Macrophages are critical for virulence of B. cenocepacia.

(A,B) Embryo survival (average inoculum 17 CFU, representative experiment) (A) and bacterial burden (total of 3 experiments) over time (B) of control (black) and pu.1 knockdown embryos (red) injected iv with B. cenocepacia K56-2. (C,D) Representative fluorescence overlay images of an mpeg1:mCherry control and mpeg1:mCherry pu.1 knockdown embryo at 30 min and 24 h after injection with ~40 CFU B. cenocepacia K56-2 (blue). See also S2 Fig. (C) mCherry-positive macrophages (red) colocalise with K56-2 at 30 mpi, and are no longer detected at 24 hpi (insets show magnification). (D) mCherry-positive macrophages are absent in knockdown embryos at 30 mpi and start to re-appear at 24 hpi (insets show magnification). Scale bars, 100 μm. (E,F) Embryo survival (average inoculum 28 CFU, representative experiment) (E) and corresponding bacterial burden (n = 10 per group per time point). (F) of mpeg1/umn⁺ embryos, untreated or treated with 5mM Mtz or 0.2% DMSO, and mpeg1/umn⁻embryos treated with 5mM Mtz iv injected with B. cenocepacia K56-2. (B,F) Geometric means with each data point representing an individual embryo. Dead embryos marked as black open circles (not recorded for 5 embryos in (B), and in (F)). (A, B, E, F) * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; **** p ≤ 0.0001. ns: not significant. See materials and methods for statistical tests. See also S1 Fig and S3 Fig.

Acute, but not persistent infection results in systemic phagocyte death.

(A) Sudan black staining of an mpx:GFP embryo (24 hpi), injected with ~45 CFU B. cenocepacia K56-2. Bright field, fluorescence and merged images showing recruited neutrophils (green) that release granules (stained by Sudan black as black deposit, white arrow) close to an infected cell containing red fluorescent bacteria. Arrow head, individual bacteria. Scale bars, 50 μm. (B) Image of the trunk region of an mpx:GFP; mpeg1:mCherry embryo 24 h post iv injection in the blood island with B. cenocepacia K56-2 (Turquoise), showing neutrophils (green) and macrophages (red) infiltrated in an infection site with multiple infected cells. BF, Bright field image, showing tissue damage. Scale bar, 50 μm. (C) Mean neutrophil numbers in non-infected control and mpx:GFP embryos injected at 50 hpf with B. cenocepacia K56-2 or B. stabilis LMG14294. See also S5B Fig and S5C Fig. (D) Mpeg1:mCherry embryos showing reduced macrophage numbers (red) at 24 hpi in B. cenocepacia K56-2-infected (~45 CFU) compared to non-infected control embryos. Scale bars, 0.5 mm. See S5D Fig for quantification. (E) Mean relative mpx and mpeg1 gene expression level (qRT-PCR) in embryos injected with on average 234 CFU of B. cenocepacia K56-2 (red bars) or 123 CFU of B. stabilis LMG14294 (pink bars) each normalised to a PBS-injected control group at each time point and analysed using Anova (error bars, SEM). Two independent experiments. Asterisks below each bar indicate significance compared to the PBS control at each time point, significance between groups per time point is indicated with a horizontal line. (F) Non-infected and B. cenocepacia K56-2 (~50 CFU, Turquoise indicated in red for better visualization) infected embryos at 24hpi with the cell-impermeable dye Sytox Green. Arrows, dead cells due to DMSO injection. Arrow heads, bacterial clusters. Scale bars, 100 μm. See also S5F Fig. (C). Each data point represents an individual embryo. (C,E) * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: non-significant. See materials and methods for statistical tests. See also S5 Fig.

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Neutrophils efficiently phagocytose surface-associated Bcc bacteria.

