INFECTION AND IMMUNITY, Aug. 2003, p. 4463–4471 Vol. 71, No. 80019-9567/03/$08.00�0 DOI: 10.1128/IAI.71.8.4463–4471.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification of the agr Locus of Listeria monocytogenes: Role inBacterial Virulence

Nicolas Autret, Catherine Raynaud, Iharilalao Dubail, Patrick Berche, and Alain Charbit*INSERM U-570, CHU Necker-Enfants Malades, 75730 Paris Cedex 15, France

Received 17 March 2003/Returned for modification 2 May 2003/Accepted 16 May 2003

Listeria monocytogenes is a gram-positive facultative intracellular food-borne pathogen that can cause severeinfections in humans and animals. We have recently adapted signature-tagged transposon mutagenesis (STM)to identify genes involved in the virulence of L. monocytogenes. A new round of STM allowed us to identify a newlocus encoding a protein homologous to AgrA, the well-studied response regulator of Staphylococcus aureus andpart of a two-component system involved in bacterial virulence. The production of several secreted proteins wasmodified in the agrA mutant of L. monocytogenes grown in broth, indicating that the agr locus influenced proteinsecretion. Inactivation of agrA did not affect the ability of the pathogen to invade and multiply in cells in vitro.However, the virulence of the agrA mutant was attenuated in the mouse (a 10-fold increase in the 50% lethaldose by the intravenous route), demonstrating for the first time a role for the agr locus in the virulence of L.monocytogenes.

Listeria monocytogenes is a gram-positive bacterium wide-spread in nature and responsible for sporadic severe infectionsin humans and other animal species (see reference 2 and ref-erences therein). This organism is a facultative intracellularparasite capable of invading most host cells, including epithe-lial cells (13), hepatocytes (7, 14), fibroblasts (20), endothelialcells (8), and macrophages (22). Each step of the intracellularparasitism by L. monocytogenes is dependent upon the produc-tion of virulence factors (4). The major virulence genes (hly,plcA, plcB, mpl, actA, inlA, and inlB) are clustered into twodistinct loci on the chromosome and are controlled by a singlepleiotropic regulatory activator, PrfA, which is required forvirulence (see reference 33 for a review).

We have recently adapted signature-tagged mutagenesis(STM) to L. monocytogenes (1). In this work, we performed anew STM screening with the liver as a target organ (see Ma-terials and Methods for details). By this procedure, we haveidentified a transposon insertion in a gene, designated agrA,encoding a protein homologous to the response regulatorAgrA of Staphylococcus aureus. The two-component regulatorysystem of which AgrA is a part has been extensively studied inS. aureus and has been shown to control the production ofvirulence factors (see reference 27 for a review). The agr locusof S. aureus expresses two primary divergent transcripts.RNAII encodes a two-component system, AgrA/AgrC, whichrecognizes the agrD-encoded secreted autoinducing octapep-tide (AIP), and AgrB, which is thought to act in the posttrans-lational processing and secretion of AIP. The second majortranscript, RNAIII, acts as the effector molecule of the agrlocus. Finally, a third, short transcript, RNAI, has also beendescribed as encoding AgrA. Upon accumulation of sufficientquantities of AIP in the growth medium, signaling via AgrA/AgrC increases transcription of both RNAII and RNAIII, re-

sulting in the down-regulation of exponential-phase proteins,such as the cell surface-associated protein A, and the increasedexpression of postexponentially secreted proteins, such as en-terotoxins B, C, and D, involved in bacterial virulence (18).AgrA, which is also required for agr activation and correspondsto the response regulator, does not appear to bind directly tothe agr promoters but may interact with SarA, the product ofa separate genetic locus, to mediate activation (25, 26). AgrCacts as a sensor for the autocrine signal provided by the AIPderived from AgrD and responds by autophosphorylation on ahistidine residue.

The genome of L. monocytogenes contains 16 putative re-sponse regulators constituting two-component regulatory sys-tems (16). This number is similar to that in Bacillus subtilis andEscherichia coli (taking into account the genome sizes). InStreptococcus pneumoniae, thirteen putative two-componentregulatory systems have been identified, and a systematic geneinactivation approach showed that most of them (8 of 13) wereimportant for bacterial virulence (31). Two of the 16 putativetwo-component systems of L. monocytogenes have been studiedexperimentally: the cheY/cheA (11) and lisR/lisK (5) systems.Transposon insertion in the promoter region of the che operonreduced flagellin expression and affected the ability of L.monocytogenes to attach to the mouse fibroblast cell line 3T3(11). The LisR/LisK system was shown to be involved in stresstolerance, and inactivation of lisRK resulted in a slight de-crease of bacterial virulence (5). A more recent study de-scribed the inactivation of five putative two-component sys-tems (19). One of them corresponded to the previously studiedLisR gene (lmo1377), and the four others corresponded tolmo2422, lmo2501, lmo2583, and lmo2678. A preliminary eval-uation of the effects of the mutations on bacterial virulence,performed with mice, showed that only the inactivation of theLisR gene reduced bacterial virulence by the different routes ofinfection used (5, 19).

