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Infection and Immunity, April 2007, p. 1964-1972, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01552-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Regulation of Exoprotein Gene Expression by the Staphylococcus aureus cvfB Gene{triangledown}

Yasuhiko Matsumoto, Chikara Kaito, Daisuke Morishita, Kenji Kurokawa, and Kazuhisa Sekimizu*

Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 3-1, 7-Chome, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received 27 September 2006/ Returned for modification 7 November 2006/ Accepted 23 January 2007


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ABSTRACT
 
We previously reported that the cvfB gene (SA1223) of Staphylococcus aureus is responsible for the virulence of this pathogenic bacterium. We show here that the cvfB gene regulates exoprotein gene expression. In a cvfB gene deletion mutant, hemolysin, DNase, and protease production were decreased, whereas protein A expression was increased. The amount of RNAIII, the transcript from the P3 promoter in the agr locus that regulates the expression of various virulence factors, was also reduced in the cvfB mutant. In addition, P2 and P3 promoter activity in the agr locus was decreased in the mutant. Under the genetic background of the agr-null mutation, cvfB gene disruption decreased the production levels of DNase and protease. Moreover, the cvfB and agr double mutant was less virulent than the agr mutant in silkworms. These results suggest that the cvfB gene product contributes to the expression of virulence factors and to pathogenicity via both agr-dependent and agr-independent pathways.


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INTRODUCTION
 
Staphylococcus aureus is an opportunistic pathogen that causes various diseases, such as localized infections of skin, endocarditis, and sepsis. S. aureus causes these diseases by controlling the expression of various virulence factors (6). Exoproteins, including hemolysin, nuclease, and protease, presumably facilitate host cell lysis, whereas cell wall-associated adhesions, such as fibronectin-binding protein and protein A, are required for colonization in host tissues and evasion of host defense mechanisms (8).

The agr locus of S. aureus has an important role in controlling the expression of virulence factors (28). The agr locus produces two divergent transcripts: RNAII and RNAIII. RNAII encodes four proteins: AgrA, AgrB, AgrC, and AgrD. AgrD is processed and secreted with the aid of AgrB and functions as an autoinducing peptide. Autoinducing peptide is recognized by AgrC, a sensor of the two-component regulatory system, and phosphorylated AgrC leads to a second phosphorylation of the activator AgrA. Phosphorylated AgrA further facilitates the transcription of RNAII and RNAIII. RNAIII regulates the expression of various virulence factors (31). RNAIII upregulates the expression of hemolysin and downregulates the expression of protein A at the transcriptional level during the postexponential phase.

In addition to the agr locus, sarA (5), sarA family genes (4), saeRS (13), srrAB (42), arlRS (10), and svrA (12) regulate the expression of virulence genes. These genes are assumed to interact with each other and to contribute to the regulation of virulence factors, although the whole picture of the regulatory system for pathogenic gene expression remains to be elucidated (28).

We previously reported a silkworm infection model using bacteria that is pathogenic against humans, such as S. aureus and Pseudomonas aeruginosa (11, 16, 20). Because antibiotics have therapeutic effects against the infection, the silkworm lethality involves bacterial growth in the body fluid of the animals (15, 20). Based on the attenuated killing ability against silkworms, we screened mutant S. aureus strains in which genes that are conserved among pathogenic bacteria (24) were deleted and identified three novel virulence-associated genes named cvfA, cvfB, and cvfC (21). Disrupted mutants of these genes also had attenuated virulence in mice. Benton et al. reported that the cvfB gene is required for the survival of S. aureus in the mouse spleen (2). We previously observed that hemolysin production was decreased in the cvfB deletion mutant (21). The cvfB gene product, with 300 amino acid residues, contains no extracellular secretion signals and has no common motifs identified in hemolysins. Therefore, CvfB protein might not be hemolysin itself. In the present study, we examined the influence of the cvfB deletion mutation on the expression of various virulence genes. The results indicate that the cvfB gene contributes to the activation of the agr locus and that this gene regulates the expression of exoproteins via an agr-independent pathway.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. The JM109 strain of Escherichia coli was used as a host for pMutinT3, pHY300, pND50, and their derivatives. E. coli strains transformed with the plasmids were cultured in Luria-Bertani broth containing 50 µg of ampicillin/ml or 12.5 µg of chloramphenicol/ml. S. aureus strains were aerobically cultured in tryptic soy broth at 37°C, and 10 µg of erythromycin, 5 µg of tetracycline, or 12.5 µg of chloramphenicol/ml was added to the medium if required. Details of bacterial strains and plasmids used in the present study are shown in Table 1. To examine the growth phase, the optical density at 600 nm (OD600) of appropriately diluted culture was measured by using a spectrophotometer (UV-1200; Shimazu, Tokyo, Japan) and was plotted onto a semilogarithmic scale. The log phase was an OD600 of 0.1 to 6. To construct the agr and cvfB double mutant (CK5), phage transduction was performed as described previously (29). Phage 80{alpha} lysates of CK3 strain was used to infect M1223 strain.


