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Previous Article | Next Article 
Infection and Immunity, October 1998, p. 4895-4902, Vol. 66, No. 10
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Surface Structure, Hydrophobicity, Phagocytosis,
and Adherence to Matrix Proteins of Bacillus cereus Cells
with and without the Crystalline Surface Protein Layer
Anja
Kotiranta,1,*
Markus
Haapasalo,2
Kirsti
Kari,3
Eero
Kerosuo,1
Ingar
Olsen,4
Timo
Sorsa,1
Jukka H.
Meurman,1 and
Kari
Lounatmaa5
Institute of
Dentistry,1
Department of Research
Laboratory, Institute of Dentistry,3 and
Institute of Biotechnology, Electron Microscopy Unit, Viikki
Biocenter,5 FIN-00014 University of Helsinki,
Finland, and
Department of Operative Dentistry and
Endodontics, University of Oslo, N-0317 Oslo2
and
Department of Oral Biology, University of Oslo, N-0316
Oslo,4 Norway
Received 12 March 1998/Returned for modification 4 May
1998/Accepted 7 July 1998
 |
ABSTRACT |
Nonopsonic phagocytosis of Bacillus cereus by human
polymorphonuclear leukocytes (PMNs) with particular attention to
bacterial surface properties and structure was studied. Two reference
strains (ATCC 14579T and ATCC 4342) and two clinical
isolates (OH599 and OH600) from periodontal and endodontic infections
were assessed for adherence to matrix proteins, such as type I
collagen, fibronectin, laminin, and fibrinogen. One-day-old cultures of
strains OH599 and OH600 were readily ingested by PMNs in the absence of
opsonins, while cells from 6-day-old cultures were resistant. Both
young and old cultures of the reference strains of B. cereus were resistant to PMN ingestion. Preincubation of PMNs
with the phagocytosis-resistant strains of B. cereus did
not affect the phagocytosis of the sensitive strain. Negatively stained
cells of OH599 and OH600 studied by electron microscopy had a
crystalline protein layer on the cell surface. In thin-sectioned cells
of older cultures (3 to 6 days old), the S-layer was observed to peel
off from the cells. No S-layer was detected on the reference strains.
Extraction of cells with detergent followed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis revealed a major 97-kDa
protein from the strains OH599 and OH600 but only a weak 97-kDa band
from the reference strain ATCC 4342. One-day-old cultures of the
clinical strains (hydrophobicity, 5.9 to 6.0%) showed strong binding
to type I collagen, laminin, and fibronectin. In contrast, reference
strains (hydrophobicity, 
1.0 to 4.2%) as well as 6-day-old cultures
of clinical strains (hydrophobicity, 19.0 to 53.0%) bound in only low
numbers to the proteins. Gold-labelled biotinylated fibronectin was
localized on the S-layer on the cell surface as well as on fragments of
S-layer peeling off the cells of a 6-day-old culture of B. cereus OH599. Lactose, fibronectin, laminin, and antibodies against the S-protein reduced binding to laminin but not to
fibronectin. Heating the cells at 84°C totally abolished binding to
both proteins. Benzamidine, a noncompetitive serine protease inhibitor,
strongly inhibited binding to fibronectin whereas binding to laminin
was increased. Overall, the results indicate that changes in the
surface structure, evidently involving the S-layer, during growth of
the clinical strains of B. cereus cause a shift from
susceptibility to PMN ingestion and strong binding to matrix and
basement membrane proteins. Furthermore, it seems that binding to
laminin is mediated by the S-protein while binding to fibronectin is
dependent on active protease evidently attached to the S-layer.
| |
INTRODUCTION |
Bacillus cereus is a
gram-positive aerobic and facultative spore-forming rod. It has been
regarded as a relatively nonpathogenic opportunist commonly associated
with enterotoxin-mediated diarrheal food poisoning (2, 3,
6). This organism has been increasingly isolated from serious
nongastrointestinal infections including endocarditis (44),
wound infection (21), and osteomyelitis (42).
Recently B. cereus has been found in the oral cavity
associated with infected root canals and periodontal pockets
(31).