In the present work, we show that inactivation of the L.monocytogenes agrA gene influences protein secretion and at-

* Corresponding author. Mailing address: Faculte de MedecineNecker, 156, rue de Vaugirard, 75730 Paris Cedex 15, France. Phone:33 1 40 61 53 76. Fax: 33 1 40 61 55 92. E-mail: charbit@necker.fr.

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tenuates the virulence of the bacterium in the mouse withoutaffecting the growth of the bacterium in cell cultures. Func-tional implications of the role of this locus in L. monocytogenesare discussed.

MATERIALS AND METHODS

Bacterial strains, plasmids, media, and DNA techniques. E. coli recombinantswere grown in Luria-Bertani medium and L. monocytogenes in brain heart infu-sion (BHI) broth (Difco Laboratories, Detroit, Mich.) at 37°C. The wild-typevirulent strain of L. monocytogenes EGD belongs to the serovar 1/2a (23). EGD-ewas transformed with the different recombinant plasmids by electroporation aspreviously described (1). Antibiotics were used at the following concentrations:kanamycin, 50 �g ml�1; erythromycin (Em), 5 �g ml�1.

Chromosomal DNA, plasmid isolation, restriction enzyme analyses, and PCRamplifications were performed as previously described (1). Oligonucleotideswere synthesized by Proligo (Paris, France). The AmpliTaq DNA polymerase ofThermus aquaticus from Finnzymes OY (Espoo, Finland) was used.

Screening procedure. STM was performed as described previously with L.monocytogenes strain EGD-e (1). Pools of 48 mutants of L. monocytogenes wereassembled, and each pool was injected into mice at a dose of 106 bacteria permouse. In the present screening, the liver was chosen as a target organ. At thisdose, the average number of bacteria reached �108 per liver at day 4, i.e.,1,000-fold higher than that in the brain. In these conditions, each mutant withina pool is represented up to 2 � 106 times, thus improving the sensitivity of thescreening. Infected livers were collected at day 4 and homogenized and culturedin BHI-Em (1).

Southern blot analysis. L. monocytogenes chromosomal DNA was prepared aspreviously described in reference 1. Briefly, chromosomal DNA was digestedwith BglII and Sau3AI and transferred onto nylon Hybond-N� membranes(Amersham) under denaturing conditions. Hybridizations were realized underhigh stringency conditions, including prehybridization at 65°C for 3 h in 6� SSC(1� SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.5% sodium dodecylsulfate (SDS)–0.05% Regilait buffer. Hybridization was carried out overnight at65°C in 6� SSC–0.1% SDS–0.05% Regilait buffer, with two 30-min washes atroom temperature in 2� SSC–0.1% SDS followed by two 30-min washes at 65°Cin 0.2� SSC–0.1% SDS. Hybridization was detected on Hyperfilm-MP films(Amersham).

The probe used corresponds to a 0.7-kb internal portion of the plasmidpAT113 located between oriRK2 and the multiple cloning site. The fragment was(i) amplified by PCR with primer RK2-F and the forward primer of mp18/pUC18(sequencing primer 1211; NE Biolabs), (ii) digested with HindIII (downstream ofthe multiple cloning site) to remove the region of the polylinker, and (iii)32P-labeled using the megaprime kit (Amersham).

Transduction. The transposon-inactivated agrA gene was transferred into awild-type background by generalized transduction with phage LMUP35 (17).Lysates of LMUP35 were prepared with the agrA mutant strain, and transductioninto wild-type EGD-e was carried out as described previously (24). The trans-ductants were checked by PCR analysis.

RT-PCR. Total RNA was extracted from L. monocytogenes cultures grownovernight in BHI broth at 37°C as described in reference 3. The primers used toamplify the mRNA corresponding to the different genes from EGD by reversetranscription-PCR (RT-PCR) are listed below. We used the procedure describedin the SuperScript One-Step RT-PCR system kit (Life Technologies, Paisley,Scotland). Prior to RT-PCR, total RNA samples were incubated for 1 h at 37°Cwith DNase I (RNase free; Boehringer, Mannheim, Germany) to eliminate anyDNA contamination. The following pairs of primers were used: agrA forwardprimer, 5�-CGAATGCCTACACATCAAGGTA-3�; agrA reverse primer, 5�-TCACCACACCTTTTGTCGTATC-3�; agrB forward primer, 5�-AAAGTCCCTTTGTCAGAAAGAATG-3�; agrB reverse primer, 5�-CACCTGAAACAAAGATCCTACCA-3�; agrC forward primer, 5�-ATTAATACGGCAACCAACGAAC-3�;and agrC reverse primer, 5�-AAATCGGTGGCATATTTACTGG-3�.

