Regulation of Staphylococcus aureus Capsular Polysaccharide

INFECTION AND IMMUNITY, Feb. 2002, p. 444–450 0019-9567/02/$04.00⫹0 DOI: 10.1128/IAI.70.2.444–450.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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Regulation of Staphylococcus aureus Capsular Polysaccharide Expression by agr and sarA Thanh Luong,1 Subrata Sau,1† Marisa Gomez,2‡ Jean C. Lee,2 and Chia Y. Lee1* Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66160,1 and Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital and Harvard University Medical School, Boston, Massachusetts 021152 Received 5 June 2001/Returned for modification 2 August 2001/Accepted 31 October 2001

This study addresses the regulation of Staphylococcus aureus type 8 capsular polysaccharide (CP8) expression by the global regulators agr and sarA. We analyzed CP8 production, cap8-specific mRNA synthesis, and blaZ reporter gene activities of the transcriptional and translational fusions in strain Becker and its agr, sarA, and agr-sarA isogenic mutants during different phases of bacterial growth. In the wild-type strain, cap8 mRNA was undetectable until the mid-logarithmic phase of growth, whereas CP8 production was undetectable until 2 h later, at the onset of stationary phase. The delay most likely reflects the time needed for completing CP8 synthesis resulting from translation of cap8 mRNA. The agr mutation caused drastic reductions in CP8 production and cap8 gene transcription, suggesting that agr is a major positive regulator of CP8 expression. The results of gene fusion studies indicated that regulation by agr is exerted at the transcriptional level. In contrast, the sarA mutation caused only a slight reduction in cap8 mRNA synthesis and reporter gene activities. By comparing CP8 production and cap8 transcription, we observed that sarA affected CP8 production both trancriptionally and posttranslationally. We showed that agr was a major activator for cap gene expression not only in type 8 strain Becker but also in strains representing the four agr groups. of the first gene, cap8A. Although several internal promoters within the cap8 gene cluster have also been identified by genetic complementation and reporter gene fusion studies, these internal promoters are much weaker than the primary promoter. Thus, regulation of the large cap8 operon likely occurs at the promoter upstream of the cap8A gene (39). Recently, we have shown that a 10-bp inverted repeat just upstream of the ⫺35 sequence of the primary cap8 promoter is required for full expression of CP8 (35), suggesting that CP8 production is regulated. Indeed, several lines of evidence indicate that CP5 and CP8 are highly regulated in various environmental conditions (23). On the other hand, studies on the transcription of cap1 genes suggest that the primary cap1 promoter is constitutively expressed (34). The virulence genes of S. aureus have been shown to be regulated by at least two global regulators, agr and sarA. The agr locus is a complex multigene system that regulates virulence genes by sensing cell density. The agr locus consists of two divergent transcriptional units. The P2 operon contains four genes, agrBDCA, in that order, whereas transcription from the P3 promoter produces a 512-nucleotide transcript, RNAIII. The RNAIII transcript encodes a gene for ␦-hemolysin (26 amino acids); however, the RNAIII itself, not its translated product, is the actual effector for the agr system. AgrD is an autoinducer propeptide which is believed to be processed and secreted by AgrB to form a small autoinducing octapeptide (reviewed in reference 28). At high cell density, binding of the octapeptide (accumulated in the medium) to the membrane receptor AgrC activates AgrA regulator by a phosphorelay mechanism typical of the bacterial two-component system (26, 30). The phosphorylated AgrA upregulates, by an unknown mechanism, both P2 and P3 promoters to produce the regulatory effector, RNAIII. Thus, the agr system is turned

More than 90% of Staphylococcus aureus strains produce capsular polysaccharide (CP). Eleven serotypes of staphylococcal CP have been identified, but only CP type 1 (CP1), CP2, CP5, and CP8 have been chemically characterized. CP1 and CP2 have been shown to be antiphagocytic virulence factors. Strains producing CP1 and CP2 are heavily encapsulated; however, these strains are rarely isolated clinically. In fact, more than 80% of clinical isolates produce either CP5 or CP8 (see reference 23 for a review). Recently, CP5 has been shown to play an important role in the pathogenesis of S. aureus, most probably by allowing the organism to resist uptake and killing by phagocytes (1, 27, 43). Because of their prevalence, CP5 and CP8 have been used as targets for vaccine development, and specific antibodies against CP5 and CP8 have been shown to be protective against S. aureus infections (13, 24). The cap1, cap5, and cap8 gene clusters, required for the synthesis of CP1, CP5, and CP8, respectively, have been cloned and sequenced. The cap5 and cap8 operons are allelic, whereas the cap1 locus is located at a different location. Twelve of the 16 genes in the cap5 and cap8 operons have high degrees of similarity, which reflects the fact that the repeating units of CP5 and CP8 are almost identical (23). Transcriptional analyses have shown that all 16 genes of the cap8 locus are transcribed as a large transcript from a major promoter upstream