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  		TABLE 1. Bacterial strains and plasmids used in this study

DNA manipulation. Transformation of E. coli, extraction of plasmid DNA from E. coli, and PCR were performed as previously described (34). Extraction of genomic DNA from S. aureus was performed by using a QIAamp DNA blood DNA kit (QIAGEN Sciences) after the cells were lysed with lysostaphin. Transformation by plasmid DNA of the S. aureus was performed by electroporation (18). To construct the plasmid for the complementation experiment, the cvfB gene of S. aureus RN4220 strain was amplified by PCR using the oligonucleotide primers FcvfB and RcvfB (Table 2). The cvfB gene corresponds to SA1223 in the S. aureus N315 genome database on GIB (the Genome Information Broker program) from the DNA Data Bank of Japan. The amplified DNA fragment was inserted into pHY300 at the SmaI site, resulting in p1223. To construct pHY300C, pHY300 without the tetracycline resistance gene was amplified from pHY300 by PCR using the primers FpHY300Tet- and RpHY300Tet- (Table 2), resulting in pHY300Tet-. The cat gene encoding chloramphenicol acetyltransferase was amplified from pND50 by PCR with the primers Fcat and Rcat (Table 2) and was inserted into pHY300Tet-, resulting in pHY300C.


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  		TABLE 2. PCR primers used in this study

Measurement of exotoxin activity. Hemolytic activity was measured by an established method (40). Briefly, a supernatant of the culture at 12 h after inoculation was sampled and incubated with sheep red blood cells at 37°C for 1 h. The reaction mixture was centrifuged (1 krpm, 5 min), and the increase in the OD405 of the supernatant was determined. The activity was expressed by hemolytic units corresponding to the reciprocal of the dilution of supernatant that yielded 50% erythrocyte lysis.

DNase activity was measured by using a previously reported method (7). In brief, a supernatant (5 µl) of the culture at 12 h after inoculation was incubated with salmon sperm DNA (500 µg) for 30 min at 37°C. The reaction mixture was added to a equal volume of 10% trichloroacetic acid and then centrifuged at 15 krpm for 10 min. The OD260 of the supernatant was measured, and the value multiplied by hundred was presented as an arbitrary unit.

To determine the protease activity, an aliquot (300 µl) filtered from the culture at 12 h after inoculation was incubated with azocasein (800 µl, 3 mg/ml) for 16 h at 37°C (36). For NCTC8325-4 strain, the mixture was incubated for 2 h. The reaction mixture was added to 50% trichloroacetic acid (800 µl) and centrifuged at 15 krpm for 3 min. The OD340 of the supernatant was measured, and the value multiplied by hundred was presented as an arbitrary unit.

Analysis of exoprotein profiles. Overnight cultures (2 ml) were inoculated into a flesh medium (200 ml) and 1.1, 0.5, and 0.3 ml of the cultures were sampled at OD600s of 5, 7 (t = 10 h), and 7 (t = 18 h), respectively. Supernatants of the centrifuged samples were trichloroacetic acid precipitated and centrifuged at 15 krpm for 15 min. The precipitates were washed twice with ice-cold ethanol and electrophoresed in a 12.5% polyacrylamide gel according to the method of Laemmli (25). The gel was stained with Coomassie brilliant blue.

