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Mutations of the Quorum Sensing-Dependent Regulator VjbR Lead_...

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Mutations of the Quorum Sensing-Dependent Regulator VjbR Lead_...

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     JOURNAL OF BACTERIOLOGY, Aug. 2007, p. 6035?C6047

    0021-9193/07/$08.00 0 doi:10.1128/JB.00265-07 Copyright ? 2007, American Society for Microbiology. All Rights Reserved.

     Vol. 189, No. 16

     Mutations of the Quorum Sensing-Dependent Regulator VjbR Lead to Drastic Surface Modi?cations in Brucella melitensis

     Sophie Uzureau, Marie Godefroid, Chantal Deschamps,? Julien Lemaire, Xavier De Bolle, and Jean-Jacques Letesson*

     Unite de Recherche en Biologie Moleculaire, Laboratoire d??Immunologie-Microbiologie, ?ä ?ä Facultes Universitaires Notre-Dame de la Paix, Namur, Belgium ?ä

     Received 16 February 2007/Accepted 21 May 2007

     Successful establishment of infection by bacterial pathogens requires ?ne-tuning of virulence-related genes. Quorum sensing (QS) is a global regulation process based on the synthesis of, detection of, and response to small diffusible molecules, called

    N-acyl-homoserine lactones (AHL), in gram-negative bacteria. In numerous species, QS has been shown to regulate genes involved in the establishment of pathogenic interactions with the host. Brucella melitensis produces N-dodecanoyl homoserine lactones (C12-HSL), which down regulate the expression of ?agellar genes and of the virB operon (encoding a type IV secretion system), both of which encode surface virulence factors. A QS-related regulator, called VjbR, was identi?ed as a transcriptional activator of these genes. We hypothesized that VjbR mediates the C12-HSL effects described above. vjbR alleles mutated in the region coding for the AHL binding domain were constructed to test this hypothesis. These alleles expressed in trans in a vjbR background behave as constitutive regulators both in vitro and in a cellular model of infection. Interestingly, the resulting B. melitensis strains, unable to respond to AHLs, aggregate spontaneously in liquid culture. Preliminary characterization of these strains showed altered expression of some outer membrane proteins and overproduction of a matrix-forming exopolysaccharide, suggesting for the ?rst time that B. melitensis could form bio?lms. Together, these results indicate that QS through VjbR is a major regulatory system of important cell surface structures of Brucella and as such plays a key role in host-pathogen interactions. Brucella spp. are gram-negative intracellular pathogens belonging to the -2 proteobacteria group, like Agrobacterium, Rhizobium, and Rickettsia, which also live in close association with a eukaryotic host (46). Bacteria of the genus Brucella are the etiologic agents of

    brucellosis, a worldwide zoonosis affecting a broad range of mammals and triggering important economic losses (63). Three Brucella species, B. melitensis, B. abortus, and B. suis, are able to infect humans, causing a chronic, debilitating disease with severe, sometimes fatal outcomes. Brucellae are remarkably well adapted to the intracellular lifestyle, being able to invade and to survive within macrophages and nonprofessional phagocytes (17, 51). This is one of the bases for the still poorly understood chronicity of brucellosis (26). This aptitude relies on the perturbation of vesicular traf?cking and the creation of a unique intracellular replication niche derived from the endoplasmic reticulum (7, 8). Brucella is confronted with very diverse environments and host defenses both in the extracellular milieu and inside host cells. It is thus expected that this pathogen has to sense external and internal signals to achieve successful adaptation throughout its infectious cycle. Among such systems, quorum sensing (QS), stringent response, and signal transduction through two-component regulators have been particularly well studied (for a review, see reference 40). In this study we focused on QS, a communication system used by a large number of bacteria to coordinate gene expression within a population according to population density (30) or limited diffusion in a restricted environment (53). In gram-negative bacteria, this communication system involves the synthesis, release, and subsequent detection of small diffusible molecules or autoinducers (commonly acyl homoserine lactones [AHLs]). As the bacterial population expands, the extracellular concentration of AHLs increases. When the autoinducer concentration reaches a threshold level, AHLs bind to LuxR-type transcriptional regulators comprising an N-terminal AHL binding domain and a C-terminal DNA binding domain containing a helix-turn-helix motif. This interaction leads to conformational changes of the regulator and subsequent modi?cations of target gene transcription. The phenotypes regulated by QS are as diverse as bioluminescence (37), competence (49), bio?lm formation (44), and virulence (50, 75, 78). We identi?ed two LuxR-type regulators in the sequenced B. melitensis genome (16), the previously described VjbR regulator (BMEI1116) (14) and a second regulator, currently undergoing characterization, called BabR (BMEI1758). Despite several attempts, we were not able to identify an AHL synthetase in B. melitensis. However, we have previously identi?ed N-dodecanoyl-DL-homoserine lactone (C12-HSL) from B. melitensis culture supernatant (73). C12-HSL represses the transcription of the ?agellar gene ?iF (14) and of the virB operon (73), whereas VjbR is a transcriptional activator of these two surface-associated virulence factors (16). The ?iF gene encodes the ?agellar MS ring monomer implicated in the establishment of chronic

