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Infection and Immunity, May 2001, p. 2996-3003, Vol. 69, No. 5
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.5.2996-3003.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Subinhibitory Clindamycin Differentially Inhibits
Transcription of Exoprotein Genes in Staphylococcus
aureus
Silvia
Herbert,
Peter
Barry, and
Richard P.
Novick*
Program in Molecular Pathogenesis, Skirball
Institute, and Department of Microbiology, New York University
School of Medicine, New York, New York 10016
Received 14 August 2000/Returned for modification 4 October
2000/Accepted 29 January 2001
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ABSTRACT |
It has long been known that certain antibiotics, at
subinhibitory concentrations, differentially inhibit the synthesis of 
-hemolysin and other staphylococcal virulence factors. In this report, we show that subinhibitory clindamycin (SBCL) eliminates production of nearly all exoproteins by Staphylococcus
aureus but has virtually no effect on cytoplasmic proteins. The
effect was abolished by a gene conferring resistance to
macrolides-lincosamides-streptogramin B, showing that differential
inhibition of protein synthesis is responsible; remarkably, however,
subinhibitory clindamycin blocked production of several of the
individual exoprotein genes, including spa
(encoding protein A), hla (encoding -hemolysin), and
spr (encoding serine protease), at the level of
transcription, suggesting that the primary effect must be
differential inhibition of the synthesis of one or more regulatory
proteins. In contrast to earlier reports, however, we found that
subinhibitory clindamycin stimulates synthesis of coagulase and
fibronectin binding protein B, also at the level of transcription.
agr and sar expression was minimally affected
by subinhibitory clindamycin. These effects varied from strain to
strain and do not seem to be responsible for the effects of
subinhibitory clindamycin on the overall exoprotein pattern.
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INTRODUCTION |
The pathogenicity of
Staphylococcus aureus, like that of other gram-positive
bacteria, depends largely on extracellular virulence factors, including
both secreted and surface proteins. These factors may be regarded as
accessory gene products, constituting a subset of proteins that are not
required for growth and cell division under ordinary conditions but
that enable the organism to adapt to special environmental conditions,
including various types of stress as well as the presence of particular
nutritional substrates. On one hand, the production of virulence
factors and other accessory gene products is carefully regulated, so
that the various genes are expressed in response to explicit
environmental contingencies. This regulation is accomplished by a
network of interlocking regulatory gene functions, often involving
signal transduction pathways that monitor the local environment. On the
other hand, the synthesis of these products is differentially
suppressed by certain antibiotics at low concentrations that have
little if any effect on overall growth. This suppression has been
reported to include secreted proteins such as - and 
-hemolysins,
DNase, lipase, coagulase, and toxic shock syndrome toxin 1 (TSST-1)
(6, 13). Such effects have also been seen with
Streptococcus pyogenes and Bacteroides fragilis,
as well as with staphylococci (9, 10, 37). Similar though
less fully documented effects are seen in response to the limitation of
essential amino acids (18). We note that suppression of
exoprotein synthesis is seen only with antibiotics that block protein synthesis; those that affect the cell envelope, such as 
-lactams and glycopeptides, have a stimulatory effect on the synthesis of most exoproteins (11, 13, 30).
It is interesting that this decrease of exoprotein expression
causes a significant attenuation of virulence. For example, the
decrease of protein A on the cell surface of S. aureus
resulted in a greater number of free receptor sites for complement C3b and an increase in phagocytosis (7, 8, 22). Reduction of
adherence to bone surfaces has also been observed (21),
and pretreatment with subinhibitory clindamycin (SBCL) or lincomycin caused a significant reduction in the severity of skin lesions in a
murine subcutaneous abscess model (5, 35). Though it may
seem strange that SBCL would attenuate virulence, one should view the
virulence factors as a subset of extracellular proteins; perhaps
suppression of exoprotein production by low concentrations of
antibiotics could be an adaptive response that evolved in relation to
soil ecology long before the development of antibiotics for clinical
use. Moreover, the environmental concentration of any antibiotic would
have been much lower than that currently encountered by the bacteria in
a typical clinical setting. In our view, the effects of low
concentrations of antibiotics such as clindamycin are likely to reflect
important principles governing the regulation of genes encoding
extracellular proteins. Accordingly, we have begun to analyze the
molecular basis of the response of S. aureus to SBCL. We
find first that with a few exceptions, SBCL essentially eliminates the
production of secreted staphylococcal proteins and stimulates the
production of certain surface proteins but has very little effect on
the production of cytoplasmic proteins. The effect is eliminated by a
standard macrolide-lincosamide-streptogramin B (MLS) resistance gene,
indicating that the basic biological activity of clindamycin,
inhibition of protein synthesis, is responsible for the observed
effects. Remarkably, however, the individual exoprotein genes
are regulated at the transcriptional level. This suggests that the
effects are mediated through one or more regulatory factors rather than
by direct translational regulation of individual exoproteins.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
The bacterial
strains used in this study are listed in Table
1. NCTC8325 is the propagating strain for
phage 47 of the international phage typing set. It is a wild-type
agr+ strain with a naturally occurring
11-nucleotide deletion in rsbU (14). RN12 is a
derivative of NCTC8325 with a silent insertion of Tn551,
conferring erythromycin resistance (26). RN9203 and RN9204
were obtained by phage transduction (80 or ø11) of plasmids pRN6832
(Phla::blaZ) and pRN6676
(Ptst::blaZ) to NCTC8325.
