A2

JAa

b

c

d

a

ARR1AA

KLGdESP

1

eut

h(ass

O

S

D

0h

Microbiological Research 168 (2013) 245– 253

Contents lists available at SciVerse ScienceDirect

Microbiological Research

jo ur n al homepage: www.elsev ier .com/ locate /micres

nchorless surface associated glycolytic enzymes from Lactobacillus plantarum99v bind to epithelial cells and extracellular matrix proteins

acob Glentinga,1,2, Hans Christian Beckb,1,3, Astrid Vranga, Holger Riemanna, Peter Ravna,nne Maria Hansenb, Martin Antonssonc,4, Siv Ahrnéd, Hans Israelsena,5, Søren Madsena,∗

Bioneer A/S, Kogle Allé 2, DK-2970 Hørsholm, DenmarkDanish Technological Institute, Kongsvang Allé 29, DK-8000 Aarhus, DenmarkProbi AB, SE-223 70 Lund, SwedenDepartment of Food Technology, Engineering and Nutrition, P.O. Box 124, SE-221 00 Lund, Sweden

r t i c l e i n f o

rticle history:eceived 15 August 2012eceived in revised form4 December 2012ccepted 8 January 2013vailable online 7 February 2013

eywords:actobacillus plantarum strain 299vlyceraldehyde 3-phosphate

a b s t r a c t

An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosalsurface. Adhesion is usually mediated by proteins or other components located on the outer cell surface ofthe bacterium. In the present study we characterized the adhesive properties of two classical intracellularenzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outercell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signalpeptides or cell surface anchoring motifs that could explain their extracellular location on the bacterialsurface. The presence of the glycolytic enzymes on the outer surface was verified by western blottingusing polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specificbinding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin.

ehydrogenasenolaseurface enzymesrobiotics

Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cellsat pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play arole in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host.Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presenceof GAPDH and ENO on the outer surface, but no correlation between the extracellular location of theseenzymes and the probiotic status of the applied strains was demonstrated.

. Introduction

The ability to adhere to the intestinal epithelial cells is consid-

red important in the selection of lactic acid bacteria for probioticse. Adhesive properties form the basis for a transient coloniza-ion of the gastrointestinal tract making the probiotic bacteria

∗ Corresponding author. Tel.: +45 45 16 04 44.E-mail addresses: jacob.glenting@alk.net (J. Glenting),

ans.christian.beck@ouh.regionsyddanmark.dk (H.C. Beck), avr@bioneer.dkA. Vrang), hkr@bioneer.dk (H. Riemann), pra@bioneer.dk (P. Ravn),mah@dti.dk (A.M. Hansen), martin.antonsson@danone.com (M. Antonsson),iv.ahrne@appliednutrition.lth.se (S. Ahrné), hi@noreba.com (H. Israelsen),ma@bioneer.dk (S. Madsen).

1 These authors contributed equally to this work.2 Present address: ALK Abelló, Bøge Allé 1, DK-2970 Hørsholm, Denmark.3 Present address: Odense Universitets hospital, Sønderboulevard 29, DK-5000dense C, Denmark.4 Present address: ProViva AB, Österlenmejeriet AB, Lunnarp 273 96 Tomelilla,

weden.5 Present address: Nordisk Rebalance, M.D. Madsensvej 14-16, DK-3450 Allerød,enmark.

944-5013/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.micres.2013.01.003

© 2013 Elsevier GmbH. All rights reserved.

able to exert positive health effects such as stimulation of theimmune system (Oelschlaeger, 2009), reduction of cholesterol lev-els (Naruszewicz et al., 2002), and lowering of higher blood pressure(Brunser et al., 2007). Furthermore, by adherence to epithelialcells, probiotic bacteria may exclude pathogenic microorganismsfrom binding and thereby preventing translocation and subsequentinfection (Mangell et al., 2006). Previous studies have describeddiverse surface molecules such as lipoteichoic acids and protein-aceous compounds as main mediators of bacterial attachment toepithelial cell lines, mucosa or extracellular matrix proteins. Forexample, lipoteichoic acid, a strongly negatively charged polyolphosphate polymer, is in Lactobacillus johnsonii NCC reported tomediate adhesion to Caco-2 cells (Granato et al., 1999). The protein-aceous surface compounds constitute a diverse group of moleculesthat includes proteins containing a C-terminal sortase recognitionmotif (LPXTG). Upon cleavage between the T and G residues bythe enzyme sortase A, the threonine carboxyl group is covalently

linked to free amino groups in the cell wall cross bridges of thepeptidoglycan. This leads to a surface-displayed protein that iscovalently linked to peptidoglycan layer of the bacterium. LPXTGmotif-containing proteins from different Lactobacillus strains have

2 ical R

bee

mTrm(Le

tiaSisebe

iasio1ebN

emilto

Lnastpa2

uLtbsntabp

2

2

oL

(Sambrook et al., 1989). All primers used for PCR or DNAsequencing were obtained from DNA Technology A/S, Aarhus,Denmark. Genomic DNA was isolated from L. plantarum aspreviously described (Johansen and Kibenich, 1992). DNA and

Table 1Strains, plasmids and cell lines.

Strains or plasmids Relevantcharacteristic(s)

Reference or source

Lactobacillus strainsplantarum 299v Human intestine Johansson et al. (1993)plantarum WCFS1 Human saliva Kleerebezem et al. (2003)plantarum ATCC14917 Pickled gabbage, type

strainATCC

plantarum B Human Lund Universityplantarum C Human Lund Universityplantarum 299 Human colon Lund Universityplantarum ATCC8014 Maize silage ATCCplantarum P Human Lund Universityplantarum Q Human Lund Universityrhamnosus CCUG21452 Type strain CCUGrhamnosus E Human Lund Universityrhamnosus R Human Lund Universityrhamnosus GG Human Lund Universityrhamnosus T Human Lund Universitygasseri DSM20243 Human, type strain DSMZgasseri Z Human Lund Universitygasseri Aa Human Lund Universitygasseri K Human Lund Universitygasseri L Human Lund Universitygasseri Y Human Lund Universitycasei ATCC334 Emmental cheese ATCCparacasei H Human Lund Universityparacasei V Human Lund Universityparacasei X Human Lund University

E. coli strainDH10B E. coli cloning host Grant et al. (1990)

PlasmidspCR®2.1-TOPO® TA-cloning vector InvitrogenpGEX-4T-3 Expression vector

(GST-tagged)GE Healthcare

Cell lineCaco-2 Human caucasian

epithelial colorectalSigma–Aldrich

46 J. Glenting et al. / Microbiolog

een shown to act as mucus adhesins (Roos and Jonsson, 2002; Buckt al., 2005; Pretzer et al., 2005) and to mediate binding to humanpithelial cells.

Other adhesins from lactobacilli bind to the extracellularatrix (ECM) proteins such as laminin, collagen and fibronectin.

he adhesins include the collagen-binding protein CnBP of L.euteri NCIB 11951 (Aleljung et al., 1994; Roos et al., 1996), theucin-binding protein MapA (mucus adhesion-promoting protein)

Miyoshi et al., 2006) and the fibronectin-binding proteins FbpA in. acidophilus (Buck et al., 2005) and SlpA from L. brevis (Hynonent al., 2002).

