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The Journal of Immunology, 2003, 170: 5607-5614.
Copyright © 2003 by The American Association of Immunologists

Subversion and Utilization of the Host Cell Cyclic Adenosine 5'-Monophosphate/Protein Kinase A Pathway by Brucella During Macrophage Infection1

Antoine Gross2,*, Monsif Bouaboula{dagger}, Pierre Casellas{dagger}, Jean-Pierre Liautard* and Jacques Dornand*

* Institut National de la Santé et de la Recherche Médicale Unité 431, IFR 56, University of Montpellier II, Montpellier, France; and {dagger} Sanofi Recherche, Montpellier, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Brucella spp. are intramacrophage pathogens that induce chronic infections in a wide range of mammals, including domestic animals and humans. Therefore, the macrophage response to infection has important consequences for both the survival of phagocytosed bacteria and the further development of host immunity. However, very little is known about the macrophage cell signaling pathways initiated upon infection and the virulence strategy that Brucella use to counteract these responses and secure their survival. In a previous study, we have shown that macrophages activated by SR141716A, a ligand of the cannabinoid receptor CB1, acquired the capacity to control Brucella and observed that the CB1 receptor-triggering engages the microbicidal activity of phagocytes. To analyze the perturbation of cell signaling pathway during macrophage infection by Brucella, we hypothesized that SR141716A provides cell signaling that interferes with the bacterial message leading to inhibition of macrophage functions. As CB1 receptor belongs to the family of G protein-linked receptors, we explored the cAMP signaling pathway. In this study, we show that the CB1 ligand inhibited the bacteria-induced cell signaling. Taking advantage of this result, we then demonstrated that Brucella infection elicited a rapid activation of the cAMP/protein kinase A pathway. This activation resulted in a prolonged phosphorylation of the transcription factor CREB. We finally demonstrate that the activation of the cAMP/protein kinase A pathway is crucial for the survival and establishment of Brucella within macrophages. For the first time in phagocytes, we thus characterized a primordial virulence strategy of Brucella involving the host signaling pathway, a novel point of immune intervention of this virulent pathogen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mononuclear phagocytes represent the first line of defense against pathogens: the nature of their response will allow the mounting of a full immune response directed against the microbes or permit their multiplication and spreading. To circumvent host defense, pathogens have established virulence strategies, which alter phagocyte response at different levels and affect the development of both innate and adaptive responses. Thus, pathogens can survive, proliferate, and avoid the establishment of an efficient immunity. Paradigms of phagocyte subversion by either extracellular or intracellular bacteria such as Salmonella, Listeria, Mycobacterium, Shigella, Yersinia, and Escherichia have been established in recent years (1, 2, 3, 4). The ability of these bacteria to exploit cell signals, transduction pathways, and cytoskeletal components to secure their survival is a well-recognized event. For this purpose, the pathogens divert the phagocyte signaling pathways that might normally set up the microbicidal responses leading to their elimination. They access cell signaling cascades through membrane receptor triggering and/or by secreting virulence factors, which are directly delivered into the cell. For instance, Yersinia and Salmonella display secretion/injection systems that perforate the cell and/or phagosome membrane and deliver toxins inside the cytoplasm, thus affecting enzymatic targets involved in the signaling cascade (5, 6).

Brucella species are facultative intracellular bacteria that induce chronic infections in a wide range of mammals, including domestic animals and humans. Infection in humans depends upon contact with infected animals or their products. After invasion of the reticuloendothelial system, the bacteria develop within mononuclear phagocytes, and the infected monocytes (or macrophages) play a crucial role in the dissemination of the bacteria in specific locations of the body (spleen, brain, heart, and bones). In comparison with other pathogenic bacteria, Brucella lack classical virulence factors, such as exotoxins, invasive proteases, toxic LPSs, capsules, virulence plasmids, and lysogenic phages, and can therefore penetrate into macrophages without inducing perturbation of the host cell membrane (7, 8, 9). Some physiological events favoring Brucella infection have been characterized, but the molecular mechanisms regulating the phenomena have not been clearly identified. Brucella inhibit neutrophil degranulation and oxidative burst (10, 11). It survives in compartments that acidify (12) and that do not fuse with the lysosome, as the bacteria affect the maturation of its vacuolar compartments (13, 14). Furthermore, in human macrophages, Brucella avoid TNF-{alpha} production (15) and protect host cells from apoptosis (16). Recently, a virB locus with homology to the type IV secretion system was identified on chromosome II of Brucella. virB is expressed during infection (17), and its products are essential for the intracellular survival of the bacteria in macrophages (18), Hela cells (19), or for maintaining persistent infection in mice (20). Nevertheless, even though some evidence exists showing that virB mediates bacterial virulence by secretion effectors within infected cells, identifying these effectors and their mode of action appears to be difficult. Direct analysis of the signaling pathways required for macrophage infection would constitute a pertinent approach to investigate the mechanisms of virulence of Brucella. However, such studies have not yet been performed, except in a recent report in which the role of GTPases of the Rho subfamily in the phagocytosis of Brucella was analyzed in nonprofessional phagocytes (7).

