ABSTRACT
Brucella abortus is a facultative extracellular-intracellular pathogen belonging to a group of Alphaproteobacteria that establishes close interactions with animal cells. This bacterium enters host cells in a membrane-bound compartment, avoiding the lysosomal route and reaching the endoplasmic reticulum through the action of the type IV secretion system, VirB. In this work, we demonstrate that the BvrR/BvrS two-component system senses the intracellular environment to mount the transcriptional response required for intracellular life adaptation. By combining a method to purify intracellularly extracted bacteria with a strategy that allows direct determination of BvrR phosphorylation, we showed that upon entrance to host cells, the regulatory protein BvrR was activated (BvrR-P) by phosphorylation at aspartate 58. This activation takes place in response to intracellular cues found in early compartments, such as low pH and nutrient deprivation. Furthermore, BvrR activation was followed by an increase in the expression of VjbR and VirB. The in vitro activation of this BvrR-P/VjbR/VirB virulence circuit rescued B. abortus from the inhibition of intracellular replication induced by bafilomycin treatment of cells, demonstrating the relevance of this mechanism for intracellular bacterial survival and replication. All together, our results indicate that B. abortus senses the transition from the extracellular to the intracellular milieu through BvrR/BvrS, allowing the bacterium to transit safely to its replicative niche. These results serve as a working model for understanding the role of this family of two-component systems in the adaptation to intracellular life of Alphaproteobacteria.
INTRODUCTION
Brucella abortus is a relevant pathogen that induces abortion in cattle and a debilitating zoonotic disease in humans (1). The pathogenesis of brucellosis resides in the ability of B. abortus to enter and multiply within host cells, since several mutants with defects in its intracellular lifestyle are highly attenuated in animal models (2–4). One of the key Brucella virulence factors involved in intracellular replication is the type IV secretion system (T4SS) VirB. This system belongs to a family of prokaryotic molecular machineries that injects proteins and/or nucleic acids into eukaryotic cells in order to modulate the interaction of bacteria with their host. The protein effectors delivered by VirB into host cells are only beginning to be discovered, and their function in modulating intracellular trafficking is largely unknown (5–9). In spite of this, it is widely accepted that the partial and transient interactions of the Brucella-containing vacuole (BCV) with lysosomes allow the expression of VirB, which in turn plays a crucial role in redirecting the intracellular trafficking to the endoplasmic reticulum (ER) (10, 11). The in vitro expression of VirB in culture media is variable and low (12–14), in contrast to the consistent and robust expression detected during the first hours of the B. abortus intracellular trafficking (13). These observations indicate that the bacterium possesses sophisticated mechanisms to control the expression of this injection machinery. Several transcription factors have been demonstrated to control the regulation of VirB (2, 15–17). Among those, the quorum-sensing transcriptional regulator VjbR has been shown to be crucial for the timely coordination of VirB expression during the B. abortus life cycle (2). VjbR is able to bind several promoters, including the virB promoter, when autoinducers like homoserine lactone (HSL) are absent. In contrast, the HSL interaction with VjbR inhibits its DNA-binding activity, allowing the expression of genes repressed by VjbR and turning off the expression of genes positively regulated by this transcriptional regulator (2). In addition to the regulation exerted on VirB, VjbR controls, both positively and negatively, a vast range of genes with functions varying from metabolism to virulence (18).
Another key element required for B. abortus intracellular replication is the BvrR/BvrS two-component system (TCS). BvrR/BvrS mutants are attenuated in cells and animals with impaired intracellular trafficking and increased susceptibility to bactericidal peptides (4). This system is composed of a sensor histidine kinase protein, BvrS, located in the plasma membrane and a cytoplasmic response regulator, BvrR, controlling genes important for the homeostasis of the outer membrane. The TCS impacts the expression of proteins like Omp25 and Omp22 and the structure of the lipopolysaccharide (LPS), specifically the fatty acids of lipid A (19, 20). Furthermore, proteomics and transcriptomics analyses of BvrR/BvrS mutants indicate the involvement of this TCS in nitrogen and carbon metabolism and the expression of VirB and its transcriptional regulator, VjbR (12, 21, 22). In this context, a direct interaction of BvrR with the virB promoter has been demonstrated, and furthermore, constitutive expression in trans of BvrR in a mutant with a transposon insertion on bvrR restores the expression of VjbR and VirB (12).
In this work, we demonstrate that BvrR/BvrS senses the transition of B. abortus from an extracellular to an intracellular environment at early stages of the intracellular life cycle. This response allows B. abortus to trigger the transcriptional response required to redirect its trafficking to the ER.