Recently, we and others have described several unexpected pro-eins on the outer surface of different Lactobacillus strains. Thesenclude glycolytic enzymes, proteins involved in stress response,nd also ribosomal proteins (Beck et al., 2009; Izquierdo et al., 2009;aad et al., 2009). In addition the elongation factor Tu (EF-Tu) wassolated from the outer surface of the probiotic bacteria L. john-onii NCC533 (Granato et al., 2004) and L. plantarum 423 (Ramiaht al., 2008) where it was found to mediate the attachment of theseacteria to human epithelial cells and mucin, and the competitivexclusion of pathogens to Caco-2 cells, respectively.

L. plantarum 299v (Probi, Sweden) is a probiotic strain whichs included in the functional food product produced and marketeds ProViva® by Skånemejerierne in Sweden. Additionally, the 299vtrain is included in products sold by Probi’s partners worldwiden the field of healthcare and functional foods. This strain wasriginally isolated from healthy intestinal mucosa (Molin et al.,993) and since then a couple of clinical trials have recognized itsffect in patients suffering from irritable bowel syndrome (IBS)y reducing bloating, flatulence and pain (Nobaek et al., 2000;iedzielin et al., 2001).

Studies by Mack et al. (1999, 2003) showed that increasedxpression of the two genes encoding the human ileocolonicucins MUC2 and MUC3 occurred when HT-29 cell were co-

ncubated with L. plantarum 299v. The increased mucin productioned to inhibition of adherence of enteropathogenic Escherichia colio the intestinal epithelial cells explaining the antagonistic effectf L. plantarum 299v.

One of the reasons for the beneficial effects of this particularactobacillus strain might be its excellent ability to adhere to man-ose residues present on the mucosal cells (Adlerberth et al., 1996)nd thereby exclude pathogenic bacteria from adhering and sub-equently translocating (Mangell et al., 2006). The gene encodinghe mannose binding adhesin was identified in the closely related L.lantarum strain WCFS1 (Pretzer et al., 2005), and recently we havelso confirmed the presence of the same gene locus in L. plantarum99v (unpublished data).

In our efforts to characterize this strain further on the molec-lar level, we here present data focusing on the identification of. plantarum 299v cell surface molecules that mediate attachmento intestinal epithelial cells, mucin and ECM proteins which coulde part of the probiotic properties of this strain. Our data demon-trates that several glycolytic enzymes are secreted and becomeon-covalent attached to the cell surface of L. plantarum 299v andhat such proteins may play a role in the adhesion to epithelial cellsnd glycoproteins. However, data does not show any correlationetween the presence of these surface associated enzymes and therobiotic status of the analyzed strains.

. Materials and methods

.1. Bacterial strains, plasmids, cell line and growth conditions

A number of Lactobacillus strains isolated from humans werebtained from a strain collection present at The University ofund. Strains, plasmids and cell line used in this study are listed in

esearch 168 (2013) 245– 253

Table 1. E. coli strain DH10B used for recombinant expression wasgrown at 37 ◦C in LB broth supplemented with 100 �g/mL of ampi-cillin when appropriate. Lactobacillus species were grown at 30 ◦Cwithout aeration in MRS medium (de Man et al., 1960) that hadbeen prepared from single components containing filter-sterilizedyeast extract and glucose added from a separately autoclaved stocksolution (medium named sMRS). Human intestinal cells (Sigma,ECACC No. 86010202, Caco-2 cells) were used between passage50 and 80. Caco-2 cells were cultured in a humidified incubator(37 ◦C, 5% CO2, 90% ambient air) using DMEM (BioWhittaker, 4.5 g/Lglucose) supplemented with 10% fetal bovine serum (BiologicalIndustries, heat-inactivated), 2 mM Glutamax (Gibco) and 1%nonessential amino acids (Gibco). Cells were fed 2–3 times a week(fresh culture medium) and sub-cultured (1:5) by trypsinizationat 70–80% confluency. Cells for binding assays were obtained byseeding Caco-2 cells at near confluency (approx. 4.0E+05 cells/cm2)followed by culturing for 15 days in a state of confluence (fullydifferentiated cells).

2.2. DNA techniques and DNA sequence analysis

DNA was manipulated according to standard procedures

adenocarcimona

ATCC, American Type Culture Collection; CCUG, Culture Collection University ofGothenburg; DSMZ, German Collection of Microorganisms and Cell Cultures; LundUniversity, Department of Food Technology.

ical R

dahlh

2a

uMNTpMgrpgDtsgcosi

2a

TashpaFcMts(ic

2a

fioalHDdEmecL1(

J. Glenting et al. / Microbiolog

educed amino acid sequences were analyzed using the nucleotidend protein BLAST programs (BLASTn and BLASTp) available atttp://www.ncbi.nlm.nih.gov/BLAST. Protein sequences were ana-

yzed for signal peptides using the Signal P 4.1 server available atttp://www.cbs.dtu.dk/services/SignalP/ (Nielsen et al., 1997).

.3. Cloning of the putative operon encoding GAPDH, PGK, TPI,nd ENO

An internal region of the GAPDH gene was PCR amplifiedsing degenerate primers (GPD-Nterm: 5′ ATHAAYGGNTTYGGN-GNATHGGN 3′ and GPD-mid REV: 5′ YTTNCCNACNGCYTTNGC-GCNCCNGT 3′) and genomic L. plantarum 299v DNA as template.he resulting 700 bp fragment was purified and inserted intoCR2.1-TOPO. The DNA sequence was determined using universal13 forward and reverse primers. The remaining part of the GAPDH

ene and the adjacent DNA regions were amplified by consecutiveounds of inverse PCR (Ochman et al., 1988). In short, total DNA of L.lantarum 299v was digested with either EcoRI or HindIII and reli-ated in a large volume. PCR amplifications were carried out usingNA primers based on DNA sequences that were obtained during

he successive rounds of inverse PCR. This cloning and sequencingtrategy was used to complete the sequencing of the four glycolyticenes encoding GAPDH, PGK, TPI and ENO. Furthermore, a trans-riptional regulator likely controlling expression of the putativeperon was identified upstream of the GAPDH gene. The completeequence of the regulator and the glycolytic operon was depositedn GenBank under accession number HQ622815.

.4. Preparation of extracts from Lb. plantarum 299v cell surfacend whole cells

L. plantarum 299v was grown in sMRS medium for 40–48 h.he bacterial cells were harvested by centrifugation (4000 × g/4 ◦C)nd resuspended in an equal volume of PBS buffer (136.9 mModium chloride, 2.68 mM potassium chloride, 8.1 mM disodiumydrogen phosphate, 1.47 mM potassium dihydrogen phosphate,H 7.2), incubated for 1–2 h at 30 ◦C (unless otherwise indicated),nd centrifuged; the supernatant constitutes the “surface extract”.or binding assays, proteins extracted from the cell surface wereoncentrated 10–20 times on 15 mL spin columns with cut-off atW 10 kDa (Millipore, MA, USA). Whole cell lysates for determina-

ion of intracellular enzyme activities were prepared from 0.5 mLuspension of washed cells in PBS sonicated with 0.7 g glass beadsSigma G-9143) in an Elma Transsonic Digital S sonication bath withce-water, full power for a total of 10 times 1.5 min. Glass beads andell debris were removed by centrifugation.

