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Antimicrob Agents Chemother. Feb 2007; 51(2): 461–467.
Published online Nov 13, 2006. doi:  10.1128/AAC.00539-06
PMCID: PMC1797754

Growth Phase-Dependent Effect of Clindamycin on Production of Exoproteins by Streptococcus pyogenes[down-pointing small open triangle]

Abstract

The administration of high-dose clindamycin plus benzylpenicillin has been recommended for the treatment of streptococcal toxic shock-like syndrome caused by Streptococcus pyogenes, and clindamycin has been found to be more effective than beta-lactams in retrospective analyses of human cases. Although therapeutic doses of clindamycin have also been shown to be effective against experimental infections and clindamycin has great efficacy against the production of bacterial exoproteins, we recently reported that the level of production of some exoproteins was unchanged or even increased by a subinhibitory dose of clindamycin when it is added upon the initiation of bacterial culture and the treated cultures were analyzed by two-dimensional gel electrophoresis. In this study we further examined the effect of clindamycin on the production of exoproteins by adding it to Streptococcus pyogenes cultures during various growth phases. We found that the levels of production of some proteins, NAD+ glycohydrolase, streptolysin O, and streptococcal inhibitor of complement, were increased when clindamycin was added at early-log-phase growth, which was the result that was seen when clindamycin was added at the beginning of culture. However, clindamycin inhibited the production of most types of proteins when it was administered to Streptococcus pyogenes cultures at mid-log-phase growth. In csrS- or mga-knockout bacterial strains, the increase in exoproteins seen in parental strains was considerably inhibited. Our study indicates that the in vitro effect of clindamycin on the production of exoproteins greatly depends on the growth phase of bacteria and some regulatory factors of Streptococcus pyogenes that are involved in this phenomenon.

Streptococcus pyogenes is a gram-positive bacterium that is one of the most common agents of upper respiratory tract infections, especially the acute pharyngitis that occurs mainly in children. It is also responsible for poststreptococcal diseases such as rheumatic fever and glomerulonephritis, in addition to increasing numbers of invasive infections, such as streptococcal toxic shock-like syndrome (TSLS), necrotizing fasciitis, bacteremia, and multiple-organ failure (15, 20, 23).

Because S. pyogenes is exquisitely susceptible to β-lactam antibiotics, benzylpenicillin (PCG) has been recommended for the treatment of most S. pyogenes infections. However, it has been reported that PCG has reduced efficacy against aggressive infections like TSLS, which is a condition in which large numbers of organisms are present. Inocula with large numbers of organisms reach the stationary phase of growth quickly, and PCG is less effective against slowly growing organisms (22, 24). Moreover, certain penicillin-binding proteins have been shown not to be expressed by S. pyogenes during the stationary phase (24). Conversely, regarding the inhibition of protein synthesis, several investigators have demonstrated that clindamycin (CLI) suppresses the production of a variety of toxins from S. pyogenes (3, 13, 19, 21, 22). CLI also has a long serum half-life and plays an important role in immune modulation (1, 21). Therefore, the administration of CLI, in addition to PCG, is now the most frequently recommended antibiotic treatment for TSLS.

We have recently shown, however, by comprehensive analysis by two-dimensional gel electrophoresis (2-DE) that the production of some exoproteins was either unchanged or increased by treatment of S. pyogenes cultures with CLI or other antibiotics that inhibit protein synthesis (25). In the previous study we did not examine the relationship between the time that the antibiotics were added and their effects on exoprotein production. Therefore, in this study we added CLI to S. pyogenes cultures at various growth phases and examined the effect of administration time on the production of exoproteins. In addition, we also examined the roles of three regulatory factors, CsrR/CsrS, Mga, and Rgg, in this phenomenon. CsrR/CsrS is the most extensively studied two-component regulatory system in S. pyogenes and is known to be one of the negative regulators of virulence genes (10); Mga regulates the M protein, which is one of the most important virulence factors; and Rgg regulates the SpeB protein, which was the most abundant protein, as detected by 2-DE in our previous study (25).

