2. 246 J. Glenting et al. / Microbiological Research 168 (2013) 245–253
been shown to act as mucus adhesins (Roos and Jonsson, 2002; Buck
et al., 2005; Pretzer et al., 2005) and to mediate binding to human
epithelial cells.
Other adhesins from lactobacilli bind to the extracellular
matrix (ECM) proteins such as laminin, collagen and fibronectin.
The adhesins include the collagen-binding protein CnBP of L.
reuteri NCIB 11951 (Aleljung et al., 1994; Roos et al., 1996), the
mucin-binding protein MapA (mucus adhesion-promoting protein)
(Miyoshi et al., 2006) and the fibronectin-binding proteins FbpA in
L. acidophilus (Buck et al., 2005) and SlpA from L. brevis (Hynonen
et al., 2002).
Recently, we and others have described several unexpected pro-
teins on the outer surface of different Lactobacillus strains. These
include glycolytic enzymes, proteins involved in stress response,
and also ribosomal proteins (Beck et al., 2009; Izquierdo et al., 2009;
Saad et al., 2009). In addition the elongation factor Tu (EF-Tu) was
isolated from the outer surface of the probiotic bacteria L. john-
sonii NCC533 (Granato et al., 2004) and L. plantarum 423 (Ramiah
et al., 2008) where it was found to mediate the attachment of these
bacteria to human epithelial cells and mucin, and the competitive
exclusion of pathogens to Caco-2 cells, respectively.
L. plantarum 299v (Probi, Sweden) is a probiotic strain which
is included in the functional food product produced and marketed
as ProViva® by Skånemejerierne in Sweden. Additionally, the 299v
strain is included in products sold by Probi’s partners worldwide
in the field of healthcare and functional foods. This strain was
originally isolated from healthy intestinal mucosa (Molin et al.,
1993) and since then a couple of clinical trials have recognized its
effect in patients suffering from irritable bowel syndrome (IBS)
by reducing bloating, flatulence and pain (Nobaek et al., 2000;
Niedzielin et al., 2001).
Studies by Mack et al. (1999, 2003) showed that increased
expression of the two genes encoding the human ileocolonic
mucins MUC2 and MUC3 occurred when HT-29 cell were co-
incubated with L. plantarum 299v. The increased mucin production
led to inhibition of adherence of enteropathogenic Escherichia coli
to the intestinal epithelial cells explaining the antagonistic effect
of L. plantarum 299v.
One of the reasons for the beneficial effects of this particular
Lactobacillus strain might be its excellent ability to adhere to man-
nose residues present on the mucosal cells (Adlerberth et al., 1996)
and thereby exclude pathogenic bacteria from adhering and sub-
sequently translocating (Mangell et al., 2006). The gene encoding
the mannose binding adhesin was identified in the closely related L.
plantarum strain WCFS1 (Pretzer et al., 2005), and recently we have
also confirmed the presence of the same gene locus in L. plantarum
299v (unpublished data).
In our efforts to characterize this strain further on the molec-
ular level, we here present data focusing on the identification of
L. plantarum 299v cell surface molecules that mediate attachment
to intestinal epithelial cells, mucin and ECM proteins which could
be part of the probiotic properties of this strain. Our data demon-
strates that several glycolytic enzymes are secreted and become
non-covalent attached to the cell surface of L. plantarum 299v and
that such proteins may play a role in the adhesion to epithelial cells
and glycoproteins. However, data does not show any correlation
between the presence of these surface associated enzymes and the
probiotic status of the analyzed strains.
2. Materials and methods
2.1. Bacterial strains, plasmids, cell line and growth conditions
A number of Lactobacillus strains isolated from humans were
obtained from a strain collection present at The University of
Lund. Strains, plasmids and cell line used in this study are listed in
Table 1. E. coli strain DH10B used for recombinant expression was
grown at 37 ◦C in LB broth supplemented with 100 g/mL of ampi-
cillin when appropriate. Lactobacillus species were grown at 30 ◦C
without aeration in MRS medium (de Man et al., 1960) that had
been prepared from single components containing filter-sterilized
yeast extract and glucose added from a separately autoclaved stock
solution (medium named sMRS). Human intestinal cells (Sigma,
ECACC No. 86010202, Caco-2 cells) were used between passage
50 and 80. Caco-2 cells were cultured in a humidified incubator
(37 ◦C, 5% CO2, 90% ambient air) using DMEM (BioWhittaker, 4.5 g/L
glucose) supplemented with 10% fetal bovine serum (Biological
Industries, 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 trypsinization
at 70–80% confluency. Cells for binding assays were obtained by
seeding Caco-2 cells at near confluency (approx. 4.0E+05 cells/cm2)
followed by culturing for 15 days in a state of confluence (fully
differentiated cells).