RESULTS
BvrR/BvrS is activated upon entrance to eukaryotic cells.Before analyzing the activation of BvrR/BvrS, we first measured the expression of the system. By Western blotting, we determined that both BvrS and BvrR are highly expressed during early times in the in vitro growth curve (Fig. 1A). The level of both proteins decreased at late exponential phase, being almost undetectable at stationary phase (Fig. 1A). This result is consistent with the high activity of the BvrR/BvrS promoter at early exponential phase and its low activity at stationary phase, measured as a luxAB transcriptional fusion (Fig. 1B). Thus, we used bacteria grown to exponential phase to determine the phosphorylation status of BvrR.
BvrR/BvrS expression varies throughout the in vitro growth curve. (A) B. abortus 2308 was grown in TSB, and samples were taken at the indicated times. Bacterial lysates were separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with anti-BvrR, anti-BvrS, or anti-Omp19 antibodies (loading control). (B) B. abortus containing an in trans transcriptional fusion of the bvrR promoter (PbvrR) with luxAB (black symbols) or a promoterless luxAB as a negative control (white symbols) were grown on TSB for the indicated times, and the optical density (OD) was determined at 420 nm (triangles). The transcription of PbvrR (black circles) was determined at the indicated times by measuring the luciferase activity (circles). These results are representative of at least three independent experiments.
To analyze the activation state of BvrR/BvrS, we measured the proportion of BvrR phosphorylation by Phos-tag SDS-PAGE (23). Recombinant BvrR separated by this method migrated as a single band, whereas recombinant BvrR previously incubated with carbamoyl phosphate migrated as two bands, indicating that the upper band corresponds to BvrR-P (Fig. 2A). The same two-band pattern was observed in lysates derived from B. abortus in exponential phase (Fig. 2A). To confirm that the upper BvrR band observed in B. abortus lysates corresponds to BvrR-P, alanine or glutamic acid was substituted for aspartate 58 (the putative phosphorylated amino acid). The wild-type BvrR version and the two aspartate 58 mutants were introduced into a mutant B. abortus strain that does not express BvrR (4). When lysates from the corresponding strains were separated by Phos-tag SDS-PAGE, wild-type BvrR migrated as two bands, following the same pattern observed in B. abortus 2308 lysates (Fig. 2B). On the other hand, both aspartate 58 mutants migrated only as a single band, with the upper band being absent (Fig. 2B). This result indicates that this upper band indeed corresponds to BvrR-P and that aspartate 58 is the phosphorylated residue in BvrR. Following this, two macrophage cell lines, J774 and RAW 264.7, were infected for 2 h with B. abortus grown to exponential phase. Intracellular bacteria were then extracted and purified (24). BvrR was phosphorylated upon entrance to both cell lines (Fig. 3A). BvrR phosphorylation was dependent on B. abortus cell entrance, since BvrR-P was barely observed in bacteria recovered from macrophages treated with cytochalasin (Fig. 3B), a treatment that inhibits phagocytosis (Fig. 4). The highest BvrR phosphorylation was achieved at 2 h after internalization (Fig. 3C and F). After this time, BvrR-P gradually diminished and was absent at 24 h postinfection (Fig. 3C). Endosome acidification is essential for BvrR intracellular phosphorylation, since its inhibition with either ammonium chloride or bafilomycin abrogates BvrR/BvrS activation (Fig. 3D and F). The intracellular activation of BvrR was followed by an increase in the expression of VjbR and VirB8 (Fig. 3E). In agreement with the differential expression of BvrR/BvrS during the growth curve, there was no increase in VjbR and VirB expression when the bacteria were at stationary phase, in which the levels of BvrR/BvrS are low (Fig. 3E).
BvrR activation can be determined by Phos-tag SDS-PAGE. (A) Recombinant BvrR (rBvrR) was phosphorylated (+), or not (−), with carbamoyl phosphate (500 mM) for 20 min. As a control, a total lysate from B. abortus 2308 grown to exponential phase was used. Samples were separated by 10% SDS-PAGE containing Phos-tag, transferred to PVDF membranes, and probed with anti-BvrR antibodies. (B) A bvrR-negative B. abortus mutant containing an empty vector (−), a plasmid encoding wild-type bvrR (+), or mutated versions of BvrR with substitutions for aspartate 58 by either alanine (D58A) or glutamic acid (D58E) was grown in TSB to exponential phase. Bacterial lysates were prepared and separated by 10% SDS-PAGE containing Phos-tag, transferred to PVDF membranes, and probed with anti-BvrR or anti-Omp19 antibodies (loading control).