MATERIALS AND METHODS

Bacterial strains and determination of antibiotic concentrations for exoprotein analysis.

The S. pyogenes M1 serotype strain used in this study, S. pyogenes 1529, was a clinical isolate from a patient in Japan with TSLS. The bacteria (strain 1529 and its derived knockout mutants) were cultured in brain heart infusion (BHI) broth (Eiken Chemical Co., Tokyo, Japan) containing 0.3% yeast extract (Difco Laboratories, Detroit, MI) at 37°C without agitation. The antibiotic used in this study was clindamycin hydrochloride (Sigma Chemical Co., St. Louis, MO). The MIC of CLI for strain 1529 was 0.03125 μg/ml, as assayed by the broth microdilution method.

Bacteria were cultured in BHI broth that contained various concentrations of CLI, as described previously (25). In brief, the maximum antibiotic concentration that did not suppress bacterial growth, as judged by the optical density (OD), was determined; and that concentration was used for this study.

Culture conditions.

An aliquot of bacterial stock solution stored frozen at −80°C was inoculated into 5 ml BHI broth and cultured overnight. Two hundred fifty microliters of overnight culture was added to 25 ml fresh BHI. The growth of the bacteria was then monitored with a colorimeter (Asahi Science, Tokyo, Japan) by determination of the OD at 660 nm (OD660). CLI was added to the medium at various growth phases: (i) when the bacterial culture was started, (ii) at early log phase (OD660, approximately 0.2), (iii) at mid-log phase (OD660, approximately 0.5), and (iv) at early stationary phase (OD660, approximately 0.8). These ODs and their correspondence to the growth phases were determined empirically. The cultures for each experimental condition were incubated for approximately 22 h before 2-DE analysis.

2-DE analysis.

Exoproteins from the culture supernatant were prepared as described previously (8, 9). In brief, all sample pellets derived from 25 ml bacterial culture supernatant were dissolved in 300 μl of immobilized pH gradient (IPG) dehydration solution (GE Healthcare Biosciences Co., Piscataway, NJ), which consisted of 7.8 M urea, 2 M thiourea, 2% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 0.6% dithiothreitol, and 0.5% IPG buffer. Aliquots (280 μl) of the samples were loaded onto 13-cm Immobiline DryStrip gels (pH 3 to 10; GE Healthcare Biosciences Co.). The first-dimensional electrophoresis conditions and second-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation were as described previously (8, 9). To compare the abundance of protein spots on the two-dimensional electrophoresis gels, the stained gels were scanned and analyzed with an ImageMaster Labscan (version 3.00) instrument. The intensity of each protein spot was measured by the software. After subtraction of the background, those intensities were compared. The experiments were repeated at least three times to confirm their reproducibilities.

Peptide mass mapping (PMM) analysis.

Sample protein mixtures were prepared as described previously (25). In brief, protein spots were excised from the gels and the peptide mixtures were extracted from the excised gel pieces with acetonitrile solution containing 0.1% (vol/vol) trifluoroacetic acid. The extracts were dried and stored at 4°C before mass analysis. Nanoelectrospray tandem mass analysis was performed as described previously (25). The proteins in the spots were identified by their conformation to the description on the Matrix Science website (http://www.matrixscience.com/).

Creation of csrS-, mga-, and rgg-knockout mutants.

