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Infection and Immunity, June 2002, p. 3004-3011, Vol. 70, No. 6
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.6.3004-3011.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of a Quorum-Sensing Signal Molecule in the Facultative Intracellular Pathogen Brucella melitensis

Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d'Immunologie et Microbiologie, Facultés Universitaires Notre-Dame de la Paix, 5000 Namur, Belgium,¹ School of Pharmaceutical Sciences, University Park, University of Nottingham, Nottingham NG7 2RD,² Institute of Infections and Immunity, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, United Kingdom,³ INSERM U431, Faculté de Médecine, 30900 Nîmes, France⁴

Received 8 November 2001/ Returned for modification 15 January 2002/ Accepted 21 March 2002

	ABSTRACT

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Brucella melitensis is a gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades and survives within both professional and nonprofessional phagocytes. A dichloromethane extract of spent culture supernatant from B. melitensis induces bioluminescence in an Escherichia coli acyl-homoserine lactone (acyl-HSL) biosensor strain based upon the activity of the LasR protein of Pseudomonas aeruginosa. HPLC fractionation of the extract, followed by mass spectrometry, identified the major active molecule as N-dodecanoylhomoserine lactone (C12-HSL). This is the first report of the production of an acyl-HSL by an intracellular pathogen. The addition of synthetic C12-HSL to an early log phase culture of either B. melitensis or Brucella suis 1330 reduces the transcription of the virB operon, which contains virulence genes known to be required for intracellular survival. This mimics events seen during the stationary phase of growth and suggests that quorum sensing may play a role in the control of virulence in Brucella.

	INTRODUCTION

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Brucella is the causative agent of brucellosis, a widely distributed zoonosis affecting a broad range of mammals and causing a chronic undulant fever in humans (reviewed by Smith and Ficht [53]). Brucellosis remains a health problem and a source of major economic losses in developing countries, where it is endemic. Brucella are nonmotile gram-negative bacteria and facultative intracellular pathogens that are able to invade and replicate in macrophages and nonprofessional phagocytes (12). In HeLa cells, virulent Brucella cells alter the intracellular trafficking of membrane-bound intracellular compartments to create a novel vacuolar niche in which they replicate (41). The mechanisms and the factors involved in this process are not well understood, with knowledge about the interactions between Brucella and the mammalian cell being mainly descriptive.

Molecular genetic analyses have identified several types of factors that are important for Brucella-host cell interactions: structural components like lipopolysaccharide (1, 16, 32) and cyclic ß(1-2) glucan (19), stress response proteins like DnaK (23) and Hfq, a homologue of Escherichia coli host factor I (HF-I) (43), and also a type IV secretion system (14, 34, 51). The VirB type IV secretion system is homologous to the VirB system of Agrobacterium tumefaciens and to the Ptl system of Bordetella pertussis (4). DNA sequence analysis of the Brucella suis virB operon has revealed 12 open reading frames (ORFs) encoding homologues of the 11 VirB proteins encoded by the pTi plasmid of Agrobacterium and a 12th ORF encoding a putative lipoprotein (34). Independent mutants of virB5, virB9, or virB10 are highly attenuated in an in vitro infection model of human macrophages (34). Two recent signature-tagged mutagenesis studies have confirmed the requirement of virB2, virB4, and virB8 for survival within macrophages and HeLa cells (14) and of virB1 and virB10 in the early stages of chronic infection in BALB/c mice (18).

The process of infection requires a complex and multifactorial regulation of virulence factors. One of the most abundant types of virulence regulatory system is the two-component family of signal transducers (7). Two-component regulatory systems that have recently been described are involved in the virulence of Brucella: the BvrR/BvrS system is critical for the intracellular growth of Brucella abortus (54), and a second system, homologous to VsrB/VsrC of Ralstonia solanacearum, has been identified in Brucella melitensis in a signature-tagged mutagenesis screening in the mouse infection model (27). If two-component regulation systems allow each single bacterium within the population to react in face of environmental changes, other systems, such as the quorum sensing, respond to signals produced by the bacteria themselves and control virulence factors at the whole-population level.

