INFECTION AND IMMUNITY, Dec. 2011, p. 4777–4783
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 12
Surface-Affinity Profiling To Identify Host-Pathogen Interactions?†
Annemarie Boleij,1Coby M. Laarakkers,1Jolein Gloerich,2
Dorine W. Swinkels,1and Harold Tjalsma1*
Department of Laboratory Medicine, Nijmegen Institute for Infection, Inflammation and Immunity and Radboud University Centre
for Oncology of the Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands,1
and Proteomics Technology Platform, Department of Laboratory Medicine, Laboratory of Genetic,
Endocrine and Metabolic Disease, Radboud University Nijmegen Medical Centre,
6500 HB Nijmegen, The Netherlands2
Received 27 June 2011/Returned for modification 7 August 2011/Accepted 18 September 2011
Proteolytic treatment of intact bacterial cells has proven to be a convenient approach for the identification
of surface-exposed proteins. This class of proteins directly interacts with the outside world, for instance, during
adherence to human epithelial cells. Here, we aimed to identify host receptor proteins by introducing a
preincubation step in which bacterial cells were first allowed to capture human proteins from epithelial cell
lysates. Using Streptococcus gallolyticus as a model bacterium, liquid chromatography-tandem mass spectrom-
etry (LC-MS/MS) analysis of proteolytically released peptides yielded the identification of a selective number
of human epithelial proteins that were retained by the bacterial surface. Of these potential receptors for
bacterial interference, (cyto)keratin-8 (CK8) was verified as the most significant hit, and its surface localiza-
tion was investigated by subcellular fractionation and confocal microscopy. Interestingly, bacterial enolase
could be assigned as an interaction partner of CK8 by MS/MS analysis of cross-linked protein complexes and
complementary immunoblotting experiments. As surface-exposed enolase has a proposed role in epithelial
adherence of several Gram-positive pathogens, its interaction with CK8 seems to point toward a more general
virulence mechanism. In conclusion, our study shows that surface-affinity profiling is a valuable tool to identify
novel adhesin-receptor pairs, which advocates its application in other hybrid biological systems.
The key to bacterial infection of host tissue is the establish-
ment of a dependable connection between the bacterium and
host surface structures. This is essential for the bacteria to
withstand mechanical cleansing processes and to compete with
other bacterial strains for microbial succession (16). After ini-
tial adherence, several pathogens can invade host cells using
intracellular structures, e.g., the cytoskeleton, to sustain growth
and prolong their survival times (8, 12). Ultimately, both ad-
hesion and internalization of pathogenic bacteria will directly
or indirectly (via induction of host responses) cause damage to
the infected tissue. It is therefore important to fully understand
the mechanisms underlying pathogenic interference so that
new methods to prevent pathogenic bacteria from initiating an
infectious process can be developed. In addition, knowledge
about pathogen-specific interactions and subsequent responses
may aid in the diagnosis of corresponding diseases.
Current advances in proteomic technologies provide oppor-
tunities to compare the protein content from different biologic
systems, making it possible to characterize host-pathogen in-
teractions in a global view. Therefore, the aim of this study was
to explore the use of a proteolytic shaving approach coupled to
liquid chromatography-tandem mass spectrometry (LC-MS/
MS) to identify potential host proteins for bacterial interfer-
ence. For this purpose, intact bacterial cells were first allowed
to selectively bind host proteins from epithelial cell lysates,
after which their surfaces were proteolytically shaved to gen-
erate small polypeptides that could be directly identified by
LC-MS/MS (36, 37). Importantly, peptides of host proteins can
be effectively recognized and discriminated from the bulk of
bacterial peptides by computer-assisted analysis of the identi-
fied peptide sequences.
To obtain proof of concept for this approach, the interaction
between the Gram-positive bacterium Streptococcus gallolyticus
subsp. gallolyticus and human colonocytes was used as a model
system. S. gallolyticus is an inefficient colonizer of the healthy
human large intestinal tract but has long been associated with
colorectal cancer (CRC) and endocarditis (4a, 7, 41). Our
recent work has indicated that malignant epithelial sites may
provide a route of infection for this bacterium via CRC-specific
adhesion and translocation mechanisms (5, 6, 21). Therefore,
knowledge of specific epithelial receptors for either invasion or
adhesion of S. gallolyticus could provide novel insight into the
association of S. gallolyticus with colonic malignancy.