In these plasmids, the -hemolysin (hla) and TSST-1 (tst) promoters are transcriptionally fused to the
promoterless -lactamase (bla) gene in the vector plasmid
pSA3800 (13). RN9202 is NCTC8325 carrying pRN6683
(agrP3::blaZ), where the accessory gene
regulator (agr) P3 promoter, driving rnaiii, is
fused to the -lactamase gene (29). RN6911 is an
agr-null strain, with a 3.3-kb deletion of the
agr locus, replaced by a tetracycline resistance gene,
tetM (29). RN9211 is RN6911 carrying pRN6788 (Pspa::blaZ), where the
protein A (spa) promoter is fused to the -lactamase
gene (13). RN9398 (agr null) was obtained by
transduction of the agr knockout from RN6911 into NCTC8325, using 80. RN9205 is NCTC8325 containing staphylococcal
pathogenicity island 1 (SaPl1) and expressing tst,
constructed by phage transduction (80) (19). COL and
WCUH29 (24) are clinical S. aureus isolates. COL is a prototypical methicillin-resistant S. aureus (MRSA)
strain whose genome is currently being sequenced and analyzed
(www.tigr.org). The genes and proteins involved in this work are
described in Table 2.
Stock cultures were maintained in CYGP broth (
25) at
70°C and cultivated overnight on GL agar (
23),
supplemented with
antibiotics as required, for use in growth studies.
In all flask
cultures, CYGP broth was used without glucose, to
eliminate the
effects of catabolite repression on exoprotein
synthesis. Thirty
milliliters of CYGP or CYGP with subinhibitory
clindamycin hydrochloride
(0.02 or 0.04 µg/ml; Sigma) in 300-ml side
arm flasks was inoculated
with
S. aureus to a density of
~3 × 10
7 cells/ml and incubated with shaking (240 rpm) at 37°C. Growth
was monitored turbidimetrically with a
Klett-Summerson photoelectric
colorimeter read at 540 nm. A Klett
reading of 100 is equivalent
to 3 × 10
8 cells/ml.
Cultures were sampled at hourly intervals from 0 to
6 h (referred
to as
T = 0 to
T = 6).
Protein analysis.
Supernatants obtained by centrifugation of
postexponential cultures (T = 5 and T = 6) were analyzed for exoproteins. Bacteria obtained by
centrifugation of late exponential cultures (T = 3 and
T = 4) were used for the analysis of cellular proteins.
Cell pellets were washed with Tris-EDTA buffer and lysed with
lysostaphin. Samples were normalized to constant cell density and then
prepared for analysis by sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis as described by Laemmli (17), using
SDS-12% polyacrylamide gels. Gels were stained with Coomassie
brilliant blue and photographed. Coagulase was assayed by the standards slide agglutination method. -Lactamase activity was determined as
described by O'Callaghan et al. (29a), modified for use
in a microtiter plate reader (12). Samples were normalized
to constant cell density unless otherwise specified.
Whole-cell lysates and Northern blotting.
Bacteria were
collected at the indicated time points (T = 0 to
T = 6), and whole-cell lysates were prepared as
described by Kornblum et al. (15). Cultures were
centrifuged, fixed in acetone-ethanol (1:1), and washed in
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid
(TES)-sucrose buffer. Equalized cell samples were incubated on ice for
30 min with lysostaphin (150 µg/ml) in TES-sucrose buffer (20%
[wt/vol] sucrose, 20 mM Tris [pH 7.6], 10 mM EDTA, 50 mM NaCl) and
shaken for 1 h with proteinase K (50 µg/ml; Sigma) and 2% SDS
at 4°C. For Northern blotting (40), the same amount of
cell lysate (10 µl) was electrophoresed through a 0.66 M
formaldehyde-1% agarose gel in MOPS (morpholinepropanesulfonic acid)
buffer (20). Nucleic acids were transferred to a
nitrocellulose membrane (Amersham) with a VacuGene apparatus
(Pharmacia) in 20× SSC (3 M NaCl, 0.3 M sodium citrate [pH 7]) and
fixed under UV light. The membrane was preincubated for 2 h at
52°C in 2× Denhardt's solution (0.02% bovine serum albumin, 0.02%
Ficoll, 0.02% polyvinylpyrrolidone, 0.05 M EDTA [pH 8], 0.2% SDS,
5× SSC, with sonicated and heat-denatured salmon sperm DNA [100
µg/ml]) and 10% (wt/vol) dextran (Sigma) and then hybridized
overnight with a 32P-labeled DNA probe in hybridization
solution. The 32P-labeled DNA probes were prepared by PCR
with gene-specific primers (Table 3). The
blot was exposed to a storage phosphor screen (Molecular Dynamics) and
quantitated with ImageQuant or NIH Image software. Quantitative values
for NCTC8325 were normalized to corresponding values for 16S rRNA,
obtained using a 16S-specific PCR product as the probe.
