The antibiotic bedaquiline activates host macrophage innate immune resistance to bacterial infection
Antibiotics are commonly used in the treatment of bacterial infections , and , in effectively combating such diseases , have substantially increased human life expectancy . As with most drugs , antibiotic treatment can also alter host metabolism , leading to adverse side-effects , including nausea , hepatotoxicity , skin reactions , and gastrointestinal and neurological disorders . Such side-effects can become critical when antibiotic treatment is long and involves several drugs , as in the treatment of tuberculosis ( TB ) , where 2–28% of patients develop mild liver injury during treatment with first-line drugs ( Agal et al . , 2005 ) . Antibiotics can interfere with the immune system , indirectly through the disturbance of the body’s microbiota ( Ubeda and Pamer , 2012 ) , or directly by modulating the functions of immune cells . Such interactions may impact treatment efficacy or the susceptibility of the host to concomitant infection . For example , after treatment completion , TB patients are more vulnerable to reactivation and reinfection of the disease , suggesting therapy-related immune impairment ( Cox et al . , 2008 ) . Drug-sensitive TB can be cured by combining up to four antibiotics in a 6-month treatment; specifically , isoniazid ( INH ) , rifampicin ( RIF ) , ethambutol ( EMB ) and pyrazinamide ( PZA ) for 2 months , and INH and RIF for additional 4 months . INH induces apoptosis of activated CD4+ T cells in Mycobacterium tuberculosis ( MTB ) -infected mice ( Tousif et al . , 2014 ) and leads to a decrease in Th1 cytokine production in household contacts with latent TB under preventive INH therapy ( Biraro et al . , 2015 ) . RIF has immunomodulatory properties and acts as a mild immunosuppressive agent in psoriasis ( Tsankov and Grozdev , 2011 ) . RIF reduces inflammation by inhibiting IκBα degradation , mitogen-activated protein kinase ( MAPK ) phosphorylation ( Bi et al . , 2011 ) , and Toll-like receptor 4 signaling ( Wang et al . , 2013 ) . PZA treatment of MTB-infected human monocytes and mice significantly reduces the release of pro-inflammatory cytokines and chemokines ( Manca et al . , 2013 ) . Recently , Puyskens et al . showed that several anti-TB drugs bind to the aryl hydrocarbon receptor and may impact host defense ( Puyskens et al . , 2020 ) . It is therefore necessary to understand how antibiotic treatment modulates macrophage functions , and more generally , how it impacts the host immune response . The worldwide rise in antibiotic resistance is a major threat to global health care . A growing number of bacterial infections , such as pneumonia , salmonellosis , and TB , are becoming harder to treat as the antibiotics used to treat them become less effective . While new antibiotics are being developed and brought to the clinic , their effects on the human immune system are not being studied in-depth . Here , we have investigated the impact of a recently approved anti-TB drug , bedaquiline ( BDQ ) , on the transcriptional responses of human macrophages infected with MTB . Macrophages are the primary cell target of MTB , which has evolved several strategies to survive and multiply inside the macrophage phagosome , including prevention of phagosome acidification ( Sturgill-Koszycki et al . , 1994 ) , inhibition of phagolysosomal fusion ( Armstrong and Hart , 1975 ) and phagosomal rupture ( Simeone et al . , 2012; van der Wel et al . , 2007 ) . They play a central role in the host response to TB pathogenesis , by orchestrating the formation of granulomas , presenting mycobacterial antigens to T cells , and killing the bacillus upon IFN-γ activation ( Cambier et al . , 2014 ) . BDQ is a diarylquinoline that specifically inhibits a subunit of the bacterial adenosine triphosphate ( ATP ) synthase , decreasing intracellular ATP levels ( Andries et al . , 2005; Koul et al . , 2007 ) . It has 20 , 000 times less affinity for human ATP synthase ( Haagsma et al . , 2009 ) . The most common side effects of BDQ are nausea , joint and chest pain , headache , and arrhythmias ( Diacon et al . , 2012; TMC207-C208 Study Group et al . , 2014 ) . However , possible interactions between BDQ and the host immune response have not been studied in detail . Understanding the impact of BDQ on the host immune response may help to develop strategies aiming at improving drug efficacy and limiting side-effects , including cytotoxicity , alteration of cell metabolism , and immunomodulation .
We treated human monocyte-derived macrophages from four healthy donors with BDQ at 5 µg/mL , which corresponds to the concentration detected in the plasma of TB patients treated with BDQ ( Andries et al . , 2005 ) . This concentration did not affect cell viability over an incubation period of 7 days ( Figure 1—figure supplement 1 ) . After 18 hr of treatment , we characterized the genome-wide gene expression profiles of BDQ-treated macrophages by RNAseq , with DMSO-treated cells serving as a control . The expression of 579 genes was affected by BDQ ( FDR < 0 . 05 , Figure 1—source datas 1 and 2 ) , with 186 being upregulated and 393 being downregulated . We classified all 579 genes by performing gene-set enrichment analysis using ClueGO cluster analysis ( Bindea et al . , 2009 ) . The gene set upregulated by BDQ was significantly enriched for genes associated with lysosome , phagocytic vesicle membrane , vacuolar lumen , hydrolase activity and lipid homeostasis ( Figure 1A ) . We next evaluated if BDQ could modulate gene expression in MTB-infected cells . In order to exclude potential differences due to the MTB bacillary load between treated and untreated cells , we generated a virulent BDQ-resistant strain of M . tuberculosis ( BDQr-MTB ) . The selected clone , which carried a Ala63→Pro mutation in subunit c of the ATP synthase ( Andries et al . , 2005; Figure 1—figure supplement 2A ) , had a similar generation time to wild-type bacteria when cultured in 7H9 liquid medium , although we observed a slower growth after 7 days of treatment . ( Figure 1—figure supplement 2B ) . We also noted no difference in virulence or in intracellular growth of both wild-type- and BDQ-resistant MTB ( Figure 1—figure supplement 2C-E ) . As expected , the MIC99 ( defined as the concentration required to prevent 99% growth ) for susceptible MTB was 0 . 