MATERIALS AND METHODS
Bacterial strains and medium. The strains used in this study were S. gallolyti-
cus subsp. gallolyticus UCN34 (here, S. gallolyticus), which was previously isolated
from a CRC patient with a concurrent endocarditis (31), and the intestinal strain
Enterococcus faecalis (ATCC 19433). Strains were cultured in brain heart infu-
sion (BHI) broth (Difco Laboratories) supplemented with 1% glucose at 37°C in
Cell lines. Adherent monolayers of HT-29, Caco-2, and HCT116 colon ade-
nocarcinoma cells were grown in Dulbecco’s modified Eagle’s medium (DMEM;
Lonza) supplemented with 10% fetal calf serum (FCS), 20 mM HEPES, 100 nM
nonessential amino acids, and 2 mM L-glutamine (Gibco) at 37°C in 5% CO2.
Cells were maintained at logarithmic growth by subculturing them every 3 to 5
* Corresponding author. Mailing address: Department of Labora-
tory Medicine /830, Radboud University Nijmegen Medical Centre,
P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: 31 24
3618947. Fax: 31 24 3668754. E-mail: H.Tjalsma@labgk.umcn.nl.
† Supplemental material for this article may be found at http://iai
?Published ahead of print on 26 September 2011.
Cell affinity profiling. For cell affinity profiling, HT-29 cells were washed three
times with phosphate-buffered saline (PBS; pH 7.4) and lysed by a 5-min incu-
bation in 2.5 ml of mammalian protein extraction reagent (M-PER) (Pierce) at
room temperature. Soluble colonocyte proteins in the supernatant were har-
vested (fraction P2), whereas insoluble proteins present in the pellet after cen-
trifugation were solubilized by PBS containing 2% Triton X-100 (fraction P1).
Both protein fractions were aliquoted and stored at ?80°C until use.
S. gallolyticus bacteria were grown overnight, resuspended at 1:20 in fresh
medium, and grown for another 5 h. Next, 109intact bacterial cells were washed
with sterile PBS and incubated with the above described HT-29-derived protein
fractions, diluted to 100 ?g/ml in sterile PBS containing 0.1% Triton X-100
(soluble protein fraction; P2) or 2% Triton X-100 (insoluble proteins; P1), for 1 h
at room temperature. As control experiments, the same procedure was per-
formed with the soluble (control fraction C2) and insoluble (control fraction C1)
fractions incubated without bacteria, while S. gallolyticus cells were also incu-
bated in the absence of HT-29 proteins (control fraction C3). Since our main
goal was to screen if this assay could be used to identify host proteins with affinity
for bacterial surface components, we used only one biological replicate per
condition, which was compensated by a thorough biochemical validation of a
selected candidate. After incubation, bacteria were collected by centrifugation
and washed twice with the corresponding incubation buffers containing 0.1% or
2% Triton X-100 and three times with PBS without detergent. Bacterial cells
were resuspended in PBS containing 10 units of trypsin (sequencing grade;
Sigma-Aldrich) and incubated for another hour at 37°C under gentle shaking.
Next, cells were pelleted by centrifugation, after which the supernatant contain-
ing released peptides and protein fragments was treated with 1 mM dithiothreitol
(DTT) and 1 mM iodoacetamide in two successive steps of 30 min at room
temperature. Fresh trypsin was added, and tryptic cleavage was continued for
18 h at 37°C.
LC-MS/MS. Prior to nano-LC-MS/MS analysis, all peptide samples were pu-
rified and desalted using C18 spin columns (Pierce), and Triton X-100 was
removed using anti-Triton beads (Calbiochem). Nano-LC-MS/MS analysis was
performed on an Agilent Nanoflow 1100 liquid chromatograph coupled online to
a 7T linear quadrupole ion trap Fourier transform (LTQ-FT) ion cyclotron
resonance mass spectrometer (Thermo Fisher, Bremen, Germany). Peptides
were loaded directly onto the analytical column using buffer A and eluted using
buffer B (see Table S1 in the supplemental material) at a flow rate of 300 nl/min.
Peptides eluting from the column tip were injected into the mass spectrometer
via a nano-spray ion source using a spray voltage of 2.1 kV. The mass spectrom-
eter was operated in positive ion mode using data-dependent fragmentation to
analyze the top four most abundant ions from each precursor scan. For detailed
information on nano-LC-MS/MS analysis and MS settings, see Table S1.
Fragmentation and precursor data were parsed from the RAW files by
ExtractMSN (ThermoFisher) and converted into a Mascot generic peak list.
