FEMS Immunology and Medical Microbiology 28 (2000) 211^218
www.fems-microbiology.org
Expression of staphylococcal protein Sbi is induced by human IgG
Lihong Zhang, Anna Rosander, Karin Jacobsson, Martin Lindberg, Lars Frykberg *
Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, 75007 Uppsala, Sweden
Received 29 December 1999 ; received in revised form 9 March 2000; accepted 10 March 2000
Abstract
Protein Sbi is an IgG- and L2 glycoprotein I-binding protein on the surface of Staphylococcus aureus. In most strains, the amount of
protein Sbi on the cell surface is very low under normal growth conditions. However, here we show that after growth in the presence of
human serum, the amount is significantly increased. In S. aureus strain 8325-4, the observed increase is concentration-dependent and the
highest level is found approximately 2 h after serum addition. The active molecule in serum was found to be IgG, which causes an increase of
surface-located protein Sbi in S. aureus strain 8325-4, in the clinical isolates tested as well as in protein A-negative mutants. Thus, the results
suggest that binding of IgG to protein Sbi upregulates protein Sbi synthesis. ß 2000 Federation of European Microbiological Societies.
Published by Elsevier Science B.V. All rights reserved.
Keywords : Protein Sbi; IgG binding ; Staphylococcus aureus; Inducible expression ; Environment-sensing receptor
1. Introduction
Staphylococcus aureus is a pathogen responsible for a
wide variety of diseases in humans and animals. The production of various extracellular proteins, such as hydrolytic enzymes and toxins, as well as cell wall-bound and
secreted proteins that interact with proteins in serum and
the extracellular matrix are important for virulence. In
vitro, the expression of most of these virulence factors is
growth phase-dependent : cell wall-associated proteins, e.g.
protein A and ¢bronectin-binding proteins, are produced
during the exponential phase while secreted proteins, e.g.
hemolysins and toxic shock syndrome toxin, are produced
post-exponentially [1]. Thus, there is a growth phase-dependent change from the production of proteins that are
important for establishing the infection and protecting
against host defence mechanisms to proteins that support
long-term survival under less favourable conditions. This
regulation has been shown to be under the control of at
least two well-characterised loci, agr and sar [2^6].
Though regulation of the expression of virulence genes
is well studied in vitro, little is known about their regulation in vivo. However, using transcriptional fusions to the
green £uorescent protein, Cheung et al. have shown that
transcription of the regulatory sar loci di¡ers in vitro and
* Corresponding author. Tel. : +46 (18) 67 32 99;
Fax: +46 (18) 67 33 92; E-mail : [email protected]
in vivo [7]. Recently, in vivo expression technology [8] and
signature-tagged mutagenesis [9] have been applied to S.
aureus to identify genes that are speci¢cally expressed, or
are important for survival, in the host [10^12].
We have previously reported on the identi¢cation of
protein Sbi, a cell surface protein in S. aureus that binds
both IgG and L2 -glycoprotein I (L2 -GPI) (Fig. 1) [13^15].
Screening of a number of S. aureus strains for the presence
of the sbi gene and its protein product showed that the
gene is present in almost all strains tested but its expression level in most strains is very low [15]. We therefore set
out to investigate whether expression might be increased
under certain growth conditions. Our hypothesis was that
sbi gene expression might be induced when the bacteria
invade the host. Since the growth of bacteria in serum
can be considered similar to the in vivo situation, we included human serum in the growth medium. This treatment resulted in a signi¢cant increase in the amount of
protein Sbi at the cell surface.
2. Materials and methods
2.1. Bacterial strains and biochemicals
The bacterial strains used are listed in Table 1. Staphylococcal strains were grown in tryptic soy broth (TSB),
when required supplemented with human serum or IgG.
The human IgG was from Sigma. The human serum
0928-8244 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 8 - 8 2 4 4 ( 0 0 ) 0 0 1 5 8 - 9
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L. Zhang et al. / FEMS Immunology and Medical Microbiology 28 (2000) 211^218
Fig. 1. Schematic drawing of staphylococcal protein Sbi aligned with
the recombinant polypeptides used in this study. S = signal peptide,
IgG = IgG-binding region, P.IgG = putative IgG-binding domain, B2 GPI = L2-glycoprotein I-binding region and Pro = proline-rich repeat region.
was from Uppsala University Hospital and the chicken
serum from ICN0 Biomedicals.
