INFECTION AND IMMUNITY,0019-9567/01/$04.00�0 DOI: 10.1128/IAI.69.11.7074–7082.2001

Nov. 2001, p. 7074–7082 Vol. 69, No. 11

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

agr Expression Precedes Escape of Internalized Staphylococcusaureus from the Host Endosome

SAARA N. A. QAZI,1,2 EMILIE COUNIL,3 JULIE MORRISSEY,2 CATHERINE E. D. REES,1 ALAN COCKAYNE,2

KLAUS WINZER,2 WENG C. CHAN,4 PAUL WILLIAMS,2,4 AND PHILIP J. HILL1,2*

School of Biosciences, University of Nottingham, Loughborough, Leicestershire LE12 5RD,1 Institute of Infections andImmunity, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH,2 and School of Pharmaceutical

Sciences, University of Nottingham, Nottingham NG7 2RD,4 United Kingdom, andInstitut National Agronomique, Paris-Grignon, France3

Received 11 April 2001/Returned for modification 1 June 2001/Accepted 23 July 2001

Staphylococcus aureus is a versatile pathogen capable of causing life-threatening infections. Many of its cellwall and exoproduct virulence determinants are controlled via the accessory gene regulator (agr). Althoughconsidered primarily as an extracellular pathogen, it is now recognized that S. aureus can be internalized byepithelial and endothelial cells. Traditional experimental approaches to investigate bacterial internalizationare extremely time-consuming and notoriously irreproducible. We present here a new reporter gene method toassess intracellular growth of S. aureus in MAC-T cells that utilizes a gfp-luxABCDE reporter operon under thecontrol of the Bacillus megaterium xylA promoter, which in S. aureus is expressed in a growth-dependent man-ner. This facilitates assessment of the growth of internalized bacteria in a nondestructive assay. The dual gfp-lux reporter cassette was also evaluated as a reporter of agr expression and used to monitor the temporalinduction of agr during the MAC-T internalization process. The data obtained suggest that agr inductionoccurs prior to endosomal lysis and that agr-regulated exoproteins appear to be required prior to the releaseand replication of S. aureus within the infected MAC-T cells.

Staphylococcus aureus is the etiologic agent of numerousinfections in humans and domesticated animals and has beenimplicated in a multitude of diseases, ranging from minorwound infections to more serious diseases, including endocar-ditis, osteomyelitis, and septic shock (reviewed by Projan andNovick [34]). The expression of many S. aureus virulence fac-tors is under the control of the accessory gene regulator (agr)which, on entering post-exponential phase, downregulates theproduction of cell surface-associated proteins and upregulatesthe expression of secreted toxins and extracellular enzymes(28, 33, 38). The role of the agr regulon is supported by in vivostudies, which show that agr mutants are greatly attenuated inseveral animal models, including intramammary infections(13), arthritis in mice (1), and endocarditis in rabbits (7). Theagr locus is a quorum-sensing-regulated system activated byautoinducing peptide pheromone (AIP) (21, 25). The agr locusconsists of two divergent transcriptional units, RNAII andRNAIII, which are under the control of the P2 and P3 pro-moters, respectively (reviewed by Novick and Muir [30]).RNAII is a polycistronic mRNA that encodes the agrB andagrD genes required for the synthesis of the AIP and also thetwo component signal transduction proteins, AgrA and AgrC,which are responsible for sensing and responding to the AIP.RNAIII is the effector molecule in the agr regulon actingprimarily at the level of gene transcription. Different S. aureusstrains produce AIPs with distinct structures, and strains canbe grouped on this basis since they will activate the agr re-sponse of strains within the same group and inhibit the agr

response of strains from different groups by competitive inhi-bition (21, 30). This inhibitory action of AIPs has identifiedthem as potential novel therapeutic and anti-infective agentsfor S. aureus.

S. aureus is primarily known as an extracellular pathogen;however, it has been shown that endothelial cells can act asnonprofessional phagocytes and promote the uptake of S. au-reus (11, 15, 31). Other groups have shown that S. aureus is ableto internalize and survive in a wide variety of mammalian cells(2, 5, 19, 45). S. aureus invades nonprofessional phagocytes viaa mechanism that requires a specific interaction between fi-bronectin-binding proteins and the host cell. This subsequentlyleads to host cell signal transduction through protein tyrosinekinases and cytoskeletal rearrangements (12, 24, 32, 41) anduptake of the bacteria into an endosome. Bayles et al. (5) haveshown that S. aureus is able to escape from this endosome,leaving the bacterial cells to survive and possibly multiply with-in the cytoplasm; however, the mechanism by which this en-dosomal membrane is breached has not been elucidated. In-ternalization experiments using a pulmonary epithelial cell linehave demonstrated the ability of internalized S. aureus to rep-licate intracellularly (22). It is now believed that intracellularreplication plays an important role in the frequency and per-sistence of invasive staphylococcal infections, perhaps by pro-viding protection against both host defenses and antibiotictreatment.