For Western blot analysis of protein A, the proteins in the gel were electroblotted onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA) and probed with mouse antistaphylococcal protein A monoclonal antibody (Sigma Chemical Co., St. Louis, MO). The bound antibody was reacted with rabbit anti-mouse immunoglobulin G monoclonal antibody conjugated with peroxidase (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) and detected by using Western Lightning (Perkin-Elmer Life Sciences, Wellesley, MA).

To identify the protein regulated by the cvfB gene, the protein band stained with Coomassie brilliant blue was excised and digested in-gel with trypsin. Nanoscale capillary liquid chromatography-tandem mass spectrometric (LC-MS/MS) analyses of in-gel digests were done by using a quadrupole time-of-flight mass spectrometer (Micromass, Manchester, United Kingdom). Database searching was performed by using the Mascot search program (33).

Northern blot analysis. Staphylococcal cells were treated with 200 µg of lysostaphin/ml, and total RNA was extracted by using RNeasy protect bacteria kit (QIAGEN). RNA (2.5 µg) was electrophoresed in a 1.2% agarose gel containing 6.6 M formaldehyde and transferred onto a nitrocellulose membrane (GeneScreen Plus; Perkin-Elmer Life Sciences). DNA fragments of the spa gene and RNAIII were amplified by PCR using the primers listed in Table 2, labeled with [{alpha}-32P]dCTP by random priming, and used as probes. Hybridization was performed at 42°C.

Reporter assay. The luc gene (1703 bp) was amplified from pGL3 (Promega, Madison, WI) by PCR using the primers Fluc and Rluc (Table 2). Fluc was designed to contain a functional ribosomal binding site and translational start codon after a series of stop codons in all reading frames, according to the green fluorescent protein reporter plasmid constructed by Charpentier et al. (3). The amplified DNA fragment was inserted into pND50 at the XbaI and HindIII site, resulting in pCK5000. The DNA fragment (299 bp) containing the P2 promoter of the agr locus was amplified from RN4220 genomic DNA by PCR using the FP2 and RP2 primers (Table 2) according to the method of Kahl et al. (19). For amplification of the DNA fragment (257 bp) containing the P3 promoter, the FP3 and RP3 primers were used. For amplification of the DNA fragments containing the hla promoter (262 bp) and the spa promoter (295 bp), the Fhla and Rhla primers or Fspa and Rspa primers were used according to the design by Fournier et al. (10). These DNA fragments were digested with KpnI and XbaI and inserted into pCK5000 at the KpnI and XbaI site, resulting in the reporter plasmids pCK5001, pCK5002, pCK5003, and pCK5004.

The staphylococcal strains transformed with each plasmid were cultured and harvested during growth. The harvested cells were snap-frozen at –80°C. After being dissolved, the cells were lysed in a lysis buffer (100 mM KH2PO4 [pH 7.8], 0.2% Triton X-100, 0.5 mM dithiothreitol, 10 mg of lysostaphin/ml). The supernatant of the cell lysate was incubated with the luciferase substrate (100 µl; Roche, Basel, Switzerland) for 10 min, and the luminescence was measured by using a luminometer (Berthold Technologies, Bad WildBad, Germany). The promoter activity was calculated as the luminescence unit per milligram of protein subtracted with the value from the cells transformed with the vector, pCK5000.

Silkworm infection experiment. The silkworm bacterial infection experiment was performed according to the previously established method (21). Silkworms were raised from fertilized eggs to fifth-instar larvae in our laboratory. The hatched fifth-instar larvae were fed antibiotic-free artificial food (Silkmate; Katakura Industries, Tokyo, Japan) for 1 day. Bacterial suspensions (0.05 ml) were injected into the hemolymph of the larvae through the dorsal surface using a 27-gauge needle. Overnight cultures of S. aureus were diluted 20-fold with saline and used for the experiment. The injected larvae were maintained without food in a safety cabinet (BHC-1303IIA; Airtech Japan, Tokyo, Japan) at 27°C with 50% humidity, and survival was monitored for 3 days after the injection. Statistical analyses of the survival curves were performed by using Kaplan-Meier survival analysis (the log-rank test, the Prism software package; Graphpad Software, San Diego, CA). P values of <0.05 were considered statistically significant.

