Quorum Sensing and Self-Quorum Quenching in the Intracellular Pathogen<i>Brucellamelitensis</i>

<div><p><i>Brucella</i> quorum sensing has been described as an important regulatory system controlling crucial virulence determinants such as the VirB type IV secretion system and the flagellar genes. However, the basis of quorum sensing, namely the production of autoinducers in <i>Brucella</i> has been questioned. Here, we report data obtained from the use of a genetic tool allowing the <i>in situ</i> detection of long-chain <i>N</i>-acyl-homoserine lactones (AHL) activity at single bacterium level in <i>Brucella melitensis</i>. These data are consistent with an intrinsic production of AHL by <i>B. melitensis</i> in low concentration both during <i>in vitro</i> growth and macrophage infection. Moreover, we identified a protein, named AibP, which is homologous to the AHL-acylases of various bacterial species. <i>In vitro</i> and during infection, expression of <i>aibP</i> coincided with a decrease in endogenous AHL activity within <i>B. melitensis</i>, suggesting that AibP could efficiently impair AHL accumulation. Furthermore, we showed that deletion of <i>aibP</i> in <i>B. melitensis</i> resulted in enhanced <i>virB</i> genes expression and VirB8 production as well as in a reduced flagellar genes expression and production of FlgE (hook protein) and FliC (flagellin) <i>in vitro</i>. Altogether, these results suggest that AHL-dependent quorum sensing and AHL-quorum quenching coexist in <i>Brucella</i>, at least to regulate its virulence.</p></div

Self-quorum quenching regulates <i>virB</i> genes expression.

<p>(<b>A</b>) <i>B. melitensis</i> wt and ▵<i>aibP</i> strains both carrying the pBBR <i>PvirB</i>-<i>gfp</i>(ASV) plasmid were grown in 2YT and GFP(ASV) fluorescence intensity was measured at indicated phases of growth by flow cytometry (5×10⁴ events acquired). Results are representative of two independent experiments. (<b>B</b>) The relative abundance of <i>virB1</i>, <i>virB2</i>, and <i>virB8</i> mRNAs was determined by qRT-PCR on RNA isolated from bacteria harvested at the early exponential phase of growth in 2YT supplemented or not with exogenous C12-HSL (5 µM). Deletion of <i>aibP</i> results in significant upregulation of <i>virB</i> genes (P<0.001 in Student’s t test), whereas exogenous C12-HSL significantly downregulates their expression (P<0.001 in Student’s t test). ▵<i>aibP PaibP</i> on the right panel is the complemented strain. Results are representative of two independent experiments. Error bars represent standard deviation from biological triplicates. (<b>C</b>) (Left panel) Western Blot analysis of VirB8 production performed on whole protein lysates of bacteria harvested at the indicated phases of growth in 2YT. ▵<i>aibP PaibP</i> is the complemented strain. (Right panel) Bacteria were harvested in the log phase of growth in 2YT in the absence (–) or in the presence of C12-HSL. The VirB8 protein was detected at its expected size (26,5 kDa). Detection of PrlR or Omp89 proteins was used to normalize total protein content.</p

Validation of the specificity and sensitivity of the QS reporter system in <i>Brucella melitensis</i> 16M.

<p>(<b>a</b>) Immunofluorescence of the <i>B. melitensis</i> control strain incubated 4 hours with C12-HSL (1 µM) and labelled with monoclonal A76-12G12 anti-LPS antibody (red). No GFP(ASV) signal is detected. (<b>from b to e</b>) Observation of GFP(ASV) production by the <i>B. melitensis</i> reporter strain after a 4h incubation with various concentrations of synthetic C12-HSL; (<b>b</b>) 1nM, (<b>c</b>) 10nM, (<b>d</b>) 100nM, (<b>e</b>) 1 µM; scale bar 5 µm. (<b>f</b>) Measurement of GFP(ASV) fluorescence intensity by flow cytometry (5×10⁴ events acquired) in the <i>B. melitensis</i> QS reporter strain fixed after a 4h-incubation with 0.1nM or 1nM of C12-HSL or 3-oxo-C12HSL. The <i>B. melitensis</i> control strain was used as a negative control (black dotted line). The results are representative of at least two independent experiments.</p

<i>B. melitensis</i> both produces and degrades long-chain AHLs during macrophage infection.

<p>(<b>A and B</b>) <i>B. melitensis</i> wt and ▵<i>aibP</i> QS reporter strains were used to infect monolayers of RAW264.7 murine macrophages. Prior to (0h) and during infection, bacteria or cells were fixed, bacteria were labelled with a monoclonal A76-12G12 anti-LPS antibody and DNA was labelled with DAPI. (A) Immunofluorescence micrographs are representative from at least two independent experiments. Bacteria LPS appears in red, DNA in blue. Scale bar, 10 µm. (B) The percentage of GFP(ASV)-positive bacteria at different times post-infection was determined as described in the Material and Methods section. Error bars represent the standard deviation from two independent experiments. Data have been analyzed by ANOVA I after testing the homogeneity of variance (Bartlett). * and ** denote significant differences (<i>P</i> < 0.05 and <i>P</i> < 0.01) in relation to wt bacteria prior to infection (0h) while # denotes a significant difference (<i>P</i> < 0.05) in relation to ▵<i>aibP</i> bacteria prior to infection. (<b>C</b>) Intracellular replication of <i>B. melitensis</i> wt and ▵<i>aibP</i> strains in RAW264.7 murine macrophages. At indicated times, cells were lysed and intracellular colony forming units (CFUs) were determined. Error bars represent the standard deviation of triplicates in one representative experiment out of three. (<b>D</b>) RAW264.7 macrophages were infected with <i>B. melitensis</i> wt in the presence of C12-HSL or ACN (negative control) and treated as described (C). ** and *** denote significant (<i>P</i> < 0.01 and <i>P</i> < 0.001 respectively) differences in relation to infection by wt bacteria in the presence of CAN (Bartlett and ANOVA I analysis).</p

Transient production of long-chain AHLs by <i>B. melitensis</i> in liquid culture.