2.2. DNA techniques and DNA sequence analysis
DNA was manipulated according to standard procedures
(Sambrook et al., 1989). All primers used for PCR or DNA
sequencing were obtained from DNA Technology A/S, Aarhus,
Denmark. Genomic DNA was isolated from L. plantarum as
previously described (Johansen and Kibenich, 1992). DNA and
Table 1
Strains, plasmids and cell lines.
Strains or plasmids Relevant
characteristic(s)
Reference or source
Lactobacillus strains
plantarum 299v Human intestine Johansson et al. (1993)
plantarum WCFS1 Human saliva Kleerebezem et al. (2003)
plantarum ATCC14917 Pickled gabbage, type
strain
ATCC
plantarum B Human Lund University
plantarum C Human Lund University
plantarum 299 Human colon Lund University
plantarum ATCC8014 Maize silage ATCC
plantarum P Human Lund University
plantarum Q Human Lund University
rhamnosus CCUG21452 Type strain CCUG
rhamnosus E Human Lund University
rhamnosus R Human Lund University
rhamnosus GG Human Lund University
rhamnosus T Human Lund University
gasseri DSM20243 Human, type strain DSMZ
gasseri Z Human Lund University
gasseri Aa Human Lund University
gasseri K Human Lund University
gasseri L Human Lund University
gasseri Y Human Lund University
casei ATCC334 Emmental cheese ATCC
paracasei H Human Lund University
paracasei V Human Lund University
paracasei X Human Lund University
E. coli strain
DH10B E. coli cloning host Grant et al. (1990)
Plasmids
pCR®
2.1-TOPO®
TA-cloning vector Invitrogen
pGEX-4T-3 Expression vector
(GST-tagged)
GE Healthcare
Cell line
Caco-2 Human caucasian
epithelial colorectal
adenocarcimona
Sigma–Aldrich
ATCC, American Type Culture Collection; CCUG, Culture Collection University of
Gothenburg; DSMZ, German Collection of Microorganisms and Cell Cultures; Lund
University, Department of Food Technology.
3. J. Glenting et al. / Microbiological Research 168 (2013) 245–253 247
deduced amino acid sequences were analyzed using the nucleotide
and protein BLAST programs (BLASTn and BLASTp) available at
http://www.ncbi.nlm.nih.gov/BLAST. Protein sequences were ana-
lyzed for signal peptides using the Signal P 4.1 server available at
http://www.cbs.dtu.dk/services/SignalP/ (Nielsen et al., 1997).
2.3. Cloning of the putative operon encoding GAPDH, PGK, TPI,
and ENO
An internal region of the GAPDH gene was PCR amplified
using degenerate primers (GPD-Nterm: 5 ATHAAYGGNTTYGGN-
MGNATHGGN 3 and GPD-mid REV: 5 YTTNCCNACNGCYTTNGC-
NGCNCCNGT 3 ) and genomic L. plantarum 299v DNA as template.
The resulting 700 bp fragment was purified and inserted into
pCR2.1-TOPO. The DNA sequence was determined using universal
M13 forward and reverse primers. The remaining part of the GAPDH
gene and the adjacent DNA regions were amplified by consecutive
rounds of inverse PCR (Ochman et al., 1988). In short, total DNA of L.
plantarum 299v was digested with either EcoRI or HindIII and reli-
gated in a large volume. PCR amplifications were carried out using
DNA primers based on DNA sequences that were obtained during
the successive rounds of inverse PCR. This cloning and sequencing
strategy was used to complete the sequencing of the four glycolytic
genes encoding GAPDH, PGK, TPI and ENO. Furthermore, a trans-
criptional regulator likely controlling expression of the putative
operon was identified upstream of the GAPDH gene. The complete
sequence of the regulator and the glycolytic operon was deposited
in GenBank under accession number HQ622815.
