Journal of Bacteriology, January 2003, p . 610-619, Vol . 185, No . 2

Global Regulation of Staphylococcus aureus Genes by Rot

B . Saïd-Salim,^1,2 P . M . Dunman,³ F . M . McAleese,⁴ D . Macapagal,³ E . Murphy,⁵ P . J . McNamara,⁶ S . Arvidson,⁷ T . J . Foster,⁴ S . J . Projan,³ and B . N . Kreiswirth^1*

Public Health Research Institute at the International Center of Public Health, Newark, New Jersey, 07103,¹ Department of Microbiology, New York University School of Medicine, New York, New York 10016,² Department of Infectious Diseases,³ Bioinformatics, Wyeth Research, Pearl River, New York 10965,⁵ Microbiology Department, Moyne Institute for Preventive Medicine, Trinity College, Dublin 2, Ireland,⁴ Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin 53706,⁶ Microbiology and Tumor Biology Center, Karolinska Institute, S-17177 Stockholm, Sweden⁷

Received 2 August 2002/ Accepted 17 October 2002

Production of S . aureus virulence determinants is controlled by several global regulatory loci, which include agr (1), sar (5), sigB (3, 41), sae (14), arl (13), and six SarA homologues (2, 24) . These regulators are parts of an important network modulating the expression of S . aureus virulence genes . One target virulence gene can be under the influence of several regulators that "cross talk" to ensure that the specific gene is expressed only when conditions are favorable . For instance, in vitro studies have demonstrated that agr negatively regulates the expression of spa, encoding protein A (32), whereas SarS binds to the spa promoter and activates its expression (38) . Interestingly, agr downregulates sarS expression (6, 38) . It has thus been proposed that agr downregulates spa expression by downregulating the expression of its activator, sarS (38) . Therefore, virulence gene regulators could affect the expression of target genes directly, by binding to their promoters, or indirectly, via other regulators .

The specific role and/or importance of a virulence factor or a virulence gene regulator in S . aureus pathogenesis may vary from one infection type to another . For instance, fibronectin-binding proteins have been shown to mediate invasion of mammalian cells by S . aureus (36) . In contrast, McElroy et al . have demonstrated that fibronectin-binding proteins decrease virulence in S . aureus-induced pneumonia (22) . Similarly, the agr locus has been shown to be important in S . aureus pathogenesis in various animal infection models such as the mouse arthritis and abscess models (28, 29) . However, a recent study by Yarwood et al . has shown that agr expression is not necessary for the development of toxic shock syndrome (40) . Taken together, these observations illustrate the multifactorial aspect of S . aureus pathogenesis . In view of the fact that an increasing number of S . aureus strains are becoming more difficult to treat due to their resistance to multiple antibiotics (39), elucidation of S . aureus pathogenesis at the molecular level is crucial in the battle against this important human pathogen, as it may help in the design of new therapeutic strategies .

Among the regulators described above, SarA and agr are the best characterized . SarA is a transcriptional regulator that binds to a consensus motif in the promoter of its target genes (8) . Recent studies have shown that SarA is a member of a large family of transcriptional regulators that share a common amino acid motif, KXRXXXDER (2) . The agr system is self-activating in a cell density-dependent manner (29) . Expression of the agr effector molecule, RNAIII, correlates with downregulation of select genes encoding cell surface proteins, such as protein A and fibronectin-binding proteins, and upregulation of genes encoding secreted proteins, such as alpha- and beta-toxins, lipases, and proteases (32) . In vitro, RNAIII-regulated factors follow a temporal trend, with surface proteins being produced during the early-exponential phase of growth and secreted proteins synthesized predominantly during the post-exponential phase . The sequential expression of these determinants correlates with the production of RNAIII, whose levels peak during the post-exponential phase (20, 32) . The mechanism by which RNAIII regulates the expression of these different genes is not completely understood . Several studies have suggested the existence of cofactors that interact directly or indirectly with RNAIII to control the expression of the different target genes (18, 24, 30) . These factors include members of the Sar family of transcriptional regulators (2) .