* Corresponding author. Mailing address: Department of Microbiology, Molecular Genetics and Immunology, Room 3025, WHW, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160. Phone: (913) 588-7156. Fax: (913) 588-7295. E-mail: clee @kumc.edu. † Present address: Department of Biochemistry, Bose Institute, Calcutta, India. ‡ Present address: School of Medicine, University of Buenos Aires, Buenos Aires, Argentina. 444

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TABLE 1. S. aureus strains and plasmids Strain or plasmid

Relevant characteristics

Strains RN4220 Becker RN6911 CYL6191 CYL608 CYL609 RN6734 RN9130 RN9120 RN8465 RN8846 RN4850 RN9121 Reynolds MBC327 Plasmids pGEMT-Easy pLL28 pSA3800 pWN1819 pCL394 pLL588

Source or reference

8325-4r⫺ CP8 strain RN6390 ⌬agr::tetM Becker ⌬agr::tetM Becker ⌬sar Becker ⌬agr::tetM ⌬sar agr group I prototype, CP nontypeable agr group II prototype, CP nontypeable RN9130 ⌬agr::tetM agr group III prototype, CP8 strain RN8465 ⌬agr::tetM agr group IV prototype, CP8 strain RN4850 ⌬agr::tetM CP5 strain Naturally occurring cap-negative strain

21

Cloning vector Temperature-sensitive cloning vector Transcriptional blaZ fusion vector Translational blaZ fusion vector cap8::blaZ transcriptional fusion in pSA3800 cap8::blaZ translational fusion in pWN1819

Promega This study 29 44 This study This study

on at high cell density, which activates extracellular proteins, such as exotoxins, and simultaneously represses surface antigens, such as protein A and surface adhesins (28). Various S. aureus strains exhibit sequence variations in AgrC, AgrD, and AgrB which affect the specificity of the receptor-ligand interaction. On the basis of autoinducer-receptor specificity, S. aureus strains have been categorized into four different agr groups (17, 18). Each of the secreted autoinducing peptides can activate the agr response within the same group but inhibit the agr response in strains belonging to the other groups (18). This type of bacterial interference may influence the dominance of a particular strain in localized infections. The sar locus contains a regulatory gene, sarA, with three different upstream promoters and only one downstream transcription terminator (28). The sarA gene can activate agr promoters, and thus it can work in concert with the agr system (4, 8, 9, 36). However, the sarA gene can also activate certain virulence genes independently of agr (2, 3, 5–7). Recently, it has been shown that SarA binds to a consensus motif upstream of the ⫺35 sequences of the promoters of sarA-dependent genes (10). Previously, agr was shown to be a positive regulator for CP5 production in various media (12). However, whether agr regulates CP5 at the transcriptional or translational level was not addressed. The regulation of staphylococcal CP by sarA has not been reported. Here we report the regulation of CP8 production by the agr and sarA loci. MATERIALS AND METHODS Strains, plasmids, and growth conditions. The S. aureus strains used in this study are listed in Table 1. Escherichia coli strain XL1-Blue was used as a host strain for plasmid constructions. S. aureus RN4220 (21) was used as the recipient in electroporations of the constructed plasmids and for direct transformations from ligated DNA. S. aureus strains were cultivated in Trypticase soy medium (Difco Laboratories, Detroit, Mich.) unless indicated otherwise. E. coli strains were cultivated in Luria-Bertani medium (Difco Laboratories). Electroporation in S. aureus was carried out by the procedure of Kraemer and Iandolo (20).