.5. Heterologous expression of GST-tagged GAPDH, PGK and ENOnd generation of polyclonal antibodies

The genes encoding GAPDH, PGK and ENO were PCR ampli-ed from the genome of Lb. plantarum 299v using individual setsf primers containing 5′ BamHI and 3′ XhoI recognition sites. Themplified DNA fragments were digested with BamHI and XhoI andigated into the same sites of the expression vector pGEX-4T-3 (GEealthcare). The ligation mixtures were transformed into E. coliH10B (Invitrogen, Carlsbad, CA, USA) according to standard proce-ures. In the pGEX-4T-3 system the recombinant GAPDH, PGK, andNO proteins were produced as translational fusions to the C ter-inal of the glutathione-S-transferase (GST) polypeptide. Protein

xpression was performed by diluting overnight cultures of E. coli

lones containing the respective plasmids 50-fold into 100 mL freshB medium containing 100 �g/mL ampicillin. Growth took place for/2 h (25 ◦C at 200 rpm) and isopropyl-�-d-thiogalactopyranosideIPTG) was added to induce expression (final concentration of

esearch 168 (2013) 245– 253 247

0.1 mM). Induction of expression continued overnight and cellswere harvested, washed in PBS and finally sonicated. Sonicatedprotein lysates containing the fusion proteins were loaded intothe Glutathione Sepharose 4B Redipack column and eluted usingglutathione according to the instructions by the supplier (Amer-sham Biosciences). Antibodies were raised against the PGK, GAPDHand ENO fusion proteins respectively, by immunizing rabbits threetimes with 100 �l 1 mg/mL of recombinant protein. The antiserawere evaluated by western blot analysis.

2.6. Western blot analysis

Pre-cast 14% Tris–glycine gels from Invitrogen were used forSDS-PAGE, and proteins were transferred to Invitrogen type 2nitrocellulose membranes using an Xcell II blot module fromInvitrogen. Membranes were blocked with a solution of 3% skimmilk powder. As primary antibody anti-GAPDH, anti-PGK oranti-ENO was diluted 1000-fold, added to the membranes andincubation took place overnight. The secondary antibody alka-line phosphatase-conjugated goat anti-rabbit antibody from Dako(Glostrup, Denmark) was diluted 5000-fold and incubation tookplace for 2 h. The membranes were washed three times in TNTsolution (20 mM Tris, pH 7.5, 0.5 M NaCl, 0.05% (v/v) Tween-20)and developed using NBT/BCIP tablets from Roche Diagnostics(Penzberg, Germany).

2.7. Enzymatic assays for the determination of GAPDH and LDHactivity

GAPDH activity assays were performed on untreated culturesamples, culture supernatants, cell surface extracts, suspensions ofwashed cell, and cell lysates, using a modification of the proceduredescribed by Gil-Navarro et al. (1997). NAD and NADH, which takepart in the GAPDH reaction, are not taken up by intact cells. There-fore, the intracellular GAPDH will not be detected without priorlysis or permeabilization of the cells. 16 �l samples were mixed ina 1 cm light path cuvette with reaction mixture to a final volumeof 0.8 mL. The reaction mixture contained 1 mM NAD and 2 mMglyceraldehyde 3-phosphate in 0.1 mM DTT, 5 mM EDTA, 50 mMsodium phosphate and 40 mM triethanolamine, adjusted to pH8.6 with HCl. A mixture without glyceraldehyde 3-phosphate wasused for control reactions. Activity of GAPDH causes an increase inabsorbance at 340 nm (A340) as NADH is formed during the reac-tion. The reaction took place at room temperature (25 ± 3 ◦C). A340was measured at intervals throughout a total incubation time of5–180 min, depending on the activity of the sample. For each sam-ple, the slope of A340 versus time was calculated, and the slopeof the control reaction without glyceraldehyde 3-phosphate wassubtracted. Further correction was made for A340 increase in reac-tion mixture with buffer added instead of sample. To obtain theactivity in units/mL, the corrected slope was multiplied by thereaction volume and divided by the sample volume and the mMextinction coefficient of NADH, 6.3 (mM cm)−1. 1 unit of GAPDHwill catalyze production of 1 �mol 1,3-diphosphoglyceric acid perminute. LDH activity was measured by a procedure which is sim-ilar to the GAPDH assay. Reduction of pyruvate at the expense ofNADH was measured as a decrease of A340 with time. The reactionmixture consisted of 10 mM sodium pyruvate and 0.2 mM NADH in63.2 mM potassium dihydrogen phosphate and 3.5 mM disodiumhydrogen phosphate (Bernard et al., 1994).

2.8. Binding of surface proteins to immobilized to fibronectin,

mucin and plasminogen

The role of GAPDH and ENO proteins as adhesins was evaluatedby an immobilized binding assay using components of the epithelial

2 ical R

l2MwMUwcUwwE2p1baETaPwA(cpfr

2

p3(t0tPTbtLAtwfit23w5fiafG

3

3o

bt

48 J. Glenting et al. / Microbiolog

ining. GAPDH and ENO were eluted from the surface of L. plantarum99v as described and concentrated 20 times using spin columns.axisorb microtitre wells (Nunc, Roskilde, Denmark) were coatedith 20 �g/mL human plasma fibronectin (Sigma–Aldrich, St. Louis,O), 20 �g/mL human plasminogen (American Diagnostica, CT,SA) or 20 �g/mL porcine mucin (Sigma–Aldrich). Wells coatedith 0.5% bovine serum albumin (BSA) alone served as negative

ontrol. Immobilization took place for 3 h at 37 ◦C with lid on.nbound components were removed by rinsing the wells five timesith PBS-0.2% (v/v) Tween-20. Free sites in the wells were blockedith 0.1% BSA containing 0.2% Tween 20 for 30 min at 500 rpm.

xcess BSA was removed by a PBS-0.2% (v/v) Tween-20 wash. A-fold dilution series of the 20 times concentrated eluted surfaceroteins were titrated onto the wells followed by incubation for

h at 37 ◦C. Plates were washed in PBS-0.2% (v/v) Tween-20 andound protein were detected after overnight incubation at 4 ◦Cnd 500 rpm with a 1:1000 dilution of rabbit anti-GAPDH or anti-NO serum diluted in PBS containing 0.1% (v/v) BSA and 0.2% (v/v)ween-20. After five further washes with PBS-0.2% (v/v) Tween-20,

1:4000 dilution of goat anti-rabbit AP-conjugated antiserum inBS with 0.1% (v/v) BSA and 0.2% (v/v) Tween-20 were added to theells and incubated at room temperature for 11/2 h and 500 rpm.fter five further washes in PBS containing 0.1% (v/v) BSA and 0.2%

v/v) Tween-20, wells were washed in H2O. Finally, bound AP-onjugated antibodies were detected by addition of p-nitrophenylhosphate (Sigma–Aldrich) in 1 M diethanolamine, 0.5 mM MgCl2or 15 min at room temperature. Plates were read in an ELISA plateeader at 405 nm.