Based on observations that agr mutants and also cells inexponential phase are internalized more efficiently, Wesson etal. (47) proposed a model for the function of agr-mediatedquorum sensing in staphylococcal invasion of cells: in an ex-tracellular environment, levels of AIP are low due to dilutioninto surrounding fluids and S. aureus expresses the cell wall-associated factors that promote binding to host cell surfaces

* Corresponding author. Mailing address: University of Nottingham,School of Biosciences, Sutton Bonington Campus, Loughborough, Lei-cestershire LE12 5RD, United Kingdom. Phone: 44-115-951-6169.Fax: 44-115-951-6162. E-mail: phil.hill@nottingham.ac.uk.

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and subsequent internalization. The expression of these sur-face proteins is known to be repressed by induction of the agrregulon. Upon internalization, bacteria are surrounded by anendosomal membrane, the presence of which may allow con-centrations of AIP to rapidly accumulate and trigger expres-sion of agr and lead to repression of the cell wall-associatedproteins and the production of agr-regulated exoproteins, suchas the hemolysins, which then facilitate bacterial release.

One way in which bacterial localization, movement, andgene expression can be studied is through the use of reportergenes. There are a number of reporters available for suchinvestigations, e.g., green fluorescent protein (GFP) and bac-terial luciferase (lux), which have their own particular advan-tages and disadvantages as reporter and/or marker genes. Themajor disadvantages of lux are that it uses reduced flavinmononucleotide as an energy source and therefore requireslive cells for signal generation (17, 27), so gene expressioncannot be assessed on fixed samples and spatial resolution byphoton-counting microscopy is poor (18). However, the shorthalf-life of Lux proteins allows gene expression and promoterkinetics to be monitored in “real time,” and signal can bedetected with great sensitivity with little background lumines-cence interfering with signal detection. GFP, on the otherhand, suffers from poor sensitivity and high-background fluo-rescence problems and cannot give a real-time representationof promoter kinetics. This is due both to the time taken for thechromophore to fold and generate fluorescent protein (8, 16)and to the long half-life of the protein once it has formed (44).Clearly, the utilization of both of these reporters represents anopportunity to capitalize on the complementary strengths ofeach so that gene expression can be assessed both in “real-time” in vivo by luminometry and/or photonic imaging and also“retrospectively” on fixed samples by fluorescence microscopyor fluorometry.

In this study we describe the construction and evaluationof lux-gfp dual operons expressed in S. aureus. The use of a

growth-phase-dependent promoter linked to bioluminescencemeasurement has led to the development of a novel, nonin-vasive technique for monitoring S. aureus internalization andsubsequent replication inside eukaryotic cells. Induction of theagr regulon in both broth culture and during internalization bybovine mammary epithelial (MAC-T) cells was also monitoredand has facilitated determination of agr expression during thisprocess.

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains and plasmids used orconstructed during this study are listed in Table 1. Throughout this study ared-shifted gfp variant, gfp mutant 3 (gfp3 [10]) was utilized. Except where stated,Luria broth and Luria plates (40) were used throughout for growth of Escherichiacoli and S. aureus. Chloramphenicol was used at 7 �g/ml and ampicillin was usedat 50 �g/ml for plasmid selection, as appropriate. Unless otherwise stated, allcultures were grown aerobically at 37°C, and growth in liquid culture was mon-itored at 600 nm (Cecil 2000 series spectrophotometer).

Preparation, manipulation, and analysis of DNA. Standard methods wereperformed as described by Ausubel et al. (4) using enzymes supplied by Boehr-inger-Mannheim in accordance with the manufacturer’s instructions. PCR prim-ers (Table 2) were supplied by Genosys Biotechnologies (Europe), Ltd. T4 DNAligase (Promega) was used for ligations. DNA fragments were isolated fromlow-melting-point agarose (FMC Bioproducts) via a freeze-thaw extractionmethod (37). PCR (39) was performed in a Techne Progene thermal cycler in50-�l reaction volumes with Taq DNA polymerase (Advanced Biotechnologies,Ltd.) in accordance with manufacturer’s instructions. E. coli JM109 cells weretransformed by electroporation as described by Sambrook et al. (40), S. aureuscells were transformed according to the method of Augustin and Gotz (3).

Construction of dual reporter expression vectors. The PCR primers are de-scribed in Table 2, and the plasmid pBluelux (Table 1) was used as a template;a luxAB amplicon was restricted with EcoRI/KpnI and inserted into pHG327 (42)to give pSB2023. A luxCD amplicon was restricted with KpnI/BamHI and aluxE amplicon with BamHI/PstI. These were ligated into pSB2023 restricted withKpnI/PstI. Recombinant plasmids (pSB2024) were selected by using ampicillinand screened for bioluminescence in the absence of exogenous aldehyde by usinga Hamamatsu VIM3 intensified video camera (Hamamatsu Photonics UnitedKingdom, Ltd.). The modified luxABCDE operon was then subcloned into thesuperlinker plasmid pSL1190 (Pharmacia), which had been restricted with MunI/PstI to give plasmid pSB2025. The luxABCDE cassette was then placed down-stream of gfp in pSB2019 (36) as a SalI/PstI fragment to generate the growth-