A regularly ordered protein or glycoprotein layer (S-layer) has been
detected as the outermost component of several gram-negative and
gram-positive organisms (3, 30, 33). The functions of the
S-layer in bacteria are not completely understood. It has been
suggested that the S-layer mediates the adhesion to avian intestinal
epithelial cells in Lactobacillus acidophilus
(41) and to collagen in Lactobacillus crispatus
(45). In Bacillus stearothermophilus DSM2358, the
S-layer has been said to function as an adhesion site for
high-molecular-weight amylase (13). Increased virulence and
resistance to phagocytosis (5, 6, 35) have been associated
with the presence of the S-layer in animal pathogens. Ellar and
Lundgren (14) described the presence of an S-layer on the
surface of B. cereus ATCC 4342. The present study was done
to investigate the functional and morphological properties of the cell
surface structures of two reference strains (ATCC 4342 and ATCC
14579T) and two clinical strains (OH599 and OH600) of
B. cereus. In this connection, cell surface hydrophobicity,
susceptibility to phagocytic ingestion, and adhesion to laminin, type I
collagen, fibrinogen, and fibronectin were examined.
| |
MATERIALS AND METHODS |
Bacterial strains.
B. cereus strains were received
from the American Type Culture Collection (ATCC 14579T and
ATCC 4342) or isolated from an infected root canal and periodontal pocket (OH599 and OH600). The clinical strains were identified on the
basis of colony morphology, Gram-stain reaction, spore formation, and
biochemical tests with the BioMerieux database system. The bacterial
strains were grown on brucella horse blood agar plates supplemented
with menadione (10 µg/ml) and hemin (5 µg/ml) in an incubator
containing 5% CO2 (water-jacketed incubators; Forma
Scientific Inc.) at 37°C for 1 to 6 days.
Electron microscopy.
The samples for both thin-sectioning
and scanning electron microscopy (SEM) were prepared as described by
Lounatmaa (29). Briefly, the samples for transmission
electron microscopy (TEM) were prefixed in phosphate-buffered (pH 7.2)
2.5% glutaraldehyde for 2 h at room temperature (RT) and
overnight at 4°C. The fixed cells were collected by centrifugation
and washed three times with phosphate buffer. All samples were
postfixed with phosphate-buffered 1% osmium tetroxide and dehydrated.
For the SEM, the bacteria were critical point dried and sputter coated
with platinum. For negative staining (TEM), the bacteria were collected
from brucella horse blood agar plates, transferred into a small amount
of phosphate buffer and stained with 1% (wt/vol) phosphotungstic acid
(pH 6.5). The transmission electron micrographs were taken with a JEOL
1200-EX transmission electron microscope at 60 kV, and the scanning
electron micrographs were taken with a Zeiss DSM 962 scanning electron microscope at 20 kV.
Hydrophobicity assay and extraction of surface proteins.
Bacterial cell surface hydrophobicities were measured by the hexadecane
method as described previously (37). One- to 6-day-old cultured cells were washed once with phosphate-buffered saline (PBS)
(pH 7.4) before adjusting the cell suspensions to an optical density of
0.6 (
= 450 nm, UltrospecII; LKB-Pharmacia) for hydrophobicity assay. Two parallel test tubes of the same samples were used for each
measurement and all strains were tested three times, with new cultures
being used each time.
For the examination of surface proteins, bacterial cells cultured for
1, 3, and 6 days were collected from the agar plates, washed once in
PBS (pH 7.4), and suspended in the same buffer; the cell suspensions
were adjusted to standard optical density. Equal volumes (4 ml) of the
cell suspensions were centrifuged (3,000 × g, 6 min).
The pellets were resuspended in 500 µl of 1% sodium dodecyl sulfate
(SDS)-Tris-HCl (pH 8) and shaken for 30 min at RT. After
centrifugation, the supernatants were boiled for 5 min in sample buffer
(60 mM Tris-HCl, 1% SDS, 10% glycerol, 1% mercaptoethanol, and
0.0005% bromophenol blue) and analyzed by SDS-10% polyacrylamide gel
electrophoresis (PAGE) as described by Laemmli (27) (Mini
Protean II; Bio-Rad, Richmond, Calif.). Cells collected from the same
experiments were boiled for 5 min in sample buffer, and the
supernatants were used for the whole-cell protein profile analysis by
SDS-PAGE.
