Figure 4. Inhibition of F_O-F₁ ATPase ‘reverse mode’ increases cell death in L. pneumophila-infected human monocyte-derived macrophages (hMDMs).

(A) hMDMs were infected with Lpp-WT-GFP and were nontreated or treated with 50 μM BTB. The presence of GFP-expressing bacteria in each cell was monitored, and the number of living infected cells in the whole population was graphed as a percentage of living infected cells. Data from three independent experiments with a total of seven replicates per condition and time point. (B) hMDMs were infected with Lpp-WT-GFP (green), the nuclei of host cells were stained with Hoechst (Nuc, blue), and Annexin-V Alexa Fluor 647 was added to the cell culture to monitor early cell death (Annexin, yellow) from 1 to 18 hr post-infection (hpi) in nontreated or BTB-treated hMDMs. Representative confocal images of nontreated and Lpp-WT-GFP-infected cells at 6, 12, and 18 hpi are shown. Intracellular bacterial replication can be observed in nontreated Lpp-WT-infected hMDMs at 12 and 18 hpi. Bar: 20 μm. (C) hMDMs stained as in (B) were infected with Lpp-WT-GFP or left uninfected (noninfected), and then were treated or not with BTB (50 μM). Percentage of Annexin-V⁺ cells at 24 hpi is shown. Data from three independent experiments with a total of seven replicates per condition (D) Single-cell analysis (12 hpi) of Annexin-V intensity of the assays described in (B). Single-cell data from one representative experiment (18 hpi shown in Figure 2—figure supplement 1A). (E) hMDMs were infected with Lpp-WT-GFP, nuclei of host cells were stained with Hoechst, and tetramethylrhodamine methyl ester (TMRM) and Annexin-V Alexa Fluor 647 were added to the cells to simultaneously monitor (1–18 hpi) Δψ_m and early cell death, respectively, in nontreated or BTB-treated hMDMs (representative multifield confocal images in Figure 2—figure supplement 1C). Single-cell analyses (12 hpi) of Δψ_m (TMRM SD/mean) and cell death (Annexin-V intensity) in more than 1600 cells per condition are shown. Single-cell data from one representative experiment. Green dots, nontreated Lpp-WT-infected single cells; orange dots, BTB-treated Lpp-WT-infected single cells. (F) Same infection conditions as in (E) but using nontreated cells. Bacterial replication was monitored in each single cell infected with Lpp-WT. Single-cell analyses (12 hpi) of Δψ_m (TMRM SD/mean), area of intracellular GFP-expressing bacteria (μm²), a proxy for intracellular replication, and cell death (Annexin-V intensity) in more than 3800 cells are shown. Single-cell data from one representative experiment; color scale (yellow) represents Annexin-V intensity per cell (AU). (G) Same as in (F) at 18 hpi. *p<0.05; **p<0.01; ***p<0.001; ****p<0.00001; ns, nonsignificant (Mann–Whitney U test).

Figure 4—figure supplement 1. Inhibition of F_O-F₁ ATPase ‘reverse mode’ delays cell death in L. pneumophila-infected human monocyte-derived macrophages (hMDMs), while transfection of LpSpl into HEK-293 cells protected transfected cells from Staurosporine (STS)-induced cell death.

(A) Noninfected hMDMs were stained with Hoechst to identify their nuclei and challenged with FCCP (10 μM) for 18 hr, while Annexin-V Alexa Fluor 647 was added to the cell culture to monitor cell death. Percentage of living cells (Annexin-V negative) is shown. These values served as reference to interpret the results shown in Figure 4A. (B) hMDMs were infected with GFP-expressing bacteria or left uninfected (noninfected), and then were treated or not with BTB (50 μM). At 24 hr post-infection (hpi), the nuclei of host cells were stained with Hoechst and Annexin-V Alexa Fluor 647 was added to the cell culture to monitor cell death. Percentage of Annexin-V+ cells at 24 hpi is shown. *p<0.1; **p<0.01. ****p<0.0001; ns, nonsignificant (Mann–Whitney U test). (C) hMDMs were infected with Lpp-WT-GFP, the nuclei of host cells were stained with Hoechst, and Annexin-V-647 was added to the cell culture to monitor early cell death from 1 to 18 hpi in nontreated or BTB-treated hMDMs. Single-cell analysis of Annexin-V intensity at 18 hpi is shown. (D) hMDMs were infected as in (A), and Hoechst intensity in the nucleus was analyzed in single cells at 12 hpi. (E) HEK-293 cells stably expressing the FcγRII receptor were transfected with a control plasmid (pGFPmax, Lonza), with a plasmid expressing LpSpl WT (harboring an Xpress tag) or with a plasmid expressing a catalytically inactive LpSpl protein (*K366A, also harboring an Xpress tag). At 24 hr post-transfection, cells were labeled with Hoechst and Annexin-V-647 to monitor cell death, and 1 μM STS was added. At 6 hr, cells were fixed, permeabilized, blocked, and stained with primary mouse antibodies against Xpress tag and secondary anti-mouse Alexa Fluor 488 antibodies. Representative confocal images of transfected cells. Blue, nucleus (Hoechst, Nuc); yellow, Annexin-V-647; green, GFP or anti-Xpress (i.e., LpSpl). Bar: 20 μm. (F) Percentage of Annexin-V-positive cells in each condition, upon addition or not of STS during 6 hr. (G) Single-cell data of Annexin-V mean fluorescence intensity. More than 400 transfected cells were analyzed in each condition. *p<0.1; **p<0.01; ***p<0.001; ****p<0.0001; ns, nonsignificant (Mann–Whitney U test).

Figure 4—figure supplement 2. BTB treatment, Δψ_m, and cell death of L. pneumophila-infected human monocyte-derived macrophages (hMDMs).

(A) hMDMs were infected with Lpp-WT-GFP (green), nuclei of host cells were stained with Hoechst (Nuc, blue), and tetramethylrhodamine methyl ester (TMRM) (red) and Annexin-V Alexa Fluor 647 (yellow) were added to the cells to simultaneously monitor Δψ_m and early cell death, respectively, in nontreated or BTB-treated hMDMs, respectively. Confocal images of living infected cells were automatically acquired in 16 fields per well (four wells per condition) using ×60 magnification. Representative images of 16 stitched fields per condition are shown at 6, 12, and 18 hr post-infection (hpi). Bar: 200 μm. (B) hMDMs were infected with Lpp-WT-GFP, nuclei of host cells were stained with Hoechst, and TMRM and Annexin-V Alexa Fluor 647 were added to the cells to simultaneously monitor (1–18 hpi) Δψ_m and early cell death, respectively, in nontreated or BTB-treated hMDMs, respectively. Single-cell analyses (18 hpi) of Δψ_m (TMRM SD/mean) and cell death (Annexin-V intensity) in more than 1600 cells per condition are shown. Single-cell data from one representative experiment, Green dots, nontreated Lpp-WT-infected single cells; orange dots, BTB-treated Lpp-WT-infected single cells.