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Relevant features and/or genotypea Source or reference

PlasmidspMK4 Gram-positive shuttle vector, Apr, Cmr 43pGC4 pMK4 derivative; 8.2-kb plasmid, contains luxAB fusion from Vibrio harveyi expressed from

Pxyn, Apr and CmrC. Rees (unpublished data)

pSL1190 Superlinker plasmid, contains 64 restriction sites, Apr PharmaciapBluelux luxCDABE from P. luminescens inserted into SmaI site of pBluescript II KS, Apr J. Throup (unpublished data)pHG327 Based on pHG165, contains pUC18 multiple cloning site, Apr 42pSB2023 luxAB inserted into multiple cloning site of pHG327, Apr This studypSB2024 luxCDE inserted downstream of luxAB in pSB2023, Apr This studypSB2025 luxABCDE operon excised from pSB2024 and ligated with pSL1190, Apr This studypSB2019 Translationally enhanced gfp3 downstream of PxylA, Apr Cmr 35pSB2030 luxABCDE excised from pSB2025 SalI/PstI and inserted downstream of PxylA::gfp (pSB2019),

Apr CmrThis study

pSB2031 P3 promoter amplified by PCR from S. aureus 8325-4, inserted into EcoRI/SmaI site ofpSB2019, Apr Cmr

This study

pSB2035 P3 amplified by PCR from S. aureus 8325-4, exchanged with PxylA from pSB2030, Apr Cmr This study

E. coli JM109 �(lac-proAB) recA1 endA1 gyrA9b thi hsdR17 supE44 relA1 F� (traD36 proAB lacIq lacZ1 �M15) 48

S. aureus8325-4 Wild-type strain, cured of known prophages and plasmids 29RN6390 Prototypical wild type 33RN4220 Restriction-deficient derivative of 8325-4 (rK

� mK�) 23

a Apr, ampicillin resistant; Cmr, chloramphenicol resistant.

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dependent reporter plasmid pSB2030. To generate the agr P3::gfp,luxABCDEexpression vector, PCR primers 7 and 8 (Table 2) were used to amplify the P3promoter from S. aureus 8325-4 chromosomal DNA. The amplicon was restrictedwith EcoRI/SmaI and ligated with pSB2019 (36) that had been restricted withEcoRI/SmaI to excise PxylA to create plasmid pSB2031. The luxABCDE operonwas excised from pSB2025 (SalI/PstI) and inserted downstream of gfp in pSB2031,generating a dual reporter designated pSB2035.

Gene expression measurement of bacterial cultures. GFP was detected usinga Nightowl CCD camera system with integrated fluorescence excitation (PerkinElmer Instruments) or by eye using a blue LED for excitation of GFP. Forquantification of GFP, overnight bacterial cultures were diluted 1/100 into pre-warmed medium containing the necessary antibiotics. A 1.5-ml sample was cen-trifuged at 13,000 � g for 2 min, washed twice in an equal volume of phosphate-buffered saline (PBS), and then concentrated 10-fold in PBS. Samples (150 �l)were transferred into microtiter plate wells, and fluorescence was measuredby using Victor 1420 multilabel counter (Perkin-Elmer Instruments). A controlsample of nontransformed bacteria was included to allow correction for back-ground fluorescence.

Bioluminescence was detected by using a Hamamatsu VIM3 camera. Forquantification of bioluminescence, overnight cultures were diluted 1/100 intoprewarmed medium containing the necessary antibiotics. Samples (200 �l) ofeach dilution were separated into aliquots in triplicate into clear-bottom 96-wellmicrotiter plates and incubated with shaking at 37°C in an Anthos Lucy 1 photo-luminometer. Both the optical density at 590 nm (OD590) and the biolumines-cence were measured every 30 min.

Preparation of cells for agr induction experiments. Bacteria harboring agr P3expression vectors were grown overnight in broth containing chloramphenicol.Cells were centrifuged (5,000 � g) and then washed with an equal volume offresh medium to remove accumulated AIPs. Bacteria were diluted 1/20 into freshmedium and grown for 2 h before the culture was again diluted 1/20 into freshmedium and grown for a further 2 h. Finally, these bacterial cultures were diluted1/50 into fresh medium to produce cells in the mid-exponential phase of growthwithout significant accumulation of AIP. To investigate the response of the agrP3reporter to exogenous AIPs, a crude preparation of the group I AIP was pre-pared as filtered spent overnight culture supernatants of RN6390 and added toa final concentration of 10% (vol/vol). Alternatively, either the activating AIP(group I peptide [21, 25]) or inhibitory AIP (S. lugdunensis) synthesized asdescribed by McDowell et al. (26) was added, and the reporter gene activity wasmonitored (as described above) over a specific time period.

Cell invasion assays. The bacterial inoculum was prepared as described for theagr assay. For the final growth cycle, bacterial cells were washed twice in an equalvolume of Dulbecco modified Eagle medium (DMEM; Sigma) and finally resus-pended in 1/10 volume DMEM and inoculated at a 1/400 dilution into medium(HEPES-buffered DMEM plus 10% [vol/vol] RPMI). These were then grown forca. 4 h until an OD600 value of 0.1 to 0.2 was reached.