07 µg/mL , a value similar to previously published study ( Andries et al . , 2005 ) , while the MIC99 of the BDQr-MTB was 36 µg/mL . We infected macrophages with BDQr-MTB . After 24 hr of infection , cells were incubated for an additional 18 hr with BDQ ( 5 µg/mL ) . The bacillary load of resistant MTB inside macrophages was the same in untreated cells as in cells after 18 hr of BDQ treatment . In contrast , in the same experiment using BDQ-susceptible MTB , there was a 70% decrease in the bacillary load ( Figure 1—figure supplement 2C ) . Following treatment , we characterized the genome-wide gene expression profiles of MTB-infected macrophages , as described above . The expression of 1 , 495 genes was affected by BDQ ( FDR < 0 . 05 , Figure 1A , Figure 1—source datas 3 and 4 ) , with 499 being upregulated and 996 being downregulated . More genes were thus affected by BDQ treatment in MTB-infected cells than in naive macrophages . This probably reflects the fact that MTB infection induces an extensive remodeling of the transcriptome ( Barreiro et al . , 2012; Tailleux et al . , 2008 ) . The genes differentially expressed by BDQ only in MTB-infected macrophages are enriched in genes related to assembly of the endoplasmic reticulum-Golgi intermediate compartment , membrane raft and cellular protein metabolic process ( Table 1 , Figure 1—source datas 5 and 6 ) . This probably reflects the cell adaptation to infection . Functional annotation of the gene set upregulated by BDQ also revealed that similar pathways were affected by BDQ in naive and BDQr-MTB-infected macrophages , with an enrichment for genes associated with glucose/phospholipid metabolism and lysosome ( Figure 1B , Figure 1—source datas 3 and 4 ) . 452 genes were differentially expressed in both naive and MTB-infected cells upon BDQ treatment with an over-representation of lysosome-associated genes ( Figure 1C–D , Figure 1—source data 7 ) . As metabolic pathways were over-represented in our RNAseq analysis , we investigated if glycolysis is affected by BDQ treatment using the Seahorse Extracellular Flux analyzer . This assay measures the rate of proton accumulation in the extracellular medium during glycolysis ( glycoPER ) and can discriminate between basal glycolysis , induced glycolytic capacity ( by addition of rotenone/antimycin A ( Rot/AA ) , an inhibitor of the mitochondrial electron transport chain ) , and non-glycolytic acidification ( by addition of the glycolytic inhibitor 2-deoxy-D-glycose ( 2-DG ) ) . After incubation with BDQ , we observed a 30% decrease in basal glycolysis and glycolytic capacity compared to untreated cells ( Figure 2A–B , Figure 2—figure supplement 1A–B ) . We assessed phospholipid metabolism , a pathway also identified in our ClueGO cluster analysis ( Figure 1B ) . Like glycolysis , lipid metabolism affects macrophage phenotype and function ( Remmerie and Scott , 2018 ) . We analyzed the lipid profile of BDQ-treated cells using MALDI-TOF mass spectrometry . We observed an increase of phosphatidylinositols upon incubation with BDQ ( Figure 2C , Figure 2—figure supplement 1C ) . No significant changes were observed in the levels of phosphatidylethanolamines , phosphatidylglycerols , or cardiolipins . Taken together , these data show that BDQ induced a significant metabolic reprogramming of both MTB-infected and resting macrophages . Macrophages are involved in innate immunity and tissue homeostasis through their detection and elimination of microbes , debris , and dead cells , which occurs in lysosomes ( Wynn et al . , 2013 ) . Lysosomes are acidic and hydrolytic organelles responsible for the digestion of macromolecules . Recent work has shown that they are also signaling platforms , which respond to nutrient and cellular stress ( Lawrence and Zoncu , 2019 ) . Functional annotations based on the GO database of the differentially expressed genes suggested a substantial impact of BDQ treatment on lysosome function ( Figure 1A–B ) . We identified 38 and 54 differentially expressed genes by BDQ , respectively in naïve- and BDQr-MTB infected cells ( FDR < 0 . 05 , Figure 3A ) . These genes are involved in lysosome biogenesis , transport and degradation of small molecules , and lysosomal acidification . They included genes coding for components of vacuolar ATPase ( V-ATPase ) , hydrolases , and SLC11A1 ( NRAMP1 ) , a divalent transition metal transporter involved in host resistance to pathogens , including MTB ( Meilang et al . , 2012 ) . To validate our transcriptomic data , we incubated BDQ-treated , BDQr-MTB-infected cells with LysoTracker Red DND-99 , a red fluorescent probe that labels acidic organelles , and analyzed them using flow cytometry . No differences were observed between control and treatment after 3 hr of BDQ treatment ( Figure 3B ) . However , at 18 hr and 48 hr post-treatment , fluorescence intensity was substantially increased in macrophages incubated with BDQ compared to DMSO-treated cells ( 1 . 7 and 5 . 4 times more , respectively ) . These results were supported by confocal microscopy , which revealed the appearance of numerous acidic compartments upon treatment ( Figure 3C ) , up to five times more in BDQ-treated macrophages than untreated cells at 48 hr post-treatment ( p<0 . 001 , Figure 3D ) . We also observed a large number of MTB phagosomes co-localized with LysoTracker-positive compartments ( Figure 3E ) . As the expression of many genes coding for hydrolases was upregulated upon BDQ treatment ( Figure 3A ) , we tested the effect of the drug on late endosomal/lysosomal proteolytic activity . BDQ-treated macrophages were incubated with DQ-Green BSA , a self-quenched non-fluorescent probe that produces brightly fluorescent peptides following hydrolysis by lysosomal proteases . At 18 hr and 48 hr post-treatment , we observed a dose-dependent increase in fluorescence intensity upon treatment with BDQ ( up to 5 . 5 times more than untreated cells , p<0 . 01 , Figure 3F ) . Similar results were obtained when we incubated naive macrophages with BDQ ( Figure 3—figure supplement 1 ) . Together , these data demonstrate that BDQ induces biogenesis of competent lysosomes . We performed additional experiments to confirm that the main effects of BDQ on lysosome were independent of infection with live bacteria . Briefly , cells were untreated or stimulated with LPS ( TLR4 agonist ) , Pam3CSK4 ( TLR1/2 agonist ) , heat-killed bacteria ( hk-MTB ) , drug-susceptible MTB or BDQr-MTB , and treated with BDQ . After 18 hr , RNA was collected and we performed RT-qPCR on a panel of lysosomal genes . We also analyzed the intensity of the LysoTracker staining using flow cytometry ( Figure 3—figure supplement 2 ) . Our results clearly show that the main effects on lysosome biogenesis/activation occurred with BDQ treatment and were not exclusively seen after infection with live MTB . The capacity of BDQ to induce acidic compartments may potentiate the efficacy of other drugs , whose activity is pH dependent . In vivo studies have suggested a synergistic interaction between BDQ and PZA ( Ibrahim et