In a previous study, we showed that macrophages activated by SR141716A, a ligand of the cannabinoid receptor CB1, acquire the capacity to control Brucella and Salmonella infection (21). In fact, CB1 receptor triggering restores the immunological process that engages the microbicidal activity of phagocytes and is neutralized by the bacteria. We took advantage of this result to analyze the perturbation of cell signaling pathway during macrophage infection by Brucella and hypothesized that SR141716A provides cell signals that interfere with those triggered by the bacteria. As the CB1 receptor belongs to the family of G protein-linked receptor (22) and is coupled to either G{alpha}i or G{alpha}s subunits, we explored the cAMP signaling pathway and established that the beneficial effects of the CB1 ligand resulted from a negative regulation of this pathway. We then demonstrated that Brucella infection elicited a rapid activation of the cAMP/protein kinase A (PKA)3 pathway and that this activation determined the establishment of the bacteria within their host cells. For the first time in phagocytes, we characterized a primordial virulence strategy of Brucella involving the host signaling pathway. Our results are discussed according to the previously published data on Brucella virulence.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

N-(Piperidin-1-yl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, the CB1 receptor antagonist SR141716A, was synthesized at Sanofi Recherche (Montpellier, France). Dibutyryl cAMP (dbcAMP) and RO-20-1724 were purchased from Sigma-Aldrich (Saint Quentin, France) and Tebu (Le Perray-en Yvelines, France), respectively. H89 and KT5823 were purchased from Biomol (Plymouth, PA). Compounds were first dissolved in DMSO (Sigma-Aldrich) and then in RPMI 1640 medium (Life Technologies, Cergy, France). The final concentration of solvent in the assays never exceeded 0.1% and had no effect on cell viability or infection.

Bacterial strains and culture medium

Brucella suis 1330 was obtained from American Type Culture Collection (ATCC 23444, Manassas, VA). Some experiments were performed with GFP-B. suis: B. suis-producing green fluorescent protein (GFP-B. suis) (23). Bacteria were grown at 37°C with vigorous shaking to a stationary phase in tryptic soy broth medium (Life Technologies).

Cell culture

THP-1 cells were obtained from the ATCC. Cells were maintained at 37°C in 5% CO2, in complete medium: RPMI 1640 medium supplemented with 5 mM glutamine (Life Technologies) and 10% (v/v) heat-inactivated FCS (Sigma-Aldrich). They were checked regularly for the absence of mycoplasma by 4,6-diamino-2-phenylindole fluorescence.

THP-1-derived monocytes

THP-1 were differentiated into monocyte-like cells with 10-7 M 1,25-dihydroxyvitamin D3 (Hoffmann-LaRoche, Bale, Switzerland) (15). Adherent VD3-THP-1 cells were then harvested, washed, and cultured overnight in serum-deprived conditions (SDM: RPMI medium supplemented with only 0.5% serum), before infection.

Infection assay

Infections were performed in 24-well plates (8 x 105 cells/well plates; Falcon; BD Biosciences, Meylan, France), or in 8-chamber culture slides (Lab-Tek, Nunc, Naperville, IL) (105 cells/well), as previously described (16). Briefly, B. suis from stationary cultures was centrifuged, washed, and then resuspended in RPMI. Cells were incubated for 30 min at 37°C with 100 µl of bacterial suspension in RPMI medium at a bacteria-to-cell ratio (multiplicity of infection (MOI)) of 20, unless mentioned otherwise, and then extensively washed with PBS to remove nonadherent bacteria. Infected cells were reincubated with fresh complete medium, supplemented with 30 µg/ml of gentamicin to kill remaining extracellular bacteria, and then cultured for different periods of time. The gentamicin concentration was sufficient to kill bacteria within 60 min and did not impair the intracellular multiplication of Brucella. At various postinfection (p.i.) times, culture supernatant was removed and the number of intracellular viable bacteria was evaluated by CFU determination from triplicate plates and serial dilutions of cell homogenates, as previously described (24). Results were expressed in CFU/well.

Infections were also performed in the presence of drugs at the indicated concentration (SR141716A, RO-20-1724, dbcAMP, H89, or KT5823). In these experiments, drugs were added to infected cells at different times p.i., as mentioned in text and figure legend.

Fluorescence microscopy assessment of infection

VD3-THP-1 cells (105 cells/well in 400 µl of SDM) were cultured in eight-chamber culture slides (Lab-Tek, Nunc), and infected with 100 µl of RPMI containing GFP-B. suis (MOI = 20). Infected cells were reincubated in 400 µl of complete medium, supplemented with 30 µg/ml gentamicin with or without the compound under study. They were then analyzed at different time periods by consecutive visualization of the GFP-B. suis-infected cells by phase-contrast microscopy and fluorescent microscopy or by confocal microscopy (Leica DM IRB, Deerfield, IL). A minimum of 800 cells was counted for each analysis.