MAC-T cells (an established bovine mammary epithelial cell line) were rou-tinely cultured as described by Hyunh et al. (20). These cells were seeded into a24-well tissue culture plate (Costar), in DMEM (without antibiotic or fetalbovine serum). These were grown overnight at 37°C in 5% CO2 to achievemonolayers. The following morning the medium was removed and MAC-T cellswere first washed with 1 ml of DMEM and then resuspended in 1 ml of DMEM.For inhibition of internalization, cytochalasin D (1 �g/ml; Sigma) was added toMAC-T cells 30 min prior to the addition of bacterial cells, and cytochalasin Dwas also present during the infection process. The MAC-T cells were infectedwith 1 ml of the prepared bacterial inoculum. The tissue culture plate wasincubated in a Victor 1420 Multilabel Counter (Perkin-Elmer Instruments), andOD600, fluorescence, and luminescence measurements were made every 10 min.

For removal of external bacterial cells from the invasion assay, the MAC-T cellswere washed, after a 2-h infection period, once with PBS beforehand and thenwith 1 ml of DMEM containing lysostaphin (10 �g/ml; Sigma). After 20-minincubation at 37°C, wells were washed again with 1 ml of PBS, and 1 ml ofHEPES-buffered DMEM was added to each well. Readings were taken in aVictor 1420 Multilabel Counter as described above.

Bacterial internalization assays for microscopic analysis. MAC-T cells thathad been seeded onto glass coverslips were incubated at 37°C with 1 ml of thebacterial inoculum. After invasion, the monolayer was washed three times withPBS and then incubated with lysostaphin (10 �g/ml) in DMEM and incubatedfor 20 min at 37°C before three washes with PBS. Immunostaining was carriedout as described by Sambrook et al. (40) with the modification that anti-�-tubulinCy3 conjugate (1/25 in PBS; Sigma) was used to visualize the microtubule net-work. During the last 10 min of the staining procedure, DAPI (4�,6�-diamidino-2-phenylindole; 50 �g/ml; Sigma) in PBS was added for visualization of the GFP-negative bacteria and eukaryotic DNA. Epifluorescent microscopy was carriedout with a Zeiss Axiovert 135TV fluorescence microscope equipped with a Priormotorized stepper stage. Excitation was done with a polychrome II monochrom-ator (T. I. L. L. Photonics) with triple-pass dichroic filter and single-pass emis-sion filters (Omega Optical) mounted in a Biopoint filter wheel. Image capturewas done with a Hamamatsu ORCA-2 cooled CCD controlled by Openlabsoftware (Improvision). Images were captured as 1-�m Z-stacks that were de-convolved by using the Openlab volume deconvolution algorithms and mergedfor presentation. A minimum of 20 fields per slide were examined for qualitativemicroscopic analysis.

RESULTS

Construction of gfp-lux dual operon and expression in S. au-reus. The native luxCDABE operon of Photorhabdus lumines-cens is expressed very poorly in S. aureus when linked to agram-positive promoter (35). To modify the lux operon forhigh expression in gram-positive bacteria, enhanced transla-tional signals (46) were introduced in front of luxA, luxC, andluxE by using PCR primers incorporating these sequences (Ta-ble 2). These primers were used to amplify luxAB (primers 1and 2), luxCD (primers 3 and 4), and luxE (primers 5 and 6).The purified amplicons were restricted using the restrictionsites introduced via the PCR primers (Table 2) and cloned intopSL1190 (Pharmacia) in the gene order luxABCDE (see Ma-terials and Methods for details). Recombinant plasmids wereidentified by their bioluminescent property in the absence ofexogenous aldehyde substrate and designated pSB2025. Tocreate a dual reporter operon, the luxABCDE operon was ex-cised from pSB2025 as a SalI/PstI fragment and inserted intothe gram-positive gfp reporter plasmid SB2019 (36), down-stream of the gfp gene and under the control of the xylA pro-moter from Bacillus megaterium to generate pSB2030.

This plasmid was introduced into S. aureus strains 8325-4and RN6390, and transformed cells were both fluorescent andbioluminescent when grown in liquid culture or on agar plates.