Confocal stacks after subcutaneous infection with B. stabilis in mpx:GFP embryos (STD intensity projection, 2 μm x 19 steps; T = 35–37 in S2 Movie). Arrows, rounded neutrophils (green) with vacuoles full of bacteria (red) at 64 minutes post injection (mpi) eject their cell contents in the surroundings (arrow heads 65.5 mpi), leaving bacterial clusters and cell debris (arrow heads, 66 min). Lower panels, MAX intensity projection of three consecutive slices (2 μm) at 65.5 mpi showing ejected cellular contents (diffuse GFP signal). Scale bars, 50 μm.

Macrophages, but not neutrophils, contribute to increased bacterial burden and pro-inflammatory responses towards subcutaneously introduced B. cenocepacia.

(A) Mpeg1/umn⁺ embryos were subcutaneously injected with B. cenocepacia K56-2 expressing Turquoise. Fluorescent overlay images were taken at 90 min and 5 h post infection, showing infected macrophages (red). Scale bars, 10 μm. (B) Images (red and blue overlay and below slightly enlarged individual fluorescence images with blue filter) of the indicated area (see drawing) of an mpx/umn⁺ (B1) and an mpeg1/umn⁺ (B2) embryo subcutaneously injected with B. cenocepacia K56-2 expressing Turquoise. B1 shows an embryo followed in time displaying neutrophil infiltration (red) and increase in bacterial burden (blue, see Fig 7A for quantification). B2 shows the image of an embryo with macrophage infiltration (red) and high bacterial burden at 24 hpi. Arrow head points at mCherry positive debris. See non-infected mpx/umn⁺ and mpeg1/umn⁺ control embryos in S6 Fig for comparison. Scale bars, 100 μm. (C) Image of the infected area of a representative mpx/umn⁺ embryo depleted of neutrophils with Mtz, and subcutaneously injected with B. cenocepacia K56-2 (Turquoise) at 24 hpi. Scale bar 100 μm, and 50 μm for inset. See also S6D Fig. (D) Image of a representative mpeg1/umn⁺ embryo depleted of macrophages with Mtz and subcutaneously injected with B. cenocepacia K56-2 (Turquoise) at 24 hpi. Scale bar 100 μm, and 50 μm for inset.

The absence of Gp91 results in increased bacterial burden and neutrophil persistence during subcutaneous infections.

(A) Bacterial burden over time after subcutaneous injection of B. cenocepacia K56-2 in control (black circles) and gp91 knockdown embryos (open red circles). Average of two independent experiments. (B) Bacterial burden over time after subcutaneous injection of B. stabilis LMG14294 in control (black circles) and gp91 knockdown embryos (open red circles). Average of three independent experiments. (C) Fluorescent overlay images of the injected area of a representative mpx:GFP control MO and gp91 knockdown embryo (neutrophils in green) in time after subcutaneous injection with B. stabilis (red). Inset shows bacterial load at ~20 hpi. % at 22 and 21 hpi indicates percentage of control and gp91 knockdown embryos that show reduced neutrophil numbers (86.8%), and persistent neutrophil infiltration (71.3%), respectively, at the infection site. Scale bar, 100 μm. (A,B) * p ≤ 0.05; **** p ≤ 0.0001; ns: non-significant. See materials and methods for statistical tests.

B. cenocepacia K56-2 induces robust pro-inflammatory Il1b expression that is dependent on macrophages.

(A,B) Mean relative il1b (A) and cxcl8 (B) gene expression levels (qRT-PCR) in embryos injected with on average 250 CFU B. cenocepacia K56-2 (red bars) or on average 111 CFU B. stabilis LMG14294 (pink bars), normalized to a PBS-injected control group at each time point. Error bars represent mean with SEM of three biological replicates. Asterisks above each bar indicate significance compared to the PBS control at each time point, significance between groups per time point is indicated with a horizontal line. (C,D) mpeg1/umn⁺ embryos were pre-treated at 34 hpf for 15 h with DMSO or 5 mM Mtz. Randomized groups were injected with either PBS or with B. cenocepacia K56-2 (on average 150 CFU). Graphs show mean relative il1b (C) and cxcl8 (D) gene expression levels (qRT-PCR) normalized to the PBS-injected DMSO-treated group at each time point. Error bars represent mean with SEM of two biological replicates. See also S8 Fig. (E) Confocal stack images (green/red overlay (left panels) and red channel (right panels)) of il1b:GFP/mpeg1:mCherry embryos 8 h post iv injection with PBS, or B. cenocepacia K56-2 (DS-Red). Due to strong fluorescence of GFP in epithelial cells in the trunk and head region, images were taken over the yolk sac valley. Scale bars 10 μm. (F) Embryo survival (average inoculum 44 CFU, representative experiment) of control (black) and il1b knockdown embryos (red) injected iv with B. cenocepacia K56-2. (G,H) Embryo survival (G) and bacterial burden over time (H) of control (black) and Anakinra-treated embryos (red) injected iv with B. cenocepacia K56-2 (average inoculum 107 CFU for both groups). Representative experiment. (A-D, F-H) * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: non-significant.

