Research ArticleHost-Pathogen Interactions

The adhesion GPCR BAI1 mediates macrophage ROS production and microbicidal activity against Gram-negative bacteria

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Science Signaling  02 Feb 2016:
Vol. 9, Issue 413, pp. ra14
DOI: 10.1126/scisignal.aac6250
  • Fig. 1 BAI1 mediates the binding and uptake of Gram-negative bacteria by primary macrophages.

    (A) The internalization of E. coli DH5α was measured in parental LR73 CHO cells and cells stably expressing exogenous BAI1 using the gentamicin protection assay as described in Materials and Methods. Data are mean fold internalization ± SEM of 10 experiments. **P < 0.01 by Mann-Whitney test. (B) Schematic of the immunofluorescence-based internalization assay. Wild-type (WT) and BAI1 knockout (BAI1-KO) BMDMs were incubated with biotinylated E. coli DH5α expressing dsRed at a multiplicity of infection (MOI) of 10 for 30 min. Cells were washed and fixed, but not permeabilized, and extracellular bacteria were labeled with Alexa Fluor 488–conjugated streptavidin (SA; green). Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (blue). In this assay, intracellular bacteria appear red (indicated by arrows), whereas extracellular bacteria appear yellow (marked by arrowheads). (C) Representative images of WT and BAI1-KO BMDMs from the immunofluorescence-based internalization assay. Scale bars, 5 μm. (D) Quantification of total cell-associated bacteria (left), extracellular bacteria (center), and intracellular bacteria (right) per cell from the experiments shown in (C). At least 125 cells per experiment were imaged, and five experiments were performed. Plots show the numbers of bacteria per cell per frame ± SEM. ***P < 0.001, ****P < 0.0001 by Mann-Whitney test.

  • Fig. 2 BAI1 is recruited to sites of bacterial engulfment.

    (A) Transgenic BMDMs expressing HA-BAI1 were fixed and stained with anti-HA antibody (green). The plasma membrane was labeled with WGA (blue), and cells were imaged by confocal microscopy. The representative image shows a single confocal section. Scale bars, 5 μm. (B and C) BMDMs expressing transgenic HA-BAI1 were infected for 30 min with either S. aureus (B) or E. coli (C) at an MOI of 10. The images show a single confocal section. The boxed areas of the merged images are magnified. White arrows indicate BAI1-positive bacteria. Scale bars, 5 μm; inset scale bars, 1 μm. (D) Quantification of the mean fluorescence intensity (MFI) of HA-BAI1 associated with bacteria. At least seven cells per condition per experiment were analyzed from a total of three experiments. A region of interest (ROI) was drawn around each bacterium, and the MFI was measured within the ROI (for details see Materials and Methods). Plot shows the MFI ± SEM of HA-BAI1 per ROI after subtraction of background MFI (Bkgd). ****P < 0.0001 by Mann-Whitney test. (E) Percentage of bacteria enriched for HA-BAI1. At least seven cells per condition were imaged. Plot shows the percentage of bacteria with an HA-BAI1 signal that was more than twofold greater than that of the background per cell ± SEM from three experiments. ****P < 0.0001 by Mann-Whitney test. (F) Schematic of the protocol for live-cell imaging analysis of BAI1 distribution. BMDMs expressing transgenic HA-BAI1 were incubated with fluorescently conjugated anti-HA antibody (green) to label extracellular receptors and then were incubated with noninvasive S. Typhimurium (∆invG) expressing dsRed. (G) Images from movie S1 are shown as a time lapse series. The white line indicates the cell periphery. Movies were generated for at least two cells from two separate experiments. Scale bars, 5 μm.

  • Fig. 3 Intracellular killing of Gram-negative bacteria is increased by BAI1-mediated bacterial recognition.

    (A) WT and BAI1-KO BMDMs were incubated for 30 min with E. coli DH5α at an MOI of 25 (t = 0) and then chased in the presence of gentamicin for the indicated times to kill extracellular bacteria. Lysates were then plated to count viable intracellular bacteria. Survival is shown relative to the bacterial counts at t = 0. All graphs display relative mean ± SEM of at least three independent experiments. Data were analyzed by two-way analysis of variance (ANOVA) with Bonferroni post hoc comparisons. P values describe the source of variation in the data set (for example, cell genotype, time, or an interaction between the cell genotype and time, which can also be considered as kinetics). Statistical information in the figure shows the results from the post hoc comparison (cell, P < 0.05; time, P < 0.001; n = 3). (B to E) Intracellular bactericidal activity by BMDMs from the indicated mice against Gram-negative bacteria was measured as described in (A). These included E. coli BW25113 (time, P < 0.01; n = 4) (B), P. aeruginosa (cell, P < 0.05; time, P < 0.001; n = 3) (C), noninvasive S. Typhimurium (∆invG) (time, P < 0.001; n = 3) (D), and the Gram-positive S. aureus (time, P < 0.05; n = 4) (E). (F and G) The survival of intracellular (F) E. coli DH5α (cell, P < 0.05; time, P < 0.01; n = 3) and (G) E. coli BW25113 (time, P < 0.001; n = 4) in PEMs from the indicated mice was measured as described in (A).