Mouse infection experiment. The experiment of staphylococcal systemic infection disseminated to the spleen was performed according to a previously established method (22, 35). Bacterial suspensions (100 µl, 8 x 108 CFU) were injected into the tail vein of female CD-1 mice, aged 8 weeks (Charles River Laboratories, Kanagawa, Japan). At 24 h postinfection, animals were sacrificed, and spleens were harvested and homogenized in phosphate-buffered saline. After appropriate dilution, the samples were spread on tryptic soy agar plates and incubated overnight, and the number of colonies was counted.


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RESULTS AND DISCUSSION
 
Decreased expression of hemolysin, DNase, and protease and increased expression of protein A in the cvfB deletion mutant. We previously reported that hemolysin production is decreased in the cvfB mutant (21). To examine whether the cvfB gene is involved in the production of other exotoxins, we measured the amounts of DNase and protease secreted from the mutant. DNase and protease activity in culture supernatant of the cvfB mutant (CK7) was significantly lower than that of the parental strain (CK6) (Table 3). The decreased production of exotoxin was restored by introducing plasmid p1223, which harbors the intact cvfB gene, indicating that the phenotype was caused by the deletion of the cvfB gene.


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  		TABLE 3. Decreased exotoxin activity in the cvfB mutant

These results led us to examine whether cvfB gene disruption affects the expression of other extracellular proteins. The cvfB mutant exoprotein expression profile was compared to that of the parental strain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intensity of the 58-kDa protein band (Fig. 1, green arrowheads) in the cvfB mutant was much stronger than that in the parental strain under different growth phases, i.e., late log phase, early stationary phase, and stationary phase. At the stationary phase, the amounts of the 40-kDa protein was much lower in the cvfB mutant than in the parental strain (Fig. 1, magenta arrowhead, compare CK6 to CK7 at the stationary phase). These changes in the exoprotein profiles were restored to the level of the parental strain by introducing the wild-type cvfB gene (Fig. 1, compare CK8 to CK6), indicating that the phenotype was caused by cvfB gene disruption. Protein A is an S. aureus exoprotein suggested to have a central role in the pathogenesis of staphylococcal pneumonia (14). Western blotting using monoclonal antibody against protein A showed a much stronger signal for the 58-kDa protein (green arrowheads) in the cvfB mutant than in the parental strain (bottom panel, compare CK6 to CK7). This result suggests that the cvfB gene downregulates the expression of protein A. The 40-kDa protein (magenta arrowheads) was analyzed by LC-MS/MS and was found to contain two proteins, staphopain B (SspB) that is a cysteine protease involved in pathogenesis (17) and autolysin (Atl) that is a peptidoglycan hydrolase involved in cell separation (37). The activity of autolysin was indistinguishable between the parent strain and the cvfB mutant (data not shown). SspB protein is a candidate that is upregulated by the cvfB gene.


Figure 1
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  		FIG. 1. Changed exoprotein profile and protein A expression in the cvfB mutant. Extracellular proteins at the late log phase, early stationary phase, and stationary phase were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Green arrowheads indicate proteins with increased amounts in the cvfB mutant. The magenta arrowhead represent proteins with decreased amounts in the cvfB mutant. Extracellular proteins at the late log phase and early stationary phase were subjected to Western blot analysis using an anti-protein A monoclonal antibody and are shown in the bottom panel. The signals were normalized with a 14-kDa band stained with Coomassie brilliant blue and correspond to the band indicated by green arrowheads.