Related to Figs 1 and 2. Role of macrophages during infection with Bcc strains causing either persistent or acute infection.

(A-D G-J) Embryo survival (A, C, G, and I) and bacterial burden (total of 2 experiments) over time (B, D, H, J) of control (black) and pu.1 knockdown embryos (red) injected iv with B. cenocepacia J2315 (A,B), B. cepacia CEP509 (C,D), B. cenocepacia J415 (G,H), and B. vietnamiensis FC441 (I,J), respectively. (E) Representative experiment (of at least three) showing embryo survival of control embryos (n = 47), pu.1 knockdown (n = 54) and nonspecific control MO (n = 52) embryos injected with B. cenocepacia K56-2 (average 53 CFU). (F) Representative fluorescence image at 24 hpi showing neutrophils (green) in an mpx:GFP pu.1 knockdown embryo injected with B. cenocepacia J2315 (red) (~50 CFU). Inset shows corresponding bright field image. Scale bar, 100 μm. (B, D, H, J) Geometric mean with each data point representing an individual embryo. Dead embryos are indicated as black open circles. (A-E and G-J) * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns: non-significant. See materials and methods for statistical tests used.

Related to Fig 1C and 1D. Pu.1 knockdown prevents B. cenocepacia K56-2 multiplication.

Fluorescence overlay images (red and green filters) of the indicated area (boxed area in embryo drawing) of two mpeg1:mCherry control embryos (left) and two mpeg1:mCherry pu.1 knockdown (right) imaged at 1, 3, 16, 20, 24, and 41 h after injection with ~50 CFU B. cenocepacia K56-2 harbouring pIN301 (green). The absence of macrophages prevents efficient bacterial replication. Bacteria in control embryos colocalise with macrophages (arrow heads). Fluorescent macrophages disappear from control embryos (>20 hpi). Appearance of macrophages in pu.1 knockdown embryos (white arrows).

Related to Fig 1. Chemical ablation of macrophages using the NTR/Mtz system.

(A) Schematic representation and treatment schedule of the chemical ablation strategy based on the nitroreductase (NTR)/ metronidazole (Mtz) system, shown for macrophage-specific ablation. See Materials and methods for details. (B) Representative fluorescence images of non-treated and Mtz-treated mpeg1/umn⁺ embryos, showing the efficacy of the Mtz treatment. Residual red fluorescence in treated embryos represented apoptotic cells. Scale bar, 0.5 mm. (C) Quantification of macrophage numbers in mpeg1/umn⁺ embryos (untreated or treated at 34 hpf with 5mM Mtz) at 0, 15 and 39 hours after treatment as indicated in (A). The efficacy of macrophage ablation by Mtz treatment was evaluated by pixel counting. The average macrophage numbers of two independent experiments (n = 6) are shown. ns, non-significant, ** p ≤ 0.01, **** p ≤ 0.0001. See material and methods for statistical analysis used.

Related to Fig 1E and 1F. Mtz-mediated ablation of macrophages prevents B. cenocepacia K56-2 multiplication.