31; A. L. Cheung This study This study This study R. Novick R. Novick R. Novick R. Novick R. Novick R. Novick R. Novick 41

Transduction was carried out as described by Shafer and Iandolo (40) using bacteriophage 52A (22). Nitrocefin was purchased from Becton Dickinson Microbiology Section (Sparks, Md.). For growth phase-dependent experiments, bacteria were grown in broth media from overnight cultures diluted to an optical density at 660 nm (OD660) of 0.05. DNA manipulations. Standard DNA manipulations were performed as described by Sambrook et al. (37). Plasmid DNA was purified with a plasmid purification kit (Qiagen, Inc., Chatsworth, Calif.). Rapid small-scale plasmid DNA purification from E. coli was done by the method of Holmes and Quigley (16). Bulk chromosomal DNA from S. aureus was purified with a chromosomal DNA purification kit (Promega, Madison, Wis.). PCR amplification was carried out with the Advantage cDNA PCR kit (Clontech, Palo Alto, Calif.). The transfer of DNA to nitrocellulose membranes was done by the method of Southern (42). Mutant strain construction. The S. aureus Becker agr mutant (CYL6191) was constructed by transducing the agr mutation (⌬agr::tetM) from RN6911 (31) to strain Becker using phage 52A and selecting for transductants with tetracycline at 5 ␮g/ml. To construct the Becker ⌬sarA mutant (CYL608), we first constructed plasmid pTL2807 by ligating two PCR-amplified fragments, a 999-bp fragment containing the upstream sequence and the N-terminal half of the sarA gene using primers GAG CTC TTG GGT AGT ATG CTT TGA CAC A and CCG CGG CTG ATG TAT GTC AAT ACA GCG and a 1,029-bp fragment containing the C-terminal half and the downstream sequence of the sarA gene using primers GGA TCC TTG TTA ATG CAC AAC AAC GTA and AAG CTT GCA ACA TCA ACT AGC ATC ATC, so that the two fragments flanked the cat gene in pLL28. The cat gene was then deleted by restriction digestion and religation. Plasmid pTL2807 was electroporated into strain RN4220 and transduced into strain Becker by using phage 52A at 30°C, with selection for tetracycline resistance at 3 ␮g/ml. The resulting strain, Becker(pTL2807), was then used for constructing the sarA mutant by temperature shift and replica-plating to screen for tetracycline-sensitive colonies as described previously (39). The desired mutant in which the 152 bp within the wild-type sarA coding region were replaced by the 18-bp sequence of the multiple cloning site of the vector was confirmed by PCR amplification and Southern hybridization (results not shown). To construct the Becker agr sarA double mutant (CYL609), the agr mutation from RN6911 was transduced by phage 52A into strain CYL608, with selection for tetracycline resistance. Construction of fusion plasmids. The bla vectors pSA3800 (29) and pWN1819 (44) were used to construct transcriptional and translational fusions, respectively. The two plasmids are essentially the same except that the ribosome-binding site of the promoterless blaZ gene in pSA3800 is intact and that in pWN1819 is deleted. Thus, a promoter fusion in pSA3800 would place the blaZ gene under the transcriptional control of the promoter, whereas an in-frame fusion of the