Phagocytic ingestion.
The ingestion of B. cereus
OH599, OH600, ATCC 14579T, and ATCC 4342 by human
polymorphonuclear leukocytes (PMNs) was studied as described by Ding et
al. (11). PMNs were separated from human blood by the
modified dextran-Ficoll method (43). One-, 3- and 6-day-old
cultures of B. cereus were collected from brucella blood agar plates, washed once with PBS (pH 7.4), and suspended in the same
buffer. Bacterial cell concentrations (in colony-forming units per
milliliter) in the experiments were calculated by serial 10-fold
dilutions, and the number of PMN cells was counted by direct
phase-contrast microscopy with a Bürker's counting chamber (Hawksley, Lancing, England). The suspended cells were mixed with PMNs
in the ratio 10:1 (3.9 × 107 to 5.5 × 107/3.3 × 106), and shaken gently at RT
for 0, 30, or 60 min. Phagocytosis was stopped by cooling the samples
on ice, and the phagocytosis was examined with a fluorescence
microscope (FITC-filter, X400; Leitz, Germany). For determination of
phagocytic ingestion, 1 ppm acridine orange (Gurr, London, England) in
PBS was added to the samples (1:2). At least 50 PMNs were counted from
the two parallel samples of each experiment, and experiments were
repeated three or four times. The percentages of PMNs which ingested at least one bacterium and the average number of ingested bacteria per
leukocyte were calculated. The clinical strain Porphyromonas gingivalis ES64 was used as a positive control, and
Eubacterium yurii subsp. margaretiae ES4C was
used as a negative control. The samples for thin sections were prepared
for electron microscopy as previously described (29).
Antibody production.
The antiserum was raised in adult
rabbits by subcutaneous injections with purified S-protein and
formalinized whole cells of B. cereus OH599. For the
isolation of the S-protein, the bacterial cells were harvested from
brucella blood agar plates after 2 days of culturing, washed in PBS,
and suspended in nonreducing sample buffer containing 0.06 M Tris-HCl,
1% SDS, 10% glycerol, and 0.0005% bromophenol blue. The samples were
kept in water at 100°C for 5 min before SDS-PAGE was performed in
10% polyacrylamide gels at 200 V as described by Laemmli
(27). The 97-kDa S-protein band was localized by means of a
prestained high-range-molecular-mass protein standard (Bio-Rad) and
detected by staining the gel with ice-cold KCl (0.25 M KCl, 5 mM
2-mercaptoethanol). The protein band was cut from the gel and washed
with distilled water, and the S-protein was eluted in the eluting
buffer (0.1% SDS, 50 mM Tris, 0.15 M NaCl, 5 mM mercaptoethanol [pH
8]) overnight at RT. The protein solution was concentrated with a
lyophilizer (Hetovac VR-1; Heto Lab Equipment A/S, Birkerød, Denmark).
The S-layer protein (300 µg of protein in 500 µl of PBS) was mixed
with complete Freund's adjuvant (1:1) and used for subcutaneous
immunization. The booster doses in incomplete Freund's adjuvant (125 µg of protein) were given after 3 and 6 weeks. The immune serum was
collected 7 days after the last booster injection. Another rabbit was
immunized by formalinized (48 h in 4% paraformaldehyde at 4°C) 1-day
cultured whole cells of B. cereus OH599 (2 × 107 cells/injection). Immunoglobulin G (IgG) was purified
from the immune sera by using an Econo-Pac Serum IgG Purification Kit
(Bio-Rad) in accordance with the manufacturer's directions and was
stored at 20°C. The specificities of anti-S IgG (antibody against
S-protein) and anti-W IgG (antibody against the whole cells) were
tested by Western blotting, and the titers were determined by
enzyme-linked immunosorbent assay (ELISA).
Binding to immobilized matrix proteins.