Next, we sought to determine whether the effect of cvfB disruption on protein A expression is at the transcriptional level. We compared the amount of transcripts of the spa gene, which encodes protein A, in the cvfB mutant with that in the parental strain by Northern blot analysis. The amount of transcripts of the spa gene was much higher in the cvfB mutant than in the parental strain at the early log phase, late log phase, and early stationary phase (Fig. 2A, compare CK7 to CK6). The increased expression of the spa gene was reduced by introducing the wild-type cvfB gene (Fig. 2A, compare CK8 to CK7), confirming that the phenotype was caused by deletion of the cvfB gene. A reporter assay was used to examine whether the increased expression of the spa gene in the cvfB mutant were caused by altered promoter activities in the spa genes. The activity of the spa promoter was increased 1.5-fold in the cvfB mutant compared to the parental strain during the exponential phase (Fig. 2B). Thus, the cvfB gene product appears to modulate the promoter activities of the spa gene.


Figure 2
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  		FIG. 2. Increased expression of the spa gene in the cvfB mutant. (A) The transcript of the spa gene was detected by Northern blot analysis. Total RNA (2.5 µg) was electrophoresed and transferred to a nitrocellulose membrane. The membrane was probed with a 32P-labeled DNA fragment that is a part of the spa gene. The amount of 23S rRNA in the sample was determined by staining the gel with ethidium bromide as shown in the bottom panel. (B) The parent strain (RN4220) and the cvfB mutant (M1223) were transformed with the plasmid harboring the spa::luc fusion (pCK5004). Luciferase activity during growth was measured. Means ± thestandard deviations from three independent experiments are shown. *, P < 0.01.

S. aureus alpha-hemolysin is encoded by the hla gene. A reporter assay was used to examine whether the decreased hemolytic activity in the cvfB mutant was caused by decreased promoter activity in the hla gene. The hla promoter activity was decreased in the cvfB mutant compared to that in the parental strain under all growth phases (Fig. 3). Therefore, the cvfB gene activates the hla promoter.


Figure 3
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  		FIG. 3. Decreased activity of the hla promoter in the cvfB mutant. The parent strain (RN4220) and the cvfB mutant (M1223) were transformed with the plasmid harboring the hla::luc fusion (pCK5003). The luciferase activity during growth was measured. Means ± the standard deviations from three independent experiments are shown. *, P < 0.01.

Decreased RNAIII expression in the cvfB deletion mutant. In S. aureus, the agr locus upregulates the hla gene and downregulates the spa gene at the transcriptional level (9). Accordingly, we reasoned that cvfB disruption might affect the expression of the agr locus. The results of Northern blot analysis indicated that the amount of RNAIII, a transcript of the agr locus, was reduced in the cvfB mutant compared to the parental strain at the mid-exponential and early stationary phases (Fig. 4A). The decrease in the amount of the transcript was restored to the level of the parental strain by introducing a plasmid harboring the cvfB gene. These results indicate that the cvfB gene product activates RNAIII expression.


Figure 4
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  		FIG. 4. Reduced expression of agr in the cvfB mutant. (A) The amount of RNAIII was determined by Northern blot analysis. Total RNA (5 µg) was isolated from cultures at the mid-log phase (OD600 = 2.5) and early stationary phase (OD600 = 7). The amount of 23S rRNA in the sample was determined by staining the gel with ethidium bromide as shown in the bottom panel. (B and C) represent P3 activity and P2 activity, respectively. The parent strain (RN4220) and the cvfB mutant (M1223) were transformed with the plasmid harboring the P2::luc fusion (pCK5001) or the plasmid harboring the P3::luc fusion (pCK5002). The luciferase activity at the mid-log phase (OD600 = 2.5) was measured. Means ± the standard deviations of three independent experiments are presented. *, P < 0.01.

We next examined whether the reduced expression of RNAIII was due to decreased promoter activity in the cvfB mutant. RNAIII is transcribed from the P3 promoter in the agr locus (31). The result of the reporter assay demonstrated that the activity of the P3 promoter in the cvfB mutant was lower than that in the parental strain (Fig. 4B), indicating that the decreased RNAIII level in the cvfB mutant was induced by decreased P3 promoter activity in the cells.