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promoter along with the 5⬘ portion of a gene to the reporter gene in pWN1819 would place blaZ under the transcriptional and translational control of the promoter. To construct Pcap8::blaZ fusions, plasmid pCL7816 with a 5.7-kb insert that contains the primary cap8 promoter region (35) was used as the template in a PCR amplification using two primers (CTG CAG GAG CTC GCA TTT GAA GAT CA and GGA TCC CCT TAG TTT GAT TCA CTA AA) to generate a 627-bp fragment containing Pcap8. The PCR-amplified fragment was first cloned into pGEMT-Easy (Promega), digested with PstI and BamHI, and ligated into the similarly digested bla vectors. The ligated DNA was electroporated into RN4220, with selection for chloramphenicol at a concentration of 10 ␮g/ml. The fusion plasmids were verified by sequencing and transduced into strain Becker and its derivatives by phage 52A transduction. ␤-Lactamase assays. BlaZ activity was assayed by the nitrocefin method (32) as described by Yoon et al. (47) with slight modifications. Briefly, culture cells were added with an equal volume of warm phosphate buffer (0.1 M, pH 7.0) and 2 volumes of warm nitrocefin solution (180 ␮g/ml in phosphate buffer). The mixture was incubated at 37°C for 10 min. Afterward, 12 volumes of ice-cold phosphate buffer were added to the mixture and incubated on ice for 5 min. The cells were pelleted, and the supernatant was measured at 482 nm. The specific BlaZ activity of a culture was expressed as the ratio of the OD482 to the OD660 of the culture. Quantification of CP8. CP8 was quantified by the immuno-slot-blotting method with rabbit anti-CP8 antiserum. Broth cultures were pelleted, washed once with phosphate-buffered saline (PBS), and resuspended in 10 ␮l of PBS per OD660 unit. The suspension was autoclaved at 121°C for 60 min (25). Cell debris was removed by centrifugation, and the supernatant was treated with proteinase K (Sigma, St. Louis, Mo.) at 50 ␮g/ml for 1 h at 37°C to remove protein A. Proteinase K was subsequently inactivated by heat treatment at 75°C for 10 min. The crude CP8 preparations were loaded on a nitrocellulose membrane in a slot-blot apparatus (Bio-Rad Laboratories, Hercules, Calif.) and washed with 2 to 4 volumes of PBS. The membrane was incubated with rabbit anti-CP8 antiserum for 1 h, washed with PBS, and then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin antiserum (Sigma) for 1 h. The membrane was finally developed using the horse radish peroxidase color development kit (Bio-Rad Laboratories). RNA analysis. Broth cultures were pelleted, washed once with PBS, and resuspended in 10 ␮l of PBS per OD660 unit. Total RNA was isolated by using the RNeasy Total RNA system (Qiagen) and treated with DNase I. Five micrograms of each sample was loaded and assayed by Northern slot-blotting as described by Sambrook et al. (37), except the washing was done at 65°C. For quantitating cap mRNA produced from strains of various agr groups and their corresponding agr mutants, dot-blotting analysis was performed. Strains cultivated in Columbia broth containing 2% NaCl at an OD650 of 0.8 to 1.0 were harvested and lysed with glass beads in a dental amalgamator (46). The RNAs were isolated by the RNeasy Total RNA system (Qiagen) and treated with DNase I and RNase OUT (Life Technologies, Gaithersburg, Md.). Serial twofold dilutions from 3 ␮g of each sample were used in dot-blotting analyses. Statistical analysis. Data from reporter gene fusion analyses were assessed by repeated measures analysis of variance with the Tukey test for multiple comparisons between means. P values of ⬍0.05 were considered statistically significant.

RESULTS Strain construction and characterization. To study the regulation of agr and sarA on cap8 gene expression in strain Becker, we first constructed agr (CYL6191) and sarA (CYL608) single mutants and an agr sarA (CYL609) double mutant from strain Becker as described in Materials and Methods. Since agr and sarA have been shown to temporally regulate their target genes, it is necessary to determine the growth curves of these mutants. Our data (results not shown) showed that although there were differences between strains in cell density at various time points, all four strains reached the stationary growth phase at about 7 h after incubation of the cultures with the initial inoculum of 0.05 OD660 at 37°C, indicating that the growth curves of the wild-type Becker and its mutant derivatives are comparable. Thus, for the growth phase-dependent experiments described below, samples were taken at specific time points.

FIG. 1. Analysis of CP8 production in Becker, CYL6191 (Becker agr), CYL608 (Becker sarA), and CYL609 (Becker agr sarA). Samples of the cultures (indicated to the left of the figure) were taken at specific times (indicated above the lanes) after inoculation and incubated with rabbit anti-CP8-specific antibody.