Bacterial adherence
to human plasma fibrinogen (Sigma Chemical Co., St. Louis, Mo.), human
plasma fibronectin (Boehringer Mannheim Biochemicals), mouse laminin
(Upstate Biotechnology, Lake Placid, N.Y.), and type I collagen from
rat tail (Sigma) was studied by a previously described assay method in
a slightly modified form (9, 26). Binding of 1-, 3- and
6-day-old cultures of B. cereus ATCC 14579T,
ATCC 4342, OH599 and OH600 was tested. Chamber slides (Nunc Inc.,
Naperville, Ill.) were coated overnight at 37°C with fibrinogen (0.1 mg/ml), fibronectin (0.1 mg/ml), laminin (0.1 mg/ml), type I collagen
(0.1 mg/ml), or 3% bovine serum albumin (BSA) (Sigma). After washing
the chamber slides twice with TPBS (Tween 20 [50 µl] and PBS [100
ml]) and once with PBS, the slides were blocked with 3% BSA at 37°C
for 3 h. The slides were rewashed as described earlier, and equal
concentrations of bacterial cell suspensions were added to the
chambers. After 2 h of incubation, the chamber slides were washed
as described above, and the adhered bacteria were fixed for 10 min with
2.5% buffered glutaraldehyde (vol/vol) at RT and visualized by
toluidine blue staining (1 g of
Na2B4O7 · 10H2O and
1 g of toluidine blue in 200 ml of distilled H2O). The
number of adhered bacteria was counted by phase-contrast microscopy
with a magnification of 1,000× in at least six microscopic fields per sample. The experiments were carried out two to four times.
ELISA was used to test the adherence of the cells of B. cereus OH599 and cell-derived material to fibronectin. Microtiter plate wells (ELISA) were coated with fibronectin (0.1 mg/ml in PBS) and
3% BSA at 37°C overnight. The wells were washed twice with TPBS and
once with PBS (pH 7.4) and blocked with 3% BSA for 2 h at 37°C.
Bacterial cells (1- and 6-day-old cultures) were collected from
brucella blood agar plates, washed once in PBS, and adjusted to equal
concentrations in PBS before transfer to the ELISA wells. After 2 h of incubation, the attached bacterial cells were labelled with anti-S
IgG (1:3,000 in 1% BSA-Tris-buffered saline [20 mM Tris, 0.5 M
NaCl]) and with alkaline phosphatase-conjugated anti-rabbit IgG
(1:1,000 in 1% BSA-TPBS; Sigma Immuno Chemicals), which was used as a
secondary antibody. After addition of p-nitrophenylphosphate (Sigma) the binding of cells or cell-derived material was measured colorimetrically at 405 nm (Titertek Multiskan Plus; Elflab, Helsinki, Finland).
The binding of B. cereus OH599 to soluble fibronectin was
examined by TEM with biotinylated fibronectin and gold-labelled ExtrAvidin (10 nm of colloidal gold-labelled ExtrAvidin in 50% glycerol; Sigma). Fibronectin was biotinylated by adding 2 µl of
biotinamidocaproate N-hydroxysuccinimide ester (50 mg/ml
dimethyl sulfoxide) to 1 ml of fibronectin solution. After 2.5 min, the reaction was stopped with Tris buffer (pH 8; final concentration, 100 mM). Biotin (2 µl of biotinamidocaproate
N-hydroxysuccinimide ester in 1 ml of PBS) in Tris buffer
without fibronectin was used in the control samples. One- and 6-day
cultured cells (1.2 × 108 cell/ml) were collected
from agar plates, washed once in PBS, and resuspended to the
biotin-fibronectin (1 µg/ml) solution or biotin solution. After
2 h of incubation at RT, the bacterial cells were washed once in
20 mM Tris buffer. Gold-labelled ExtrAvidin (1:20 in PBS) was added,
and the samples were prepared for TEM as described above.
Inhibition of binding to matrix proteins.
One-day-old cells
of B. cereus OH599 were preincubated with 10 and 100 mM
lactose, anti-S IgG (1:10 dilution in PBS), anti-W IgG (1:10 dilution
in PBS), fibronectin (0.1 mg/ml in PBS), and laminin (0.1 mg/ml in PBS)
for 1 h at RT. After incubation, the cells were repeatedly washed
in PBS, adjusted to equal concentrations in the same buffer, and
transferred to glass slide wells previously coated with fibronectin and
laminin as described above. Adherence to fibronectin and laminin of
heat-treated 1-day-old cultures of OH599 was also tested. The heat
treatment was done by keeping the bacterial cell suspension at 84°C
in a heat bloc (Grant Instruments Ltd., Cambridge, England) for 30 min
before the attachment assay. The number of adhered bacteria was
determined as described above and compared to those from assays of
bacterial binding to fibronectin and laminin without any inhibition.