RNAII is transcribed from the P2 promoter in the agr locus and encodes four proteins constituting a quorum-sensing system in S. aureus (30). We investigated whether cvfB disruption decreased P2 promoter activity. The results of the reporter assay indicated that P2 promoter activity in the cvfB mutant was decreased to half that in the parent strain (Fig. 4C), suggesting that the cvfB gene disruption decreases promoter activity in the agr locus, which results in a change in the expression of the downstream genes.

We then examined whether the agr disruption affects expression of the cvfB gene. The result of Northern blot analysis demonstrated that the amount of the cvfB transcript was not altered by deletion of the agr locus (data not shown), suggesting that disruption of the agr locus does not affect the expression of the cvfB gene, although the cvfB gene activates expression of the agr locus.

Regulation of exotoxin gene expression by the cvfB gene via an agr-independent pathway. To determine whether the product of the cvfB gene also regulates exoprotein gene expression via the agr-independent pathway, we examined the effect of cvfB disruption on the exoprotein expression under the agr-null background. Protein A expression was not altered by the cvfB deletion under the agr-null background (Fig. 1, bottom panel, compare CK3 to CK5), and protein A expression in the cvfB agr double mutant was reduced by a plasmid harboring the agr locus (compare CK5 to CK9). The amount of spa transcript was not altered by the cvfB deletion under the agr-null background (Fig. 2, compare CK3 to CK5) and spa expression in the cvfB agr double mutant was reduced by a plasmid harboring the agr locus (compare CK5 to CK9). Thus, the cvfB gene does not downregulate protein A expression via the agr-independent pathway. In contrast, DNase and protease activity in the culture supernatant of the cvfB agr double mutant was much lower than that in the agr mutant (Table 3). This result suggests that the cvfB gene contributes to the expression of these exotoxins via an agr-independent pathway.

We previously reported that the cvfB mutant attenuated virulence in a silkworm infection model. Here, we examined whether the cvfB deletion attenuated virulence in an agr-null background. We compared the virulence of the parent strain, the cvfB mutant, the agr mutant, and the cvfB agr double mutant. Half of the silkworm larvae injected with the parent strain were killed at 30 h after injection, whereas half of those injected with agr mutant were killed at 60 h after the injection (Fig. 5). The cvfB mutant showed less virulence than the agr mutant. Moreover, more than 70% of the silkworm larvae injected with the cvfB agr double mutant survived even at 72 h after the injection, which was a higher survival rate than for those injected with the agr mutant. In a murine model of systemic infection, the number of viable cells of the cvfB mutant was 10 times lower than for the parental strain (Fig. 6). The number of viable cells of the cvfB agr double mutant was 10 times lower than that of the agr mutant (Fig. 6). Thus, the attenuation of virulence by cvfB disruption was observed even under the agr-null background. These results suggest that the attenuated virulence by cvfB disruption was caused not only by the decreased expression of the agr locus but also by a functional disorder of the agr-independent pathway.


Figure 5
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  		FIG. 5. Attenuated virulence caused by the cvfB disruption under the agr-null background in silkworm infection model. Ten silkworms were injected with 10-fold-diluted overnight cultures (50 µl, 3.5 x 107 CFU) of the parent strain (RN4220) and the cvfB mutant (M1223), the agr mutant (CK3), and the cvfB agr double mutant (CK5), and larval survival was monitored. The curves are representative of at least three independent experiments. P values between RN4220 and M1223 and between CK3 and CK5 were <0.05, respectively.


Figure 6
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  		FIG. 6. Attenuated virulence caused by the cvfB disruption in an agr-null background in mouse infection model. Five or six CD-1 mice were injected via the tail vein with bacterial suspensions (100 µl, 8 x 108 CFU) of parent strain (RN4220), the cvfB mutant (M1223), the agr mutant (CK3), and the cvfB agr double mutant (CK5). The numbers of bacteria in the spleens were determined at 24 h after infection. The horizontal bar shows the group mean. Student t test P values between RN4220 and M1223 and between CK3 and CK5 are shown.