TABLE 2. Sequences of PCR primers used in this study

Primer Sequence (5�-3�) Incorporated site

Primer 1 (luxA forward) GCA CGA ATT CGT CGA CAG GAG GAC TCT CTA TGA AAT TTG GAA AC EcoRISalI

Primer 2 (luxB reverse) CAA CTC GGT ACC TAT TAG GTA TAT TTC ATG TGG KpnIPrimer 3 (luxC forward) CCC CGG TAC CAG GAG GAA GGC AAA TAT GAC TAA AAA AAT TTC KpnIPrimer 4 (luxD reverse) CTC AGG ATC CTT TAA GAC AGA GAA ATT GC BamHIPrimer 5 (luxE forward) CCC GGA TCC TGA GGA GGA AAA CAG GTA TGA CTT CAT ATG BamHIPrimer 6 (luxE reverse) GGG TTA GCT GCA GGA TAT CAA CTA TCA AAC GC PstI

EcoRVPrimer 7 (P3 forward) CAC CGA ATT CCT CAC TGT CAT TAT ACG EcoRIPrimer 8 (P3 reverse) CAT CAA CCC CGG GCC ATC ACA TCT CTG TCA TCT AG SmaI

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When the reporter output was monitored from S. aureusRN6390(pSB2030) grown in liquid culture, the biolumines-cence data showed that the B. megaterium xylA promoter wasonly expressed in actively growing (logarithmic-phase) cultures(Fig. 1A). In contrast, GFP fluorescence was seen to inducelater, and the signal was maintained at a maximal level for alonger period (Fig. 1B). These differences can be explained bythe time required for posttranslational modification of GFPnecessary before the functional fluorophore is formed and theextremely long half-life of mature GFP (�24 h [36]). The GFPsignal generated, therefore, peaks later than the biolumines-cence signal and remains at a higher level. These data confirmthe utility of lux as a real-time reporter of promoter kineticswhich, in contrast to GFP, allows the downregulation as well asthe induction of promoter activity to be assessed.

Expression of the dual gfp-lux operon from agrP3. To con-struct a dual expression vector to study agr gene expression,

PCR primers 7 and 8 (Table 2) were used to amplify the P3promoter from S. aureus 8325-4, and this was used to replacethe xylA promoter in the gram-positive gfp plasmid pSB2019(35); luxABCDE was then inserted SalI/PstI downstream of gfp,creating plasmid pSB2035. To confirm that this new reporterconstruct accurately reflected P3 activity, pSB2035 was intro-duced into S. aureus 8325-4 (agr group I). Cells were grown tomid-log phase after repeated subculture and washing to re-move naturally produced AIP from the culture supernatant.Synthetic group I AIP (Fig. 2A) was added to these cells, andbioluminescence was measured as a reporter of P3 induction.The results of these experiments indicate that the agrP3 pro-moter fusion is activated by the synthetic group I AIP (Fig.3A). The plasmid-encoded agr-P3 promoter exhibits a dose-dependent response to the activator molecule, with lower lev-els of the AIP activator leading to a lower luminescent output.When no activator was added to the bacterial culture, thelevels of luminescence observed were ca. 10-fold lower thanthe induction seen after addition of even the lowest concen-trations of AIP. Since S. aureus 8325-4 is not an agr� mutant,it is able to produce its natural group I AIP; hence, someexpression of P3 is expected in the absence of exogenous AIP.These data indicated that the bioluminescence genes are ef-fective reporters of agrP3 induction.

It has been shown that the AIP from S. lugdunensis (Fig. 2B)can inhibit the agr response in other S. aureus groups (21, 30).Since we showed that the P3 promoter induction could bemeasured by using the dual gfp-lux operon, we then testedthe competitive inhibition of P3 induction by the addition ofsynthetic S. lugdunensis AIP. When S. aureus 8325-4(pSB2035)was grown in the presence of S. lugdunensis AIP, the expected

FIG. 1. Growth-dependent expression of the gfp-luxABCDE dualreporter from PxylA. S. aureus RN6390(pSB2030) was grown in 1-mlvolumes in a 24-well microtiter plate in DMEM supplemented with10% RPMI. Samples were incubated at 37°C in a Victor 2 MultilabelCounter, and growth (OD600; E), luminescence (counts/second, panelA; F), and fluorescence (panel B; f) were measured for 10 h.

FIG. 2. Structures of S. aureus group I AIP (25) (A) and S. lug-dunensis AIP (26) (B).

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inhibition of the P3 promoter was seen, and bioluminescencelevels were much lower than when S. lugdunensis AIP was notadded to the culture medium (Fig. 2B). The competitive na-ture of this inhibition was demonstrated by adding S. lugdunen-sis AIP to cells grown in the presence of their natural group IAIP activator (added in the form of 10% [vol/vol] spent culturesupernatant). Levels of the S. lugdunensis AIP of �1 �M re-duced the luminescence output, suggesting a decrease in P3activity (Fig. 3B). Concentrations of the S. lugdunensis AIP ofbetween 5 to 10 �M completely blocked the induction of S. au-reus group I agrP3 expression, as indicated by bioluminescencereadings.

