Department of Microbiology, Molecular Biology
and Biochemistry, University of Idaho, Moscow, Idaho
83844,1 and Inhibitex Inc., Norcross,
Georgia 300922
Received 12 June 2000/Returned for modification 24 July
2000/Accepted 7 August 2000
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INTRODUCTION |
Bacteria have developed various
mechanisms for inducing internalization into nonprofessional phagocytes
(8, 9). A shared requirement is that a molecular interaction
must occur between a bacterial surface adhesin and a host ligand in the
cytoplasmic membrane. This interaction must be of sufficiently high
affinity to induce signal transduction through the membrane, resulting in cytoskeletal rearrangements and uptake of the organism (14, 37). For some organisms, a simple model involving the binding of
a bacterial adhesin to its host receptor is sufficient to induce uptake. Yersinia species and Listeria
monocytogenes are examples of organisms with this pattern of
uptake. The adhesins on these facultative intracellular pathogens bind
directly to integrins or E-cadherin, respectively, with an affinity
that is sufficient to induce internalization (15, 24).
A number of reports have described bacterial surface adhesins, which
adhere to host extracellular matrix (ECM) proteins. The ECM binding
proteins are termed MSCRAMMS, and Staphylococcus aureus expresses several of these proteins with different ligand specificities (17, 18, 27). We demonstrated previously that the S. aureus surface adhesin responsible for stimulating signal
transduction upon uptake by nonprofessional phagocytes is one of its
MSCRAMMs, fibronectin (Fn) binding protein (FnBP) (6). Our
data were confirmed in two additional publications (22, 28).
This finding raised several questions regarding the nature of the
molecular interactions at the host cell surface.
Fn is the ECM protein commonly associated with integrins. It is known
that Fn is bivalent and can serve as a bridging molecule between FnBP
and the host cell integrins (17, 26, 39). Although other
bacteria use Fn as a link to adhere to host tissues, the mechanisms by
which this linkage could induce internalization are less clear. For
example, Tran Van Nhieu and Isberg showed that coating of S. aureus with Fn did not lead to efficient internalization (37). It was proposed that the binding affinity between Fn
and integrins was not sufficient to induce uptake. Interestingly, at
least one organism, Neisseria gonorrhoeae has overcome this limitation by employing a heparin-containing accessory coreceptor, which is necessary to induce maximal internalization by HEp-2 cells
(39).
The present study was initiated to identify potential cellular ligands
and other molecular requirements for uptake of S. aureus by
nonprofessional phagocytes. Using a variety of methods, we found that
FnBP binds directly to heat shock protein 60 (Hsp60) on the membranes
of human and bovine epithelial cells. Fn and 
1 integrins
are also required for maximal uptake. Based on these combined results,
a potential model to explain the molecular interactions leading to
uptake of S. aureus is proposed.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
The S. aureus strains used in this study are listed in Table
1. All strains were grown in Todd-Hewitt
broth (Difco Laboratories) for 18 h at 37°C with aeration, in
the presence of antibiotics when necessary. The bacterial cells were
grown to stationary phase, collected by centrifugation, and washed with
sterile 150 mM phosphate-buffered saline (PBS) (pH 7.2). The washed
pellets were resuspended to their original volumes in invasion medium
(described below), diluted 1:100 with the same medium, and returned to
37°C for 1 to 2 h to induce logarithmic growth.
Cell culture.
An established bovine mammary epithelial cell
line (MAC-T) (12) was cultured as described previously
(6). Briefly, the cells were grown in high-glucose
Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL), supplemented
with 10% heat-inactivated fetal bovine serum (HyClone), insulin (5 µg/ml), hydrocortisone (5 µg/ml), penicillin (100 U/ml), and
streptomycin sulfate (100 µg/ml). HEp-2 (ATCC CCL 23) and Caco-2
(ATCC HTB 37) cells were cultured in the same medium except that
insulin and hydrocortisone were omitted. The cells were seeded to
24-well culture plates (6 × 104/well) or to
100-mm-diameter culture dishes (2 × 106/dish)
(Costar) and incubated at 37°C under 6% CO2. When nearly confluent, the monolayers were washed once with sterile PBS and incubated overnight (at 37°C under 6% CO2) in DMEM (1.0 ml/well).
Internalization assays.