2.2. Antibodies
To avoid non-speci¢c binding of antibodies to protein A
or protein Sbi, antibodies developed in chicken were used
throughout the study except for the secondary antibody
used in the £uorescence microscopy-study. Antibodies
against recombinant protein Sbi were obtained through
Immunsystem AB [15]. After growth in the presence of
serum, IgG and L2 -GPI are bound by protein Sbi. Therefore, in most serum induction assays, antibodies against
the full-length protein were used to minimise the blocking
e¡ect of bound IgG and L2 -GPI. These antibodies were
a¤nity-puri¢ed on immobilised Mal-Sbi, i.e. amino acids
33^436 fused to the Mal protein [14] (Fig. 1). To remove
any cross-reactivity to protein A, the antibody preparation
was passed over protein A-Sepharose (Pharmacia Biotech).
For the IgG induction assays, antibodies against the L2 GPI-binding domain were used since this domain should
not be blocked by bound IgG. For this purpose, a polypeptide, L2G (Fig. 1), consisting of amino acids 145^267
in protein Sbi, was expressed and puri¢ed using the IMPACT-system (New England BioLabs) according to the
manufacturer's instructions and used for the a¤nity puri¢cation of antibodies (K-L2G). Both antibodies were 125 Ilabelled using the IODO-BEADS Iodination Reagent Kit
(Pierce) according to the manufacturer's instructions.
For immuno£uorescence studies an FITC-labelled
mouse monoclonal antibody against chicken light chains
(Sigma) was used.
For the Western blot experiment, horseradish-peroxidase (HRP)-labelled human IgG and K-L2G antibodies,
respectively, were used.
2.3. Binding assay
Unless otherwise stated, the following procedure was
used in all binding assays. Overnight cultures of bacteria
were diluted to OD600 = 0.2 in TSB and, when required,
serum or IgG was added to ¢nal concentrations as indicated in Figs. 2^7. The cultures were incubated for 2 h on
a shaker at 37³C. For the binding assay, an amount of
cells corresponding to 1 ml of cells at OD600 = 1 (approximately 109 cfu) were collected by centrifugation. To release serum components bound during growth, pellets
were washed once in 0.25 M acetic acid (pH 2.8) and
once in phosphate bu¡ered saline (PBS) with 0.05% Tween
20 and 0.1% bovine serum albumin (BSA) (PBS-T-BSA).
The cells were then resuspended in 400 Wl PBS-T-BSA and
incubated with 100 Wl 125 I-labelled antibodies (K-Mal-Sbi:
approximately 100 000 cpm, speci¢c activity 1.2U106 cpm
Wg31 or K-L2G: approximately 30 000 cpm, speci¢c activity 1.4U106 cpm Wg31 ) for 1 h at room temperature. Finally, the cells were washed twice in PBS with 0.05%
Tween 20 (PBS-T) and the binding of iodine-labelled antibodies against protein Sbi was measured in an LKB gamma counter.
To investigate the time dependence of Sbi induction,
10% human serum or 0.1 mg IgG ml31 was used and
samples were collected at intervals as shown in Figs. 2B
and 4B. For determining the e¡ect of di¡erent concentrations of human serum and IgG on Sbi synthesis, 0.1^20%
serum or 0.01^1 mg IgG ml31 was used. In most studies,
Staphylococcus epidermidis strain 247 was included as a
negative control.
2.4. Fluorescence microscopy
S. aureus 8325-4 were grown with or without IgG (0.1
mg ml31 ) for 2 h and washed as described above. The
bacteria were incubated with K-L2G antibodies for 1 h,
washed twice and further incubated with an FITC-labelled
K-chicken antibody for 1 h. After washing, £uorescence
was visualised using a Zeiss Axioskop with a standard
100U magni¢cation lens and a Zeiss MC63 photosystem.