al . , 2007 ) , and it is commonly assumed that a low pH is required for PZA activity against MTB ( Zhang and Mitchison , 2003 ) . We thus infected macrophages with BDQr-MTB and treated them with BDQ and PZA . After 7 days of treatment , cells were lysed and bacteria counted . PZA showed moderate bactericidal activity , with 50 µg/mL PZA resulting in a 36% decrease in bacterial numbers compared to untreated cells ( Figure 4A ) . We confirmed that the combination of PZA with BDQ was highly bactericidal on MTB , leading to a 83% decrease in colony forming units using 50 µg/mL PZA . This decrease was not a result of an additive effect between the two drugs , as BDQ alone at 1 µg/mL had no antibacterial activity . We also found no synergy between BDQ and PZA on the BDQ-resistant mutant cultivated in Middlebrook 7H9 liquid medium ( Figure 4B ) . Thus , the potentiation of PZA activity by BDQ is most likely due to the effect of BDQ on the host cell , and in particular on the increase of lysosomal acidification . We next tested whether BDQ would synergize with the other first-line anti-TB drugs in liquid culture or in BDQr-MTB-infected macrophages . We found that BDQ did not potentiate the activity of ethambutol ( EMB ) , isoniazid ( INH ) and rifampicin ( RIF ) in either case . While we cannot exclude the possibility that BDQ may have additive effects with other anti-TB drugs , as has been described ( Ibrahim et al . , 2007 ) , we saw no evidence of synergism with any of the first-line agents ( Figure 4—figure supplement 1 ) . Bacterial hydroxyl radical released after treatment with INH or PZA can directly induce host cell autophagy ( Kim et al . , 2012 ) . We thus tested whether other antibiotics might have similar effects to BDQ . We characterize the genome-wide gene expression profiles of naïve macrophages and macrophages stimulated with hk-MTB , and treated with amikacin ( AMK ) , EMB , INH , PZA or RIF for 18 hr . We chose drug concentrations based on the concentrations detected in the plasma of treated TB patients . Following treatment , only RIF and PZA significantly modulate gene expression in macrophages . 556 and 752 genes were differentially expressed in cells stimulated with heat-killed bacteria and exposed to RIF and PZA , respectively ( Figure 5A , Figure 5—figure supplement 1 , Figure 5—source datas 1–5 ) . We classified these genes by performing gene-set enrichment analysis and confirmed that the lysosomal pathway was not induced upon RIF or PZA treatment ( Table 2 ) . The expression of only two genes belonging to this pathway was upregulated by RIF , and only one by PZA , compared to 46 whose expression was modulated by BDQ ( Figure 5B–C ) . Consistent with these results , none of these antibiotics were able to increase LysoTracker staining ( Figure 5D ) . Given BDQ’s effect on lysosomal acidification we asked whether it promoted lysosome formation . Lysosome biogenesis is linked to the endocytic and autophagic pathways . Autophagy delivers cytoplasmic material and organelles for lysosomal degradation and is implicated in the immune response to microbes ( Germic et al . , 2019 ) . We therefore tested three inhibitors of the autophagy pathway on BDQ activity: bafilomycin ( BAF ) , which inhibits the V-ATPase; chloroquine ( CQ ) , a lysomotropic agent which prevents endosomal acidification and impairs autophagosome fusion with lysosomes; and 3-methyladenine ( 3-MA ) which blocks autophagosome formation by inhibiting of the type III phosphatidylinositol 3-kinases ( PI-3K ) . We infected macrophages with BDQr-MTB and incubated the cells with BDQ in the presence or absence of the different inhibitors . These molecules were not toxic at the concentrations tested ( Figure 1—figure supplement 1B ) . After 2 days , we analyzed LysoTracker staining as a read-out of lysosome activation using flow cytometry and observed that all three inhibitors prevented the increase in staining upon BDQ treatment ( Figure 6A ) . Microtubule-associated protein light-chain 3B ( LC3B ) is involved in the formation of autophagosomes and autolysosomes . We observed an increase of LC3B puncta per cell at 18 hr and 48 hr post-BDQ treatment using confocal microscopy ( Figure 6B–C ) , which was associated with the detection of lipidated LC3 ( LC3-II ) , the form of LC3 recruited to autophagosomal membranes , and with a decrease in sequestosome 1 ( SQSTM1 ) or p62 levels ( Figure 6D–E ) . p62 is a ubiquitin-binding scaffold protein , which is degraded upon autophagy induction , and which is used as a marker of autophagic flux ( Liu et al . , 2016 ) . Given we have previously observed that some mycobacterial phagosomes colocalized with lysosomes in BDQ-treated cells ( Figure 3E ) , we tested whether BDQ promotes MTB killing , independently of its bactericidal activity on MTB by autophagy . BDQ significantly reduced the number of bacteria ( measured by CFU ) in cells infected with BDQ-resistant MTB . This effect was completely inhibited by the autophagy inhibitors 3-MA and BAF ( Figure 6F ) . Overall , these data show BDQ activates the autophagy pathway in human macrophages and this is involved in its anti-TB activity . Autophagy plays numerous roles in innate immunity and in host defenses against intracellular pathogens , including MTB ( Gutierrez et al . , 2004 ) . We thus asked if BDQ conferred protection to bacterial infections naturally resistant to BDQ . To test this hypothesis , we infected macrophages with two different bacterial species: a gram-positive bacterium , Staphylococcus aureus and a gram-negative bacterium , Salmonella Typhimurium . We confirmed that these two species are resistant to BDQ , even when exposed to high concentration of the drug ( 20 µg/mL , Figure 7A ) . However , when macrophages were incubated with BDQ and then infected with S . aureus and S . Typhimurium for 24 hr , we observed a substantial decrease in bacterial survival rates ( Figure 7B ) . To determine if autophagy is involved in this antibacterial activity , we incubated the infected cells with the autophagy inhibitor , 3-MA , and were unable to revert the macrophage resistance to S . aureus infection upon BDQ treatment ( Figure 7C ) . Macrophages are professional phagocytes , which have evolved a myriad of defense strategies to contain and eradicate bacteria , such as radical formation , phagosome maturation , and metal accumulation ( Weiss and Schaible , 2015 ) . Upon incubation with BDQ , we detected an increase in the amount of NO2- , a stable derivative of NO , in the culture supernatant of macrophages ( Figure 7D ) . When the cells were treated with N ( G ) -nitro-L-arginine methyl ester ( L-NAME ) , an inhibitor of nitric oxide ( NO ) synthesis , S . aureus-infected cells were unable