For most bacterial internalization
assays, the following standard assay was employed. Confluent cell
monolayers (approximately 2.3 × 105 MAC-T cells/well
or 4.0 × 105 HEp-2 cells/well) were washed once with
DMEM containing 1% bovine serum albumin (BSA) and inoculated with
bacteria suspended in the same medium (multiplicity of infection
[MOI], 10 to 50). After incubation for 1 h (at 37°C under 6%
CO2), the wells were washed once with PBS and 1.0 ml of
invasion medium, supplemented with 100 µg of gentamicin per ml, was
added to each well. The plates were incubated for an additional 1 h to kill extracellular bacteria. The monolayers were washed four times
with sterile PBS, detached from the plates by treatment for 5 min at
37°C with 100 µl of trypsin solution (0.25%) in Hanks' balanced
salt solution (HBSS) (GibcoBRL), and then lysed by adding 900 µl of
sterile deionized water. The cell lysates were serially diluted 10-fold
and plated in triplicate on Todd-Hewitt agar plates to quantify
intracellular staphylococci.
To assess internalization in the presence of various potential
effectors, the standard assay described above was modified as follows.
Prior to inoculation with bacteria, the nearly confluent monolayers
were preincubated for 30 min (at 37°C under 6% CO2) in
DMEM containing 1% BSA, plus one of the following: bovine Fn (1 to 100 nM) (Calbiochem), protein G-purified immunoglobulin G1 LK-1 murine
monoclonal antibody (MAb) specific for eukaryotic Hsp60 (recombinant
human Hsp60 was used as the immunogen; the epitope is located between
residues 383 and 447 of human Hsp60, so that the antibody identifies
Hsp60 in eukaryotes but not in prokaryotes) (StressGen Corp.), bovine
Fn antiserum prepared in rabbits (Biogenesis Inc.), nonimmune rabbit
serum (Sigma), human 1 integrin ascites prepared in mice
(clone P4C10; Gibco-BRL), or nonimmune murine ascites. The cultures
were then infected with bacteria and processed by the standard
internalization assay method.
An additional modification was made in some experiments to assess the
effect of ligand-induced receptor translocation on internalization. The
protocol for these experiments was based on the receptor downmodulation method described by Rozdzinski and Tuomanen (30). Wells in
96-well microtiter plates (A1/2; Costar) were coated by adding 50 µl
of one of the following reagents dissolved in PBS containing 2 mM CaCl2 and 1 mM MgCl2, (DPBS): LK-1 Hsp60 MAb
(10 µg/m), Du-D4 functional FnBP Fn binding fragment (16)
(100 µg/ml), or 0.1% BSA. The plates were incubated for 1 h at
room temperature and then overnight at 4°C. After the plates were
washed three times with DPBS, MAC-T cells (5 × 104)
in 100 µl of invasion medium-0.1% BSA, were added to each well. The
plates were incubated (at 37°C under 6% CO2) for 1 h to allow attachment and spread of the cells. Subsequently 100 µl of
bacterial cell suspension (approximately 3.5 × 105
cells) in DMEM-0.1% BSA was added to achieve a MOI between 20 and 30. The plates were incubated for 30 min at 37°C under 6% CO2 to allow internalization of the bacterial inoculum and
then processed by the standard internalization assay method.
SDS-PAGE and blotting.
Proteins were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 7.5 or 10% acrylamide gel slabs. Proteins were visualized within the gels
by staining with Coomassie blue R-250 or by silver staining.
For some experiments, the resolved proteins were transferred to
nitrocellulose (pore size, 0.45 µm; Schleicher & Schuell) or
Immobilon P (Millipore Corp.) membranes in a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad), using standard methods, and
were detected by immunoblotting or ligand blotting. For ligand blotting, potential membrane receptors were probed with Du-D4 peptide
(10 µg per ml of blocking solution [3% BSA, 150 mM Tris HCl (pH
7.5), 150 mM NaCl]) followed by a rabbit polyclonal Du-D4 antiserum.
Hsp60 immunoblot analyses were conducted using the LK-1 MAb (see above).
Purification of staphylococcal FnBP.
Wild-type S. aureus expresses two highly conserved forms of FnBP, FnBPA and
FnBPB (10). Each is encoded by a separate gene, designated
fnbpA and fnbpB, respectively. S. aureus DU5885(pFnBPA4) is a complemented deletion mutant that
harbors the fnbpA gene on a recombinant plasmid and
overexpresses FnBPA, and it was used to purify the protein for this
present study.