Effect of agr and sarA on production of CP8. To analyze agr and sarA regulation of capsule production by strain Becker, we first measured the production of CP8 from the wild-type strain, agr, sarA, and agr sarA mutants. Samples of each culture were taken at 2 h (early log phase), 4 h (mid-log phase), 6 h (logstationary transition phase), 8 h (early stationary phase), and 24 h (late stationary phase). The data from one of the experiments are shown in Fig. 1. These results showed that in the wild-type strain, CP8 was not produced during log phase (2 and 4 h) but was significantly produced at the onset of stationary phase (6 h). CP8 production peaked at 8 h, and no significant increase was found after 24 h of incubation. In the agr mutant and agr sarA double mutant, the amounts of CP8 were reduced to almost undetectable levels at all time points. These results indicate that agr is a strong activator of CP8 production. Moreover, CP8 appears to be regulated similarly to staphylococcal exotoxins in that high bacterial cell density induces their production due to activation of the agr system. In contrast to the agr mutant, we found that the production of CP8 by the sarA mutant increased at 2 h, decreased to undetectable levels at 4 h, and gradually increased from 6 h to the maximal level at 24 h. The amount of CP8 produced in the sarA mutant was about fourfold higher at 2 h, twofold less at 6 h, slightly less at 8 h, but slightly more at 24 h than that in the wild-type strain. Thus, the effect of the sarA mutation differed significantly from that of the agr mutation and seemed to be dependent on the growth phase of the bacterium. These results were reproducible, indicating that they are not due to an artifact. Effect of agr and sarA on synthesis of cap8 mRNA. To determine whether the effect of agr and sarA occurs at the transcriptional level, we measured cap8-specific mRNA synthesis by RNA slot-blotting. Samples of the cultures were taken for total RNA preparation at the same time points as for the CP8 measurements above. The RNAs were probed with either the cap8D or the cap8N gene probe (we found no difference between the two probes), and a duplicate set were probed with the 16S rRNA gene. An example of the blots is shown in Fig. 2. In the wild-type strain, significant amounts of cap8 transcript could be detected as early as 4 h. Transcription reached the peak level at about 6 h, and the level remained constant even after 24 h of incubation. This pattern was generally similar to that of the CP8 production (Fig. 1) except that there was a 2-h difference between mRNA synthesis and CP8 production. The difference may be a result of the time needed for translation of the enzymes required for the synthesis of the monosaccharide precursors, assembly of the repeating units, transport of the

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FIG. 2. Analysis of cap8-specific mRNA taken from the same strains and at the same time points as in Fig. 1. The membrane was probed with the cap8D gene (top panel). A duplicate set of samples were probed with the S. aureus 16S rRNA gene (bottom panel) to indicate equal loading of the samples.

repeating units across the membrane, and anchoring of the polysaccharides to the cell wall. The agr mutant and the agr sarA double mutant showed drastically reduced cap8 gene transcription, as very limited amounts of cap8-specific mRNA could be detected. These results almost mirrored those of CP8 production shown in Fig. 1, indicating that agr is a major activator, which regulates CP8 production primarily at the transcriptional level. In contrast, the sarA mutation reduced transcription only moderately. Densitometric measurements showed that the sarA mutation resulted in about a twofold reduction in cap8 mRNAs compared with the wild-type strain Becker. The synthesis of cap8 mRNA by the sarA mutant was reduced at various growth phases compared to that of the wild type, indicating that sarA is a minor activator that commences its activation at mid-log phase through stationary phase but to a lesser extent than agr. Interestingly, however, the pattern of CP8 production at different growth phases shown in Fig. 1 was very different from that of cap8 mRNA synthesis shown in Fig. 2. In particular, we found that a significant amount of CP8 was detected at 2 h even though no cap8 mRNA was detected and that CP8 production increased from 6 to 24 h but the cap8 transcript level remained constant over this period. Thus, the pattern of CP8 production at different growth phases did not match that from the mRNA analyses, suggesting that sarA can also affect CP8 production at the posttranscriptional level. agr and sarA regulate cap8 gene expression at the transcriptional level. The above results suggest that agr mostly regulates the CP8 production at the level of cap8 gene transcription and that sarA regulates production both transcriptionally and posttranscriptionally. To confirm the transcriptional regulation of cap8 gene expression by agr and sarA and to determine whether sarA exerts its additional posttranscriptional effect on CP8 production at the translational or the posttranslational level, we constructed blaZ reporter gene fusions which resulted in transcriptional fusion plasmid pCL394 and translational fusion plasmid pLL588. The insert in these constructs contains the primary cap8 promoter and the 5⬘ end of the cap8A gene (429 bp upstream and 186 bp downstream of the transcriptional start site of the primary transcript). In pLL588, the construction resulted in an in-frame fusion of the first 55 codons of the first gene, cap8A, to the blaZ gene in pWN1819. The fusion plasmids were then transferred into strain Becker and its mutant derivatives. BlaZ activities were measured at 2,