Escherichia coli JM109 was used to propagate the plasmid constructions. Mutants with nonpolar inactivated csrS, mga, and rgg genes were constructed by double-crossover allelic replacement in the chromosome of S. pyogenes 1529. To construct the plasmid for the csrS-knockout mutant, the 5′ end of csrS (fragment 1) was amplified with oligonucleotide primer csrS-n4 with an NheI restriction site and primer csrS-c6 with an SmaI restriction site, and the 3′ end of csrS (fragment 2) was amplified with primer csrS-n3 with an SmaI restriction site and primer csrS-c7 with an SpeI restriction site. Fragment 2 was digested with SmaI and SpeI for insertion into multicloning site 2 of plasmid pFW12 (12). The resulting plasmid was then digested with NheI and SmaI; and both the spc1 DNA fragment containing aad9 (promoterless spectinomycin resistance gene), which was obtained from an SmaI-digested fragment of pSL60-1 (12), and the NheI-SmaI-digested fragment 1 were inserted. This plasmid, csrS::aad9/pFW12, was a suicide vector for S. pyogenes. The construction of the plasmid for mga was performed in a similar manner. The 5′ end of mga (fragment 3) was amplified with primer mga-n3 with an NheI restriction site and primer mga-c3. The 3′ end (fragment 4) was amplified with primer mga-n4 with an SpeI restriction site and primer mga-c4 with an SmaI restriction site. Fragment 3 was digested with NheI and HincII for insertion into multicloning site 1 of plasmid pFW12. The resulting plasmid was then digested with SpeI and SmaI, and the SpeI-SmaI-digested fragment 4 was inserted. Finally, the resulting plasmid was digested with HincII and SmaI, and spc1 was inserted. For the rgg knockout, a DNA fragment was amplified with primer rgg-n1 with an NheI restriction site and primer rgg-c1 with an SpeI restriction site. The resulting PCR fragment was digested with NheI and HindIII and inserted into multicloning site 1 of plasmid pFW12 (pFW12-rggn). The same PCR fragment was also digested with SspI and SpeI and was then inserted into the SmaI-SpeI site of multicloning site 2 of plasmid pFW12-rggn (pFW12-rggnc). Finally, plasmid pFW12-rggnc was digested with HincII and HindIII and blunt ended with the Klenow enzyme, and the spc3 DNA fragment from SmaI-digested pSL60-3 (12) was inserted. For the preparation of competent cells, strain 1529 was harvested at early to mid-log phase (OD660, 0.4) and washed twice with 0.5 M sucrose buffer. The suicide vectors constructed, csrS::aad9/pFW12, mga::aad9/pFW12, and rgg::aad9/pFW12, were each transformed into strain 1529 by electroporation. The conditions of electroporation were 1.25 kV/mm, 25-μF capacitance, and 200-Ω resistance; and electroporation was performed with a GenePulser II instrument (Bio-Rad, Hercules, CA). After incubation at 37°C for 3 h, competent cells were spread onto BHI agar plates containing 0.3% yeast extract and spectinomycin (final concentration, 100 μg/ml). Selected colonies on the plates were cultured. The cultured bacteria were washed once with saline, resuspended in 10 mM Tris-1 mM EDTA, and boiled for 10 min. Genomic DNA was obtained from the supernatant of the boiled bacteria. The double-crossover replacement was analyzed by PCR of the genomic DNA. Successful double-crossover replacement was further confirmed by DNA sequencing. The sequences of all PCR primers used are shown in Table Table11.

TABLE 1.
Oligonucleotides used in this study

Assessment of mRNA levels by semiquantitative reverse transcription-PCR (RT-PCR).

Total RNA was extracted and purified as described previously (25). In brief, bacterial cells were cultured in 5 ml BHI broth, to which a subinhibitory concentration of CLI had been added at the beginning of culture, and were harvested when the OD660 was approximately 0.8 (late log phase). Total RNA was extracted and purified with an RNA-protected bacterial reagent (QIAGEN, Hilden, Germany), followed by treatment with RNase-free DNase (QIAGEN). Total RNA (2 μg) was reverse transcribed in the presence of sic- and gyrA-specific primers with the SuperScript first-strand synthesis system, according to the instructions of the manufacturer (Invitrogen Co.), in a 10-μl reaction volume. The primers used for amplification were sic-n6 and sic-c4 for sic and gyrA-F and gyrA-R for gyrA. A total of 0.4 μl from the first 10 μl reverse transcriptase reaction was used for the next PCR (reaction volume, 10 μl). Amplification and detection of the specific products were performed with the following cycle profile: 25 thermal cycles of 30 s at 94°C, 30 s at 55°C for annealing, and 20 s (gyrA) or 30 s (sic) at 72°C for extension, with an additional extension of 300 s at 72°C. The amount of contaminating chromosomal DNA in each sample was determined in control reactions that did not contain reverse transcriptase. The quantity of cDNA for each experimental gene was normalized to the quantity of gyrA cDNA in each sample.