S. aureus DU5883(pFnBPA4) was grown in Todd-Hewitt broth
plus chloramphenicol (5 µg/ml) to a density of ca. 5 × 107 cells per ml. The cells were collected by
centrifugation, washed twice with PBS, and resuspended (12 g of wet
weight) in 50 ml of 0.7 M sucrose solution (pH 6.5) containing 0.02 M
maleate, 0.02 M MgCl2, 2× Bacto Antibiotic Medium 3 (Difco
Laboratories), and 200 µg of lysostaphin (Sigma). The suspension was
incubated for 15 to 30 min, until cell wall lysis was complete. The
suspension was clarified by centrifugation for 30 min at
14,000 × g and then dialyzed exhaustively against
water (4°C). FnBP was partially purified from the retentate by
preparative isoelectric focusing in crushed Sephadex G-50, using
Ampholine (pH 3.5 to 10.0) (Amersham Pharmacia Biotech AB)
(11). Gel fractions were analyzed by SDS-PAGE (10%
acrylamide) and immunoblotting with rabbit polyclonal serum specific
for FnBP (see above). Fractions containing FnBP with only minor
impurities were subjected to further purification (to homogeneity) by
precipitation with (NH4)2SO4 (60%
saturation). The purified protein was dialyzed against 50 mM ammonium
acetate and lyophilized.
Isolation of epithelial cell membrane proteins.
MAC-T and
HEp-2 cells were grown to near confluency (2.8 × 107
MAC-T cells/dish and 5.5 × 107 HEp-2 cells/dish) in
100-mm-diameter culture dishes, washed twice with HBSS, and then
detached by addition of 1 mM EDTA in HBSS for 20 min at 37°C. The
detached cells were washed twice with HBSS-5 mM MgCl2-0.5
mM CaCl2 and were either processed immediately or frozen at
80°C until use.
To isolate crude membrane protein preparations, the cell pellet
was suspended in 10 volumes of 10 mM Tris (pH 7.5) containing 1 mM EDTA
and protease inhibitor cocktail (Boehringer GmbH, Mannheim, Germany).
After being incubated for 20 min on ice, the suspension was transferred
to, and lysed using, a Dounce homogenizer. Cell lysis was monitored by
light microscopy; 10 strokes were usually sufficient. Cell debris was
removed by centrifugation initially for 10 min at 1,000 × g, and the membrane fraction was recovered by centrifugation of
the supernatant at 100,000 × g for 1 h at 4°C.
Proteins in the membrane fraction pellet (derived from 2 × 107 cells) were dissolved by incubation (3 h at 4°C) in
50 mM HEPES buffer (pH 7.5) containing 100 mM NaCl, 1% Triton X-100,
1% sodium deoxycholate, 1 mM EDTA, and protease inhibitor cocktail.
The preparation was clarified by centrifugation for 20 min at
100,000 × g and analyzed by SDS-PAGE and
electroblotting or used in ligand affinity experiments.
Identification of potential epithelial cell membrane receptors by
using an affinity gel matrix.
Affi-Gel 102 was purchased from
Bio-Rad, and purified FnBP (see above) was ligated to the gel as
specified by the manufacturer. Typically, 0.25 ml of Affi-Gel 102 and
200 µg of FnBP were used. An aliquot of solubilized membrane proteins
(100 µl) derived from 2 × 107 cells was diluted
10-fold with 50 mM HEPES buffer (pH 7.5) containing 100 mM NaCl, 1%
Triton X-100, 1% sodium deoxycholate, 1 mM EDTA, and protease
inhibitor cocktail. The protein preparation was then mixed with 100 µl of FnBP-Affi-Gel 102 in a 1.7-ml centrifuge tube and incubated
for 3 h at 4°C on a rocker. The gel was washed extensively with
50 mM HEPES buffer (pH 7.5) containing 100 mM NaCl, 1% Triton X-100,
1% sodium deoxycholate, 1 mM EDTA, and protease inhibitor cocktail.
The bound proteins were dissociated from the gel by adding SDS-PAGE
sample buffer and analyzed by SDS-PAGE.
Biotinylation of surface membrane proteins.