BvrR/BvrS is activated upon entrance to host cells. (A) RAW 264.7 or J744 macrophages were infected with B. abortus in exponential phase. After 2 h, intracellular bacteria were purified. Bacterial lysates were prepared and separated by 10% SDS-PAGE containing Phos-tag, transferred to PVDF membranes, and probed with anti-BvrR or anti-Omp19 antibodies (loading control). Bacteria grown in vitro in TSB were used as a control for extracellular (Ext) B. abortus. (B) RAW 264.7 macrophages were treated with cytochalasin D (Cyt) for 2 h before infection or were left untreated. Cells were then infected with B. abortus in exponential phase for the indicated times. After infection, intracellular bacteria were purified and processed as described for panel A. (C) RAW 264.7 macrophages were infected with B. abortus in exponential phase for the indicated times. After infection, intracellular bacteria were purified and processed as described for panel A. Since the amount of total BvrR varied significantly throughout the intracellular curve, the intensity of each lane was adjusted to obtain the same signal of unphosphorylated BvrR in all the samples. (D) RAW 264.7 macrophages were treated with 30 μM ammonium chloride (Ac) or 50 nM bafilomycin (Bm) or were left untreated (−). Cells were then infected with B. abortus 2308 at exponential growth phase. After 2 h, intracellular bacteria were purified (Int) and processed as described for panel A. All the lanes correspond to duplicates of the same condition and belong to the same gel. (E) RAW 264.7 macrophages were infected with B. abortus in exponential or stationary growth phase for the indicated times. After infection, intracellular bacteria were purified and processed as described for panel A for the detection of BvrR-P or for the detection of VjbR and VirB8 by Western blotting. (F) The percentage of BvrR-P from total BvrR was calculated for each indicated condition by densitometry from at least three independent experiments. **, P < 0.005, compared to results for extracellular (Ext) bacteria (Student's t test). p.i., postinfection.
Cytochalasin inhibits phagocytosis of macrophages. Macrophages incubated with (10 μM) or without cytochalasin were infected with B. abortus. After 2 h, cells were extensively washed and living nonpermeabilized macrophages were incubated with an FITC-conjugated anti-B. abortus antibody for 30 min at 4°C. Cells were then fixed and permeabilized, and bacteria were further detected with a rabbit-anti-Brucella antibody and an anti-rabbit antibody–Texas Red conjugate. Nuclei were stained with DAPI. Intracellularly located bacteria are visible in red, whereas extracellular bacteria are green and red.
BvrR/BvrS is activated at low pH and under nutrient-limited conditions.To explore the signals that might activate the BvrR/BvrS TCS, we exposed B. abortus grown to exponential phase to different pHs (5.0 and 7.0) and different nutrient concentrations, rich medium (tryptic soy broth [TSB]), and minimal medium (MM). It was possible to activate BvrR only when bacteria were exposed to the combination of low pH and limited nutrient availability (Fig. 5A), conditions that mimic the environment found by B. abortus during its early interaction with eukaryotic host cells. None of the other combinations tested induced activation of BvrR/BvrS (Fig. 5A). The in vitro conditions that induced BvrR phosphorylation also increased VjbR and VirB8 expression (Fig. 5B). In vitro incubation of B. abortus under low-nutrient conditions and at different pHs indicated that the strongest activation of the system was achieved under acidic conditions (pH 4.0 and pH 5.0) with negligible activation at neutral pHs (pH 6.0 and pH 7.0) (Fig. 6A). Direct acidification of TSB with citric acid did not induce BvrR phosphorylation (Fig. 6B). When bacteria grown at stationary phase, expressing low levels of BvrR/BvrS, were exposed to low-nutrient conditions and acidic pH, we did not detect activation of the BvrR/BvrS nor an increase in the expression of VjbR or VirB8 (Fig. 7A and B). The expression of VjbR and VirB8 was not induced in a bvrR-negative strain grown to exponential phase and exposed to a low-nutrient medium in acidic pH (Fig. 8).
In vitro conditions mimicking the intracellular environment induce activation of BvrR/BvrS and expression of VjbR and VirB. B. abortus 2308 in exponential phase was incubated in minimal medium (MM) or rich medium (TSB) at pH 5.0 or pH 7.0 for the indicated times. After incubation, bacterial lysates were prepared and separated by 10% SDS-PAGE containing Phos-tag to evaluate BvrR phosphorylation (A) or by 10% SDS-PAGE to determine VjbR and VirB expression by Western blotting (B). Omp19 detection was used as a loading control. These results are representative of at least three independent experiments.