to effectively control infection upon incubation with BDQ ( Figure 7E ) . Thus , our results suggest that BDQ confers innate resistance to bacterial infection through different mechanisms . BDQ affects cardiac electrophysiology by prolonging the QT interval ( TMC207-C208 Study Group et al . , 2014 ) and it has been suggested that BDQ inhibits the cardiac potassium channel protein encoded by the human ether-a-go-go-related gene ( hERG ) ( Janssen Therapeutics , 2012 ) . Therefore , to further understand the molecular mechanisms underpinning macrophage activation by BDQ , we determined if human monocyte-derived macrophages expressed hERG , but were unable to detect hERG RNA by RT-qPCR ( Figure 8—figure supplement 1 ) . We investigated if BDQ might interfere with other activities of mitochondria . Conflicting reports suggest that BDQ inhibits the mitochondrial ATPase ( Fiorillo et al . , 2016; Haagsma et al . , 2009 ) . We have already shown that there were no significant differences in the amount of cardiolipin , a constituent of inner mitochondrial membranes , between BDQ-treated cells and control cells ( Figure 2C ) . We quantified changes in mitochondrial membrane potential using flow cytometry in cells incubated with BDQ or with oligomycin , a positive control , which hyperpolarizes the mitochondrial membrane potential , and stained with TMRM . TMRM is a fluorescent cell-permeant dye that accumulates in active mitochondria with intact membrane potentials . No changes were observed when macrophages were incubated with the BDQ for 6 , 24 and 48 hr ( Figure 8A ) . We obtained similar results when mitochondria were stained with MitoTracker Red FM whose accumulation in mitochondria is dependent upon membrane potential ( Figure 8B ) . We also measured the oxygen consumption rate ( OCR ) , and detected no change in basal respiration , ATP-linked respiration , maximal respiration , and non-mitochondrial respiration in cells treated with BDQ for 24 hr and 48 hr as compared to untreated cells ( Figure 8C , Figure 8—figure supplement 2 ) . Mitochondrial reactive oxygen species ( ROS ) are involved in the regulation of several physiological and pathological processes , including autophagy ( Sena and Chandel , 2012 ) . We thus stained for mitochondrial superoxide using the MitoSOX dye in BDQ-stimulated cells . Again , we saw no difference upon antibiotic treatment ( Figure 8D ) . Incubation with the antioxidant glutathione ( GSH ) or with its precursor N-Acetyl cysteine ( NAC ) , which prevent the formation of mitochondrial ROS and reactive nitrogen species ( RNS ) , did not prevent lysosome activation and the killing of S . aureus by BDQ ( Figure 8E ) . Based on these results , it is unlikely that BDQ alters mitochondrial function in human macrophages . Given that BDQ induced a lysosomal gene expression signature in macrophages , we wondered whether BDQ could activate the basic helix-loop-helix transcription factor EB ( TFEB ) . TFEB is a master regulator of autophagy and lysosome biogenesis ( Settembre et al . , 2011 ) . In resting cells , TFEB is largely cytosolic and inactive , but upon activation , it translocates into the nucleus and activates the transcription of many autophagy and lysosomal genes ( Settembre et al . , 2011 ) . We therefore analyzed the cellular localization of TFEB , using confocal microscopy . At 18 hr post-treatment , TFEB was mainly localized in the nucleus of BDQ-treated cells ( Figure 9A–B ) . The activity of TFEB is regulated by phosphorylation on specific amino acid residues , and its activation is mediated by calcineurin , an endogenous serine/threonine phosphatase , through Ca2+ release from the lysosome ( Medina et al . , 2015 ) . In agreement with these studies , we observed an increase in intracellular Ca2+ concentration in macrophages treated for 18 hr with BDQ ( Figure 9C ) , and confirmed that this intracellular calcium accumulation was required for antibiotic-induced TFEB translocation to the nucleus and lysosomal gene expression . Upon treatment with BAPTA-AM , a cell permeable Ca2+ chelator , TFEB remained localized in the cytoplasm of BDQ-treated cells ( Figure 9D ) , and we were unable to detect changes in the expression of a panel of lysosomal genes , previously identified as differentially expressed in macrophages incubated with BDQ ( Figure 9E ) . The increased bactericidal activity against S . aureus was also abrogated in the presence of BAPTA-AM ( Figure 9F ) . To study the role of TFEB in the enhancement of bactericidal activity upon BDQ treatment in more depth , we inactivated TFEB expression in human macrophages using siRNA-mediated gene silencing ( Troegeler et al . , 2014 ) . The level of TFEB expression was decreased by about 85% upon silencing ( Figure 9—figure supplement 1A ) . After treating the cells with BDQ , we analyzed the intensity of the LysoTracker staining using flow cytometry and performed RT-qPCR on a panel of lysosomal genes . In TFEB-silenced cells , BDQ increases slightly the LysoTracker staining ( Figure 9G ) , and the expression of atp6v0a1 , mcoln1 , scarb1 or trpm2 ( Figure 9H ) . We also infected macrophages with S . aureus and MTB , and showed that that TFEB activation is required for better control of both pathogens upon BDQ treatment ( Figure 9I–J ) . We next evaluated the recruitment of TFEB to the promoters of some lysosomal genes . As expected , BDQ induces the recruitment of TFEB to these promoters ( Figure 9—figure supplement 1B ) . Collectively , our data indicate that BDQ activates TFEB in macrophages and in this way modulates innate immune resistance to bacterial infection .
The emergence of bacterial strains resistant to antibiotics requires the constant development of new antibiotics , which , beyond their bactericidal activity , may have a significant impact on cellular functions . Here , we have analyzed the effects of the new anti-TB drug BDQ on human macrophages . We found that in addition to its antibacterial activity , BDQ induces cell reprogramming , increasing macrophage bactericidal activity . Gene expression profiling revealed that 1495 genes were differentially expressed in MTB-infected macrophages incubated with BDQ , with over-representation of genes involved in metabolism , lysosome biogenesis , and acidification . Recent work has highlighted the role of metabolic reprogramming in controlling immunological effector functions , emphasizing the close connection between cell function and metabolism ( Wang et al . , 2019 ) . In agreement with these results , we observed a substantial increase in both the number of acidic compartments and proteolytic activity of macrophages upon BDQ treatment . BDQ is a cationic amphiphilic drug , consisting of a hydrophobic