Peptides and proteins were identified using the Mascot algorithm (Mascot,
version 2.2; Matrix Science) to search a local copy of human RefSeq database
(release 33) combined with the S. gallolyticus protein database (provided by P.
Glaser) (see Table S1 in the supplemental material for data analysis settings).
The following modifications were allowed in the search: carbamidomethylation
of cysteines (fixed), oxidation of methionine (variable), and deamidation of
asparagine and glutamine (variable). After a search, the peptides matching to
human proteins (see Table S2 in the supplemental material) were validated using
the criteria that are summarized in Table S1.
CK8 binding assay, SDS-PAGE, and Western blot analysis. To monitor af-
finity of cytokeratin-8 (CK8), intact bacteria (S. gallolyticus and E. faecalis) were
incubated with colonocyte fractions as indicated before. After three washes with
PBS, bacterial cells were directly incubated in SDS sample buffer (50 mM
Tris-HCl, pH 6.0, 2% SDS, 5% ?-mercaptoethanol, and 10% glycerol) and
incubated for 15 min at 95°C prior to 10% glycine SDS-polyacrylamide (PAA)
gel electrophoresis (SDS-PAGE) (38).
To determine subcellular localization of CK8, the colon adenocarcinoma cell
lines were fragmented, using an S-PEK kit (subcellular proteome extraction kit;
Calbiochem). into four protein fractions: cytosolic (FI), membrane and organelle
(FII), nuclear (FIII), and cytoskeletal (FIV). Subcellular protein fractions (20
?g) were separated by SDS-PAGE. For detection of CK8, proteins were trans-
ferred to polyvinylidene difluoride (PVDF) membranes (Amersham) by Western
blotting (38). Blots were incubated with monoclonal rabbit anti-keratin-8 anti-
bodies (Abcam) diluted 1:25,000 or with polyclonal rabbit anti-CYP1A1 antibody
diluted 1:5,000 (Millipore). CYP1A1, a typical membrane protein with an amino-
terminal membrane anchor that resides in the endoplasmic reticulum, was used
to determine the effectiveness of subcellular proteome fractionation. Bound
antibodies were visualized with an enhanced chemiluminescence (ECL) detec-
tion system (Amersham) using anti-rabbit IgG-horseradish peroxidase (HRP)
conjugates diluted 1:25,000 (Jackson ImmunoResearch).
In-cell Western (ICW) analysis. Ninety-six-well plates were coated with intact
S. gallolyticus or E. faecalis cells (1 ? 109cells/ml) that were harvested during the
exponential growth phase (optical density at 600 nm [OD600] of 0.6) to investi-
gate the binding of CK8. Briefly, bacteria were incubated with 10 ?g of protein
fraction FII of HT-29, HCT116, and Caco-2 cells in PBS at room temperature for
1 h with gentle mixing. Next, bacteria were pelleted and resuspended in PBS and
allowed to attach to a poly-L-lysine-coated 96-well plate at 4°C overnight. The
coated plates were washed with PBS and fixed with 3.7% formaldehyde before
being blocked with 5% BSA. CK8 bound to bacterial cells was detected with
monoclonal rabbit anti-CK8 antibody and visualized with an Odyssey instrument
(Li-Cor Biosciences) using a secondary anti-rabbit antibody conjugated with
Alexa Fluor 800 (Molecular Probes). The DNA stain DRAQ5 (Invitrogen) was
used as a reference to control for the amount of cells.
Confocal microscopy. HT-29, Caco-2, and HCT116 cells were seeded onto
coverslips in six-well plates for 24 h at 37°C. Then, cells were fixed with 3.7%
formaldehyde and either left intact or made permeable by four washes with
PBS–0.3% Triton. Next, cells were blocked with 5% BSA in PBS and subse-
quently incubated with rabbit anti-cytokeratin-8 antibodies (1:500) overnight at
4°C. Cells were washed three times with PBS–0.3% Triton and stained with goat
anti-rabbit antibodies conjugated with Alexa Fluor 488. The DNA stain DRAQ5
was used to stain the nucleus. CK8 was visualized with laser scanning confocal
microscopy (Olympus FV1000).
