JOURNAL OF BACTERIOLOGY, Feb. 2002, p. 1095–1101 Vol. 184, No. 40021-9193/02/$04.00�0 DOI: 10.1128/JB.184.4.1095–1101.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Repression of the Staphylococcus aureus Accessory Gene Regulatorin Serum and In Vivo

Jeremy M. Yarwood,1 John K. McCormick,1 Michael L. Paustian,2 Vivek Kapur,2and Patrick M. Schlievert1*

Department of Microbiology, Medical School, University of Minnesota, Minneapolis,1 and Department of VeterinaryPathobiology and Biomedical Genomics Center, University of Minnesota, St. Paul,2 Minnesota

Received 28 September 2001/Accepted 8 November 2001

Subgenomic DNA microarrays were employed to evaluate the expression of the accessory gene regulator (agrlocus) as well as multiple virulence-associated genes in Staphylococcus aureus. Gene expression was examinedduring growth of S. aureus in vitro in standard laboratory medium and rabbit serum and in vivo in subcuta-neous chambers implanted in either nonimmune rabbits or rabbits immunized with staphylococcal enterotoxinB. Expression of RNAIII, the effector molecule of the agr locus, was dramatically repressed in serum and invivo, despite the increased expression of secreted virulence factors sufficient to cause toxic shock syndrome(TSS) in the animals. Statistical analysis and clustering of virulence genes based on their expression profilesin the various experimental conditions demonstrated no positive correlation between the expression of agr andany staphylococcal virulence factors examined. Disruption of the agr locus had only a minimal effect on theexpression in vivo of the virulence factors examined. An effect of immunization on the expression of agr andvirulence factors was also observed. These results suggest that agr activation is not necessary for developmentof staphylococcal TSS and that regulatory circuits responding to the in vivo environment override agr activity.

A leading cause of nosocomial infections worldwide, Staph-ylococcus aureus is the etiologic agent of numerous diseases,ranging from relatively benign skin infections to life-threaten-ing illnesses, such as toxic shock syndrome (TSS), septicemia,and osteomyelitis (reviewed in reference 25). Its ability tocause this range of disease is due, in part, to the expression ofa wide array of secreted and cell surface-associated virulencefactors. The expression of most of these virulence factors hasbeen described as being regulated by the quorum-dependentaccessory gene regulator (agr locus). The present model of agractivity and its effect on virulence factor expression is wellreviewed elsewhere (15, 19). Briefly, the agr locus expressestwo primary, divergent transcripts. RNAII (agrBDCA) encodesa two-component system (AgrA-AgrC), recognizing the agrD-encoded secreted autoinducing octapeptide (AIP), and AgrB,which is thought to act in the posttranslational processing andsecretion of the AIP. The second transcript, RNAIII, acts asthe effector molecule of the agr locus. A third, short transcript,RNAI, has been described as encoding AgrA. Upon accumu-lation in the growth medium of sufficient quantities of the AIP,usually during the transition from the exponential to the sta-tionary phase of growth, signaling via AgrA-AgrC acts to in-crease transcription of both RNAII and RNAIII. In vitro, thisresults in the increased expression of secreted virulence fac-tors, including the pyrogenic toxin superantigens and hemo-lysins, and the repression of numerous surface-associated vir-ulence factors, typified by protein A, which binds the Fccomponent of immunoglobulin G.

Since the identification of agr (20), models of staphylococcalvirulence have assigned it a central role in the organism’s

ability to cause disease (15, 19). Numerous studies using agrmutants have implicated this system in infections ranging frommurine arthritis to endocarditis in rabbits (reviewed in refer-ence 3). It has also been proposed that ligand-based inhibitionof agr activity might form the basis of antistaphylococcal che-motherapy (11). However, very few studies have examined theexpression of agr in vivo. In this study, we employed sub-genomic DNA microarrays to describe the expression of agr,together with numerous associated virulence factors thought tobe regulated by this system. Gene expression was examinedduring growth of S. aureus in serum and in vivo using a sub-cutaneous infection model in staphylococcal enterotoxin B(SEB)-immune and nonimmune rabbits. We found that agractivation was not necessary for the development of TSS andhypothesize that signals generated by the in vivo environmentact to override agr activity and increase expression of secretedvirulence factor genes by S. aureus.