Quorum sensing (QS) is a regulatory system for controlling gene expression in response to increasing cell density (reviewed in references 13, 15, 58, and 66). This cell-to-cell signaling is achieved through the production and release of a small signaling molecule or pheromone. With the growth of the bacterial population, there is an accumulation of this pheromone until a threshold concentration is reached, indicating that a quorum of bacteria is present. At this point, the pheromone activates a transcriptional regulator, leading to the activation or, in some cases, the repression of target genes (49, 55, 64, 65, 70). In a large number of gram-negative bacterial species, the signal molecule is an N-acyl homoserine lactone (acyl-HSL). In most cases, the acyl-HSL synthase is a member of the LuxI family and the response regulator is a member of the LuxR family (reviewed by Swift [59]). Here, the concentration of the signal molecule reflects the numbers of bacterial cells and the perception of a threshold level of the signal indicates a quorate population, ready to employ multicellular adaptive responses involved in, for example, virulence (21, 37, 46, 64), symbiosis (29, 63), and biofilm formation (10, 30) as well as individual survival strategies, such as the induction of stationary phase responses (25) and motility for colony escape (2, 42).

Acyl-HSL signals are composed of a homoserine lactone ring linked via an amide bond to an acyl chain of variable length and with variable substitutions. In vitro and in vivo studies have demonstrated that acyl-HSL synthesis couples amino acid and fatty acid biosynthesis, with the HSL moiety derived from S-adenosyl methionine and the acyl moiety from acylated-acyl carrier protein (acyl-ACP) (20, 33, 35, 48, 61). Signals with shorter acyl chains (C4 and C6) are freely diffusible (22, 38); however, in Pseudomonas aeruginosa, the long acyl chain signal (3-oxo-C12-HSL) requires an active efflux pump for its release, although it may enter cells by diffusion (38).

Surprisingly, acyl-HSL signals with long acyl chains exert significant effects upon the cells and tissues of eukaryotic organisms. An immunosuppressive effect of 3-oxo-C12-HSL upon the host immune system has been observed by Telford et al. (60), an effect that will prejudice the host against antibacterial action and so favor bacteria. Additionally, Lawrence et al. have described a vasorelaxant activity for 3-oxo-C12-HSL, which may contribute to the ability of the bacteria to maintain the supply of key nutrients to a site of infection by increasing the local blood flow (26).

The advantages to an intracellular pathogen of using QS to regulate genes within the closed environment of the replication vacuole and also to be able to modulate the activity of the host's immune responses are clear. We therefore sought to determine whether Brucella employs acyl-HSL-mediated QS, and in this paper, we report the identification and purification of N-dodecanoyl-homoserine lactone (C12-HSL) and its role in the control of the transcription of the virB operon, thus suggesting a putative link between QS and virulence in Brucella.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. Table 1 describes the bacterial strains and plasmids used in this study. E. coli and Chromobacterium violaceum strains were routinely cultured at 30°C in Luria-Bertani (LB) medium (NaCl, 10 g liter^-1; yeast extract, 5 g liter^-1; peptone, 10 g liter^-1) supplemented with tetracycline (20 µg ml^-1) where appropriate. Brucella strains were routinely cultured in 2YT medium (NaCl, 5 g liter^-1; yeast extract, 10 g liter^-1; peptone, 10 g liter^-1). For acyl-HSL extraction, a liquid culture of Brucella sp. was grown at 37°C in RPMI 1640 medium (Life Technologies, Merelbeke, Belgium) supplemented with 0.1% glucose (RPMI-glucose). 2YT medium, yeast extract solution, and peptone solution used in competition assays with HSL were not autoclaved but were sterile filtered. As high temperature may promote the formation of cyclic dipeptides (diketopiperazines) (17), this ensured that inhibitory compounds found in these solutions were not the result of such treatment. To examine the influence of acyl-HSLs upon virB expression, B. melitensis 16M Nal^r and B. suis were grown in modified minimal E medium (24).