2.4. Preparation of extracts from Lb. plantarum 299v cell surface
and whole cells
L. plantarum 299v was grown in sMRS medium for 40–48 h.
The bacterial cells were harvested by centrifugation (4000 × g/4 ◦C)
and resuspended in an equal volume of PBS buffer (136.9 mM
sodium chloride, 2.68 mM potassium chloride, 8.1 mM disodium
hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate,
pH 7.2), incubated for 1–2 h at 30 ◦C (unless otherwise indicated),
and centrifuged; the supernatant constitutes the “surface extract”.
For binding assays, proteins extracted from the cell surface were
concentrated 10–20 times on 15 mL spin columns with cut-off at
MW 10 kDa (Millipore, MA, USA). Whole cell lysates for determina-
tion of intracellular enzyme activities were prepared from 0.5 mL
suspension of washed cells in PBS sonicated with 0.7 g glass beads
(Sigma G-9143) in an Elma Transsonic Digital S sonication bath with
ice-water, full power for a total of 10 times 1.5 min. Glass beads and
cell debris were removed by centrifugation.
2.5. Heterologous expression of GST-tagged GAPDH, PGK and ENO
and generation of polyclonal antibodies
The genes encoding GAPDH, PGK and ENO were PCR ampli-
fied from the genome of Lb. plantarum 299v using individual sets
of primers containing 5 BamHI and 3 XhoI recognition sites. The
amplified DNA fragments were digested with BamHI and XhoI and
ligated into the same sites of the expression vector pGEX-4T-3 (GE
Healthcare). The ligation mixtures were transformed into E. coli
DH10B (Invitrogen, Carlsbad, CA, USA) according to standard proce-
dures. In the pGEX-4T-3 system the recombinant GAPDH, PGK, and
ENO proteins were produced as translational fusions to the C ter-
minal of the glutathione-S-transferase (GST) polypeptide. Protein
expression was performed by diluting overnight cultures of E. coli
clones containing the respective plasmids 50-fold into 100 mL fresh
LB medium containing 100 g/mL ampicillin. Growth took place for
1/2 h (25 ◦C at 200 rpm) and isopropyl--d-thiogalactopyranoside
(IPTG) was added to induce expression (final concentration of
0.1 mM). Induction of expression continued overnight and cells
were harvested, washed in PBS and finally sonicated. Sonicated
protein lysates containing the fusion proteins were loaded into
the Glutathione Sepharose 4B Redipack column and eluted using
glutathione according to the instructions by the supplier (Amer-
sham Biosciences). Antibodies were raised against the PGK, GAPDH
and ENO fusion proteins respectively, by immunizing rabbits three
times with 100 l 1 mg/mL of recombinant protein. The antisera
were evaluated by western blot analysis.
2.6. Western blot analysis
Pre-cast 14% Tris–glycine gels from Invitrogen were used for
SDS-PAGE, and proteins were transferred to Invitrogen type 2
nitrocellulose membranes using an Xcell II blot module from
Invitrogen. Membranes were blocked with a solution of 3% skim
milk powder. As primary antibody anti-GAPDH, anti-PGK or
anti-ENO was diluted 1000-fold, added to the membranes and
incubation took place overnight. The secondary antibody alka-
line phosphatase-conjugated goat anti-rabbit antibody from Dako
(Glostrup, Denmark) was diluted 5000-fold and incubation took
place for 2 h. The membranes were washed three times in TNT
solution (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 LDH
activity
GAPDH activity assays were performed on untreated culture
samples, culture supernatants, cell surface extracts, suspensions of
washed cell, and cell lysates, using a modification of the procedure
described by Gil-Navarro et al. (1997). NAD and NADH, which take
part in the GAPDH reaction, are not taken up by intact cells. There-
fore, the intracellular GAPDH will not be detected without prior
lysis or permeabilization of the cells. 16 l samples were mixed in
a 1 cm light path cuvette with reaction mixture to a final volume
of 0.8 mL. The reaction mixture contained 1 mM NAD and 2 mM
glyceraldehyde 3-phosphate in 0.1 mM DTT, 5 mM EDTA, 50 mM
sodium phosphate and 40 mM triethanolamine, adjusted to pH
8.6 with HCl. A mixture without glyceraldehyde 3-phosphate was
used for control reactions. Activity of GAPDH causes an increase in
absorbance at 340 nm (A340) as NADH is formed during the reac-
tion. The reaction took place at room temperature (25 ± 3 ◦C). A340
was measured at intervals throughout a total incubation time of
5–180 min, depending on the activity of the sample. For each sam-
ple, the slope of A340 versus time was calculated, and the slope
of the control reaction without glyceraldehyde 3-phosphate was
subtracted. Further correction was made for A340 increase in reac-
tion mixture with buffer added instead of sample. To obtain the
activity in units/mL, the corrected slope was multiplied by the
reaction volume and divided by the sample volume and the mM
extinction coefficient of NADH, 6.3 (mM cm)−1. 1 unit of GAPDH
will catalyze production of 1 mol 1,3-diphosphoglyceric acid per
minute. LDH activity was measured by a procedure which is sim-
ilar to the GAPDH assay. Reduction of pyruvate at the expense of
NADH was measured as a decrease of A340 with time. The reaction
mixture consisted of 10 mM sodium pyruvate and 0.2 mM NADH in
63.2 mM potassium dihydrogen phosphate and 3.5 mM disodium
hydrogen 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 evaluated
by an immobilized binding assay using components of the epithelial
4. 248 J. Glenting et al. / Microbiological Research 168 (2013) 245–253
lining. GAPDH and ENO were eluted from the surface of L. plantarum
299v as described and concentrated 20 times using spin columns.