RESULTS

Determination of subinhibitory concentration of clindamycin.

S. pyogenes 1529 was cultured with several concentrations of CLI, which were added at different bacterial growth phases. The concentration of CLI used in this study was 0.006 μg/ml. This concentration was similar to that used in the previous study (25).

Effect of CLI addition at early log phase of culture on production of exoproteins, as analyzed by 2-DE.

In the previous study, the bacteria were cultured with CLI for 16 h. In this study we decided to expose the bacteria to CLI for the same duration of time, even for the growth phase when CLI was added at an OD660 of 0.8 (early stationary phase). For this reason the total incubation time was prolonged compared with that in the previous study (25). To confirm the previous results (25), CLI was added to the medium upon initiation of the bacterial culture. The bacteria were harvested after 22 h of culture (the bacteria were exposed to CLI for 22 h), when the bacteria were in the late stationary phase of growth. After separation by 2-DE, several protein spots were identified by PMM analysis. The profiles of the exoproteins and their corresponding spots are shown in Fig. Fig.1b1b (compare Fig. Fig.1b1b with Fig. Fig.1a)1a) and Table Table2.2. Figure Figure1a1a shows the 2-DE profile without antibiotics. The increased level of some proteins was not as great as that seen in the previous study, possibly due to degradation because of the longer incubation time. However, the increases in Sic (spot 3), Slo (spot 7), and Nga (spot 8) and the decrease of SpeB (spot 1) were confirmed.

FIG. 1.
Analysis of exoproteins from S. pyogenes 1529 identified by PMM analysis after separation by 2-DE. S. pyogenes 1529 was cultured and CLI was added to the medium (b) upon initiation of the bacterial culture, (c) when the bacterial culture reached early ...
TABLE 2.
Identities and densities of protein spots in strain 1529 after administration of CLI at several bacterial growth phasesa

CLI was then added to the culture medium when the growth of the bacteria reached early log phase (OD660, approximately 0.2). The total incubation time was also 22 h, including 20 h of exposure to CLI. The profiles of the exoproteins and their corresponding spots are shown in Fig. Fig.1c1c and Table Table2.2. The effect of the addition of CLI at early log phase was similar to the effect of CLI added at the beginning of culture (compare Fig. Fig.1c1c with Fig. Fig.1b).1b). The levels of cysteine protease (SpeB; spot 1) and secreted endoglycosidase (EndoS; spot 6) were significantly decreased. The levels of M1 protein (spot 5) and streptococcal pyrogenic exotoxin F (SpeF; spot 9) were decreased. The level of AmyA (cyclomaltodextrin glucanotransferase; spot 2) was decreased, although it did not change when CLI was added at the beginning of the culture. The level of Mf3 (spot 4) was also decreased, although it was slightly increased when CLI was added at the beginning of the culture. The levels of Sic (spot 3) and streptolysin O (Slo) (spot 7) were increased. Nga (spot 8) was barely detected, but its level was slightly increased when CLI was added at the beginning of the culture.

Effect of addition of CLI at mid-log phase on production of exoproteins.

CLI was also added to the culture medium when the growth of the bacteria reached mid-log phase (OD660, approximately 0.5). The total incubation time was also 22 h, including 18 h of exposure to CLI. The profiles of the exoproteins and their corresponding spots are shown in Fig. Fig.1d1d and Table Table2.2. The levels of almost all exoproteins, SpeB (spot 1), AmyA (spot 2), Mf3 (spot 4), M1 protein (spot 5), EndoS (spot 6), and SpeF (spot 9), were significantly decreased. The levels of Sic (spot 3) and Slo (spot 7) were also significantly decreased, although they were increased when CLI was added before early log phase (compare Fig. Fig.1d1d with Fig. 1b and c). Nga (spot 8) was not detected. Thus, we found that the production of almost all exoproteins was inhibited when CLI was added at the mid-log phase of growth.