Cell signaling determination

cAMP assays. VD3-THP-1 cells were cultured overnight in 24-well plates (8 x 105 cells in 1 ml/well) in RPMI 1640. They were washed and cultured in the same medium supplemented or not with 1 µM SR141716A for 30 min. Then 900 µl of medium was removed, and the cells were incubated with B. suis (MOI = 100) (or not) in the remaining medium (100 µl) for 15 or 30 min at 37°C. The cells were then rapidly washed and lysed, and their intracellular cAMP content was determined with a Biotrak cAMP enzyme immunoassay system (Amersham Pharmacia Biotech, Buckinghamshire, U.K.) (21). Values are means of triplicate determinations ± SEM

PKA assay. VD3-THP-1 cells were cultured overnight in six-well plates (6 x 106 cells/well/6 ml) with RPMI 1640. They were then infected with B. suis (MOI = 100) in 500 µl of RPMI for 15 or 30 min, or treated with 1 mM dbcAMP for 30 min. Following treatment, the cells were washed with PBS to remove extracellular bacteria and detached with 0.5 ml of 20 mM Tris-HCl (pH 7.5), 5 mM EDTA, 10 mM EGTA, 1 mM PMSF, and 10 mM benzamide. They were then lysed by multiple freeze-thawing cycles, followed by centrifugation at 20,000 x g for 60 min at 4°C. Supernatants were then collected, and 8 µg of protein from each lysate was assayed using a protein kinase assay kit (Calbiochem-Novabiochem, San Diego, CA). PKA activity was quantified using mouse rPKA as the standard, according to the manufacturer’s protocol. One PKA unit is defined as the amount of enzyme that transfers 1 nmol of phosphate in 1 min from ATP to a synthetic pseudosubstrate at 37°C, pH 7.5. In each experiment, the mean of triplicate samples is shown ± SEM. PKA specificity was demonstrated by adding 10 µM H89 or KT5823 to the reaction medium during the pseudosubstrate phosphorylation assay.

CREB analysis by Western blot. VD3-THP-1 cells were cultured overnight in 24-well plates (8 x 105 cells in 1 ml/well) in RPMI 1640. They were then incubated in 100 µl RPMI for 15 or 30 min with B. suis (MOI = 100), with B. suis killed by gentamicin treatment (100 µg/ml for 1 h at 37°C) (MOI = 100), or B. suis treated for 30 min at 37°C with 25 µg/ml chloramphenicol (15) in the absence or presence of different additives, as indicated. They were then rinsed with PBS and directly lysed in wells with Laemmli buffer and analyzed by Western blot analysis, as described (24). Briefly, lysates were first boiled, before electrophoresis on SDS-10% acrylamide gel, and transferred to nitrocellulose membrane by using a semidry electroblotting Millipore (Bedford, MA) system. The membranes were then incubated at 4°C overnight with an anti-phospho (Ser133) CREB Ab (Cell Signaling Technology, Beverly, MA) at 0.1%. The blots were then treated with a 1/2000 dilution of HRP-conjugated goat anti-rabbit IgG (Amersham Life Science, Les ulis, France) for 1 h at room temperature and developed by using an ECL kit (Amersham), as recommended by the manufacturer. The immunoblots were then dehybridized, and an anti-pan CREB mAb (Cell Signaling Technology) was used to control total CREB protein in each sample.

Kinetic determinations. Cell cAMP levels, PKA activities, and CREB phosphorylation were measured at different times p.i. For time periods that exceeded 30 min (i.e., from 1 to 24 h, depending on the experiment), after 30 min of cell-bacteria contact, the extracellular bacteria were washed and the infected cells were cultured in RPMI 1640 supplemented with 30 µg/ml of gentamicin for the desired period of time before treated, as indicated, in each protocol.

Toxicity of H89, KT5823, dbcAMP, RO-20-1724, and SR141716A. None of the different drugs used affected cell viability. This was established: 1) by measuring lactate dehydrogenase activity in 100 µl culture supernatants using the CytoTox96 Promega kit (Promega France, Charbonnières, France), and 2) by microscopy, apoptotic and necrotic cells were analyzed, using fluorescein-conjugated annexin V and propidium iodide, as previously described (16). In parallel, B. suis cultures in tryptic soy medium (or in complete medium) in the presence of different drugs at concentrations 5-fold higher than those used in our experiments did not reveal any significant effect of the drugs on bacterial development.

Statistical analyses. Statistical analyses were performed using the multiway ANOVA test (Statistical Software SigmaStat version 2.02; SPSS, Chicago, IL), according to programmer instructions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activators of cAMP/PKA pathway reverse bactericidal activity of SR141716A-stimulated macrophages

VD3-differentiated THP-1 cells phagocytose bacteria and constitute a convenient model to assess human macrophage infection by Gram-negative bacteria (15). It was used to establish that the CB1 cannabinoid receptor antagonist SR141716A is a potent inhibitor of macrophage infection by B. suis (21).