Development of a staphylococcal internalization assay. Thestrain of S. aureus chosen for internalization assays was RN6390since it has previously been shown to be both virulent in severalanimal models (6, 7) and internalized successfully by MAC-Tcells (5, 47). To determine whether we could use the reportergenes to monitor S. aureus growth during the invasion of MAC-T

cells, S. aureus RN6390(pSB2030) was used. Bioluminescencefrom all samples peaks at 120 min, regardless of the presenceof MAC-T cells, and then decreases (Fig. 4). In the wells con-taining MAC-T monolayers, bioluminescence increases againafter 200 min. This is in marked contrast to the bacteria incu-bated in wells without MAC-T cells, where bioluminescencedecreases to background levels (Fig. 4). The luminescencefrom the cultures seen over the first 120 min represents thegrowth of S. aureus in the tissue culture medium, followed bythe expected decrease in bioluminescence when these bacteriaenter stationary-phase growth and downregulate PxylA. The ob-served increase in bioluminescence after 200 min when MAC-T cells are present is believed to be due to replication ofS. aureus on the surface of, or within, MAC-T cells. To test thishypothesis, cytochalasin D, which inhibits F-actin polymeriza-tion in the MAC-T cells and compromises their ability to in-ternalize S. aureus (5, 22), was added to the infected MAC-Tcells prior to and during infection. In this case, the level ofbioluminescence in the second peak is lower than that in wellswith no cytochalasin D, commensurate with a reduction in thenumber of internalized bacteria (Fig. 4).

To validate this conclusion and to specifically measure thegrowth of internalized S. aureus, similar experiments were per-formed with S. aureus RN6390(pSB2030) and MAC-T cells,but with the incorporation of a lysostaphin treatment after 2 hof infection. This removes extracellular and adherent bacteria,so reporter activity measured must be due solely to intracellu-larly replicating bacteria. Measurements of bioluminescencewere not taken during the 2-h infection period; hence, no ini-tial peak of bioluminescence can be seen (Fig. 5). In the lyso-staphin-treated wells containing MAC-T cells, a luminescentsignal is detectable after ca. 1 h, indicating that the signal is dueto bacteria that are replicating intracellularly. This is supportedby the fact that the luminescent signal generated by bacteria in

FIG. 3. S. aureus 8325-4(pSB2035) response to activating and in-hibitory AIPs. S. aureus 8325-4(pSB2035) was grown at 37°C in 96-wellplates in an Anthos Lucy 1 photoluminometer. OD600 and lumines-cence readings (in relative light units [RLU]) were taken over a periodof 20 h. These data are plotted as specific luminescence (RLU/OD)versus time to normalize changes in cell density. (A) Cells were grownin Luria broth alone ({) or Luria broth supplemented with group IAIP at 5 �M (E), 25 �M (�), 50 �M (Œ), 75 �M (f), or 100 �M (}).(B) Cells were grown in Luria broth supplemented with 10% spentculture supernatant (F) or also supplemented with S. lugdunensis AIPat 1 �M (�), 5 �M (�), 10 �M (‚), or 50 �M (�).

FIG. 4. Invasion of MAC-T cells by S. aureus RN6390(pSB2030). S.aureus RN6390(pSB2030) were used to inoculate a 24-well plate con-taining MAC-T monolayers, in DMEM supplemented with 10% RPMIwith (�) or without (f) 1 �g of cytochalasin D/ml. As controls, bac-teria were also inoculated into wells containing medium alone with (Œ)or without (}) 1 �g of cytochalasin D/ml. All samples were incubatedat 37°C, and luminescence readings (in counts/second [cps]) taken overa 10-h period in a Victor 2 Multilabel Counter.

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the wells containing MAC-T plus cytochalasin D is not abovebackground levels (i.e., wells with no MAC-T monolayer; Fig.5). An additional conclusion from this study is that the intra-cellular S. aureus were replicating since we know that the PxylA

is only expressed in actively growing cells (Fig. 1A). This con-clusion was confirmed by both bacterial enumeration afterMAC-T lysis (viable count assay) and also by microscopic ex-amination (cell enumeration) of samples taken at t 60, 180,300, and 480 min (data not shown).

These observations have allowed us to develop a simple,rapid, and noninvasive assay of S. aureus internalization. Theinvasion assay is based on three principle observations: (i) onlyactively growing S. aureus(pSB2030) cells are luminescent, (ii)MAC-T cells treated with cytochalasin D are compromised intheir ability to internalize S. aureus, and (iii) lysostaphin treat-ment effectively removes extracellular and adherent bacteria.Therefore, the level of reporter activity from S. aureus(pSB2030)incubated with MAC-T cells in the presence of cytochalasin-Dand after lysostaphin treatment reflects only the intracellularreplication of the bacteria.

Analysis of patterns of agrP3 expression by S. aureus uponMAC-T invasion. To examine agr expression in an intracellularenvironment, MAC-T cell invasion assays were performed withthe agrP3 reporter strain S. aureus RN6390(pSB2035). As above,MAC-T monolayers were incubated with bacteria both in thepresence and in the absence of cytochalasin D with lysostaphintreatment after a 2-h infection period. As controls, bacteriawere incubated in assay wells alone, with or without cytocha-lasin D. In these control experiments no induction of eitherpromoter was seen (Fig. 6). In the MAC-T internalizationassay, bioluminescence from S. aureus RN6390(pSB2035) wasat a maximum after 100 min (Fig. 6). In parallel infectionexperiments performed with S. aureus RN6390(pSB2030)(PxylA), luminescence did not peak until 370 min (Fig. 6), afinding which indicates bacterial replication (Fig. 1). It isknown that internalized S. aureus are surrounded by an endo-somal membrane (5, 19), from which they then escape by lysis

(5). Our data now suggest that agr is induced to high levelswhile the bacteria are within the endosome (as illustrated byhigh luminescence output during the first 100 min of internal-ization) and is followed by replication on release into thecytoplasm. Hence, it is likely that production of agr-regulatedexoproteins results in endosomal lysis.