2.5. Inhibition study
Ig-L (amino acids 32^267, i.e. the IgG- and L2 -GPIbinding domains of protein Sbi, see Fig. 1) were expressed
and puri¢ed using the IMPACT-system. Ig-L was serially
diluted to ¢nal concentrations of 1^50 Wg ml31 and pre-
Table 1
Staphylococcal strains used in this study
Strain
S. aureus
8325-4
DU5723
Cowan 1
GH34
GH3401
ClI1-5
Characteristics
Refs.
NCTC 8325 cured of prophages
Protein A-negative mutant of 8325-4
NCTC 8350, high in protein A production
MRSA clinical isolate
Spontaneous mutant of GH34 that has lost the
mec and spa genes
Human clinical isolates from Bertil Christensson,
University of Lund
[21]
[22]
[23]
[24]
[24]
S. epidermidis
247
Negative control
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[25]
L. Zhang et al. / FEMS Immunology and Medical Microbiology 28 (2000) 211^218
213
Fig. 2. A: The e¡ect of di¡erent concentrations of human serum on the amount of protein Sbi in S. aureus strain 8325-4 after growth for 2 h. The
twitch in the curve between 0.5 and 1% serum was reproducibly observed also in other experiments. B: Accumulation of protein Sbi at the cell surface
over time. Cultures of S. aureus strain 8325-4 were grown in TSB alone or TSB supplied with 10% human serum. Samples were collected immediately
after serum addition (time zero) and at the times indicated in the ¢gure. In both experiments, protein Sbi on the cell surface was detected with iodinelabelled K-Mal-Sbi antibodies. All numbers are mean values of duplicate samples in one typical experiment.
incubated with the IgG (10 Wg ml31 ) in the culture
medium for 10 min prior to addition to the bacteria.
Apart from this, the assay was carried out as described
above.
2.6. Removal of IgG from human serum
Human serum was passed twice over a protein GSepharose column (Pharmacia Biotech) to remove the
Fig. 3. The e¡ect of human and chicken serum on the amount of cell
surface located protein Sbi. S. aureus strain 8325-4 was cultured in TSB
or TSB supplied with 10% serum. Western blot of cell lysates detected
with (a) HRP-labelled K-L2G antibodies and (b) HRP-labelled human
IgG. Cell lysates of staphylococci grown in lane 1, TSB with chicken serum ; lane 2, TSB with human serum and lane 3, TSB alone. Size
markers are shown on the left.
IgG. Bound IgG was eluted in 0.1 M glycine (pH 3.0)
and neutralised with 0.1 M Tris (pH 8.2). The concentration was determined spectrophotometrically and, for
the reconstitution experiment, puri¢ed IgG was added
to the IgG-depleted serum to a ¢nal concentration of
10 mg ml31 .
2.7. Western blot
For Western blot analysis, proteins were released from
S. aureus by boiling in sample bu¡er. Protein was prepared from cultures of S. aureus 8325-4 grown in TSB
or in TSB supplemented with 10% human or chicken serum. An overnight culture of S. aureus strain 8325-4 was
diluted to OD600 = 0.5 and cultured at 37³C for 2 h, after
which an amount of cells corresponding to 10 ml of
OD600 = 1 (approximately 1010 cells), were collected. Pellets were washed once in 0.25 M acetic acid (pH 2.8), once
in PBS and then resuspended in 20 Wl PBS. After addition
of 20 Wl 2Usample bu¡er (1Usample bu¡er = 62.5 mM
Tris^HCl pH 6.8, 10% glycerol, 2% SDS, 5% L-mercaptoethanol and 0.01% bromophenol blue), samples were
boiled for 3 min and centrifuged at 14 000 rpm for
15 min. Supernatants were collected and analysed by
SDS^PAGE using the Phast-system (Pharmacia Biotech)
with PhastGel Gradient 8^25% gels and PhastGel SDS
Bu¡er Strips. Proteins were blotted onto nitrocellulose ¢lters. The presence of protein Sbi was detected using HRPlabelled antibodies against amino acids 145^267 of protein
Sbi (L2G) and HRP-labelled human IgG. Bound antibodies were detected with 4-chloro-1-naphthol.