Synthesis of HSLs. The general method described by Chhabra et al. (5) was used to synthesize N-octanoyl-L-homoserine lactone (C8-HSL), N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL), N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL),and N-dodecanoyl-L-homoserine lactone (C12-HSL). Each compound was purified to homogeneity by semi-preparative HPLC, and its structure was confirmed by mass spectrometry and proton NMR spectroscopy.

RNA extraction and slot blot analysis. Total RNA was extracted with the RNAeasy total RNA isolation kit (QIAGEN, Courtaboeuf, France) according to the manufacturer's protocol. Samples were treated with DNase (Roche, Bruxelles, Belgium) to remove contaminating DNA. The preparation was heated to 95°C for 5 min and extracted with phenol to remove DNase. The RNA was precipitated with ethanol, collected by centrifugation, dried to near completion, dissolved in an appropriate volume of DEPC double-distilled water, and quantified by spectrophotometry. Slot blot analysis was performed as described by Ausubel et al. (3). Serial dilutions were immobilized on positively charged nylon membrane (Roche), ranging from 1 µg to 15.625 ng of total RNA. The virB5 and l7/l12 probes were labeled by PCR, using 0.1 mM digoxigenin-UTP (Roche) according to the manufacturer's instructions. Probe hybridization, stringency washes, probe detection by an anti-digoxigenin alkaline phosphatase (AP) conjugate, and visualization using the chemiluminescent AP substrate CSPD (Roche) were as directed by the manufacturer (14).

	RESULTS

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Acyl-HSL activity is present in B. melitensis culture supernatant. T-streak assays of B. melitensis 16M Nal^r against the biosensor strains C. violaceum CV026, E. coli JM109 (pSB401), and E. coli JM109(pSB1075) (Table 1) on 2YT at 37°C failed to detect any acyl-HSL activity. To obtain a concentrated sample of any putative Brucella acyl-HSLs that could be assayed at the recommended temperature of 30°C (31, 68), dichloromethane extracts of spent Brucella culture supernatants (2YT [E_16M] and RPMI [E_R16M] grown) were prepared and concentrated 10,000-fold in acetonitrile. A twofold end-point dilution assay was performed for each extract with the biosensors E. coli JM109 (pSB401) and E. coli JM109 (pSB1075) by using dilutions of purified 3-oxo-C6-HSL and 3-oxo-C12-HSL, respectively, as positive controls. As a negative control for these assays, an equivalent extract of the noninoculated medium (denoted E_2YT and E_R, respectively, for 2YT and RPMI 1640). No acyl-HSL activity was detected by E. coli JM109 (pSB401) in extracts from 2YT- or RPMI-cultured B. melitensis 16M Nal^r. No acyl-HSL activity was detected by E. coli (pSB1075) in extracts from 2YT; however, a clear activation of the LasR-based biosensor was observed in an extract of RPMI-cultured B. melitensis 16M Nal^r (Fig. 1), suggesting that a long-chain acyl-HSL was present. No activation of the biosensors could be seen when incubated with the extract of noninoculated RPMI 1640.

View larger version (18K): [in this window] [in a new window]   FIG. 1. RPMI-cultured B. melitensis 16M Nalr cells produce an acyl-HSL activity detected by E. coli (pSB1075). The activation of LasR and the lasI promoter by acyl-HSL activities are shown in twofold dilution series of (left to right) synthetic C12-HSL (5 ng in the first column), synthetic C12-HSL plus solvent extract from 2YT medium (E2YT) (5 ng of C12-HSL plus 5 µl of E2YT [corresponding to 50 ml of medium] in the first column), solvent extraction from RPMI medium (ER; 5 µl [corresponding to 50 ml of medium] in the first column), and solvent extraction from RPMI-cultured B. melitensis 16M Nalr (ER16M; 5 µl [corresponding to 25 ml of medium] in the first column). The expression of lasI'::luxCDABE was measured in relative light units (RLU).