Recently, a novel SarA homologue, Rot, was identified as a repressor of toxins based on the following observation . An agr-negative strain produces low levels of alpha-toxin and protease activity . However, in a rot agr double mutant, expression of these proteins is increased due to the lack of repression by Rot . Understanding how Rot regulates these and other virulence factors is important in order to further discern S . aureus virulence gene regulation . Most S . aureus virulence gene modulators have both negative and positive effects on their target genes (8, 32, 38) . For these reasons, we hypothesized that Rot may have both up- and downregulatory functions . To identify additional target genes regulated by Rot, we compared the transcriptional profile of a rot agr double mutant to that of its parental agr strain during the late-exponential phase of growth by use of the Affymetrix GeneChip . The GeneChip used in this study covers 86% of the S . aureus COL genome and contains approximately 4,600 DNA fragments, representing 2,339 individual S . aureus genes (12) .

In this study, we demonstrate that Rot is a global regulator that negatively regulates the transcription of several known virulence genes . Some of these genes encode secreted proteins such as lipase, hemolysins, and proteases, all of which are postulated to play a role in tissue invasion . Rot also positively regulates the expression of a number of genes including those encoding cell surface adhesins . In addition, Rot modulates the expression of genes not previously implicated in pathogenesis . A relationship between Rot and agr in the regulation of S . aureus virulence genes is proposed .

 
Construction of rot agr and sarH1 (sarS) agr mutants. The rot agr double mutant (BK9986) was constructed by bacteriophage 11-mediated transduction of the rot::Tn917 insertion from strain PM614 into the agr mutant strain RN6911 . The sarH1 agr double mutant (BK9987) was made by transduction of the agr::tmn mutation from RN6911 into KT201 (sarH1 mutant) . Transfer of the rot and agr mutations into strains BK9986 and B9987 was verified by Southern blot analysis (data not shown) .

RNA isolation and Northern blot analysis. Overnight cultures of S . aureus were diluted 1:50 in CY-GP medium and grown to the late-exponential (5 h) phase of growth . Since our aim was to determine genes whose expression is affected by Rot at any time point, we chose the late-exponential-growth phase because at this time point, we can still observe transcripts of genes expressed early, such as cell surface protein genes, as well as those expressed later, such as genes encoding secreted enzymes and toxins . Cells were harvested by centrifugation, and RNA was isolated by the FastPrep system (developed by Savant [Farmingdale, N.Y.] and Bio 101 [Vista, Calif.]) (4) . RNA samples (10 µg) were electrophoresed through a 1.5% agarose-0.66 M formaldehyde gel in morpholinepropanesulfonic acid (MOPS) running buffer . RNA was blotted onto a Hybond N⁺ membrane (Amersham Pharmacia Biotech Inc., Piscataway, N.J.) by using the VacuGene XL blotting system (Amersham Pharmacia) . The transfer was performed with 20x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate),, pH 7.0, for 2 h . After the RNA transfer, the RNA ladder (Invitrogen, Carlsbad, Calif.) was manually marked before the hybridization step . Membranes were hybridized overnight with a PCR-amplified probe derived from the target gene (Table 2) . Specific transcripts were detected by using the enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia) . In all the Northern blot experiments, the same RNA samples were hybridized with 16S rRNA as an internal control (38) . New membranes were used for each Northern blot experiment .

 
RNA labeling. The integrity of RNA preparations was analyzed by electrophoresis in 1.2% agarose-0.66 M formaldehyde gels . Samples with defined 16S and 23S rRNA bands were subjected to a series of reactions that resulted in 5' biotin-labeled fragmented RNA for GeneChip analysis, as previously described (12) . Briefly, total bacterial RNA was first enriched for mRNA . This was accomplished by reverse transcription with rRNA-specific primers, followed by RNase H digestion to remove the rRNA of RNA-cDNA hybrid molecules and DNase I treatment . Enriched mRNA was then chemically fragmented and 5'-thiolated by T4 polynucleotide kinase treatment in the presence of -S-ATP . Samples were then biotinylated by addition of polyethylene oxide (PEO)-iodoacetyl-biotin .