ring structure and a hydrophilic side chain with a charged cationic amine group ( Diacon et al . , 2012 ) . Cationic amphiphilic drugs can accumulate in lysosomes through ion trapping ( de Duve et al . , 1974 ) . At neutral pH , they passively diffuse across cell and organelle membranes but when they enter the luminal space of acidic compartments such as lysosomes , the amine group ionizes and becomes membrane-impermeable ( MacIntyre and Cutler , 1988 ) . Such lysosomotropic compounds usually increase the lysosomal pH and thus decrease lysosomal enzyme activity ( Kazmi et al . , 2013 ) . However , our results reveal instead that BDQ triggers lysosomal activation , upregulating the expression of genes coding for hydrolases and for subunits of the lysosomal proton pump v-ATPase . Consistent with these observations , we observed that BDQ-treated cells significantly increase their ability to degrade DQ-BSA . Pre-clinical studies have shown that BDQ may induce phospholipidosis , potentially explaining some of the drug’s observed toxicities ( Diacon et al . , 2012 ) . Phospholipidosis , which is characterized by the accumulation of phospholipids in lysosomes , resulting in impaired lysosome function , is common upon treatment with cationic amphiphilic compounds ( Shayman and Abe , 2013 ) . Various phospholipid species have been described including phosphatidylcholine , phosphatidylethanolamine , phosphatidylserine , and lysobisphosphatidic acid ( Reasor , 1984; Yamamoto et al . , 1971a; Yamamoto et al . , 1971b; Yoshikawa , 1991 ) . In BDQ-treated macrophages , we only observed an increase in the amount of phosphatidylinositol and phosphatidylinositol-4-phosphate . The quantity of cardiolipin , phosphatidylethanolamine , and phosphatidylglycerol remained unchanged upon treatment . These observations do not indicate lysosomal dysfunction , but rather a targeted regulation of certain phospholipids by BDQ . In accordance with this idea , 28 genes involved in phospholipid metabolism were differentially expressed in BDQ-treated macrophages . Phosphatidylinositol phosphates regulate many cellular functions , including endosomal trafficking , endoplasmic reticulum ( ER ) export , autophagy , and phagosome-lysosome fusion ( De Matteis et al . , 2013; Levin et al . , 2017 ) . These phospholipids may thus be involved in the increase of autophagy and mycobacterial phagosome-lysosome fusion upon BDQ treatment . Lysosomes are both digestive organelles of the endocytic and autophagic pathways and signaling hubs involved in nutrient sensing , cell growth and differentiation , transcriptional regulation , and metabolic homeostasis ( Lamming and Bar-Peled , 2019; Lawrence and Zoncu , 2019 ) . In response to nutrients and growth factors , the mechanistic target of the rapamycin complex 1 ( mTORC1 ) is recruited and activated at the lysosomal surface , where it promotes ribosomal biogenesis , translation , and biosynthesis of lipids ( Lamming and Bar-Peled , 2019; Lawrence and Zoncu , 2019 ) . mTORC1 binds to and phosphorylates TFEB , resulting in its cytosolic sequestration ( Roczniak-Ferguson et al . , 2012; Settembre et al . , 2012 ) . Upon starvation or lysosomal stress , mTORC1 is released from the lysosomal membrane and becomes inactive ( Lamming and Bar-Peled , 2019; Lawrence and Zoncu , 2019 ) . The release of lysosomal Ca2+ activates the phosphatase calcineurin , which de-phosphorylates TFEB and promotes its nuclear translocation ( Medina et al . , 2015 ) . TFEB then binds to CLEAR ( coordinated lysosomal expression and regulation ) elements within the promoters of genes involved in autophagy and lysosomal biogenesis and activates their expression ( Lamming and Bar-Peled , 2019; Lawrence and Zoncu , 2019 ) . We found that TFEB translocates from the cytoplasm to the nucleus in BDQ-treated cells , with the concomitant up-regulation of 85 genes containing CLEAR elements 18 hr after incubation with the drug . Finding the direct host-cell target of BDQ would have been a major breakthrough , but unfortunately , we were unable to identify the target . However , it is possible that BDQ could modulate the host response by interfering with ion homeostasis inside the lysosome . Greenwood et al . recently showed that BDQ accumulated primarily in host cell lipid droplets ( Greenwood et al . , 2019 ) . It is tempting to speculate that these droplets are actually lysosomes . Due to its high hydrophobicity , BDQ might accumulate in lysosomal membranes and thereby change the transmembrane ion permeability . In agreement with this hypothesis , it has been shown that BDQ can accumulate at the lipid membrane of liposomes and act as a H+/K+ ionophore ( Hards et al . , 2018 ) . The ensuing lysosomal stress could then facilitate the dissociation of mTOR from the lysosomal membrane ( Plescher et al . , 2015 ) and the activation of TFEB . Interestingly , BDQ increases the expression of mucolipin 1 gene ( mcoln1 , Figure 3A , Figure 3—figure supplement 2A ) , which in turn can activate TFEB . Medina et al . showed indeed that lysosomal Ca2+ is released through MCOLN1 and activates calcineurin , which binds and dephosphorylates TFEB ( Medina et al . , 2015 ) . A striking feature of BDQ-treated macrophages is their capacity to control pathogenic bacterial infection . BDQ enhances macrophage innate defense mechanisms , including induction of antimicrobial effectors such as nitric oxide , phagosome-lysosome fusion , and autophagy . Other anti-TB drugs have been described to regulate autophagy . INH and PZA promote autophagy activation and phagosomal maturation in MTB-infected murine macrophages ( Kim et al . , 2012 ) . This process was dependent of bacterial factors and was suggested to be essential for antimycobacterial drug action and for dampening proinflammatory cytokines ( Kim et al . , 2012 ) . In our system , we did not detect increased autophagy in cells treated with INH , which may also be due to differences in the autophagy response in murine and human macrophages . Altogether , we demonstrate that BDQ is able to boost the innate defenses of human cells . A growing number of pathogenic bacteria are becoming resistant to antibiotics , making their use less effective . In addition to the development of ‘classical’ drugs targeting key factors in bacterial physiology , host-directed therapy ( HDT ) has emerged as approach that could be used in adjunct with existing or future antibiotics ( Machelart et al . , 2017 ) . For example , metformin , an FDA-approved drug for type II diabetes , increases the production of mitochondrial reactive oxygen species and stimulates phagosome-lysosome fusion by activating the 5′-adenosine monophosphate-activated protein kinase ( AMPK ) ( Singhal et al . , 2014 ) , and recent studies suggest that metformin provides better outcomes in TB patients , especially those with diabetes mellitus ( Yew et al . , 2019 ) . Pathogens manipulate host-signaling pathways to subvert innate and adaptive immunity . It might thus be possible to reprogram the host immune system to better control or even kill bacteria . For instance , MTB has developed several strategies to counteract autophagy , including the product of the enhanced intracellular survival ( Eis ) gene , which limits ROS generation ( Shin et al . , 2010 ) . Our results clearly show that BDQ can bypass these escape mechanisms and allow more effective control of bacterial infection . We also showed that BDQ potentiates the activity of other anti-TB drugs , independently of its bactericidal activity on MTB . Hence , our work opens new avenues for downstream evaluation of the potential use of BDQ as a potent drug in HDT .
