Bacterial strains and culture conditions
Lactobacilli (Table 1) were grown at 37°C in MRS medium (Difco) or in Lactobacilli AOAC medium (Difco) in non-shaking conditions. CMPG10200 was grown in the presence of 5 μg/ml erythromycin for stable maintenance of the integrative plasmid. In addition, Escherichia coli strains TOP10 [F- mcrA Δ(mrr-hsdRMS-mcrBC) F80lacZ ΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG] and BL21(DE3)/pLysS [F- ompT hsdSB(rB- mB-) gal dcm (DE3) pLysS (CamR)] were used for plasmid preparation and protein expression, respectively. E. coli was routinely grown in Luria-Bertani (LB) medium at 37°C with agitation and was supplemented with 50 μg/ml kanamycin when required.
Standard DNA protocols were used for molecular cloning and related procedures as described previously . Sequencing of the msp1 gene from different L. rhamnosus and L. casei strains (Table 1) was performed on PCR products obtained by colony PCR with primers Pro-0997 (5'ATGAATTCACAGGGACGGTCAGTTACAAATCC-3', EcoRI site underlined) and Pro-0998 (5'-ATGAATTCGTTGTTCGGCAATGGCAATCACTG-3', EcoRI site underlined) for L. rhamnosus and Pro-0679 (5'-AGAAAGTATTGTCAGTAACGGCAGG-3') and Pro-0680 (5'-TTGGCAAGCCGATACCATGTTTCAC-3') for L. casei strains by Macrolon (The Netherlands). These DNA sequences were translated to amino acid sequence and a multiple alignment around the S106-S107 dipeptide of LGG was constructed using the Geneious Pro™ software.
Production of purified recombinant Msp1 protein
The coding sequence for msp1 (LGG_00324) not including the sequence for the N-terminal signal peptide was amplified by PCR from LGG genomic DNA by means of two flanking 5'- and 3'-end oligoprimers, one with an EcoRI restriction site (5'- TACCGTTTCGAATTCGACAGGGACGGTC-3', EcoRI site is underlined]) and another with an XhoI restriction site (5'- GATCATTAAGCCTCGAGTGACGGGCGAAC-3', XhoI site is underlined) (Oligomer, Finland). The EcoRI- and XhoI-digested PCR fragment was ligated into the pET28b+ expression vector (Novagen) and the resulting recombinant plasmid (pKTH5316) then propagated in the E. coli strain BL21 (DE3)/pLysS for cytosolic production of C-terminal hexahistidine-tagged protein. Recombinant Msp1 (48.4 kDa) also consists of an additional seven N-terminal- and two C-terminal-located amino acids, all of which originate from the coding sequence of the expression vector. Recombinant Msp1 production was carried out as described previously . Briefly, E. coli cells with pKTH5316 were cultivated at 37°C in LB-kanamycin (50 μg/ml) broth, and at the mid-log phase they were then induced by 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown for another 3 h to allow the accumulation of recombinant Msp1. After cells were pelleted by centrifugation, they were sonicated in lysis buffer (50 mM NaH2PO4 [pH 8.0], 300 mM NaCl, 10 mM imidazole) and the broken cell suspension that resulted was then clarified by centrifugation and membrane-filtering (0.45-μm pore-size). The clarified suspension was added to a nickel nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) column and, following rinsing with wash buffer (50 mM NaH2PO4 [pH 8.0], 300 mM NaCl, 20 mM imidazole), recombinant Msp1 was recovered with elution buffer (50 mM NaH2PO4 [pH 8.0], 300 mM NaCl, 250 mM imidazole). Elution buffer was exchanged to 10 mM Tris-HCl (pH 8.0) with a Bio-Rad EconoPac 10 DG desalting column and purified Msp1 was then concentrated using a 30-kDa Microsep filter (Pall Life Sciences).
Production of anti-Msp1 serum
Polyclonal antiserum was raised against purified recombinant Msp1 in rabbit as described previously . In brief, a 1:1 mixture of 400 μg Msp1 and Freund's complete adjuvant (1-ml volume) was first administered subcutaneously and then followed by additional 1 ml booster injections (200 μg Msp1 and Freund's incomplete adjuvant as a1:1 mix) every 3 weeks for a total of 9 weeks. Fourteen days after the final booster injection, blood was recovered for antiserum preparation as described elsewhere .
For Western blot experiments, equivalent amounts, determined by a bicinchoninic acid (BCA) assay, of protein were separated by SDS-PAGE and subsequently electroblotted onto polyvinyldifluoride (PVDF) membrane. The PVDF sheets were blocked with 3% bovine serum albumine (BSA) in TBS (20 mM Tris-HCl, 500 mM NaCl, pH 7.5) and incubated at room temperature with Msp1 antiserum (1:7500 to 1:15000) for 1 h. After several washings with TBS, blots were incubated for 1 h with goat anti-rabbit antibodies conjugated with alkaline phosphatase (Sigma) at a dilution of 1:1000. Detection was performed with nitro blue tetrazolium and bromo-chloro indolyl phosphate as substrate and the blue coloring reaction was monitored.