     6035

     * Corresponding author. Mailing address: Facultes Universitaires ?ä Notre-Dame de la Paix, Namur, Belgium, Unite de Recherche en ?ä Biologie Moleculaire, Laboratoire d??Immunologie-Microbiologie, rue ?ä de Bruxelles 61, 5000-Namur, Belgium. Phone: (32) 81 72 44 02. Fax: (32) 81 72 42 97. E-mail: jean-jacques.letesson@fundp.ac.be. ? Current address: Departement de Biologie Cellulaire, Institut Cochin, F-75014 Paris, France. Published ahead of print on 8 June 2007.

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     UZUREAU ET AL. TABLE 1. Bacterial strains and plasmids used in this study

     J. BACTERIOL.

     Strain or plasmid

     Relevant characteristicsa

     Source or reference

     B. melitensis strains 16M

     Wild type, Nalr

     CD100 CD110 SB200 B. abortus strains 2308 RMD100 E. coli strains DH10B S17-1( pir) Plasmids pSK-oriT cat pSB001 pJD27 PvirB pDONR201 pSB101 pSB102 pSB103 pMR10-cat pRH001 pSB201 pSB202 pSB203 pBBR1-MCS1 pRH002 pSB301 pSB302 pSB303 pSB305 pRH018 pSB401 pSB402 pSB403

     a

     vjbR::Kanr vjbR::Kanr, pJD27 PvirB Ampr vjbR::Kanr omp31::Cmr Wild type, Nalr vjbR::Kanr F mcrA (mrr-hsdRMS-mcrBC) 80lacZ M15 lacX74 recA1 endA1 ara 139 (ara leu)7697 galU galK rps (Strr) nupG recA thi pro hsdR [res mod ][RP4::2-Tc::Mu-Km::Tn7] pir phage lysogen Suicide vector, Cm

     r

     A. Macmillan, Central Veterinary Laboratory, Weybridge, United Kingdom 14 14 This study 54 R.-M. Delrue, unpublished data Gibco BRL 61 I. Danese and P. Lestrate, unpublished data This study 14 Invitrogen This study This study This study A. A. Bourniquel 34 This study This study This study 38 34 This study This study This study This study 34 This study This study This study

     pSK-oriT cat derivative carying a 500-bp internal fragment of BMEII0844 PvirB-luxAB, Ampr Gateway vector, Kanr pDONR carrying the PCR product vjbR pDONR carrying the PCR product vjbR( 1?C180) pDONR carrying the PCR product vjbR(D82A) Broad-host-range cloning vector, RK2 OriV Cmr Kanr pMR10 derivative gateway destinantion vector, medium copy number, Cmr pRH001 derivative, Plac-controlled synthesis of VjbR pRH001 derivative, Plac-controlled synthesis of VjbR( 1?C180) pRH001 derivative, Plac-controlled synthesis of VjbR(D82A) Broad-host-range cloning vector, Cmr pBBR1-MCS1 derivative gateway destination vector, high copy number, rep, Cmr pRH002 derivative, Plac-controlled synthesis of VjbR pRH002 derivative, Plac-controlled synthesis of VjbR( 1?C180)

    pRH002 derivative, Plac-controlled synthesis of VjbR(D82A) pRH002 derivative, Plac-controlled synthesis of Omp31 (BMEII0844) pRH002 derivative allowing C-terminal fusion with 13Myc tag pRH018 derivative, Plac-controlled synthesis of VjbR-13Myc pRH018 derivative, Plac-controlled synthesis of VjbR( 1?C180)-13Myc pRH018 derivative, Plac-controlled synthesis of VjbR(D82A)-13Myc