GeneChip analysis. The custom-made S . aureus GeneChips used in this study have been described previously (12) . Each chip contains 4,528 open reading frames (ORFs), 12 tRNAs, and 3 rRNAs, with an average of 25 probe sets per ORF . Due to the preliminary state of the genome sequence at the time of design, many genes are represented on the chip as partial, duplicate, or overlapping fragments . Subsequent analyses based on the updated S . aureus genomic sequences suggest that these 4,528 tilings represent approximately 2,700 to 2,900 individual genes and that >86% of the S . aureus COL genome is represented on the chip . The missing genes (14%) include those encoding staphylococcal accessory regulator A (sarA), the sensor histidine kinase SaeS (saeS), D-alanine-D-alanyl carrier protein ligase (dltA), serine protease (sspA), leukotoxin D (lukD), sigma factor B regulators (rsbU, rsbV, and rsbW), and staphylococcal accessory regulator R (sarR) .

GeneChip analysis was performed by the method described by Dunman and colleagues (12) . Accordingly, to address the issue of reproducibility, two separate RNA samples from each strain were prepared from two different experiments, and for each sample, 1.5 µg of biotinylated RNA was hybridized to at least two separate GeneChips for 14 h at 45°C . GeneChips were then washed and subjected to a series of staining procedures that included the successive binding of streptavidin, biotinylated anti-streptavidin, and phycoerythrin-conjugated streptavidin . Each GeneChip was washed and scanned at a 570-nm wavelength and a 3-µm resolution in an Affymetrix GeneChip scanner . Affymetrix Microarray Suite 4.0 algorithms calculated signal intensities (average difference) and present or absent determinations for each ORF (19) . GeneChips were then normalized, and background was defined by using GeneSpring 4.0 (Silicon Genetics) as previously described (12) . GeneSpring software was used to further analyze the transcription patterns of genes . Genes negatively regulated by Rot were identified as ORFs with transcript titers at least twofold higher in BK9986 (rot negative) than in RN6911 (rot positive) . Genes whose transcript levels were at least twofold higher in RN6911 (rot positive) than in BK9986 (rot negative) were categorized as being positively regulated by Rot . These lists were further filtered to include only genes with signal intensities above background level in the rot agr (rot-negative) strain for Rot-downregulated genes and in the agr (rot-positive) strain for Rot-upregulated genes . Genes represented more than once on the chip are represented only once on the list .

Urease activity. Urease production was assayed on urea agar slants (Remel, Lenexa, Kans.) as described by the manufacturer . In this test, urease-positive strains show a color change from orange to pink while urease-negative strains have an intact orange color .

 
Virulence genes negatively regulated by Rot. Initial characterization of Rot has indicated that it is a repressor of hla, encoding alpha-toxin (24) . Our GeneChip data showed that Rot negatively regulates another toxin gene, hlb, which encodes beta-toxin (Tables 3 and 5) . The GeneChip result for hlb was confirmed by Northern blot analysis using the rot agr double mutant (rot negative) and its parental agr strain (rot positive) . As shown in Fig . 1A, the hlb transcript is present at higher levels in the rot agr strain than in the agr mutant . The detection of two bands that hybridize with hlb is consistent with previous findings (7, 41) . The 16S rRNA transcript (internal control) is present at similar levels in the two strains (Fig . 1B) .

 
Rot was previously shown to have a phenotypic effect on protease production . (24) . Unlike alpha-toxin and beta-toxin, which are solely encoded by hla and hlb, respectively, staphylococcal protease activity is associated with the gene products of at least two operons, splABCDEF (33) and sspABC (34) . Our GeneChip results demonstrated that Rot negatively regulates both operons (Tables 3 and 5) . The sspA gene, encoding V8 protease (34), is not listed in Table 3 because it is not represented on the GeneChip used in this study .