.9. Binding of surface proteins to Caco-2 cells

Cell wall associated proteins were obtained by harvesting a L.lantarum 299v culture after growth in 50 mL sMRS medium at0 ◦C for 48 h. The bacterial cells were washed twice in 50 mL PBSpH 7.2). The eluted surface proteins were subsequently concen-rated 16 times. 500 �l was added to 6 mL PBS (pH 7.2) and to 6 mL.05 M NaAc buffer (pH 5.0) containing 0.1 M NaCl. T75 flasks con-aining differentiated Caco-2 cells were washed twice with eitherBS (pH 7.2) or 0.05 M NaAc buffer (pH 5.0) containing 0.1 M NaCl.he Caco-2 cells were then incubated for 2 h at 37 ◦C with 6 mL PBSuffer or 6 mL NaAc buffer containing the released cell surface pro-eins from L. plantarum 299v. As negative controls buffers without. plantarum cell surface proteins were added to the Caco-2 cells.fter 2 h, unbound cell wall material was removed and followed by

hree washes using the respective buffers. The Caco-2 cell layersere then harvested and transferred into 50-mL Falcon tubes. Anal wash took place and the cells were transferred into Eppendorfubes. Elution of 299v surface proteins that have bound to the Caco-

cells was performed by addition of 200 �l 0.1 M glycine–HCl (pH.0) and incubation for 30 min at room temperature. Eluted proteinsere separated from Caco-2 cells by centrifugation at 1500 × g for

min and the supernatants containing the eluted proteins werenally neutralized using 200 �l 1 M Tris–HCl (pH 8.0). The elutednd the unbound protein samples were analyzed by SDS-PAGE andurther analyzed by western blot analysis using antibodies againstAPDH and ENO as described previously.

. Results

.1. Identification, cloning and transcriptional analysis of anperon encoding surface associated glycolytic enzymes

Genes encoding surface located proteins are usually identifiedy genome sequencing followed by bioinformatics tools such ashe Signal P 4.1 prediction server or experimentally by use of signal

esearch 168 (2013) 245– 253

peptide selection vectors which carries an export-specific reporterprotein allowing cloning of genes harboring signal peptides neededfor exported proteins (Smith et al., 1987). However, these methodsdo not reveal atypical proteins, which are transported and surfaceattached by so far unknown mechanisms. In a recent study we useda proteomic based approach aiming at identification of extracellularproteins that do not exploit the conventional secretion pathways oranchoring mechanisms (Beck et al., 2009). In this study we investi-gated the adhesion characteristics for GAPDH, enolase (ENO), andphosphoglycerate kinase (PGK). They were identified as the mostabundant of the proteins that were found to be non-covalentlyattached to the cell surface of L. plantarum 299v.

Using PCR techniques we cloned the gene encoding GAPDH.DNA sequencing revealed that beside the gene encoding GAPDH,genes encoding the glycolytic enzymes, enolase (ENO), triose phos-phate isomerase (TPI), and phosphoglycerate kinase (PGK) werelocated in the same operon and in the following order: GAPDH,PGK, TPI and ENO. The prediction of the specific enzyme functionwas based on E-values (Expect value) of 0.0 for all four proteins.The Northern blot analysis (data not shown) showed that the fourgenes are transcribed into several units corresponding to a GAPDHtranscript, a GAPDH-PGK transcript and a complete GAPDH-PGK-TPI-ENO transcript. Upstream of GAPDH a gene encoding a putativetranscriptional regulator controlling the glycolytic operon wasidentified. The predicted function of this gene is based on thehigh similarity to annotated transcriptional regulators from otherLactobacillus species (E-value of 0.0). The protein sequences wereanalyzed for potential signal peptides using the Signal P 4.1 pre-diction server and for cell wall anchoring motifs consisting of aC-terminally located LPXTG amino acid motif (Schneewind et al.,1993), which could explain their extracellular presence. However,as expected, none of the genes encoded signal peptides or LPXTGanchoring motifs that could explain their extracellular presenceand/or attachment to the bacterial surface.

3.2. Detection of glycolytic enzymes using anti-sera and activityassay

The presence of these glycolytic enzymes on the surface wasverified by western blotting analysis on PBS surface extracts fromstationary phase cultures. Recombinant GAPDH, ENO and PGK wereexpressed and purified using GST affinity chromatography. Thepurified recombinant proteins were used to generate specific rab-bit polyclonal antibodies against GAPDH, PGK and ENO. The useof these antisera demonstrated recognition of specific proteinsof the expected molecular weights in surface extracts of strain299v whereas much lower levels were detected in surface extractsof strain WCFS1 (Fig. 1). To test whether the surface-locatedGAPDH protein was enzymatically active, activity assays were per-formed on untreated culture samples, culture supernatants, cellsurface extracts, and washed cell suspensions. These measure-ments showed that the extracellular GAPDH enzyme was active,and was released to the supernatant by washing at pH 7.2 (PBS) butnot at pH 5 (10 mM KH2PO4, 140 mM NaCl). More than 90% of theextracellular GAPDH activity was found in the surface extract andless than 10% was found in the supernatant and washed cells (datanot shown). However, the presence of active surface associatedGAPDH could still be due to cell lysis. Therefore, we also deter-mined the ratio of extracellular/intracellular GAPDH and comparedthis with the ratio of extracellular/intracellular lactate dehydroge-nase (LDH). LDH is a typical intracellular enzyme and therefore anindicator of potential cell lysis. Despite small amounts of extracellu-

lar LDH the extracellular/intracellular ratio of GAPDH was at least10 times higher than the same ratio of LDH, clearly indicating aspecific mechanism for transportation of GAPDH and maybe otherglycolytic enzymes to the bacterial surface rather than lysis. The

J. Glenting et al. / Microbiological Research 168 (2013) 245– 253 249

Fig. 1. Identification of glycolytic surface proteins by western blot analysis. Extracellular surface extracts were isolated from L. plantarum strains 299v and WCSF1. Surfacee lue® Pw

eetTdtd

3

tcaeaateptaassb

Fsa

xtracts from strain 299v was loaded in lane 2 and from strain WCFS1 in lane 3. SeeBere detected using specific antibodies against GAPDH (A), ENO (B) and PGK (C).

nzymatic analysis of GAPDH and LDH showed that much morextracellular active GAPDH enzyme is present on cells of L. plan-arum strain 299v compared to L. plantarum strain WCFS1 (Fig. 2).he surface-located activity of GAPDH in growing cultures was alsoetermined, however, the activity was very low indicating thathe extracellular activity is growth phase dependent and increasesuring transition to stationary phase.