     Nalr, nalidixic acid resistant; Kanr, kanamycin resistant; Cmr, chloramphenicol resistant; Ampr, ampicillin resistant.

     infection (28), while the virB operon encodes a type IV secretion system (TFSS) involved in the control of Brucella-containing vacuole maturation into a replication-permissive organelle (11). In the current study, we investigated whether VjbR could mediate the C12-HSL repressor effect. To achieve this objective, we used VjbR polypeptides mutated in the N-terminal AHL binding domain of the regulator. These mutant polypeptides behave as signal-independent regulators both in B. melitensis cultures and during cellular infection. Strains expressing these mutated regulators displayed a clumping phenotype that led us to investigate the role of VjbR in the regulation of cell surface components. Our data show that VjbR regulates exopolysaccharide (EPS) synthesis or export and also the production of several outer membrane proteins (Omps), some of which are involved in virulence.

     MATERIALS AND METHODS Bacterial strains and culture conditions. All strains and plasmids used in this study are listed in Table 1. Brucella strains were grown with shaking at 37?ãC in 2YT medium (10% yeast extract, 10 g liter 1 tryptone, 5 g liter 1 NaCl) con-

     taining appropriate antibiotics from an initial optical density at 600 nm (OD600) of 0.05. The Escherichia coli DH10B (Gibco BRL), S17-1 (60), and DB3.1 (Invitrogen) strains were grown in Luria-Bertani medium with appropriate antibiotics. Chloramphenicol, nalidixic acid, and ampicilin were used at 20 g/ml, 25 g/ml, and 100 g/ml, respectively. Synthetic C12-HSL from Fluka was prepared in acetonitrile (ACN) and was added to bacterial growth media at a ?nal concentration of 5 M. The same volume of ACN was used for a negative control. Plasmid construction. DNA manipulations were performed according to standard techniques (1). Restriction enzymes were purchased from Roche, and primers were purchased from Sigma-Aldrich. Derivatives of the replicative plasmids pRH001 and pRH002 (34) harboring vjbR mutant alleles were constructed using the Gateway technique (Invitrogen). The destination vectors pRH001 and pRH002 harbor a chloramphenicol resistance (cat) marker and the toxic cassette ccdB. This group of genes is ?anked by attR1 and attR2 recombination sites. The wild-type (wt) control allele corresponding to the total VjbR protein (amino acids 1 to 260) was ampli?ed with primers VjbR-B1 (5

    -GGGGACAAGTTTGTACAAAAAAGCAGGCTCGAT GAGTCTTGATCTCGTTCAT-3) and

    VjbR-B2 (5 -GGGGACAAGTTTGTA CAAAAAAGCAGGCTACACGAGATGCTGTACCTCG-3 ). Gateway primers HTH-B1 (5 -GGGGACAAGTTTGTACAAAAAAGCAGGCTCGATGAA GGATGCAAATTCAGTTGCAAG-3 ) and VjbR-B2 were used for ampli?cation of the predicted C-terminal DNA binding domain corresponding to amino acids 181 to 260 of VjbR [vjbR( 1?C180)]. B. melitensis 16M genomic DNA was used as the template for all ampli?cations. The resulting PCR products (vjbR-wt and vjbR-HTH,