MATERIALS AND METHODS

Bacterial strains. S. aureus MN NJ is an isolate from a recent case of non-menstrual TSS and produces SEB and two novel superantigens, SEK (16) andSEQ (P. M. Orwin, D. Y. Leung, H. L. Donahue, R. P. Novick, and P. M.Schlievert, unpublished data). S. aureus MN8, used as a source of genomictemplate for amplification of virulence gene probes for the DNA microarrays,was isolated before 1980 from a case of menstrual TSS (23). S. aureus RN4282and RN4256 are isogenic strains in which expression of the agr locus in RN4256has been disrupted by insertion of the erythromycin resistance transposon Tn551into agrA (13, 18, 20).

Growth of S. aureus in vitro. Fifty-milliliter cultures of S. aureus MN NJ weregrown aerobically with shaking at 37°C in either Todd-Hewitt (TH) medium(Becton Dickinson, Sparks, Md.) or rabbit serum (Gibco BRL, Carlsbad, Calif.).TH medium contains (per liter) 9 g of beef heart digest, 11 g of pancreatic digestof casein, 3 g of soybean peptone, 2 g of dextrose, 2.5 g of Na2CO3, 0.5 g ofNaH2PO4, and 2 g of NaCl. Two independent cultures in each medium weregrown in parallel, and samples were removed at the exponential, postexponen-tial, and stationary phases of growth (2, 3, and 8 h, respectively, after inoculation

* Corresponding author. Mailing address: MMC 196, 420 DelawareSt., S.E., Minneapolis, MN 55455. Phone: (612) 624-9471. Fax: (612)626-0623. E-mail: pats@lenti.med.umn.edu.

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with an initial optical density at 600 nm of 0.1). Expression of agr and associatedvirulence factors was quantified using DNA microarrays.

Immunization of Dutch-belted rabbits. The use of animals in this study com-plied with all relevant federal and institutional guidelines. Two Dutch-beltedrabbits were immunized with SEB, which is made in high concentrations (�10�g/ml) by MN NJ grown in vitro. Rabbits were immunized by three subcutaneousinjections at 2-week intervals, with each injection containing 25 �g of purifiedSEB resuspended in 0.5 ml of phosphate-buffered saline and emulsified in 0.5 mlof incomplete Freund’s adjuvant. Development of antibody to SEB was deter-mined by enzyme-linked immunosorbent assay of serum samples taken 1 weekafter the final immunization. The two rabbits developed anti-SEB (immunoglob-ulin G) titers of 1:5,120 and 1:10,240, respectively, compared to preimmune titersof �1:20.

Growth of S. aureus in vivo. Sterilized perforated hollow polyethylene golf ballswere implanted subcutaneously in four Dutch-belted rabbits (24). Implantationof the polyethylene balls and subsequent healing created transudate-filled cavi-ties in the rabbits with volumes of approximately 15 ml. Six weeks after implan-tation of the polyethylene balls, �1010 CFU of S. aureus grown in TH mediumwas collected by centrifugation from the late exponential phase of growth, re-suspended into 2 ml of phosphate-buffered saline, and injected into the im-planted polyethylene balls. Two milliliters of transudate containing S. aureus wasthen removed from the infection chambers at the indicated times after inocula-tion, using a sterile syringe, S. aureus was enumerated by plating, and expressionof agr and associated virulence factors was quantified using DNA microarrays.