Buffy coats were obtained from healthy donors after informed consent . The blood collection protocols were approved by both the French Ministry of Research and a French Ethics Committee . The blood collection was carried out in accordance with these approved protocols by the Etablissement Français du Sang ( EFS , n°12/EFS/134 ) . Blood mononuclear cells were isolated from buffy coats by Lymphocytes Separation Medium centrifugation ( Eurobio , Les Ulis , France ) . CD14+ monocytes were isolated by positive selection using CD14 microbeads ( Miltenyi Biotec , Bergisch Gladbach , Germany ) and were allowed to differentiate into macrophages in the presence of granulocyte macrophage colony-stimulating factor ( GM-CSF , 20 ng/mL; Miltenyi Biotec ) over a 6-day period . To exclude potential differences due to the MTB bacillary load between treated and untreated cells , macrophages were infected with BDQ-resistant MTB strain H37Rv ( BDQr-MTB ) expressing green-fluorescent protein ( GFP ) . Briefly , exponentially growing MTB carrying the pEGFP plasmid ( Tailleux et al . , 2003 ) was plated during 4 weeks on Middlebrook 7H11 agar supplemented with OADC ( Becton Dickinson , Franklin Lakes , New Jersey ) and containing 0 . 3 µg/mL BDQ . Some clones were then selected . Resistance to BDQ was confirmed ( i ) by bacterial culture in Middlebrook 7H9 Broth ( Becton Dickinson ) supplemented with albumin-dextrose-catalase ( ADC , Becton Dickinson ) and 0 . 3 µg/mL BDQ , and ( ii ) by confirming the mutation in the ATP synthase gene . The atpE gene was PCR-amplified using primers ( forward: 5-TCGTGTTCATCCTGATCTCCA-3; reverse: 5-GACAATCGCGCTCACTTCAC-3 ) and the PCR products were sent to Eurofins for sequencing . All the selected mutants carried a mutation in the atpE gene as described previously ( Andries et al . , 2005 ) . Only mutant with similar growth rate ( in liquid medium and in macrophages ) as the wild-type strain has been used for further experiments . Before infection , bacteria were washed and resuspended in 1 mL PBS . Clumps were disassociated by 50 passages through a needle , and then allowed to sediment for 5 min . The density of bacteria in the supernatant was verified by measuring the OD600 and aliquot volumes defined to allow 0 . 5 bacterium-per-cell infections . After 2 hr of incubation at 37°C , infected cells were thoroughly washed in RPMI 1640 to remove extracellular bacteria and were incubated in fresh medium . Cell viability was determined using the LDH Cytotoxicity Assay kit or the MTT assay kit ( Abcam ) , according to manufacturer’s instructions . The microdilution test was performed in 96-well plates as previously described ( Palomino et al . , 2002 ) . Briefly , BDQr-MTB and H37Rv were cultured in 7H9 liquid medium containing 2-fold dilutions of BDQ ( from 80 to 0 . 039 µg/mL ) during 6 days . The dye resazurin ( Sigma ) was then added to each well at a final concentration of 0 . 003% . After 24 hr , the absorbance was measured at 570 nm . Mφs ( 105 cells/mL ) were plated in 96-well tissue culture plates ( CellCarrier-96 Ultra Microplates ) and were infected with drug-susceptible H37Rv or BDQr-MTB expressing the GFP protein . Cells were then treated with BDQ ( 5 µg/mL ) for 18 hr or 5 days . Cells were fixed with 4% paraformaldehyde for 1 hr at room temperature and were strained with HCS CellMask Blue stain ( HCS , Invitrogen Molecular Probes , 2 µg/mL ) and Hoechst 33342 ( 5 µg/mL ) at 4°C in PBS . Confocal images were acquired using the automated fluorescence microscope Opera Phenix High Content Screening System ( Perkin Elmer Technology , Waltham , Massachusetts ) , with a 20x air objective . Images were analyzed using Columbus Conductor Database ( Perkin Elmer Technologies ) . Total RNA from macrophages was extracted using QIAzol lysis reagent ( Qiagen , Hilden , Germany ) and purified over RNeasy columns ( Qiagen ) . The quality of all samples was assessed with an Agilent 2100 bioanalyzer ( Agilent Technologies , Santa Clara , CA ) to verify RNA integrity . Only samples with good RNA yield and no RNA degradation ( ratio of 28S to 18S , >1 . 7; RNA integrity number , >9 ) were used for further experiments . cDNA libraries were prepared with the Illumina TruSeq RNA Sample Preparation Kit v2 and were sequenced on an llumina HiSeq 2500 at the CHU Sainte-Justine Integrated Centre for Pediatric Clinical Genomics ( Montreal , Canada ) . STAR v2 . 5 . 0b ( Dobin et al . , 2013 ) was used to map RNA-seq reads to the hg38 reference genome and quantify gene expression ( option-quantMode GeneCounts ) by counting the fragments overlapping the Ensembl genes ( GRCh38 v . 83 ) . Differential expression analysis was performed using a generalized linear model with the R Bioconductor package DESeq2 v1 . 18 . 1 ( Love et al . , 2014 ) on the 12 , 584 genes with at least one count-per-million ( CPM ) read in at least four samples . The model formula used in DESeq2 ( ~Donor + Infection + Infection:Donor + Infection:Treatment + Donor:Treatment ) contained: the main effects for Donor and Infection , interactions of Donor with Infection and Treatment to adjust for various responses to infection and treatment between donors , and a nested interaction of Infection with Treatment because we were interested in the infection-status-specific treatment effects . The latter was used to extract differentially expressed genes between treated and untreated samples under the infected and uninfected conditions . p-Values were adjusted for multiple comparisons using the Benjamini-Hochberg method producing an adjusted P-value or false-discovery rate ( FDR ) . Gene ontology ( GO ) enrichment analyses were performed using the Cytoscape app ClueGO ( version 2 . 5 . 3 ) ( Bindea et al . , 2009 ) . The following parameters were used: only pathways with pV ≤0 . 