  • Fig. 4 Early microbicidal activity against Gram-negative bacteria is enhanced by BAI1 in macrophages.

    (A) WT and BAI1-KO BMDMs were incubated for 15 min with E. coli BW25113 at an MOI of 25. After extensive washing, cells were either lysed immediately (t = 0) or were chased in complete medium for 30 or 60 min. For each time point, lysates were plated on LB agar to enumerate viable bacteria. Survival is shown relative to total cell-associated bacteria at t = 0. All graphs display relative means ± SEM. Data were analyzed by two-way ANOVA with Bonferroni post hoc comparisons (cell, P < 0.0001; time, P < 0.0001; n = 8). (B to E) Cell-associated bactericidal activity of BMDMs from the indicated mice against P. aeruginosa PAO3 (cell, P < 0.01; time, P < 0.01; n = 5) (B), B. cenocepacia BC7 (cell, P < 0.01; n = 4) (C), B. cenocepacia K56-2 (cell, P < 0.01; n = 5) (D), and S. aureus (n = 5) (E) was measured as described in (A). (F) WT-flx and transgenic BAI1-RKR-AAA BMDMs were incubated with E. coli BW251113 at an MOI of 25. Bacterial killing was measured as described in (A) (cell, P < 0.01; n = 3).

  • Fig. 5 Loss of BAI1 impairs Rac activation in response to E. coli.

    (A and B) Rac1 activation was measured by a standard pull-down assay. Unprimed BMDMs were incubated with E. coli BW25113 for 10 or 30 min. Cells were then lysed, and GTP-bound Rac was precipitated with glutathione S-transferase (GST)–p21-binding domain (PBD) beads as described in Materials and Methods. Precipitates were then analyzed by Western blotting to detect Rac1. Band intensities were quantified by densitometry. Aliquots of each cell lysate were analyzed by Western blotting for total Rac1 (bottom) to demonstrate equal total Rac1 protein in control and BAI1-KO lysates. (B) Quantitation of Western blotting data from six separate experiments. Data are mean fold changes in Rac1-GTP abundance ± SEM. Two-way ANOVA with Bonferroni post hoc comparison was used for analysis (cell, P < 0.05).

  • Fig. 6 BAI1-deficient macrophages exhibit attenuated ROS production in response to Gram-negative bacteria.

    (A) LDCL assays were performed to measure ROS production by WT and BAI1-KO BMDMs after incubation with E. coli BW25113. DPI (10 μM) was added to replicate wells to inhibit NADPH oxidase activity. Graph shows a representative example of ROS activity and kinetics as mean relative light units (RLUs) ± SEM. Repeated-measures two-way ANOVA with Bonferroni post hoc comparison was used for analysis (interaction, P < 0.0001; cell, P < 0.0001; time, P < 0.0001). (B) The mean fold change in peak ROS production ± SEM of WT or BAI1-KO BMDMs treated with E. coli BW25113 from eight experiments was analyzed by Student’s t test. (C to J) BMDMs from WT or BAI1-KO mice were treated with the indicated inflammatory stimuli and analyzed as described in (A) and (B). The stimuli are listed, followed by the corresponding analysis of a representative experiment and the mean fold change in peak ROS production. (C) P. aeruginosa: interaction, P < 0.01; cell, P < 0.01; time, P < 0.0001. (D) P < 0.05; n = 5. (E) B. cenocepacia: interaction, P < 0.0001; cell, P < 0.05; time, P < 0.0001. (F) P < 0.05; n = 2. (G) S. aureus: time, P < 0.0001. (H) n = 5. (I) PMA: interaction, P < 0.0001; cell, P < 0.01; time, P < 0.0001. (J) n = 4. (K) ROS was measured in WT or gp91phox-KO BMDMs incubated with E. coli BW25113 using LDCL and analyzed as described in (A) (interaction, P < 0.0001; cell, P < 0.0001; time, P < 0.0001). (L) The mean fold change in peak ROS production ± SEM from three experiments is shown for WT and gp91phox-KO BMDMs treated with E. coli BW25113. Data were analyzed by Student’s t test.

  • Fig. 7 ROS-mediated microbicidal activity in BAI1-expressing macrophages.