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L. Zhang et al. / FEMS Immunology and Medical Microbiology 28 (2000) 211^218
Fig. 4. A: The e¡ect of di¡erent concentrations of human IgG on the amount of protein Sbi in S. aureus strain 8325-4 after growth for 2 h. B: Accumulation of protein Sbi at the cell surface over time. Cultures of S. aureus strain 8325-4 were grown in TSB alone or TSB supplied with IgG (0.1 mg
ml31 ). Samples were collected immediately after IgG addition (time zero) and at the times indicated in the ¢gure. C: Expression of protein Sbi visualised with anti-L2G antibodies and an FITC-labelled secondary antibody. Left: S. aureus grown in TSB and right: S. aureus grown in TSB supplied
with IgG, as seen in visual light (top) and UV-light (bottom). In A and B, protein Sbi was detected with iodine-labelled K-L2G antibodies. The numbers
are mean values of duplicate samples in one typical experiment.
3. Results
3.1. Human serum causes an increase of protein Sbi at the
cell surface
Fig. 2A shows the e¡ect of di¡erent concentrations of
human serum on protein Sbi synthesis in strain 8325-4.
Already at low serum concentrations there is a signi¢cant
increase in the amount of protein Sbi on the cell surface.
However, an increase in the serum concentration to more
than 10% did not signi¢cantly increase the abundance of
Sbi. Therefore, 10% serum in TSB was used in subsequent
experiments.
The e¡ect of serum in the growth medium of S. aureus
was seen within 0.5 h after addition. However, the highest
amount of Sbi proteins on the bacterial surface was de-
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L. Zhang et al. / FEMS Immunology and Medical Microbiology 28 (2000) 211^218
Fig. 5. Human serum depleted of IgG does not cause an accumulation
of protein Sbi at the cell surface. S. aureus strain 8325-4 was cultured
for 2 h in TSB, or TSB supplied with human serum, serum depleted of
IgG by protein G chromatography, depleted serum restored with IgG
or puri¢ed IgG. The amount of protein Sbi at the cell surface was detected with iodine-labelled K-L2G antibodies. The numbers are mean
values of duplicate samples in one experiment.
tected after 2 h (Fig. 2B). In a culture without serum, only
a small increase in Sbi levels was observed at the transition
from lag to early exponential growth phase.
To investigate whether more general factors, such as
concentration of salt, metal ions or a factor present in
all sera, were responsible for the increased amount of
Sbi, the e¡ect of chicken serum was studied. While human
serum was able to increase the amount of Sbi (from
811 þ 58 cpm in TSB to 8037 þ 353 cpm in the presence
of 10% serum), chicken serum had no e¡ect (466 þ 108
cpm). This strongly suggests that one or several components in human serum interact with S. aureus and cause
the induction. These components may either be absent in
chicken serum or may be present as species-speci¢c variants that fail to interact with S. aureus.
To con¢rm that the amount of protein Sbi is increased
when the bacteria are grown in the presence of serum,
proteins were released from the cells by boiling in sample
bu¡er, a procedure that does not release protein A [14].
Protein Sbi was detected both with anti-L2G antibodies
(Fig. 3a) and by its ability to bind human IgG (Fig. 3b).
Again, the results clearly show that the amount of Sbi at
the cell surface is increased in the presence of human serum but not by chicken serum.
3.2. Human IgG is the active molecule in serum
The increase in protein Sbi synthesis after addition of
serum to the growth medium could either be mediated by
215
protein Sbi itself or another surface component(s). If the
induction is mediated by protein Sbi, serum proteins
bound by protein Sbi should be responsible for the induction. As shown in Fig. 4A, a very low concentration of
human IgG (0.01 mg ml31 ) in the growth medium resulted
in a highly elevated level of protein Sbi at the bacterial cell
surface. Increasing the IgG concentration above 0.1 mg
ml31 did not seem to further increase the amount of protein Sbi. Thus, the staphylococci respond to IgG concentrations considerably lower than those found in blood (5^
15 mg ml31 ).
Similar to the serum-induced culture, the highest
amount of protein Sbi was found 1.5^2 h after IgG addition (Fig. 4B). After 10 h, an increased amount of protein
Sbi, compared to the non-induced culture, was observed in
the serum- but not in the IgG-induced culture.