Maxisorb microtitre wells (Nunc, Roskilde, Denmark) were coated
with 20 g/mL human plasma fibronectin (Sigma–Aldrich, St. Louis,
MO), 20 g/mL human plasminogen (American Diagnostica, CT,
USA) or 20 g/mL porcine mucin (Sigma–Aldrich). Wells coated
with 0.5% bovine serum albumin (BSA) alone served as negative
control. Immobilization took place for 3 h at 37 ◦C with lid on.
Unbound components were removed by rinsing the wells five times
with PBS-0.2% (v/v) Tween-20. Free sites in the wells were blocked
with 0.1% BSA containing 0.2% Tween 20 for 30 min at 500 rpm.
Excess BSA was removed by a PBS-0.2% (v/v) Tween-20 wash. A
2-fold dilution series of the 20 times concentrated eluted surface
proteins were titrated onto the wells followed by incubation for
1 h at 37 ◦C. Plates were washed in PBS-0.2% (v/v) Tween-20 and
bound protein were detected after overnight incubation at 4 ◦C
and 500 rpm with a 1:1000 dilution of rabbit anti-GAPDH or anti-
ENO serum diluted in PBS containing 0.1% (v/v) BSA and 0.2% (v/v)
Tween-20. After five further washes with PBS-0.2% (v/v) Tween-20,
a 1:4000 dilution of goat anti-rabbit AP-conjugated antiserum in
PBS with 0.1% (v/v) BSA and 0.2% (v/v) Tween-20 were added to the
wells and incubated at room temperature for 11/2 h and 500 rpm.
After 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-
conjugated antibodies were detected by addition of p-nitrophenyl
phosphate (Sigma–Aldrich) in 1 M diethanolamine, 0.5 mM MgCl2
for 15 min at room temperature. Plates were read in an ELISA plate
reader at 405 nm.
2.9. Binding of surface proteins to Caco-2 cells
Cell wall associated proteins were obtained by harvesting a L.
plantarum 299v culture after growth in 50 mL sMRS medium at
30 ◦C for 48 h. The bacterial cells were washed twice in 50 mL PBS
(pH 7.2). The eluted surface proteins were subsequently concen-
trated 16 times. 500 l was added to 6 mL PBS (pH 7.2) and to 6 mL
0.05 M NaAc buffer (pH 5.0) containing 0.1 M NaCl. T75 flasks con-
taining differentiated Caco-2 cells were washed twice with either
PBS (pH 7.2) or 0.05 M NaAc buffer (pH 5.0) containing 0.1 M NaCl.
The Caco-2 cells were then incubated for 2 h at 37 ◦C with 6 mL PBS
buffer or 6 mL NaAc buffer containing the released cell surface pro-
teins from L. plantarum 299v. As negative controls buffers without
L. plantarum cell surface proteins were added to the Caco-2 cells.
After 2 h, unbound cell wall material was removed and followed by
three washes using the respective buffers. The Caco-2 cell layers
were then harvested and transferred into 50-mL Falcon tubes. A
final wash took place and the cells were transferred into Eppendorf
tubes. Elution of 299v surface proteins that have bound to the Caco-
2 cells was performed by addition of 200 l 0.1 M glycine–HCl (pH
3.0) and incubation for 30 min at room temperature. Eluted proteins
were separated from Caco-2 cells by centrifugation at 1500 × g for
5 min and the supernatants containing the eluted proteins were
finally neutralized using 200 l 1 M Tris–HCl (pH 8.0). The eluted
and the unbound protein samples were analyzed by SDS-PAGE and
further analyzed by western blot analysis using antibodies against
GAPDH and ENO as described previously.