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RESULTS |
SBCL affects the synthesis of exoproteins but not
cytoplasmic proteins.
Although many antibiotics that inhibit
protein synthesis have been observed to suppress the synthesis of
virulence factors at subinhibitory concentrations, we have studied
primarily the effects of clindamycin, because it seems to have the
greatest effect (29; unpublished data) and because its
effects were seen at concentrations that had little or no effect on
growth. A general picture of the effects of SBCL is seen in Fig.
1 and 2.
Clindamycin at a concentration of 0.02 µg/ml had a very slight effect
(<5%) on the exponential doubling time of NCTC8325 and some of its
derivatives (Fig. 1 and 3D), for others,
there was no effect (not shown). Additionally, all of the strains
tested multiplied and formed colonies of normal size on agar containing
clindamycin at 0.02 or 0.04 µg/ml. This is typical of results
obtained by other investigators (33). Figure 2A shows the
exoprotein profiles of a postexponential culture of NCTC8325
after growth in the presence (lanes 3 and 4) or absence (lanes 1 and 2)
of clindamycin at a concentration of 0.02 µg/ml. As can be seen,
there was a dramatic inhibition by SBCL of general exoprotein
synthesis, in general agreement with earlier reports on the
effects of SBCL on specific exoproteins, including - and
-hemolysins, lipase, DNase, and TSST-1 (4, 13, 30, 32,
34). Given that NCTC8325 has a deletion in rsbU
(16), which encodes an anti-anti-
B factor
that could affect the clindamycin response (S. Herbert, B. Peter, and
R. P. Novick, unpublished data; see below), we analyzed two
rsbU+ clinical S. aureus isolates,
WCUH29 and the MRSA strain COL, as well as NCTC8325. Similar, though
less dramatic, results were obtained with both WCUH29 (Fig. 2B) and COL
(data not shown). An example of an exoprotein whose synthesis
is not blocked by SBCL is -lactamase (Fig. 3D; see below). This is
consistent with the possibility that the effects of SBCL are mediated
through specific pleiotropic regulatory proteins (see below), since
-lactamase is regulated independently of other extracellular
proteins.
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FIG. 2.
Effect of SBCL on exocellular and cellular protein
expression. S. aureus strains NCTC8325 (A and C) and WCUH29
(B) were incubated with CYGP (A, lanes 1 and 2; B, lane 1; C, lanes 1 and 2) and CYGP with SBCL (0.02 µg/ml) (A, lanes 3 and 4; B, lane 2;
C, lanes 3 and 4). M, molecular size markers; Hla, -hemolysin (1 µg, 33 kDa). (A) Exoproteins in NCTC8325 culture supernatants; (B)
exoproteins in WCUH29 culture supernatants; (C) cellular
(cytoplasmic and membrane-bound) proteins of NCTC8325. (D) Effect of
MLS resistance. RN12 was grown in CYGP with (lane 2) or without (lane
1) SBCL (0.02 µg/ml). Supernatants from 5-h cultures were analyzed as
above.
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FIG. 3.
Effect of SBCL on exoprotein gene transcription
(gene fusion analysis). Cultures of strains containing
plasmid-carried exoprotein gene promoter-blaZ
fusions were grown with or without SBCL and assayed for -lactamase
activity at hourly intervals. (A) Phla-blaZ
(RN9203); (B) Ptst-blaZ (RN9204); (C)
Pspa-blaZ (RN9211). (D) Strain RN0024,
containing plasmid pRN3038, a derivative of pl258 that produces
-lactamase constitutively. Here, the absolute -lactamase values
were plotted (linear scale), not normalized to cell density, in
comparison to growth of the culture (semilog scale).
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In striking contrast to these results, we find that the profiles of
cellular (cytoplasmic and membrane-bound) proteins with
or without
clindamycin are virtually indistinguishable (Fig.
2C,
lanes 3 and 4 versus lanes 1 and 2), with the exception of two
protein bands
that were increased in the presence of SBCL (see
below). This is
consistent with the observed normal growth of
the bacteria. Thus, it is
clear that SBCL has a dramatic differential
effect on the synthesis of
secreted versus cellular proteins.
For reasons described below, we
predict that there is at least
one cytoplasmic protein whose synthesis
is inhibited by SBCL.
Current experiments are directed toward the
identification of
such
proteins.
SBCL acts by inhibiting protein synthesis.
Because the
differential effect of clindamycin on extracellular proteins was so
striking, it was important to determine whether the effect was a
consequence of the standard activity of clindamycin or of inhibition of
protein synthesis by binding to the 50S ribosomal subunit or was a
manifestation of another, novel activity of the antibiotic. To test
this, we used strain RN12, which has a silent insertion of
Tn551, carrying a classical MLS resistance determinant, in
strain NCTC8325, comparing the exoprotein profiles in the
presence and absence of SBCL. As shown in Fig. 2D, the profiles
are indistinguishable, indicating that Tn551 eliminates the
effect of SBCL on exoprotein production, thus demonstrating
that its effect on sensitive strains is, in fact, a
consequence of protein synthesis inhibition. To evaluate the
specificity of the Tn551 effect, we tested derivatives of NCTC8325 resistant to chloramphenicol and tetracycline, which also
inhibit protein synthesis. We found that these strains are as sensitive
to SBCL as NCTC8325 (data not shown), indicating that MLS resistance
specifically abolishes the clindamycin effect.
