Host cell egress of Brucella abortus requires BNIP3L-mediated mitophagy

Abstract

The facultative intracellular pathogen Brucella abortus interacts with several organelles of the host cell to reach its replicative niche inside the endoplasmic reticulum. However, little is known about the interplay between the intracellular bacteria and the host cell mitochondria. Here, we showed that B. abortus triggers substantive mitochondrial network fragmentation, accompanied by mitophagy and the formation of mitochondrial Brucella-containing vacuoles during the late steps of cellular infection. Brucella-induced expression of the mitophagy receptor BNIP3L is essential for these events and relies on the iron-dependent stabilisation of the hypoxia-inducible factor 1α. Functionally, BNIP3L-mediated mitophagy appears to be advantageous for bacterial exit from the host cell as BNIP3L depletion drastically reduces the number of reinfection events. Altogether, these findings highlight the intricate link between Brucella trafficking and the mitochondria during host cell infection.

Keywords: Brucella; BNIP3L; intracellular trafficking; iron; mitophagy.

Figure 1. B. abortus triggers mitochondrial network fragmentation during the late steps of infection in HeLa cells

1. A

   Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 for the indicated times, then fixed and immunostained for TOMM20 (Alexa Fluor 488—green) and B. abortus LPS (Alexa Fluor 568—Magenta). DNA was stained with Hoechst 33258 (blue). Scale bars: 20 μm.

2. B

   Schematic summary of the calculation of the aspect ratio (AR) and the end‐point/branched‐point ratio (EBR) of the mitochondrial network. See the “Quantification of mitochondrial network morphology” paragraph in the Methods section for further information. Created with Biorender.com.

3. C, D

   Quantification of the mitochondrial population morphology by assessing the AR (C) and EBR (D) of the mitochondria of HeLa cells infected or not (NI) with B. abortus 544 for the indicated times from micrographs shown in (A). Data are presented as means ± SD from n = 5 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a multiple Mann–Whitney test followed by a Holm‐Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); *P < 0.05.

Source data are available online for this figure.

Figure 2. B. abortus triggers mitophagy in HeLa cells

1. A

   Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP for 48 h, then fixed and immunostained for the β‐subunit of the ATP synthase (Alexa Fluor 633—magenta) and LC3 (Alexa Fluor 568—green). DNA was stained with Hoechst 33258 (blue). Arrows indicate LC3‐ATP synthase β‐positive punctae. Scale bars: 20 μm. Inset scale bars: 5 μm.

2. B

   Quantification of the number of LC3‐β‐subunit of the ATP synthase‐positive punctae per HeLa cell infected or not (NI) with B. abortus 544 GFP for 24, 48 and 72 h from micrographs shown in (A) and (Fig EV3A–C). Data are presented as means ± SD from n = 3 (biological replicates independent experiments; the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a two‐way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; ***P < 0.001; ****P < 0.0001; hashtags indicate significant differences between infected conditions throughout time; ^# P < 0.05; ^## P < 0.01.

3. C

   Representative confocal micrographs of HeLa cells transfected with a FIS1‐GFP (green)‐mCherry (magenta) expression construct, infected or not with B. abortus 544 for 48 h, then fixed and immunostained for B. abortus LPS (Alexa Fluor 633—Red). DNA was stained with Hoechst 33258 (blue). Arrows indicate FIS1‐mCherry‐positive‐GFP‐negative punctae. Scale bars: 20 μm. Inset scale bars: 5 μm.

4. D

   Quantification of the number of FIS1‐mCherry‐positive‐GFP‐negative punctae per HeLa cell infected or not (NI) with B. abortus 544 for 48 h from micrographs shown in (C). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; ***: P < 0.001 (P = 0.0008).

Source data are available online for this figure.

Figure 3. B. abortus induces HIF‐1α stabilisation and BNIP3L expression in HeLa cells

1. A

   Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (red) for 24, 48 and 72 h then fixed and immunostained for HIF‐1α (Alexa 568—green). DNA was stained with Hoechst 33258 (Blue). Scale bars: 20 μm.

2. B

   Quantification of the percentages of cells positive for a nuclear localisation of HIF‐1α from HeLa cells infected or not (NI) with B. abortus 544 GFP for 24, 48 and 72 h from micrographs shown in (A). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a two‐way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ****P < 0.0001.

3. C

   Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (red) for 48 h, then fixed and immunostained for BNIP3L (Alexa 568—green) and TOMM20 (Alexa 633—magenta). DNA was stained with Hoechst 33258 (blue). Scale bars: 20 μm.