Effect of addition of CLI at early stationary phase on production of exoproteins.

Finally, CLI was added to the culture medium when the growth of the bacteria reached early stationary phase (OD660, approximately 0.8). The total incubation time was also 22 h, including 16 h of exposure to CLI. The profiles of the exoproteins and their corresponding spots are shown in Fig. Fig.1e1e and Table Table2.2. As in mid-log phase, the levels of almost all exoproteins, SpeB (spot 1), AmyA (spot 2), Sic (spot 3), Mf3 (spot 4), M1 protein (spot 5), EndoS (spot 6), Slo (spot 7), and SpeF (spot 9), were decreased; but the amount of the decrease was not as great as that in mid-log phase (compare Fig. Fig.1e1e with Fig. Fig.1d1d).

A summary of the effects of CLI on the production of exoproteins is shown in Table Table2.2. The most effective time of administration of CLI was mid-log phase growth, when the production of almost all proteins was inhibited. In contrast, the production of Nga, Slo, Mf3, and Sic was increased when CLI was added before mid-log phase growth.

Influences of the regulators on alteration of production of exoproteins by CLI.

To clarify the mechanism for the increased production of some exoproteins when CLI was added before mid-log phase growth, we established csrS-, mga-, and rgg-knockout mutants of strain 1529 and analyzed their roles by 2-DE (Fig. (Fig.2,2, ΔcsrS; Fig. Fig.3,3, Δmga; and Fig. Fig.4,4, Δrgg). In all experiments, CLI was added at the beginning of the culture and 2-DE was performed after 22 h of culture.

FIG. 2.
Analysis of exoproteins from S. pyogenes strain 1529 ΔcsrS identified by PMM analysis after separation by 2-DE. S. pyogenes 1529 ΔcsrS was cultured, and CLI was added to the medium upon initiation of the bacterial culture. The protein ...
FIG. 3.
Analysis of exoproteins from S. pyogenes strain 1529 Δmga identified by PMM analysis after separation by 2-DE. S. pyogenes 1529 Δmga was cultured, and CLI was added to the medium upon initiation of the bacterial culture. The protein spots ...
FIG. 4.
Analysis of exoproteins from S. pyogenes strain 1529 Δrgg identified by PMM analysis after separation by 2-DE. S. pyogenes 1529 Δrgg was cultured, and CLI was added to the medium upon initiation of the bacterial culture. The protein spots ...

The production of exoproteins was first estimated in the ΔcsrS mutant. The production of many exoproteins, including Sic, Slo, and Nga, was increased by the CsrS-knockout mutant even without the addition of CLI (Fig. (Fig.2a).2a). In addition, the level of production seen in the ΔcsrS mutant was much greater than that seen in strain 1529 with CLI (compare Fig. Fig.2b2b with Fig. Fig.1b).1b). However, in contrast to the results seen for the parental strain, the levels of production of these exoproteins, especially Sic and Nga, with the addition of CLI were lower (Fig. (Fig.2b)2b) than those without CLI (Fig. (Fig.2a).2a). The experiment was then repeated with the Δmga strain. Mga is an important regulator for bacterial colonization of host tissues and the subversion of the immune response (11). In the mga mutant, Sic was barely detected in the presence or absence of CLI (Fig. 3a and b). The levels of Nga and Slo were increased, similar to the results seen for the parental strain with CLI (Fig. (Fig.3b3b).

The Δrgg mutant was then analyzed. Rgg is a regulator that controls the expression of extracellular products. Unlike the results seen with the other mutant strains, with the addition of CLI, the levels of production of Nga, Slo, and Sic were increased more for the Δrgg strain than for the parental strain (Fig. (Fig.4b).4b). The expression of the M1 protein by the Δrgg strain was also increased (Fig. (Fig.4b),4b), although it was decreased by the parental strain (Fig. (Fig.1b).1b). A summary of qualitative estimates of the semiquantitative differences in the protein spots with the knockout of regulatory genes is shown in Table Table33.

