Ohmyungsamycins promote antimicrobial responses through autophagy activation via AMP-activated protein kinase pathway

The induction of host cell autophagy by various autophagy inducers contributes to the antimicrobial host defense against Mycobacterium tuberculosis (Mtb), a major pathogenic strain that causes human tuberculosis. In this study, we present a role for the newly identified cyclic peptides ohmyungsamycins (OMS) A and B in the antimicrobial responses against Mtb infections by activating autophagy in murine bone marrow-derived macrophages (BMDMs). OMS robustly activated autophagy, which was essentially required for the colocalization of LC3 autophagosomes with bacterial phagosomes and antimicrobial responses against Mtb in BMDMs. Using a Drosophila melanogaster–Mycobacterium marinum infection model, we showed that OMS-A-induced autophagy contributed to the increased survival of infected flies and the limitation of bacterial load. We further showed that OMS triggered AMP-activated protein kinase (AMPK) activation, which was required for OMS-mediated phagosome maturation and antimicrobial responses against Mtb. Moreover, treating BMDMs with OMS led to dose-dependent inhibition of macrophage inflammatory responses, which was also dependent on AMPK activation. Collectively, these data show that OMS is a promising candidate for new anti-mycobacterial therapeutics by activating antibacterial autophagy via AMPK-dependent signaling and suppressing excessive inflammation during Mtb infections.

immune cells by directly ameliorating pro-inflammatory signaling and limiting the synthesis of certain lipid intermediates relevant to inflammation [13][14][15] .
We previously reported that ohmyungsamycins (OMS) A and B are novel cyclic peptides that were isolated from a marine bacterial strain belonging to the Streptomyces genus collected from Jeju Island in Korea 16 . We previously showed that OMS-A and -B are cyclic peptides that exhibit inhibitory effects against a diverse range of cancer cells and bacteria such as Bacillus subtilis, Kocuria rhizophila, and Proteus hauseri 16 . In this study, we demonstrated that OMS-A and -B are robust activators of autophagy that lead to antimicrobial responses against Mtb. We used a Drosophila melanogaster (D. melanogaster)-Mycobacterium marinum infection model to show that OMS treatment elicited anti-mycobacterial effects through autophagy activation in vivo. We further investigated the mechanisms by which OMS activated antimicrobial responses and showed that AMPK-dependent signaling was involved in the OMS-mediated activation of autophagy in murine bone marrow-derived macrophages (BMDMs). In addition, OMS treatment inhibited macrophage inflammatory responses during Mtb infection by activating the AMPK pathway. Together, these data suggest that the OMS-induced activation of autophagy and suppression of excessive pathologic inflammation may contribute to the innate host defenses against mycobacterial infection.

OMS-A and OMS-B stimulate the killing of mycobacteria in vitro and in vivo. OMS-A and
OMS-B have inhibitory activities against Bacillus subtilis, Kocuria rhizophila, and Proteus hauseri 16 . To examine the anti-mycobacterial effects of OMS-A and OMS-B, we first assessed the antibacterial properties of OMS-A and OMS-B against Mtb. The minimum inhibitory concentration (MIC) of OMS-A and OMS-B against Mtb was determined using the resazurin microtiter assay (REMA) plate method. Isoniazid and ethambutol (known antimicrobial drugs) and SQ109 (an anti-Mtb drug candidate) were selected as positive controls 17 . After incubating Mtb for 5 days with the compounds, a decrease in fluorescence was observed, indicating a dose-dependent killing effect. As shown in Fig. 1a, OMS-A and OMS-B were very potent, with MICs lower than those of isoniazid, ethambutol, and SQ109. The MIC50 values for OMS-A and OMS-B were 57 nM and 117 nM, respectively.