    (A) BMDMs were pretreated with either vehicle or the ROS scavenger NAC before being incubated with E. coli BW25113 for the indicated times. Bacterial survival was measured as described in Fig. 3A. All graphs show mean survival ± SEM from four experiments. Data were analyzed by two-way ANOVA with Bonferroni post hoc comparisons. WT versus BAI1-KO: cell, P < 0.001; time, P < 0.001. WT versus WT-NAC: cell, P < 0.05; time, P < 0.05. (B) WT and gp91phox-KO BMDMs were infected with E. coli BW25113 for the indicated times, and the survival of the associated bacteria was measured and analyzed as described in Fig. 3A (cell, P < 0.01; time, P < 0.001; n = 6 experiments). (C) Incubation of WT cells with the ROS scavenger NAC reduces bacterial killing to the extent exhibited by gp91phox KO cells. WT and gp91phox KO BMDMs were incubated with E. coli BW25113 for 60 min in the presence or absence of NAC. Bacterial survival was measured as described in Fig. 3A. One-way ANOVA with Bonferroni post hoc comparison was used for analysis. n = 2 experiments.

  • Fig. 8 BAI1 mediates bacterial clearance in vivo.

    (A) WT, BAI1-KO, and gp91phox-KO mice were infected intraperitoneally with E. coli BW25113 and analyzed on the basis of several parameters of susceptibility to bacterial challenge. Bacterial dose, length of infection, and type of analysis are shown in schematic form. (B) WT, BAI1-KO, and gp91phox-KO mice were infected intraperitoneally (IP) with 5 × 108 CFU E. coli, and disease severity was analyzed 4 hours later. Graph displays mean score ± SEM of three experiments. ***P < 0.001, **P < 0.01 by one-way ANOVA Kruskal-Wallis test with Dunn’s post hoc comparisons. (C) Survival was measured in WT and BAI1-KO mice after intraperitoneal infection with 1 × 108 CFU E. coli. Survival was blindly scored on the basis of the criteria in (B). Mantel-Cox log rank was used to compare survival. **P < 0.01; n = 2 experiments. (D to F) Bacterial burden 4 hours after infection: CFUs were measured in the peritoneum (D), liver (E), and spleen (F) of the indicated mice 4 hours after challenge with 5 × 108 CFU E. coli. Each data point is representative of a single animal. Data are mean CFUs per tissue ± SEM of four experiments. Analysis was performed by one-way ANOVA Kruskal-Wallis test with Dunn’s post hoc comparison. ***P < 0.001, **P < 0.01, *P < 0.05. (G to I) Bacterial burden at 24 hours after infection: CFUs were measured in the peritoneum (G), liver (H), and spleen (I) 24 hours after challenge with 5 × 105 CFU E. coli. Data are mean CFUs per tissue ± SEM of three experiments. Analysis was performed by one-way ANOVA Kruskal-Wallis test with Dunn’s post hoc comparison. ***P < 0.001, **P < 0.01, *P < 0.05.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/413/ra14/DC1

    Fig. S1. BAI1 localizes to the cell periphery, the perinuclear region, and the sites of bacterial cell association.

    Fig. S2. BAI1 promotes cellular microbicidal activity in J774 macrophages.

    Fig. S3. Loss of BAI1 impairs Rac activation in response to E. coli in IFN-γ–primed BMDMs.

    Fig. S4. Loss of BAI1 impairs intracellular ROS generation but does not affect IFN-γ priming or iNOS production induced by E. coli.

    Fig. S5. Mitochondrial ROS produced in response to Gram-negative bacteria is independent of BAI1-mediated recognition and signaling.

    Fig. S6. Measurement of disease activity analysis.

    Movie S1. BAI1 is enriched at the phagocytic cup.

  • Supplementary Materials for:

    The adhesion GPCR BAI1 mediates macrophage ROS production and microbicidal activity against Gram-negative bacteria

    Emily A. Billings, Chang Sup Lee, Katherine A. Owen, Ryan S. D'Souza, Kodi S. Ravichandran, James E. Casanova*

    *Corresponding author. E-mail: jec9e{at}virginia.edu

    This PDF file includes:

    • Fig. S1. BAI1 localizes to the cell periphery, the perinuclear region, and the sites of bacterial cell association.
    • Fig. S2. BAI1 promotes cellular microbicidal activity in J774 macrophages.
    • Fig. S3. Loss of BAI1 impairs Rac activation in response to E. coli in IFN-γ–primed BMDMs.
    • Fig. S4. Loss of BAI1 impairs intracellular ROS generation but does not affect IFN-γ priming or iNOS production induced by E. coli.
    • Fig. S5. Mitochondrial ROS produced in response to Gram-negative bacteria is independent of BAI1-mediated recognition and signaling.
    • Fig. S6. Measurement of disease activity analysis.
    • Legend for movie S1

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    Format: Adobe Acrobat PDF

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). BAI1 is enriched at the phagocytic cup.

    Citation: E. A. Billings, C. S. Lee, K. A. Owen, R. S. D'Souza, K. S. Ravichandran, J. E. Casanova, The adhesion GPCR BAI1 mediates macrophage ROS production and microbicidal activity against Gram-negative bacteria. Sci. Signal. 9, ra14 (2016).

    © 2016 American Association for the Advancement of Science

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