The antibiotic bedaquiline activates host macrophage innate immune resistance to bacterial infection

Antibiotics are widely used in the treatment of bacterial infections. Although known for their microbicidal activity, antibiotics may also interfere with the host’s immune system. Here, we analyzed the effects of bedaquiline (BDQ), an inhibitor of the mycobacterial ATP synthase, on human macrophages. Genome-wide gene expression analysis revealed that BDQ reprogramed cells into potent bactericidal phagocytes. We found that 579 and 1,495 genes were respectively differentially expressed in naive- and M. tuberculosis-infected macrophages incubated with the drug, with an over-representation of lysosome-associated genes. BDQ treatment triggered a variety of antimicrobial defense mechanisms, including phagosome-lysosome fusion, and autophagy. These effects were associated with activation of transcription factor EB, involved in the transcription of lysosomal genes, resulting in enhanced intracellular killing of different bacterial species that were naturally insensitive to BDQ. Thus, BDQ could be used as a host-directed therapy against a wide range of bacterial infections.


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We assessed phospholipid metabolism, a pathway also identified in our ClueGO cluster 122 analysis ( Figure 1B). Like glycolysis, lipid metabolism affects macrophage phenotype and 123 function (Remmerie and Scott, 2018). We analyzed the lipid profile of BDQ-treated cells using 124 MALDI-TOF mass spectrometry. We observed an increase of phosphatidylinositols upon 125 incubation with BDQ ( Figure 1E and figure supplement 3E). No significant changes were 126 observed in the levels of phosphatidylethanolamines, phosphatidylglycerols, or cardiolipins.

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Taken together, these data show that BDQ induced a significant metabolic reprogramming of

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To validate our transcriptomic data, we incubated BDQ-treated, BDQr-MTB-infected cells with 146 LysoTracker Red DND-99, a red fluorescent probe that labels acidic organelles, and analyzed 147 them using flow cytometry. No differences were observed between control and treatment after 148 3 h of BDQ treatment ( Figure 2B). However, at 18 h and 48 h post-treatment, fluorescence 149 intensity was substantially increased in Mφs incubated with BDQ compared to DMSO-treated 150 cells (1.7 and 5.4 times more, respectively). These results were supported by confocal 151 microscopy, which revealed the appearance of numerous acidic compartments upon 152 treatment ( Figure 2C), up to 5 times more in BDQ-treated Mφs than untreated cells at 48 h 153 post-treatment (p < 0.001, Figure 2D). We also observed a large number of MTB 154 phagosomes co-localized with LysoTracker-positive compartments ( Figure 2E).

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As the expression of many genes coding for hydrolases was up-regulated upon BDQ non-fluorescent probe that produces brightly fluorescent peptides following hydrolysis by 160 lysosomal proteases. At 18 h and 48 h post-treatment, we observed a dose-dependent 161 increase in fluorescence intensity upon treatment with BDQ (up to 5.5 times more than 162 untreated cells, p < 0.01, Figure 2F). Together, these data demonstrate that BDQ induces 163 biogenesis of competent lysosomes.

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The capacity of BDQ to induce acidic compartments may potentiate the efficacy of other 172 drugs, whose activity is pH dependent. In vivo studies have suggested a synergistic 173 interaction between BDQ and PZA (Ibrahim et al., 2007), and it is commonly assumed that a 174 low pH is required for PZA activity against MTB (Zhang and Mitchison, 2003). We thus 175 infected Mφs with BDQr-MTB and treated them with BDQ and PZA. After 7 days of treatment, 176 cells were lysed and bacteria counted. PZA showed moderate bactericidal activity, with 50 177 µg/mL PZA resulting in a 36% decrease in bacterial numbers compared to untreated cells 178 ( Figure 2H). We confirmed that the combination of PZA with BDQ was highly bactericidal on 179 MTB, leading to a 83% decrease in colony forming units using 50 µg/mL PZA. This decrease 180 was not a result of an additive effect between the two drugs, as BDQ alone had no 181 antibacterial activity, given that we used a drug-resistant strain of MTB. Thus, the potentiation      it has been suggested that BDQ inhibits the cardiac potassium channel protein encoded by 241 the human ether-a-go-go-related gene (hERG) (Therapeutics, 2012). Therefore, to further 242 understand the molecular mechanisms underpinning Mφ activation by BDQ we determined if 243 human monocyte-derived Mφs expressed hERG, but were unable to detect hERG RNA by

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We thus stained for mitochondrial superoxide using the MitoSOX dye in BDQ-stimulated cells.

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Again, we saw no difference upon antibiotic treatment ( Figure 5D). Incubation with the 266 antioxidant glutathione (GSH) or with its precursor N-Acetyl cysteine (NAC), which prevent 267 the formation of mitochondrial ROS and reactive nitrogen species (RNS), did not prevent 268 lysosome activation and the killing of S. aureus by BDQ ( Figure 5E). Based on these results,

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it is unlikely that BDQ alters mitochondrial function in human Mφs.

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At neutral pH, they passively diffuse across cell and organelle membranes but when they

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Our results clearly show that BDQ can bypass these escape mechanisms and allow more

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The analysis was performed using the FlowJo software.  Numbers correspond to mass-to-charge ratio (m/z). Cells derived from 3 donors were analyzed. Error bars represent the mean ± SD and significant differences between treatments are indicated by an asterisk, in which * p < 0.05, ** p < 0.01, *** p < 0.001.  Figure 5. Basal respiration, ATP production, maximal respiration, respiratory reserve and nonmitochondrial respiration were followed by sequential additions of oligomycin (OM, an inhibitor of the ATPase), the mitochondrial oxidative phosphorylation uncoupler FCCP, and the inhibitors of electron transport antimycin A/rotenone (Rot/AA). Error bars represent the mean ± SD of 3 technical replicates. One representative experiment (out of two) is shown.