Biotinylation
reactions were performed on nearly confluent cultures of MAC-T cells or
HEp-2 cells. The cells were treated with
sulfo-N-hydroxysuccinimide-long-chain spacer arm
(sulfo-NHS-LC-biotin; Pierce), which is not permeable to the cell
membrane, or with the negative control reagent sulfo-NHS-acetate as
recommended by the manufacturer. Membrane proteins from cells exposed
to the biotinylation reagents were extracted by the method described above for isolation of membrane proteins. The solubilized and biotinylated membrane proteins were analyzed by SDS-PAGE and
electroblotting. For the localization of proteins, the blots were
incubated with a streptavidin-alkaline phosphatase conjugate (Sigma) or
with specific antibodies.
Microsequencing.
For N-terminal sequencing reactions,
proteins were resolved by a double SDS-PAGE separation (20).
Crude membrane fractions from MAC-T and HEp-2 cells were resolved first
in SDS-PAGE preparative gels (10% acrylamide), and the proteins were
localized by staining with Coomassie brilliant blue. The desired
protein bands were excised. After the gel had been shrunk by soaking in
50% ethanol-50% stacking gel buffer, the excised band was subjected
to an additional electrophoresis step, using an SDS-PAGE gel (7.5%
acrylamide). Proteins were electroblotted to Immobilon P membranes
(Millipore Corp.) backed by nitrocellulose membranes. Proteins on the
Immobilon P membrane were localized by staining with Coomassie
brilliant blue, and those on the nitrocellulose membrane were probed
with Du-D4 peptide and Du-D4 rabbit antiserum. The protein band
colocalizing on both blots was excised from the Immobilon P membrane,
and N-terminal sequencing was performed by the Edman procedure using a
477A Sequencer.
Interaction between FnBP and Hsp60 on whole cells.
Nearly
confluent MAC-T cell monolayers (in 100-mm-diameter culture dishes)
were inoculated with S. aureus DU5883 or S. aureus DU5883(pFnBPA4) at a MOI of 50. After 1 h of
incubation at 37°C under 6% CO2, the monolayers were
washed three times with HBSS and the cells were lysed by being
suspended in 1.5 ml of 50 mM HEPES buffer containing 1% Triton X-100,
1% deoxycholate, 1 mM EDTA, and protease inhibitor cocktail. The
lysate was transferred to a 2.0-ml centrifuge tube, and insoluble
material was recovered by centrifugation for 10 min at
10,000 × g. The pellet was washed three times with
lysing solution, solubilized with SDS-PAGE sample buffer, and analyzed
by SDS-PAGE, immunoblotting, or ligand blotting, using Hsp60 MAb or
Du-D4 peptide probes, as described above.
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RESULTS |
A functional fragment of FnBP binds to Hsp60 from epithelial cell
membrane fractions.
Previous studies in our laboratory have
demonstrated that FnBP is essential for efficient internalization
within mammary epithelial cells (6). To identify the cell
receptor for S. aureus, a ligand-blotting method was
employed. Cell membrane proteins, obtained from MAC-T and HEp-2 cells
lysates, were resolved by SDS-PAGE (10% acrylamide) and transferred to
nitrocellulose. Proteins on the nitrocellulose were probed with Du-D4,
a peptide fragment corresponding to the functional ECM binding
molecular regions of FnBP. The location of the bound Du-D4 was
determined by immunoblotting with Du-D4 rabbit antiserum. This assay
revealed that Du-D4 bound significantly to only one major protein band,
with an apparent size of ~55 kDa (Fig.
1A).
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FIG. 1.
Ligand blotting for FnBP receptors on epithelial cells.
Proteins in MAC-T or HEp-2 cell membrane preparations were resolved by
SDS-PAGE and transferred to membranes. The proteins were probed with
Du-D4 peptide followed by Du-D4 rabbit antiserum (A) or with Hsp60 MAb
(B).
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Membrane preparations were obtained from MAC-T, HEp-2, or Caco-2 cells.
The partially purified membrane proteins were solubilized and incubated
with an affinity gel matrix prepared by ligation of FnBP to Affi-Gel
102. From all three cell types, a protein with an apparent size of
~55 kDa was retained by the gel (Fig. 2). Control Affi-Gel 102 preparations,
derivatized with BSA, did not retain proteins from cell lysates
(results not shown).
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FIG. 2.
