Figure 2. The mitochondrial F_OF₁-ATPase works in the ‘reverse mode’ during infection of human monocyte-derived macrophages (hMDMs) by L. pneumophila.

(A) In the ‘forward mode’ of the mitochondrial ATPase, the Δψ_m generated by the electron transport chain is used by the F_OF₁-ATPase to synthesize ATP. The ‘reverse mode’ of the F_OF₁-ATPase leads to ATP hydrolysis to pump H⁺ to the intermembrane space (IMS). IMM, inner mitochondrial membrane. (B) When the F_OF₁-ATPase is inhibited by oligomycin or dicyclohexylcarbodiimide (DCCD), an increase in Δψ_m indicates that the ATPase was working in the ‘forward mode’ (H⁺ accumulate in the IMS), while a decrease in Δψ_m indicates functioning in the ‘reverse mode’ (H⁺ cannot be translocated to the IMS by the F_OF₁-ATPase to sustain the Δψ_m). (C) hMDMs were infected with GFP-expressing bacteria (green) or left uninfected (noninfected). At 5.5 hr post-infection (hpi), cells were labeled with Hoechst to identify the cell nucleus (Nuc, blue) and tetramethylrhodamine methyl ester (TMRM) (red) to quantify Δψ_m. At 6 hpi, addition of medium (no changes) or FCCP (complete depolarization) was used as controls. Representative confocal images of Lpp-WT-infected hMDMs (6 hpi) at 5 min before the addition of medium (top) or FCCP (bottom), and at 5, 25, and 50 min after addition of medium or FCCP. Bar: 20 μm. (D) Quantification of (C) before (baseline) and after the addition of medium. Each dot represents mean ± SD of three independent experiments with a total of eight replicates. (E) Same as (D) but FCCP was added. (F) Same as (D) but oligomycin was added. (G) Same as (D) but DCCD was added. (H) Same as (C) but infection was performed with Lpp-WT, Lpp-ΔdotA, Lpp-ΔlncP, Lpp-Δspl, L. pneumophila strain Philadelphia JR32 (JR32)-WT, JR32-ΔicmT or JR32-ΔmitF. TMRM values (SD/mean) at 50 min after DCCD addition are shown. Data from a minimum of three experiments per strain with 10 or more replicates per strain. (I) hMDMs were infected with GFP-expressing bacteria or left uninfected (noninfected). At 5.5 hpi, cells were labeled with Hoechst to identify the cell nucleus and TMRM to quantify Δψ_m. At 6 hpi, addition of DCCD revealed whether the F_OF₁-ATPase works in the ‘reverse mode.’ Infection was performed with Lpp-WT expressing empty pBCKS vector (empty vector), Lpp-Δspl-expressing empty vector and Lpp-Δspl-expressing pBCKS-spl vector, which express LpSpl (complemented strain, Lpp-Δspl:: spl). Data from three donors are shown. Each dot represents a replicate. *p<0.1; **p<0.01; ***p<0.001; ****p<0.00001; ns, nonsignificant (Mann–Whitney U test).

Figure 2—figure supplement 1. Basal respiration of human monocyte-derived macrophages (hMDMs) infected with L. pneumophila Δspl mutant and validation of the model in HEK-293 cells.

(A) hMDMs were infected with L. pneumophila strain Paris WT (Lpp-WT), a type 4 secretion system (T4SS)-deficient ΔdotA mutant, a LpSpl-deficient Δspl mutant, or left uninfected (noninfected). At 6 hr post-infection (hpi), basal oxygen consumption rate (OCR) was measured using the Seahorse technology. Each dot represents the value of one replicate from a representative experiment. (B) HEK-293 cells stably expressing the FcγRII receptor to efficiently internalize IgG-opsonized L. pneumophila were labeled with Hoechst to identify the cell nucleus (Nuc, blue) and tetramethylrhodamine methyl ester (TMRM) (red) to quantify Δψ_m. Addition of medium (no changes) or FCCP (complete depolarization) to noninfected (NI) HEK-293 cells was used as controls. Representative confocal images at 5 min before the addition of medium (top) or FCCP (bottom), and at 50 min after addition of medium or FCCP. Bar: 20 μm. (C) Quantification of (B) before (baseline) and after the addition of medium/FCCP (dotted line). Each dot represents mean ± SD of three independent experiments. (D) HEK-293 cells were infected with GFP-expressing bacteria (WT, ΔdotA mutant) opsonized using an anti-flagellin antibody, or left uninfected (noninfected). At 6 hpi, dicyclohexylcarbodiimide (DCCD) was added to the cells and changes Δψ_m were monitored. (E) Representative confocal images at 5 min before the addition of DCCD and at 50 min after addition of DCCD. Green: GFP-expressing bacteria. Bar: 20 μm. (F) Same as (D) but infection was performed with opsonized Lpp-WT, Lpp-ΔdotA, or Lpp-Δspl strains. TMRM values (SD/mean) at 50 min after DCCD addition are shown. Data from three experiments per strain with six or more replicates per strain. *p<0.1; **p<0.01; ***p<0.001; ns, nonsignificant (Mann–Whitney U test).