RN4220 is a strain mutagenized by nitrosoguanidine exposure to accept foreign DNA. There might be uncharacterized mutations emphasizing the involvement of the cvfB gene in exotoxin gene expression. We examined the effect of the cvfB disruption in the nonmutagenized strain NCTC8325-4. Disruption of the cvfB gene in NCTC8325-4 did not cause decrease in hemolysin and DNase (Table 4, compare CK10 to CK11). The protease activity was decreased in the cvfB mutant, but it was not complemented by a plasmid harboring the intact cvfB gene. Disruption of the cvfB gene in the agr mutant of NCTC8325-4 caused a decrease in hemolysin, DNase, and protease activities (Table 4, compare CK13 to CK14). The decreased exotoxin production in the cvfB agr double mutant was restored by a plasmid harboring the intact cvfB gene (Table 4, compare CK14 to CK15). These results indicate that the cvfB gene contributes to the expression of hemolysin, DNase, and protease production via an agr-independent pathway, but its effect is masked by an agr-dependent pathway in NCTC8325-4 strain. The different effects of the cvfB disruption on the exotoxin expression between RN4220 and NCTC8325-4 could be due to mutations in the RN4220 strain. For example, RN4220 contains a mutation in the agrA gene, which results in the ineffective expression of hemolysin (38). The cvfB disruption might cause a decrease in exotoxin when the function of the agr locus is impaired.


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  		TABLE 4. Involvement of the cvfB gene in exotoxin production in NCTC8325-4 strain

The present study demonstrated that the cvfB gene positively regulates expression of the agr locus, a virulence regulator in S. aureus. P2 and P3 promoter activity in the agr locus and the amount of RNAIII, the transcript from the P3 promoter, were reduced in the cvfB mutant of the RN4220 strain. An increase in the amount of protein A caused by agr disruption was also observed in the cvfB deletion mutant, and the cvfB disruption did not increase the expression of protein A in an agr-null background of the RN4220 strain. Thus, the cvfB gene regulates the expression of protein A in an agr-dependent manner.

In contrast, the effects of cvfB disruption under the agr-null backgrounds of RN4220 and NCTC8325-4 suggest that the cvfB gene regulates the expression of protease and DNase in an agr-independent manner. The results obtained from analysis using the silkworm infection model and the mouse infection model also indicated that the cvfB gene contributes to virulence via the agr-independent pathway. The simultaneous regulation of the two pathways, agr dependent and agr independent, by the cvfB gene is presumed to be important for the regulation of virulence in S. aureus. The sarA gene also contributes to the expression of virulence genes via agr-dependent and agr-independent pathways (41). The presence of unidentified pathways that regulate both the expression of the agr locus and other regulatory transcription factors according to extracellular signals such as nutrients, oxygen, and NaCl was predicted (28). The cvfB gene might regulate the expression of virulence genes through such a pathway.

The cvfB gene is conserved among various pathogenic bacteria, such as Streptococcus pyogenes and Listeria monocytogenes (21). A recent study suggested that the fas locus, a homolog of the agr locus, contributes to virulence in S. pyogenes (23). Also, a gene homologous with the agr locus of S. aureus is involved in virulence in L. monocytogenes (1). The cvfB genes in these bacteria are expected to contribute to the expression of the agr homologs and virulence. An understanding of the mechanism by which the cvfB gene regulates exoprotein expression will help to define the fundamental mechanism by which various pathogenic bacteria regulate the expression of virulence genes.


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ACKNOWLEDGMENTS
 
We thank R. P. Novick, K. Hiramatsu, T. J. Foster, and N. Ogasawara for kindly providing bacterial strains and plasmids.

This study was supported in part by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science.


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FOOTNOTES
 
* Corresponding author. Mailing address: Graduate School of Pharmaceutical Sciences, University of Tokyo, 3-1, 7-Chome, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Phone: 81 3 (5841) 4820. Fax: 81 3 (5684) 2973. E-mail: sekimizu{at}mol.f.u-tokyo.ac.jp. Back

{triangledown} Published ahead of print on 5 February 2007. Back

Editor: J. B. Bliska


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Infection and Immunity, April 2007, p. 1964-1972, Vol. 75, No. 4
0019-9567/07/$08.00+0     doi:10.1128/IAI.01552-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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