B. melitensis produces C12-HSL. A dichloromethane extract of 18 liters of B. melitensis 16M Nal^r-spent RPMI culture supernatant was dried and reconstituted in a final volume of 1.2 ml of acetonitrile. The extract was fractionated using reverse-phase HPLC with a linear gradient of acetonitrile in water. HPLC fractions F5 and F6 activated E. coli (pSB1075) in agar well assays (Fig. 2). Fractionation by TLC on silica plates followed by biosensor overlay (Fig. 3) revealed that each fraction contained an active compound. Moreover, these molecules seem to be different as the active compound present in F5, but not the one in F6, exhibits a characteristic tailing which is commonly observed with 3-oxo-substituted acyl-HSLs. F5 and F6 were therefore subfractionated by HPLC in isocratic conditions (60% for F5 and 70% for F6), and the biologically active subfractions F5.2 and F6.3 were identified. Liquid chromatography coupled to mass spectrometry (LC-MS) analysis of subfraction F6.3 revealed a molecular ion (M+H) peak at m/z 284 and two fragmentation ions at m/z 183 (corresponding to the acyl chain) and 102 (corresponding to the homoserine lactone ring) (Fig. 4). These data indicate that this acyl-HSL is C12-HSL. C12-HSL was synthesized and shown to possess the same HPLC retention time and mass spectrum as the natural product (Fig. 4). For fraction 5.2, no significant spectra containing a peak at m/z 102, indicative of the homoserine lactone ring, was identified.

View larger version (35K): [in this window] [in a new window]   FIG. 2. HPLC fractionation of the acyl-HSL activity produced in RPMI. (A) Induction of bioluminescence from E. coli (pSB1075) by fractions 5 and 6, collected after HPLC separation of ER16M. For each sample, 5 µl was diluted to 50 µl with LB medium and added to wells cut into the LB agar as described in Materials and Methods. +, positive control (5 ng of 3-oxo-C12-HSL). Fractions 1, 2, 3, 4, and 7 showed no activity. (B) Key to the loading of the plate.

View larger version (67K): [in this window] [in a new window]   FIG. 3. TLC fractionation of the acyl-HSL activity produced in RPMI. Normal-phase TLC of the acyl-HSLs present in cell-free supernatant of B. melitensis 16M Nalr (ER16M) was revealed by overlay with E. coli (pSB1075). Samples were spotted on a silica plate and developed with 45% acetone in N-hexane. Lane A, positive control (5 ng of C8-HSL); lanes B to D, fractions 4 to 6 of ER16M, respectively; lane E, negative control (ER); lane F, ER16M.

View larger version (30K): [in this window] [in a new window]   FIG. 4. MS of F6.3 identifies C12-HSL. (A) The chemical structure of C12-HSL showing the two fragmentation ions having major peaks in electrospray MS (m/z 102 and m/z 183). (B) Comparison of the MS spectrum of synthetic C12-HSL and of the C12-HSL found in subfraction F6.3 of the culture supernatant extract of B. melitensis 16M Nalr.

View larger version (89K): [in this window] [in a new window]   FIG. 5. Total RNA was extracted from lag (A600= 0.05), early log (A600 = 0.15), and transition (A600 = 1) phase cultures of B. melitensis 16M Nalr in the presence (+) or absence (-) of 50 ng of synthetic C12-HSL ml-1. A twofold dilution series of total RNA for each treatment was immobilized on a positively charged nylon membrane. Blots were hybridized with digoxigenin-labeled virB5 probe (A) and digoxigenin-labeled l7/l12 probe (B) as controls.