01 , Minimum GO level = 3 , Maximum GO level = 8 , Min GO family >1 , minimum number of genes associated to GO term = 5 , and minimum percentage of genes associated to GO term = 8 . Enrichment p-values were calculated using a hypergeometric test ( p-value<0 . 05 , Bonferroni corrected ) . Measurement of glycolysis was done using the Glycolytic rate assay kit ( Seahorse , Agilent Technologies ) , following the manufacturer’s protocol . Briefly , cells were seeded in Xe96 plates treated with BDQ for 24 hr . The cells were then incubated in the assay medium ( Seahorse XF Base Medium without phenol , 2 mM glutamine , 10 mM glucose , 1 mM pyruvate and 5 . 0 mM HEPES ) at 37°C , during 1 hr . Extracellular acidification rate ( ECAR , milli pH/min ) and oxygen consumption rate ( OCR , pmol/min ) were measured using the Seahorse Bioscience XFe96 Analyzer . Cells were treated with BDQ during 18 hr and them lysed in water during 10 min at 37°C . Samples were heated at 90°C during 40 min in order to inactivate MTB , and were then washed three times to remove salts and contaminants that could preclude the analysis . Prior to mass spectrometry analysis , the 2 , 5-dihydroxybenzoic acid ( Sigma-Aldrich , Saint-Louis , Missouri ) matrix was added at a final concentration of 10 mg/mL in a chloroform/methanol mixture at a 90:10 ( v/v ) ratio; 0 . 4 μL of a cell solution at a concentration of 2 × 105 to 2 × 106 cells/mL , corresponding to ∼100–1 , 000 cells per well of the MALDI target plate ( 384 Opti-TOF 123 mm ×84 mm , AB Sciex ) , and 0 . 6 μL of the matrix solution were deposited on the MALDI target plate , mixed with a micropipette , and left to dry gently . MALDI-TOF MS analysis was performed on a 4800 Proteomics Analyzer ( with TOF-TOF Optics , Applied Biosystems , Foster City , California ) using the reflectron mode . Samples were analyzed operating at 20 kV in the negative and positive ion mode . Mass spectrometry data were analyzed using Data Explorer version 4 . 9 from Applied Biosystems . Cells were incubated with LysoTracker DND-99 ( 100 nM; Thermo Fisher , Waltham , Massachusetts ) during 1 hr at 37°C . Cells were then fixed with 4% paraformaldehyde at room temperature ( RT ) for 1 hr . Fluorescence was analyzed using a CytoFLEX Flow Cytometer ( Beckman Coulter , Brea , California ) . More than 10 , 000 events per sample were recorded . The analysis was performed using the FlowJo software . LysoTracker staining was also analyzed using a Leica TCS SP5 Confocal System . Briefly , cells were washed twice with PBS after incubation with LysoTracker DND-99 ( 1 µM ) , fixed with 4% paraformaldehyde for 1 hr at RT , stained with DAPI ( 1 µg/mL , Thermo Fisher ) during 10 min mounted on a glass slide using Fluoromount mounting medium ( Thermo Fisher ) . Quantification of LysoTracker staining was performed using Icy software . Macrophages were activated with heat-killed MTB and treated with BDQ during 18 hr or 48 hr . Cells were then incubated with DQ-Green BSA ( 10 µg/mL; Thermo Fisher ) for 1 hr at 37°C . The hydrolysis of the DQ-Green BSA by lysosomal proteases produces brightly fluorescent peptides . Cells were washed and incubated further in culture medium for 3 hr to ensure that DQ BSA had reached the lysosomal compartment . Cells were detached and were fixed with 4% paraformaldehyde and the fluorescence was analyzed using a CytoFLEX Flow Cytometer ( Beckman Coulter ) . Macrophages were lysed in distilled water with 0 . 1% Triton X-100 . MTB was enumerated as previously described Tailleux et al . ( 2003 ) and plated on 7H11 . CFUs were scored after three weeks at 37°C . S . aureus and S . Typhimurium were plated on Luria-Bertani agar and CFUs were counted after 1 day at 37°C . Macrophages ( 4 × 105 cells/mL ) were grown on 12 mm circular coverslips in 24-well tissue culture plates for 24 hr in cell culture medium , followed by BDQ treatment . Cells were fixed with 4% paraformaldehyde for 1 hr at RT , and were then incubated for 30 min in 1% BSA ( Sigma-Aldrich ) and 0 . 075% saponin ( Sigma-Aldrich ) in PBS , to block nonspecific binding and to permeabilize the cells . Cells were incubated with anti-LC3 ( MBL , Woburn , MA ) during 2 hr at RT . Alternatively , cells were fixed with cold methanol for 5 min , and were then incubated for 10 min in PBS containing 0 . 5% saponin . Cells were stained with anti-TFEB ( Thermo Fisher ) overnight at 4°C . Cells were washed and incubated with Alexa Fluor 555 secondary antibody ( Thermo Fisher ) for 2 hr . Nuclei were stained with DAPI ( 1 µg/mL ) during 10 min . After labeling , coverslips were set in Fluoromount G medium containing 1 µg/ml 4′ , 6-diamidino-2-phenylindole ( DAPI ) ( SouthernBiotech , Birmingham , Alabama ) on microscope slides . Fluorescence was analyzed using Leica TCS SP5 . Quantification of TFEB staining was performed using Icy software . LC3B puncta were analysed by confocal microscopy and quantified using ImageJ . Infected cells were manually segmented , thresholded and puncta counted using Analyze Particles . Dot plots represent the mean values of at least 83 cells from two donors . Error bars depict the SD . Reverse transcription of mRNA to cDNA was done using SuperScript III Reverse Transcriptase ( Thermo Fisher ) followed by amplification of cDNA using Power SYBR Green PCR Master Mix ( Thermo Fisher ) . All primers used in this study are listed in Supplementary file 1 . Reactions were performed using a StepOnePlus Real-Time PCR System Thermal Cycling block ( Applied Biosystems ) . The relative gene expression levels were assessed according to the 2-ΔCt method ( Pfaffl , 2001 ) . Cells were lysed with RIPA buffer ( Thermo Fisher ) containing protease inhibitor cocktails ( Roche ) and stored at −80°C . Protein concentration was determined using the BCA protein assay kit ( Thermo Fisher ) according to the manufacturer instructions . 20 µg of total protein were loaded on a NUPAGE 4–12% Bis-Tris polyacrylamide gel ( Thermo Fisher ) and transferred to PVDF membranes ( iBlot , Thermo Fisher ) . The membranes were blocked with TBS-0 . 1% Tween20 , 5% non-fat dry milk for 30 min at RT and incubated overnight with primary antibodies against α-β-Tubulin , p-62 ( Cell Signaling , Danvers , Massachusetts ) and LC3 ( Abcam , Cambridge , United Kingdom ) . Membranes were washed in TBS-Tween and incubated with secondary HRP-conjugated antibody ( GE Healthcare , Chicago , IL ) at RT for 1 hr . Membranes were washed and exposed to SuperSignal West Femto Maximum Sensitivity Substrate ( Thermo Fisher ) . Detection and quantification of band intensities was performed using Azure Imager C400 ( Azure Biosystems , Dublin , CA ) and ImageJ software ( version 1 . 