.3. GAPDH and Eno from L. plantarum 299v act as adhesins

Previous studies in pathogenic microorganisms have shown thathe two surface associated enzymes GAPDH and ENO bind to hostomponents such as fibronectin, mucin and plasminogen (Pancholind Fischetti, 1992, 1998). It is anticipated that the glycolyticnzymes are virulence factors involved in colonization, persistencend invasion of the host tissue (Pancholi and Chhatwal, 2003). Thebility of surface bound GAPDH and ENO of L. plantarum 299vo bind to plasminogen, fibronectin, and mucin was analyzed bynzyme-linked-immuno-sorbent-assays. Surface proteins from L.lantarum were released by a mild PBS wash at neutral pH, concen-rated and subsequently allowed to bind to immobilized ligands in

microtiter plate. Specific binding was detected using anti-GAPDH

nd anti-ENO polyclonal antibodies followed by incubation with aecondary antibody conjugated with alkaline phosphatase. Fig. 3Ahows the binding of GAPDH and ENO to plasminogen, and theinding of GAPDH and ENO to fibronectin is shown in Fig. 3B.

ig. 2. Surface located GAPDH and LDH enzyme activities in Lactobacillus spp. The extraceurface protein fractions as described in Section 2. Each of the Lactobacillus strains was tectivities are represented by the black bars and surface located LDH activities are represe

lus2 pre-stained protein standard was loaded in lanes 1. Glycolytic surface proteins

Additionally it was shown that surface released GAPDH but notsurface released ENO was able to bind to mucin with very lowaffinity (Fig. 3C). However, binding of GAPDH to mucin was lessefficient than binding to plasminogen and fibronectin. In conclu-sion we demonstrated that GAPDH and ENO from L. plantarum 299vhave adhesive properties, and they could be involved in host cellprotein binding.

3.4. pH dependent binding of GAPDH and ENO from L. plantarum299v to Caco-2 intestinal epithelial cells

Previous studies have shown that binding of elongation fac-tor Tu from L. johnsonii to intestinal Caco-2 cells and mucus ispH dependent and higher binding at pH 5.0 than at pH 7.2. It ispostulated that pH of the gut lumen is neutral but becomes moreacidic at the mucus-covered surface due to the presence of sialicacid residues (Granato et al., 2004). Binding at pH 5.0 thereforesimulates the physiological situation in the body. DifferentiatedCaco-2 cells were washed at pH 5 or pH 7 and then incubated withreleased surface proteins of L. plantarum 299v. Unbound proteinswere removed by repeated washing at the appropriate pH values,before bound proteins were eluted. Caco-2 binding of GAPDH and

ENO were demonstrated by western blot analysis using specificantibodies against GAPDH and ENO (Fig. 4). For both enzymes wedemonstrated specific binding to Caco-2 cells at pH 5, but not atpH 7.

llular GAPDH and LDH activity in 23 Lactobacillus spp. were measured in the elutedsted twice in independent cultures on two different days. Surface located GAPDH

nted by the white bars.

250 J. Glenting et al. / Microbiological Research 168 (2013) 245– 253

Fig. 3. Binding of GAPDH and ENO to plasminogen and fibronectin. (A) Humanplasminogen was coated in microtiter plates and increasing amounts of elutedsurface proteins were added to the wells. (B) Human fibronectin was coated inmicrotiter plates and increasing amounts of eluted surface proteins were added tothe wells. (C) Porcine mucin was coated in microtiter plates and increasing amountsof eluted surface proteins were added to the wells. Specific binding of GAPDHand ENO was detected by addition of antisera against GAPDH and ENO obtainedfrom immunized rabbits. Detection of bound GAPDH and ENO was measuredby the addition of a goat anti-rabbit alkaline phosphatase conjugated antiserumfollowed by reading of the absorbance at 405 nm in an ELISA reader. The open sym-bols show the non-specific binding of GAPDH and ENO (BSA was coated insteadof plasminogen/fibronectin/mucin). The closed symbols show binding of GAPDHtpd

3L

p2ssgpaELpaj

Fig. 4. Binding of surface located GAPDH and ENO to Caco-2 cells. Panel A: unboundGAPDH after incubation with Caco-2 cells at pH 7 (lane 1). GAPDH eluted from Caco-2 cells after binding at pH 7 (lane 4). Binding of GAPDH to Caco-2 cells at pH 5 (lane6). Lanes 3 and 5 are negative controls where no surface extract was incubated withthe Caco-2 cells. Panel B: unbound ENO after incubation with Caco-2 cells at pH 7(lane 1). Binding of ENO to Caco-2 cells at pH 7 (lane 4). Binding of ENO to Caco-2cells at pH 5 (lane 6). Lanes 3 and 5 are negative controls where no surface extract

®

o plasminogen, fibronectin and mucin (diamonds) and the binding of ENO tolasminogen, fibronectin and mucin (triangles). Results shown are means of twoeterminations. The variation was <10%.

.5. The presence of glycolytic surface enzymes is widespread inactobacillus species

Other studies have demonstrated that GAPDH and ENO areresent on the surface of Lactobacillus species (Antikainen et al.,007; Hurmalainen et al., 2007). In order to investigate how dis-eminated this feature is among different Lactobacillus species wecreened a collection of Lactobacillus species for surface associatedlycolytic enzymes using either enzymatic assays on stationaryhase cultures (GAPDH and LDH activity, Fig. 2) or Western blotnalysis of surface extracts of using antibodies against GAPDH andNO (Fig. 5). Most of the strains were from a strain collection at

und University, and among the tested Lactobacillus strains only L.lantarum 299v, L. plantarum B, and L. rhamnosus GG are considereds probiotic strains. L. plantarum B (HEAL9) is used in a probioticuice (Bravo Friscus, Skånemejerierne and Probi), which has been

was incubated with the Caco-2 cells. SeeBlue Plus2 pre-stained protein standard(lane 2). Surface proteins were detected using specific polyclonal antibodies againstGAPDH or ENO.

shown to strengthen the immune system (Berggren et al., 2011).We found significant activity of extracellular GAPDH in eight out ofnine tested L. plantarum strains, two out of five L. rhamnosus, twoout of five L. gasseri strains, one out of three L. paracasei, whereasno GAPDH activity was found in the L. casei ATCC 334 strain (Fig. 2).GAPDH could be measured in cultures of some of the remainingstrains, but as there was also increased extracellular LDH activity,the GAPDH could originate from lysed cells. This was the case forL. gasseri strains DSM20243, K and Y, indicating lysis or differen-tial extracellular stability of the two enzymes. Generally GAPDHenzyme activity correlated with recognition of both GAPDH andENO by Western blot analysis. This demonstrated that the pres-ence of surface associated GAPDH and ENO is widespread amonglactobacilli species and especially a common trait among the ana-lyzed L. plantarum strains. Furthermore, the amount and GAPDHactivity among the different strains within the same species variedconsiderably.

4. Discussion

The presence of glycolytic enzymes on the surface is observedin a variety of Gram positive pathogenic bacteria and yeast/fungi,where research has shown that they are implicated in adhesionto ECM, plasminogen binding and subsequent tissue/cell invasion.In the present study we have demonstrated a similar surface-localization of the glycolytic enzymes GADPH, ENO, and PGKin Lactobacillus species and their pH-dependent interaction withhuman epithelial cells and binding to ECM proteins.

GAPDH is a multifunctional enzyme that – besides its functionin glycolysis also exhibits several other functions. These includeactivation of transcription (Zheng et al., 2003), initiation of apopto-sis (Hara et al., 2005), and pathogen–host interactions (Bergmannet al., 2004; Hara et al., 2005) where this protein has also been iden-tified on the outer surface of several pathogens such as group Astreptococci (Lottenberg et al., 1992; Pancholi and Fischetti, 1992),staphylococci (Modun and Williams, 1999), and pathogenic fungiand parasites such as Candida albicans and Schistosoma mansoni(Goudot-Crozel et al., 1989).