RNA preparation and DNA microarrays. Analysis of staphylococcal geneexpression in vitro and in vivo using DNA microarrays was performed as de-scribed elsewhere (http://www.agac.umn.edu/microarray/protocols/protocols.htm).In brief, a library of targets representing 68 genes from S. aureus MN NJ andMN8 was constructed with primers designed to amplify fragments of �300 bp ofeach gene open reading frame from genomic DNA. Two successive rounds ofPCR were performed to minimize genomic DNA contamination in the amplifi-cation products, and the final 100-�l reaction mixtures were checked for qualityon agarose gels and purified with the QIAquick PCR Purification Kit (Qiagen,Valencia, Calif.). The purified products were printed in triplicate using a TotalArray System robot (BioRobotics, Boston, Mass.). Cell pellets from centrifugedsamples of S. aureus cultures were flash-frozen in liquid nitrogen. Total RNA wasprepared using the RNeasy Mini Kit (Qiagen) according to the manufacturer’sdirections. DNA was removed from the RNA preparations using the RNase-FreeDNase Set (Qiagen) according to the manufacturer’s directions. cDNA preparedfrom 10 �g of RNA from S. aureus cultures to be compared was labeled witheither Cy3 or Cy5 fluorescent dye (Amersham Pharmacia Biotech, Piscataway,N.J.) and competitively hybridized with the printed microarrays. Images of thehybridized arrays were obtained with a Scanarray 5000 microarray scanner (GSILumonics, Watertown, Mass.). One independent hybridization (in triplicate) wasconducted for each of two independent experiments. Fluorescence intensities forindividual spots were normalized based on the total intensity of fluorescence inthe Cy3 and Cy5 channels. Fluorescence intensity was determined as the averageintensity of the triplicate spots for each gene. Total fluorescence for each genewas normalized between arrays for independent experiments, the data werecombined from both experiments, and statistical significance was determinedusing the Student t test to compare expression data from the two growth condi-tions of interest. To account for possible bias in labeling of cDNA by either Cy3or Cy5, dye labeling was reversed in the second independent experiment for eachof several experimental conditions. No dye bias was detected. A link to additionalresults for the microarray studies is posted (http://www.micab.umn.edu/faculty/Schlievert.html).

One-step real-time RT-PCR. Real-time reverse transcription-PCR (RT-PCR)analysis of the RNA samples used in DNA microarrays assays was used toconfirm relative expression changes in RNAIII, hla, and spa. RT-PCRs wereperformed using the SYBR Green PCR reagents and TaqMan reverse transcrip-tion reagents (Applied Biosystems, Foster City, CA) according to the manufac-turer’s directions with an ABI Prism 7700 Sequence Detection System. Briefly, 25ng of total RNA from the same preparations as used for the microarray analyseswas added to each of 25-�l PCR mixtures containing 400 nM concentrations offorward and reverse primers (RNAIII, 5�-GATGTTGTTTACGATAGCT-3�and 5�-TTCAATGGCACAAGATATC-3�; hla, 5�-GCAAATGTTTCGATTGGT-3� and 5�-TGTTTGTTGTTTGGATGC-3�; spa, 5�-AGAACAACGCAATGGTTT-3� and 5�-GGCTTGTTGTTGTCTTCC-3�; 16S rRNA, 5�-CTGTGCACATCTTGACGGTA-3� and 5�-TCAGCGTCAGTTACAGACCA-3�). Reactionmixtures were incubated for 30 min at 48°C, followed by an incubation step for10 min at 95°C and then by 30 cycles of 30 s at 95°C, 30 s at 54°C, and 1 min at72°C. The increase in fluorescence between extension steps was used to monitorthe increase in the amount of amplified product and to determine a fractional

cycle number (CT) required to achieve a set threshold of amplification. CT valuesfor the genes of interest were normalized using the CT of 16S rRNA for thecorresponding sample.

RESULTS

To model staphylococcal virulence factor expression in se-rum and in vivo, we chose S. aureus strain MN NJ, an isolatefrom a case of nonmenstrual TSS. Our laboratory has se-quenced a novel staphylococcal pathogenicity island (SaPI3)(J. M. Yarwood, J. K. McCormick, M. L. Paustian, P. M.Orwin, V. Kapur, and P. M. Schlievert, submitted for publica-tion) in this strain that encodes SEB and two novel superan-tigenic enterotoxins, SEK (16) and SEQ (Orwin et al., unpub-lished data). Expression of virulence factors by this strain whengrown in standard laboratory medium is as predicted by thepresent model of agr regulation (Table 1). Levels of RNAIIIand exotoxins increased in the stationary phase of growth com-pared to the late exponential phase of growth, while the ex-pression of the surface-associated protein A was repressed.