4. D

   Relative median fluorescence intensity (MFI) of BNIP3L immunostaining from HeLa cells infected or not (NI) with B. abortus 544 GFP for 48 h as measured by flow cytometry. Data are presented as means ± SD from n = 4 (biological replicates) independent experiments (8,296 cells analysed in total per condition). Statistical analyses were performed using a one sample t‐test; *P < 0.05 (P = 0.0351).

Source data are available online for this figure.

Figure EV1. B. abortus triggers Parkin‐independent mitophagy

1. A

   Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (red) for 48 h, stained with 100 nM of MTO fluorescent probe (green) for 30 min before analysis, then fixed and immunostained for TOMM20 (Alexa Fluor 633—Magenta). DNA was stained with Hoechst 33258 (blue). HeLa cells treated with 20 μM FCCP for 30 min were used as a positive control. Scale bars: 20 μm.

2. B

   Relative median fluorescence intensity (MFI) of the MTO fluorescent probe of HeLa cells infected or not (NI) with B. abortus 544 GFP for 48 h as measured by flow cytometry. HeLa cells treated with 20 μM FCCP for 30 min were used as a positive control. Data are presented as means ± SD from n = 5 (biological replicates) independent experiments (9,718 cells analysed in total per condition). Statistical analyses were performed using a one‐way ANOVA followed by a Tukey's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); *P < 0.05; ***P < 0.001.

3. C

   Representative confocal micrographs of HeLa cells transfected with a Parkin‐mCherry (green) expression construct, infected or not with B. abortus 544 GFP (red) for 48 h, then fixed and immunostained for TOMM20 (Alexa Fluor 647—magenta). DNA was stained with Hoechst 33258 (blue). HeLa cells treated with FCCP (20 μM for 30 min) were used as a positive control. Scale bars: 20 μm.

Source data are available online for this figure.

Figure EV2. B. abortus induces HIF‐1α stabilisation in a hypoxia‐independent manner

1. A–C

   Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (red) for 24 h (A), 48 h (B) and 72 h (C), treated with 150 μM of the EF5 compound for 3 h before analysis, then fixed and immunostained for EF5 (Anti‐EF5 Cy5 conjugate—magenta) and HIF‐1α (Alexa 568—green). DNA was stained with Hoechst 33258 (blue). Scale bars: 20 μm. Figure panels EV2A–C, reuse the same experiment as described in Fig 3A.

2. D

   Representative confocal micrographs of HeLa cells treated with 150 μM of the EF5 compound, exposed to normoxia (21% O₂), hypoxia (1% O₂) or an intermediate hypoxia (between 21% and 1% O₂) for 3 h, then fixed and immunostained for EF5 (anti‐EF5 Cy5 conjugate—green). DNA was stained with Hoechst 33258 (blue). Scale bars: 20 μm.

Source data are available online for this figure.

Figure 4. Iron prevents B. abortus‐induced HIF‐1α/BNIP3L pathway activation in HeLa cells and iBMDM

1. A

   Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (red) treated or not (ctrl) with FeCl₂ (500 μM) for 48 h, then fixed and immunostained for HIF‐1α (Alexa 568—green). DNA was stained with Hoechst 33258 (blue). Scale bars: 20 μm.

2. B

   Quantification of the percentages of cells positive for a nuclear localisation of HIF‐1α from HeLa cells infected or not (NI) with B. abortus 544 GFP (red) and treated or not (ctrl) with 500 μM FeCl₂ for 48 h from micrographs shown in (A). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a two‐way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ***P < 0.001; ****P < 0.0001; hashtags indicate significant differences compared to the infected condition without FeCl₂; ^#### P < 0.0001.

3. C

   Relative median fluorescence intensity (MFI) of HIF‐1α immunostaining from HeLa cells infected or not (NI) with B. abortus 544 GFP treated or not (ctrl) with 500 μM FeCl₂ for 48 h as measured by flow cytometry. Data are presented as means ± SD from n = 3 independent experiments (10,093 cells analysed in total per condition). Statistical analyses were performed using a two‐way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns, not significant; ****P < 0.0001; hashtags indicate significant differences compared to the infected condition without FeCl₂; ^#### P < 0.0001.

4. D

   Representative confocal micrographs of HeLa cells infected or not (NI) with B. abortus 544 GFP (red) treated or not (ctrl) with 500 μM FeCl₂ for 48 h, then fixed and immunostained for BNIP3L (Alexa 568—green). DNA was stained with Hoechst 33258 (blue). Scale bars: 20 μm.