Real-time quantitative Taqman PCR assay. (i) Extraction of L. monocytogenesRNA. Bacteria were grown to an optical density at 600 nm (OD600) of 0.4. Cellswere broken in a solution of Trizol (1 ml; Life Technologies) with mini glassbeads by using a Bead Beater apparatus (Polylabo) set at maximum speed. RNAwas extracted with 300 ml of chloroform-isoamyl alcohol. After 10 min of cen-trifugation at 13,000 � g, the aqueous phase was transferred to a tube containing270 ml of isopropanol. Total RNA was then precipitated overnight at 4°C andwashed with 1 ml of a 75% ethanol solution before suspension in diethyl pyro-carbonate-treated water. Contaminating DNA was removed by digestion withDNase I according to the instructions of the manufacturer (Roche).

(ii) Real-time quantitative PCR. The assay was carried out with the ABI Prism7700 sequence detection system by using Taqman Universal PCR master mix (PEApplied Biosystems). The primers were designed by using the Primer Expresssoftware and obtained from PE Applied Biosystems. The sequences were asfollows: agrA forward primer, 5�-AATGTTTTGAATTAGCTCAGGAAA-3�;agrA reverse primer, 5�-ACTCCGCATGTGTTGTAATAAAAATAA-3�; agrBforward primer, 5�-AGGTACATTTGGATTTATACTGCTCAAC-3�; agrB re-verse primer, 5�-TCTTCACCGATTAAAGGCAAACT-3�; agrC forwardprimer, 5�-ATTGACAAGATTTCGATGGATAGTATAGATT-3�; agrC reverseprimer, 5�-CACAAGTTAACGCCGCTTCA-3�; gyr forward primer, 5�-AAATGCGGACATCATTCCTAGACT-3�; gyr reverse primer, 5�-TTTAACCCGTCACGAACATCAG-3�; 16S forward primer, 5�-CATAATGGTGCAAAACCATCTGA-3�; and 16S reverse primer, 5�-TTGGTTATGGAATTTTTCCATGATGATAC-3�.

RT-PCR experiments were carried out with 1 �g of RNA and 2.5 pmol ofprimers specific for agr, gyr, and 16S in a volume of 8 �l. After denaturation at65°C for 10 min, 12 �l of the mixture containing 2 �l of deoxynucleosidetriphosphate (25 mM), 4 �l of 4� buffer, 2 �l of dithiothreitol, 1 �l of RNasin(Promega), and 1.5 �l of Superscript II (Invitrogen) was added. Samples wereincubated for 60 min at 42°C, heated at 75°C for 15 min, and then chilled on ice.Samples were diluted with 40 �l of H2O and stored at �20°C. PCR conditionswere identical for all reactions. The 25-�l reaction mixtures consisted of 12.5 mlof PCR master mix (PE Applied Biosystems) containing Sybr Green, 4 �l oftemplate, and 5 pmol of each primer. The reactions were carried out in sealedtubes. Results were normalized to the amount of gyr mRNA and 16S (16SrRNA). The gyr and 16S genes were chosen as standards because they had beenpreviously shown to be constant under different conditions in several gram-positive bacteria. Our assays confirmed that these two reporter genes also re-mained constant in L. monocytogenes under all the growth conditions used here(for the same concentrations of RNA under different conditions of growth, weobtained the same amounts of mRNA for gyr and 16S; data not shown). All theexperiments have been done in triplicate.

Sequence analysis and identification of transposon insertion site in Listeriagenome. (i) Inverse PCR. The DNA sequence flanking the transposon wasdetermined as described previously (1). Chromosomal DNA was digested withSau3A. Digestion products were ligated, generating circular molecules contain-ing either the right end or the left end of Tn1545. DNA was amplified by PCRwith the primers SeqL (5�-GGATAAATCGTCGTATCAAAG-3�) and SeqR(5�-CGTGAAGTATCTTCCTACAGT-3�).

(ii) PCR sequencing. The PCR products were sequenced with the automatedABI Prism 310 sequencer (Perkin Elmer; Applied Biosystems) using the BigDyeTerminator Cycle Sequencing Ready Reaction kit. Sequences were analyzed withthe Sequence Navigator software program (Perkin Elmer). Similarity searcheswere then performed via the Internet with BLAST software (1) from the Na-tional Center for Biotechnology Information home page (www.ncbi.nlm.gov./BLAST/) by launching the sequences in the complete 2,900,000-bp Listeria ge-nome database (BLASTn search). The sequences of the identified open readingframes (ORFs) were then launched in the general databases (nonredundantBLASTp search).