TABLE 3.
Change in densities of protein spots for strains 1529, ΔcsrS, Δmga, and Δrgg after administration of CLI at the beginning of culturea

Assessment of transcription level of sic in the Δmga strain exposed to a subinhibitory concentration of CLI.

Because the Sic protein was barely detected in the Δmga strain by 2-DE analysis (Fig. (Fig.3),3), we used semiquantitative RT-PCR to determine whether sic is expressed in the Δmga strain and whether the level of transcription of sic is increased with the addition of a subinhibitory concentration of CLI. Sic was expressed in the Δmga strain (Fig. (Fig.5a,5a, lane 3), but its level did not increase when CLI was added at the beginning of bacterial growth (Fig. (Fig.5a,5a, lane 4), although it increased in the parental strain under those conditions (Fig. (Fig.5a,5a, lanes 1 and 2). A non-reverse-transcribed control showed no amplification product, suggesting that the reverse-transcribed cDNA did not contain genomic DNA (data not shown). We also confirmed that the internal control gyrA mRNA level was unchanged (Fig. (Fig.5b5b).

FIG. 5.
RT-PCR analysis of the sic (a) and gyrA (b) genes. Total RNA was isolated from the S. pyogenes 1529 parental strain (lanes 1, 2, 5, and 6) and the Δmga strain (lanes 3, 4, 7, and 8). Lanes 1, 3, 5, and 7, controls (no antibiotics); lanes 2, 4, ...

DISCUSSION

In experimental models of necrotizing fasciitis and myonecrosis, CLI has been reported to be effective, regardless of the size of the inoculum or the stage of bacterial growth (22, 24). Moreover, it has been reported that CLI alone or in combination with penicillin optimizes the treatment of S. pyogenes infections by reducing the bacterial burden and inhibiting streptococcal pyrogenic exotoxin A (SpeA) release and M-protein production (3, 6). Thus, CLI has been recommended for use with PCG for the treatment of TSLS. Contrary to those previous reports, however, our previous comprehensive studies of exoproteins by 2-DE showed that the levels of production of some exoproteins and some protein synthesis inhibitors were increased by CLI when it was added at the beginning of the bacterial culture (25).

It has not been completely proved how the growth phases of bacteria in each patient with different S. pyogenes infections in vivo are correlated to the growth phases in vitro; possibly, there are many kinds of bacteria with different growth phases, and the bacteria from the TSLS patient may have been past the log phase of growth. Also, the concentration of CLI may vary in each patient and each tissue; for example, the tissues where the bloodstream is limited may have a subinhibitory concentration of CLI and insufficient CLI might induce the production of some exoproteins, as we reported previously (25). For these reasons, we examined the relevance of growth phases on the effect and the subinhibitory effect of CLI.

In this study we further demonstrated that addition of CLI to a bacterial culture at the early log phase of growth increased the production of these proteins. Conversely, we also presented data that CLI inhibited protein production efficiently when it was added to S. pyogenes cultures at the mid-log or early stationary phase of growth, suggesting that the effect of CLI depends on the timing of administration in regard to the bacterial growth phase and that CLI can inhibit most protein production by bacteria whose growth phase is already past the log phase in most TSLS patients. The mechanisms by which subinhibitory doses of CLI and other protein inhibitor antibiotics cause increased production of exotoxins are unknown at present. It has been reported that S. pyogenes can switch virulence phenotypes in response to environmental stimuli (16). CLI may have a direct or an indirect effect on the regulator proteins that are important in the production of exoproteins. Several multigene regulons that respond to the bacterial growth phase and alteration of host environments through “stand-alone” transcriptional regulator proteins, such as Mga, RofA-like protein, and Rgg/RopB, in addition to two-component signal transduction systems, such as CsrR/CsrS, FasBCAX, and Ink/Irr, have been reported to be involved with the switch of virulence phenotypes (11). It has been reported that the expression of these regulators is susceptible to the growth phase and several environmental stresses and that all these regulators form a regulatory network and influence each other (11).