SBCL inhibits exoprotein gene transcription.
In
principle, clindamycin could affect the synthesis, secretion, or
stability of the exoproteins. The most direct way to determine the mechanism seemed to be to test for inhibition of transcription, since if transcription were blocked, the other possibilities
would automatically be eliminated. For this test, we used
transcriptional fusions to -lactamase, at clindamycin
concentrations of 0.02 µg/ml and sometimes 0.04 µg/ml, and Northern
blot hybridization analysis of the transcripts at a clindamycin
concentration of 0.02 µg/ml. As spa is expressed very
poorly in NCTC8325, owing to its down-regulation by
agr (40), we also used agr-null
derivatives, RN9211 and RN9398, for the analysis of spa. The
Phla and Ptst
-lactamase fusions (14) in strain NCTC8325 were
dramatically inhibited by SBCL at either concentration (Fig. 3A and B),
whereas SBCL had no effect on the synthesis of -lactamase encoded by
a constitutive mutant of plasmid p1258 (Fig. 3D). The Pspa -lactamase fusion in RN9211 was
also inhibited, but only partially (Fig. 3C).
To confirm the promoter fusion results, we measured the gene-specific
transcripts by Northern blot hybridization. To broaden
the study, we
included WCUH29 and three additional genes,
coa, fnbB, and
spr. Figure
4A shows Northern
blot analysis of whole-cell
RNAs prepared from NCTC8325 and probed
for
hla, spr, coa, and
fnbB and from RN9205 and
probed for
tst. In Fig.
4B is a graphical
representation of
these results after normalization to 16S rRNA,
which was detected on
the same blots by hybridization with a 16S-specific
probe (not shown).
Also shown are Northern blotting patterns for
RNA isolated from the
isogenic
agr-null strain, RN9398, and probed
for
spa (Fig.
4A) and for WCUH29 RNA probed for
hla, spr,
spa, coa, and
fbnB (Fig.
4C). In agreement with the
results of the
promoter activity tests, SBCL (0.02 µg/ml) sharply
decreased
hla and
tst mRNA transcripts in
NCTC8325. However, the effects of
clindamycin on the
spa
transcript (Fig.
4A) seem considerably
stronger than its effects on the
P
spa -lactamase fusion
(Fig.
3), whereas the
inhibition of
tst transcription was greater
as measured by
the P
tst -lactamase fusion (Fig.
3B)
than by
Northern blotting (Fig.
4A). We note that the Northern
blotting pattern
with a
spa probe was somewhat different for RN9398
than for
other
agr-null strains such as RN6911, in that the
spa transcript level remained high throughout the
postexponential
phase for RN9398, whereas it decreased considerably for
the others
(not shown). There was no difference between the two strains
in
their response to SBCL. We note also that the clindamycin inhibition
of
hla and
spr transcription was greater in
WCUH29 than in NCTC8325
(Fig.
4A and B). This is especially striking
for
spr, whose expression
was delayed rather than
blocked by clindamycin in NCTC8325.
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FIG. 4.
Effect of SBCL on exoprotein gene transcription
as analyzed by Northern blot hybridization. Cultures were grown in CYGP
with or without SBCL (0.02 µg/ml) and sampled hourly for analysis.
(A) NCTC8325 and derivatives; (B) graphs of data from panel A,
normalized to 16S RNA; (C) WCUH29.
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Transcription of
coa and
fnbB seemed to be
stimulated in NCTC8325 and WCUH29 by SBCL. The
coa and
fbnB transcription signals
seemed stronger in WCUH29
than in NCTC8325, and we suggest that
this and other differences
observed between NCTC8325 derivatives
and WCUH29 could be related to
the
rsbU deletion in the former
(
16). Because
these results are inconsistent with earlier reports
of inhibition of
coagulase production and fibronectin binding
by SBCL (
4,
31), we determined coagulase activity in culture
supernatants as
well as transcription of
coa. Since the
coa mRNA
signal was present only at the earliest time points, we measured
coagulase activity in T1-h as well as in T5-h samples. In T1-h
samples,
activity was detected at a 1:32 dilution, whereas in
the T5-h sample,
activity was detected only in the undiluted supernatants.
In neither
case was there any detectable effect of SBCL. We suggest
that the
decrease in coagulase activity may be a consequence of
increased
protease activity late in growth. We have no explanation
for the
apparent disparity between
coa transcription and coagulase
activity. Note also that, as we show elsewhere (
11),
expression
of both
coa and
fbnB is dependent on
the alternative sigma factor,
B, and it is known
that
coa has a
B-dependent promoter
(
23). Moreover, considerable interstrain
differences have
been observed for these proteins. In any case,
we suggest that the
effects of SBCL seen in Fig.