Cross-linking of recombinant CK8 to bacterial surface proteins. S. gallolyticus
and E. faecalis were grown overnight, diluted 1:10 in fresh culture medium, and
incubated for 4 h at 37°C in 5% CO2. After centrifugation at 4,000 ? g for 10
min, the bacterial pellet was washed three times with PBS to remove loosely
bound proteins. The cells were resuspended in 500 ?l of PBS containing 8 ?g of
recombinant CK8 (Progen Biotechnik) for 1 h at room temperature (RT) with
gentle shaking. Next, bacterial cells were washed three times with incubation
buffer to remove loosely bound proteins. Bound recombinant CK8 protein was
cross-linked to the bacterial cell wall with 2 mM 3,3?-dithiobis(sulfosuccinimi-
dylpropionate) (DTSSP; Pierce), washed three times with incubation buffer, and
subsequently boiled in SDS sample buffer with or without DTT to either disrupt
or conserve the cross-linked protein complexes. Cross-linked recombinant CK8
was visualized with SDS-PAGE and Western blotting.
Immunoprecipitation of cross-linked protein complexes containing CK8. For
the immunoprecipitation of CK8 containing cross-linked protein complexes,
bacteria were lysed in 10 mM Tris-buffer containing 2 mM MgCl2, 3 mg/ml
lysozyme (Fluka), and 100 ?g/ml mutanolysin (Sigma-Aldrich) for 60 min at
37°C. Then, 1% Triton and protease inhibitors were added to the lysate, and
samples were sonicated on ice with intervals of 0.3 s for 20 s (two runs of 30
pulses each). To remove bacterial DNA, samples were incubated with Benzonase
for 30 min on ice and subsequently spun at maximum speed for 30 min at 4°C.
Supernatants were incubated with anti-rabbit CK8 antibody (1/100) overnight at
4°C with shaking. Next, antibodies were precipitated with protein G magnetic
beads (Thermo Scientific) for 2 h at RT. Beads were washed three times with
PBS–0.1% Triton, after which immunoprecipitated cross-linked protein com-
plexes were boiled in electrophoresis buffer at 100°C for 5 min and analyzed by
7.5% SDS-PAGE after staining with blue-silver Coomassie (9). Alternatively,
proteins were blotted on PVDF membrane to control the cross-linking with
monoclonal rabbit anti-keratin-8 antibodies (Abcam) diluted 1:25,000 or with
polyclonal rabbit anti-enolase antibodies (generously provided by V. Pancholi)
diluted 1:10,000. Antibodies were visualized using an ECL detection system as
Analysis of protein complexes by vMALDI-LTQ MS/MS. Cross-linked protein
complexes were excised from SDS-PAA gels for in-gel trypsin digestion as de-
scribed previously (15). After digestion, peptides were extracted by sonication in
a water bath for 15 to 20 s, concentrated using a centrifugal evaporator, and
diluted 1:1 with 2% trifluoroacetic acid (TFA). Next, samples were spotted on a
stainless steel matrix-assisted laser desorption ionization (MALDI) plate. Sam-
ple analysis was performed on a linear ion trap fitted with an intermediate-
pressure MALDI source (vMALDI-LTQ; Thermo Fisher Scientific) (18). In
total, five full MS runs were analyzed, each resulting in a selection of the 10
highest peaks, which were further analyzed by collision-induced dissociation
fragmentation analysis. Generated RAW data files were analyzed with
SEQUEST (version 28, revision 12), and identifications were considered signif-
icant with a peptide probability of ?1E?002 and a protein probability of
?1E?003. For further details on MALDI-MS analysis, data analysis settings,
and validation criteria see Table S1 in the supplemental material.
4778BOLEIJ ET AL.INFECT. IMMUN.
Profiling eukaryotic proteins with affinity for the bacterial
cell surface. The main aim of this study was to provide proof of
concept for the use of a surface-shaving LC-MS/MS approach
to identify host proteins with affinity for intact bacterial cells.
To this purpose, eukaryotic proteins from HT-29 intestinal
epithelial cells were first allowed to interact with intact S.