5. E

   Relative median fluorescence intensity (MFI) of BNIP3L immunostaining from HeLa cells infected or not (NI) with B. abortus 544 GFP treated or not (ctrl) with 500 μM FeCl₂ for 48 h as measured by flow cytometry. Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (10,199 cells analysed in total per condition). Statistical analyses were performed using a two‐way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns, not significant; *P < 0.05; hashtags indicate significant differences compared to the infected condition without FeCl₂; ^# P < 0.05.

6. F

   Representative confocal micrographs of iBMDM infected or not (NI) with B. abortus 544 GFP (red) treated or not (ctrl) with 500 μM FeCl₂ for 48 h, then fixed and immunostained for BNIP3L (Alexa 568—green). DNA was stained with Hoechst 33258 (blue). Scale bars: 20 μm.

7. G

   Relative median fluorescence intensity (MFI) of BNIP3L immunostaining from iBMDM infected or not (NI) with B. abortus 544 GFP treated or not (ctrl) with 500 μM FeCl₂ for 48 h as measured by flow cytometry. Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (14,152 cells analysed in total per condition). Statistical analyses were performed using a two‐way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns, not significant; *P < 0.05; hashtags indicate significant differences compared to the infected condition without FeCl₂; ^### P < 0.001.

Source data are available online for this figure.

Figure EV3. B. abortus‐induced HIF‐1α stabilisation is not mediated by mitochondrial ROS in HeLa cells

1. A

   Representative wide‐field micrographs of HeLa cells infected or not (NI) with B. abortus 544 GFP (green) for 48 h, treated or not (ctrl) with 10 μM MTEMPOL for 24 h before analysis, and then stained with 0.5 μM MitoSOX™ fluorescent probe (red) for 30 min. Samples were observed under live‐imaging conditions with the Nikon Eclipse Ti₂‐inverted epifluorescence microscope. Scale bars: 20 μm.

2. B

   Representative confocal micrographs of HeLa cells infected or not (NI) with B. abortus 544 GFP (red) for 48 h, treated or not (ctrl) with 10 μM MTEMPOL for 24 h before analysis, and then fixed and immunostained for HIF‐1α (Alexa 568—green). DNA was stained with Hoechst 33258 (blue). Scale bars: 20 μm.

3. C

   Quantification of the percentages of cells positive for a nuclear localisation of HIF‐1α from HeLa cells infected or not (NI) with B. abortus 544 GFP and treated or not (ctrl) with 10 μM MTEMPOL for 24 h before analysis from micrographs shown in (B). Data are presented as means from n = 1 experiment (the numbers indicated in the columns represent the number of cells analysed per condition).

4. D

   Representative confocal micrographs of HeLa cells infected or not (NI) with B. abortus 544 GFP (red) for 48 h, treated or not (ctrl) with 5 mM NAC for 24 h before analysis, and then fixed and immunostained for HIF‐1α (Alexa 568—green). DNA was stained with Hoechst 33258 (blue). Scale bars: 20 μm.

Source data are available online for this figure.

Figure 5. BNIP3L depletion prevents B. abortus‐induced mitochondrial network fragmentation and mitophagy in HeLa cells

1. A

   Representative confocal micrographs of HeLa cells transfected with a non‐targeting siRNA pool (siNT—40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L—40 nM), infected or not (NI) with B. abortus 544 GFP (red) for 48 h, then fixed and immunostained for BNIP3L (Alexa Fluor 568—magenta) and TOMM20 (Alexa Fluor 633—green). DNA was stained with Hoechst 33258 (blue). Scale bars: 20 μm.

2. B, C

   Quantification of the mitochondrial population morphology by assessing the AR (B) and EBR (C) of the mitochondria of HeLa cells from micrographs shown in (A). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a multiple Mann–Whitney test followed by a Holm‐Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); *P < 0.05; **P < 0.01; hashtags indicate significant differences compared to the infected condition transfected with a siNT; ^# P < 0.05.

3. D

   Representative confocal micrographs of HeLa cells transfected with a FIS1‐GFP (green)‐mCherry (magenta) expression construct, infected or not (NI) with B. abortus 544 for 48 h while being transfected with a non‐targeting siRNA pool (siNT—40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L—40 nM), then fixed and immunostained for B. abortus LPS (Alexa Fluor 633—red). DNA was stained with Hoechst 33258 (blue). Arrows indicate FIS1‐mCherry‐positive‐GFP‐negative punctae. Scale bars: 20 μm. Inset scale bars: 5 μm.

4. E

   Quantification of the number of FIS1‐mCherry‐positive‐GFP‐negative punctae per cell of HeLa cells from micrographs shown in (D). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using a two‐way ANOVA followed by a Šidàk's multiple comparisons test; asterisks indicate significant differences compared to the control (NI); ns: not significant; **P < 0.01; hashtags indicate significant differences compared to the infected condition transfected with a siNT; ^# P < 0.05.