Affinity purification of potential host cell receptors
on FnBP-Affi-Gel 102. Solubilized membrane fractions from MAC-T cells,
HEp-2 cells, or Caco-2 cells were incubated with FnBP-Affi-Gel 102. The gel was then washed extensively. Proteins on the gel were eluted,
analyzed by SDS-PAGE, stained, and in some experiments subjected to
N-terminal sequence determinations. Control BSA-Affi-Gel 102 conjugates did not retain proteins from cell lysates (results not
shown). std, protein standards.
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The identity of this protein was determined by N-terminal
microsequencing after being transferred to an Immobilon P membrane. The
unambiguous and identical sequence AKDVKFGADA was obtained for the
proteins isolated from either MAC-T cells or HEp-2 cells. A search of
PIR International Pattern Match, protein databases, and Bioinformatics
Tool (www-nbrf.georgetown.edu/pirwww/search/patmatch.html) revealed an
exact match with the 10 N-terminal amino acids of human and bovine
Hsp60. The identity of the proteins from MAC-T and HEp-2 cells was
further confirmed by immunoblotting with the LK-1 Hsp60 MAb (Fig. 1B).
Hsp60 is exposed on the cytoplasmic membrane of epithelial
cells.
A potential role for Hsp60 in mediating bacterial adherence
and uptake would require its presence on the host cell surface. Although initial experiments (described above) demonstrated the binding
of FnBP to Hsp60 in a membrane fraction, it was possible that Hsp60
originated from contamination by a mitochondrial fraction. Thus, to
confirm the cell membrane localization and surface exposure of Hsp60,
human and bovine epithelial cells were treated with sulfo-NHS-LC-biotin, a biotin derivative which is not permeable to cell
membrane. Therefore, any biotinylation observed is attributed to
proteins on the cell surface. Furthermore, addition of a biotin tag
specifically to the cell surface proteins would be expected to generate
two pools of Hsp60 (biotinylated and nonbiotinylated), one derived from
the cytoplasmic membrane and the other derived from the intracellular
location. As expected, two bands were detected in immunoblots of
lysates from surface-biotinylated MAC-T and HEp-2 cells probed with an
Hsp60 MAb (Fig. 3). The two bands
corresponded to nonbiotinylated Hsp60 (lower band) and its biotinylated
derivative (upper band). The latter band is absent in the blot of a
control MAC-T cell preparation treated with sulfo-NHS-acetate. These
results confirm the cell surface membrane localization of Hsp60 and are consistent with a potential for Hsp60 to interact with FnBP on S. aureus cells.
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FIG. 3.
Immunoblot of biotin-tagged and nontagged Hsp60. Intact
MAC-T or HEp-2 cells were biotinylated with sulfo-NHS-LC-biotin (lanes
2 and 3) or treated with sulfo-NHS-acetate (for MAC-T cells only) as a
negative control (lane 1). Cell lysates were resolved by SDS-PAGE and
probed with an Hsp60 MAb.
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Interaction of FnBP and Hsp60 on whole cells.
In an experiment
to determine the specificity of the Hsp60 interaction with FnBP, MAC-T
cell monolayers were incubated with S. aureus DU5883, a
mutant deficient in FnBP synthesis, or its isogenic strain complemented
with a plasmid harboring fnbpA, the structural gene for
FnBPA. The monolayers and bacteria were coincubated for 30 min. The
cells were washed to remove nonadherent bacteria, and the MAC-T cells
were lysed. The cell lysate was centrifuged to pellet bacterial cells
plus any attached MAC-T cell proteins. The resulting pellet was washed
exhaustively and analyzed by SDS-PAGE and immunoblotting. The results
in Fig. 4 indicate that Hsp60 binds to
the surface of intact S. aureus cells expressing FnBP. A
protein, which migrated in gels with an apparent size of ~55 kDa and
was identified as Hsp60 by immunoblotting, was consistently detected in
cell lysates generated using this technique. This interaction was
specific for FnBP since S. aureus DU5883, the FnBP-deficient
mutant, did not bind to Hsp60. Similarly, preincubation of S. aureus (pFnBPA4) with Du-D4 antiserum blocked the association of
Hsp60 with the bacterial cell surface.
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FIG. 4.