We demonstrated a pH-dependent specific binding of GAPDHand ENO to Caco-2 cells; GAPDH and ENO adhered Caco-2 cells

at pH 5 but not at pH 7. We also observed that more ENO thanGAPDH was eluted from the Caco-2 cells, which could reflect thateither ENO had a higher affinity for Caco-2 cells or that the elu-tion using glycine at low pH was more effective in releasing the

J. Glenting et al. / Microbiological Research 168 (2013) 245– 253 251

Fig. 5. Detection of surface located GAPDH and ENO in Lactobacillus spp. by western blot analysis. The presence of extracellularly located GAPDH and ENO in selectedLactobacillus spp. was analyzed in the eluted surface protein fractions by western blot analysis. The upper panel shows the western blot using an antibody against GAPDHand the lower panel shows the western blot using an antibody against ENO. Eluted surface proteins from the following strains were analyzed L. plantarum 299v (lanes 1 and3), L. plantarum WCFS1 (lane 4), L. gasseri Y (lane 5), L. gasseri L (lane 6), L. gasseri K (lane 7), L. gasseri Aa (lane 8), L. gasseri Z (lane 9), L. gasseri DSM20243 (lane 10), L. caseiATCC 334, (lane 11), L. paracasei X (lane 12), L. paracasei V (lane 13), L. paracasei H (lane 14), L. rhamnosus T (lane 15), L. rhamnosus GG (lane 16), L. rhamnosus R (lane 17), L.rhamnosus E (lane 18), L. rhamnosus CCUG 21452 (lanes 19 and 20), L. plantarum 299v (lane 21), L. plantarum WCFS1 (lane 22). SeeBlue® Plus2 pre-stained protein standardw

ameicscrpst

atsateds(cgbolMlw

gavagio1oltnt

as loaded in lanes 2 and 23.

ttached ENO vs GAPDH. The Caco-2 cell type expresses severalarkers distinct of normal small intestinal villi and has proven

xcellent as in vitro model for probiotic adhesion and pathogenicnvasion (Lehto and Salminen, 1997; Granato et al., 2004). Also,ell wall association of GAPDH and ENO in probiotic bacteria havehown to be pH-dependent; these enzymes associate to the outerell surface of probiotic and pathogenic bacteria at pH 5 but areeleased into the growth medium at neutral and slightly alkalineH (Antikainen et al., 2007). At low pH these enzymes thereforeeems to play a role in mediating attachment of probiotic bacteriao intestinal epithelial cells.

Another aspect that has to be considered is how GAPDH, ENO,nd PGK are secreted and attached to the outer surface of the bac-erial cell. Analysis of these enzymes revealed no binding motifsuch as the C-terminal LPXTG motif that is typical for covalentlyssociated proteins. Nor did the genes corresponding to the iden-ified proteins encode signal sequences that could explain theirxtracellular location. In a recent study Antikainen et al. (2007)emonstrated that GAPDH and ENO are anchored on the outer cellurface of L. crispatus through the interaction with lipotechtoic acidLTA). This binding was due to the interaction of the negativelyharged carboxylic acid of LTA with the positively charged aminoroups of GAPDH and ENO at low pH. Similar findings were doney Granato et al. (2004) that identified EF-Tu as a component of theuter surface of L. johnsonii NCC 533 (La1) where it mediates a simi-ar pH-dependent binding to epithelial cells and human gut mucin.

oreover, the heat shock protein GroEL from the same Lactobacil-us strain also exhibited a pH-dependent binding to epithelial cells

ith a more efficient binding at acidic pH (Bergonzelli et al., 2006).We found a specific binding of GAPDH to fibronectin, plasmino-

en, and – with somewhat lower affinity – also to mucin. ENO wasble to bind to fibronectin and plasminogen but not to mucin. Pre-ious studies have shown that GAPDH from L. plantarum LA318dheres to human colonic mucin (Kinoshita et al., 2008b) and theene sequence of the identified protein from this bacterium wasdentical to the protein from L. plantarum WCFS1. Sequence analysisf the glycolytic enzymes present in strain 299v revealed an almost00% identity to the corresponding enzymes from the strain WCFS1n the amino acid level. However, we could not only detect very

ow extracellular GAPDH activity in the WCSF1 strain, indicatinghat despite identical protein sequences other factors or mecha-isms differing among the two strains are likely responsible forhis extracellular localization.

We analyzed several Lactobacillus strains and observed varyingamounts of extracellular GAPDH, and in all cases a good correla-tion between GAPDH enzyme activity and band intensity on thewestern blots was observed. A number of research groups havealso described the presence of surface-associated GAPDH in a vari-ety of other Lactobacillus strains (Antikainen et al., 2007; Kinoshitaet al., 2008a, b; Izquierdo et al., 2009) indicating that this is alsoa widespread phenomenon in lactic acid bacteria. Among the ana-lyzed Lactobacillus strains only L. plantarum 299v, L. plantarum B,and L. rhamnosus GG are considered probiotic strains and used incommercial probiotic products. While the two plantarum strains299v and B showed high extracellular GAPDH activity only littleGAPDH activity was found extracellularly in strain GG. Therefore,based on this limited dataset we do not find a correlation betweenthe presence of surface located GAPDH and the probiotic status ofthe analyzed strains.

Enolase is also reported to be located on the outer surfaceof micro-organisms such as yeast, fungi, and bacteria includingA streptococci (Pancholi and Fischetti, 1998), Streptococcus pneu-moniae (Whiting et al., 2002), Staphylococcus aureus (Molkanenet al., 2002), and the fungus C. albicans (Jong et al., 2003) and likesurface-associated GAPDH, acts as a strong plasminogen and plas-min binding protein. A similar role of enolase isolated from theouter surface of L. plantarum 299v is speculated. In E. coli enolaseis modified by the covalent attachment of 2-phosphoglycerate toLys341. Replacement of this amino acid residue for arginine, ala-nine, glutamine or glutamate hindered the transport of enolase tothe outer bacterial surface demonstrating that this modificationmay play a significant role in the transport of enolase to the outersurface (Boel et al., 2004).