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4, 6, 8, and 24 h. The BlaZ activities of the control strains (i.e., Becker and its derivatives each containing pSA3800 or pWN1819) were also measured simultaneously, which was necessary because we found that strain Becker and its isogenic mutants expressed a significant background level of BlaZ activity. As shown in Fig. 3, the agr mutation reduced cap8 gene expression considerably in both transcriptional fusion and translational fusion analyses. In the sarA mutant, the mutation only reduced about 50% of the activities at all time points compared to that of the wild type in both fusions. Statistical analyses showed that the differences were significant at 6 h in the transcriptional fusion assay and at all time points except 2 h in the translational fusion assay. The agr sarA double mutation, like the agr single mutation, greatly reduced the BlaZ activities at all time points. However, the activities in the agr mutant were consistently less than those of the agr sarA double mutant, although there is no statistically significant difference between the two mutants in the two assays. These results were generally consistent with those of the RNA slot-blotting described above. The fact that the blaZ activities in these mutants decreased in a similar pattern in both transcriptional and translational fusions suggests that the regulation of cap8 genes by agr and sarA is exerted at the transcriptional level rather than at the translational level. Since we found no translational regulation of cap8 gene expression by sarA, the additional level of sarA regulation on CP8 must be exerted posttranslationally. Taking the fusion results together with the fact that CP8 in the sarA mutant was produced in gradually increasing amounts from 6 to 24 h and yet the mRNA remained at the same level during that period (comparing Fig. 1 and 2), we concluded that sarA also posttranslationally affected the stability of CP8. agr is a major activator of cap gene expression in different agr groups of S. aureus strains. The above results indicate that agr is a major regulator for cap8 gene expression in strain Becker. Since virulence genes in different agr groups may be regulated differently (18), we sought to determine whether agr in strains belonging to different agr groups exerts its regulatory effects differently on cap gene expression. To this end, we determined the capsular serotypes and genotypes of isogenic pairs of S. aureus strains representing each of the four agr groups. As shown in Table 1, strains RN8465 (group III) and RN4850 (group IV) produced CP8, whereas strains RN6734 (group I) and RN9130 (group II) were nontypeable. However, the results of hybridization experiments indicated that both the RN6734 and RN9130 strains carried an intact cap5 locus. Total RNA from these strains and their respective isogenic agr mutants was isolated and used in RNA dot-blotting analyses with cap5ABCD (common to the cap5 and cap8 gene clusters) as a probe. In addition, strain Reynolds (CP5 positive with undetermined agr group) and strain MBC327, lacking the cap5(8) locus in the genome, were used as positive and negative controls, respectively. The RNAs were also probed with the housekeeping recA gene. The results (Fig. 4) showed that, in all cases, the agr mutation resulted in dramatic reductions in cap mRNA expression, although the effect of the agr mutation in the group II and group IV strains was slightly less than that in

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FIG. 3. Specific BlaZ activities of plasmid pCL394 containing the cap8A::blaZ transcriptional fusion (A), and of plasmid pLL588, containing the cap8::blaZ tanslational fusion (B), in the same strains as shown in Fig. 1. Data were obtained by subtracting the basal levels produced from control strains containing the vectors at each time point. Results represent averages of at least three independent experiments. Error bars indicate standard error of the mean.

other strains. These data indicate that agr is a strong activator of cap gene expression regardless of the agr group. DISCUSSION In this study, we evaluated the regulation of S. aureus CP8 expression by the global regulators agr and sarA. An examina-

FIG. 4. Analysis of cap5- and cap8-specific mRNA in strains from different agr groups and their agr isogenic mutants. RNA samples with two fold serial dilutions were probed with cap5ABCD, common genes between the cap5 and cap8 operons (top panel). A duplicate set of samples were probed with the S. aureus recA gene (bottom panel) to indicate equal loading of the samples. Representatives of agr groups were used. Group I, RN6734 (agr⫹) and RN6911 (agr); group II, RN9130 (agr⫹) and RN9120 (agr); group III, RN8465 (agr⫹) and RN8846 (agr); group IV, RN4850 (agr⫹) and RN9121 (agr). Type 5 strain Reynolds (lane 1) and a cap mutant strain, MBC327 (lane 2), were used as positive and negative controls, respectively. ND, not determined. Both cap and agr genotypes are also indicated.