It was previously reported that the M. marinum-D. melanogaster infection system is an alternative model host for evaluating Mycobacterium infections 18 . Therefore, we evaluated whether this infection model could be used to effectively assess the antimicrobial effects of OMS-A in vivo. As shown in Fig. 1d, flies challenged with M. marinum died within ~20 days (500 CFU/50 nL), consistent with previous reports by Kim et al. 19 . We then monitored the survival of flies treated with OMS-A (1, 10 μM), which exhibited a significant decrease in lethality compared with those treated with the solvent control. Antibiotic control flies, which received food containing amikacin (1 μg/ml), showed comparable survival to those treated with OMS-A after M. marinum injection (Fig. 1d). In addition, the viable bacterial counts in surviving flies infected with M. marinum were monitored in the control and OMS-treated groups. The in vivo bacterial counts were consistently higher in control flies compared with flies treated with OMS-A 12 days after infection with M. marinum (n = 20 per group; Fig. 1e). These findings suggest that OMS-A exhibits in vivo antimicrobial activities against mycobacteria.

OMS-A and OMS-B increase autophagy activation and autophagic flux in murine macrophages.
We previously showed that treatment with antibiotics against Mtb infection resulted in autophagy, which is required for antimicrobial effects in the host 19 . Recent studies demonstrated that the important immunosuppressive agent cyclosporine A, a cyclic peptide, induces autophagic cell death in canine lens epithelial cells 20 . Thus, we assessed whether OMS-A or OMS-B enhances the activation of autophagy in BMDMs. Treatment with OMS-A (10 μM) or OMS-B (10 μM) caused no hazardous effects in BMDMs (data not shown). We next assessed LC3 puncta formation and lipidation, which are well-known indicators of autophagy induction 21 , in BMDMs treated with OMS-A (10 μM) or OMS-B (10 μM). As shown in Fig. 2a, fluorescent staining of LC3 puncta was increased in BMDMs treated with OMS-A (10 μM) or OMS-B (10 μM) for 24 h. Quantitative analysis showed that LC3 puncta formation was significantly increased by OMS-A and OMS-B treatment (Fig. 2b). It was noted that OMS-A and OMS-B had comparable effects on autophagy activation in BMDMs, similar to those observed in BMDMs treated with rapamycin (200 nM) or Torin1 (10 μM), the known autophagy activators 4,22 (Fig. S2).
We then examined the effects of autophagy in OMS-induced LC3 punctate formation. To examine this, BMDMs from Atg7 fl/fl LysM-Cre + (Atg7 KO) mice and their Atg7 + littermates control (Atg7 wildtype; Atg7 WT) were treated with OMS-A (10 μM) or OMS-B (10 μM), and LC3 punctate formation was compared. As shown in Fig. 2h and i, the OMS-A or OMS-B-induced increase in LC3-positive autophagosome formation in Atg7 WT BMDMs was significantly decreased in Atg7 KO BMDMs. Together, these data suggest that OMS-A or OMS-B treatment enhances autophagy activation and autophagic flux in BMDMs.

OMS-A and OMS-B enhance Mtb phagosome maturation in BMDMs.
Mtb is a highly adapted pathogen that arrests the maturation of phagosomes into phagolysosomes 23 . Recent studies also showed that  the Mtb virulent strain H37Rv significantly inhibited autophagic flux in macrophages 24 . The activation of antibacterial autophagy leads to mycobacterial phagosomal maturation, thus reducing the bacterial burden in macrophages 25 . To further evaluate OMS-A (10 μM) or OMS-B (10 μM) treatment-induced phagosome maturation, BMDMs were infected with enhanced red fluorescent protein (ERFP)-Mtb and then treated with OMS-A (10 μM) or OMS-B (10 μM). As shown in Fig. 3a-d, treating Mtb-infected BMDMs with OMS-A or OMS-B increased the co-localization of LC3, an autophagosomal marker, with Mtb phagosomes (Fig. 3a,b). Next, the co-localization of Mtb phagosomes with lysosomes was assessed in Mtb-infected BMDMs after OMS-A (10 μM) or OMS-B treatment (10 μM). Treating Mtb-infected BMDMs with OMS-A or OMS-B significantly enhanced the co-localization of Mtb phagosomes with LAMP2 ( Fig. 3c,d).