     VOL. 189, 2007

     respectively) were cloned into pDONR201 (Invitrogen Life Technologies) by the BP reaction as described previously (21). The resulting entry clones pSB101 and pSB102 were con?rmed by PCR using primers VjbR-B1 and VjbR-B2 and primers HTH-B1 and VjbR-B2, respectively. Aspartate 82 of VjbR was mutated into alanine via PCR-based site-directed mutagenesis (QuikChange site-directed mutagenesis kit; Stratagene), using pSB101 as the template. The resulting plasmid, pSB103, was sequenced to con?rm the mutation using primers VjbR-B1 and VjbR-B2. Entry clones containing vjbR alleles were used together with the destination vectors pRH001 and pRH002 during Gateway LR reactions as described previously (21). For quanti?cation of the autoinducer response of VjbR mutants, the resulting vectors pSB201, pSB102, and pSB103 were transferred into the CD110 strain (B. melitensis vjbR::Kanr containing a PvirB-luxAB transcriptional fusion) by mating. For a cellular infection experiment the resulting vectors pSB301, pSB302, and pSB303 were transferred into the CD100 strain (B. melitensis vjbR::Kanr). For characterization of the clumping phenotype in B. abortus, the vectors pSB201, pSB202, and pSB203 were transferred into the RMD100 strain (B. abortus vjbR::Kanr). For construction of vjbR::Kanr omp31::Cmr, an internal fragment of omp31 (BMEII0844) was initially ampli?ed by PCR from B. melitensis 16M genomic DNA with primers F-Omp31 (5 -CTCGGCATTGCGGCTATTTTC-3 ) and ROmp31 (5

    -CAGGTTGAACGCAGATTT-3 ). The R-Omp31 primer contains a TGA stop codon to avoid production of a functional truncated protein. The 341-bp ampli?ed product was then inserted into the EcoRV-digested pSK-oriT cat vector in the opposite orientation relative to the Plac promoter to avoid expression of the 3 fragment of the disrupted coding sequences in Brucella. The construct was introduced into B. melitensis 16M (Nalr) from E. coli S17-1 by mating. A single crossover then led to disruption of the wt locus on the chromosome. Integrative mutants were selected on a medium containing kanamycin and nalidixic acid. Plasmids used to assess mutated polypeptide stability were constructed using the Gateway technology with the pSB101 (pPlac-vjbR), pSB102 [pPlac-vjbR( 1?C 180)], and pSB103 [pPlac-vjbR(D82A)] entry clones and the pRH018 destination vector (34). The resulting plasmids pSB401, pSB402, and pSB403 allowed a C-terminal fusion of the regulator with the 13Myc tag. The

    complementation plasmid carrying the omp31 open reading frame under Plac control (pSB305) was constructed from pRH002 and pDONR-BMEII0844 from the ORFeome (21) using the Gateway technology. Mating. Mating was performed by mixing equal volumes (100 l) of liquid cultures of E. coli S17-1 donor cells (OD600 0.6) and the B. melitensis 16M Nalr recipient strain (overnight culture) on a 0.22- m-pore-size ?lter. The ?lter was left for 1 h on a 2YT medium plate without antibiotics and then transferred onto a 2YT medium plate containing chloramphenicol and nalidixic acid. After 3 days of incubation at 37?ãC, the exconjugates were replicated on a 2YT medium plate containing nalidixic acid and chloramphenicol. Measurement of luciferase activity. Bacterial strains were grown overnight in 2YT medium with aeration at 37?ãC. Cultures were centrifuged, and the bacterial pellets were resuspended and washed twice in 2YT medium. For each strain, three 10-ml portions of cultures in 2YT medium (initial OD600 of 0.05) were incubated at 37?ãC with shaking. After 20 h (PvirB expression peak) the OD600 was measured, and 0.2-ml samples were harvested. N-decanal substrate was added to a ?nal concentration of 0.145 mM (stock concentration, 5.8 mM in 50% ethanol). After 10 min, light production was measured for 5 s using a Microlumat LB96P luminometer (EG and Berthold). Luciferase activity was expressed in relative luminescence units per OD600 unit at a given time point. Measurement was performed in triplicate. Cellular infections. Survival of Brucella strains was evaluated in an immortalized cell line of bovine peritoneal macrophages (67) as described previously (15). A vjbR mutant was used as a negative control for replication defects during the cellular infection. C12-HSL was added at a ?nal concentration of 5 M together with gentamicin. The same volume of ACN, the C12-HSL resuspension solvent, was used as a negative control. Scanning electron microscopy. B. melitensis strains were grown overnight in 2YT medium with aeration at 37?ãC. For each strain three 1-ml portions of cultures in 2YT medium (initial OD600 of 0.05) were incubated at 37?ãC with shaking in a 24-well plate containing an insert plate with a porous membrane (diameter, 1.0 m) (BD Falcon). After 24 h, brucellae were ?xed for 20 min with 4% paraformaldehyde (PFA), and plates were centrifuged for 10 min at 1,000 rpm. Membranes were cut and dehydrated for 5 min in 25, 50, 75, 95, and 100% ethanol at room temperature. They were ?nally prepared by critical-point drying, mounted on an aluminum stub, and covered with a thin layer of gold (20 to 30 nm). Examination was carried out with a scanning electron microscope (XL-20;