A key feature of probiotics is their capability to adhere to epithe-lial cells and the intestinal mucosa. In recent studies a rangeof proteins including GAPDH, ENO, and PGK were identified assurface-associated proteins of lactobacilli. Here, we demonstratedthat GAPDH and ENO could play a role in the adhesion of L. plan-tarum 299v to extracellular matrix proteins and to mucin. Thisstudy did not allow us to prove that the presence of these glycolyticenzymes on the outer surface of L. plantarum 299v is a prerequi-site for binding of this strain to the mucosal surface. Ideally, those

three genes should have been knocked-out and the binding capac-ity should have been compared to the wild type strain. However,the generation of such isogenic mutants are probably not possi-ble as the three genes are essential for normal metabolism. Even if

2 ical R

scs(opisspfbals

A

Afig

R

A

A

A

B

B

B

B

B

B

B

B

d

G

G

G

G

52 J. Glenting et al. / Microbiolog

uch mutants were constructed their metabolism would have beenhanged in many other ways making comparisons to the wild typetrain very difficult. However, our and very recent published dataBeck et al., 2009; Izquierdo et al., 2009) indicate that the processf adhesion is multifactorial that cannot be attributed to a singlerotein alone but to an extensive overlap between several proteins

ncluding GAPDH, ENO, EF-Tu, and GroES. This battery of unusualurface-located proteins most likely provides markers for the adhe-ive properties of lactobacilli that are potential probiotics. Theroteomic based approach used here and in recent studies may beurther developed to predict other probiotic effects or specific pro-iotic properties such as immunomodulation, anticancer-effectsnd cure of irritable bowel syndrome and deciphering the under-ying mechanisms of probiotic action, thus providing markers forelection of novel probiotic lactobacilli strains.

cknowledgments

For excellent technical assistance we thank Annemette Brix,nne Cathrine Steenbjerg and Ulla Poulsen. This work was partlynanced by the Ministry of Science, Technology, and Innovation,rant 603/4024-9.

eferences

dlerberth I, Ahrne S, Johansson ML, Molin G, Hanson LA, Wold AE. A mannose-specific adherence mechanism in Lactobacillus plantarum conferring binding tothe human colonic cell line HT-29. Appl Environ Microbiol 1996;62(7):2244–51.

leljung P, Shen W, Rozalska B, Hellman U, Ljungh A, Wadstrom T. Purification ofcollagen-binding proteins of Lactobacillus reuteri NCIB 11951. Curr Microbiol1994;28(4):231–6.

ntikainen J, Kuparinen V, Lahteenmaki K, Korhonen TK. pH-dependentassociation of enolase and glyceraldehyde-3-phosphate dehydrogenase ofLactobacillus crispatus with the cell wall and lipoteichoic acids. J Bacteriol2007;189(12):4539–43.

eck HC, Madsen SM, Glenting J, Petersen J, Israelsen H, Norrelykke MR, et al. Pro-teomic analysis of cell surface-associated proteins from probiotic Lactobacillusplantarum. FEMS Microbiol Lett 2009;297(1):61–6.

erggren A, Lazou Ahren I, Larsson N, Onning G. Randomised, double-blind andplacebo-controlled study using new probiotic lactobacilli for strengthening thebody immune defence against viral infections. Eur J Nutr 2011;50(3):203–10.

ergmann S, Rohde M, Hammerschmidt S. Glyceraldehyde-3-phosphate dehydro-genase of Streptococcus pneumoniae is a surface-displayed plasminogen-bindingprotein. Infect Immun 2004;72(4):2416–9.

ergonzelli GE, Granato D, Pridmore RD, Marvin-Guy LF, Donnicola D, Corthesy-Theulaz IE. GroEL of Lactobacillus johnsonii La1 (NCC 533) is cell surfaceassociated: potential role in interactions with the host and the gastric pathogenHelicobacter pylori. Infect Immun 2006;74(1):425–34.

ernard N, Johnsen K, Ferain T, Garmyn D, Hols P, Holbrook JJ, et al. NAD(+)-dependent D-2-hydroxyisocaproate dehydrogenase of Lactobacillus delbrueckiisubsp. bulgaricus. Gene cloning and enzyme characterization. Eur J Biochem1994;224(2):439–46.

oel G, Pichereau V, Mijakovic I, Maze A, Poncet S, Gillet S, et al. Is 2-phosphoglycerate-dependent automodification of bacterial enolases implicatedin their export? J Mol Biol 2004;337(2):485–96.

runser O, Gotteland M, Cruchet S. Functional fermented milk products. Nestle NutrWorkshop Ser Pediatr Program 2007;60:235–47, discussion 247–50.

uck BL, Altermann E, Svingerud T, Klaenhammer TR. Functional analysis of puta-tive adhesion factors in Lactobacillus acidophilus NCFM. Appl Environ Microbiol2005;71(12):8344–51.

e Man JD, Rogosa M, Sharpe ME. A medium for the cultivation of Lactobacilli. J ApplBacteriol 1960;23(2):130–5.

il-Navarro I, Gil ML, Casanova M, O’Connor JE, Martinez JP, Gozalbo D. The gly-colytic enzyme glyceraldehyde-3-phosphate dehydrogenase of Candida albicansis a surface antigen. J Bacteriol 1997;179(16):4992–9.

oudot-Crozel V, Caillol D, Djabali M, Dessein AJ. The major parasite surface antigenassociated with human resistance to schistosomiasis is a 37-kD glyceraldehyde-3P-dehydrogenase. J Exp Med 1989;170(6):2065–80.

ranato D, Bergonzelli GE, Pridmore RD, Marvin L, Rouvet M, Corthesy-Theulaz IE.Cell surface-associated elongation factor Tu mediates the attachment of Lac-tobacillus johnsonii NCC533 (La1) to human intestinal cells and mucins. Infect

Immun 2004;72(4):2160–9.

ranato D, Perotti F, Masserey I, Rouvet M, Golliard M, Servin A, et al. Cellsurface-associated lipoteichoic acid acts as an adhesion factor for attachment ofLactobacillus johnsonii La1 to human enterocyte-like Caco-2 cells. Appl EnvironMicrobiol 1999;65(3):1071–7.

esearch 168 (2013) 245– 253

Grant SG, Jesse J, Bloom FR, Hanahan D. Differential plasmid rescue from transgenicmouse DNAs into Escherichia coli methylation-restriction mutants. PNAS USA1990;87(12):4645–9.

Hara MR, Agrawal N, Kim SF, Cascio MB, Fujimuro M, Ozeki Y, et al. S-nitrosylatedGAPDH initiates apoptotic cell death by nuclear translocation following Siah1binding. Nat Cell Biol 2005;7(7):665–74.

Hurmalainen V, Edelman S, Antikainen J, Baumann M, Lahteenmaki K, KorhonenTK. Extracellular proteins of Lactobacillus crispatus enhance activation of humanplasminogen. Microbiology 2007;153(Pt 4):1112–22.

Hynonen U, Westerlund-Wikstrom B, Palva A, Korhonen TK. Identification byflagellum display of an epithelial cell- and fibronectin-binding function inthe SlpA surface protein of Lactobacillus brevis. J Bacteriol 2002;184(12):3360–7.

Izquierdo E, Horvatovich P, Marchioni E, Aoude-Werner D, Sanz Y, Ennahar S. 2-DE and MS analysis of key proteins in the adhesion of Lactobacillus plantarum,a first step toward early selection of probiotics based on bacterial biomarkers.Electrophoresis 2009;30(6):949–56.

Johansen E, Kibenich A. Isolation and characterization of IS1165, an insertionsequence of Leuconostoc mesenteroides subsp. cremoris and other lactic acidbacteria. Plasmid 1992;27(3):200–6.

Jong AY, Chen SH, Stins MF, Kim KS, Tuan TL, Huang SH. Binding of Can-dida albicans enolase to plasmin(ogen) results in enhanced invasion ofhuman brain microvascular endothelial cells. J Med Microbiol 2003;52(Pt 8):615–22.