In addition, we infected Atg7 WT and Atg7 KO BMDMs with ERFP-Mtb in the presence or absence of OMS-A or OMS-B. Notably, the LC3 punctate formation in Mtb-infected/OMS-A/OMS-B (10 μM) treated Atg7 WT BMDMs was markedly decreased in Atg7 KO BMDMs (Fig. S4a). In addition, the co-localization of bacterial phagosomes with the autophagosomal marker LC3 was significantly decreased in Atg7 KO BMDMs compared to Atg7 WT BMDMs (Fig. S4a,b). Collectively, these data indicate that OMS-A and OMS-B induce the phagosomal maturation of Mtb through autophagy activation in BMDMs.

OMS-A and OMS-B activate the AMPK pathway, which is required for Mtb phagosomal maturation and antimicrobial responses in macrophages.
Our previous studies showed that activation of AMPK contributes to autophagy activation and antimicrobial responses against Mtb 12 . We thus examined whether OMS-A or OMS-B activates AMPK by measuring the phosphorylation of Thr172 of the catalytic α-subunit of AMPK 26 . As shown in Fig. 4a,b, OMS-A and OMS-B induced the phosphorylation of AMPK at Thr172, as well as its downstream target acetyl-CoA carboxylase (ACC), in a time-dependent manner. Treating BMDMs with OMS-A (10 μM) or OMS-B (10 μM) rapidly increased AMPK phosphorylation within 0.5-1 h after stimulation. The AMPK phosphorylation levels were then slightly decreased in BMDMs after OMS-B stimulation, however, they were sustained in BMDMs in response to OMS-A and OMS-B at later time points (Fig. 4a,b).
To further define the role of AMPK in enhancing phagosome maturation, BMDMs were transduced with short hairpin RNAs (shRNA) lentiviral vectors that specifically target Ampkα (shAmpk). The efficiency of Ampkα silencing was confirmed 48 h after transduction; the mRNA levels of AMPK were reduced compared with those in cells transduced with lentiviruses expressing nonspecific shRNA (shNS). To examine the effects of Ampkα silencing on the colocalization of Mtb phagosomes and LC3 autophagosomes, BMDMs were transduced with shAmpk or shNS, infected with ERFP-Mtb, and then treated with or without OMS-A (10 μM) or OMS-B (10 μM). The colocalization of LC3 autophagosomes and Mtb phagosomes was markedly increased in shNS-transduced BMDMs by treatment with OMS-A (10 μM) or OMS-B (10 μM). However, there was no significant difference between the effects of OMS-A and OMS-B on the co-localization of phagosomes and autophagosomes. In addition, silencing Ampkα dramatically attenuated OMS-induced colocalization of autophagosomes and Mtb phagosomes in BMDMs compared with those under the control conditions (Fig. 4c,d). Next, the ability of AMPK to promote the intracellular killing activities induced by OMS-A or OMS-B was examined. As shown in Fig. 4e, the inhibitory effects of OMS-A and OMS-B against Mtb in shNS-transduced macrophages were significantly counteracted by shAmpk. These findings suggest that AMPK is the major signaling pathway mediating OMS-A and OMS-B-induced Mtb phagosome maturation and antimicrobial responses in macrophages.

OMS-A and OMS-B activate autophagy to enhance antimicrobial responses against Mtb in vitro and in vivo.
To further examine the effects of autophagy in the antimicrobial responses by OMS, we performed additional cfu experiments using macrophages from Atg7 KO mice. BMDMs from Atg7 WT and Atg7 KO were infected with Mtb, followed by treatment with or without OMS-A (10 μM) or OMS-B (10 μM). As shown in Fig. 5a, Atg7 WT macrophages showed significantly increased killing effects against Mtb, whereas this was significantly inhibited in Atg7 KO BMDMs following treatment with OMS-A (10 μM) or OMS-B (10 μM). It was also noted that OMS-A-or OMS-B-induced antimicrobial effects were substantially higher than those induced by another autophagy activators, such as rapamycin (100 nM) or Torin1 (10 μM) (data not shown). We next examined whether hATG5 is required for the OMS-mediated antimicrobial response against intracellular Mtb by transducing human monocyte-derived macrophages (MDMs) with shRNA against hATG5 (shATG5) and determining whether knockdown of hATG5 affected the intracellular survival of Mtb in human MDM. Silencing of ATG5 significantly counteracted the intracellular killing effects of OMS-A and OMS-B against Mtb in human MDMs ( Fig. 5b; shNS vs. shATG5 under OMS-A (10 μM)or OMS-B (10 μM)-treated conditions; P < 0.001, for both; moi = 10). These data indicate that OMS-mediated autophagy enhances the antimicrobial response in murine and human macrophages.