To approximate the expression of virulence factors in vivoand determine those genes whose expression is enhanced orrepressed by growth in a standard laboratory medium, culturesof S. aureus were grown in either TH medium or rabbit serum.Expression profiles for virulence-associated genes reflectingthe fold increase or decrease induced by growth in rabbitserum versus TH medium are shown in Fig. 1 (lanes 1 to 3),and a summary of results for selected genes is shown in Fig. 2.The most dramatic effect of growth in serum was the repres-sion of agr, as indicated by the 34-, 13-, and 24-fold repressionof RNAIII in the exponential, postexponential, and stationaryphases of growth, respectively. As would be predicted by thepresent model of agr regulation, the level of spa transcript,encoding the surface molecule protein A, was increased duringgrowth in serum. However, levels of hla transcript, encodingthe secreted alpha-hemolysin, which would be predicted todecrease in response to repression of agr, were not significantlyaffected (P � 0.05 by Student’s t test). Furthermore, the ex-pression of other exotoxins was somewhat inconsistent with therepression of agr. Although RNAIII was strongly repressedduring all phases of growth in serum, sek and seq transcriptlevels were increased during exponential-phase growth, while

TABLE 1. Comparison of gene expression by S. aureus MN NJin stationary-phase growth versus late-exponential-phase

growth in TH mediuma

Gene Description or product Fold expression changeb

RNAII agrBDCA 2.2 � 0.2RNAIII Effector molecule of agr locus 5.8 � 1.3spa Protein A �33.6 � 6.3hla Alpha-hemolysin 6.6 � 0.9seb Enterotoxin B 8.2 � 1.1hlgA Gamma-hemolysin, a-component 2.7 � 0.5

a Strain MN NJ was grown aerobically at 37°C with shaking, cells were har-vested 2 and 8 h after inoculation (initial optical density at 600 nm of 0.1), andgene expression was determined using DNA microarrays as described in the text.

b Values represent the fold change of gene expression � standard error of themean in the stationary-phase cultures compared to the late-exponential-phasecultures. Positive values indicate genes with increased expression in stationary-phase growth; negative values indicate genes repressed in stationary-phasegrowth. The results represent averages of data for two independent experiments,each of which was evaluated by arrays conducted in triplicate. All results weresignificant (P � 0.01 by Student’s t test).

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expression of hlgA was increased in the stationary phase ofgrowth. (Unlike seb, sek and seq are constitutively expressedthroughout the growth phases of S. aureus in vitro [unpub-lished data].) Transcription of RNAI was also repressed bygrowth in serum during the postexponential and stationaryphases of growth, although it was not nearly as repressed as thedivergent RNAIII transcript. RNAII levels were unaffected bygrowth in serum. The agr regulon is thought to operate in apositive-feedback mechanism to increase transcriptional activ-ity from both the agr P2 (RNAII) and P3 (RNAIII) promoters.

If this model is correct, these data indicating differential reg-ulation of transcriptional activity from the P2 and P3 promot-ers suggest that the AIP is secreted and recognized by theAgrA-AgrC two-component system in serum as it is in THmedium and that a secondary, unknown factor (or factors) actsto repress transcriptional activity from the P3 promoter. Thisfactor is unlikely to be SarA, whose expression did not corre-late with that of agr (Fig. 1 [cluster analysis]) and which isgenerally thought to activate transcription of the agr locus (19).

To evaluate expression of S. aureus virulence factors in a

FIG. 1. Expression profiles and hierarchical clustering of 31 staphylococcal virulence-associated genes and significance of results. The red andgreen colors of the expression profiles (lane numbers in boldface) represent fold decreases and increases, respectively, in gene expression inresponse to growth in serum compared to TH medium (lanes 1 to 3) or growth in vivo compared to the inoculum (lanes 4 to 6). Results are shownfor cultures in the exponential (lane 1), postexponential (lane 2), and stationary (lane 3) phases of growth in TH medium versus serum. Resultsare also shown for S. aureus cultures removed from the subcutaneous infection chambers in nonimmune rabbits at 2 h after inoculation (lane 4)and in SEB-immune rabbits at 2 h (lane 5) and 8 h (lane 6) after inoculation. Dots (lane numbers in italic) indicate those fold changes that werefound to be significant (Student’s t test; P � 0.05) in the corresponding lane of the expression profiles. Corresponding gene names and descriptionsare shown as follows: black type, gene regulators; blue type, secreted proteins; magenta type, surface-associated molecules; and gold type,two-component systems. Clustering based on similarity of expression profiles and visualization was performed using the software program SpotfireDecisionSite 6.1 (http://www.spotfire.com). Similarities between expression profiles of individual genes in all six experimental conditions weredetermined using the “city-block distance” method (http://www.spotfire.com).