Macrophage infection in the presence of SR141716A resulted in a large decrease in the number of viable intracellular Brucella measured at 48 h p.i. (Fig. 1). CB1 receptors are associated to different cell signaling pathways, including the cAMP/PKA pathway (22). Therefore, to investigate the mechanisms regulating Brucella infection at the molecular level, the SR141716A effect was assessed after modulation of the host cell cAMP/PKA pathway. Fig. 1 shows that the capacity of SR141716A to inhibit Brucella infection was totally or partially reversed (p < 0.001) when the intracellular concentration of cAMP was enhanced during infection by either blocking cAMP degradation with the phosphodiesterase inhibitor RO-20-1724 or by exogeneous addition of dbcAMP, a stable cell-permeable cAMP analog. These observations established that the SR141716A-induced inhibition of Brucella infection correlated to a modification of the intracytoplasmic concentration of cAMP. In contrast, in infections performed in the presence of RO-20-1724 (or dbcAMP) and in the absence of SR141716A, compounds enhancing the cAMP/PKA pathway always potentiated Brucella infection compared with control infections. In fact, in these experiments, 2- to 3-fold increase in the number of live intracellular bacteria was always observed at 48 h p.i. This difference, which had a small impact in terms of exponential growth of the pathogen (Fig. 1), was reproducible in four different experiments. Altogether, these convergent results showed that agents that elevated the intracellular concentration of cAMP favored macrophage invasion by Brucella. Thus, we assessed the possibility that a similar issue was implicated in the virulence strategy of Brucella.



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  		FIGURE 1. cAMP-elevating agents restore B. suis proliferation in THP-1 cell-derived macrophages treated with SR141716A. Differentiated THP-1 cells (8 x 105/well) were exposed to 1 µM of SR141716A for 30 min (gray bars) or not (filled bars). They were then infected with Brucella (MOI = 20) in the absence or presence of 100 µM RO-20-1724 or 1 mM dbcAMP and cultured in gentamicin-supplemented complete medium with or without these compounds. The intracellular development of Brucella was analyzed by measuring the number of CFU/well at 48 h p.i. for each condition. Histograms represent means of triplicate determinations in one experiment representative of four similar other ones. Neither SR141716A (21 ) nor the cAMP-elevating agents (not shown) affected Brucella phagocytosis, which ranged at ~25,000 ± 3,750 CFU/well in the different assays. As reported previously (21 ), the number of intracellular bacteria in the SR141716A-treated cells was always statistically different from the number of intracellular bacteria in untreated control cells (p always <0.05).

 
B. suis infection causes a significant cAMP increase in macrophagic cells

Using a competitive enzyme immunoassay, we first measured the intracellular levels of cAMP during Brucella infection. When cultured with macrophagic cells, B. suis penetrated rapidly within these cells (9). At the same time, a rapid up-regulation of the cellular cAMP concentrations could be measured. cAMP was transiently increased to moderate levels, 250–300% of the basal level after 30 min of infection. Such an increase was shown to be significant in 10 different experiments (p < 0.001). The cAMP concentration then returned to its basal level at 3 h p.i. (Fig. 2A). On the contrary, in SR141716A-treated macrophages, Brucella was unable to up-regulate the intramacrophagic concentration of cAMP, and there was significantly less cAMP in infected SR141716A-treated cells than in infected control cells (p < 0.015) (Fig. 2B); SR141716A had no effect on the basal cAMP level of the resting cells. These findings were in line with an effect of SR141716A mediated by the cAMP pathway.



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  		FIGURE 2. cAMP concentration in B. suis-infected VD3-THP-1 cells. A, A total of 8 x 105 THP-1-derived macrophages was infected with B. suis (MOI = 100) and cultured in RPMI 1640. At different time p.i., the cellular cAMP concentration of the infected cells was measured. B, A total of 8 x 105 THP-1-derived macrophages (or THP-1-derived macrophages pretreated with 1 µM SR141716A) was infected with B. suis (MOI = 100) for 30 min, in the presence (SR141716A-pretreated cells) or absence of 1 µM SR141716A. The cellular cAMP concentration of the infected cells was measured. The cAMP level was also determined in noninfected SR141716-treated cells. Results were expressed as percentage of the cAMP concentration of resting untreated cells (control). Ten (A) or four (B) independent experiments were performed. Histograms are means of percentages ± SEM obtained in the different experiments. The level of cAMP was 0.9 ± 0.2 pmol/well in control.

 
Elevated PKA activity in macrophagic cells infected with B. suis

Following the increase in intracellular cAMP concentration of B. suis-infected cells, PKA activity of the cytosolic compartment was determined by measuring the phosphorylation of a pseudosubstrate. As shown in Fig. 3A, VD3-THP-1-infected cells displayed a significantly enhanced PKA activity. In accordance with the cAMP level increase, this activity was optimal between 90 min and 4 h p.i. and reached 20-fold the activity of noninfected controls, depending on the experiment. A relatively high level of PKA activity was then maintained in infected cells during a minimum of 4 h, after which it decreased to basal levels at 24 h p.i. The specificity of the phosphorylation of the pseudosubstrate by PKA was demonstrated using H89 and KT5823, which are PKA and protein kinase G inhibitors, respectively. At 10 µM, H89 totally inhibited Brucella-induced PKA activity in infected cells, while KT5823 had no effect (Fig. 3B). Furthermore, the H89-induced inhibition was dose dependent (data not shown), with an IC50 = 3.2 ± 0.4 µM, and the PKA activity of B. suis-infected cells corresponded to 20% of that observed at 90 min in 1 mM dbcAMP-treated cells (125 U/mg).