Isolation of different protein fractions from LGG
(1) supernatant. LGG bacteria were grown for 24 h in AOAC-medium. After centrifugation (6000 g, 20 min), proteins were precipitated from supernatant by incubation at 4°C for 30 min in the presence of trichloro acetic acid (20% final concentration). After centrifugation (9000 g, 20 min), precipitated proteins were washed twice with cold acetone. Pellet was air-dried and proteins were resuspended in lysis buffer (2 M thio-urea, 7 M urea, 4% CHAPS, 2% DTT). (2)cell wall. After centrifugation (6000 g, 20 min) and washing with PBS, bacteria were sonicated in PBS buffer (Branson sonifier) until lysis was observed (several minutes at 10% power). Lysate was cleared by centrifugation (3 times at 4000 g). Cell wall proteins were precipitated by ultracentrifugation (22000 g for 45 min). Pellet was washed 3 times with 50 mM Tris pH8, 500 mM NaCl and dissolved in a small volume PBS. (3) cytosol. After centrifugation (6000 g, 20 min), the pellet was washed 2× with 0.9% NaCl. Subsequently, the pellet was dissolved in 1 ml 100 mM Tris, 1% SDS, pH 9.5 and sonicated for 3 min (0.5" on, 1.5" off) with a Branson sonifier. Subsequently, cell debris was removed by centrifugation and proteins were precipitated with TCA precipitation, two times washed with ice-cold 100% acetone, air-dried and then dissolved in PBS.
Purification of the L. rhamnosus GG Msp1 protein
The p75/Msp1 protein was partially purified by cationic exchange as described previously . Hereto, L. rhamnosus GG was grown for 24 h in AOAC medium, after which spent culture medium was collected (6000 g, 20 min). The culture supernatant was then loaded onto a SP Sepharose HighPerfomance column (GE Healthcare), equilibrated with 60 mM lactate buffer pH 4.0. Bound proteins were eluted using lactate buffer containing sequential NaCl concentrations (100-1000 mM). Fractions positive for the presence of Msp1 were identified using SDS-PAGE and spin concentrated using Vivaspin filters with MW cut off 10.000 (Sartorius Stedim biotech GmbH, Germany). For the MS/MS experiments, Hi-trap ConA 4B prepacked columns (GE Healthcare) were used according to manufacturer's instructions to further purify positive fractions.
Characterization of glycan nature of the p75/Msp1 protein
(i) ProQ Emerald glycoprotein stain. Samples were loaded onto the wells of the SDS-containing gel after adding buffer and reducing agent according to manufacturer's instructions from the Pro-Q Emerald 488 Glycopotein Gel and Blot stain kit (Molecular Probes, Invitrogen). (ii)Treatment with TFMS. Msp1 was chemically deglycosylated by the TFMS method as described before  at -20°C for 30 min. After treatment, the proteins were extensively dialyzed and then analyzed by SDS-PAGE. (iii) Lectin blotting. The proteins were electroblotted onto PVDF membrane similar as for Western blot. After blocking, the PVDF sheets were incubated at room temperature with lectins conjugated with biotin (biotinylated ConA, WGA or Ulex europaeus, Sigma-Aldrich or GNA, HHA, MAA
SNA-I purified and biotinylated according to  for 1 h at final concentrations of 0.25 μg/ml in the presence of 1 mM Mg2+ and 1 mM Ca2+ ions. After washing, blots were incubated for 1 h with streptavidin conjugated with alkaline phosphatase (Roche) at a dilution of 1:1000. Detection was performed as described for Western blotting.
The LGG Msp1 protein purified by cation exchange chromatography was applied to SDS-PAGE using Nu-Page 4-12% gradient Bis-Tris (Invitrogen) gels. The excised gel pieces containing Msp1 were reduced, alkylated and digested with trypsin (Promega), as previously described  or with Proteinase K (from T. album; Sigma-Aldrich) . After digestion, (glyco-)peptides were collected using two rounds of extraction with 20 μl of 0.1% trifluoroacetic acid and stored at -20°C prior to analysis by mass spectrometry. The total tryptic digest of Msp1 was applied to a reverse phase column (PepMap, 3 μm, 75 μm·x 100 mm; Dionex/Thermo Fisher, Waltham, MA) using an Ultimate 3000 nano-LC system (Dionex/Thermo Fisher). The column was equilibrated at RT with eluent A (0.1% formic acid in water) at a flow rate of 300 nl/min. After injection of the sample a linear gradient was applied (15 min 25% eluent B (95% acetonitrile), 25 min 70% B, 30 min 70% B). The eluate was monitored by absorption at 215 nm. The LC column was coupled to an Esquire HCT-Ultra ESI-ion trap-MS (Bruker Daltonics, Bremen, Germany) equipped with an online nanospray source operated in the positive ion mode. For electrospray (1100-1250 V), electropolished, stainless steel LC-MS emitters (150 μm OD, 30 μm ID) from Proxeon A/S (Odense, Denmark) were used. The solvent was evaporated at 170°C employing a nitrogen stream of 6 l/min. When operated in the auto MS/MS mode, monitoring ions from m/z 500 to 1600, each MS scan was followed by the acquisition of MS/MS spectra of up to five of the most abundant ions in the MS spectrum.