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subcutaneous infection model of TSS, sterilized perforatedhollow polyethylene golf balls were implanted subcutaneouslyin four Dutch-belted rabbits (24). The rabbit model was chosenover a mouse model because rabbits develop symptoms of TSSmore consistent with those described for humans (17). This isdue in part to the relative resistance of mice to the lethaleffects of staphylococcal superantigens without coadministra-tion of a hepatotoxin, while in humans and rabbits, superanti-gens alone are lethal (5, 6). Two of the four rabbits wereimmunized with SEB.

All four rabbits developed symptoms consistent with TSS.These included hypotension, respiratory distress, erythema,and obvious discomfort in the vicinity of the infection chamber.However, the SEB-immunized rabbits experienced less severeand delayed onset of symptoms and were euthanized at 22 hafter inoculation, while the nonimmune rabbits died from theinfection several hours after inoculation. This indicates thatimmunity against SEB provided only partial protection againststaphylococcal TSS, likely due to the fact that S. aureus MN NJproduces multiple superantigens. Cell densities from the re-covered abscess samples did not vary more than 0.22 log unitfrom the inoculum cell density (Table 2), indicating that theobserved effects on gene expression were not due to alterationsin cell density but rather were due to a response of the organ-ism to the in vivo environment.

Expression profiles for virulence-associated genes reflectingfold increases or decreases during incubation in vivo comparedto the inoculum are shown in Fig. 1 (lanes 4 to 6), and a

summary of results for selected genes is shown in Fig. 3. Asobserved during growth in serum, transcription of RNAII andRNAIII was repressed by growth in vivo in both the nonim-mune and immunized animals. Interestingly, a significant (P �0.05 by Student’s t test) effect of immunization on agr activitywas observed, with RNAIII being much less repressed in theimmune animals than in the nonimmune animals (4- versus17-fold, respectively, at 2 h). This effect of immunization wasmuch less dramatic for RNAI and RNAII transcription. Aneffect of immunization on the expression of alpha-toxin (hla),SEB (seb), and the A component of gamma-hemolysin (hlgA),all of whose fold activation was much less at 2 h in the immu-nized rabbits than the nonimmune rabbits, was also observed.

No positive correlation between RNAIII levels and the ex-pression of exotoxins in vivo was observed. The present model

FIG. 2. Activation or repression of selected S. aureus genes by growth of the bacterium in serum compared to growth in TH medium in theexponential, postexponential, and stationary phases of growth. Data are plotted against a logarithmic scale. Values are averages of data from twoindependent experiments, each of which was evaluated using triplicate DNA microarrays. Standard errors of the means are indicated.

TABLE 2. CFU recovered from subcutaneous infectionchambers 2 and 8 h after inoculationa

Rabbit ImmunizedbCFU/ml (108) at

2 h 8 h

1 No 8.82 No 7.23 Yes 4.4 4.24 Yes 4.6 4.0

a The cell density of cultures used for inoculum prior to concentration forinjection was 6.7 108 CFU/ml.

b Rabbits were either nonimmune or immunized against SEB.

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of agr activity would have predicted that transcription of thealpha-hemolysin gene (hla), which has been shown to bestrongly and positively regulated by agr in vitro (15), would berepressed in response to decreased RNAIII levels. Instead, hlaexpression was increased by 10-fold in the nonimmune rabbits,while RNAIII transcription levels were reduced 17-fold. Theexpression of SEB (seb) and gamma-hemolysin (hlgA) wasincreased in these animals as well. While RNAIII levels weresomewhat restored in the SEB-immune animals, the fold acti-vation of hla, seb, and hlgA transcription was reduced. Othervirulence factor genes, such as those for capsule (cap5A) andcoagulase (coa), were generally not affected by growth in vivoand the observed agr repression, despite having been reportedas being regulated by agr (4, 14). Meanwhile, transcription ofother virulence genes behaved as predicted from repression ofagr. Expression of fibronectin-binding protein (fnbA) and pro-tein A (spa), both surface-associated virulence factors, wasincreased in vivo, while that of V8 protease (sasp) and staphy-lokinase (sak) was repressed in vivo.