The experiments were carried out at least three times with new cultures
each time.
To determine the role of the proteinase activity of B. cereus in adhesion to matrix proteins, 100 mM benzamidine (final
concentration) was used to inhibit the proteinase activity of B. cereus OH599. Effectiveness of protease inhibition by benzamidine
was verified by Azocoll assay. One- and 6-day cultured cells of
B. cereus OH599 were adjusted to equal concentrations in
PBS. The bacterial cell suspension (0.3 ml) was incubated with 1.5 ml
of Azocoll suspension (1 mg/ml in PBS) in the absence or presence of
100 mM benzamidine. After incubation for 3, 5, and 23 h, Azocoll
degradation was measured spectrophotometrically and compared to
positive (no inhibition) and negative (no bacteria) controls.
| |
RESULTS |
Surface properties and extraction of surface proteins.
Cells
of 1-, 3- and 6-day-old cultures of both reference strains were
hydrophilic. Young cells of the clinical strains were also hydrophilic,
whereas 3- and 6-day-old cultures were hydrophobic, as measured by the
hexadecane method (Table 1).
Whole cells of B. cereus cultured for 1, 3, and 6 days were
extracted with 1% SDS to examine the protein profiles by SDS-PAGE. A
major 97-kDa band was obtained from the two clinical strains of
B. cereus. No corresponding band was detected in the
reference strains; however, a weak 97-kDa band could be seen on SDS
extract from 6-day-old cultures of strain ATCC 4342. Anti-whole cells and anti-S-layer antibodies detected the 97-kDa band in strains OH599
and OH600 as determined by Western blotting. A weak staining of the
97-kDa band of strain ATCC 4342 was also seen by treatment with
anti-S-layer antibody. No differences in the relative intensities of
the protein bands obtained from clinical strains of 1-, 3- or 6-day-old
cultures in the whole-cell protein profiles could be seen (Fig. 1A, B,
and C).
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FIG. 1.
SDS-PAGE analysis of cell surface proteins of B. cereus OH599, OH600, ATCC 14579T, and ATCC 4342. Whole-cell protein profiles (lanes 2 to 5) and 1% SDS extracts (lanes
6 to 9) of 1- (A), 3- (B), and 6- (C) day-old cultures are shown. Lane
1, prestained high-molecular-mass standard (myosin, 206 kDa;
 -galactosidase, 117 kDa; BSA, 89 kDa; ovalbumin, 47 kDa). Lanes 2 and 6, OH599; lanes 3 and 7, OH600; lanes 4 and 8, ATCC
14579T; lanes 5 and 9, ATCC 4342.
|
|
Ultrastructure of B. cereus.
The ultrastructure of all
strains was studied. A crystalline cell surface protein layer was
observed by negative staining and by thin sectioning of the cells of
the clinical isolates of B. cereus (Fig.
2 and 3).
In 1-day-old cultures, the S-layer was covering the entire cell surface
whereas in specimens from 6-day-old cultures, the S-layer was seen
peeling off from the cells (see Fig. 9). No periodic cell surface
structures were detected in B. cereus ATCC
14579T or ATCC 4342.
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FIG. 2.
Negatively stained fragment of an S-layer of B. cereus OH599. The periodic structure is clearly seen. Bar, 0.2 µm.
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Phagocytosis of B. cereus strains.
The
susceptibilities to nonopsonic PMN-phagocytosis of 1-, 3-, and
6-day-old cultures of B. cereus were assessed by
fluorescence microscopy. One-day-old cultures of the clinical strains
were ingested in 30 min by human PMNs while the reference strains and 3- and 6-day-old cultures of the clinical strains were resistant (Table
2). Phagocytosis of the sensitive strain
OH599 was not affected by 30 min of preincubation of PMNs with a
1-day-old culture of ATCC 14579T or a 6-day-old culture of
OH599, which were resistant to phagocytosis (data not shown). The
phagocytic ingestion of B. cereus cells and spores was also
demonstrated by TEM and SEM, and thin sections made from 6-day cultured
cells showed sheets of detached S-layer attached to PMN-cells (Fig.