We further examined whether OMS-mediated antimicrobial effects were decreased in flies administered chloroquine, an inhibitor of the autophagy pathway 27 . We thus injected flies with M. marinum and treated them with OMS-A (10 μM) in the presence or absence of chloroquine (1 μM). We then monitored fly survival for 20 days. As shown in Fig. 5c, the flies treated with chloroquine exhibited increased lethality to M. marinum challenge compared to control flies, which were administered food containing OMS-A (10 μM) only. Consistent with these findings, viable bacterial counts in surviving flies were significantly increased by chloroquine administration relative to those in control flies treated with OMS-A only (after 7 days; n = 20 per group; Fig. 5d).
We then injected control and Atg7 mutant flies with M. marinum in the presence or absence of OMS-A (10 μM) and monitored survival for 27 days. As shown in Fig. 5e, the Atg7 mutant flies increased lethality to M. marinum challenge when compared to control flies. In addition, administration of control flies with food containing OMS-A (10 μM) led to a significant increase of survival after M. marinum injection. However, OMS-A-mediated protective effects were not significant in Atg7-mutant flies after M. marinum infection (Fig. 5e). Furthermore, viable bacterial counts of M. marinum were significantly higher in atg7 mutant flies than those in control flies infected with M. marinum (after 3 days; n = 20 per group; Fig. 5f). OMS-A treatment significantly decreased the bacterial loads in control flies, but did not decrease the viable bacteria in Atg7 mutant flies to the levels observed in control flies (Fig. 5f). These findings suggest that host autophagy activation is required for the OMS-induced antimicrobial effects against mycobacterial infection in vitro and in vivo.
Finally, we examined whether AMPK is involved in the attenuation of proinflammatory cytokine production in BMDMs. BMDMs were transduced with shAmpk or shNS, infected with Mtb, and then treated with OMS-A or OMS-B. As shown in Fig. 6c, the OMS-mediated inhibition of proinflammatory cytokine production was nearly completely counteracted by shAmpk in BMDMs. Collectively, these data suggest that OMS-induced AMPK activation is required for suppression of Mtb-induced inflammatory responses in BMDMs.  . The error bars indicate 95% confidence intervals. Log-rank analysis of the survival curves showed that the survival rates of Atg7 control flies were significantly increased by OMS-A treatment (***p < 0.001), whereas these were not significant in Atg7 mutant flies with the same treatment. (f) Each group (n = 20) of flies was harvested at 3 days, homogenized, and quantified by CFU assay. All data represent the means ± SD of triplicates from each sample. **p < 0.01, ***p < 0.001, compared with control conditions (a,b,d,f). SC, solvent control; AMK, amikacin; CQ, chloroquine.

Discussion
Much of the recent focus regarding therapeutic strategies against Mtb has been on the development of host-directed therapies that enhance antimicrobial mechanisms, such as autophagy inducers, cathelicidin  (a and c) The supernatants were harvested and subjected of ELISA analysis of TNF-α, IL-6, IL-1β, and IL-12p40 production. All data represent the means ± SD of triplicates from each sample. **p < 0.01, ***p < 0.001, compared with SC (a-c). U, uninfected/untreated; SC, solvent control; ns, no significant.
Scientific RepoRts | 7: 3431 | DOI:10.1038/s41598-017-03477-3 inducers, and anti-inflammatory agents 29,30 . Novel functions and mechanisms by which various autophagy inducers enhance antibacterial autophagy and antimicrobial responses have been reported 31,32 . Early studies showed that interferon (IFN)-γ 5 , vitamin D 10,33,34 , and both treatments synergize the elimination of Mtb via autophagy 10 . Various innate signals and pathogen-and damage-associated molecular patterns enhance the Mtb-induced inhibition of intracellular growth by activating autophagic machinery, interacting with innate signaling pathways, and delivering antimicrobial peptides to phagosomes 10,33,[35][36][37] . Here, we demonstrated that the newly identified cyclic peptides OMS-A and OMS-B activate autophagy via the AMPK pathway, to promote antimicrobial effects against Mtb. In addition, OMS-induced autophagy leads to antimicrobial responses to Mtb in macrophages, as shown by intracellular survival assays.