SDS-PAGE and Western blot analysis. Proteins from culture supernatants andtotal bacterial extracts were prepared as follows. Fractions of bacterial culturesgrown in BHI rich medium at different OD600s were centrifuged (exponentialphase, OD600 of 0.6; stationary phase, OD600 of 10). Supernatants were passedthrough a 0.22-�m-pore-size filter (Millipore) and further concentrated withultrafree columns (Millipore) with a cutoff of 30 kDa. Samples were finallysuspended in 1� SDS-PAGE sample buffer (130 mM Tris-HCl [pH 6.8], 1%SDS, 7% 2-�-mercaptoethanol, 7% sucrose, 0.01% bromophenol blue). Thebacterial pellets were suspended in cold water, and bacteria were disrupted byusing a Fastprep FP120 apparatus (BIO101, La Jolla, Calif.) with three pulses of30 s at a speed rating of 6.5. After centrifugation for 3 min at 8,000 � g, lysateswere collected and suspended in 1� SDS-PAGE sample buffer. Electrophoresisand Western blotting were carried out as described previously (15) in SDS–8%polyacrylamide minigels (Mini Protean II; Bio-Rad). Silver staining of gels wasadapted from that described in reference 32.

Nitrocellulose sheets were probed either with anti-listeriolysin O (anti-LLO)monoclonal antibodies (MAbs) SE1 and SE2 (9) or with polyclonal anti-ActA oranti-PlcB antibodies, along with anti-mouse or anti-rabbit horseradish peroxi-dase-conjugated secondary antibody. Antibodies were used at a final dilution of1:1,000, except for anti-PlcB (1:200). Antibody binding was revealed by adding0.05% diaminobenzidine-tetrahydrochloride (Sigma) and 0.03% hydrogen per-oxide (Sigma).

Culture of cell lines. The human colon carcinoma cell line Caco-2 (ATCCHTB37) and the human hepatocellular carcinoma cell line HepG-2 (ATCC HB

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8065, kindly provided by S. Dramsi and P. Cossart, Institut Pasteur, Paris,France) were propagated as previously described (7) in Dulbecco modified Eaglemedium (Invitrogen) containing 10% fetal bovine serum (FBS). Cells wereseeded at ca. 2 � 105 cells cm�2 in 24-well tissue culture plates (Falcon).Monolayers were used 24 h after seeding.

Invasion assays. The invasion assays were carried out essentially as describedpreviously (15). Briefly, cells were inoculated with bacteria at a multiplicity ofinfection of approximately 100 bacteria per cell. The cells were incubated for 1 hat 37°C to allow the adherent bacteria to enter. Cells were washed six times inphosphate-buffered saline (PBS) medium and inoculated with fresh Dulbeccomodified Eagle medium-fetal bovine serum. At 2, 3, and 4 h, cells were washedthree times in PBS medium containing MgCl2 and CaCl2 and processed forcounting of infecting bacteria. For processing, cells were lysed by adding asolution of 0.1% Triton X-100. The titer of viable bacteria released from the cellswas determined by spreading bacteria onto BHI plates. Each experiment wascarried out in triplicate and repeated twice.

Bone marrow-derived macrophages. Bone marrow-derived macrophages fromBALB/c mice were cultured as described previously (6) and then infected asfollows: bacteria grown overnight were diluted in cell culture medium to give amultiplicity of infection of one bacteria per macrophage. Bacteria were allowedto adhere to cells by incubation on ice for 15 min and then to enter cells byplacing cells at 37°C for 15 min. After the medium was removed, the infectedcells were washed six times with 1 ml of PBS medium containing MgCl2 andCaCl2 and once with RPMI medium to remove extracellular bacteria. The cellswere then replaced in fresh medium and incubated at 37°C.

Infection of mice. Specific-pathogen-free 6- to 8-week-old female Swiss mice(Janvier, Le Genest St. Isle, France) were used. Bacteria were grown for 18 h inBHI broth, centrifuged, appropriately diluted in 0.15 M NaCl, and inoculated(0.5 ml) intravenously (i.v.) into mice via the lateral tail veins. Groups of fivemice were challenged i.v. with various doses of bacteria, and mortality wasmonitored for 10 days. The virulence of the mutant was estimated by using the50% lethal dose (LD50) with the Probit method (10).