51 ) . S . aureus and S . Typhimurium were grown in Luria-Bertani broth . Bacteria were washed three times and resuspended in PBS . The density of bacteria was estimated by measuring the OD600 . Cells were then infected at a multiplicity of infection of 2:1 . After 1 hr of infection , cells were extensively washed and incubated for 1 hr in culture medium supplemented with gentamicin ( 100 µg/mL ) . After washing , cells were cultured with different concentrations of BDQ and gentamicin ( 5 µg/mL ) . NO was measured by Griess reaction assay ( Promega , Madison , Wisconsin ) according to the manufacturer's instructions . Briefly cell culture supernatants were incubated with sulfanilamide solution during 10 min followed by additional 10 min with N-1-napthylethylenediamine dihydrochloride . The absorbance was measured at 520 nm . Cells were stained with Image-IT TMRM ( 10 nM , Thermo Fischer ) during 30 min at 37°C or with MitoTracker Deep Red ( 100 nM , Thermo Fisher ) during 45 min at 37°C . Cells were washed in PBS and detached from culture plates with 0 . 05% Trypsin-EDTA . Fluorescence was analyzed using a CytoFLEX Flow Cytometer ( Beckman Coulter ) . The oxygen consumption rate was measured using the XF Cell Mito Stress Test Kit ( Seahorse , Agilent Technologies ) according to the manufacturer’s protocol . Briefly , cells were seeded in Xe96 plates and treated with BDQ for 24 hr . The test was performed by adding oligomycin ( 1 µM ) , FCCP ( 1 µM ) , rotenone and antimycin ( 0 . 5 µM ) at the indicated time points . Cells were incubated with MitoSOX Red ( 5 µM , Thermo Fisher ) during 10 min at 37°C . Cells were washed in PBS and detached from culture plates with 0 . 05% Trypsin-EDTA . Fluorescence was analyzed using a CytoFLEX Flow Cytometer ( Beckman Coulter ) . Cells were treated with BDQ for 1 to 18 hr , then labeled with Fluo-8 AM ( 4 µM , Abcam ) during 1 hr . Cells were washed twice with PBS and fluorescence was analyzed using FLUOstar Omega ( BMG Labtech , Ortenberg , Germany ) . siRNA transfection was performed as previously described ( Troegeler et al . , 2014 ) . Briefly , macrophages were transfected using the lipid-based HiPerfect system ( Qiagen ) and an ON-TARGETplus SMARTpool siRNA targeting TFEB ( 50 nM ) and a non-targeting siRNA ( scramble ) ( Dharmacon ) . After 6 hr , cells were washed and incubated in complete culture medium for an additional 2 days . The inactivation of TFEB was confirmed RT-qPCR at 48 hr post-transfection . The protocol was adopted from Blecher-Gonen et al . ( 2013 ) . Briefly , cells treated were fixed in 1% formaldehyde during 10 min . Glycine was then added to a final concentration of 0 . 15 M . Cells were washed with PBS and were lysed with ice-cold RIPA buffer supplemented with protease inhibitors ( Roche ) . The chromatin fraction was sonicated to obtain fragments from 100 to 500 bp and were coupled with DynaBeads protein G coupled to the anti-TFEB antibody ( D2O7D , Cell Signaling ) overnight at 4°C . Immune complexes were eluted from the beads with 1% SDS in TE . After treatment with RNaseA and proteinase K , protein-DNA cross-links were reversed by adding NaCl 5M and incubated at 65°C overnight . Chromatin immunoprecipitation analysis was performed by qPCR using Power SYBR Green PCR Master Mix ( Thermo Fisher ) . The primers for gla and mcoln1 were used from Palmieri et al . ( 2011 ) . Data are expressed as means ± standard deviations ( SD ) . Statistical analyses were performed with Prism software ( GraphPad Software Inc ) , using the t test and one-way analysis of variance ( ANOVA ) as indicated in the figure legends . A p value of < 0 . 05 was considered to be significant . The raw fastq files of BDQ-treated cells have been deposited in NCBI's Gene Expression Omnibus ( Edgar et al . , 2002 ) and are accessible through GEO Series accession number GSE133145 . The raw fastq files of cells stimulated with heat-killed MTB or treated with different antibiotics are accessible through GEO Series accession numbers GSE143627 and GSE143731 .
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 .
The discovery of antibiotic drugs , which treat diseases caused by bacteria , has been a hugely valuable advance in modern medicine . They work by targeting specific cellular processes in bacteria , ultimately stopping them from multiplying or killing them outright . Antibiotics sometimes also affect their human hosts and can cause side-effects , such as gut problems or skin reactions . Recent evidence suggests that antibiotics also have an impact on the human immune system . This may happen either indirectly , by affecting ‘friendly’ bacteria normally present in the body , or through direct effects on immune cells . In turn , this could change the effectiveness of drug treatments . For example , if an antibiotic weakens immune cells , the body could have difficulty fighting off the existing infection – or become more vulnerable to new ones . However , even though new drugs are being introduced to combat the worldwide rise of antibiotic-resistant bacteria , their effects on immunity are still not well understood . For example , bedaquiline is an antibiotic recently developed to treat tuberculosis infections that are resistant to several drugs . Giraud-Gatineau et al . wanted to determine if bedaquiline altered the human immune response to bacterial infection independently from its direct anti-microbial effects . Macrophages engulf foreign particles like bacteria and break them down using enzymes stored within small internal compartments , or ‘lysosomes’ . Initial experiments using human macrophages , grown both with and without bedaquiline , showed that the drug did not harm the cells and that they grew normally . A combination of microscope imaging and genetic analysis revealed that exposure to bedaquiline not only increased the number of lysosomes within macrophage cells , but also the activity of genes and proteins that increase lysosomes’ ability to break down foreign particles . These results suggested that bedaquiline treatment might make macrophages better at fighting infection , even if the drug itself had no direct effect on bacterial cells . Further studies , where macrophages were first treated with bedaquiline and then exposed to different types of bacteria known to be resistant to the drug , confirmed this hypothesis: in every case , the treated macrophages became efficient bacterial killers . In contrast , older anti-tuberculosis drugs did not have any such potentiating effect on the macrophages . This work sheds new light on our how antibiotic drugs can interact with the cells of the human immune system , and can sometimes even boost our innate defences . Such immune-boosting effects could one day be exploited to make more effective treatments against bacterial infections .