FnBP-Hsp 60 interaction on cell surfaces. MAC-T cells
were incubated for 30 min with no bacteria (lane 1), S. aureus DU5883 (lane 2), S. aureus DU5883(pFNBPA4)
preincubated with Du-D4 rabbit antiserum (lane 3), or S. aureus DU5883(pFNBPA4) (lane 4). After lysis of the MAC-T cells,
the residual bacterial cell suspension and attached proteins were
recovered by centrifugation and the pellets were solubilized in
SDS-PAGE sample buffer, resolved by SDS-PAGE, transferred to a
membrane, and probed with Hsp60 MAb (A) or Du-D4 followed by Du-D4
antiserum (B). The protein bands shown in the figure correspond to Hsp
60 (apparent size, ~55 kDa) (A and B) and S. aureus FnBPA
(104 kDa) (B).
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Interaction with membrane Hsp60 is required for maximal
internalization of S. aureus by epithelial cells.
To
determine whether interaction of FnBP with Hsp60 could affect
internalization of the organism, the effect of a MAb specific for human
Hsp60 was assessed. First, MAC-T cell cultures were preincubated with
the Hsp60 MAb or a murine acites control and then infected with
S. aureus DU5875, a strain expressing FnBP (but not protein
A). The results of these experiments are summarized in Fig.
5A and indicate that the addition of the
Hsp60 MAb to culture supernatants significantly reduced the efficiency
of internalization by the epithelial cells in a dose-dependent manner.
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FIG. 5.
Effect of Hsp60 MAb (A) or bovine Fn rabbit antiserum
(B) on internalization of S. aureus DU5875 by MAC-T cells.
MAC-T cell monolayers were preincubated for 30 min with serum or
ascites proteins at the dilution or quantities indicated (MOI = 20). The number of internalized bacteria was determined in a standard
internalization assay, and the results are presented as a percentage of
the number in identical control cultures treated with nonimmune ascites
(A) or normal rabbit serum (B).
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Second, we determined the effect of ligand-induced receptor
translocation of Hsp60 from the epithelial-cell apical surface. The
ability of some membrane proteins to translocate toward their ligands
can be used to differentially affect the receptor density on the apical
and basal surfaces of cells. As such, it is often possible to reduce
the receptor density on the apical membranes of cells by incubating
them on surfaces coated with specific ligands or antibodies
(30). We predicted that if interaction of Hsp60 with
S. aureus FnBP was involved in internalization,
translocation of Hsp60 from the apical surfaces of the epithelial cell
monolayers should reduce internalization. To address this possibility,
MAC-T cells were seeded into wells coated with Hsp60 MAb, Du-D4 peptide (positive control), or BSA (negative control). The results summarized in Table 2 consistently demonstrated, as
expected, that Du-D4, the functional fragment of FnBP, reduced
internalization by causing translocation of its receptor. A significant
reduction was also caused by the Hsp60 antibody, suggesting that an
interaction of FnBP with Hsp60 on monolayers is essential for the most
efficient internalization of S. aureus.
Fn and integrins are required for efficient internalization of
S. aureus by MAC-T and HEp-2 epithelial cells.
FnBP
was initially recognized for its ability to promote adherence to host
tissues through its high affinity for Fn. Since integrins also bind Fn
and since other bacteria are internalized though 1
integrin signaling (26, 39), it was important to assess the
role of Fn in staphylococcal internalization. The effects of adding
exogenous Fn varied depending on the cell types used and whether they
expressed Fn. For example, initial experiments in which MAC-T cell
cultures were first depleted of exogenous Fn and then supplemented with
known quantities of Fn showed that internalization was blocked in the
presence of very small quantities of exogenous Fn (Fig.
6A). It was suspected that this result
was due to saturation of host cell integrins with bovine Fn produced by
the MAC-T cells. Supplementing cultures with additional soluble Fn
presumably results in its binding to FnBP, making it impossible for the
staphylococcal adhesin to interact with Fn on the MAC-T cell surface.
We subsequently confirmed this prediction by using two different
methods. First, we showed that MAC-T cells express their own endogenous
Fn by immunoblot analyses of membrane proteins using Fn antiserum
(results not shown). Second, additional experiments showed that uptake
of S. aureus by MAC-T cells was reduced in a dose-dependent
manner when cultures were preincubated with a commercially available Fn
antiserum (Fig. 5B).
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FIG. 6.