tion of cap8 mRNA production by the wild-type strain Becker and its isogenic mutants at various growth phases showed that agr was the main positive regulator of CP8 production. These results confirmed a previous report from Dassy et al. (12), who showed that CP5 production was reduced in an agr mutant of strain Newman in various media. Since the sequences of the promoter regions of the cap5 and cap8 operons are almost identical (38), it is not surprising that CP5 and CP8 are regulated similarly. However, it has been reported that microcapsules could be regulated differently by carbon dioxide depending on the strain being studied (15). For this reason, we examined members of the four agr groups (17, 18) to determine whether the agr locus positively regulated CP expression in strains with different genetic backgrounds. In each case, a mutation in the agr locus resulted in abrogation or a drastic reduction in cap5- or cap8-specific mRNA levels. The results were similar among strains carrying either the cap5 or cap8 gene. It is noteworthy that two of the strains were phenotypically capsule negative, although they made wild-type levels of cap5-specific mRNA. Like strain NCTC 8325 (45), these nontypeable strains may have point mutations in any one of the cap5 genes that are essential for CP5 production. We also studied the agr and sarA regulation of cap8 gene expression by transcriptional and translational reporter gene fusions. These fusion studies are possible because we previously found that the cap8 operon was transcribed primarily from a major promoter located upstream of the first gene, cap8A. The results of these fusion studies along with those of

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the CP8 assays and mRNA analyses indicate that the regulation of CP8 by agr is mostly exerted at the level of transcription. In the case of sarA, the results indicate that sarA also activates the cap8 gene expression at the transcriptional level, though the effect is only minor compared to that of agr. Most interestingly, we found that sarA also exerted its regulation on CP8 production posttranslationally by affecting CP8 stability. The increased stability of CP8 in the sarA mutant could explain why a significant amount of CP8 was detected at early logarithmic phase (Fig. 1)—the stable CP8 would result in a large amount of CP8 accumulated in overnight cultures so that the carryover CP8 from the inoculation could still be detected even after 2 h of incubation. However, how sarA affects CP8 stability requires further studies. agr is a quorum-sensing system which is activated by high cell density (28). This mode of virulence gene regulation by agr is thought to reflect actual expression of virulence factors during bacterial infections. At the initial stage of infection, when cell density is low, cell surface proteins, including adhesins, are derepressed, which allows initial attachment or colonization of the bacterial cells onto the host tissues at an infection site. At the later stage of infection, when cell density is high, exoproteins such as membrane-damaging toxins which degrade surrounding tissues and allow spread of the bacterial cells to other infection sites are upregulated. Our results in this study indicate that CP8, unlike the surface-associated proteins, is hardly expressed until the onset of stationary phase. This mode of expression is similar to that of exoproteins, suggesting that capsule is important for spreading rather than initial colonization. Indeed, Kiser et al. (19) demonstrated that a mutant defective in CP5 production colonized the nares of mice as well as the parental strain at 1 week, but it showed reduced levels of colonization 2 weeks after inoculation. Furthermore, CP1 and CP2 have been implicated in the masking of cell surface adhesins (11, 14, 33). Thus, CP5 and CP8 may not have a role in initial colonization, but they may play a role during spreading from the initial site to the secondary sites. Several recent studies have suggested that CP5 confers bacterial virulence as an antiphagocytic factor (1, 43). As such, one could postulate that the upregulated CP5 or CP8 in the highcell-density condition could serve as an important protection for the bacterial cells from phagocytosis during translocation from the initial infection site. However, once staphylococcal cells leave the initial infection site, they would encounter lowcell-density environments, such as the host blood stream, in which the agr system is turned off, resulting in loss of cap gene expression. Nonetheless, as demonstrated in this study, CP production could continue for a period of time even after transcription of the cap genes had been terminated by agr inactivation. Continued synthesis of CP after transcription termination could enable the bacterial cells to reach secondary infection sites while still covered with enough CP to evade phagocytosis. We previously showed that a 10-bp inverted repeat just upstream of the ⫺35 site was required for primary cap8 promoter activity. The inverted repeat could serve as a DNA-binding site for an activator or a complex of activators in regulating cap8 gene transcription (35). Since agr and sarA positively regulate cap8 gene transcription, it is possible that agr or sarA exerts the regulation through this 10-bp inverted repeat either by directly