3. Results
3.1. Identification, cloning and transcriptional analysis of an
operon encoding surface associated glycolytic enzymes
Genes encoding surface located proteins are usually identified
by genome sequencing followed by bioinformatics tools such as
the Signal P 4.1 prediction server or experimentally by use of signal
peptide selection vectors which carries an export-specific reporter
protein allowing cloning of genes harboring signal peptides needed
for exported proteins (Smith et al., 1987). However, these methods
do not reveal atypical proteins, which are transported and surface
attached by so far unknown mechanisms. In a recent study we used
a proteomic based approach aiming at identification of extracellular
proteins that do not exploit the conventional secretion pathways or
anchoring mechanisms (Beck et al., 2009). In this study we investi-
gated the adhesion characteristics for GAPDH, enolase (ENO), and
phosphoglycerate kinase (PGK). They were identified as the most
abundant of the proteins that were found to be non-covalently
attached 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) were
located in the same operon and in the following order: GAPDH,
PGK, TPI and ENO. The prediction of the specific enzyme function
was based on E-values (Expect value) of 0.0 for all four proteins.
The Northern blot analysis (data not shown) showed that the four
genes are transcribed into several units corresponding to a GAPDH
transcript, a GAPDH-PGK transcript and a complete GAPDH-PGK-
TPI-ENO transcript. Upstream of GAPDH a gene encoding a putative
transcriptional regulator controlling the glycolytic operon was
identified. The predicted function of this gene is based on the
high similarity to annotated transcriptional regulators from other
Lactobacillus species (E-value of 0.0). The protein sequences were
analyzed for potential signal peptides using the Signal P 4.1 pre-
diction server and for cell wall anchoring motifs consisting of a
C-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 LPXTG
anchoring motifs that could explain their extracellular presence
and/or attachment to the bacterial surface.
3.2. Detection of glycolytic enzymes using anti-sera and activity
assay
The presence of these glycolytic enzymes on the surface was
verified by western blotting analysis on PBS surface extracts from
stationary phase cultures. Recombinant GAPDH, ENO and PGK were
expressed and purified using GST affinity chromatography. The
purified recombinant proteins were used to generate specific rab-
bit polyclonal antibodies against GAPDH, PGK and ENO. The use
of these antisera demonstrated recognition of specific proteins
of the expected molecular weights in surface extracts of strain
299v whereas much lower levels were detected in surface extracts
of strain WCFS1 (Fig. 1). To test whether the surface-located
GAPDH protein was enzymatically active, activity assays were per-
formed on untreated culture samples, culture supernatants, cell
surface 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) but
not at pH 5 (10 mM KH2PO4, 140 mM NaCl). More than 90% of the
extracellular GAPDH activity was found in the surface extract and
less than 10% was found in the supernatant and washed cells (data
not shown). However, the presence of active surface associated
GAPDH could still be due to cell lysis. Therefore, we also deter-
mined the ratio of extracellular/intracellular GAPDH and compared
this with the ratio of extracellular/intracellular lactate dehydroge-
nase (LDH). LDH is a typical intracellular enzyme and therefore an
indicator of potential cell lysis. Despite small amounts of extracellu-
lar LDH the extracellular/intracellular ratio of GAPDH was at least
10 times higher than the same ratio of LDH, clearly indicating a
specific mechanism for transportation of GAPDH and maybe other
glycolytic enzymes to the bacterial surface rather than lysis. The
5. 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. Surface
extracts from strain 299v was loaded in lane 2 and from strain WCFS1 in lane 3. SeeBlue®
Plus2 pre-stained protein standard was loaded in lanes 1. Glycolytic surface proteins
were detected using specific antibodies against GAPDH (A), ENO (B) and PGK (C).
enzymatic analysis of GAPDH and LDH showed that much more
extracellular active GAPDH enzyme is present on cells of L. plan-
tarum strain 299v compared to L. plantarum strain WCFS1 (Fig. 2).
The surface-located activity of GAPDH in growing cultures was also
determined, however, the activity was very low indicating that
the extracellular activity is growth phase dependent and increases
during transition to stationary phase.