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  		FIGURE 3. PKA activity in B. suis-infected VD3-THP-1 cells. A total of 8 x 105 THP-1-derived macrophages was infected with B. suis (MOI 100) and cultured in RPMI 1640 for the indicated periods of time (A) or for 90 min (B). Cell lysates were then prepared, and PKA activity of cytosols was measured with 8 µg of cytosolic protein. PKA activity was expressed in U/mg of cytosolic protein. In B, when indicated, PKA determinations were performed in the presence of 10 µM H89 or KT5823. Results are means ± SEM of three independent experiments.

 
CREB phosphorylation in macrophagic cells infected with B. suis

Several cell signaling pathways such as the cAMP-induced activation of PKA activate the trans activator CREB by phosphorylating the serine residue 133 (25). Such an effect was observed in macrophagic cells after addition of 1 mM dbcAMP or RO-20-1724 (Fig. 4A). Therefore, we examined whether CREB is phosphorylated in Brucella-infected VD3-THP-1 cells and noticed a potent phosphorylation of this transcription factor (Fig. 4B). The phosphorylation, which was maximal at 45 min p.i., was maintained at high levels in infected cells 75 min after the onset of infection. It then decreased, but remained above basal levels 2 h p.i. Once again, the use of H89 and KT5823 demonstrated that B. suis-induced phosphorylation of CREB was dependent on PKA activation and consequently on the enhancement of intracellular cAMP level (Fig. 4C).



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  		FIGURE 4. CREB phosphorylation in B. suis-infected VD3-THP-1 cells. The following treatment was applied to 8 x 105 THP-1-derived macrophages. A, Cells were cultured in RPMI 1640 in the presence or absence (control) of 100 µM RO-20-1724 (or 1 mM dbcAMP) for 15 min. B, Cells were infected with B. suis (MOI = 100) and cultured in RPMI 1640 for the indicated periods of time. C, Cells pretreated or not (control) with 10 µM H89 or KT5823 for 30 min were infected for 45 min with B. suis (MOI = 100) in the absence (control) or presence of the corresponding drugs. D, Cells were infected with live B. suis, gentamicin-killed B. suis (GN-B. suis), or chloramphenicol-treated B. suis (CM-B. suis) (MOI = 100) and cultured in RPMI 1640 for the indicated periods of time. The cells were then lysed, loaded on electrophoresis SDS gels, blotted, and analyzed with an anti-phospho-CREB Ab. The results are representative of 10 (B), 4 (C and D), or 3 (C) different experiments. In each experiment (A, B, C, or D), the immunoblots were then dehybridized, and analyzed with an anti-pan-CREB Ab that did not reveal significant differences between the different lines (not shown).

 
Gentamicin-killed Brucella and chloramphenicol-treated Brucella, which display a reduced capacity to synthetize proteins, also induced CREB phosphorylation in VD3-THP-1 cells (Fig. 4D). However, the phosphorylation kinetics did not correspond to those promoted by live bacteria and were similar to that observed with Escherichia coli LPS (data not shown) (26, 27). In these experiments, CREB phosphorylation was transient and then rapidly decreased after 30 min. Even if the discrepancies in kinetics of CREB phosphorylation were not a direct prove, they greatly suggested that the signaling pathways triggered by live B. suis were specific to a process of virulence of the bacteria and were poorly dependent on bacterial LPS.

PKA activation is required for the intramacrophagic development of B. suis

To investigate the role of the cAMP/PKA pathway in the virulence strategy of Brucella, the ability of the bacteria to proliferate intracellularly was evaluated in the presence (or absence) of 10 µM H89. The drug was added to cell cultures concomitantly with gentamicin, which killed extracellular bacteria to avoid any effect of H89 on Brucella penetration. Fig. 5 shows the development of B. suis in infected THP-1-derived macrophages. This experiment confirmed that after a limited decrease of their intracellular number during the first 7 h p.i., the phagocytosed Brucella exponentially replicated within their host cells. In H89-treated cells, the initial decrease of intracellular Brucella was much more pronounced than in untreated cells. In 10 different experiments at 7 h p.i., the number of residual bacteria was 100- to 200-fold less important than in controls (p = 0.08). This finding demonstrated an important killing of the phagocytosed bacteria, 99% of them being rapidly eliminated. The small number of surviving bacteria that escaped the bactericidal activity of H89-induced cells was first unable to proliferate during the first 24 h of infection. At 24 h p.i., there were 1000 less Brucella in these conditions than in H89-untreated cells (102 vs 105 CFU/well). However, after this delay, residual Brucella proliferated. The H89 effect on Brucella intracellular multiplication was observed starting from a concentration 100 nM and was dose dependent. On the contrary to H89, KT5823 did not exert any action on B. suis infection (data not shown).



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  		FIGURE 5. B. suis growth in THP-1-derived macrophages treated with H89. A total of 8 x 105 THP-1-derived macrophages was incubated with B. suis (MOI = 20) for 30 min, washed, and cultured for different periods of time in gentamicin-supplemented complete medium in the absence or presence of 10 µM H89. The intracellular development of the bacteria was then followed by measuring the number of CFU/well at different times p.i., as mentioned in Materials and Methods. Data are means ± SEM of 10 different experiments. CFU at 30 min represents the number of viable Brucella phagocytosed before drug addition.