FIG. 1. Genetic organization of the agr locus and transcriptional analysis. (A) Genetic organization of the agr locus. (Upper panel) agr locusof S. aureus. The grey arrows indicate the orientations and approximate sizes of the different genes. The dotted lines ending in arrows indicate thetwo main divergent transcripts RNAII and RNAIII. To the right, a schematic representation of the various components of the agr locus is shown,with letters representing the proteins encoded A, AgrA; B, AgrB; C, AgrC; D, AgrD; asterisk, phosphorylated group; P2 and P3, promoters ofRNAII and RNAIII, respectively. (Lower panel) agr locus of L. monocytogenes. The arrows indicate approximate sizes and orientations of thedifferent genes. The predicted length of each protein is indicated below (in amino acids [aas]). The stop sign-shaped symbols indicate putativetranscription terminators. The values in percentages below agrB, agrC, and agrA indicate the percentage of amino acid identity between the L.monocytogenes and S. aureus orthologues encoded by the genes of the agr loci. Numbers between parentheses indicate the sizes (in base pairs) ofthe intergenic regions. The site of Tn1545 insertion is represented by an inverted black triangle. (B) Transcriptional analysis by RT-PCR. Thedotted lines enclosed by arrows in the lower half of panel A indicate the positions of the primers and PCR products used in the RT-PCR analysis.The amplified products, numbered 1 to 5, were subjected to Tris acetate-EDTA-agarose gel electrophoresis. The arrows preceded by numbers (inkilobases) to the left of the panel correspond to the molecular weight (MW) DNA ladder. (C) Expression of the agr genes in stationary (Stat) andexponential (Exp) phases. The expression was measured by real-time quantitative RT-PCR. The amount of agr mRNA relative to that of thenormalizing gene gyr (upper panel) and the 16S gene (lower panel) was determined by real-time quantitative RT-PCR with bacteria grown in BHIin the exponential or stationary phase of growth. The amounts of gyr mRNA and 16S rRNA were constant under these conditions (data not shown).The values shown are means of results from three assays; the error bars indicate the standard deviations.

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Kinetics studies. Thirty mice per mutant were inoculated i.v. in the lateral tailveins. At days 1, 2, 3, 4, 7, and 10, groups of five mice were sacrificed and theorgans (spleens, livers, and brains) were aseptically removed and separatelyhomogenized in 0.15 M NaCl. Bacterial numbers in organ homogenates weredetermined at various intervals on BHI plates containing Em. Five hundredmicroliters of bacterial suspension containing 2 � 104 bacteria were injected permouse.

In all the assays, bacteria were stored in 1-ml fractions at �80°C after growthand the number of bacteria per milliliter in each defrosted culture was deter-mined before and after inoculation.

RESULTS

Isolation and characteristics of the agrA mutant of L. mono-cytogenes. Among the candidates identified by this new STMscreen, one mutant contained a Tn1545 insertion in a gene,designated agrA by analogy to the agr two-component regula-

tory system of S. aureus. The particular attention devoted re-cently to the role of two-component systems in the control ofbacterial pathogenesis (30), and the fact that the agr system ofS. aureus is one of the most studied two-component systems ingram-positive organisms, prompted us to focus, in the presentwork, on the properties of this mutant.

Characterization of the mutant. The transposon insertionsite in the chromosome of the agrA mutant was determined asdescribed previously (1). The Tn1545 insertion occurred 276bases downstream of the first ATG of agrA, thus leading to thecomplete inactivation of its expression. We first checked bySouthern blot hybridization that the mutant corresponded to asingle Tn1545 insertion. Then, in order to eliminate possibleadditional mutations in other regions of the chromosome, thetransposon-inactivated agrA gene was transferred into a wild-

FIG. 2. Multiple alignment of AgrA (A) and AgrC (B) from L. monocytogenes and S. aureus. Alignments were performed by using CLUSTALWand AlignP programs. Lismo, L. monocytogenes (EGD-e); Stau, S. aureus. Identical residues are boxed and shaded. (S. aureus AgrA is from strainN315, accession no. P13131; S. aureus AgrC is from strain KSI54, accession no. gi:1916243). In panel B, the transmembrane N-terminal domain(boxed) and the histidine kinase domain of AgrC are indicated.

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type background by generalized transduction (17). All furtheranalyses were performed with the transduced strain.

Organization of the agr locus of L. monocytogenes and tran-scriptional analysis. (i) In silico analyses. Analysis of the L.monocytogenes genome (16) revealed that it contains a com-plete agr locus, comprising the four genes agrB, agrD, agrC, andagrA in the same arrangement found in S. aureus (Fig. 1). TheagrB-encoded protein of L. monocytogenes shares only 28%identity with AgrB of S. aureus. AgrC of L. monocytogenesshares 30% identity with AgrC of S. aureus. Between L. mono-cytogenes agrB and agrC, a short ORF encodes a putative AgrDpolypeptide of 53 amino acids from which an analogue to theAIP of S. aureus may derive. Aside from comparable sizes, nosignificant similarity was found between the two polypeptides.This observation concurs with the fact that the AgrD polypep-tides are highly variable among various S. aureus isolates (18).With 41% identity between those of L. monocytogenes and S.aureus, the AgrA proteins were the most conserved (Fig. 2).AgrB, AgrD, AgrC, and AgrA are highly conserved in theother pathogenic serogroup, that of L. monocytogenes 4b (pre-liminary sequence data were obtained from the Institute forGenomic Research website at http://www.tigr.org). The AgrA,AgrC, and AgrD protein sequences were identical in thestrains. AgrB of L. monocytogenes 4b is 14 residues shorter

than its counterpart in EGD-e, but the conserved portions had99% amino acid identity. The loci were comparably organizedand conserved in the nonpathogenic species L. innocua (over90% identity).