To further determine whether a functional agr locus wasnecessary for the expression of virulence factors in vivo, iso-genic S. aureus strains RN4282 and RN4256 (agrA::Tn551)were each used to inoculate the subcutaneous infection cham-bers of two nonimmune rabbits. All four animals developedsymptoms consistent with TSS and died within 16 h after in-

oculation. As expected, insertion of the transposon resulted inrepression of RNAIII transcription (Table 3). However, thedisruption of agr expression had only a minimal effect (foldchange of �1.6) on the expression of representative surface-associated and secreted virulence factors both 2 and 8 h afterinoculation of the infection chambers.

FIG. 3. Activation or repression of selected genes in S. aureus removed from subcutaneous infection chambers at the indicated times comparedto gene expression by S. aureus in the inoculum. Expression values are plotted for S. aureus removed from the nonimmmune rabbits 2 h afterinoculation and from the SEB-immune rabbits 2 and 8 h after inoculation. Data are plotted against a logarithmic scale. Values are averages of datafrom experiments conducted with four rabbits (two immune and two nonimmune), each of which was evaluated using triplicate DNA microarrays.Standard errors of the means are indicated. Those genes whose expression was significantly affected (P � 0.05 by Student’s t test) at 2 h afterinoculation by immunization against SEB are indicated by an asterisk.

TABLE 3. Comparison of virulence gene expression by S. aureusstrains RN4256 (agrA::Tn551) and RN4282 (agr�) recovered from

subcutaneous infection chambers at 2 and 8 h after inoculation

Gene Description or product

Fold expressionchangea at:

2 h 8 h

RNAIIb agrBDCA �2.9 � 0.5 �1.6 � 0.4RNAIII Effector molecule of agr locus �15.1 � 3.4 �7.1 � 1.0spa Protein A 1.6 � 0.2 1.4 � 0.2hla Alpha-hemolysin 1.3 � 0.3 1.3 � 0.1tstH TSS toxin 1 �1.5 � 0.5 1.5 � 0.2hlgA Gamma-hemolysin, a-component �1.4 � 0.2 �1.5 � 0.1

a Values represent the fold change in gene expression � standard error of themean in strain RN4256 compared to strain RN4282 as determined using tripli-cate DNA microarrays. Results are averages of data obtained using four rabbits,two infected with RN4256 and two infected with RN4282.

b Insertion of Tn551 did not interfere with the ability to detect RNAII, as themicroarray probe for RNAII was specific for a region upstream of agrA.

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The relative changes in expression for RNAIII, hla, and spain vivo versus the inoculum were confirmed using TaqManRT-PCR with the same RNA samples as prepared for microar-ray analysis as templates (Fig. 4). The results demonstrated astrong positive correlation (r 0.95) between the data ob-tained using the two different techniques.

DISCUSSION

Taken together, the data from both the serum and in vivoexperiments suggest that expression of the agr locus, particu-larly its effector molecule RNAIII, is not critical for the ex-pression of exotoxins important in the development of staph-ylococcal TSS associated with an abscess or bacteremia.Despite dramatic repression of agr both in serum and in vivo,the expression of alpha-hemolysin, SEB, and gamma-hemoly-sin was increased or unaffected. A cluster analysis of genesbased on the similarity of their expression profiles under thevarious experimental conditions confirmed that expression ofagr did not correlate with the expression of staphylococcalexotoxins (Fig. 1). Indeed, RNAI and RNAIII comprised anexpression profile group separate from any of the virulencefactors examined. Furthermore, disruption of the agr locus hadonly a minimal effect on the expression of both surface-asso-ciated and secreted virulence factors in vivo (Table 3).