The major regulatory activity of CsrS/CsrR is exerted during the exponential and stationary growth phases (7). CsrR/CsrS is one of the repressors of virulence genes (10), influences 15% of all chromosomal genes of S. pyogenes (7), and also regulates itself (5). The knockout of CsrS itself caused the increase in the levels of production of many exoproteins. However, under the condition when a subinhibitory CLI concentration is added at the beginning of bacterial culture, the production of exoproteins, including Nga, Slo, and Sic, was increased in parental strain 1529 but was decreased in the ΔcsrS strain (Fig. (Fig.2).2). Our results demonstrate that it may be possible that CsrS/CsrR functions as a direct positive regulator in the presence of CLI or that it is located upstream of the positive regulators that function to increase the production of exoproteins by CLI. Another possibility is that CsrS/CsrR functions as a repressor after all and down-regulates the other negative regulators which play a role in CLI stress. At present it is unknown whether CsrS is involved directly or indirectly in the mechanism of action.

Mga is expressed maximally during the exponential phase (14) and regulates genes which encode the proteins that are required for bacterial adhesion and invasion. Bacterial avoidance of host immune systems is under the control of the Mga regulon and regulatory networks (4, 18). Unlike the results seen in the experiments with the parental strain, the increase in the level of Sic brought about by CLI was not detected in the Δmga strain culture (Fig. (Fig.33 and and5).5). This finding suggests that Mga is an important regulator for increasing the production of Sic under the condition in which a subinhibitory concentration of CLI is present.

Rgg is maximally expressed in the stationary phase of culture (17). Rgg affects the expression of many virulent proteins, especially SpeB, SpeF, and Mf3, at the level of transcription (2). Rgg also influences several regulatory genes and especially represses expression of the Mga regulon and promotes CsrR/CsrS expression (2). In our study, the levels of production of Nga, Slo, and Sic were increased more and the level of production of EndoS was decreased less in the Δrgg strain compared to the levels of production in the parental strain. In addition, when CLI was added at the beginning of the culture, the level of production of the M1 protein was increased in the Δrgg strain, whereas it was decreased in the parental strain (Fig. (Fig.4).4). These results suggest that Rgg is also involved in the increased production of some exoproteins; however, its function appears to be in opposition to the functions of CsrS and Mga.

Our results suggest that if bacteria express some proteins for adaptation in the presence of antibiotic stress, like that from CLI, and that if the increased levels of Nga, Slo, and Sic are critical for S. pyogenes, CsrS/CsrR and Mga have important roles; but the role of Rgg is unclear. Also unknown are why exoprotein production is increased only when CLI is added before mid-log phase and what the sensor protein for CLI is. One possible explanation is that because both Mga and CsrS (seemingly “positive regulators”) work most efficiently in log phase, the administration of CLI at mid-log phase diminishes their functions. Another explanation is that the CLI sensor is expressed before mid-log phase, and therefore, the effect of CLI cannot be demonstrated if the antibiotic is added after mid-log phase. Another possibility is that with a very complicated regulatory network, the balance between positive regulation and negative regulation, including regulation with other regulators which we did not analyze in this study, can vary according to the growth phase and can also vary according to the effect of the administration of CLI at different growth phases.

Additional in vivo and in vitro studies are necessary to confirm our in vitro results.

Acknowledgments

This work was supported by a grant for comprehensive research on aging and health (H16-choju-004) from the Ministry of Health, Labor and Welfare of Japan and a Grant-in-Aid (no. 16590356) for Science Research from the Ministry of Education, Culture, Sports, Science, and Technology.

We thank S. Lukomski for providing plasmids pFW12, pSL60-1, and pSL60-3.

Footnotes

[down-pointing small open triangle]Published ahead of print on 13 November 2006.

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