Source data are available online for this figure.

Figure 6. BNIP3L depletion limits aBCV formation and prevents reinfection events in HeLa cells

1. A

   Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (green) and transfected with a non‐targeting siRNA pool (siNT—40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L—40 nM) for 48 h, then incubated under reinfection‐permissive conditions for 24 h before analysis at 72 h pi. Cells were fixed and DNA was stained with Hoechst 33258 (blue). Arrows indicate reinfected cells. Scale bars: 50 μm.

2. B

   Quantification of the percentages of reinfection foci per infected cell at 72 h pi of HeLa cells from micrographs shown in (A). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; **P < 0.01 (P = 0.0032).

3. C

   CFU assay expressing Log (CFU/ml) from the supernatant of HeLa cells infected with B. abortus 544 GFP (green) that were previously transfected with a non‐targeting siRNA pool (siNT—40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L—40 nM) for 48 h, then incubated under reinfection‐permissive conditions for 24 h before collecting the supernatant at 72 h pi for analysis. Data are presented as means ± SD from n = 5 (biological replicates) independent experiments; Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; **P < 0.01 (P = 0.0042).

4. D

   Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Red) and transfected with a non‐targeting siRNA pool (siNT—40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L—40 nM) for 72 h pi, then fixed and immunostained for LAMP‐1 (Alexa Fluor 568—green). DNA was stained with Hoechst 33258 (blue). Arrows indicate LAMP‐1‐positive BCVs (aBCVs). Scale bars: 20 μm. Inset scale bars: 5 μm.

5. E

   Quantification of the number of LAMP‐1‐positive BCVs (aBCVs) per infected HeLa cells from micrographs shown in (E). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; *P < 0.05 (P = 0.0117).

Source data are available online for this figure.

Figure EV4. BNIP3L depletion does not impair B. abortus intracellular replication in HeLa cells

CFU assay expressing Log (CFU/ml) from HeLa cells transfected with a non‐targeting siRNA pool (siNT—40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L—40 nM), then infected with B. abortus 544 GFP for the indicated times. Data are presented as means ± SD from n = 3 (biological replicates) independent experiments; Statistical analyses were performed using a two‐way ANOVA followed by a Šidàk's multiple comparisons test; no significant differences were found. Source data are available online for this figure.

Figure 7. Iron limits aBCV formation and prevents reinfection events in HeLa cells and iBMDM

1. A

   Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (Green) treated or not (ctrl) with FeCl₂ (500 μM) for 48 h, then incubated under reinfection‐permissive conditions (with or without FeCl₂) for 24 h before analysis at 72 h pi. Cells were fixed and DNA was stained with Hoechst 33258 (blue). Arrows indicate reinfected cells. Scale bars: 50 μm.

2. B

   Quantification of the percentages of reinfection foci per infected cell at 72 h pi of HeLa cells from micrographs shown in (A). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; ***P < 0.001 (P = 0.0008).

3. C

   Representative confocal micrographs of HeLa cells infected or not with B. abortus 544 GFP (Magenta) treated or not (ctrl) with FeCl₂ (500 μM) for 72 h, then fixed and immunostained for LAMP‐1 (Alexa Fluor 568—green). DNA was stained with Hoechst 33258 (blue). Arrows indicate LAMP‐1‐positive BCVs (aBCVs). Scale bars: 20 μm. Inset scale bars: 5 μm.

4. D

   Quantification of the number of LAMP‐1‐positive BCVs (aBCVs) per infected HeLa cells from micrographs shown in (C). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; *P < 0.05 (P = 0.0368).

5. E

   Representative confocal micrographs of iBMDM infected with B. abortus 544 GFP (Green) treated or not (ctrl) with FeCl₂ (500 μM) for 48 h, then incubated under reinfection‐permissive conditions (with or without FeCl₂) for 24 h before analysis at 72 h pi. Cells were fixed and DNA was stained with Hoechst 33258 (blue). Arrows indicate reinfected cells. Scale bars: 50 μm.

6. F

   Quantification of the percentages of reinfection foci per infected cell at 72 h pi of iBMDM from micrographs shown in (E). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; **P < 0.01 (P = 0.0043).

Source data are available online for this figure.

Figure 8. B. abortus is observed inside a fraction of swollen mitochondria in HeLa cells and iBMDM during the late steps of infection

1. A

   Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (magenta) for 48 and 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 647—green). DNA was stained with Hoechst 33258 (blue). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 μm. Inset scale bars: 5 μm.

