SEARCH

SEARCH BY CITATION

FilenameFormatSizeDescription
cmi12223-sup-0001-fig_s1.tiff8824K

Fig. S1. Replication and cytosolic residence of B. cenocepacia J2315 in macrophages.

A. Intracellular growth kinetics of the WT J2315 strain in THP-1, hMDMs, and BMDMs. The infection was carried out in triplicate with MOI 1, for 1 h, followed by 2 h of gentamicin/ceftazidime treatment to kill extracellular bacteria. The infected monolayers were lysed at different time intervals and plated onto agar plates for colony enumeration.

B. Defining the optimal MOI used for THP-1 infection. The infection and colony enumeration was carried out as described for (A) with an increasing MOI of 0.1, 1.0, 10 and 30.

C. Quantification of the percentage of THP-1 cells harbouring bacteria at the indicated times post infection with MOI 1. Uncountable bacteria in heavily infected cells were considered as > 15 bacteria. Error bars represent the standard deviation of the % infected cells mean value averaged from 100 cells over 3 experiments.

D. Confocal images of THP-1 cells infected with DsRed-expressing J2315 B. cenocepacia at MOI of 1.0 for the indicated time points.

E. Confocal images of THP-1 cells infected with DsRed-expressing J2315 B. cenocepacia at MOI of 1.0 for 1 h post infection. Cells were labelled with Gal-3 (green) prior to visualization. Bacterial colocalization with Gal-3 was quantified using Imaris and is shown in the fourth panel.

F. Gal-3 staining in non-infected THP-1 cells.

cmi12223-sup-0002-fig_s2.tiff7382K

Fig. S2.B. cenocepacia resides in close proximity to ER-derived membranes.

A. TEM of THP-1 cells infected with B. cenocepacia at MOI of 1 for 1 h. The ER-derived membrane is indicated by the dashed white arrowheads.

B. Colocalization of the bacteria with the KDEL ER marker at the indicated time points post infection. At least 100 infected cells from multiple coverslips were examined in each experiment. Results shown are representative of three independent experiments performed in triplicates. Data represent means ± standard deviation. Asterisks represent statistically significant difference (P value < 0.05).

C. Confocal images of THP-1 macrophages infected with WT or FK DsRed B. cenocepacia at an MOI of 1.0 at 1 h post infection. Infected cells were labelled with the anti-KDEL ER membrane marker antibody in green prior to visualization. Bacteria colocalization with KDEL was quantified using Imaris and is shown in the fourth panel. The surface rendering of a three-dimensional reconstruction of the infected cell is shown in the 5th panel. In the last panel, the surface was clipped from the top to show how the bacterium is surrounded by KDEL+ signal only in the WT bacteria-infected cell. Representative images from three independent experiments are shown. Data represent means ± standard deviation.

cmi12223-sup-0003-fig_s3.tiff6065K

Fig. S3. Ubiquitin acquisition by B. cenocepacia precedes p62 recruitment, while LC3B colocalization requires p62.

A. Western blot analysis of p62 from THP-1 cells treated with either non-targeting control siRNA or p62-siRNA. Actin was used as a loading control.

B. Confocal images of GFP-LC3B expressing THP-1 macrophages transfected with non-targeting control siRNA (upper row) or p62-siRNA (lower two rows) for 48 h then infected with the WT DsRed B. cenocepacia at an MOI of 1, for 3 h. Cells are immunostained with anti-ubiquitin antibody (magenta). Data represent means ± standard deviation.

cmi12223-sup-0004-fig_s4.tiff5523K

Fig. S4. Autophagic flux assessments: B. cenocepacia infection disrupts autophagic flux of p62 while maintaining its expression levels.

A. THP-1 cells were infected for the indicated time points and the p62 mRNA expression levels relative to b-actin were assessed. Data represent means ± standard deviation. Asterisks represent statistically significant difference (P value < 0.05).

B. Western blot of p62 levels and RhoGDI used as a loading control are shown. Cells were either left untreated or treated with 400 nM of bafilomycin A before infection for the indicated time points.

cmi12223-sup-0005-fig_s5.tiff9012K

Fig. S5. Autophagy induction decreases the replication of B. cenocepacia within macrophages.

A–D. Confocal images of THP-1 macrophages expressing GFP-LC3B left untreated, pre-starved, or pretreated with rapamycin for 2 h. Cells were infected with WT DsRed B. cenocepacia at an MOI of 1.0. At the indicated times post infection, cells were fixed and processed for confocal microscopy. Infected cells from multiple coverslips were examined in each experiment. Representative images from three independent experiments are shown.

cmi12223-sup-0006-fig_s6.tiff7446K

Fig. S6. Autophagy induction promotes bacterial clearance from infected macrophages.