The GeneChip data for spl were confirmed by Northern blot analysis, using splA as a probe . The transcript of the spl operon (5.5 kb) is detectable only in the rot agr strain, BK9986, not in the agr strain, RN6911 (Fig . 1C) . We tested ssp operon expression in the two strains by Northern blot analysis, using sspA as a probe, and found that the transcript levels of the ssp operon (2.8 kb) were higher in the rot agr double mutant than in the agr strain (Fig . 1D) .

Rot also negatively regulates the expression of geh, encoding lipase (Tables 3 and 5) . The negative regulation of geh expression by Rot was confirmed by Northern blot analysis in which RNA samples from the rot agr double mutant and the agr strain taken at late-exponential growth were hybridized with a geh-specific probe . The results demonstrated that geh mRNA is present at higher levels in the rot agr strain than in the agr strain (Fig . 2A) .

 
As shown in Table 3, GeneChip data indicated that Rot negatively regulates various urease genes such as ureA, ureB, and ureC, encoding different subunits of the urease enzyme, as well as accessory genes such as ureD and ureE, required for the enzyme activity (17) (GenBank accession number AP003136) . We performed Northern blot analysis using the ureC gene as a probe and found that the rot agr strain had higher levels of ureC transcripts than the agr strain (Fig . 2B) . One of the urease genes downregulated by Rot, ureD, was previously identified as a potential virulence gene, because a ureD transposon mutant was strongly attenuated in an animal model (10) . We also tested for urease activity on urea agar slants for the two strains . As shown in Fig . 2C, the rot agr strain was urease positive but the agr strain was urease negative .

Virulence genes upregulated by Rot. Although Rot was originally identified as being a repressor of certain virulence factors, our results indicate that Rot also acts as a positive modulator of staphylococcal virulence genes . Among the genes upregulated in the presence of Rot are those encoding cell surface proteins . One of these genes is the well-studied spa gene, which encodes protein A (Tables 3 and 5) . To validate this result, Northern blot analysis was performed on rot agr and agr strains, and the results demonstrated that spa mRNA is detected only in the agr (rot-positive) strain, not in the rot agr double mutant (rot negative) (Fig . 3A) .

 
Previous studies have demonstrated that SarH1, also called SarS, positively regulates the expression of the protein A gene, spa (6, 38) . Since our results indicate that spa is also positively regulated by Rot, we sought to determine if Rot positively regulates spa expression by positively regulating the expression of the spa activator SarS . Using Genespring software analysis, we found that the sarS transcript was considered present in the rot-positive strain (RN6911) but absent in the rot mutant (BK9986), suggesting that Rot may positively regulate sarS expression .

To validate the sarS result, and to analyze the effects of Rot and SarS on each other, we created a sarH1 agr double mutant (BK9987) and compared the transcription of both rot and sarS in the two strains, i.e., BK9986 (rot agr) and BK9987 (sarS agr), as well as in RN6911 (agr) and RN6390 (wild-type strain), in a Northern blot assay . We found that rot is expressed in all the strains tested except the rot mutant, as expected (Fig . 3B) . On the other hand, sarS is expressed only in the presence of Rot and in the absence of RNAIII (Fig . 3C) . The fact that the sarS transcript is detectable in RN6911 (agr) but not in BK9986 (rot agr) indicates that Rot is indeed required for sarS expression . The higher levels of sarS transcript in RN6911 compared to RN6390 are in good agreement with previous data that showed that agr represses sarS expression (6, 38) .

Rot and agr have opposing effects on the expression of target genes. Our results indicate that Rot-regulated factors follow a general expression pattern opposite that of agr, that is, secreted proteins generally upregulated by agr are negatively regulated by Rot (Table 5) . For example, Rot negatively regulates hla and hlb while agr positively regulates them (32) . Since Rot negatively regulates the expression and activity of urease, we tested to see if agr will upregulate them . For this, we tested the urease activity of an agr mutant (RN6911) and its parental wild-type strain (RN6390) on urea agar slants and found that indeed the agr-positive strain was urease positive whereas the agr-negative strain was urease negative (data not shown) . Conversely, cell surface proteins generally downregulated by agr appear to be positively modulated by Rot (Table 5) . For instance, Rot positively regulates spa, encoding protein A, whereas agr negatively regulates it . In addition to structural genes, Rot and agr also have opposing effects on the expression of the virulence gene regulator sarS . Our results indicate that Rot positively regulated the expression of sarS, which has been shown to be repressed by agr (6, 38) .