Since issues regarding multidrug-resistant Mtb are increasing worldwide, there is an urgent need to develop new antitubercular drugs. The current data suggest that OMS-A and OMS-B exhibit at least three attractive anti-tubercular drug activities: 1) direct antimicrobial killing activity against Mtb, 2) direct activation of autophagy, which was essential for antimicrobial effects, in host cells, and 3) inhibition of Mtb immunopathology. REMAs revealed that OMS-A and OMS-B have excellent MICs and MBCs against Mtb, comparable to those of isoniazid and ethambutol, which are the antibiotics currently used against human tuberculosis. Our previous studies revealed that OMS-A and OMS-B are cyclic peptides that harbor activity against several bacteria, as well as exhibit an inhibitory effect on cancer cell proliferation 16 . Recently, chemical investigations identified several cyclic peptides as promising anti-Mtb agents due to strong antitubercular activities in vitro 38 . In addition, a high-throughput screening study revealed that ecumicin is an excellent drug candidate with activity against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb strains 39 . Lassomycin, a basic and ribosomally encoded cyclic peptide, also exhibits bactericidal activity against mycobacteria 40 . The mycobacterial ClpC1 ATPase complex was identified as a drug target for both ecumicin and lassomycin 39,40 . Although we did not try to identify the targets of OMS-A and OMS-B, we speculate that the same ClpC1 ATPase complex could be a potential target. Indeed, significant effort in developing new cyclic antimicrobial peptides has been devoted to designing analogs of drugs that reduce cytotoxic side effects but promote antitubercular and antibacterial properties using conformational analyses based on structure-activity relationships 41 . Importantly, we did not observe any toxic effects on host macrophages and flies in vivo treated with OMS-A and OMS-B, at least at the doses and time periods evaluated. Nevertheless, further studies are needed to develop a large scale of OMS and design safe and effective chemical analogs for future clinical trials.
Both OMS-A and OMS-B activated autophagy, which is an essential part of the antimicrobial defense against Mtb. Our data demonstrated that OMS-A and OMS-B-induced autophagy is essential for phagosomal maturation, and for antimicrobial effects against Mtb infection, both in vitro and in flies. The findings of this study were in partial agreement with our previous studies in showing that isoniazid and pyrazinamide, two antibiotics used to treat tuberculosis in humans, activate the antibacterial autophagy required for successful antimicrobial activity during mycobacterial infection 19 . In a recent report, thiostrepton, a thiopeptide antibiotic possessing a quinaldic acid moiety, was used successfully in the treatment of M. marinum, through ER-stress-mediated autophagy 42 . In addition, a search for active compounds to enhance interferon-gamma responses in macrophages identified several such compounds within the flavagline (rocaglate) family, which effectively induce autophagy to combat intracellular mycobacteria and inhibit pathologic inflammation during infection 43 .
These OMS-A and OMS-B activities were related to their ability to activate AMPK in macrophages. AMPK is an energy sensor that regulates energy balance at a cell-autonomous level in living organisms 11 . Much attention has been given to promising adjunctive host-directed therapeutic candidates that affect cell signaling pathways associated mainly with immunometabolism (i.e., the core pathways intersecting immunologic and metabolic responses) 44 . Understanding the mechanisms that regulate AMPK and mammalian target of rapamycin signaling will likely facilitate significant advances in the development of adjunctive options for several unmet needs regarding tuberculosis, including new drugs against MDR and XDR strains, shortening the chemotherapeutic treatment duration, and targeting various clinical stages, even in combination with human immunodeficiency viral infection 44,45 . Recent studies demonstrated that AMPK plays a key role in antimicrobial autophagy against mycobacteria 44 and that several AMPK activators have the potential to activate autophagy and intracellular killing in response to Mtb 12,46 . In addition, the widely used anti-diabetes drug metformin, which exhibits AMPK-activating activity, was identified as an excellent adjunct antituberculous therapy 47 . The current data demonstrate that AMPK activation by OMS-A and OMS-B could impact maturation. These studies collectively suggest that activation of AMPK activation in host cells enhances the cell-autonomous and in vivo killing effects against mycobacteria.