     MUTATIONS OF QS-DEPENDENT REGULATOR VjbR

     6037

     Eindhoven, Philips, The Netherlands) at the Unite Interfacultaire de Microsco?ä pie Electronique (University of Namur, Belgium). EPS

    staining. Bacteria in a late-exponential-phase culture (OD600, 1.0) were ?xed with 4% PFA for 20 min before staining. (i) Calco?uor white staining. For detection of polysaccharides, 1 ml of 0.05% calco?uor white (?uorescent whitener 28; Sigma) was added to 0.1 ml of PFA?xed cells. Visualization was accomplished with an epi?uorescence microscope (Nikon Eclipse E1000) with the appropriate ?lter sets. Pictures were captured using a Hamamatsu ORCA-ER camera. (ii) ConA-FITC staining. For confocal microscopy, 0.1 ml of concanavalin A (ConA)-?uorescein isothiocyanate (FITC) (1 mg/ml) was added to 0.2 ml of PFA-?xed cells. One microliter of propidium iodide (10 mM) was added for visualizing bacteria. After incubation for 30 min in the dark, cells were washed in phosphate-buffered saline (PBS) (pH 8.5), resuspended in 100 l of the same buffer, and examined immediately using a Leica SP-1 confocal laser scanning microscope. Dot blot analysis. Brucellae were grown for 48 h in 2YT medium at 37?ãC. Crude extracts were prepared as follows. After being washed in fresh 2YT medium, bacteria were concentrated 10-fold in PBS and inactivated for 1 h at 80?ãC. Equivalent amounts of proteins for each crude extract were used for serial twofold dilutions. Two microliters of each dilution was applied to a nitrocellulose membrane (Hybond; Amersham). Omp immunodetection was performed with the following monoclonal antibodies (MAbs) (10): anti-Omp25 MAb (A68/4B10/ F5) at 1/100, anti-Omp31 MAb (A59/10F9/G10) at 1/100, anti-Omp36 MAb (A68/25G5/A5) at 1/100, anti-Omp 89 MAb (A53/10B2) at 1/1,000, anti-Omp10 MAb (A68/7G11/C10) at 1/5, anti-Omp19 MAb (A68/25H10/A5) at 1/5, and anti-Omp16 (A68/08C03/G03) at 1/10. Horseradish peroxidase-conjugated goat anti-mouse antibodies (Amersham) were used at 1/5,000 along with the ECL system (Amersham) to develop blots for chemoluminescence before visualization on ?lm. Dot blots using MAbs speci?c for Omp16 (PAL lipoprotein) were used as internal loading controls. This protein did not show any change in the conditions tested. Dot blots were quanti?ed using a PhosphorImager. The values used for the graph corresponded to the ?rst dilutions at which differences between samples could be seen. Polymyxin B test. Bacterial survival after controlled exposure to polymyxin B (7,870 U/mg; Sigma-Aldrich, Germany) was assayed essentially as described by Sola-Landa et al. (64). Brie?y, serial dilutions of polymyxin B prepared in 1 mM HEPES (pH 8.0) were prepared in 96-well microtiter-type plates. Bacteria resuspended at 2 104 CFU/ml were dispensed into triplicate rows, and plates were incubated for 1 h at 37?ãC. Viable bacteria were counted by spreading 20 l from each well onto 2YT agar. The results were expressed as percentages of survival; 100% corresponded to the control incubated without the peptide. ELLSA applied to culture supernatants. A peroxidase-labeled ConA solution stored at 20?ãC was diluted in PBS containing 0.05% (vol/vol) Tween 20 diluting buffer to