gallolyticus cells, after which firmly attached host proteins were
released by tryptic digestion of surface-exposed proteins and
protein complexes (Fig. 1). As expected, shotgun MS/MS anal-
ysis of the released peptides resulted in the identification of
both bacterial and eukaryotic proteins. Candidate targets for
bacterial interference (samples P1 and P2) were discriminated
from protein precipitates originating from eukaryotic cell ly-
sates that could cofractionate with intact bacterial cells during
centrifugation (samples C1 and C2) and “contaminating” pro-
teins (sample C3) (see “Cell affinity profiling” above for details
on these samples). A total of 27 eukaryotic proteins fulfilled
the protein identification criteria (see Tables S1 and S2 in the
supplemental material). Proteins were ranked according to the
change in exponentially modified protein abundance index
(delta emPAI) values that were generated by subtracting the
emPAI value of control sample C1 or C2 from the correspond-
ing experimental sample P1 or P2 (Table 1). Next, proteins that
were identified by at least two peptides in sample P1 or P2, but
not in sample C3, were assigned as potential bacterial inter-
ference factors. These stringent identification criteria nar-
rowed the results to a total of eight identified proteins from the
soluble fraction (P2) and three proteins in the insoluble frac-
tion (P1) (Table 1). Among these proteins were several cyto-
skeleton proteins as well as ribonucleoproteins. Interestingly,
cytokeratin-8 (CK8) and CK18 received the most significant
emPAI values and peptide hits. Importantly, literature sup-
ports a role for CK8 as a potential bacterial adherence factor
(17, 20) but also indicates that it is expressed at increased levels
by colon tumor cells (2), which could be relevant for the in-
creased incidence of infection of S. gallolyticus in CRC patients
(4a). For these reasons CK8 was selected as a target for further
evaluation in this study.
Presence of CK8 at the eukaryotic cell surface. In general,
keratins are intracellular cytoskeleton proteins. To validate a
possible role of CK8 as an adherence receptor for S. gallolyti-
cus, it was important to know whether this protein is also
present at the cell surface of human epithelial cells. To inves-
tigate this, HT-29 cells were fractionated and evaluated by
Western blotting. As expected, these experiments showed that
CK8 is most abundant in the cytoskeleton fraction; however,
this protein was also detectably present in the cytosolic, mem-
brane, and nuclear fractions (Fig. 2A). Antibodies against the
membrane protein CYP1A1 were used to control for correct
fractionation, which confirmed that this protein was detected
only in the membrane fraction (Fig. 2B). However, as this
fraction also includes intracellular membranes, confocal mi-
croscopy was used to further investigate the cell surface ex-
pression of CK8 (Fig. 2C). This revealed a speckle-like mem-
brane staining of CK8 in nonpermeabilized epithelial cells. In
contrast, cell permeabilization results in a more equal staining
that mainly presents the contours of the cell. Overall, these
experiments indicate that CK8 is primarily an intracellular
protein that also has an exposed component at the epithelial
cell surface of colorectal cancer cells.
CK8 binding to the bacterial cell surface. After showing that
CK8 also has a possible surface-exposed component in addi-
FIG. 1. Schematic representation of the bacterial surface-affinity profil-
were incubated with host protein P1/P2 fractions or without eukaryotic host
proteins. Control conditions concerned the incubation of the respective host
protein fractions without S. gallolyticus cells (C1/C2) to check for protein
precipitates and a control for contaminating proteins that are not derived
from the added host protein pool (C3). After a washing step, the bacterial
surface was shaved with trypsin to release peptides from accessible proteins.
Protein fragments in the supernatants were further cleaved with trypsin for
resulted in the identification of 27 proteins, of which 11 proteins were as-
signed as candidate receptors for bacterial interference (B) (Table 1).
VOL. 79, 2011INTERACTION BETWEEN HOST CK8 AND BACTERIAL ENOLASE4779
tion to the intracellular component, we confirmed the binding
of CK8 to the bacterial cell surface by Western blotting and
ICW analysis. After incubation with the epithelial membrane
fraction, S. gallolyticus cells were washed and boiled in SDS-
PAGE sample buffer to release extracellular bound proteins.
Western blot analysis confirmed that CK8 from HT-29 and
Caco-2 cells was detectably retained by the bacterial cell sur-
face under the applied conditions (Fig. 3A). To evaluate
whether this feature is restricted to S. gallolyticus, the same
procedure was performed with E. faecalis cells. As shown in the
lower panel of Fig. 3B, E. faecalis cells retain CK8 to a much
higher extent than observed for S. gallolyticus. The latter ob-
servation was confirmed with ICW analysis (Fig. 3B), suggest-
ing that bacterial binding to CK8 is a rather general phenom-
CK8 interacts with bacterial enolase. Evaluation of the bac-
terial cell surface confirmed that CK8 binds to S. gallolyticus
and E. faecalis cell surfaces. To reveal which bacterial adhesin
is involved in CK8 binding, recombinant CK8 was incubated
with intact bacterial cells and subsequently cross-linked to the
TABLE 1. Eukaryotic proteins identified by LC-MS/MSa
Protein group and identification no.
Protein name and/or description
Delta peptide no.