Johansson ML, Molin G, Jeppsson N, Nobaek S, Ahrné S, Bengmark S. Administrationof different Lactobacillus strains in fermented oatmeal soup: in vivo colonizationof human intestinal mucosa and effect on the indigenous flora. Appl EnvironMicrobiol 1993;59(1):15–20.

Kinoshita H, Wakahara N, Watanabe M, Kawasaki T, Matsuo H, Kawai Y, et al. Cellsurface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Lactobacillusplantarum LA 318 recognizes human A and B blood group antigens. Res Microbiol2008a;159(9–10):685–91.

Kinoshita H, Uchida H, Kawai Y, Kawasaki T, Wakahara N, Matsuo H, et al.Cell surface Lactobacillus plantarum LA 318 glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) adheres to human colonic mucin. J Appl Microbiol2008b;104(6):1667–74.

Kleerebezem M, Boekhorst J, van Kranenburg R, Molenaar D, Kuipers OP, et al.Complete genome sequence of Lactobacillus plantarum WCFS1. PNAS USA2003;100(4):1990–5.

Lehto EM, Salminen SJ. Inhibition of Salmonella typhimurium adhesion to Caco-2 cellcultures by Lactobacillus strain GG spent culture supernate: only a pH effect?FEMS Immunol Med Microbiol 1997;18(2):125–32.

Lottenberg R, DesJardin LE, Wang H, Boyle MD. Streptokinase-producing strepto-cocci grown in human plasma acquire unregulated cell-associated plasminactivity. J Infect Dis 1992;166(2):436–40.

Mack DR, Michail S, Wei S, McDougall L, Hollingsworth MA. Probiotics inhibitenteropathogenic E. coli adherence in vitro by inducing intestinal mucin geneexpression. Am J Physiol 1999;276(4 (Pt 1)):G941–50.

Mack DR, Ahrne S, Hyde L, Wei S, Hollingsworth MA. Extracellular MUC3 mucinsecretion follows adherence of Lactobacillus strains to intestinal epithelial cellsin vitro. Gut 2003;52(6):827–33.

Mangell P, Lennernas P, Wang M, Olsson C, Ahrne S, Molin G, et al. Adhesive capabilityof Lactobacillus plantarum 299v is important for preventing bacterial transloca-tion in endotoxemic rats. APMIS 2006;114(9):611–8.

Miyoshi Y, Okada S, Uchimura T, Satoh E. A mucus adhesion promoting protein,MapA, mediates the adhesion of Lactobacillus reuteri to Caco-2 human intestinalepithelial cells. Biosci Biotechnol Biochem 2006;70(7):1622–8.

Modun B, Williams P. The staphylococcal transferrin-binding protein is acell wall glyceraldehyde-3-phosphate dehydrogenase. Infect Immun1999;67(3):1086–92.

Molin G, Jeppsson B, Johansson ML, Ahrne S, Nobaek S, Stahl M, et al. Numericaltaxonomy of Lactobacillus spp. associated with healthy and diseased mucosa ofthe human intestines. J Appl Bacteriol 1993;74(3):314–23.

Molkanen T, Tyynela J, Helin J, Kalkkinen N, Kuusela P. Enhanced activation ofbound plasminogen on Staphylococcus aureus by staphylokinase. FEBS Lett2002;517(1–3):72–8.

Naruszewicz M, Johansson ML, Zapolska-Downar D, Bukowska H. Effect of Lacto-bacillus plantarum 299v on cardiovascular disease risk factors in smokers. Am JClin Nutr 2002;76(6):1249–55.

Niedzielin K, Kordecki H, Birkenfeld B. A controlled, double-blind, randomized studyon the efficacy of Lactobacillus plantarum 299v in patients with irritable bowelsyndrome. Eur J Gastroenterol Hepatol 2001;13(10):1143–7.

Nielsen H, Engelbrecht J, Brunak S, von Heijne G. Identification of prokaryotic andeukaryotic signal peptides and prediction of their cleavage sites. Protein Eng1997;10(1):1–6.

Nobaek S, Johansson ML, Molin G, Ahrne S, Jeppsson B. Alteration of intesti-nal microflora is associated with reduction in abdominal bloating and painin patients with irritable bowel syndrome. Am J Gastroenterol 2000;95(5):1231–8.

Ochman H, Gerber AS, Hartl DL. Genetic applications of an inverse polymerase chainreaction. Genetics 1988;120(3):621–3.

Oelschlaeger TA. Mechanisms of probiotic actions—a review. Int J Med Microbiol2009;300(1):57–62.

Pancholi V, Fischetti VA. A major surface protein on group A streptococci is aglyceraldehyde-3-phosphate-dehydrogenase with multiple binding activity. JExp Med 1992;176(2):415–26.

ical R

P

P

P

R

R

R

Streptococcus pneumoniae that binds plasminogen and is immunogenic. J MedMicrobiol 2002;51(10):837–43.

J. Glenting et al. / Microbiolog

ancholi V, Fischetti VA. alpha-Enolase, a novel strong plasmin(ogen) bind-ing protein on the surface of pathogenic streptococci. J Biol Chem1998;273(23):14503–15.

ancholi V, Chhatwal GS. Housekeeping enzymes as virulence factors for pathogens.Int J Med Microbiol 2003;293(6):391–401.

retzer G, Snel J, Molenaar D, Wiersma A, Bron PA, Lambert J, et al. Biodiversity-basedidentification and functional characterization of the mannose-specific adhesinof Lactobacillus plantarum. J Bacteriol 2005;187(17):6128–36.

amiah K, van Reenen CA, Dicks LM. Surface-bound proteins of Lactobacillusplantarum 423 that contribute to adhesion of Caco-2 cells and their role in com-petitive exclusion and displacement of Clostridium sporogenes and Enterococcusfaecalis. Res Microbiol 2008(6):470–5.

oos S, Jonsson H. A high-molecular-mass cell-surface protein from Lactobacil-

lus reuteri 1063 adheres to mucus components. Microbiology 2002;148(Pt2):433–42.

oos S, Aleljung P, Robert N, Lee B, Wadstrom T, Lindberg M, et al. A collagen bindingprotein from Lactobacillus reuteri is part of an ABC transporter system? FEMSMicrobiol Lett 1996;144(1):33–8.

esearch 168 (2013) 245– 253 253

Saad N, Urdaci M, Vignoles C, Chaignepain S, Tallon R, Schmitter JM, et al. Lactobacil-lus plantarum 299v surface-bound GAPDH: a new insight into enzyme cell wallslocation. J Microbiol Biotechnol 2009;19(12):1635–43.

Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed.Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989.

Schneewind O, Mihaylova-Petkov D, Model P. Cell wall sorting signals in surfaceproteins of Gram-positive bacteria. EMBO J 1993;12(12):4803–11.

Smith H, Bron S, Van Ee J, Venema G. Construction and use of signalsequence selection vectors in Escherichia coli and Bacillus subtillis. J Bacteriol1987;169(7):3321–8.

Whiting GC, Evans JT, Patel S, Gillespie SH. Purification of native alpha-enolase from

Zheng L, Roeder RG, Luo Y. S phase activation of the histone H2B promoter byOCA-S, a coactivator complex that contains GAPDH as a key component. Cell2003;114(2):255–66.