    obtain a ?nal concentration of 10 g ml 1. One hundred microliters of the peroxidase-labeled lectin solution was added to wells previously coated for 16 h with 100- l portions of supernatants of stationary-phase cultures vortexed for 1 min at full speed. At least three parallel experiments per sample dilution were run in each assay. Wells covered with PBS for the same contact time that was used for supernatants were subjected to the same treatment and used to estimate the nonspeci?c binding in the enzyme-linked lectin sorbent assay (ELLSA) response. Microtiter plates (Maxisorp; Nunc) were placed at room temperature for 1 h to allow the lectin to bind to the polysaccharides. The peroxidase-labeled lectin solution was removed from the wells by inverting the plates and tapping them on absorbent paper. Following ?ve successive washes with 200 l of diluting buffer to eliminate unbound enzyme conjugate, the linked peroxidase conjugate was visualized following addition of 100 l of K-Blue as recommended by the manufacturer (Neogen). The reaction was allowed to develop for 15 min in the dark, and the absorbance at 650 nm and 450 nm was measured was a microplate reader. Statistical analysis. Anova I was used for data analysis after the homogeneity of variance was tested (Barlett test). Average comparisons were performed by using pairwise Scheffe??s test (55). The error bars in ?gures indicate the 95% con?dence intervals of the means (computed from the residual mean square using Student??s t test, 0.05).

     RESULTS Negative effect of C12-HSL on PvirB expression is mediated by VjbR. As previously described, the LuxR-type regulator VjbR and C12-HSLs share common targets (14). VjbR is required for virB expression, and C12-HSLs repress the transcrip-

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     UZUREAU ET AL.

     J. BACTERIOL.

     FIG. 1. Schematic representation of the VjbR mutated polypeptides. The pSB201-encoded wt VjbR polypeptide is shown at the top; the proposed autoinducer (AI) binding region is indicated by a solid bar, and the DNA binding region is indicated by a cross-hatched bar. Mutations in the VjbR polypeptide are indicated in the middle. The D82A substitution is indicated by an arrowhead, and conserved regions of deletants are represented. The relative levels of luciferase activity are indicated on the right. The values are expressed as percentages of the PvirB activity in the B. melitensis CD110 strain containing the pSB201 plasmid (top) grown without C12-HSL. The average PvirB activity in the vjbR mutant was 40%. The values are the means of at least three experiments (the variation coef?cients were between 1 and 8%).

     tion from the virB promoter (PvirB). These observations led to the hypothesis that the C12-HSL repressor effect on the PvirB promoter could be linked to its inhibitory effect on the VjbR regulator. To test this

    hypothesis, we constructed two vjbR alleles mutated in the predicted AHL binding domain. The structure of TraR (the LuxR-type regulator of Agrobacterium tumefaciens) bound to its autoinducer led to prediction of several conserved amino acids directly involved in the binding of the pheromone (76, 82). These studies suggest that several hydrogen bonds, between the AHL and some conserved amino acids within the AHL binding hydrophobic pocket, are involved in the binding of the AHL. These residues are highly conserved in LuxR-type regulators (76, 82). One of them, Asp70, is conserved in VjbR (Asp82). Mutation of this amino acid has been described to inactivate the AHL binding to LuxR-type regulators (42, 57, 62). Consequently, we constructed the vjbR(D82A) allele encoding replacement of aspartate 82 with alanine. The vjbR( 1?C180) allele results in the complete deletion of the predicted autoinducer binding domain (14). wt as well as mutant alleles of vjbR were cloned under Plac control into the medium-copy-number plasmid pRH001 to generate pSB201 (pPlac-vjbR), pSB202 [pPlacvjbR( 1?C180)], and pSB203 [pPlac-vjbR(D82A)] (Table 1). For the following experiments, the plasmids containing the wt vjbR, vjbR( 1?C180), and vjbR(D82A) alleles were designated pSBN01, pSBN02, and pSBN03, respectively. To assess the effect of mutated VjbR regulators on PvirB activity, pSB201, pSB202, and pSB203 were introduced into CD110, a B. melitensis vjbR::Kanr strain containing a PvirBluxAB transcriptional fusion as a reporter (Table 1). As shown in Fig. 1, the activity of PvirB-luxAB was reduced twofold in the presence of the wt vjbR allele and C12-HSL, analogous to the effect of this signal molecule in the wt strain. We were not able to detect any repression effect on PvirB upon addition of C12HSL with the vjbR(D82A) or vjbR( 1?C180) allele. Since both mutant regulators should be unable to bind C12-HSL, these results suggest that the LuxR-type regulator VjbR mediates the repression of PvirB by C12-HSL. As PvirB is insensitive to AHL repression in the presence of the vjbR(D82A) or vjbR( 1?C180) allele, we propose that the VjbR polypeptides encoded by these alleles are defective in AHL binding and therefore behave like constitutive regulators.