 
Cultures of bacteria performed in the presence of 10 µM H89 showed that the compound had no direct effect on Brucella growth. In contrast, the inhibitory effect observed in the presence of H89 did not result from cell cytotoxicity that could expose intracellular bacteria to gentamicin killing. This was verified by vital staining of the noninfected and GFP-B. suis-infected cells (data not shown).

The development of intramacrophagic Brucella at different times p.i. was also followed by using GFP-B. suis in coordination with video microscopy (Fig. 6, A and C) or confocal microscopy (Fig. 6, B and D) analysis. In accordance with previous results (8, 16), we observed that only 9–10% of cells were infected and displayed an intense green fluorescence at 48 h p.i. (Fig. 6A). Observation at higher magnification showed that fluorescent cells were invaded by a relatively high number of intracellular Brucella (Fig. 6B). In H89-treated macrophages, the percentage of fluorescent cells was much lower (Fig. 6C, red arrows). Furthermore, confocal microscopy analysis revealed that Brucella development was strongly reduced in infected cells that contained a very low number of bacteria or sometimes only isolated Brucella (Fig. 6D, red arrows). This confirmed the analysis of Brucella development reported in Fig. 5. Similar results were observed in infections performed with Brucella abortus 2308 or Brucella melitensis 16 M, two other virulent species of Brucella (data not shown). Taken together, these results demonstrated an involvement of the cAMP/PKA pathway in Brucella development within their preferential host cells and consequently in Brucella virulence.



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  		FIGURE 6. Microscopic visualization of the inhibition of the intracellular development of B. suis in THP-1-derived macrophages treated with H89. A total of 105 THP-1-derived macrophages was infected with GFP-B. suis (MOI = 20) in eight-chamber culture slides and cultured in complete medium supplemented with gentamicin in the presence of 10 µM H89 (C and D) or not (A and B). Forty-eight hours after the onset of infection, the green fluorescence of the GFP B. suis was visualized by either video microscopy (A and C) or confocal microscopy (B and D). The results are representative of more than 10 different experiments.

 
PKA activation is required during the early phase of infection

To better analyze the relationship between Brucella-induced cAMP/PKA activation and H89-promoted inhibition of infection, H89 was added to Brucella-infected cells at different times p.i., and the kinetics of bacterial multiplication was then analyzed (Fig. 7). Addition of H89 1.5 h p.i. still induced a pronounced decrease in the number of viable intracellular bacteria 7 h following phagocytosis. However, the number of Brucella recovered was significantly greater than in experiments in which H89 was added to infected cells at 30 min p.i. Moreover, the surviving bacteria were more rapidly able to establish and proliferate within their host cells. When it was added later (at 3 h p.i.), H89 had only a poor effect on both intracellular Brucella survival and proliferation, compared with control.



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  		FIGURE 7. B. suis growth in THP-1-derived macrophages treated with H89 at different time p.i. A total of 105 THP-1-derived macrophages was incubated with B. suis (MOI = 20) for 30 min. They were then washed and cultured for different periods of time in gentamicin-supplemented complete medium in the absence or presence of 10 µM H89. H89 was added to infected cell cultures 30 min, 1.5 h, or 3 h after the onset of infection. The intracellular development of the bacteria was then followed by measuring the number of CFU/well at different times p.i. Data presented are means ± SEM of 10 similar experiments. CFU value at 30 min represents number of viable intracellular bacteria before drug addition.

 
These data were in line with the early activation of the cAMP/PKA pathway triggered by Brucella: the bacteria engaged an early cAMP/PKA-dependent virulence process to counteract the macrophage defense in the first few hours of infection. After this crucial step, once the process was induced, PKA was not required for the further development of Brucella.

Brucella-induced PKA activation is not sufficient to allow an optimal infection

Macrophage infection resulted in a small percentage of permissive cells (<10%) that could be quantified using GFP-B. suis. The pathogen proliferated efficiently within these cells. When dbcAMP was added to infected cells at the onset of infection, the percentage of permissive cells increased 3-fold at 48 h compared with control (p = 0.003) (Fig. 8A). Moreover, in agreement with the data of Fig. 1, CFU determination demonstrated that at 7 and 48 h p.i., there were 3- and 2-fold more intracellular Brucella, respectively, in dbcAMP- or RO-20-1724-treated cells than in controls (Fig. 8B). This showed that the cAMP-elevating agents favored Brucella proliferation, and, consequently, the bacteria-triggered cAMP/PKA pathway was not sufficient to protect all the internalized bacteria from the macrophage microbicidal activity.



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  		FIGURE 8. cAMP-elevating agents improve B. suis survival in the first hours of infection. THP-1-derived macrophages were infected with GFP-B. suis (MOI = 20) in eight-chamber culture slides (A) or with B. suis (MOI = 20) (B) in 24-well plates (MOI 20) in the absence or presence of 1 mM dbcAMP or 100 µM RO-20-1724. They were then cultured in gentamicin-supplemented medium in the presence or absence of the corresponding drugs. A, The percentage of permissive cells (i.e., where Brucella proliferated) was determined 48 h p.i. in a fluorescence microscopy assay evaluating the percentages of GFP B. suis-infected cells. At least 800 cells visualized by phase-contrast microscopy were analyzed. Data presented are means ± SEM of eight different experiments. B, The intracellular development of B. suis was followed by measuring the number of CFU/well at different times p.i. Data presented are mean ± SEM of five different experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To develop within macrophages, Brucella spp. must rapidly promote virulence mechanisms that divert cell pathways that normally manage the early microbicidal attack on the host. In this study, we describe a mechanism by which virulent Brucella spp. exploit a physiologic signal transduction mechanism of macrophages to their own advantage. We then characterize molecular targets of the cell important for Brucella establishment within their host.