Effect of Fn on internalization of staphylococci by
HEp-2 or MAC-T cells. Monolayers were incubated for 30 min with Fn, at
the concentrations shown, prior to infection with the bacterial
suspension (MOI = 25). The number of internalized bacteria was
determined by the standard invasion assay, and the results are
presented as the percentage of the inoculum used.
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Interestingly, a different effect was observed with HEp-2 cells, a cell
line noted for lack of Fn expression (26). Experiments using
HEp-2 cells showed a clear dose-response effect in which small
quantities of Fn (up to 5 nM) stimulated uptake (Fig. 6). These results
were consistent with a model in which Fn helps link S. aureus FnBP to the epithelial-cell surface by binding to
integrins. Higher Fn concentrations blocked uptake. This effect is
consistent with the function of Fn in this system as a bifunctional
molecule in which a blocking effect at high concentrations would be due to saturation of the integrin-FnBP system.
1 integrins were also required for efficient
internalization of S. aureus DU5875 by HEp-2 cells.
Preincubation of monolayers with the 1 integrin MAb
caused a dramatic reduction in internalization to levels similar to
those in the absence of exogenous Fn (Fig. 7). We were unable to demonstrate a
similar requirement for integrins with the bovine MAC-T cells since
MAbs raised against either bovine or human 1 integrins
did not block internalization (results not shown). The reason for this
is unclear and is currently a topic of investigation by our
laboratories.
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FIG. 7.
Effect of 1 integrin MAb on
internalization of S. aureus DU5875 by HEp-2 cells. HEp-2
monolayers were preincubated with or without 5 nM Fn plus nonimmune
ascites (1:200) or 1 integrin MAb (1:200) for 30 min
prior to infection with the bacterial suspension (MOI = 17). The
number of internalized bacteria was determined by the standard invasion
assay, and the results are presented as the percentage of inoculum
used.
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DISCUSSION |
We previously demonstrated that S. aureus could
be internalized by nonprofessional phagocytes through a process
requiring FnBP binding to the host cell surface. Our results showed
that binding of FnBP to the host cell induced a genistein-sensitive signal transduction and cytoskeletal rearrangement (6),
effects similar to those seen with other well-characterized
intracellular organisms (29). This present study was
performed to identify potential ligands for S. aureus on
epithelial cells. Using a variety of techniques, we obtained evidence
for a complex interaction at the bacterial and epithelial cell surfaces
involving at least four molecules, FnBP, Fn, 1
integrins, and Hsp60. The interactions of FnBP with Fn and integrins
have been demonstrated previously and are considered to be crucial for
binding to host tissues (17, 26). However, the extension of
these observations to defining a role for Fn and integrins in
internalization of S. aureus plus the potential contribution
of Hsp60 in this process were less obvious prior to the beginning of
this study.
To identify host cell ligand proteins that could bind directly to FnBP,
we used standard ligand affinity binding techniques that had been used
to identify receptors for a number of other intracellular bacterial
adhesins (15, 30, 37, 38). The only protein that
consistently bound to purified FnBP, cell-associated FnBP, or Du-D4, a
functional fragment of FnBP, was bovine or human Hsp60, depending on
the cell source. Although the primary sequences of FnBPA and FnBPB are
not identical, the predicted functional residues within the region
encompassed by the Du-D4 peptide are conserved (17). This
suggests that both FnBPs have the ability to interact with Hsp60,
although only purified FnBPA was used in the present study.
Interestingly, these results are consistent with those of Tompkins et
al. (36), who reported the isolation of a 50-kDa protein
from human endothelial cells that was capable of binding to S. aureus cells in vitro. Although the protein in that former study
was not identified, its properties, including the ability to interact
directly with the surface of S. aureus, resembled those of
Hsp60 in the present study.
Its direct binding to FnBP, the staphylococcal adhesin
responsible for internalization, raises the possibility that Hsp60 is
involved in the adherence and internalization processes. In support of
this possibility was our demonstration that a MAb raised against Hsp60
significantly reduced internalization of S. aureus by MAC-T
cells. Hsp60 is a 57-kDa protein that was originally well characterized
because of its function as a molecular chaperonin and its association
with the mitochondrial matrix. However, a number of recent reports have
documented the presence of Hsp60 not only in the mitochondria but also
in several extramitochondrial sites including the cytoplasmic membrane
(3, 5, 13, 19, 21, 31, 32, 42). Although many studies have
associated the surface expression of Hsp60 with stressed, apoptotic, or
transformed cells, membrane Hsp60 is now considered to be typical of
eukaryotic cells in general (33, 34).