2. B

   Quantification of the percentages of infected HeLa cells displaying TOMM20‐positive BCVs (mBCVs) at the indicated times, from micrographs shown in (A). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; ***P < 0.001 (P = 0.0001).

3. C

   Quantification of the number of TOMM20‐positive BCVs (mBCVs) per infected HeLa cells, at the indicated times, from micrographs shown in (E). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; *P = 0.05.

4. D

   Representative confocal micrographs of iBMDM infected with B. abortus 544 GFP (magenta) for 48 and 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 647—green). DNA was stained with Hoechst 33258 (blue). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 μm. Inset scale bars: 5 μm.

5. E

   Quantification of the percentages of infected iBMDM displaying TOMM20‐positive BCVs (mBCVs) at the indicated times, from micrographs shown in (D). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; ***P < 0.001 (P = 0.0009).

6. F

   Quantification of the number of TOMM20‐positive BCVs (mBCVs) per infected HeLa cells, at the indicated times, from micrographs shown in (E). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; ns: not significant (P = 0.3739).

7. G

   FIB/SEM micrographs of HeLa cells infected with B. abortus 2308 RFP for 48 h showing bacteria within mitochondria. Scale bars: 600 nm.

8. H

   Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (red) for 72 h, then fixed and immunostained for LAMP‐1 (Alexa Fluor 568—green) and TOMM20 (Alexa Fluor 633—magenta). DNA was stained with Hoechst 33258 (blue). Arrows indicate LAMP‐1‐positive BCVs (aBCVs). Stars indicate TOMM20‐positive BCVs (mBCVs). Scale bars: 20 μm. Inset scale bars: 5 μm.

Source data are available online for this figure.

Figure EV5. Neither BNIP3L depletion nor iron supplementation alters mBCV occurrence in HeLa cells

1. A

   STED micrographs of a HeLa cell infected with B. abortus 544 GFP (magenta) for 72 h, then fixed and immunostained for TOMM20 (Abberior® STAR 635—green). The presented cell displays a rare but spectacular event of enlarged mBCVs with colonisation of the bacteria. Scale bars: 5 μm.

2. B

   Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (magenta) and transfected with a non‐targeting siRNA pool (siNT—40 nM) or a BNIP3L siRNA SMARTpool (siBNIP3L—40 nM) for 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 647—green). DNA was stained with Hoechst 33258 (blue). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 μm. Inset scale bars: 5 μm.

3. C

   Quantification of the number of TOMM20‐positive BCVs (mBCVs) per infected HeLa cells, from micrographs shown in (B). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; ns: not significant (P = 0.6742).

4. D

   Representative confocal micrographs of HeLa cells infected with B. abortus 544 GFP (magenta), treated or not (ctrl) with FeCl₂ (500 μM) for 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 647—green). DNA was stained with Hoechst 33258 (blue). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 μm. Inset scale bars: 5 μm.

5. E

   Quantification of the number of TOMM20‐positive BCVs (mBCVs) per infected HeLa cells, from micrographs shown in (D). Data are presented as means ± SD from n = 3 (biological replicates) independent experiments (the numbers indicated in the columns represent the number of cells analysed per condition). Statistical analyses were performed using an unpaired two‐tailed Student's t‐test; ns, not significant (P = 0.4724).

6. F

   Representative confocal micrographs of HeLa cells infected with B. abortus 544 WT for 72 h, then fixed and immunostained for TOMM20 (Alexa Fluor 633—magenta) and LC3 (Alexa Fluor 568—green). DNA (from the HeLa nucleus and B. abortus) was stained with Hoechst 33258 (red). Arrows indicate when B. abortus was found inside a mitochondrion (mBCVs). Scale bars: 20 μm. Inset scale bars: 5 μm.

Source data are available online for this figure.

Figure 9. Summary of the interactions between Brucella abortus and the mitochondria of the host cell

In this study, we showed that until 24 h pi (A), Brucella abortus affects neither the mitochondrial morphology nor the endogenous degradation of HIF‐1α. However, at 48 h pi (B), B. abortus induces an iron starvation response (1) that stabilises and activates HIF‐1α (2) which therefore translocates to the nucleus (3). There, HIF‐1α upregulates the expression of the mitophagy receptor BNIP3L (4) which mediates two phenotypes during the late stages of B. abortus intracellular trafficking. B. abortus‐induced BNIP3L expression mediates mitochondrial network fragmentation (5), one hallmark of mitophagy (6) that is characterised by the engulfment of mitochondrial fragments inside phagophores to specifically degrade and recycle them. Eventually, BNIP3L helps to fulfil B. abortus intracellular cycle as it is necessary for bacterial egress (7) and reinfection of neighbouring cells (8). Created with Biorender.com.