	DISCUSSION

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In comparison with other bacteria studied, Brucella appears to produce low levels of acyl-HSLs. To obtain sufficient C12-HSL for mass spectrometry, it was necessary to extract 18 liters of spent culture medium (compared with around 4 to 6 liters reported in the literature for other bacteria [e.g., as seen in references 44 and 56]). One explanation for this observation may be found in the hydrophobic nature of C12-HSL. Pearson et al. have shown that the efficiency of extraction depends upon the hydrophobicity of the acyl-HSL (38). For synthetic [³H]3-oxo-C12-HSL incubated with a P. aeruginosa culture, only 75% of the signal is recovered by the extraction process, with 25% remaining trapped in bacterial membranes. C12-HSL is more hydrophobic than 3-oxo-C12-HSL, so we expected that in our extractions of the Brucella spent supernatant that less than 75% of the total production would have been recovered.

The intracellular lifestyle of Brucella may provide a second explanation for the production of low levels of acyl-HSL. Brucella cells survive intracellularly in mammalian host cells but do not begin to replicate until they have modified intracellular trafficking to produce the replication vacuole. At this point and depending on the type of cell infected, some heterogeneity of behavior has been observed. In placental trophoblasts, for example, the bacteria multiply within membrane-bound intracellular compartments until the infected cell lyses. In chronic infection, however, Brucella cells survive in macrophages and can be seen in low numbers. In either case, however, Brucella will be found within a confined compartment and any C12-HSL produced by Brucella will accumulate, especially given the slow diffusion of hydrophobic acyl-HSLs across membranes, leading to the activation of QS at a lower cell density than in an open environment (38). Given that Brucella cells are unlikely to encounter any other sources of C12-HSL within their specialized intracellular niche, it is probable that they have evolved a system sensitive to low levels of signal.

An alternative third explanation for low levels of acyl-HSL production in vitro is a possible regulation mechanism controlling the HSL synthesis. A mechanism that could rely on the presence of a typical environment or a key host cell-derived signal to activate acyl-HSL production. This may be particularly relevant given the influence of the host cell-derived signals (like opines and flavonoids) on QS in other alpha subgroup proteobacteria (Agrobacterium and Rhizobium, respectively) (8, 9, 39) and the possibility that C12-HSL may be produced for both its effects upon the bacterial gene regulation and its effects upon the host organism.

Extracellular pathogens, such as P. aeruginosa, Erwinia carotovora, and Aeromonas hydrophyla, use QS to control the expression of virulence factors. This was explained by the necessity to prevent an early release of these factors that would elicit an inflammatory or immune response until the population density is high enough to cope successfully with this host response (21, 36, 40, 56). We have found that QS could be linked to Brucella virulence by downregulating expression of the virB operon. The transcription of the virB genes is reduced in the late exponential phase. Slot blot experiments probing for virB mRNA reveal that this phenomenon can be mimicked in lag and early exponential phase by the addition of exogenous C12-HSL but also enhanced in the transition phase of growth. This effect is observed in cultures of both B. melitensis 16M Nal^r and B. suis, suggesting a common role for C12-HSL in gene regulation in both species. The VirB system has been shown to be essential for virulence in both in vitro and in vivo infection models, possibly playing a role in the establishment of the replication niche (6, 11, 45). This control of the expression of virB by QS suggests that expression of VirB is required only in the early stages of the infection and that it is perhaps detrimental at later stages. This hypothesis is supported by our observation that the presence of the virB operon on a pBBR1MCS derivative (10 copies per cell) complements the defect in intracellular replication of Brucella virB mutants only in the early stages of the infection. Furthermore, the presence of this plasmid inhibits intracellular multiplication of wild-type B. suis between 24 and 48 h postinfection (34).

The regulation of gene expression by QS is most commonly described as a process of activation. However, negative regulation has also been described, e.g., for Burkholderia cepacia, where QS induces protease production but also represses siderophore production (28), and Yersinia pseudotuberculosis, where QS appears to repress flagellin production (and hence flagellar-mediated motility) (2). Analysis of the QS regulon in Brucella may therefore identify genes positively and/or negatively regulated and will give important information regarding the factors required during different phases of the infection process.