The digest generated by Proteinase K was separated using an Ultimate 3000 nano-LC system (Dionex/Thermo Fisher) equipped with a graphitized carbon trap column (Hypercarb, 5 μm, 170 μm × 10 mm; Thermo Scientific) and nano column (75 μm x·100 mm) packed by Grom Analytik (Rottenburg, Germany). The column was equilibrated at RT with eluent A (0.1% formic acid in water) at a flow rate of 400 nl/min. After injection of the sample a linear gradient was applied (15 min, 25% eluent B (95% acetonitrile); 25 min, 70% B; 30 min, 70% B). The eluate was monitored by absorption at 215 nm. Eluates were analyzed by ESI-ion trap-MS as described above. The ions were monitored from m/z 300 to m/z 1500 in the MS mode. The MS/MS spectra were analyzed with Data Analysis (Bruker Daltonics), converted to MGF files and searched against NCBI database, with search limitation "bacteria" using the MASCOT search algorithm. Carbamidomethylation of cysteines was set as a fixed modification and oxidized methionines were set as a variable modification. Trypsin was specified as the proteolytic enzyme and missed cleavages were not allowed. The mass tolerance of the precursor ion was set to 2 Da and that of the fragment ions was set to 0.5 Da. In addition, MS/MS spectra were used for manual interpretation and de
Activation of Akt in Caco-2 cells
Caco-2 cells were cultured as described before . 24 h before the experiments, the Caco-2 cells were deprived of FBS. LGG wild-type was grown overnight in AOAC medium, centrifuged at 4000 × g and 4°C for 10 min, and washed with cold PBS. Caco-2 cells were incubated with 1.5 ml of DMEM without FBS containing either 1 × 107 CFU/ml LGG wild-type or msp1 mutant CMPG10200, 100 ng/ml wild-type, recombinant or TFMS-treated Msp1. DMEM without FBS was used as a negative control. After 16 h incubation at 37°C, cell lysates were made with HEPES-lysis buffer containing 25 mM HEPES pH 7.5, 0.3 M NaCl, 1.5 mM MgCl2, 20 mM β-glycerol-phosphate, 2 mM EDTA, 2 mM EGTA, 1 mM DTT, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 10 μg/μl leupeptin, 5 μg/μl aprotinin, 1 mM PMSF, 1 mM Na3VO4 and 50 mM NaF. Protein concentration was determined using BCA assay (Perbio, Thermo Fisher Scientific). Samples with loading buffer were prepared and processed on the CriterionTM system (Bio-Rad Laboratories, Hercules, CA, USA) on a 4-12% Bis-TRIS gel (Biorad) and Protran 2 μm-pored nitrocellulose membrane (Perkin-Elmer, Wellesley, MA, USA). Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween-20 and 5% BSA. The membrane was incubated overnight at 4°C with the primary antibody diluted in 5% BSA in 1× TBS plus 0.1% Tween 20, then washed 3 times with TBS plus 0.1% Tween 20, followed by incubation with the second polyclonal antibody for 2 h at 4°C. Antibodies against phospho-AKT (Ser473) and total AKT were obtained from Cell Signaling Technology (Beverly, MA).
The cell wall hydrolyzing activity was investigated by zymogram analysis as described previously by Lepeuple et al. (14). SDS-PAGE was performed with 10% (w/v) polyacrylamide separating gels (NuPage, Invitrogen). Autoclaved LGG cells treated with 10% TCA were added to the gels as enzyme substrates and the gels were loaded with 10 to 20 μg of protein samples. After sample migration, the gels were washed for 30 min in deionized H2O at room temperature and then incubated in a phosphate buffer, pH 6.2, 1 mM dithiothreitol (DTT) containing 0.1% (v/v) Triton X-100, overnight at 37°C. The gels were subsequently washed for 30 min in deionized H2O, then stained with 0.1% methylene blue in 0.01% (w/v) KOH for 2 h at room temperature and destained in deionized H2O.