3.3. GAPDH and Eno from L. plantarum 299v act as adhesins
Previous studies in pathogenic microorganisms have shown that
the two surface associated enzymes GAPDH and ENO bind to host
components such as fibronectin, mucin and plasminogen (Pancholi
and Fischetti, 1992, 1998). It is anticipated that the glycolytic
enzymes are virulence factors involved in colonization, persistence
and invasion of the host tissue (Pancholi and Chhatwal, 2003). The
ability of surface bound GAPDH and ENO of L. plantarum 299v
to bind to plasminogen, fibronectin, and mucin was analyzed by
enzyme-linked-immuno-sorbent-assays. Surface proteins from L.
plantarum were released by a mild PBS wash at neutral pH, concen-
trated and subsequently allowed to bind to immobilized ligands in
a microtiter plate. Specific binding was detected using anti-GAPDH
and anti-ENO polyclonal antibodies followed by incubation with a
secondary antibody conjugated with alkaline phosphatase. Fig. 3A
shows the binding of GAPDH and ENO to plasminogen, and the
binding of GAPDH and ENO to fibronectin is shown in Fig. 3B.
Additionally it was shown that surface released GAPDH but not
surface released ENO was able to bind to mucin with very low
affinity (Fig. 3C). However, binding of GAPDH to mucin was less
efficient than binding to plasminogen and fibronectin. In conclu-
sion we demonstrated that GAPDH and ENO from L. plantarum 299v
have adhesive properties, and they could be involved in host cell
protein binding.
3.4. pH dependent binding of GAPDH and ENO from L. plantarum
299v 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 is
pH dependent and higher binding at pH 5.0 than at pH 7.2. It is
postulated that pH of the gut lumen is neutral but becomes more
acidic at the mucus-covered surface due to the presence of sialic
acid residues (Granato et al., 2004). Binding at pH 5.0 therefore
simulates the physiological situation in the body. Differentiated
Caco-2 cells were washed at pH 5 or pH 7 and then incubated with
released surface proteins of L. plantarum 299v. Unbound proteins
were 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 specific
antibodies against GAPDH and ENO (Fig. 4). For both enzymes we
demonstrated specific binding to Caco-2 cells at pH 5, but not at
pH 7.
Fig. 2. Surface located GAPDH and LDH enzyme activities in Lactobacillus spp. The extracellular GAPDH and LDH activity in 23 Lactobacillus spp. were measured in the eluted
surface protein fractions as described in Section 2. Each of the Lactobacillus strains was tested twice in independent cultures on two different days. Surface located GAPDH
activities are represented by the black bars and surface located LDH activities are represented by the white bars.
6. 250 J. Glenting et al. / Microbiological Research 168 (2013) 245–253
Fig. 3. Binding of GAPDH and ENO to plasminogen and fibronectin. (A) Human
plasminogen was coated in microtiter plates and increasing amounts of eluted
surface proteins were added to the wells. (B) Human fibronectin was coated in
microtiter plates and increasing amounts of eluted surface proteins were added to
the wells. (C) Porcine mucin was coated in microtiter plates and increasing amounts
of eluted surface proteins were added to the wells. Specific binding of GAPDH
and ENO was detected by addition of antisera against GAPDH and ENO obtained
from immunized rabbits. Detection of bound GAPDH and ENO was measured
by the addition of a goat anti-rabbit alkaline phosphatase conjugated antiserum
followed 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 instead
of plasminogen/fibronectin/mucin). The closed symbols show binding of GAPDH
to plasminogen, fibronectin and mucin (diamonds) and the binding of ENO to
plasminogen, fibronectin and mucin (triangles). Results shown are means of two
determinations. The variation was <10%.