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binding to this repeat or by indirectly regulating the putative activator. However, the DNA-binding target of RNAIII, the effector of the agr system, has not been identified, whereas there is no resemblance between the sequence of the 10-bp inverted repeat and the sarA consensus binding sequence (10). Thus, it is unlikely that agr or sarA binds directly to this inverted repeat. Instead, it is most likely that agr or sarA regulates CP8 expression by indirectly regulating the putative DNA-binding activator. Identification of the putative activator gene, which is currently under way, is needed to test this possibility. ACKNOWLEDGMENTS We thank A. L. Cheung for providing strain RN6911 and R. P. Novick for providing the four agr mutant pairs. This work was supported by grant AI37027 to C.Y.L. and by grant AI29040 to J.C.L. from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. Bhasin, N., A. Albus, F. Michon, P. J. Livolsi, J.-S. Park, and J. C. Lee. 1998. Identification of a gene essential for O-acetylation of the Staphylococcus aureus type 5 capsular polysaccharide. Mol. Microbiol. 27:9–21. 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. 3. Chan, P. F., and S. J. Foster. 1998. Role of sarA in virulence determinant production and environmental signal transduction in Staphylococcus aureus. J. Bacteriol. 180:6232–6241. 4. Cheung, A. L., M. G. Bayer, and J. H. Heinrichs. 1997. sar genetic determinants necessary for transcription of RNAII and RNAIII in the agr locus of Staphylococcus aureus. J. Bacteriol. 179:3963–3971. 5. Cheung, A. L., K. J. Eberhardt, E. Chung, M. R. Yeaman, P. M. Sullam, M. Ramos, and A. S. Bayer. 1994. Diminished virulence of a sar⫺/agr⫺ mutant of Staphylococcus aureus in the rabbit model of endocarditis. J. Clin. Investig. 94:1815–1822. 6. Cheung, A. L., K. Eberhardt, and J. H. Heinrichs. 1997. Regulation of protein A synthesis by the sar and agr loci of Staphylococcus aureus. Infect. Immun. 65:2243–2249. 7. Cheung, A. L., and P. Ying. 1994. Regulation of alpha- and beta-hemolysins by the sar locus of Staphylococcus aureus. J. Bacteriol. 176:580–585. 8. Chien, Y., and A. L. Cheung. 1998. Molecular interactions between two global regulators, sar and agr, in Staphylococcus aureus. J. Biol. Chem. 273: 2645–2652. 9. Chien, Y., A. C. Manna, and A. L. Cheung. 1998. SarA level is a determinant of agr activation in Staphylococcus aureus. Mol. Microbiol. 30:991–1001. 10. Chien, Y., A. C. Manna, S. J. Projan, and A. L. Cheung. 1999. sarA, a global regulator of virulence determinants in Staphylococcus aureus, binds to a conserved motif essential for sar-dependent gene regulation. J. Biol. Chem. 274:37169–37176. 11. Cifrian, E., A. J. Guidry, C. N. O’Brien, and W. W. Marquardt. 1995. Effect of alpha-toxin and capsular exopolysaccharide on the adherence of Staphylococcus aureus to cultured teat, ductal and secretory mammary epithelial cells. Res. Vet. Sci. 58:20–25. 12. Dassy, B., T. Hogan, T. J. Foster, and J. M. Fournier. 1993. Involvement of the accessory gene regulator (agr) in expression of type 5 capsular polysaccharide by Staphylococcus aureus. J. Gen. Microbiol. 139:1301–1306. 13. Fattom, A., J. Sarwar, A. Ortiz, and R. Naso. 1996. A Staphylococcus aureus capsular polysaccharide (CP) vaccine and CP-specific antibodies protect mice against bacterial challenge. Infect. Immun. 64:1659–1665. 14. Gillaspy, A. F., C. Y. Lee, S. Sau, A. L. Cheung, and M. S. Smeltzer. 1998. Factors affecting the collagen capacity of Staphylococcus aureus. Infect. Immun. 66:3170–3178. 15. Herbert, S., S. W. Newell, C. Y. Lee, K. P. Wieland, B. Dassy, J. M. Fournier, C. Wolz, and G. Do ¨ring. 2001. Regulation of Staphylococcus aureus type 5 and type 8 capsular polysaccharides by CO2. J. Bacteriol. 183:4609–4613. 16. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193–197. 17. Jarraud, S., G. J. Lyon, A. M. S. Figueiredo, L. Gerard, F. Vandenesch, J. Entienne, T. W. Muir, and R. P. Novick. 2000. Exfoliative-producing strains define a fourth agr specificity group in Staphylococcus aureus. J. Bacteriol. 182:6517–6522. 18. Ji, G., R. Bervis, and R. P. Novick. 1997. Bacterial interference caused by autoinducing peptide variants. Science 276:2027–2030. 19. Kiser, K., J. M. Cantey-Kiser, and J. C. Lee. 1999. Development and char-

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