As shown in Fig. 4C, using K-L2G antibodies and an
FITC-labelled secondary antibody, almost no £uorescence
was seen when the bacteria were grown in TSB alone.
However, after growth in the presence of IgG, all cells
were intensely £uorescent. The secondary antibody alone
did not give any visible signal under either of the growth
conditions (data not shown).
To further con¢rm that IgG is responsible for the observed increase in protein Sbi, human serum was passed
over a protein G column to remove the IgG. This completely removed the inducing ability in the serum while the
puri¢ed IgG strongly induced the synthesis of protein Sbi.
When the depleted serum was reconstituted with puri¢ed
Fig. 6. Recombinant protein Sbi (Ig-L domain) inhibits the IgG induction of protein Sbi. S. aureus strain 8325-4 was cultured for 2 h in TSB
supplied with 10 Wg ml31 IgG that had been preincubated with various
concentrations of Ig-L or human serum albumin. b = S. aureus strain
8325-4 cultured in TSB alone. The amount of protein Sbi at the cell
surface was detected with iodine-labelled K-L2G antibodies. The numbers are mean values of duplicate samples in one typical experiment.
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L. Zhang et al. / FEMS Immunology and Medical Microbiology 28 (2000) 211^218
protein Sbi synthesis in response to the binding of IgG to
protein Sbi.
3.4. Induction of protein Sbi synthesis in di¡erent strains
Fig. 7. The e¡ect of human IgG on the amount of protein Sbi on the
cell surface in di¡erent strains of S. aureus. Protein Sbi on the cell surface was detected with iodine-labelled K-L2G antibodies. The numbers
are mean values of duplicate samples from one experiment. S. e 247 =
S. epidermidis, included as a negative control.
IgG to a ¢nal concentration of 10 mg ml31 serum, the
inducing ability was restored (Fig. 5). Bacteria grown in
the presence of the same amount of puri¢ed IgG as is
present in the restored serum, bound twice as high
amounts of K-L2G antibodies. Most likely, parts of the
binding sites for K-L2G antibodies on the Sbi protein,
are blocked by L2 -GPI. The fact that IgG-depleted serum
does not induce Sbi synthesis indicates that human L2 -GPI
is unable to cause an increase of protein Sbi despite its
binding to protein Sbi.
3.3. Recombinant protein Sbi can inhibit the induction of
protein Sbi synthesis
To further study the induction process, Ig-L, i.e. the
IgG- and L2 -GPI-binding part of protein Sbi, was puri¢ed.
At an IgG concentration of 10 Wg ml31 , the induction of
protein Sbi synthesis could be inhibited by Ig-L in a concentration-dependent manner, whereas human serum albumin has no e¡ect (Fig. 6). This shows that the IgG must
be able to bind to protein Sbi on the cell surface to cause
the accumulation of Sbi. The most likely explanation for
the accumulation is an increased expression of Sbi. Two
alternative explanations are conceivable: (i) binding of extracellular protein Sbi from the supernatant via IgG as a
bridging molecule or (ii) a transient repression of a protease that degrades protein Sbi in the absence of IgG. In
both cases, an increased amount of protein Sbi at the cell
surface would be expected after the addition of Ig-L. Instead, Ig-L causes a complete inhibition of the IgG-mediated induction of Sbi. Furthermore, no protein Sbi can be
detected in the supernatant (our unpublished observation).
Taken together, this suggests that there is an increase in
Of the strains tested, 8325-4 shows the highest induction
which, after subtracting the background value obtained
with S. epidermidis, was found to be approximately ten
times higher in the culture with IgG compared to the
non-induced (Fig. 7). Furthermore, protein Sbi synthesis
was induced by human IgG in all clinical isolates included
in the study, but at levels varying between strains.
Strain DU5723, a protein A-negative mutant of 8325-4,
binds slightly more K-L2G antibodies than the parent
strain when grown in TSB, but Sbi synthesis is induced
to a similar level as in the parent strain in the presence of
IgG. Also in strain GH3401, a spontaneous protein A
deletion mutant of strain GH34, protein Sbi synthesis is
induced by IgG but to a level lower than in the parent
strain. These results clearly preclude that binding of IgG
to protein A could induce Sbi synthesis.