Additional Rot-regulated genes. Table 3 lists additional virulence genes negatively regulated by Rot as determined by GeneChip analysis, and Table 4 lists all genes shown to be positively modulated by Rot . These tables include genes which encode factors previously shown to promote the growth and survival of S . aureus in an infection model . For instance, Rot downregulates hlgB and hlgC, which encode gamma-hemolysins B and C, respectively . Gamma-hemolysin is an S . aureus virulence factor that has been shown to play a role in S . aureus endophthalmitis and corneal pathogenesis (11, 37) . Rot positively regulates thrB, a gene involved in threonine biosynthesis . Disruption of this gene reduces virulence in animal infection models (25) . From both lists, it is clear that in addition to virulence genes, Rot also regulates the expression of genes that play different biological roles, including amino acid and carbohydrate metabolism, cell wall biosynthesis, and transport .

Virulence factors whose expression is controlled by the different regulators generally fall into two categories: cell surface proteins and secreted proteins . The agr system generally downregulates cell surface proteins such as protein A and upregulates secreted proteins such as alpha-toxin . By use of DNA array technology, we have clearly demonstrated in this report that Rot does not act exclusively as a repressor . The work presented here indicates that Rot also positively regulates the expression of genes, many of which have previously been determined to be virulence factors . We also demonstrated that Rot and agr have opposing effects on the expression of certain virulence determinants . Collectively, these findings suggest that Rot is a more global virulence regulator than previously recognized . Whether the effects of Rot on its target genes are direct or indirect is currently unknown .

Rot positively regulates the expression of sarS, a transcriptional modulator of virulence gene expression (6, 38) . Arvidson and colleagues have shown that SarS binds to the promoter of spa (38), a gene activated by both Rot and SarS . Thus, Rot may affect spa transcription via SarS . Other SarA homologues have also been shown to regulate the expression of virulence gene regulators . For instance, SarR represses sarA expression (21) and SarA represses sarT expression (35) . We could postulate that these modulators affect certain target genes by influencing the expression of regulators of these genes .

Besides genes with well-characterized or postulated virulence functions, Rot also regulates genes not previously linked to virulence . These genes could either be potential virulence genes or additional accessory genes with other biological functions . In the latter case, as Dunman and colleagues point out, the regulators involved in the agr-SarA regulatory pathway may be more global than previously recognized and may affect processes that have yet to be established in S . aureus pathogenesis (12) . Given the ability of S . aureus to cause a broad range of clinical manifestations, it is likely that many of these gene products are involved in various, as yet unrecognized stages of infection .

Although this study identified numerous members of the Rot regulon, it is very likely that the list is not complete due to the fact that 14% of the staphylococcal genes are missing from the chip . For example, the sspA gene from the serine protease operon is missing from the GeneChip and hence from Table 3 . However, Northern blot analysis showed that the whole operon is downregulated by Rot . With the complete genome available, new GeneChips are being designed to cover 100% of the genome, and these will be used in future studies .