Fluorescence overlay images (red and green filter) of two DMSO-treated (left panels) and two Mtz-treated (right panels) mpeg1/umn⁺ embryos imaged at 1, 3, 24, and 48 h after iv injection with ~50 CFU B. cenocepacia K56-2 harbouring pIN301 (green). Bacterial replication in control embryos resulted in macrophage cell death, seen as mCherry positive debris (>24 hpi) (see also S5F Fig). At 28 and 48 hpi individual fluorescence images (green filter) are shown below the overlay images. Drawing indicates imaged area, marking the embryo sac extension (green) prone to autofluorescence. Arrow head indicates mCherry positive macrophages in apoptosis.

Related to Fig 3. Behaviour and fate of neutrophils and macrophages during acute and persistent infection. (A). Confocal stack images of a time series, first image ~20 hours after infection of an mpx:GFP embryo with B. cenocepacia K56-2. Patrolling neutrophil inspects heavily infected macrophage (white arrow). Individual bacteria (arrow head), possibly released from infected cell nearby (open arrow), are being moved around by the neutrophil. The last image shows the same area 90 minutes later with the infected macrophage still intact. (B,C) Mpx:GFP embryos (50 hpf) were injected with B. stabilis LMG14294. (B) Representative images of control and B. stabilis infected mpx:GFP embryos with increased neutrophil numbers. Neutrophils were more dispersed in B. stabilis-infected compared to non-infected control embryos, where most neutrophils were resting in the caudal hematopoietic tissue. (C) The number of neutrophils in infected and non-infected control embryos was determined by pixel counting at different time points after injection. Each data point represents one embryo. The graph represents one of the three experiments represented in Fig 3C, but includes an additional time point at 5 days post infection, which was not determined in the other 2 experiments. A percentage of embryos injected with B. stabilis contained more neutrophils than control embryos (see also (B)). *, p ≤ 0.05. (D) Mpeg1:mCherry embryos were injected with B. cenocepacia K56-2 (~45 CFU) and the number of macrophages was evaluated by pixel counting at 0 and 24 hpi (30 and 54 hpf, respectively). The results are related to the corresponding experiment shown in Fig 3D. Each data point represents one embryo. Significance was determined using a one-way Anova with Sidak’s Multiple Comparisons test. **** p ≤ 0.0001. Two independent experiments (n = 5). (E) Image showing B.cenocepacia K56-2 (red) in an L-plastin labelled macrophage (blue) at 24 hpi. Scale bar, 50 μm. Inset, magnification red/ blue filters, scale bar 10 μm. (F) Images (bright field, green/red overlay, and detailed image with red/green filter), of a non-infected mpeg1/umn⁺ embryo (left panels) and mpeg1/umn⁺ embryo (right panels) iv infected with B. cenocepacia K56-2 (green, arrows) at 24 hpi. The images are similarly treated to enhance the red fluorescence to visualize the mCherry positive debris. The close up shows individual macrophages (left panel, red), and red fluorescent debris (arrow heads). Related to Fig 3F.

Related to Figs 6 and 7. Macrophages, but not neutrophils, contribute to increased bacterial burden and pro-inflammatory responses towards subcutaneous B. cenocepacia.

(A) mpeg/umn⁺ embryo infected subcutaneously at 2 dpf with B. cenocepacia K56-2 (green). Confocal stack (59 slices, 1 μm) at 24 hpi showing bacteria in macrophages. A close up of the indicated area is shown in 3D, with volume rendering for GFP signal and surface rendering of mCherry signal without (middle panel) and with 50% surface transparency (right panel). Scale bar 50 μm. (B) Fluorescence images showing non-infected mpx/umn⁺ and mpeg1/umn⁺ control embryos at 3 dpf, the time point that resembles 24 h post subcutaneous injection in Fig 6. (C) Image overlay (bright field, red and blue filters) of the tail region of an mpx/umn⁺ embryo after subcutaneous injection with B. cenocepacia K56-2 expressing Turquoise, presenting tissue damage by 24 hpi. Scale bar, 100 μm. (D) Image overlay (bright field, red, blue and green filters) of the tail region of an Mtz-treated mpeg1/umn⁺; mpx:GFP embryo 24 h after subcutaneous injection with B. cenocepacia K56-2 expressing Turquoise. Scale bar, 100 μm.