2-aminoimidazoles collapse mycobacterial proton motive force and block the electron transport chain

There is an urgent need to develop new drugs against tuberculosis. In particular, it is critical to target drug tolerant Mycobacterium tuberculosis (M. tuberculosis), responsible, in part, for the lengthy antibiotic regimen required for treatment. We previously postulated that the presence of in vivo biofilm-like communities of M. tuberculosis could contribute to this drug tolerance. Consistent with this hypothesis, certain 2-aminoimidazole (2-AIs) molecules with anti-biofilm activity were shown to revert mycobacterial drug tolerance in an in vitro M. tuberculosis biofilm model. While exploring their mechanism of action, it was serendipitously observed that these 2-AI molecules also potentiated β-lactam antibiotics by affecting mycobacterial protein secretion and lipid export. As these two bacterial processes are energy-dependent, herein it was evaluated if 2-AI compounds affect mycobacterial bioenergetics. At low concentrations, 2B8, the lead 2-AI compound, collapsed both components of the proton motive force, similar to other cationic amphiphiles. Interestingly, however, the minimum inhibitory concentration of 2B8 against M. tuberculosis correlated with a higher drug concentration determined to interfere with the mycobacterial electron transport chain. Collectively, this study elucidates the mechanism of action of 2-AIs against M. tuberculosis, providing a tool to better understand mycobacterial bioenergetics and develop compounds with improved anti-mycobacterial activity.