Acidification is required but not sufficient to induce BvrR phosphorylation. (A) B. abortus 2308 in exponential growth phase was incubated in vitro in minimal medium (MM) at the indicated pH for the indicated times or in TSB at pH 7.0. After incubation, bacterial lysates were prepared, separated by 10% SDS-PAGE containing Phos-tag, transferred to PVDF membranes, and probed with anti-BvrR or anti-Omp19 antibodies (loading control). (B) B. abortus 2308 was grown in TSB to exponential growth phase. The pH of the culture medium was then decreased to 5.0 by the addition of citric acid. Control bacteria were not treated, and the pH of the medium remained at pH 7.0. Samples were taken at the indicated times after treatment. Bacterial lysates were prepared, separated by 10% SDS-PAGE containing Phos-tag, transferred to PVDF membranes, and probed with anti-BvrR or anti-Omp19 antibodies (loading control). These results are representative of at least three independent experiments.
In vitro conditions mimicking the intracellular environment do not activate BvrR/BvrS or induce the expression of VjbR and VirB when B. abortus is in stationary growth phase. B. abortus 2308 in stationary growth phase was incubated in minimal medium (MM) or rich medium (TSB) at pH 5.0 or pH 7.0 for the indicated times. Untreated bacteria grown in TSB were used as a control (lane C). After incubation, bacterial lysates were prepared and separated by 10% SDS-PAGE containing Phos-tag to evaluate BvrR phosphorylation (A) or 10% SDS-PAGE to determine VjbR and VirB expression by Western blotting (B). Omp19 detection was used as a loading control. These results are representative of at least three independent experiments.
BvrR controls the expression of VjbR and VirB in response to low pH and minimal medium. A bvrR-negative B. abortus strain in exponential phase was incubated in minimal medium (MM) or rich medium (TSB) at pH 5.0 or pH 7.0 for the indicated times. Untreated bacteria grown in TSB were used as a control (lane C). After incubation, bacterial lysates were prepared and separated by 10% SDS-PAGE to determine VjbR and VirB expression by Western blotting. Omp19 detection was used as a loading control.
Activation of the BvrR-P/VjbR/VirB virulence circuit impacts the ability of B. abortus to survive intracellularly.As shown above, activation of BvrR/BvrS both ex vivo and in vitro was followed by an increase in the expression of VjbR and VirB. Whereas the direct VirB regulation by VjbR has been well documented (25), no direct interaction has been reported between BvrR and the vjbR promoter (PvjbR). To test this possibility, electrophoretic mobility shift assay assays (EMSAs) were performed. Recombinant BvrR was in vitro phosphorylated (BvrR-P) using carbamoyl phosphate. When BvrR-P was incubated with a labeled PvjbR probe, a clear retardation in the migration was observed, in contrast to an internal region of the ribosomal L7/L12 gene used as a negative control (Fig. 9A). This interaction was specific, since unlabeled PvjbR outcompeted labeled PvjbR (Fig. 9B). In contrast, unlabeled PL7/L12 was unable to outcompete PvjbR (Fig. 9B). To determine the sequence of events, BvrR/BvrS was activated in vitro by low pH and nutrient-limited conditions in the presence or absence of homoserine lactone (HSL), a molecule that binds to VjbR and inhibits its DNA-binding ability (26). In the presence of HSL, BvrR/BvrS was still activated (Fig. 9C). However, under these conditions, VirB expression did not increase (Fig. 9C). All together, these results indicate the existence of a virulence circuit in which BvrR/BvrS activation leads to the expression of VjbR and subsequently to increased levels of VirB.
BvrR-P, VjbR, and VirB form a virulence circuit. (A) Digoxigenin-labeled probes containing the promoter region of vjbR (PvjbR) or the negative-control L7/L12 were incubated with increasing concentrations of phosphorylated BvrR (BvrR-P). Samples were then separated in nondenaturing gels, transferred to a membrane, and developed by chemiluminescence using anti-digoxigenin antibodies. (B) Digoxigenin-labeled probes of PvjbR were incubated with BvrR-P (1 mM) and an excess of the indicated nonlabeled probes. Samples were then processed as described for panel A. (C) B. abortus 2308 in exponential phase was incubated in minimal medium at pH 5.0 for the indicated times in the presence (50 μM) or absence of homoserine lactone. After incubation, bacterial lysates were prepared and separated by 10% SDS-PAGE containing Phos-tag to evaluate BvrR phosphorylation or by 10% SDS-PAGE to determine VjbR and VirB expression by Western blotting. Omp19 detection was used as a loading control. These results are representative of at least three independent experiments.