NF-κB activation and inflammatory responses during Mtb infections appear to be a double-edged sword, i.e., a defense reaction against microbial insults and immunopathologic responses in tuberculosis. Indeed, the design of immunomodulatory agents has been focused on host-directed therapeutics that facilitates protective immune responses while simultaneously reducing deleterious inflammatory responses 45 . The current data revealed that OMS-induced AMPK activation inhibited inflammatory responses in macrophages induced by Mtb infection. Activation of the AMPK signaling pathway promotes anti-inflammatory and anti-cancer effects by inducing oxidative metabolism 28 . AMPK activators such as metformin and salicylate suppress inflammatory responses in various cell types by modulating NF-κB or other signaling pathways 28,48,49 . In addition, autophagy controls excessive inflammatory responses and type I interferon activation, particularly during microbial infections 50 . Previous studies showed that autophagy modulates NF-κB signaling pathways during innate immune responses to attenuate exacerbated inflammation. It was reported that inhibition of NF-κB signaling in human macrophages increased apoptosis and autophagy, which subsequently decreased intracellular Mtb survival 51 . Previous studies revealed the mechanisms by which autophagy regulates NF-κB signaling. Specifically, hepatoma tumor cell-conditioned medium induced TLR2 signaling, which stimulated the degradation of NF-κBp65 by SQSTM1/ p62-mediated selective autophagy 52 . Thus, OMS-induced anti-inflammatory responses may be the result of at least two mechanisms: AMPK-dependent-and autophagy-activating pathways.
Scientific RepoRts | 7: 3431 | DOI:10.1038/s41598-017-03477-3 Taken together, these results provide a novel mechanism underlying the antimicrobial effects of the novel cyclic peptides OMS-A and OMS-B, via AMPK-dependent autophagy activation, thus overcoming the inhibition of phagolysosomal fusion caused by pathogens. OMS-A and OMS-B induced a direct killing activity against Mtb and, interestingly, promoted host defenses; they did not only activate autophagy but also regulated pathologic inflammatory responses during Mtb infection. These efforts may contribute to the development of new therapeutic drugs and biologics by enhancing dual effects on both the bacteria and host immunity.

Materials and Methods
Cultivation and extraction of the actinobacterial strain SNJ042 to produce OMS-A and B. Isolation of OMS-A and OMS-B were performed as described previously 16 . The actinobacterial strain SNJ042 (99% identity with Streptomyces cheonanensis based on 16 S ribosomal DNA analysis) was isolated from a sand beach in Jeju Island, Republic of Korea, and was cultivated to produce OMS-A and OMS-B. It was incubated on solid YEME medium (10 g malt extract, 4 g yeast extract, 4 g glucose, and 18 g agar powder in 1 L sterilized 3.4% artificial seawater) at 25 °C to acquire spores. The spores were inoculated into 125 ml liquid A1/C medium (10 g starch, 4 g yeast extract, 2 g peptone, and 1 g CaCO 3 in 1 L sterilized 2.4% artificial seawater) in a 500 ml Erlenmeyer flask. The liquid culture was incubated at 30 °C with shaking at 200 rpm. After 2 days, 10 ml of the liquid culture were transferred to 1 L liquid A1/C medium in a 2.8 L Fernbach flask and further cultivated on a rotary shaker at 30 °C and 180 rpm (108 × 1 L, total culture volume 108 L). After 7 day cultivation, 1.5 L ethyl acetate (EtOAc) were added to each 1 L bacterial culture and extracted twice using a separation funnel. The EtOAc layer was collected in an empty 2.8 L Fernbach flask, and anhydrous sodium sulfate was added to the organic extract to remove residual water. The extract in EtOAc was concentrated in vacuo to yield 12 g dry material.