(no. of peptide hits)
P1 ? C1P2 ? C2P1 ? C1P2 ? C2
High detergent concn (insoluble proteins)
Cell division cycle 10 isoform 2
Heterogeneous nuclear ribonucleoprotein M isoform b
Ribophorin I precursor
Low detergent concn (soluble proteins)
Tubulin alpha 6
Tubulin alpha, ubiquitous
Ribosomal protein S3
Myosin, heavy chain 14 isoform 2
aEukaryotic proteins identified by LC-MS/MS with corresponding reference number, emPAI values, and the number of peptide hits per fraction (see Table S2 in
the supplemental material for peak lists and peptide sequences). Proteins that were identified in fraction P1 and P2 but not in the control fraction C3 were evaluated
based on delta emPAI values above 0 and a delta peptide number of at least 2 with the respective control fractions C1 and C2. The 11 proteins matching these criteria
were marked as potential candidate target proteins for bacterial interference. All other identified proteins can be found in Table S2. P1, S. gallolyticus plus the HT-29
insoluble fraction; P2, S. gallolyticus plus the HT-29 soluble fraction; C1, HT-29 insoluble fraction without S. gallolyticus; C2, HT-29 soluble fraction without S.
gallolyticus; C3, S. gallolyticus without HT-29 proteins.
FIG. 2. Localization of CK8 in epithelial cells. The presence of CK8
(A) and CYP1A1 (B) in HT-29 cell fractions was evaluated by immuno-
blotting. Fractions are as follows: FI, cytosol; FII, membrane; FIII, nu-
cleus; FIV, cytoskeleton. (C) Subcellular localization of CK8 was visual-
ized in permeable cells and cells that were nonpermeabilized by confocal
microscopy, showing a speckle-like membrane staining in nonpermeabi-
lized Caco-2 and HT-29 cells (Membrane), whereas a more equal staining
along the cell contours is observed in permeable cells (Total).
FIG. 3. CK8 binding to the bacterial surface. (A) S. gallolyticus and
E. faecalis cells were incubated with HT-29 and Caco-2 cell lysates
(membrane fraction) to allow binding of CK8, which was evaluated by
SDS-PAGE and immunoblotting. (B) ICW analysis was used to mon-
itor binding of CK8 to immobilized bacteria in 96-well plates. Results
are expressed as the number of fluorescent counts/mm2relative to the
amount of bacterial cells as measured by DNA staining with DRAQ5
(see “In-cell Western analysis”).
4780BOLEIJ ET AL.INFECT. IMMUN.
bacterial cell surface using the membrane-impermeable agent
DTSSP. Next, protein complexes containing CK8 were isolated
through immunocapture experiments, after which potential ad-
hesion-receptor pairs were visualized by SDS-PAGE (Fig. 4A).
The cross-linked adhesion-receptor pairs that were either left
intact or disrupted with DTT were evaluated with Western
blotting for their containment of CK8. Fig. 4B clearly shows
that the cross-linked complex (CL) is retained at approxi-
mately 155 to 160 kDa. Disrupting the cross-linked proteins
with DTT reduced CK8 to its non-cross-linked form at 58 kDa
(reduced cross-linked complex [RCL]). In-gel trypsin digestion
and subsequent vMALDI-LTQ MS/MS analysis of six possible
complexes resulted in the identification of bacterial enolase (47
kDa) in relatively faint bands with estimated sizes of 155 (two
peptides) and 160 (one peptide) kDa (Fig. 4C). LC-MS/MS
analysis of this peptide sample confirmed the identification of
enolase in this 155-kDa protein band (data not shown). The
positions of other identified bacterial proteins on SDS-PAGE
were in accordance with their theoretical native molecular
weights, and these proteins were therefore considered not to
be in complex with CK8 (see Table S3 in the supplemental
To further investigate the interaction between enolase and
CK8, cross-linked complexes were analyzed by Western blot-
ting. As already shown in Fig. 4B, CK8 is present in multiple
protein complexes, of which the ?155-kDa complex also reacts
with anti-enolase antibodies (Fig. 4D). Importantly, the eno-
lase-CK8 complex is detected only in the cross-linked nonre-
duced sample (CL) and not in the reduced sample in which
cross-links have been disrupted (RCL). Taken together, these
FIG. 4. Identification of bacterial interaction partners of CK8. (A) Cross-linked adhesin-receptor pairs were immunoprecipitated with anti-CK8
antibodies and visualized by SDS-PAGE. The position of native CK8 at approximately 58 kDa is indicated. After in-gel tryptic digestion and
MS/MS analysis, bacterial enolase could be identified in the protein complex at 155 kDa. The other protein products at 120, 110, 85, and 75 kDa
were all identified as bacterial proteins with matching protein sizes, indicating that these proteins are not part of a complex (see Table S3 in the