To analyze whether the activation of this virulence circuit plays a role in the intracellular survival of B. abortus, we determined the fate of the bacteria in cells whose endosomal acidification was inhibited. BvrR was not phosphorylated in cells previously treated with bafilomycin (Fig. 3D and F), in agreement with the requirement of a low pH for the activation of BvrR/BvrS. Consistent with previous reports (27), B. abortus was unable to replicate in bafilomycin-treated cells (Fig. 10A). However, if B. abortus was previously exposed to low pH in nutrient-limited medium, then the bacterium was able to replicate in bafilomycin-treated cells (Fig. 10A). In contrast, bacteria exposed to a neutral pH and nutrient-rich medium were rapidly cleared (Fig. 10A). Exposure to low pH under nutrient-limited conditions was not able to rescue a bvrR-negative mutant (Fig. 10B), indicating that this phenomenon is dependent on a functional BvrR/BvrS system. In agreement with the high activation of BvrR/BvrS in bacteria grown to exponential phase and the low activation in bacteria grown to stationary phase, the former achieved a significantly higher intracellular replication in RAW 264.7 macrophages (Fig. 10C). Thus, induction of the virulence circuit formed by BvrR/BvrS, VjbR, and VirB positively modulated the ability of B. abortus to survive intracellularly.
Activation of the BvrR-P/VjbR/VirB virulence circuit promotes intracellular survival of B. abortus. B. abortus 2308 (A) or a bvrR-negative strain (B) in exponential phase was incubated in vitro with MM at pH 5.0 (white circles) or TSB at pH 7.0 (black circles) for 3 h. Untreated bacteria grown in TSB were used as a control. Before infection of RAW 264.7 macrophages, the acidification of endosomes was inhibited with 50 nM bafilomycin (Bm). Control cells (black triangles) remained untreated. The intracellular growth was determined at the indicated times using a gentamicin protection assay. *, P < 0.05, compared to results with nontreated B. abortus replicating on Bm-treated cells (white triangles) (Student's t test). (C). Macrophages (RAW 264.7) were infected with B. abortus in exponential (Exp) or stationary (St) phase and incubated in the presence of gentamicin (5 μg/ml) for the indicated times. Cells were then lysed, and intracellular bacteria were determined by plate counting. *, P < 0.05, and **, P < 0.005, compared to the results for the corresponding time in stationary phase (Student's t test). These results are representative of at least three independent experiments.
DISCUSSION
Previous evidence indicates that BvrR/BvrS impacts the expression level of VirB and its transcriptional regulator, VjbR. Furthermore, a direct interaction of BvrR and VjbR with the virB promoter has been previously demonstrated (12, 25). Since these three systems are crucial for B. abortus virulence and intracellular survival, we postulate the existence of a virulence transcriptional circuit in which BvrR/BvrS induces the expression of VjbR and, in turn, VirB is expressed. Considering the existence of this virulence circuit and that VirB expression is significantly induced upon entrance to the eukaryotic host cell (28), we hypothesized that BvrR/BvrS is responsible for sensing the intracellular location of the bacterium. In the present work, and by combining a method to extract and purify intracellular bacteria with the direct detection of the phosphorylation state of BvrR, we unambiguously determined the activation of BvrR/BvrS during the early stages of the interaction of B. abortus with its eukaryotic host cell. Thus, our direct experimental evidence indicates that BvrR/BvrS functions as a sensor that detects the transition of B. abortus from the extracellular milieu to the intracellular niche. This activation during the early stages of the B. abortus intracellular cycle results in the expression and assembly of the T4SS system, allowing the escape of the bacterium from the lysosomal route and the redirection of its trafficking to the endoplasmic reticulum (Fig. 11). Interestingly, we observed a high expression of BvrR/BvrS in exponential phase and an almost negligible expression in stationary phase, correlating with the ability to switch on, or not, the virulence circuit described above. This difference might be responsible for the higher intracellular replication achieved by bacteria in exponential phase than that achieved by bacteria in stationary phase.
Model showing the coordination by BvrR/BvrS of the intracellular response of B. abortus. Once B. abortus enters the cells, BvrS detects intracellular environment cues such as low pH and a nutrient-limited medium that trigger its autophosphorylation. The phosphate group is then transferred to BvrR, which in turn will bind its regulated promoters. Among those, the transcription of vjbR will be induced and both VjbR and BvrR will cooperate to induce the expression of the virB operon. The assembly of this T4SS will allow B. abortus to modify its intracellular trafficking in order to reach the ER, its replicative niche. PM, plasma membrane; IM, inner membrane; OM, outer membrane.