Our previous report (21), which described a protective effect of SR141716A on macrophage infection by Brucella, showed that the drug manipulates macrophage signaling through CB1 receptors that can be associated to the cAMP/PKA pathway (22). The reversibility of the SR141716A effect by compounds, which enhanced the cAMP intracellular concentration, demonstrated that cAMP was a mediator of the SR141716A-triggered inhibition of Brucella development. The underlying proposal was that Brucella could induced a cAMP rise, which might be beneficial for their intracellular development. Brucella infection resulted in an enhanced intramacrophagic concentration of cAMP, which in turn activated host cell PKA and induced the phosphorylation of the trans activator CREB. Furthermore, in line with a negative effect of cannabinoids on specific adenylate cyclase isoenzymes (22), the Brucella-induced cAMP rise was inhibited by SR141716A. This was the first observation of the induction of a second messenger during an early step of macrophage infection by Brucella that was related to bacterial virulence.

Recent reports from our laboratory (28) and others (29) have claimed that Brucella phagocytosis and replication require lipid rafts. These microdomains that engulf the bacteria contain lipidic structures avoiding fusion with lysosome (30). These structures carry membrane receptors including mediators of bacteria recognition such as CD14, heat shock protein 70, and heat shock protein 90 (31), and are associated with different signaling pathways, including the cAMP/PKA pathway (32). Thus, when immersed in lipid rafts, Brucella, whose receptors are still unknown (33), could affect the cAMP/PKA pathway through a receptor(s) directly or indirectly coupled to macrophage adenylate cyclase. Brucella could also corrupt the cAMP/PKA pathway by injecting host cells with molecules capable of modulating this pathway. For instance, the Brucella type IV secretion system (18, 19, 34), which is rapidly activated in acidic phagosome (17), could secrete molecules that affect the cAMP/PKA pathway. These substances could be toxins, as in Bordetella pertussis (35), or compounds modeling phosphodiesterase or PKA activity, such as the endogeneous nucleotides that Brucella release under stress conditions (36).

The long-lasting activation of PKA and CREB suggested that Brucella exerted an active process on the cAMP/PKA pathway; such a process should be in accordance with the discrepancies in the kinetics of CREB phosphorylation induced by live Brucella, killed Brucella, or chloramphenicol-treated Brucella. Furthermore, it seemed unlikely that the Brucella LPS participated in CREB phosphorylation; it displays poor activation properties (37), and LPS signaling involves the p38 mitogen-activated protein kinase (26, 27) that is inhibited by intracellular cAMP-elevating agents (38).

The cAMP concentration increase resulting from Brucella infection was modest, but significant. This could be due to: 1) the low percentage of cells permissive to Brucella infection (<10%) and/or 2) the low number of Brucella that bound to permissive phagocytes (1–2 bacteria per cell), even if these bacteria were all internalized (9). It explains why dbcAMP-treated cells displayed a much higher PKA activity than infected cells, the cAMP agonist being able to optimally activate PKA in the whole macrophagic population. Nevertheless, Brucella-induced increase in cAMP level was of the same order of magnitude than in Ehrlichia risticii-infected macrophages (39), and Brucella-induced PKA activation resembled PKA activation in Ehrlichia chaffeensis-invaded THP-1 cells (40). Raising the activity of the cAMP/PKA pathway at these levels is a crucial mechanism for Ehrlichiae survival within macrophages (39, 40). Therefore, such a Brucella-induced effect could also be important for macrophage infection by this bacteria.

Infection of H89-treated macrophages demonstrated that the cAMP/PKA pathway activation belonged to a strategy of virulence, crucial for Brucella spp. development in their host. When PKA was inhibited, Brucella could not evade host cell bactericidal activity, >99% of intracellular bacteria being rapidly eliminated. The kinetics of bacterial elimination and the experiments involving H89 addition at different times p.i. showed that Brucella development required PKA activation in the first hours of infection. On the contrary, bacteria that have engaged a virulence program linked to the cAMP/PKA pathway (i.e., at 3 h. p.i.) proliferated independently of PKA. Furthermore, Brucella behavior in cells treated with H89 at 1.5 h p.i. showed that all internalized Brucella did not concomitantly activate this virulence program. Brucella could have adapted themselves and developed strategies to: 1) activate the cell cAMP/PKA pathway at the onset of infection and 2) circumvent the macrophage response driven by this pathway for several hours. This should explain the unusual long-lasting PKA and CREB activation induced by Brucella. The impact of RO-20-1724 (or dbcAMP) both on the percentage of infected cells and the number of bacteria showed that several intracellular Brucella did not activate the cAMP/PKA-dependent program of virulence. It is possible that, among the Brucella receptors (8, 33), only some of them settled the bacteria in situation of activating the cAMP/PKA pathway. Moreover, despite a dramatic RO-20-1724-triggered cAMP rise, several intracellular bacteria did not proceed further. This meant that in addition to the subversion of cAMP/PKA pathway, Brucella need to develop other mechanisms to successfully invade macrophages.

