtained by eluting with NaCl gradient (0–2 M) in 20 mM Tris buffer, pH 8.2 at a flow rate 0.3 ml/min, monitored at 220 nm with an UV–vis absorbance detector and Knauer differential refractometer. The eluates were analyzed for the carbohydrate content by the phenol/sulfuric acid reaction.29 The amounts of phosphate were determined by the method of Chen et al.30 The fractions containing neutral EPS were pooled, desalted by dialysis against water at 4 °C for 24 h, and lyophilized. The average molecular mass of the neutral EPS was determined by gel filtration chromatography on a TSK HW-55S column (1.6  100 cm, Amersham Pharmacia Biotech), calibrated with dextran standards (Mw 10, 70, 200, 500 kDa) with 0.1 M ammonium acetate buffer as eluent. The column eluate was monitored by absorbance detector at k = 230 and 280 nm and with a differential refractometer (Knauer). 4.3. Chemical analyses For sugar analysis the polysaccharide sample (0.5 mg) was subjected to hydrolysis with 10 M HCl at 80 °C for 25 min followed by evaporation under a stream of N2.31 The resulting monosaccharides were converted into alditol acetates according to Sawardeker et al.32 and analyzed by gas-liquid chromatography–mass spectrometry (GLC–MS) as previously,31 using a Hewlett–Packard 5971A system, equipped with an HP-1 capillary column with a temperature gradient from 150 to 270 °C at 8 °C/min. For methylation analysis, peracetylation has been performed before permethylation, because of the difficulties to dissolve EPS in DMSO. As a result of this procedure higher solubility of this polymer in DMSO was obtained. The polysaccharide samples were first peracetylated with a mixture of trifluoroacetic anhydride and acetic acid (2:1 vol/ vol, 25 °C, 10 min)33 and then permethylated according to the method of Ciukanu and Kerek.34 The product was purified by water–chloroform extraction. The methylated polysaccharide was hydrolyzed with 2 M TFA at 120 °C for 2 h and evaporated under N2. Finally methylated monosaccharides were reduced with NaBD4 and acetylated for GLC–MS analysis using the same conditions as for sugar analysis described above. For determination of the absolute configuration of the monosaccharides35 sample of material (0.5 mg) was transformed into (S)-2-butyl glycosides by butanolysis (300 ll (S)-2-butanol and 20 ll AcCl, 100 °C, 3 h). The resulting mixture was trimethylsilylated (1:3:9 trimethylchlorosilane/hexamethyldisilazane/pyridine, 22 °C, 30 min) and then analyzed by GLC–MS using the same conditions as for sugar analysis described above. 4.4. NMR spectroscopy The NMR spectra were obtained on a Bruker 600 MHz Avance II spectroscope. The NMR spectra were obtained for 2H2O solution of the polysaccharide at 25 °C using acetone (dH 2.225, dC 31.05) as an internal reference. The polysaccharide (10 mg) was repeatedly exchanged with 2H2O with intermediate lyophilization. The data were acquired and processed using standard Bruker software. The processed spectra were assigned with the help of SPARKY NMR analysis program.36 The signals were assigned by one- and twodimensional experiments (COSY, TOCSY, NOESY, HSQC with and without carbon decoupling), HMQC-TOCSY and HMBC. TOCSY experiments were carried out with mixing times of 30, 60, and 100 ms. The delay time in HMBC was 60 ms and the mixing time for NOESY was 200 ms. The HSQC-TOCSY experiment was carried out with 80 ms TOCSY mixing time.

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4.5. Preparation of rabbit serum and serological assays Serum was obtained from rabbits immunized with bacterial mass of L. johnsonii 142. Rabbits (5–6 months old) were immunized twice a week with a dried cell mass suspended in phosphate-buffered saline (PBS). After the first subcutaneous injection with 100 lg/ml, the succeeding doubly increasing amounts (200– 6400 lg/ml) were injected intravenously. After the last injection rabbits were bled, the separated antisera were decomplemented (56 °C, 30 min.) and stored at 20 °C. Preimmune serum was used as a control. Titers of antisera were checked by passive hemagglutination test. Uncoated sheep red blood cells were used as a control. Erythrocytes in citric buffer were washed twice with PBS. The pellets (150 ll) were resuspended in solution of the dry bacterial cells (10 mg in 5 ml PBS). Samples were incubated at 37 °C for 2 h, then centrifuged and washed twice with PBS. The 1% suspension of the coated and uncoated erythrocytes in PBS was prepared. About 50 ll of these suspensions was added to plastic microtiter plates with doubly decreasing dilution (from 1/2 to 1/4096) of rabbit sera. The plates were incubated at 37 °C for 2 h and then at room temperature overnight. Hemagglutination was observed macroscopically. Double immunodiffusion test37 was carried out in 1% agarose dissolved in PBS. Gel was poured onto plates, prepared central well was filled with serum and external wells with exopolysaccharides. The antigens were used at the concentration of 1 mg/ml. Plates were examined after 24, 48, and 72 h of incubation. The precipitation lines were recorded after 48 h or 72 h. Quantitative immunoprecipitation test was performed acc. to Kabat and Meyer.38 Rabbit serum anti-L. johnsonii 142 was centrifuged (1800g, 4 °C, 1 h) and diluted with 1% polyethylene glycol (PEG) in PBS. The antigens were used with doubly decreasing dilution (from 100 to 6.25 lg). Antigens were mixed with serum (1:1 v/v) and incubated first at 37 °C for 1 h, then at 4 °C for 72 h. Precipitate was centrifuged (24,000g, 4 °C, 5 min) and washed with 400 ll 1% PEG in PBS. The protein content in precipitate was determined by Lowry method.39 4.6. Transmission electron microscopy For transmission electron microscopy, bacteria at the stationary phase were collected by centrifugation at 6000 rpm at 4 °C for 10 min. They were fixed at 4 °C for 2 h with 2.5% (v/v) glutaraldehyde, 0.075% (w/v) ruthenium red, in 0.1 M cacodylate buffer (with 1 mM CaCl2, 1 mM MgCl2, pH 7.4). The cells were washed twice with cacodylate buffer and then post-fixed with 1% OsO4 and in 0.1 M cacodylate buffer at 22 °C for 1 h. After washing in MiliQ water and 1% uranyl acetate (22 °C, 2 h) the cells were dehydrated in an ethanol series (25%, 60%, 95%, and 100%, v/v) at 22 °C for 5 min, each step and then washed with acetone (22 °C, 10 min). Cells were left in a mixture of acetone–resin (1:1 v/v) for 16 h. Polymerization was done at 45 °C for 24 h and then at 60 °C for 5 h. Ultrathin sections were cut and stained with 1% methylene blue. Electron micrographs were obtained using a Zeiss EM900 transmission electron microscope. Acknowledgments The authors thank Professor Janusz Kubrakiewicz from Zoology Institute, University of Wrocław for the imaging of bacteria by transmission electron microscopy instrumentation. The work was supported to S.G. from Ministry of Sciences and Higher Education by Grant No. N401 332036. References 1. Salminen, S.; von Wright, A.; Morelli, L.; Marteau, P.; Brassart, B.; de Vos, W. M.; Fondén, R.; Saxelin, M.; Collins, K.; Mogensen, G.; Birkeland, S.; MattilaSandholm, T. Int. J. Food Microbiol. 1998, 44, 93–106.

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