Several two-component system orthologues to BvrR/BvrS have been postulated to mediate the interaction of Alphaproteobacteria with their corresponding eukaryotic hosts. For instance, the BatR/BatS TCS from Bartonella henselae controls the adaptive response during its life cycle by being activated at a neutral pH upon entrance to eukaryotic host cells to trigger the expression of a T4SS crucial for intracellular replication (29). The ChvG/ChvI TCS from Agrobacterium tumefaciens controls the expression of acid-induced genes essential for the formation of plant tumors (30, 31). Finally, the ExoS/ChvI TCS from Sinorhizobium meliloti is essential for symbiosis with host plants (32, 33). The role of these systems has been mainly deduced from indirect evidence, since mutants in these TCSs are defective in establishing an interaction with host cells (4, 31). The activation of the systems by host-derived signals has also been determined indirectly by measuring the expression of target genes (30, 34–36). Thus, the direct results presented here contribute to the understanding of the interaction of Alphaproteobacteria with their eukaryotic hosts.
The precise nature of the signal sensed by BvrS remains to be determined. Our results indicate that acidity is essential but not sufficient for the activation of BvrR, since treatment of cells with bafilomycin or ammonium chloride partially abrogated the activation of the system but acidification alone did not induce BvrR phosphorylation. We could activate in vitro BvrR/BvrS by combining low pH with a nutrient-limiting environment. Furthermore, this in vitro activation was functional since it rescued B. abortus from the inhibition of intracellular replication exerted by bafilomycin. However, the intracellular milieu encountered by the bacterium is far more complex, and other factors may contribute to a complete activation of the system. In this context, it is tempting to speculate that rather than sensing a single signal, BvrS responds to a biophysical environment composed by several factors. This environment would induce conformational changes in BvrS, leading to its autophosphorylation and the subsequent activation of BvrR. Alternatively, as demonstrated for related alphaproteobacteria, the periplasmic protein ExoR could sense the intracellular environment leading to its degradation. In S. meliloti, ExoR physically interacts with ExoS, the sensor of the ExoS/ChvI TCS, keeping it in an off state. Upon proteolysis of ExoR within the periplasm, the negative effect on ExoS/ChvI is released, and this system then upregulates crucial phenotypes as succinoglycan production (37). In A. tumefaciens, the ChvG/ChvI TCS is repressed by ExoR at neutral pH, but when the bacterium encounters acid pH, ExoR is proteolyzed, allowing the transcription of motility and chemotaxis genes, succinoglycan biosynthesis genes, a type VI secretion system (T6SS), and various virulence genes (35).
It would be interesting in the future to monitor the state of BvrR phosphorylation at late stages of the intracellular life cycle, when the bacteria have moved from the ER to autophagosome-like compartments and are ready to exit the cell (38). In fact, a VirB requirement in postreplication events has recently been demonstrated (39). Thus, it is likely that under these circumstances, bacteria again increase their competence for intracellular survival and would then activate the virulence circuit formed by BvrR/BvrS, VjbR, and VirB to initiate another cycle of replication within host cells.
MATERIALS AND METHODS
Bacterial culture and strain construction.The plasmids, strains, and primers used in this study are detailed in Tables 1, 2, and 3, respectively. B. abortus strains were maintained and grown using previously described protocols (40). When needed, B. abortus strains were supplemented with antibiotics to maintain plasmid selection. Escherichia coli strains used for cloning were grown in LB (37°C) supplemented with 50 μg · ml−1 kanamycin (Kan), 100 μg · ml−1 ampicillin (Amp), or 30 μg · ml−1 chloramphenicol (Cm) when required. B. abortus strains were grown in tryptic soy broth (TSB) at 37°C supplemented with 5% CO2.