Structural predictions and sequence alignments of AgrCof L. monocytogenes predict the presence of five or six mem-brane-spanning helices in the N-terminal part of the protein,suggesting an organization similar to that of the S. aureusprotein. Secondary structure predictions were performed withthe TOPPRED 2 program (available on the Internet at http://www.sbc.su.se/�erikw/toppred2/), and sequence alignmentswere performed with the CLUSTALW algorithm (http://www.infobiogen.fr/services/analyseq/cgi-bin/clustalw_in.pl).Indeed, in S. aureus, AgrC contains two distinct domains: anN-terminal receiver module, which is highly variable amongdifferent groups of S. aureus (18), and a C-terminal cytoplasmichistidine kinase domain that is conserved in the same subgroup(with 44% identity in the region from amino acids 236 to 431).The transmembrane topology model of AgrC of S. aureus (21),the receiver module comprises five or six transmembrane he-lices followed by the cytoplasmic domain.

However, no equivalent to the divergent transcript RNAIIIof S. aureus was found upstream of the agr operon of L.monocytogenes (Fig. 1A). The ORF (lmo0047) preceding agrB

FIG. 3. Western blot analyses. Culture supernatants and membrane fractions from cells grown overnight at 37°C with agitation in BHI mediumwere tested in exponential phase (EP) and stationary phase (SP). Identical amounts of each culture supernatant were loaded onto SDS–10%polyacrylamide gels. Proteins were transferred electrophoretically onto nitrocellulose and detected with specific antibodies. (Upper panel)Anti-LLO MAb. Decreasing amounts of supernatant were loaded, corresponding to 107, 5 � 106, and 2.5 � 106 bacteria (b). The anti-LLO MAbsSE1 and SE2 were used at a final dilution of 1/1,000. (Middle panel) Anti-PC-PLC. Decreasing amounts of supernatant were loaded, correspondingto 107 and 5 � 106 bacteria. The anti-PlcB polyclonal antibody was used at a final dilution of 1/200. (Lower panel) Anti-ActA. Membrane fractionscorresponding to 107 bacteria were loaded. The anti-ActA polyclonal antibody was used at a final dilution of 1/1,000. WT, strain EGD-e; agr,agrA-Tn1545 insertion mutant.

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is in the same orientation as agrB and encodes a 203-amino-acid-long hypothetical protein of unknown function. TheagrBDCA gene cluster is flanked on both sides by putativetranscription terminators (in the 257-bp intergenic region be-tween lmo0047 and agrB and downstream of the agrBDCAoperon). The ORF (lmo0052) located 167 bp downstream ofagrA encodes a hypothetical protein of 657 amino acids, highlysimilar to YybT, a hypothetical protein of B. subtilis. A poten-tial promoter region is predicted immediately upstream of agrB(with the promoter prediction program available on the Inter-net at http://www.fruitfly.org/seq_tools/promoter.html).

(ii) Transcriptional analyses. First, we tested by RT-PCRwhether the agr region was transcribed in the wild-type strain(EGD-e) grown in laboratory conditions (see Materials andMethods). Our RT-PCR analysis demonstrated that the threegenes agrB, agrC, and agrA were transcribed, suggesting thatthey encode functional proteins. Moreover, we found thatagrB, agrD, and agrC, as well as agrC and agrA, were cotrans-cribed (Fig. 1B), confirming that that the four genes constitutean operon.

Then, we quantified by real-time quantitative RT-PCR thevariations in the levels of expression of the mRNA from agrB,agrC, and agrA genes during the exponential and stationaryphases of growth. Bacteria were grown at 37°C in BHI richmedium. Two internal control genes were used: gyr, encodinggyrase, and the gene determining 16S rRNA (see Materialsand Methods for details). The levels of expression of agrB,agrC, and agrA were comparable in exponential-phase growth(data not shown). In the stationary phase of growth, the ex-pression of agrB and agrC remained similar to that in theexponential phase, the ratio of the values recorded for the twoconditions of growth being around one. In contrast, a twofoldincrease in the amount of agrA mRNA was recorded in thestationary phase compared to that in the exponential phase

(Fig. 1C). These data indicate that, in contrast to those of S.aureus, the levels of expression of agrB and agrC of L. mono-cytogenes are not up-regulated in the stationary phase. Theyalso demonstrate that, as reported for S. aureus, a short tran-script encoding AgrA is expressed in L. monocytogenes.

Properties of the agrA mutant of L. monocytogenes. At all thetemperatures tested (4, 30, 37, and 42°C), growth of the agrAmutant was identical to that of wild-type EGD-e in BHI me-dium, indicating that the Tn1545 insertion had no effect onbacterial multiplication in culture (data not shown).