A–D. Quantification of the percentage of B. cenocepacia replication in THP-1 cells. Cells were either left untreated, pre-starved or pretreated with rapamycin 2 h prior to infection. Cells were infected with the WT and FK B. cenocepacia at an MOI of 1. Uncountable bacteria present in a heavily infected cell were considered as > 15 bacteria. Infected cells from multiple coverslips were examined in each experiment. The results are representative of three independent experiments, each performed in triplicate. Error bars represent standard deviations. Asterisks represent statistically significant difference (P value < 0.05).

cmi12223-sup-0007-fig_s7.tiff8545K

Fig. S7.B. cenocepacia resides in autophagosome-like compartments in starved macrophages.

A and B. TEM of THP-1 cells pre-starved for 2 h then infected with B. cenocepacia at MOI of 1 for 4 h and 24 h respectively. At both time points bacteria are surrounded by multiple cellular membranes. At the 24 h time point, there is evidence of autophagosome-lysosome fusion.

cmi12223-sup-0008-fig_s8.tiff4872KFig. S8. Induction of autophagy by starvation promotes maturation of B. cenocepacia-containing phagosomes to lysosomes. Confocal images showing the localization of WT B. cenocepacia in THP-1 macrophages relative to late endocytic and lysosomal markers. THP-1 cells were either left untreated [non-starved (NS)] or pre-starved (PS) for 2 h, then infected with WT B. cenocepacia at MOI 1 for 24 h. Bacteria were labelled with B. cenocepacia-specific antisera (Red) and the endocytic markers LAMP-2 (A) or Cathepsin-D (B) were labelled with specific antibodies (green) prior to visualization. Bacterial colocalization with each marker was quantified using Imaris software, and shown in the third panel. Infected cells from multiple coverslips were examined in each case, and images are representative of three independent experiments. Data represent means ± standard deviation.
cmi12223-sup-0009-fig_s9.tiff7318K

Fig. S9. Macrophages derived from the CGD mouse model support increased B. cenocepacia survival and replication but autophagy stimulation prior to infection improves its bacterial clearance. Mouse BMDMs from WT C57BL/6 (Normal) or gp91phox−/− (CGD) mice were either left untreated or pretreated with rapamycin for 2 h prior to infection with WT J2315. The infection was carried out in triplicate with MOI 1 for 1 h, followed by 2 h of gentamicin/ceftazidime treatment to kill extracellular bacteria.

A. Confocal images of BMDMs fixed at various times post infection and labelled with B. cenocepacia-specific antibody (green) prior to visualization. Scale bar shown is 5 μm.

B. Intracellular growth kinetics of WT J2315 in normal and CGD BMDMs infected as described above. The infected monolayers were lysed at different time intervals and plated onto agar plates for colony enumeration.

C and D. Quantification of the percentage of cells from normal (C) or CGD mice (D) harbouring various bacterial loads at 24 h post infection. Uncountable bacteria present in a heavily infected cell were considered as > 15 bacteria. For all imaging experiments shown, infected cells from multiple coverslips were examined in each experiment. All data are representative of three independent experiments, each performed in triplicate. Graphs represent mean ± standard deviation. Asterisks represent statistically significant difference (P value < 0.05).

cmi12223-sup-0010-movie_s1.mov2839KMovie S1. 3D Reconstruction of GFP-LC3B localization with the WT B. cenocepacia. THP-1 cells expressing GFP-LC3B were infected with the DsRed-labelled WT J2315 at MOI 1. Three-dimensional confocal imaging of these cells reveals that GFP-LC3B envelops the majority of the live WT bacteria imaged at 3 h post infection.
cmi12223-sup-0011-movie_s2.mov1889KMovie S2. WT B. cenocepacia replicates despite being targeted by autophagy. Time-lapse imaging of GFP-LC3B expressing THP1 cells infected with DsRed-labelled WT J2315 at MOI 1 starting at 3 h post infection, The images were taken at 20 min intervals. This movie demonstrates that the GFP-LC3B puncta appear initially in close contact with B. cenocepacia, however as replication begins at 6–8 h post infection, the bacteria clearly separate from the LC3B and the localized GFP-LC3B signal dissipates as the bacteria rapidly multiply. Around 20–24 h post infection, the heavily replicating bacteria appear to begin exiting the cell.
cmi12223-sup-0012-movie_s3.mov1108KMovie S3. Increasing autophagy flux by starvation blocks bacterial replication. GFP-LC3B expressing THP1 cells were pre-starved for 2 h then infected with DsRed-labelled WT J2315 at MOI 1. The time-lapse imaging started at 3 h post infection and the images were taken at 20 min intervals. This movie demonstrates that the GFP-LC3B puncta appeared in close contact with B. cenocepacia similar to the non-starved cells. However, over the course of a 16 h infection, the bacteria remained within the LC3B positive compartment, without any evidence of replication.
cmi12223-sup-0013-file_s1.docx125KFile S1. Extended experimental procedures.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.