To confirm that the increase in bioluminescence was due tospecific induction of agr and not to an increase in staphylococ-cal numbers, a microscopic study was carried out utilizing GFPsignal from the pSB2035 reporter plasmid. MAC-T cells seed-ed onto coverslips were incubated with S. aureus RN6390(pSB2035). After various infection periods, lysostaphin wasapplied to remove extracellular bacteria, and samples werestained (as described in Materials and Methods) prior to anal-ysis by fluorescence microscopy. An anti-�-tubulin-Cy3 conju-gate was used to visualize microtubules for orientation withinthe cells to confirm that the staphylococci imaged were indeedintracellular. After 2 h, S. aureus RN6390(pSB2035) cells wereclearly visible within epithelial cells, although the numberswere low. At this time most of the bacteria were not exhibitinga GFP� phenotype (blue cells due to DAPI staining of thebacterial nucleoid; Fig. 7a and b). This is expected even if agrP3 had been induced because of the time required for thematuration of GFP. However, we have evidence that at thispoint agr expression has not been induced since the red stain-ing seen around the individual staphylococcal cells is due to thebinding of the anti-�-tubulin-Cy3 conjugate to protein A in thecell wall. Since protein A is downregulated upon agr induction,this indicates that the agr regulon has not been induced inthese cells at this time point.

FIG. 5. Measurement of intracellular growth of S. aureus RN6390(pSB2030). S. aureus RN6390(pSB2030) were used to inoculate a 24-well plate containing MAC-T monolayers, in DMEM supplementedwith 10% RPMI with (�) or without (}) 1 �g of cytochalasin D/ml. Ascontrols, bacteria were also inoculated into wells containing mediumalone with (Œ) or without (�) 1 �g of cytochalasin D/ml. After a 2-hinfection period, all samples were treated with lysostaphin, and thenthe medium was replaced with DMEM. The plate was incubated at37°C, and luminescence readings (in counts/second [cps]) were takenover a 10-h period in a Victor 2 Multilabel Counter.

FIG. 6. Growth and expression of agr by S. aureus RN6390 in MAC-T cell invasion assay. To measure agrP3 expression, S. aureus RN6390(pSB2035) was inoculated into a 24-well plate containing MAC-T mono-layers, in DMEM supplemented with 10% RPMI with (Œ) or without(}) 1 �g of cytochalasin D/ml. As controls, bacteria were also inocu-lated into wells containing medium alone with (�) or without (f) 1 �gof cytochalasin D/ml. To monitor staphylococcal replication, S. aureusRN6390(pSB2030) was used to inoculate wells containing MAC-Tmonolayers with (�) or without (F) 1 �g of cytochalasin D/ml. Aftera 2-h infection period, all wells were treated with lysostaphin and thenthe medium was replaced with DMEM. The plate was incubated at37°C, and luminescence readings were taken over a 10-h period in aVictor 2 Multilabel Counter. The data are plotted as the percent maxi-mum luminescence for each reporter construct to compensate forvariations in promoter strength.

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As the infection process proceeds to 4 h (Fig. 7c), bacteriaare still seen to be internalized within the epithelial cells, butnow more of the bacteria are GFP�, suggesting that agrP3 hasnow been induced within the MAC-T cells. This coincides withthe loss of the red staining of protein A as a phenotypic indi-cator of agr induction. As the infection proceeds to 6 h (Fig.7d), the number of internalized bacteria increases, as does thenumber of bacteria expressing GFP and, again, no red halosare observed. The high level of GFP suggests that the majorityof the internalized bacteria have expressed agr. As the infec-tion proceeds through to the latter stages, the bacteria are seenin pairs or tetrads, resulting from replication, throughout theMAC-T cells (Fig. 7c and d). These cells stain primarily bluedue to DAPI staining of the nucleoid. The loss of GFP signalis probably due to the downregulation of agrP3 on release of

cells into the cytoplasm and then dilution of accumulated GFPafter bacterial cell division.

DISCUSSION

The lux operon from P. luminescens has been reconstructedto contain enhanced translational signals for genes whose se-quence analysis suggested might be poorly translated. Threegenes in this operon, luxA, -C, and -E, were engineered to con-tain optimized gram-positive translational initiation sequences.This was combined with gfp that had been similarly modified(36) to construct a dual reporter operon and expressed inS. aureus to allow studies of bacterial growth and gene expres-sion during cell invasion.