2 involve transcription
rather than
protein degradation or
secretion.
Since the primary mode of action of clindamycin is blockage of protein
synthesis at the level of the ribosome, the blockage
of
exoprotein synthesis by SBCL would have been expected to be
at
this step. It is therefore especially significant that the
low
concentrations of clindamycin used in these experiments block
transcription of the exoprotein genes rather than translation
of their products. The possibility that the primary effect is
translational and that the results shown in Fig.
4 represent mRNA
destabilization seems unlikely on the basis of the stimulation
of
certain extracellular proteins and of the gene fusion results
shown in
Fig.
3.
Effects of SBCL on global regulators.
Although the observed
effects of SBCL are clearly mediated by translational inhibition, which
is the basis of the antibiotic activity of clindamycin, the results
presented so far suggest that these effects are not at the level of
direct interference with exoprotein translation. In other
words, clindamycin must interfere with translation of one or more
regulatory gene products that, in turn, affect transcription of the
exoprotein genes. Accordingly, we have begun to address this
question by determining the effects of SBCL on agr and
sar, well-characterized global regulators of exoprotein synthesis in S. aureus. The
agr locus specifies two transcripts, RNAII (2.3 kb) and
RNAIII (514 nucleotides), of which the former determines activation of
agr transcription (28) and the latter is the
effector of agr regulation (29).
It is clear that
agr cannot be the sole mediator of the
clindamycin effect, since SBCL blocks transcription of genes such
as
hla that are up-regulated as well as genes, such as
spa, that
are reciprocally down-regulated by
agr.
As noted earlier (Fig.
3C and
4A), SBCL strongly inhibited
spa transcription in the
agr-null
strain,
confirming that its effects on
spa are independent of
agr. Nevertheless, it remained possible that SBCL
could act through
agr, at least on some of the
exoprotein genes. Accordingly, we
tested an
agrP3-
blaZ fusion in NCTC8325 for the effects of
SBCL
and also analyzed the
agr transcription patterns
directly by Northern
blotting with
agr-specific probes. As
seen in Fig.
5A, the onset
of
-lactamase synthesis is delayed by SBCL at either dose and
is
somewhat inhibited by the higher dose but not by the lower
one. The
Northern blotting patterns (0.02 µg of clindamycin/ml)
(Fig.
5B) are
fully consistent with the gene fusion results for
NCTC8325. Note that
there is a 1-h delay in both cases and that
RNAIII synthesis is roughly
parallel with and without clindamycin
thereafter. This delay was not
seen with WCUH29, and there was
even a modest stimulation of
RNAIII transcription at the early
time points. One possible explanation
for the apparent difference
in strains is that NCTC8325 and its
derivatives are mutated in
rsbU, as noted. Since
hla,
spr, and
tst transcription are inhibited
at all time
points in both strains, it is clear that the effects
of SBCL on RNAIII
cannot be responsible for the effects of the
drug on transcription of
these three genes.
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FIG. 5.
Effect of SBCL on agr and
sar expression. (A) Strain RN9202, containing a
plasmid-carried agrP3-blaZ fusion, was grown
in CYGP with or without SBCL at 0.2 or 0.4 µg/ml, and
hourly samples were assayed for -lactamase. (B)
Clm, clindamycin. Strains NCTC8325 and WCUH29 were grown as
above, with or without SBCL (0.02 µg/ml), and hourly
samples were analyzed by Northern blotting for the two major
agr transcripts, RNAII and RNAIII. (C) Strains NCTC8325,
WCUH29, and COL were grown as above, and hourly samples were analyzed
by Northern blotting for the three sar transcripts, using a
probe specific for the 3' end of the sar locus.
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The effects of SBCL on transcription of
sar in these same
two strains and also in COL are shown in Fig.
5C.
sar
is transcribed
from three tandem promoters,
P
sarA,, P
sarB,
and
P
sarC, producing three overlapping transcripts,
all
of which encode SarA, a transcriptional regulator (
1).
SBCL
stimulated each of these promoters but did so differently in each
of the three strains. The strongest effects were on
P
sarA in NCTC8325 and in WCUH29 and on
P
sarC in COL and in
WCUH29. These effects are
difficult to interpret since the roles
of the three
sar
promoters in the production of SarA have not
been clearly defined.
Since
sar strongly enhances the expression
of
agr
RNAIII (
3) and therefore contributes significantly to
the
agr response, it is clear that the general effects of SBCL
on exoprotein synthesis cannot be mediated through the effects
of
sar on
agr. Nevertheless,
sar has
effects on several exoprotein
genes independently of
agr (
2,
41), and it is conceivable
that SBCL
could act partly through
sar in some
cases.