Recently, Sieira et al. (51) described the virB operon in B. abortus 2308 and reported that expression is induced in the stationary phase by monitoring ß-galactosidase activity in a virB10::lacZ fusion strain. This result, shown for B. abortus, is in contradiction to the data we present here and elsewhere for B. melitensis and B. suis (Boschiroli et al., submitted). However, the experimental conditions, and hence the species, used by Sieira et al. are different from those described in this paper. B. abortus cells were cultured in tryptone soy broth, a rich medium similar to 2YT and not to RPMI or E medium. We suggest that compounds present in tryptone soy broth may inhibit the QS regulation of virB expression in Brucella. Indeed, we have not been able to demonstrate any significant effect of C12-HSL upon virB transcription in rich medium (data not shown).

The nature of the inhibitory compound is not known, however; Holden et al. (17) demonstrated the presence of small cyclic dipeptides or diketopiperazines (DKPs) in Pseudomonas culture supernatant that are able to activate or to antagonize QS biosensor systems. They showed that several DKPs are able to antagonize the induction of bioluminescence in E. coli (pSB401) by 3-oxo-C6-HSL without interfering with light emission from E. coli harboring constitutively expressed lux genes. Moreover, DKPs have been isolated from protein digests used as media components, and so the inhibitory effects observed with 2YT could be attributed to these molecules.

The immunosuppressive effect of 3-oxo-C12-HSL upon the host immune system observed by Telford et al. (60) prejudices against antibacterial action and so favors the bacteria. Additionally, the vasorelaxant activity of 3-oxo-C12-HSL observed by Lawrence et al. (26) may contribute to the ability of the bacteria to maintain the supply of key nutrients to the site of infection by increasing local blood flow. This acyl-HSL, produced by P. aeruginosa, can also modulate the expression of membrane receptors of human tracheal gland cells, aggravating Pseudomonas infections in cystic fibrosis patients (47). Given the chemical proximity between 3-oxo-C12-HSL and those produced by Brucella, a possible effect of C12-HSL on the immune system should not be ignored, especially given the biology of Brucella and its ability to deal with cellular actors of the immune system. Indeed, 3-oxo-C12-HSL has been shown to inhibit interleukin-12 and tumor necrosis factor alpha production, thus creating a situation potentially leading to a TH2 immune response (60). As an intracellular pathogen, Brucella has been shown to be more susceptible to a TH1-oriented response. For instance, depletion of IL-12 leads to an exacerbated infection by Brucella (69). Taken together, the possibility that C12-HSL and 3-oxo-C12-HSL could have a similar effect on cytokine production would obviously benefit Brucella.

We plan to extend our investigation of the role of QS in Brucella infections. We benefit now from the availability of the B. melitensis genome sequence for the search of the luxI/luxR homologs. The deletion of these genes will allow us to evaluate their impact upon the virulence of Brucella. Moreover, it will be interesting to identify other genes under the control of QS in Brucella.

	ACKNOWLEDGMENTS

 
We are grateful to J.-M. Verger (Laboratoire de Pathologie Infectieuse et d'Immunologie, Institut National de Recherche Agronomique, Nouzilly, France) for the B. melitensis 16M Nal^r nalidixic acid-resistant strain.

Bernard Taminiau holds a specialization grant from the Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture (FRIA). This work was supported in part by a grant (to P. Williams) from the Biotechnology and Biological Sciences Research Council U.K. and in part by the Commission of the European Communities, contract no. QLK2-CT-1999-00014 (Anne Tibor), which are gratefully acknowledged.

	FOOTNOTES

 
* Corresponding author. Mailing address: Unité de Recherche en Biologie Moléculaire (URBM), Laboratoire d'Immunologie et Microbiologie Facultés, Universitaires Notre-Dame de la Paix, Rue de Bruxelles 61, 5000 Namur, Belgium. Phone: 3281724409. Fax: 3281724420. E-mail: Bernard.Taminiau{at}fundp.ac.be.

Editor: V. J. DiRita

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