<p>The <i>B. melitensis</i> QS reporter strain was grown in 2YT and cell density was determined when fluorescence intensity of GFP(ASV) was assessed by flow cytometry (5×10⁴ events acquired). (<b>A</b>) Growth curve of the <i>B. melitensis</i> QS reporter strain. Numbers represent the 4 distinct phases of growth. OD₆₀₀, optical density at 600nm. (<b>B</b>) Histograms of GFP(ASV) fluorescence intensity representative of the growth phases represented in A. The <i>B. melitensis</i> control strain was used as a negative control. In (<b>B2</b>), the peak of GFP(ASV) fluorescence intensity due to endogenous AHLs was compared with results obtained after a 4h-incubation of the <i>B. melitensis</i> QS reporter strain with synthetic C12-HSL or 3-oxo-C12-HSL (bacteria from the early log phase were used). The insets show differential interference contrast (DIC) and FITC fluorescence microscopy of (from top to bottom) the negative control strain, the QS reporter strain in the absence of exogenous AHL and incubated with C12-HSL 0.1nM, with C12-HSL 1nM, and with 3-oxo-C12-HSL 0.1 nM respectively. Results are representative of three independent experiments.</p

Primers used in this work.

<p>Primers used in this work.</p

Characterization of the cell surface phenotype of infected cells in the spleen of IL12p40-deficient BALB/c mice.

<p>IL12p40^-/- STAT6^+/+ BALB/c mice were injected i.n. with 2x10⁷ CFU of mCherry-Br. The mice were sacrificed at 28 days post-infection and the spleens were collected and examined by immunohistofluorescence. <b>A</b>. The left panels show the overall distribution of the CD11c-, ERTR9 and MOMA-1-expressing cells in the spleen. The panels to the right of the first ones show mCherry-Br co-localization with negative cells and positive cells for CD11c, ER-TR9 and MOMA-1. The panels are color-coded with the text for phalloidin, the antigen examined or mCherry-Br. Scale bar = 200 and 20 μm, as indicated. r.p.: red pulp; w.p.: white pulp. Yellow arrowheads indicate the presence of bacteria. The data are representative of at least three independent experiments. <b>B</b>. Representative confocal images of mCherry-Br infected CD11c⁺ cells. The panels are color-coded with the text for DAPI, mCherry-Br or CD11c. Scale bar = 10 μm, as indicated. w.p.: white pulp.</p

Course of <i>B</i>. <i>melitensis</i> infection in organs of wild-type (wt), STAT6-, IL12p40- and STAT6/IL12p40-deficient BALB/c mice.

<p>The mice were injected intranasally (i.n.) with 2x10⁷ CFU of mCherry-Br <i>B</i>. <i>melitensis</i> and sacrificed at the indicated times. The data represent the number of CFU per gram of lung, spleen and liver. Grey bars represent the medians. “n” is the number of mice used. These results are representative of at least two independent experiments. ns, non-significant.</p

Characterization of infected cells expressing CD11c, CD205 and MOMA-1 in the spleen of IL12p40-deficient BALB/c mice.

<p>IL12p40^-/- STAT6^+/+ BALB/c mice were injected i.n. with 2x10⁷ CFU of mCherry-Br. The mice were sacrificed at 28 days post-infection and the spleens were collected and examined by immunohistofluorescence. <b>A</b>, The left panel shows mCherry-Br co-localization with cells expressing CD11c. The middle panel shows mCherry-Br co-localization with cells expressing CD205 and the right panel shows co-localization of CD11c- and CD205-expressing cells. <b>B</b>, The upper panels show distribution of MOMA-1-expressing cells and co-localization with mCherry-Br (left), distribution of DEC205-expressing cells and co-localization with mCherry-Br (middle), and co-localization of MOMA-1- and CD205-expressing cells (right). The panels below are higher magnification views of the same stainings. The panels are color-coded with the text for phalloidin, the antigen examined or mCherry-Br. Scale bar = 50 and 20 μm, as indicated. r.p.: red pulp; w.p.: white pulp. Yellow arrowheads indicate the presence of bacteria. The data are representative of at least two independent experiments.</p

Analysis of CD205, Arginase1, Fizz1 and CD301 expression on infected spleen cells from IL12p40- and STAT6/IL12p40-deficient BALB/c mice.

<p>IL12p40^-/- STAT-6^+/+ and IL12p40^-/- STAT-6^-/- BALB/c mice were injected i.n. with 2x10⁷ CFU of mCherry-Br. The mice were sacrificed at 28 days post-infection and the spleens were collected. The figure shown the percentage of mCherry-Br that co-localizes or not with Dec205-, Arg1-, Fizz1- and CD301-expressing cells in the spleen of IL12p40^-/- STAT-6^+/+ and IL12p40^-/- STAT-6^-/- BALB/c mice. The percentage of co-localization between mCherry-Br and positive cells for the antigen in IL12p40^-/- STAT-6^+/+ mice is indicated. The data are representative of at least two independent experiments.</p