The activation of the cAMP/PKA pathway occurring at the onset of infection could control two early Brucella-dependent events, crucial for the survival and the further development of the bacteria, i.e., 1) the acidification of the bacterial phagosomes and 2) the inhibition of the fusion of these compartments with lysosomes (12, 13). However, in the first hours that followed infection in the presence of H89 (or SR141716A), these drugs neither prevented the acidification of the bacterial phagosomes nor allowed the fusion of these compartments with lysosomes (data not shown). Therefore, it was unlikely that, in early phases of infection, these events were dependent on the cAMP/PKA pathway. Although several Brucella were rapidly killed in the presence of drugs, their localization in such phagosomes was not surprising. At the onset of infection, killed Brucella behave as live Brucella and stay in acidic phagosomes that did not fuse with lysosomes (8, 10, 12), the inhibition of the fusion being controlled by the lipidic rafts that allowed the penetration of the bacteria (28, 41). On the contrary, in later phases of infections performed in the presence of H89, Brucellae were observed in phago-lysosomes (data not shown), while they develop in phagosomes in the absence of H89. Nevertheless, it cannot be concluded from these results that the cAMP/PKA pathway controlled the late phagosome-lysosome fusion. These differences could be a consequence rather than a cause of the drug effects. At 24 h p.i.: 1) in macrophages that have ingested killed B. suis, bacterial phagosomes fuse with lysosomes; 2) in B. suis-infected macrophages, H89 allows the killing of 99% of the bacteria.

It was tempting to connect the cAMP/PKA pathway to different known mechanisms that favor Brucella infection and were described in our group. 1) Brucella infection does not promote any oxidative burst (42), a result in line with the absence of control of infection by NADPH oxidase (43). Receptor-triggered oxidative burst being inhibited by cAMP-elevating drugs (44, 45), the cAMP/PKA protective effect that originates at the onset of infection could be due to an inhibition of the macrophage oxidative burst. However, because in the presence of H89 Brucella did not trigger any oxidative burst (data not shown), it was unlikely that the cAMP/PKA pathway was involved on Brucella evasion of the reactive oxygen intermediates. 2) Brucella favor their own development by preventing host cell elimination and rendering macrophages resistant to apoptotic signals (16). As cAMP and CREB transcription factors are antiapoptotic mediators (46, 47), Brucella-induced elevation in cAMP/PKA activity could control the protection of macrophages to apoptosis. However, if H89 inhibited the antiapoptotic signals given by B. suis to freshly isolated monocytes or Fas ligand-stimulated macrophages (data not shown), this effect could result from the rapid killing of the bacteria (Fig. 5): Brucella strains that did not develop within macrophages did not protect cells from apoptosis. 3) cAMP being an inhibitor of TNF-{alpha} production (48), the cAMP rise could strengthen the inhibition of TNF-{alpha} that occurred during human macrophage infection by Brucella and involved Omp25 (15, 49). 4) Brucella-infected macrophages are resistant to IFN-{gamma} activation (16). A similar resistance in E. chaffeensis infection involved a direct control of the Janus kinase/STAT pathway by PKA (40), and in Mycobacterium tuberculosis infection a disruption in CBP/P300 binding to STAT1 that could result from CREB phosphorylation (50).

Besides these responses, the cAMP elevation could also lower the innate and adaptive responses of the host (48, 51, 52) by altering cytokine production (53) and/or Ag presentation (48, 52), and thus favor Brucella dissemination in their host.

In summary, our results point out that, as already described for infections with Vibrio cholerae virus or B. pertussis bacteria (35), Brucella uses the host cell cAMP/PKA pathway to their own advantage. These results define a virulence process that determines, at least in part, the evolution of infection. Later investigations with virulent and nonvirulent Brucella might allow us to determine the molecular mechanisms implicated upstream and downstream of the cAMP/PKA pathway, and thus, to better characterize relationships between the host cell physiology and Brucella virulence.


   Acknowledgments
 
We are grateful to Annie Terraza for technical assistance, and to Sherri Dudal for the careful reading of the manuscript.


   Footnotes
 
1 This work has been supported by grants from Institut National de la Santé et de la Recherche Médicale, European Union (Project QLK2-CT-1999-0001), and Evaluation Orientation de la Cooperation Scientifique Nord program (Grant M99S01). Back

2 Address correspondence and reprint requests to Dr. Gross Antoine, Institut National de la Santé et de la Recherche Médicale Unité 431, Université Montpellier II, cc100, Place E. Bataillon, 34095 Montpellier, France. E-mail address: gross{at}univ-montp2.fr Back

3 Abbreviations used in this paper: PKA, protein kinase A; dbcAMP, dibutyryl cAMP; GFP, green fluorescent protein; MOI, multiplicity of infection; p.i., postinfection. Back

Received for publication October 9, 2002. Accepted for publication March 28, 2003.


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