Effect of agrA inactivation on protein secretion. In S. aureus,surface proteins such as protein A, a major surface antigen, areproduced early in growth in vitro and their production is down-regulated at a later time. Most secreted proteins are producedat the end of exponential growth. The production of all thesefactors (the virulence response) is controlled primarily by theglobal regulatory locus agr (see reference 28 for a review).Therefore, we tested whether inactivation of agrA of L. mono-cytogenes would alter the production of known virulence fac-tors. We chose two virulence factors secreted in the culturemedium, LLO and the phosphatidylcholine phospholipase(PlcB), and the membrane-anchored protein ActA. No differ-ences in the production of ActA and PC-PLC were observed inthe agrA mutant compared to that in the wild-type strain (Fig.3). However, a moderate reduction in the amount of LLOsecreted was observed in the stationary phase (ca. 2.5-fold, asdetermined by densitometry scanning of the Western blot withthe NIH Image software version 1.61). This observation was inagreement with the AgrA/AgrC-dependent increase in expres-sion of postexponentially secreted proteins reported for S.aureus (18).

We then tested whether the agr locus would influence theproduction of other proteins by comparing the protein con-

FIG. 4. Protein secretion in the agrA mutant. Culture supernatants and membrane fractions from cells grown overnight at 37°C with agitationin RPMI medium were tested in exponential phase (EP) and stationary phase (SP). The black arrowheads point to protein bands present in thesupernatant of the agr mutant in the exponential phase of growth that are missing in that of the wild-type (WT) strain. Numbers to the rightcorrespond to the molecular weight (MW) markers.

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tents of culture supernatants or envelope fractions of the agrAmutant to those of the wild-type strain.

Silver staining of SDS-polyacrylamide gels loaded with su-pernatants of exponentially grown cells revealed the presenceof several additional protein bands in the agrA mutant (Fig. 4),indicating that the agr locus plays a role in regulating as yetunidentified secreted proteins of L. monocytogenes. The addi-tional bands observed with the agrA mutant (mainly between20 and 40 kDa) might correspond to the release of normallycell wall-associated proteins that would be down-regulated inthe wild-type background. Further proteomic analyses will berequired to address this point. In contrast, we did not observesignificant differences between the protein patterns of the mu-tant and those of the wild-type strain in the stationary phase,either in bacterial supernatants or in envelope fractions (Fig.4). Since agr of S. aureus has been shown to up-regulate proteinsecretion in the stationary phase, we expected to see the dis-appearance of bands in the supernatant of the agr mutant. Ourdata suggest that the agr locus represses expression of proteinin the exponential phase. They imply that the role of the agrlocus of L. monocytogenes in regulating protein secretion isdistinct from that of the corresponding locus of S. aureus.

Effect of agrA inactivation on intracellular multiplicationand bacterial virulence. The ability of the mutants to penetrateinto and replicate within cells was studied first with two non-professional phagocytic mammalian cell lines: epithelial cells(Caco-2) and hepatocytes (HepG-2), previously used as modelsystems to study infection by L. monocytogenes (7, 22, 28). Thenumber of viable mutant bacteria and that of wild-type bacte-ria were not statistically different (although the number ofmutant bacteria was slightly lower) at any time in the growthcurve and in both cell types (Fig. 5A and B). We also moni-tored the intracellular multiplication of the agrA mutant inbone marrow-derived macrophages from BALB/c mice. TheagrA mutant did not show any growth defect in macrophages,indicating that the agrA gene product does not participate inintracellular survival of L. monocytogenes.

In vivo properties. We determined the LD50 of the agrAmutant by i.v. inoculation of Swiss mice. The LD50 of the agrAmutant was of 105.6, i.e., 10-fold higher than that of the paren-tal strain (104.6), reflecting a moderate attenuation. We mon-itored the kinetics of bacterial survival in the spleens and liversof mice infected with a sublethal dose of 104 bacteria permouse. As shown in Fig. 6, the capacity of the agrA mutant tosurvive and multiply in vivo was only slightly lower than that ofthe wild-type strain, both in the spleens and in the livers ofinfected animals. These data favor the idea that the agr locusmight have only a global indirect influence on the pathogenesis

FIG. 5. Invasiveness of the agrA mutant was evaluated in two dif-ferent cell lines, Caco-2 cells (A) and HepG-2 cells (B), and in bonemarrow macrophages (C). Cell monolayers were incubated for 1 h at37°C with approximately 100 bacteria per cell. After washing, the cellswere reincubated for 4 h in fresh culture medium. At 2, 3, and 4 h, thecells were washed again and lysed and viable bacteria were counted onBHI plates. Values and error bars represent the means and standarddeviations of the numbers of bacteria per well (three wells per assay,two different assays). F, EGD-e; �, agrA mutant.

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of L. monocytogenes rather than a direct effect on the temporalcontrol of key virulence factors.

DISCUSSION

We report for the first time the participation of a locushomologous to the agr locus of S. aureus in the virulence of L.monocytogenes. Inactivation of the agrA homologue reducedbacterial virulence without impairing intracellular survival,suggesting an indirect participation of this locus in the patho-genicity of the bacterium.