4
through 7).
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FIG. 4.
Numerous S-layer fragments (OH600) peeled off from the
cell wall between a PMN and bacteria. Bar, 0.2 µm.
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FIG. 5.
Two PMN-ingested bacteria (OH600). Notice that two PMNs
are trying to phagocytize the same cell. Bar, 0.2 µm.
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Adherence of B. cereus strains to matrix proteins.
The adherence of B. cereus strains to matrix proteins
immobilized on glass slides was examined by direct counting by
phase-contrast microscopy. One-day-old cultures of the clinical strains
adhered efficiently to laminin, type I collagen, and fibronectin, while binding to fibrinogen was only slightly better than binding to BSA
(Fig. 8). The number of attached cells
was considerably lower when 3- and 6-day-old cultures of the clinical
strain or 1-, 3-, and 6-day-old cultures of the reference strains were
tested (Fig. 8).
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FIG. 8.
Adhesion of B. cereus OH599, OH600, ATCC
4342, and ATCC 14579T (1- and 6-day-old [1d and 6d]
cultures) to the indicated matrix proteins. Bar lengths indicate the
mean numbers of attached cells per microscopic field of two to four
separate experiments. Inset bars shows standard deviations.
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The adhesion of biotinylated fibronectin to the clinical strain of
B. cereus OH599 was also detected by gold-labelled
ExtrAvidin. Fibronectin adhered to the S-layer on the bacterial cell
surface and to the S-layer which was peeling off from the cells of a
6-day-old culture (Fig. 9). No label was
seen on the cell surfaces of the reference strain ATCC
14579T or on the control samples incubated without
fibronectin. The IgG against purified S-protein of B. cereus
OH599 was used as a primary antibody when the adherence of 1- and
6-day-old cultures of B. cereus OH599 was tested by the
ELISA method. The binding of the cells or cell-derived material to
fibronectin was equal in both young and old cultures. The average
absorbances ± standard deviations as determined colorimetrically
by ELISA ( = 405 nm) were as follows: 1-day-old culture of OH599,
0.93 ± 0.12; 6-day-old culture of OH599, 1.13 ± 0.21; and
BSA control, 0.36 ± 0.15 (averages of three separate
experiments).
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FIG. 9.
A pre-embedding immunoelectron micrograph showing (with
gold particles) the localization of the biotinylated fibronectin on the
S-layer (partly peeling off) of a 6-day cultured cell of B. cereus OH599. Bar, 0.2 µm.
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Binding of 1-day cultured cells of B. cereus OH599 to matrix
proteins was inhibited by heat treatment of the cells at 84°C (Table
3). Pretreatment of the cells with
antisera made against purified S-protein of OH599 did not inhibit the
adhesion to fibronectin but reduced the number of cells attached to
laminin by 32% (Table 3). Fibronectin (0.1 mg/ml), laminin (0.1 mg/ml), and lactose (10 and 100 mM) pretreatment reduced the bacterial
cell adhesion to laminin but had no effect on adhesion to fibronectin.
When the proteinase activity of B. cereus OH599 was
inhibited by 100 mM benzamidine, the adherence to fibronectin was
reduced by 69% and an increase was detected in the adhesion to laminin
(Table 3).
| |
DISCUSSION |
Crystalline cell surface protein, S-layer, has been detected in
several bacterial species isolated from infections of the oral cavity
(23, 24, 30, 34). In recent years, the involvement of the
S-layer in bacterial adhesion and in bacteria-host interactions has
attracted interest due to the potential association with bacterial virulence (6, 20).