Based on the virulence function of Rot-regulated genes, we hypothesize that Rot plays an important role in the early steps of infection . Factors positively modulated by Rot include cell surface adhesins such as ClfB (clumping factor B), which binds fibrinogen (26), and SdrC, a cell surface adhesin (15) . Rot also positively regulates the expression of a gene represented by ORF 1175 on the chip, which corresponds to N315 ORF 2284 (Table 4) . This ORF contains a cell wall sorting signal motif, LPXTG, which is characteristic of most adhesins (17) . Rot positively regulates the production of protein A, a factor involved in evasion of the host immune system (32) . Another gene upregulated by Rot is dltD (Table 4) . This gene is a member of the dlt operon, which consists of dltA, dltB, dltC, and dltD (31) . This operon encodes proteins involved in D-alanine incorporation into teichoic acid in the S . aureus cell wall . Interestingly, dltA is not present on the list because it is among the genes missing from the GeneChip used in this study (see Materials and Methods) . In addition, dltB and dltC are not listed because their fold difference (1.3 for dltB and 1.4 for dltC) did not make the cutoff . The dlt operon has been shown to confer resistance to human antimicrobial agents such as defensins (31) . A recent study by Collins et al . has demonstrated that dlt mutants are more efficiently killed by human neutrophils than wild-type strains (9) . The same study has also shown that mortality and arthritis frequencies were significantly lower in mice infected with dlt mutants than in mice infected with the wild type strains, indicating that the strains lacking the dlt operon were less virulent than dlt-positive strains . All these factors positively modulated by Rot are hypothesized to be required at an early stage of infection . Some factors, such as the adhesins, allow the bacteria to attach to host cells, while others, such as protein A and DltA, -B, -C, and -D facilitate establishment of the infection through inhibition of the host immune response .

Simultaneously, Rot negatively regulates factors that may interfere with the establishment of the infection . For instance, Rot negatively regulates the production of proteases . One role for proteases is to degrade surface proteins such as protein A, fibronectin-binding proteins, and clumping factor B, which facilitate adherence of the bacteria to host tissues . Degradation of these factors will allow the bacteria to detach from the initial site of infection and start the infection at another location (16, 23) . Rot also represses the synthesis of hla and hlb, which encode alpha- and beta-hemolysin, respectively . These hemolysins are known to disrupt a variety of mammalian cells, and because of their function, they are classified as virulence factors involved in invasion and tissue penetration (32) . Thus, Rot promotes early stages of infection by stimulating factors needed at this stage and by inhibiting those that interfere with the early processes of infection or that are needed for later processes .

During late stages of infection, which correspond to the post-exponential growth phase, there is a peak of the levels of RNAIII, the agr effector molecule (32) . Since our results indicate that Rot and agr have opposing effects on the expression of S . aureus virulence genes, it is likely that RNAIII either directly or indirectly exerts an inhibitory effect on Rot . Since rot is transcribed throughout the growth curve (data not shown) and the rot transcript is present in both RNAIII-positive and RNAIII-negative strains (Fig . 3B), Rot is likely to be inhibited by RNAIII posttranscriptionally . The inhibitory effects of RNAIII on Rot result in the repression of the cell surface proteins and the activation of the secreted proteins that promote the spread of the bacteria and allow them to initiate infection at other sites . Given that Rot was identified in an agr-negative strain, we proceeded to determine the extent of S . aureus gene regulation by Rot in the same background . However, preliminary results indicate that Rot effects on select target genes such as ureC, geh, spl, and ssp operons could be validated in an agr-positive background as well .

In conclusion, the present body of work has employed transcription profiling technology to investigate the spectrum of genes that are regulated by the S . aureus repressor of toxin, Rot . The results demonstrate that Rot is a global transcriptional modulator of virulence genes and that its influence on the expression of certain target genes is opposite that of agr . Genes whose expression is influenced by Rot can be grouped in three major categories: (i) genes that have been extensively studied and have well-defined virulence functions, (ii) genes postulated to play a role in virulence, as their disruption results in attenuation of the mutant in a specific infection model, and (iii) genes not previously known to play a role in S . aureus pathogenesis . By unveiling additional regulatory levels, this study gives us better insight into the agr-SarA regulatory network of S . aureus virulence genes . Additional studies are needed to investigate the molecular interaction of Rot with its target genes and with other regulators, as well as the role of the novel genes regulated by Rot in S . aureus virulence .

T.J.F . and F.M.M . acknowledge the Health Research Board of Ireland for support .

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