Role of the agr locus in protein secretion and bacterialvirulence. We show here that the agr locus of L. monocytogenesinfluences the production of several secreted proteins, includ-ing LLO. However, these variations do not correlate with amajor defect in virulence, suggesting that the regulatory func-tions of the agr locus might be less critical for the developmentof the infectious cycle than for the saprophytic life of thebacterium. Strikingly, inactivation of agrA attenuated the vir-ulence of L. monocytogenes in the mouse model (a 10-foldreduction of the LD50) without significantly impairing bacterialgrowth in vitro. This unexpected phenotype suggests that theagrA mutation might exert an effect on the overall infectiousprocess.

Possible roles of the agr locus in L. monocytogenes. In addi-tion to agr, S. aureus was shown to possess a second globalregulatory locus, designated sar, that also influences exopro-tein and cell surface protein expression. The sarA locus con-tains three overlapping transcripts designated sarA, sarC, andsarB. SarA binds to conserved regions termed “Sar boxes”within promoter regions of genes encoding secreted and cellsurface proteins as well as to agr. SarA binding to agr promot-ers (26) increases both RNAII and RNAIII and thereforecontributes indirectly to the regulation of some virulencegenes. SarA also affects the expression of certain virulencegenes directly, independently of its effects on agr.

No homologue of the RNAIII transcript or SarA could befound in the genome of L. monocytogenes by using BLASTexperiments. The fact that L. monocytogenes lacks the equiva-lent of RNAIII, and a SarA homologue, strongly suggests thatthe regulation of the agr locus and agr-dependent protein ex-pression are quite different in the two organisms. Since S.aureus is an extracellular pathogen while L. monocytogenes isable to multiply intracellularly, the two organisms have prob-ably developed distinct strategies to temporally control theirproduction of virulence factors.

A third two-component regulatory system of S. aureus, theArlS/ArlR system, has also been recently shown to influenceprotein secretion. The data suggest that the three systemsinteract to modulate the virulence regulation network in S.aureus (12). It is likely that other yet unknown two-componentsystems are involved in these regulatory networks. Notably, byreexamining the sequence of LisR/LisK proteins of L. mono-cytogenes (5), we found that they shared significant similaritieswith ArlR/ArlS of S. aureus; LisR and LisK share 53 and 34%amino acid identity with ArlR and ArlS, respectively.

Of interest, a series of SarA homologues have been identi-fied by computer-assisted analysis of the recently released S.aureus genomes (www.ncbi.nlm.nih.gov/genome and www.TIGR.org) (23, 29). The four closest homologues of SarA are

SarT (or SarH3), SarS (or SarH1), SarU (or SarH2), and SarR.As in S. aureus, RNAIII is up-regulated in a sarT mutant andit is believed that sarT may down-regulate agr RNAIII expres-sion. Thus, in addition to the cooperative effect of the AIPs ofagr secreted by the bacteria, the sarT pathway may represent asecond level of control of the agr signal. By searching forparalogs of SarT, SarS, SarU, and SarR in the genome of L.monocytogenes, we identified a single ORF, lmo1225, encodinga protein of 150 amino acids that shares 29.2% identity and62.5% similarity with SarT (SA2506). Thus, the possibility can-not be excluded that in L. monocytogenes, this SarT homologuemay influence expression of the agr locus.

At this stage it is possible to speculate that the modest defectcaused by the inactivation of agrA on the virulence of L. mono-cytogenes might be due to a compensatory role of another (ormore) yet to be determined two-component regulatory sys-tem(s). Alternatively, it is also possible that a response regu-lator module from another two-component regulatory systemmay be able to interact functionally with the sensor AgrC totransduce the agr-dependent signals.

A recent study showed that, although the agr locus of S.

FIG. 6. In vivo survival of the agrA mutant. The kinetics of bacterialgrowth were monitored in mice infected either with EGD-e or with theagrA mutant. Mice were inoculated with 2 � 104 bacteria (indicated byan arrow to the left of the ordinate). Bacterial survival in the spleens(A) and livers (B) was monitored over a 4-day period. F, EGD-e; �,agrA mutant.

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aureus can modulate the expression of secreted proteins in cellcultures, it has little or no regulatory role in vivo (34), suggest-ing the existence of complex regulatory networks sensing andresponding to the variations of environmental conditions ofgrowth. The present work constitutes the first preliminary re-port on the role of the agr locus of L. monocytogenes. Themolecular mechanisms by which the agr locus participates in orinfluences the pathogenicity of L. monocytogenes in vivo re-main to be elucidated.

ACKNOWLEDGMENTS

We thank Salete Newton, Phillip Klebba, and Colin Tinsley forcareful reading of the manuscript and helpful comments on this work.

This work was supported by CNRS, INSERM, Universite Paris V,Universite Paris VII, and the EEC (BMH-4 CT 960659). NicolasAutret received a fellowship from the Ministere de l’Education Natio-nale de la Recherche et de la Technologie and from Universite ParisV. Catherine Raynaud was supported by a fellowship from the FRM.

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