To be involved in S. aureus internalization by
nonprofessional phagocytes, Hsp60 would have to possess several
important properties. The first, localization on the cell surface, was
confirmed by using several techniques. The topology of Hsp60 in the
cytoplasmic membrane is not well understood, but a transmembrane
prediction program (Prediction of Transmembrane Regions and
Orientation, ISREC Bioinformatics Server, Lausanne, Switzerland
[www.ch.embnet.org/cgibin.TMPRED_form.html]) showed that Hsp60
has at least two potential transmembrane helices (results not shown).
Although mitochondrial Hsp60 is believed to form heptameric rings
(2, 7, 35, 40), it is unclear if membrane Hsp60 aggregates
in a similar fashion.
A second requirement for internalization is that Hsp60 should be able
to either (i) facilitate signaling by another membrane protein or (ii)
to transduce a signal directly. The direct binding of FnBP to Hsp60
does not preclude a potential role for other host ligands or ECM such
as Fn and fibrinogen, both of which bind to FnBP (41). In
fact, the results of the present study, in addition to the documented
ability of Fn and integrins to mediate the adherence of S. aureus (4), suggest that Hsp60 could function as a
coreceptor with integrins linked through Fn (Fig.
8A). A similar model has been proposed by
van Putten et al. (39) for Neisseria gonorrhoea
internalization. OpaA, a gonococcal FnBP, mediates uptake through
1 integrin signaling. The gonococcal model involves a Fn
bridge linking OpaA and the integrin, with the Fn linkage stabilized by
a host cell glycosaminoglycan coreceptor. The coligand is suspected to
bind simultaneously to OpaA and Fn. Binding of bacterial FnBPs to a
coligand (i.e., Hsp60 or glycosaminoglycan) would be one potential
mechanism for organisms to overcome the low-affinity interaction
between Fn and 1 integrins. This notion is consistent
with the results of Tran Van Nhieu and Isberg (37) indicating that Fn alone is insufficient to induce the uptake of
microorganisms including S. aureus. It was postulated that this inability was due to a low-affinity binding of Fn to the integrin,
in contrast to high-affinity bacterial ligands such as
Yersinia invasin, which binds directly to integrins.
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FIG. 8.
Potential models for FnBP receptor binding leading to
internalization. (A) FnBP interacts with integrin and Hsp60 coreceptors
through a Fn linkage. (B) FnBP interacts independently with Hsp60 and
the Fn-integrin complex.
|
|
Another possibility is that the binding of FnBP to Hsp60 occurs
independently of 1 integrins (Fig. 8B). This scenario
would most probably require a direct or indirect role for Hsp60 in
signal transduction. Although Hsp60 has several potential
phosphorylation sites and can be phosphorylated by protein kinase A
(21), it has not been reported to directly transduce a
signal following binding to extracellular ligands. However, Hsp60 is
known to control the activity of Src tyrosine kinases. Phosphorylation
of E, A, and Y random polymers by c-Src kinase was stimulated 20-fold
in the presence of Hsp60 (1). Also, Hsp60 interacts with
p21ras, a signaling molecule in the cytoplasmic
membrane (5, 13). Whether any of these activities is
involved in internalization of S. aureus into epithelial
cells is currently under investigation in our laboratories.
This work was supported by USDA (NRICGP) (G.A.B.) and PHS grants
AI28401 (G.A.B.) and AI38901 (K.W.B.), the United Diarymen of Idaho
(G.A.B.), and the Idaho Agricultural Experiment Station.
We are grateful to Timothy Foster, who supplied the strains used in
this study. N-terminal sequencing was performed by Laurey Steinke,
Protein Structure Core Facility, University of Nebraska Medical Center,
Omaha, Nebr.
We recently became aware of a supporting publication (B. Sinha, P. P. François, O. Nüße, M. Foti, O. M. Hartford, P. Vaudaux, T. J. Foster, D. L. Lew, M. Hermann, and K.-H. Krause, Cell. Microbiol. 1:101-117, 1999) in which the authors provided evidence of
a role for host cell integrins and fibronectin in internalization of
S. aureus by a variety of nonprofessional phagocytes.
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Abstract
Introduction