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DISCUSSION |
It has long been known that the synthesis of many staphylococcal
exoproteins, including virulence factors, is inhibited by subinhibitory concentrations of antibiotics whose mode of action is to
block protein synthesis. The clindamycin effect has been observed for
extracellular proteins in a variety of organisms, including
streptolysin S and M protein production by Streptococcus pyogenes (9, 37) and extracellular lipase production
by Propionibacterium acnes and P. granulosum
(39). Similar effects have been observed for
tetracycline and aminoglycosides, which down-regulate protease production by Pseudomonas aeruginosa
(36) and hemolysin production by Escherichia
coli (38). In some cases, disparate results have been
reported by different investigators, which may well represent interstrain differences. In any case, we suggest that the observed effects of subinhibitory antibiotics have profound implications for the
overall regulatory strategies that are used by bacteria in their
interactions with the environment, and we have begun to study the
phenomenon with this idea in mind.
In this report, we have confirmed that the synthesis of many
exoproteins is inhibited by SBCL. We have shown that the
effects of SBCL on exoprotein synthesis are manifested as
effects on transcription of the exoprotein genes. This is
puzzling since the standard effect of clindamycin is blockage of
protein translation at the level of the ribosome. Moreover, the effects
on exoprotein gene transcription were abolished by a standard
MLS resistance determinant. The suggested explanation of this seeming
paradox is that clindamycin specifically interferes with translation of
one or more proteins that regulate transcription of the
exoprotein genes. For this to be true, translation of these
regulatory proteins would have to be differentially sensitive to SBCL.
The basis of such differential sensitivity would be an important
subject for further study. An alternative possibility is that SBCL
differentially blocks transcriptionally coupled translation of proteins
that use (membrane-bound) ribosomes dedicated to exoprotein synthesis. This possibility is ruled out by gene fusion experiments using -lactamase as a reporter, since -lactamase is an
extracellular protein whose synthesis is not blocked by SBCL.
We have shown here that agr cannot be responsible for the
clindamycin effect, although sar could conceivably have a
role with respect to certain exoproteins. We show elsewhere
that SBCL up-regulates the B operon (Herbert et al.,
unpublished), which is clearly important for the observed effects of
the drug on exoprotein synthesis.
Finally, it may be instructive to consider the biological context in
which the effects of subinhibitory antibiotics could have a role. As
has been noted, there are profound effects on virulence
as would be
expected, since many virulence factors are extracellular proteins.
However, it is suggested that the role of subinhibitory antibiotics
must be viewed within the competitive environment where the production
of antibiotics must have evolved, namely, the soil. Since antibiotic
concentrations comparable to those used therapeutically are probably
never encountered in the soil, one must assume that the evolution of
antibiotics has been driven by forces based on very low environmental,
i.e., subinhibitory, concentrations. Further, it is very difficult to
imagine that inhibition of the production of exoproteins
involved in virulence for metazoan hosts could have provided the
evolutionary driving force. Rather, it would seem instructive to
consider other types of exoproteins, namely, (i) enzymes
that degrade macromolecules and thus enable the organism to obtain
importable nutrients and (ii) bacteriocins and other antibacterials. It
is possible that other types of secondary metabolism, such as the
production of antibiotics, are also affected. Could it be that the
development of antibiotics by soil organisms has been driven by the
selective advantage gained from the inhibition by low antibiotic
concentrations of the production by competitors of degradative enzymes,
bacteriocins, or antibiotics?
| |
ACKNOWLEDGMENTS |
We thank Hope F. Ross for discussion and critical reading of the
manuscript. We also thank D. McDevitt for S. aureus strain WCUH29.
This work was supported by National Institutes of Health grant
RO1-AI30138 to R.P.N.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Program in
Molecular Pathogenesis, Skirball Institute, and Department of
Microbiology, New York University School of Medicine, 540 First Ave.,
New York, NY 10016. Phone: (212) 263-6290. Fax: (212) 263-5711. E-mail: RICHARD.NOVICK{at}MCSKA.MED.NYU.EDU.
Editor:
E. I. Tuomanen
| |
REFERENCES |
| 1.
|
Bayer, M. G.,
J. H. Heinrichs, and A. L. Cheung.
1996.
The molecular architecture of the sar locus in Staphylococcus aureus.
J. Bacteriol.
178:4563-4570[Abstract/Free Full Text].
|
| 2.
|
Blevins, J. S.,
A. F. Gillaspy,
T. M. Rechtin,
B. K. Hurlburt, and M. S. Smeltzer.
1999.
The staphylococcal accessory regulator (sar) represses transcription of the Staphylococcus aureus collagen adhesin gene (cna) in an agr-independent manner.
Mol. Microbiol.
33:317-326[CrossRef][Medline].
|
| 3.
|
Cheung, A. L.,
J. H. Heinrichs, and M. G. Bayer.
1996.
Characterization of the sar locus and its interaction with agr in Staphylococcus aureus.
J. Bacteriol.
178:418-423[Abstract/Free Full Text].
|
| 4.
|
Gemmel, C. G., and A. M. A. Shibl.
1976.
The control of toxin and enzyme biosynthesis in staphylococci by antibiotics, p. 657-664.
In
J. Jeljaszewicz (ed.), Staphylococci and staphylococal diseases. Gustav Fischer Verlag, Stuttgart, Germany.
|
| 5.
|
Gemmell, C.
1978.