Ellar and Lundgren (14) have previously described the
presence of an S-layer on the surface of B. cereus ATCC
4342. In the present study, however, no S-layer was detected on the
cell surface of reference strains ATCC 14579T and ATCC
4342. Obviously, several years of preservation and culturing in
laboratories can suppress the S-protein expression of the reference strains (7). The S-layer of strain ATCC 4342 has earlier
been shown to have tetragonal symmetry (14). The symmetry in
our clinical isolates was periodic but not tetragonal. The observed structural differences of the S-layer protein may also result from
differences in culture conditions. The synthesis of an S-layer protein
with p6 (hexagonal) symmetry in B. stearothermophilus was
shown to be inhibited, and this protein was replaced by a new type of
S-layer protein with oblique lattice when the oxygen supply was
increased and glucose was used as the sole carbon source (38,
39).
Different collagen types and glycoproteins, such as fibronectin and
laminin, are constituents of the extracellular matrix, basement
membrane, and body fluids acting in the cell anchorage and migration as
well as in blood clot formation (15). Adherence to these
proteins and other host molecules is an important step for bacteria to
express their virulence (16, 25, 40). Recently, attachment
to laminin and fibronectin in Aeromonas salmonicida (12), to avian intestinal epithelial cells in L. acidophilus (41), and to collagen in L. crispatus (45) has been shown to require a crystalline
surface protein layer. In the present study, cells of 1-day-old
cultures of the clinical strains OH599 and OH600, both with an S-layer,
attached in high numbers to type I collagen, laminin, and fibronectin.
Three- and 6-day cultured cells of strains OH599 and OH600 showed low
or practically no ability to attach to these matrix proteins as
revealed by direct microscopy of toluidine blue-stained cells. However,
when the adherence to fibronectin and laminin was examined by the ELISA
method, we found no significant differences in binding between the 1- and 6-day-old cells of B. cereus OH599. Interestingly,
samples from parallel cell cultures examined by electron microscopy
showed that the S-layer was strongly peeling off of older cells but to
only a minor extent from 1-day-old cultures. The results suggest that
the detached crystalline surface protein from older cells adhered to
the matrix proteins, and that this material was detected by anti-S
antibodies as well as with whole-cell antibodies that were also
produced mainly against the S-protein, but not by toluidine blue
staining showing only whole cells.
Proteases of P. gingivalis and Treponema
denticola have earlier been shown to have a role in the binding of
these bacteria to fibronectin and hyaluronic acid, respectively
(18, 22, 28). B. cereus possesses a strong
pattern of proteases, including collagenolytic enzymes (32).
Binding to laminin and fibronectin was slow as only <60% of the 1-day
cultured cells of B. cereus OH599 had adhered after 30 min.
Inhibition of binding to fibronectin by 100 mM benzamidine, a
noncompetitive and noncovalent serine proteinase inhibitor, indicated
that B. cereus proteinase is involved in binding to
fibronectin. Binding to laminin was not, however, decreased but was
increased by the presence of 100 mM benzamidine. Although not shown by
the present study, it could be possible that the cell
surface-associated serine-type proteinase of B. cereus
processes the S-layer binding sites so that binding to laminin is
decreased and binding to fibronectin is enhanced.
Pretreatment of B. cereus OH599 cells with laminin,
fibronectin, lactose, or purified anti-S IgG or anti-W IgG of this
strain also had different effects on attachment to fibronectin and
laminin. Precoating the bacteria with specific antibodies did not
affect the binding to fibronectin, but the adherence to laminin
decreased by 32% when the bacteria were precoated with anti-S IgG and
by 15% when they were precoated with anti-W IgG. This indicates the involvement of the S-protein in binding to laminin. The inhibition experiments indicate that the binding of B. cereus to
fibronectin and laminin occurs by different mechanisms. The receptors
mediating adherence to fibronectin seemed to be present in the S-layer
sheets peeling off the cells from older cultures as shown by the ELISA method. Biotinylated fibronectin was also localized by electron microscopy in the S-layer by gold-labelled ExtrAvidin. It is thus possible that the proteinase involved in fibronectin binding is embedded in the S-layer. Inhibition of binding by over 90% to both
matrix glycoproteins by heat treatment of B. cereus may also indicate the involvement of protein-glycoprotein structures.
Attachment to laminin was not totally inhibited by antibody made
against S-protein, possibly because it did not block all receptors
available for binding. Extraction of surface proteins with 1% SDS and
by subsequent boiling could also lead to protein that raises an
antiserum with limited affinity to native protein.