     VjbR mediates C12-HSL inhibitory effect on B. melitensis intracellular replication. Since C12-HSLs are known to repress PvirB expression in bacteriological cultures, we tested whether this is also the case during cellular infection. Bovine macrophages were infected with a wt B. melitensis strain in the presence or in the absence of C12-HSL. These signal molecules were added at the beginning of the infection at a ?nal concentration of 5 M. After 1 h and 48 h of infection the number of intracellular bacteria was evaluated. As shown in Table 2, C12-HSL addition did not affect B. melitensis internalization (log CFU at 1 h postinfection) but strongly reduced its intracellular replication (log CFU at 48 h postinfection). Interestingly, this effect

    was not observed when C12-HSLs were added at 24 h postinfection (data not shown). These results suggest that perturbation of the QS network impaired intracellular replication or traf?cking of the bacteria within macrophages. To assess whether the effect of C12-HSL during infection is also dependent on VjbR, bovine macrophages were infected in the presence or in the absence of C12-HSL with strain CD100/ pSB301 (B. melitensis vjbR::Kan r /pPlac-vjbR) or CD100/ pSB303 [B. melitensis vjbR::Kanr/pPlac-vjbR(D82A)]. B.

     TABLE 2. Intracellular replication of B. melitensis in macrophagesa

     Log CFU/well Strain Conditions 1 h postinfection 48 h postinfectionb

     wt CD100 CD100/pSB301 CD100/pSB303

     ACN C12-HSL ACN C12-HSL ACN C12-HSL ACN C12-HSL

     3.19 3.28 3.26 3.07 2.89 3.01 2.95 2.96

     0.01 0.01 0.11 0.02 0.10 0.03 0.03 0.01

     5.00 3.60 2.92 2.96 3.84 2.12 3.82 3.82

     0.12 A 0.23 A 0.07 0.09 0.07 0.04 B 0.04 B 0.03

     a Infections were performed in triplicate. At different time points, the cells were lysed and the numbers of intracellular bacteria were determined by plating the cell lysates on agar plates and expressed in log CFU per well standard deviations. ACN is the C12-HSL solvent. b Values followed by the same letter were signi?cantly different (P 0.001).

     VOL. 189, 2007

     MUTATIONS OF QS-DEPENDENT REGULATOR VjbR

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     FIG. 2. Observation of the clumping phenotype (left panels) and phasecontrast images of B. melitensis (right panels) in exponential growth phase. (A) B. melitensis 16M; (B) strain CD100/pSB203 harboring vjbR(D82A).

     melitensis 16M and the CD100 vjbR defective strain were used as infection controls. As shown in Table 2, addition of exogenous C12-HSL to the CD100 (B. melitensis vjbR::Kanr) strain complemented with

     the wt vjbR allele (CD100/pSB301) reduced the intracellular replication approximately 1.7 log, which is similar to the 1.4 log reduction observed with the wt strain. This repression effect was not observed with strain CD100/pSB303 expressing the mutated allele vjbR(D82A). These results suggest that VjbR mediates the effect of C12-HSL on intracellular replication. B. melitensis VjbR mutants display a clumping phenotype. Interestingly, the CD100 strain expressing the vjbR(D82A) or vjbR( 1?C180) allele exhibits a striking phenotype. As the bacterial cultures reached a high density, cells aggregate and form clumps (Fig. 2). This clumping phenotype was also observed with the CD100 (B. melitensis vjbR::Kanr) strain (clumps were smaller and generally observable only by microscopy), suggesting that

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