Effect of subinhibitory concentrations of antibiotics on experimental pyogenic infections in mice., p. 512-514.
In
Chemotherapy.
|
| 6.
|
Gemmell, C. G.
1995.
Antibiotics and the expression of staphylococcal virulence.
J. Antimicrob. Chemother.
36:283-291[Abstract/Free Full Text].
|
| 7.
|
Gemmell, C. G.
1984.
Clindamycin and its action on the susceptibility of pathogenic bacteria to phagocytosis.
Scand. J. Infect. Dis. Suppl.
43:17-23[Medline].
|
| 8.
|
Gemmell, C. G., and A. O'Dowd.
1983.
Regulation of protein A biosynthesis in Staphylococcus aureus by certain antibiotics: its effect on phagocytosis by leukocytes.
J. Antimicrob. Chemother.
12:587-597[Abstract/Free Full Text].
|
| 9.
|
Gemmell, C. G.,
P. K. Peterson,
D. Schmeling,
Y. Kim,
J. Mathews,
L. Wannamaker, and P. G. Quie.
1981.
Potentiation of opsonization and phagocytosis of Streptococcus pyogenes following growth in the presence of clindamycin.
J. Clin. Investig.
67:1249-1256.
|
| 10.
|
Gemmell, C. G.,
P. K. Peterson,
D. Schmeling,
J. Mathews, and P. G. Quie.
1983.
Antibiotic-induced modification of Bacteroides fragilis and its susceptibility to phagocytosis by human polymorphonuclear leukocytes.
Eur. J. Clin. Microbiol.
2:327-334[CrossRef][Medline].
|
| 11.
|
Hallander, H. O.,
G. Laurell, and G. Lofstrom.
1966.
Stimulation of staphylococcal haemolysin production by low concentrations of penicillin.
Acta Pathol. Microbiol. Scand.
68:142-148[Medline].
|
| 12.
|
Ji, G.,
R. Beavis, and R. Novick.
1995.
Cell density control of staphylococcal virulence mediated by an octapeptide pheromone.
Proc. Natl. Acad. Sci. USA
92:12055-12059[Abstract/Free Full Text].
|
| 13.
|
Kobayasi, A.,
J. Barnett, and J. Sanford.
1966.
Effect of antibiotics on the in vitro production of alpha hemolysin by Staphylococcus aureus.
J. Lab. Clin. Med.
68:890.
|
| 14.
|
Kornblum, J.,
B. Kreiswirth,
S. J. Projan,
H. Ross, and R. P. Novick.
1990.
Agr: a polycistronic locus regulating exoprotein synthesis in Staphylococcus aureus, p. 373-402.
In
R. P. Novick (ed.), Molecular biology of the staphylococci. VCH Publishers, New York, N.Y.
|
| 15.
|
Kornblum, J. S.,
S. J. Projan,
S. L. Moghazeh, and R. P. Novick.
1988.
A rapid method to quantitate non-labeled RNA species in bacterial cells.
Gene
63:75-85[CrossRef][Medline].
|
| 16.
|
Kullik, I.,
P. Giachino, and T. Fuchs.
1998.
Deletion of the alternative sigma factor B in Staphylococcus aureus reveals its function as a global regulator of virulence genes.
J. Bacteriol.
180:4814-4820[Abstract/Free Full Text].
|
| 17.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[CrossRef][Medline].
|
| 18.
|
Leboeuf-Trudeau, T.,
J. deRepentigny,
R. M. Frenette, and S. Sonea.
1969.
Tryptophan metabolism and toxin formation in S. aureus Wood 46 strain.
Can. J. Microbiol.
15:1-7[Medline].
|
| 19.
|
Lindsay, J. A.,
A. Ruzin,
H. F. Ross,
N. Kurepina, and R. P. Novick.
1998.
The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus.
Mol. Microbiol.
29:527-543[CrossRef][Medline].
|
| 20.
|
Maniatis, T.,
E. F. Fritsch, and J. Sambrook.
1982.
Molecular cloning: a Laboratory Manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 21.
|
Mayberry-Carson, K. J.,
W. R. Mayberry,
B. K. Tober-Meyer,
J. W. Costerton, and D. W. Lambe, Jr.
1986.
An electron microscopic study of the effect of clindamycin on adherence of Staphylococcus aureus to bone surfaces.
Microbios
45:21-32[Medline].
|
| 22.
|
Milatovic, D.,
I. Braveny, and J. Verhoef.
1983.
Clindamycin enhances opsonization of Staphylococcus aureus.
Antimicrob. Agents Chemother.
24:413-417[Abstract/Free Full Text].
|
| 23.
|
Miyazaki, E.,
J.-M. Chen,
C. Ko, and W. Bishai.
1999.
The Staphylococcus aureus rsbW (orf159) gene encodes an anti-sigma factor of SigB.
J. Bacteriol.
181:2846-2851[Abstract/Free Full Text].
|
| 24.
|
Nicholas, R. O.,
T. Li,
D. McDevitt,
A. Marra,
S. Sucoloski,
P. L. Demarsh, and D. R. Gentry.
1999.