Plasmids and vectors used in this study
Strains used in this study
Primers used in this study
Construction of plasmids prH002bvrR, prH002bvrRD58A, and prH002bvrRD58E.To determine BvrR phosphorylation site and to validate SDS-PAGE containing Phos-tag as a technique that allows the detection of phosphorylation of this response regulator, we generated two point mutations in the BvrR putative phosphorylation aspartate residue. To generate the point mutations in the bvrR gene, the GeneArt site-directed mutagenesis system kit (Invitrogen) was used in accordance with the manufacturer's specifications. Briefly, in order to amplify a 1,093-bp fragment containing 198 bp of bvrR promoter, 720 bp of bvrR, and 175 bp of the bvrR-bvrS intergenic region, a PCR was carried out using primers 3-200bvrR3 and revtrSinterg1.5 (Table 3). The PCR product was cloned into plasmid pCR8/GW/TOPO (Table 1), generating plasmid GWEntrybvrR. GWEntrybvrR was methylated, and its bvrR region was mutated specifically in aspartate residue 58 (BvrR phosphorylation site). The products were transformed into the E. coli One Shot MAX Efficiency DH5α-T1R strain. In order to confirm the mutations, the plasmids were sequenced according to the manufacturer's instructions (BigDye Terminator v.3.1 cycle sequencing kit; Applied Biosystems), using primers 3-200bvrR3 and revtrRSinterg1.5. The plasmid with the bvrR point mutation in the putative phosphorylation site in which a glutamate residue was substituted for the aspartate residue at position 58 was named GWEntrybvrRD58E, and when an alanine residue was substituted for the aspartate residue, it was named GWEntrybvrRD58A. Using primers HisbvrRF and HisbvrRR, bvrR, bvrRD58A, and bvrRD58E (738-bp fragment), sequences were subcloned into the entry plasmid pCR8/GW/TOPO according to the manufacturer's instructions (pCR8/GW/TOPO TA cloning kit; Invitrogen). Chemically competent cells of E. coli One Shot Mach1-T1 (Invitrogen) were transformed with the plasmids pTOPObvrR, pTOPObvrRD58A, and pTOPObvrRD58E. To confirm the correct sense of the inserts, M13F and M13R primers were hybridized, respectively, with upstream and downstream regions from the insert. Additionally, M13F and M13R primers were used with bvrR-bv1R and bvrR-bv1F primers, respectively, to determine the orientation of the insert. An amplicon from each allele with the right sense of insertion was selected and sequenced (BigDye Terminator v.3.1 cycle sequencing kit; Applied Biosystems) using primers M13F and M13R. The obtained plasmids, pTOPObvrR, pTOPObvrRD58A, and pTOPObvrRD58E, were subcloned into the vector prH002 by site-specific recombination using the Gateway LR recombination cloning procedure in accordance with manufacturer's instructions (Invitrogen Life Technologies) (41, 42). The vectors prH002bvrR, prH002bvrRD58A, and prH002bvrRD58E were transformed and selected with chloramphenicol into chemically competent E. coli TOP10F′ cells to preserve the plasmid frozen. The plasmids were then extracted, transformed in the chemically competent E. coli S17-1 cells, and selected with chloramphenicol. The resulting plasmids were introduced into the B. abortus bvrR-negative (4) strain by mating with E. coli S17-1 as previously described (43). Conjugants carrying the plasmid were selected by plating the mating mixture onto tryptic soy agar (TSA)-Nal-Cm plates, which were incubated at 37°C for 3 days.
Construction of plasmid pBvrR-luxAB transcriptional fusion (pJD27-PbvrR).To study the bvrR promoter (PbvrR) activity, a luciferase transcriptional fusion approach was used. Briefly, in order to amplify a 225-bp fragment containing PbvrR genomic DNA, primers 3-200bvrR3 and 3-200bvrR5 were used (Table 3). The PCR product was cloned into plasmid pJET1.2 in accordance with the manufacturer's instructions (CloneJET PCR cloning kit; Thermo Scientific). The resulting plasmid was named pJET1.2 PbvrR. Plasmid pJET1.2 PbvrR was electroporated into E. coli XL1-Blue electrocompetent cells, digested with BamHI and XhoI restriction enzymes, and ligated into the pJD27 BamHI and XhoI restriction sites (R. C. Essenberg, Department of Biochemistry, Oklahoma State University). Primers pjd27seqF and pjd27seqR were used to sequence and confirm the insert orientation of pJD27-PbvrR (Table 3). Plasmid pJD27-PbvrR was then electroporated in B. abortus 2308 (40), and the resulting strain was named B. abortus PbvrR-luxAB. For a negative control, B. abortus 2308 was electroporated with the empty vector pJD27 and the strain was named B. abortus luxAB.
BvrR and BvrS detection and determination of BvrR phosphorylation.The in vitro growth curve was made as described elsewhere (12). The bacteria were concentrated by centrifugation (10,000 × g, 10 min), resuspended in Laemmli sample buffer, and heated at 100°C for 20 min. The protein concentration was determined by the Bio-Rad DC method according to the manufacturer's instructions. Equal amounts of protein (20 μg) were loaded onto a 10% gel for SDS-PAGE. Separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and probed with the indicated antibodies. To analyze the phosphorylated status of BvrR, samples were solubilized in Laemmli sample buffer without heating, and equal amounts of protein (20 μg) were loaded onto a 10% gel for SDS-PAGE containing Phos-tag (100 mM) and MnCl2 (0.2 mM). Recombinant BvrR phosphorylated with the phosphate universal donor carbamoyl phosphate was used as a positive control (44).