supplemental material). (B) Immunoprecipitated proteins were analyzed by Western blotting either in cross-linked (CL) or reduced (RCL) form;
recombinant CK8 was loaded in lane 1. The positions of recombinant CK8 at 58 kDa (native) and cross-linked products between 110 and 160 kDa
are indicated. (C) Protein sequence and peptides of S. gallolyticus enolase that were identified by MS/MS in the ?155-kDa complex. (D) Visu-
alization of enolase by Western blot analysis of the same samples that were loaded in panel B along with the enolase control. The positions of
enolase at 50 kDa (native) and an enolase-containing cross-linked complex at ?155 kDa are indicated. Importantly, this complex had the same
mobility in the SDS-PAA gel as the CK8-containing product with the highest molecular mass, as indicated in the CL lane of panel B. Note that
the cross-linked product containing CK8 and enolase (CL lanes) can be disrupted by DTT (RCL lanes), indicating that it concerns a genuine
VOL. 79, 2011 INTERACTION BETWEEN HOST CK8 AND BACTERIAL ENOLASE4781
experiments reveal that epithelial CK8 and bacterial enolase
have the potential to form an adhesin-receptor pair, which may
contribute to the interaction between bacteria and host epi-
Advances in proteomic tools provide the opportunity to ex-
plore interactions between pathogens and the host in a broad
manner. The fact that bacterial shaving has previously been
proven to be a successful method to identify bacterial proteins
at the cell surface of Gram-positive bacteria (14, 30, 33, 34, 36,
40) inspired us to explore the ability to use a modified ap-
proach to profile potential host-pathogen interactions. Here,
we report that it is indeed possible to identify host factors that
are candidate receptors for bacterial cells by proteolytically
shaving the bacterial surface after preincubation with complex
mixtures of eukaryotic proteins. Surprisingly, the most signifi-
cant hits were limited to soluble proteins, whereas no classical
membrane proteins were identified. However, in addition to
the difficulties of solubilizing transmembrane proteins, the
identification of this class of membrane proteins is more
challenging. Transmembrane proteins naturally contain fewer
cleavage sites, which results in larger peptides upon proteolytic
cleavage. Despite these apparent limitations, we have shown
that this approach allows the identification of cytoskeletal pro-
teins, such as CK8 and CK18, that may be important mediators
of bacterial infection by either functioning as surface receptors
or by hijacking the protein during invasion of epithelial cells.
Keratins are part of the cytoskeleton of eukaryotic cells that
is composed of three different types of morphologically distinct
filamentous structures: microfilaments, intermediate filaments,
and microtubules (3). This cytoskeletal network is responsible
for the mechanical integrity of the cell. CK8 and CK18 are the
major constituents of intermediate filaments of simple epithe-
lia, such as those of the intestine. In addition, both keratin-18
and -19 can form heterodimers with CK8. Interestingly, CK8
and CK18 have continuous and increased expression in tumor
cells, while the expression of other cytokeratins is lost (2, 29).
CK8, CK18, and CK19 are the most abundant intermediate
filaments expressed in malignancy and can be detected in a
number of body fluids of cancer patients, while the level of
cytokeratins in the circulation of healthy individuals is low (25).
Our experiments suggest that CK8 has a surface-exposed com-
ponent in colon adenocarcinoma cells that allows its interac-
tion with intestinal pathogens. This theory is supported by
several reports on CK8/CK18 membrane localization in colon
tumor cells (10, 13, 17, 20, 24). Furthermore, Pankov et al.
found that this surface localization was present only in tumor
cells and not in healthy tissue (29). Since our results have
identified CK8 and CK18 as possible receptors for S. gallolyti-
cus adherence, this might reflect the association between CRC
and infections with this bacterium. However, our data also
suggest that binding to these keratins is not a very specific
feature of S. gallolyticus, which is in line with our recent finding
that this association is more likely to be dominated by the
specific ability of S. gallolyticus to adhere to (intestinal) colla-
Cytosolic proteins and, in particular, cytoskeleton proteins
have been described to be important for adhesion, invasion,
and transcytosis of several bacteria, e.g., Clostridium difficile,
Neisseria gonorrhoeae, and Staphylococcus aureus (26, 32, 39).