3.5. The presence of glycolytic surface enzymes is widespread in
Lactobacillus species
Other studies have demonstrated that GAPDH and ENO are
present on the surface of Lactobacillus species (Antikainen et al.,
2007; Hurmalainen et al., 2007). In order to investigate how dis-
seminated this feature is among different Lactobacillus species we
screened a collection of Lactobacillus species for surface associated
glycolytic enzymes using either enzymatic assays on stationary
phase cultures (GAPDH and LDH activity, Fig. 2) or Western blot
analysis of surface extracts of using antibodies against GAPDH and
ENO (Fig. 5). Most of the strains were from a strain collection at
Lund University, and among the tested Lactobacillus strains only L.
plantarum 299v, L. plantarum B, and L. rhamnosus GG are considered
as probiotic strains. L. plantarum B (HEAL9) is used in a probiotic
juice (Bravo Friscus, Skånemejerierne and Probi), which has been
Fig. 4. Binding of surface located GAPDH and ENO to Caco-2 cells. Panel A: unbound
GAPDH 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 (lane
6). Lanes 3 and 5 are negative controls where no surface extract was incubated with
the 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-2
cells at pH 5 (lane 6). Lanes 3 and 5 are negative controls where no surface extract
was incubated with the Caco-2 cells. SeeBlue®
Plus2 pre-stained protein standard
(lane 2). Surface proteins were detected using specific polyclonal antibodies against
GAPDH or ENO.
shown to strengthen the immune system (Berggren et al., 2011).
We found significant activity of extracellular GAPDH in eight out of
nine tested L. plantarum strains, two out of five L. rhamnosus, two
out of five L. gasseri strains, one out of three L. paracasei, whereas
no GAPDH activity was found in the L. casei ATCC 334 strain (Fig. 2).
GAPDH could be measured in cultures of some of the remaining
strains, but as there was also increased extracellular LDH activity,
the GAPDH could originate from lysed cells. This was the case for
L. gasseri strains DSM20243, K and Y, indicating lysis or differen-
tial extracellular stability of the two enzymes. Generally GAPDH
enzyme activity correlated with recognition of both GAPDH and
ENO by Western blot analysis. This demonstrated that the pres-
ence of surface associated GAPDH and ENO is widespread among
lactobacilli species and especially a common trait among the ana-
lyzed L. plantarum strains. Furthermore, the amount and GAPDH
activity among the different strains within the same species varied
considerably.
4. Discussion
The presence of glycolytic enzymes on the surface is observed
in a variety of Gram positive pathogenic bacteria and yeast/fungi,
where research has shown that they are implicated in adhesion
to 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 PGK
in Lactobacillus species and their pH-dependent interaction with
human epithelial cells and binding to ECM proteins.
GAPDH is a multifunctional enzyme that – besides its function
in glycolysis also exhibits several other functions. These include
activation of transcription (Zheng et al., 2003), initiation of apopto-
sis (Hara et al., 2005), and pathogen–host interactions (Bergmann
et al., 2004; Hara et al., 2005) where this protein has also been iden-
tified on the outer surface of several pathogens such as group A
streptococci (Lottenberg et al., 1992; Pancholi and Fischetti, 1992),
staphylococci (Modun and Williams, 1999), and pathogenic fungi
and parasites such as Candida albicans and Schistosoma mansoni
(Goudot-Crozel et al., 1989).
We demonstrated a pH-dependent specific binding of GAPDH
and 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 than
GAPDH was eluted from the Caco-2 cells, which could reflect that
either 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
7. 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 selected
Lactobacillus spp. was analyzed in the eluted surface protein fractions by western blot analysis. The upper panel shows the western blot using an antibody against GAPDH
and 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 and
3), 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. casei
ATCC 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 standard
was loaded in lanes 2 and 23.
attached ENO vs GAPDH. The Caco-2 cell type expresses several
markers distinct of normal small intestinal villi and has proven
excellent as in vitro model for probiotic adhesion and pathogenic
invasion (Lehto and Salminen, 1997; Granato et al., 2004). Also,
cell wall association of GAPDH and ENO in probiotic bacteria have
shown to be pH-dependent; these enzymes associate to the outer
cell surface of probiotic and pathogenic bacteria at pH 5 but are
released into the growth medium at neutral and slightly alkaline
pH (Antikainen et al., 2007). At low pH these enzymes therefore
seems to play a role in mediating attachment of probiotic bacteria
to intestinal epithelial cells.
Another aspect that has to be considered is how GAPDH, ENO,
and PGK are secreted and attached to the outer surface of the bac-
terial cell. Analysis of these enzymes revealed no binding motifs
such as the C-terminal LPXTG motif that is typical for covalently
associated proteins. Nor did the genes corresponding to the iden-
tified proteins encode signal sequences that could explain their
extracellular location. In a recent study Antikainen et al. (2007)
demonstrated that GAPDH and ENO are anchored on the outer cell
surface of L. crispatus through the interaction with lipotechtoic acid
(LTA). This binding was due to the interaction of the negatively
charged carboxylic acid of LTA with the positively charged amino
groups of GAPDH and ENO at low pH. Similar findings were done
by Granato et al. (2004) that identified EF-Tu as a component of the
outer surface of L. johnsonii NCC 533 (La1) where it mediates a simi-
lar pH-dependent binding to epithelial cells and human gut mucin.