Fibronectin or laminin pretreatment of bacteria decreased their
adhesion to laminin, but had little or no effect on the adhesion to
fibronectin. This is in accordance with observations by Dawson and
Ellen (10), who reported an enhanced binding of
fibronectin-pretreated T. denticola cells to fibronectin.
Fibronectin and laminin consist of multiple domains with distinct
binding functions (19). It is possible that these proteins
possess complementary receptors which affect the adherence to laminin
but not to fibronectin to which the attachment was mediated by a
proteolytic enzyme on the cell surface in the S-layer. It has also been
presumed that immobilized and soluble fibronectins have
differences in receptor conformations (17).
Bacterial surface properties and structures are important in the
regulation of bacterium-phagocyte interactions. Blaser et al.
(4) have reported phagocytosis resistance in
Campylobacter fetus strains possessing an S-layer. We have
earlier studied nonopsonophagocytosis of three subspecies of E. yurii, all hydrophobic and expressing S-protein. The results
indicated that the S-layer of the only resistant subspecies, E. yurii subsp. margaretiae, lacked receptors necessary
for leukocyte binding and phagocytosis (24). In the present
study, we examined the susceptibility to phagocytosis of clinical and
reference strains of B. cereus cultured for different time
periods. Cells from 1- and 6-day-old cultures of the American Type
Culture Collection reference strains were all resistant to PMN
phagocytosis without the presence of opsonizing antibodies and
complement proteins. However, young cultures of B. cereus OH599 and OH600 with an entire S-layer covering the cells were sensitive to nonopsonic PMN-phagocytosis, whereas cells from older cultures of the same strains were resistant. By TEM, sheets of S-layer
in 3- and 6-day-old cultures of strains OH599 and OH600 were seen in
contact with the PMNs, and only insignificant ingestion of the cells
was detected. When 6-day-old cultured bacterial cells were preincubated
with purified anti-S IgG or anti-W IgG, they were rapidly ingested by
the PMNs. Resistance of bacterial cells to nonopsonophagocytosis by
PMNs can in general be explained by a lack of specific receptors
mediating the binding to PMNs or by a direct inhibitory effect by the
bacteria on their function. Preincubation of PMNs with the
phagocytosis-resistant strain ATCC 14579T or OH599 from
6-day-old cultures had no inhibitory effect on the subsequent
phagocytosis of the sensitive OH599 from 1-day-old cultures. This
strongly indicates that resistance to phagocytosis of B. cereus cells from old cultures was not due to inhibition of the
PMN functions.
Cell surface hydrophobicity of the clinical strains of B. cereus increased with aging cultures. Hydrophobicity is important in nonopsonophagocytosis of several bacteria, and a high level of
hydrophobicity facilitates phagocytosis (1, 8). However, in
our earlier study with E. yurii, we found that high
hydrophobicity alone without receptor mediated-binding resulted in low
phagocytic ingestion (24). In the present study, hydrophobic
cells from older cultures were resistant whereas hydrophilic strains
from young cultures were sensitive to PMN phagocytosis. Increase of hydrophobicity and apparent loss of PMN-binding sites are probably both
results of the detachment of the S-layer in older cells.
These results indicate that a protease, evidently attached to the
S-layer, up-regulates the binding of B. cereus to
fibronectin and on the other hand down-regulates laminin binding,
reflecting matrix protein-specific actions in proteolytic virulence
properties of B. cereus. Also, changes during aging of the
cultures related to the S-layer expression and the cell wall
conformation of the clinical isolates of B. cereus are
followed not only by changes in hydrophobicity but also by changes in
the binding sites available for PMN-attachment.
| |
ACKNOWLEDGMENTS |
This study was financially supported by the Finnish Dental
Society (to A.K.) and the Academy of Finland (to K.L.).
We thank Arja Strandell for preparing the samples for electron
microscopy and Maire Holopainen for Azocoll assays.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Dentistry, P.B. 41, University of Helsinki, FIN 00014 University of
Helsinki, Helsinki, Finland. Phone: 358-9-19127354. Fax:
358-9-19127519. E-mail:
kotiranta{at}hammas.helsinki.fi.
Editor:
E. I. Tuomanen
| |
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Abstract
Introduction