Interestingly, our study is not the first to provide evidence for
the role of CK8 as a mediator of bacterial adherence. Previ-
ously, Tamura and Nittayajarn (35) showed a role of CK8 as an
adherence factor for different serotypes of group B streptococ-
cus (GBS) and concluded that this represents a shared mech-
anism among these strains. Our results underscore a more
general role for CK8 as a bacterial interference factor since S.
gallolyticus as well as E. faecalis cells were able to interact with
CK8. Also, the observation that other bacteria like Streptococ-
cus macedonicus, Lactobacillus plantarum (our unpublished
observations), Staphylococcus aureus, and Streptococcus pyo-
genes (35) were able to interact with CK8 strengthens the
notion that CK8 is a more general adherence mediator for
Gram-positive bacteria. Notably, the observed high-CK8-bind-
ing capacity of E. faecalis cells compared to that of S. gallolyti-
cus is consistent with their relative adherence characteristics to
HT-29 monolayers (5).
The previous observation of Tamura and Nittayajarn (35),
who showed that proteolytic treatment of the GBS surface
abolished its interaction with CK8, is of particular importance
for our findings as this bacterial proteinaceous compound re-
mained previously unidentified. Our current investigations on
CK8 interaction show for the first time that bacterial enolase is
a bacterial counterpart of this eukaryotic protein. Notably, S.
gallolyticus enolase was among the abundant bacterial proteins
released by the proteolytic shaving procedure (our unpub-
lished data). These findings fit with a common mechanism for
bacterial adherence to CK8 as enolase is a conserved bacterial
protein with great sequence conservation between different
bacterial species (22). Furthermore, enolase has already been
shown to be expressed at the bacterial cell surface and to be of
importance for the binding of, e.g., S. pneumoniae to airway
epithelial cells (1, 4, 11, 27, 28). Our current data suggest that
surface-exposed bacterial enolase can, in addition to plasmin-
ogen activator (27), also bind to epithelial CK8. It should be
kept in mind, however, that our data suggest that besides
enolase, CK8 could also interact with other presently uniden-
tified bacterial adhesins. In addition, our profiling approach
also resolved other candidate eukaryotic receptors for bacte-
rial adherence, like CK18, the role of which needs further
In conclusion, our study shows that surface-affinity profiling
is a promising approach to discover eukaryotic mediators of
bacterial interference. However, further refinement of this ap-
proach is necessary to apply this procedure also to eukaryotic
membrane proteins. Furthermore, bacterial adherence to host
tissue might also involve human or bacterial proteins, such as
fimbriae, that are not cleaved by trypsin (19). Thus, it cannot
be excluded that some important host-bacteria interactions are
being missed by this procedure. In future investigations, the
number of detected interactions might be enhanced by using
complementary proteases that cleave surface proteins at other
It goes without saying that future follow-up experiments
should further confirm the biological relevance of the CK8-
enolase interaction for bacterial infection. Such experiments
could consist of, but not be limited to, an examination of the
influence of CK8 downregulation on adherence to epithelial
4782 BOLEIJ ET AL.INFECT. IMMUN.
cells and of the virulence characteristics of an S. gallolyticus
enolase knockout in vitro and in vivo. Our first experiments to
reduce the expression of CK8 by okadaic acid treatment (23)
appeared not to be compatible with bacterial adherence assays
due to a general loss of cell attachment (our unpublished
observations). It might be anticipated that an RNA-silencing
approach would turn out to be more successful in this matter.
Cork et al. have previously shown that enolase mutants of group
wild-type strain, stressing the importance of enolase for bacterial
adherence. Unfortunately, however, it was not possible until now
to generate an S. gallolyticus enolase mutant strain to perform
similar experiments in our model systems. Nevertheless, our pres-
ent data clearly illustrate that a straightforward surface-affinity
profiling procedure can provide leads for the identification of
novel adhesin-receptor pairs. Thus, this approach demonstrates
its potential to improve our understanding of host-pathogen in-
teractions at human epithelial surfaces.
A.B was supported by the Dutch Cancer Society (project KUN
The funder had no involvement in the design, analysis, and inter-
pretation of the data. We declare that we have no conflicts of interest.
We thank P. Glaser of the Institut Pasteur for providing the S.
gallolyticus intestinal strain, V. Pancholi for providing anti-enolase
antibodies, R. Roelofs, G. Kortman, R. Schaeps, P. Hermans, H.
Wessels, W. Pluk, and our other colleagues from the Department of
Laboratory Medicine and Proteomics Technology Platform for tech-
nical assistance and useful discussions.
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Editor: A. Camilli
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