Moreover, the heat shock protein GroEL from the same Lactobacil-
lus strain also exhibited a pH-dependent binding to epithelial cells
with a more efficient binding at acidic pH (Bergonzelli et al., 2006).
We found a specific binding of GAPDH to fibronectin, plasmino-
gen, and – with somewhat lower affinity – also to mucin. ENO was
able to bind to fibronectin and plasminogen but not to mucin. Pre-
vious studies have shown that GAPDH from L. plantarum LA318
adheres to human colonic mucin (Kinoshita et al., 2008b) and the
gene sequence of the identified protein from this bacterium was
identical to the protein from L. plantarum WCFS1. Sequence analysis
of the glycolytic enzymes present in strain 299v revealed an almost
100% identity to the corresponding enzymes from the strain WCFS1
on the amino acid level. However, we could not only detect very
low extracellular GAPDH activity in the WCSF1 strain, indicating
that despite identical protein sequences other factors or mecha-
nisms differing among the two strains are likely responsible for
this extracellular localization.
We analyzed several Lactobacillus strains and observed varying
amounts of extracellular GAPDH, and in all cases a good correla-
tion between GAPDH enzyme activity and band intensity on the
western blots was observed. A number of research groups have
also described the presence of surface-associated GAPDH in a vari-
ety of other Lactobacillus strains (Antikainen et al., 2007; Kinoshita
et al., 2008a, b; Izquierdo et al., 2009) indicating that this is also
a 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 in
commercial probiotic products. While the two plantarum strains
299v and B showed high extracellular GAPDH activity only little
GAPDH activity was found extracellularly in strain GG. Therefore,
based on this limited dataset we do not find a correlation between
the presence of surface located GAPDH and the probiotic status of
the analyzed strains.
Enolase is also reported to be located on the outer surface
of micro-organisms such as yeast, fungi, and bacteria including
A streptococci (Pancholi and Fischetti, 1998), Streptococcus pneu-
moniae (Whiting et al., 2002), Staphylococcus aureus (Molkanen
et al., 2002), and the fungus C. albicans (Jong et al., 2003) and like
surface-associated GAPDH, acts as a strong plasminogen and plas-
min binding protein. A similar role of enolase isolated from the
outer surface of L. plantarum 299v is speculated. In E. coli enolase
is modified by the covalent attachment of 2-phosphoglycerate to
Lys341. Replacement of this amino acid residue for arginine, ala-
nine, glutamine or glutamate hindered the transport of enolase to
the outer bacterial surface demonstrating that this modification
may play a significant role in the transport of enolase to the outer
surface (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 range
of proteins including GAPDH, ENO, and PGK were identified as
surface-associated proteins of lactobacilli. Here, we demonstrated
that GAPDH and ENO could play a role in the adhesion of L. plan-
tarum 299v to extracellular matrix proteins and to mucin. This
study did not allow us to prove that the presence of these glycolytic
enzymes 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
8. 252 J. Glenting et al. / Microbiological Research 168 (2013) 245–253
such mutants were constructed their metabolism would have been
changed in many other ways making comparisons to the wild type
strain very difficult. However, our and very recent published data
(Beck et al., 2009; Izquierdo et al., 2009) indicate that the process
of adhesion is multifactorial that cannot be attributed to a single
protein alone but to an extensive overlap between several proteins
including GAPDH, ENO, EF-Tu, and GroES. This battery of unusual
surface-located proteins most likely provides markers for the adhe-
sive properties of lactobacilli that are potential probiotics. The
proteomic based approach used here and in recent studies may be
further developed to predict other probiotic effects or specific pro-
biotic properties such as immunomodulation, anticancer-effects
and cure of irritable bowel syndrome and deciphering the under-
lying mechanisms of probiotic action, thus providing markers for
selection of novel probiotic lactobacilli strains.
Acknowledgments
For excellent technical assistance we thank Annemette Brix,
Anne Cathrine Steenbjerg and Ulla Poulsen. This work was partly
financed by the Ministry of Science, Technology, and Innovation,
grant 603/4024-9.
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