The gfp-lux dual reporter downstream of the B. megaterium

FIG. 7. Microscopic evaluation of growth and agrP3 expression by S. aureus RN6390 invading MAC-T cells. MAC-T monolayers on coverslipswere inoculated with S. aureus RN6390(pSB2035) in DMEM supplemented with 10% RPMI. The specimens were treated with lysostaphin after2 h and stained with anti-�-tubulin-Cy3 conjugate (red) and DAPI (blue) at 2 h (a and b), 4 h (c), and 6 h (d) postinfection. The specimens werevisualized by fluorescence microscopy.

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xylA promoter, which is expressed in a growth-dependent man-ner, has been used to investigate ex vivo the intracellular rep-lication of S. aureus in MAC-T cells. When MAC-T monolay-ers were incubated with the bacteria for the duration of theinvasion assay, two peaks of bioluminescence were observed.The first corresponds with luminescence emitted from bacteriaincubated in the absence of MAC-T cells and represents bac-terial replication in the cell culture medium. The second peakof bioluminescence was shown to be due to replicating S. au-reus both on and within the eukaryotic cells. To measure thereplication of intracellular S. aureus alone, the assay was fur-ther modified by the inclusion of a lysostaphin treatment aftera 2-h invasion period. In this improved assay, only wells con-taining MAC-T monolayers inoculated with S. aureus in theabsence of cytochalasin D exhibited a bioluminescent outputsince cytochalasin D inhibits the uptake of S. aureus by MAC-Tcells (5). Thus, by using the new dual-reporter operons, wehave developed an assay to study intracellular replication ofS. aureus, monitoring the event in real time by using the luxgenes and in fixed samples by using the GFP signal. When thislatter reporter is used, time is needed to allow the posttrans-lational modification required to produce the functional chro-mophore; however, we have demonstrated its effectiveness inthe microscopic analysis of cell invasion.

For S. aureus to replicate within MAC-T cells, the bacteriamust first escape from the encapsulating endosomal membrane(5, 19). agr expression has previously been reported to beimportant for S. aureus intracellular survival (47), although thespecific role of agr in either endosomal escape or intracellularreplication has not been elucidated. The use of the new non-destructive dual reporters to measure agr expression in ournovel cell invasion assay system has now allowed this questionto be addressed. Previously, Wesson et al. (47) demonstratedthat the agr mutant, RN6911, was internalized at higher effi-ciencies in MAC-T cells than the wild-type strain RN6390. Thiswas not unexpected, since the agr mutants were known toproduce increased levels of the cell surface binding proteinssuch as the fibronectin-binding proteins. However, althoughthis agr mutant was internalized, it was unable to replicate in-tracellularly; in fact, the numbers of viable bacteria decreasedover time. Wesson et al. hypothesized that agr-regulated prod-ucts are necessary for escape from the endosome and furthergrowth in the cytoplasm (47). Furthermore, these authors sug-gested that, due to the confined space within the endosome,the accumulation of AIP is rapid and lysis of the endosomemay be due to the induction of agr-regulated exoproteins. Thishypothesis has now been substantiated by our data with theagrP3 dual reporter in which bioluminescence from the agrreporter is induced within the first 100 min of internalizationprior to bacterial replication, as monitored by the expression ofPxylA, which is not induced until the cells are released into thehost cell cytoplasm. Recently, it has been found that the S. au-reus strains from the NCTC 8325 lineage (including RN6390and 8325–4) are downregulated in sigB expression due to adefect in rsbU (14), which may in turn affect the level of agrexpression if internalization is perceived as a stress condition.To address this, we carried out preliminary studies using aclinical isolate (WCUH29) with an intact sigB locus, and in thisbackground the promoter kinetics are in agreement with thoseseen with RN6390.

The involvement of agr in internalization and subsequentreplication in MAC-T cells has also been demonstrated micro-scopically by utilizing the GFP protein in combination withfluorescent dyes. By using the agr dual reporters, it has beenshown that agr expression is low upon internalization, as indi-cated by low gfp expression. Cell surface-associated proteinsare highly expressed under these conditions, as illustrated bythe binding of the antitubulin conjugate to protein A in thebacterial cell wall, which gives a phenotypic indicator of agrexpression to further validate the reporter data. At both 4 hand 6 h postinfection, the number of GFP� staphylococci in-creases significantly. At these time points, no detectable levelsof protein A were observed by immunofluorescence, a findingagain indicative of upregulation of agr and a correspondingdecrease in cell surface-associated proteins.

The data gathered during the course of this study show thatthe novel dual-reporter gene system is a powerful, nondestruc-tive tool for gene expression studies ex vivo and potentially invivo. For example, lux expression coupled with low-light imag-ing can allow the visualization of bacterial gene expression incomplex environments such as whole animals (9), providinginformation about the temporal and spatial regulation of par-ticular genes. Coupling this approach with histologic studies us-ing the GFP signal to give a “retrospective” measure of whichbacterial genes were expressed in which tissues or cells wouldgive a more detailed picture of staphylococcal pathogenesis.

ACKNOWLEDGMENTS

We thank Stewart Wood for synthesis of the AIPs.We are grateful to the Biotechnology and Biological Sciences Re-

search Council (96/A1/F/02414) and the Medical Research Council(G9219778) for funding.

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