Determining the MIC by REMA. The REMA was performed as described previously to determine the MICs of OMS-A or OMS-B, isoniazid (INH; I3377), ethambutol (E4630), and SQ-109 (SML1309) (all from Sigma, St. Louis, MO, USA) against H37Rv 17 . Briefly, a 100 µl inoculum was used to inoculate each well of the plate, and two-fold serial dilutions of each test compound were prepared in 96-well plates in triplicate. An inoculum at an optical density 600 of 0.005 was prepared by diluting mid-log cultures and then added to each well. Growth controls containing no drug and a sterile control were also prepared in each experiment. Plates were incubated at 37 °C for 5 days, and 40 µL 0.025% resazurin (Sigma, R7017) solution were then added to each well. After an overnight incubation, the fluorescence of the resazurin metabolite resorufin was determined by excitation at 560 nm and emission at 590 nm using the Synergy H1 microplate reader (Biotechnology Inc, Dallas, TX, USA). The MIC50 (the MIC required to inhibit the growth of 50% of the organism) was determined using GraphPad Prism 5.0 software.
Generation of tandem LC3B retrovirus. Tandem LC3B plasmid (mCherry-EGFP-LC3B) was performed as described previously 21 . To produce tandem LC3 retrovirus, human Phoenix amphotropic (ATCC, CRL-3213) cells were seeded at 70-80% confluence into a 6-well plate and co-transfected with 0.75 μg of the packaging plasmid pCL-Eco (Addgene, 12371), 0.25 μg of the envelope plasmid pDM2.G (Addgene, 12259) and 1 μg pBABE-puro Tandem LC3B plasmid using Lipofectamine 2000 for 6 h. Subsequently, the media containing tandem LC3B retrovirus were replaced with fresh media and cultured for 48 h. The tandem LC3B retrovirus was then harvested and filtered through a 0.45 μm syringe filter. CFU assay. To quantify intracellular bacteria, CFU assays were performed as described previously 12 . BMDMs were plated on 7H10 at a concentration of 2 × 10 5 cells per well and infected with Mtb for 4 h. The cells were then washed with phosphate buffered saline (PBS) to remove extracellular bacteria and treated with OMS-A, OMS-B, or INH in medium for 3 days. Thereafter, the intracellular bacteria were harvested, and the lysates were diluted two-fold in PBS. Each sample was plated on 7H10 agar plates and incubated at 37 °C in a 0.5% CO 2 incubator for 3 weeks.
Western blotting and enzyme-linked immunosorbent assay (ELISA). Western blotting and ELISA were performed as described previously 55 . For western blotting, cell lysates were denatured by boiling and separated on 12% acrylamide SDS-PAGE gels. Then, proteins were transferred to polyvinyl difluoride membranes (Millipore, Boston, MA, USA) and incubated with primary Abs (LC3, p-AMPKα, and p-ACC) diluted at a ratio of 1:1000. The bands were visualized using ECL (Millipore, Danvers, MA, USA) and exposure to chemiluminescence film (Fujifilm, Tokyo, Japan).
Flow cytometry. Intracellular LC3B levels were analyzed as described previously 12 . Stimulated cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature (RT), and permeabilized using 0.25% Triton X-100 in PBS for 10 min at RT. Cells were stained with primary Abs against LC3B (2775 S; Cell Signaling, 1:100) for 1-2 h at 4 °C followed by anti-rabbit IgG secondary Abs (7074 S; Cell Signaling, 1:100) for 1 h on ice. After two washes with PBS, cells were fixed in 4% paraformaldehyde and assayed immediately. The cells were examined using a FACSCanto II flow cytometer (Becton Dickinson, San Jose, CA, USA). Data were collected from 10,000-30,000 cells and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
LC3 punctate dot fluorescence intensity was measured using ImageJ analysis software. For colocalization analysis, the co-distribution of LC3 autophagosomes or LAMP2 phagolysosomes with bacterial phagosomes were quantified using the ImageJ analysis software. Each condition was assayed in triplicate, and at least 100 cells per well were counted.