Brucella abortus Senses the Intracellular Environment through the BvrR/BvrS Two-Component System, Which Allows B. abortus To Adapt to Its Replicative Niche

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.

Keywords: brucellosis; two-component system; type IV secretion system.

FIG 1

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 (P_bvrR) 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 P_bvrR (black circles) was determined at the indicated times by measuring the luciferase activity (circles). These results are representative of at least three independent experiments.

FIG 2

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).

FIG 3

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.

FIG 4

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.

FIG 5

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.

FIG 6

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.

FIG 7

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.

FIG 8

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.

FIG 9

BvrR-P, VjbR, and VirB form a virulence circuit. (A) Digoxigenin-labeled probes containing the promoter region of vjbR (P_vjbR) 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 P_vjbR 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.

FIG 10

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.

FIG 11

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.