4. Discussion
The results presented here show that the level of protein
Sbi on the surface of cells grown in TSB is very low, but
increases signi¢cantly after growth in the presence of human serum. The component in serum responsible for the
induction of protein Sbi synthesis has been identi¢ed as
IgG. Induction of protein Sbi synthesis was transient and
peaked approximately 2 h after the addition of serum or
IgG. At this time, all cells had high amounts of Sbi at the
cell surface (Fig. 4C). Two hours later the abundance of
protein Sbi was reduced in all cells (data not shown). The
decline in expression was not due to ligand limitation since
it could not be prevented by the addition of more serum
(data not shown). These results suggest a co-ordinated
process, which after initiation, is not dependent on ligand
concentration.
Serum depleted of IgG had lost all inducing capacity,
but it was restored by the addition of puri¢ed IgG. This
suggests that the second human serum protein bound by
protein Sbi, L2 -GPI, cannot induce protein Sbi synthesis.
The increased amount of Sbi at the cell surface will however lead to an increased capacity to bind L2 -GPI, which
as such may be important for virulence. Although the biological function of L2 -GPI is not clear, it is interesting to
note that it is structurally related to regulators of complement system [16]. L2 -GPI has been reported to have several biological functions, such as promoting clearance of
liposomes and possibly foreign particles from the bloodstream [17] and inhibiting blood coagulation [18].
The induction seems to be a general mechanism in
S. aureus since all clinical isolates tested respond to IgG
in the growth medium. Protein A is not involved in the
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L. Zhang et al. / FEMS Immunology and Medical Microbiology 28 (2000) 211^218
induction, instead binding of IgG to protein Sbi appears
to be required. Cleary and Retnoningrum have proposed
that streptococcal immunoglobulin-binding proteins
(IGPs) such as M-proteins, may act as sensory proteins
[19]. In their model, a conformational change is induced
upon binding of plasma proteins to these IGPs which, by
phosphorylation, could transmit a signal resulting in the
repression or induction of other genes. However, this
would require the involvement of a second intracellular
protein considering that these IGPs lack a cytoplasmic
tail ; they become cleaved at the LPXTG motif and are
anchored to the cell wall [20]. In this context, it is interesting to note that Sbi di¡ers from typical S. aureus adhesins.
It contains a repetitive proline-rich region, which is a characteristic feature of a cell wall-spanning domain. However,
this proline-rich domain is not localised at the C-terminal
end. Instead, the protein extends another 134 amino acids
and, furthermore, lacks the cell wall-anchoring LPXTG
motif. Therefore, protein Sbi is not expected to be processed proteolytically. Although no predicted membranespanning region is found, the C-terminal part may be localised in the cytoplasm of the cell, or may transmit a
signal by interacting with a transmembrane protein. Interestingly, the amino acid composition of the C-terminal
part of Sbi di¡ers from the rest of the protein in that it
is rich in tyrosine and threonine, amino acids known to be
potential targets for phosphorylation.
It is possible that IgG only induces expression of Sbi,
thus increasing the bacterial binding of L2 -GPI. A more
attractive hypothesis is that the induction of Sbi is one of
the ¢rst responses after entry into the host and this could
then promote altered expression of other genes, ultimately
resulting in adaptive changes appropriate for life in a
di¡erent environment. Hence, we propose that protein
Sbi is an environment-sensing receptor by which the bacteria can sense the entry into and/or localisation within the
host.
Acknowledgements
We thank Dr Bertil Christensson, University of Lund,
Sweden, for supplying the clinical isolates and Professor
Timothy Foster, University of Dublin, Ireland, for
DU5723, GH34 and GH3401. The helpful comments on
the manuscript provided by Professor Gerhardt Wagner
are gratefully acknowledged. This study was supported
by grants from the Swedish Medical Research Council
(16X-03778), the Swedish Council for Engineering Sciences (96-759) and from Carl Tryggers Foundation.
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