Isolation and characterization of a sigB deletion mutant of Staphylococcus aureus.
Infect. Immun.
67:3667-3669[Abstract/Free Full Text].
|
| 25.
|
Novick, R. P.
1991.
Genetic systems in staphylococci.
Methods Enzymol.
204:587-636[Medline].
|
| 26.
|
Novick, R. P.,
I. Edelman,
M. D. Schwesinger,
D. Gruss,
E. C. Swanson, and P. A. Pattee.
1979.
Genetic translocation in Staphylococcus aureus.
Proc. Natl. Acad. Sci. USA
76:400-404[Abstract/Free Full Text].
|
| 27.
|
Novick, R. P.,
E. Murphy,
T. J. Gryczan,
E. Baron, and I. Edelman.
1979.
Penicillinase plasmids of Staphylococcus aureus: restriction-deletion maps.
Plasmid
2:109-129[CrossRef][Medline].
|
| 28.
|
Novick, R. P.,
S. Projan,
J. Kornblum,
H. Ross,
B. Kreiswirth, and S. Moghazeh.
1995.
The agr P-2 operon: an autocatalytic sensory transduction system in Staphylococcus aureus.
Mol. Gen. Genet.
248:446-458[CrossRef][Medline].
|
| 29.
|
Novick, R. P.,
H. F. Ross,
S. J. Projan,
J. Kornblum,
B. Kreiswirth, and S. Moghazeh.
1993.
Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule.
EMBO J.
12:3967-3975[Medline].
|
| 29a.
|
O'Callaghan, C. H.,
A. Morris,
S. M. Kirby, and A. H. Shingler.
1972.
Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate.
Antimicrob. Agents Chemother.
1:283-288[Abstract/Free Full Text].
|
| 30.
|
Ohlsen, K.,
W. Ziebuhr,
K. P. Koller,
W. Hell,
T. A. Wichelhaus, and J. Hacker.
1998.
Effects of subinhibitory concentrations of antibiotics on alpha-toxin (hla) gene expression of methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolates.
Antimicrob. Agents Chemother.
42:2817-2823[Abstract/Free Full Text].
|
| 31.
|
Proctor, R. A.,
P. J. Olbrantz, and D. F. Mosher.
1983.
Subinhibitory concentrations of antibiotics alter fibronectin binding to Staphylococcus aureus.
Antimicrob. Agents Chemother.
24:823-826[Abstract/Free Full Text].
|
| 32.
|
Schlievert, P. M., and J. A. Kelly.
1984.
Clindamycin-induced suppression of toxic-shock syndrome-associated exotoxin production.
J. Infect. Dis.
149:471[Medline].
|
| 33.
|
Shibl, A.
1977.
Effect of antibiotics on enzyme and toxin production by S. aureus. Ph.D. dissertation.
Council for National Academic Awards, Glasgow, Scotland.
|
| 34.
|
Shibl, A. M.
1983.
Effect of antibiotics on production of enzymes and toxins by microorganisms.
Rev. Infect. Dis.
5:865-875[Medline].
|
| 35.
|
Shibl, A. M.
1982.
Subcutaneous staphylococcal infections in mice: the influence of antibiotics on staphylococcal extracellular products.
Chemotherapy
28:46-53[Medline].
|
| 36.
|
Shibl, A. M., and I. A. Al-Sowaygh.
1980.
Antibiotic inhibition of protease production by Pseudomonas aeruginosa.
J. Med. Microbiol.
13:345-348[Abstract/Free Full Text].
|
| 37.
|
Shibl, A. M., and I. A. Al-Sowaygh.
1979.
Differential inhibition of bacterial growth and hemolysin production by lincosamide antibiotics.
J. Bacteriol.
137:1022-1023[Abstract/Free Full Text].
|
| 38.
|
Shibl, A. M., and C. G. Gemmell.
1983.
Effect of four antibiotics on haemolysin production and adherence to human uroepithelial cells by Escherichia coli.
J. Med. Microbiol.
16:341-349[Abstract/Free Full Text].
|
| 39.
|
Unkles, S. E., and C. G. Gemmell.
1982.
Effect of clindamycin, erythromycin, lincomycin, and tetracycline on growth and extracellular lipase production by propionibacteria in vitro.
Antimicrob. Agents Chemother.
21:39-43[Abstract/Free Full Text].
|
| 40.
|
Vandenesch, F.,
J. Kornblum, and R. Novick.
1991.
A temporal signal, independent of agr, is required for hla but not for spa transcription in Staphylococcus aureus.
J. Bacteriol.
173:4204-4209.
|
| 41.
|
Wolz, C.,
P. Pohlmann-Dietze,
A. Steinhuber,
Y. T. Chien,
A. Manna,
W. van Wamel, and A. Cheung.
2000.
Agr-independent regulation of fibronectin-binding protein(s) by the regulatory locus sar in Staphylococcus aureus.
Mol. Microbiol.
36:230-243[CrossRef][Medline].
|
Infection and Immunity, May 2001, p. 2996-3003, Vol. 69, No. 5
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.5.2996-3003.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