Determination of bvrR promoter activity.The bvrR promoter (PbvrR) activity was determined by a luciferase transcriptional fusion construct introduced in trans in B. abortus 2308. A promotorless luxAB construct was used as a negative control. The strains were grown in TSB at 37°C, and at the times indicated in the figures, the optical density at 420 nm (OD420) was measured, the bacterial load was calculated by plate counting, the luciferase activity was determined by mixing 0.1 ml of samples with 10 μl of ethanol-decanal, and, after 10 min, light production (relative luminescence units [RLU]) was measured for 5 s using a BioTek Synergy HT luminometer. Luciferase activity is expressed as the number of RLU per CFU/0.1 ml at a given time point.
In vitro BvrR/BvrS activation assay.The different B. abortus strains at the growth phases indicated in the figures were centrifuged (10 000 × g, 3 min) and exposed to minimal medium (33 mM KH2PO4, 60.3 mM K2HPO4, and 0.1% yeast extract) or nutrient-rich medium (TSB) at pH 5.0 or 7.0 (adjusted with citric acid) for the times indicated in the figures at 37°C at 200 rpm. After incubation, bacteria were concentrated by centrifugation (10,000 × g, 3 min) and resuspended in Laemmli sample buffer. Total lysates were then processed for electrophoretic and immunochemical analyses as described above. To inhibit VjbR DNA-binding activity, B. abortus 2308 in exponential phase had been previously incubated in the presence (50 μM) of homoserine lactone for 1 h. Bacteria were then processed as described above.
Cell culture and gentamicin protection assay.Murine macrophage-like J774.A1 cells (ATCC, TIB-67), murine RAW264.7 macrophages (ATCC TIB-71), or HeLa epithelial cells (ATCC clone CCL-2) were cultivated and infected with B. abortus in exponential or stationary phase as previously described (24). For the intracellular replication experiments, 2 days before infection, cells were seeded in 24-well tissue culture plates to obtain a final concentration of 5 × 105 cells per well and multiplicities of infection (MOI) of 500 and 100 were used for HeLa cells and macrophages, respectively. For the intracellular bacterial isolation experiments, 2 days before infection, cells were seeded in 6-well tissue culture plates to obtain a final concentration of 3 × 106 cells per well and an MOI of 2,000 was used (24). Drugs to inhibit phagosome acidification were used as described by Porte (27). To inhibit macrophage phagocytosis, cytochalasin was used at concentrations and incubation times described by Palmer et al. (45).
Intracellular replication quantitation.The number of intracellular viable B. abortus CFU was determined at different hours postinfection. Cells were washed twice with phosphate-buffered saline (PBS) and treated for 10 min with Triton X-100 (0.1%). Lysates were serially diluted and plated on tryptic soy agar dishes for quantitation of CFU.
Immunofluorescence microscopy.In order to differentiate extracellular bacteria from internalized bacteria, at 2 h postinfection, a double immunofluorescence was performed. To determine extracellular bacteria, infected cells were washed three times with PBS and incubated with a cow anti-B. abortus antibody (conjugated with fluorescence isothiocyanate [FITC]) for 30 min at 4°C. Then, to stain all bacteria, cells were fixed and permeabilized, and bacteria were stained with a rabbit anti-Brucella antibody and anti-rabbit antibody–Texas Red. Nuclei were stained with DAPI, and slides were mounted using ProLong gold medium (Invitrogen). Intracellularly located bacteria are exclusively red, whereas extracellular bacteria are stained red and green.
Purification of intracellular bacteria.Intracellular B. abortus bacteria were purified at the hours postinfection in the figures using a previously standardized protocol based on sucrose gradients and ultracentrifugation (24). Afterwards, the pellet was stored at −70°C for electrophoretic and immunochemical analyses described above.
Statistical analysis.Statistical analyses were performed using SPSS software. All results are presented as means ± SD from at least three independent experiments, unless otherwise stated. Statistical significance was determined by Student's t test.
ACKNOWLEDGMENTS
This work was funded by Fondos de Recursos del Sistema FEES/CONARE, Red Temática de Brucelosis, Vice Presidency for Research, University of Costa Rica (project 803-B3-761) (www.vinv.ucr.ac.cr/), the National Council of Science and Technology of Costa Rica through FORINVES (grant FV-0004-13) (www.conicit.go.cr/), and the International Center for Genomic Engineering and Biotechnology (contract CRP/12/007) (www.icgeb.trieste.it).
The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
FOOTNOTES
- Received 17 October 2017.
- Returned for modification 10 November 2017.
- Accepted 16 January 2018.
- Accepted manuscript posted online 29 January 2018.
- Copyright © 2018 American Society for Microbiology.