Although contradictory to present models of staphylococcalvirulence, our results are not entirely unprecedented, as someprevious studies have suggested a less critical role for agr. Inone of the few studies to quantify RNAIII levels in humaninfections, Goerke et al. directly quantified RNAIII transcriptlevels in sputum samples from cystic fibrosis patients infectedwith S. aureus (9). Those authors found that RNAIII waspoorly expressed in vivo and concluded that agr activity wasnonessential for S. aureus infection of the cystic fibrosis lung.More recently, the same group determined that expression ofalpha-hemolysin in a guinea pig model of device-related infec-

tion was unaffected by disruption of agr expression (10). Fur-thermore, Cheung et al. were unable to demonstrate signifi-cantly attenuated virulence for an agr mutant in a rabbit modelof endocarditis, although a sar agr double mutant was dimin-ished in both infectivity and intravegetation bacterial densities(2). Additional studies have found that although agr mutantsare somewhat attenuated in virulence, they remain clearly ca-pable of causing infection in a rabbit model of osteomyelitis (8)and a rat model of endophthalmitis (7). These studies, com-bined with the data presented here, suggest a much morecomplicated picture of staphylococcal virulence than that mod-eled with agr as a global regulator of virulence factors.

The ability of staphylococci to sense and respond to the invivo environment now seems to be of paramount importance inthe regulation of virulence factor expression. In part, our dataindicate that unknown regulatory elements act to repress tran-scription of RNAIII in vivo. The results also suggest the use byS. aureus of environmental sensing mechanisms that regulatevirulence factors in response to multiple in vivo signals. Thesesignals appear to override agr activity (perhaps even acting inits repression) to increase expression of secreted virulencefactors. Indeed, numerous environmental conditions, consis-tent both in vitro and in vivo, have been shown to be necessaryfor exotoxin production (1, 12, 15, 21–23, 26, 27). For example,even in the presence of a functional agr system, decreasedoxygen and carbon dioxide levels will result in repression ofexotoxin production (21, 26, 27). However, there likely remainsto be identified an additional in vivo environmental signal, orcombination of signals, to which staphylococci respond in theirregulation of virulence factors.

It is not yet clear why the rabbit immune status affects theexpression of staphylococcal virulence genes (Fig. 3). The pri-mary difference between the in vivo environments of the im-mune and nonimmune animals may be due to the superanti-genic activity of SEB. SEB activity in the nonimmune animalswould be expected to result in fairly rapid activation of andcytokine release by antigen-presenting cells, which was re-flected in the visible erythema surrounding the infection cham-ber upon autopsy of the animals. On the other hand, neutral-ization of SEB by host antibody in the immunized animalslikely eliminated its superantigenic activity. Thus, the immuneresponse in the SEB-immune animals would more closely re-semble the very early stages of a natural secondary immuneresponse to conventional antigens. It is also possible that thepresence of high antibody titers to SEB might interfere withsecretion of SEB, and perhaps other proteins, by S. aureus. Theability of S. aureus to sense these various immune responseseither directly or indirectly may be responsible for the ob-served changes in virulence gene expression.

In light of the findings presented here and elsewhere (9, 10),the proposed use of therapeutic agents to inhibit signaling byagr (11) may be ineffective and irrelevant, as the expression ofagr appears to have little or no effect on the expression ofexotoxins in vivo. Instead, considering the demonstrated im-portance of environmental conditions in the expression ofstaphylococcal virulence factors, more effective therapeuticstrategies might seek to interfere with the ability of S. aureus tosense and respond to the in vivo environment.

FIG. 4. Correlation of microarray and Taqman RT-PCR assays.The fold difference in the number of cDNA molecules present in vivocompared to the inoculum as determined by both microarrays andRT-PCR was log transformed, and values were plotted. Closed dia-monds represent samples removed 2 h after inoculation of nonimmunerabbits, and closed and open circles represent samples removed 2 and8 h, respectively, after inoculation of SEB-immune rabbits. The line ofbest fit is shown (r 0.95).

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ACKNOWLEDGMENTS

This research was funded in part by the Procter & Gamble Co. Invivo experiments were supported by NHLBI grant HL36611 toP.M.S. J.M.Y. was supported by a Howard Hughes Medical Institutepredoctoral fellowship.

We thank G. M. Dunny for his helpful review of the manuscript,T. K. Leonard for assistance in preparation of figures, B. J. May forassistance with RT-PCR, and J. Rucker for technical assistance.

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