2B8 collapses both components of the mycobacterial PMF. The MIC values against M. tuberculosis
are provided in Table 1 and the structures for 2B8 and RA13 in Supplementary Fig. S1. Based on our previous findings that 2B8 inhibits M. tuberculosis cell wall lipid export and protein secretion 29 , it was hypothesized that 2B8 could collapse the PMF required for these cellular processes 30,31 . PMF is collectively established by two parameters: Δψ and ΔpH 33 , with Δψ having a preponderant role in mycobacteria 13,34 . Thus, the effect of 2-AI compounds on M. smegmatis PMF was evaluated using the membrane potential and pH gradient-sensitive dyes DiSC 3 (5) and ACMA, respectively. Upon treatment, the three tested concentrations of 2B8 (31.25-125 µM), rapidly depolarized the membrane potential of live M. smegmatis (Fig. 1a) and collapsed the ΔpH generated by M. smegmatis IMVs energized with NADH (Fig. 1c). A similar result was observed with TRZ and CCCP (Fig. 1b,d).
2B8 increases mycobacterial oxygen consumption. In normoxic conditions, oxygen, the final electron acceptor, is reduced by electrons flowing through the ETC to generate water. The energy released by electron flow is harnessed to pump protons outside the cell membrane and form a proton gradient (or PMF) that drives  29 . b MIC of Carbenicillin, Meropenem and 2B8 against M. tuberculosis were previously reported in 29 . Experiments were repeated three times and performed in triplicate.
Scientific RepoRts | (2019) 9:1513 | https://doi.org/10.1038/s41598-018-38064-7 the enzymatic phosphorylation of ADP by ATP synthase 37 . In this process, known as oxidative phosphorylation, oxygen consumption is coupled to ATP synthesis 37 . Conversely, uncoupling occurs when increased oxygen consumption is not conducive to ATP synthesis. The prototypical uncoupler CCCP collapses the PMF by shuttling protons back from outside the cell membrane, to the inside 38 . In an effort to reestablish the PMF, the ETC is driven into a futile cycle in which oxygen consumption is increased but ATP synthesis is not commensurate. Knowing 2B8 collapsed components of the PMF, it was predicted 2B8 would increase mycobacterial oxygen consumption. Indeed, M. tuberculosis OCR increased in the presence of 2B8 (Fig. 2), leveling off at 62.5 µM 2B8. Thereafter, increasing concentrations of 2B8 resulted in decreasing M. tuberculosis OCR, such that no statistical difference was seen between basal OCR (without 2B8) and that induced by 250 µM 2B8 (highest tested concentration). A similar bell-shaped, dose-response effect was also observed for CCCP (Fig. 2). However, CCCP's effect was more potent than 2B8 as evidenced by the greater magnitude in fold-change and absolute OCR levels, as well as the lower compound concentration required to increase M. tuberculosis OCR. Meanwhile, BDQ (as previously shown in 35,36 ) and TRZ also significantly increased M. tuberculosis OCR, and this change persisted in the face of increasing compound concentration (Fig. 2). The maximum, absolute OCR levels induced by BDQ and TRZ were comparable to those induced by CCCP and 2B8, respectively (Fig. 2, bottom right panel). Finally, and consistent with the feeble effects on mycobacterial PMF, RA13 induced minimal changes in OCR (Fig. 2). These experiments were primarily performed in media without BSA, as its presence shifted the dose-response curve to the right and increased compounds' MICs (Supplementary Table S1), possibly due to drug-protein interactions.
2B8 depletes intracellular ATP levels in M. tuberculosis. To confirm that 2B8 has uncoupling activity (PMF collapse, increase OCR, ATP deficit), the amount of intracellular ATP was determined by luminescence in treated M. tuberculosis (luminescence was normalized to bacterial counts remaining after treatment). After 2 h treatment, 2B8 did not significantly affect intracellular ATP levels in M. tuberculosis (Fig. 3). Similarly, ATP levels were also unchanged in bacteria treated for 2 h with CCCP, despite rapidly collapsing both components of the PMF (Fig. 1), and potently increasing OCR (Fig. 2). At 24 h of treatment, however, ATP levels were lower in M.  (5) and ACMA were used to evaluate 2B8's effect on mycobacterial Δψ (a,b) and ΔpH (c,d), respectively. Uptake by live M. smegmatis slowly quenched DiSC 3 (5) fluorescence (a,b). Likewise, ACMA fluorescence was quenched upon energizing M. smegmatis IMVs with NADH (arrow), to create a ΔpH (c,d). Thereafter, bacteria or IMVs were treated with different drugs and monitored for fluorescence reversal. As indicated by fluorescence reversal, treatment with 31.25-125 µM 2B8 abruptly depolarized M. smegmatis membrane potential (a) and collapsed the ΔpH generated by M. smegmatis IMVs (c). Both parameters were also affected by 15 µM CCCP and 80 µM TRZ (b,d). The ΔpH (d) but not Δψ (b), was collapsed with 18 µM BDQ. A similar pattern was observed for 125 µM RA13 (a,c). 5 µM valinomycin and 10 µM nigericin were used as positive controls for Δψ and ΔpH. Experiments were repeated three separate times and representative data are shown. ***p < 0.001 by ANOVA. tuberculosis treated with CCCP or increasing concentrations of 2B8 (Fig. 3). As expected based on its mechanism of action (mycobacterial ATP-synthase inhibitor 12 ), 18 µM BDQ significantly reduced ATP levels at both time points (Fig. 3). A similar effect was also observed for 80 µM TRZ (Fig. 3). In contrast, ATP levels were not affected at either time point when M. tuberculosis was treated with RA13 ( Fig. 3). Altogether, the effects on components of the PMF, OCR and ATP confirmed that 2B8 has uncoupling activity in mycobacteria.

2B8 increases the NADH/NAD + ratio in M. tuberculosis.
To determine if 2B8 affected mycobacterial redox homeostasis, the NADH/NAD + ratio was evaluated. Indeed, the intracellular NADH/NAD + ratio was significantly increased as early as 2 h after treating M. tuberculosis with 62.5-125 µM 2B8 (Fig. 4) and this effect persisted at 24 h (Fig. 4). This ratio was also increased at both time points when M. tuberculosis was treated with TRZ ( Fig. 4), an expected finding taking in consideration that TRZ and other phenothiazines inhibit NADH oxidation by NDH-2 21 , the dominant mycobacterial complex I 18 . Interestingly, treatment with CCCP did not increase the NADH/NAD + ratio at either time point (Fig. 4), despite having similar uncoupling effects as 2B8 (Figs 1-3). Again, 125 µM RA13 was inactive. Consistent with previous results 36,39 , BDQ increased the NADH/ NAD + ratio at both time points (Fig. 4). Altogether, these results suggested that when tested at higher concentrations, the mechanism of action of 2B8 is not limited to an uncoupling effect. The acute effects of 2-AI compounds on mycobacterial redox homeostasis were confirmed by observing a concentration-dependent decrease in the rate of alamarBlue ® reduction ( Supplementary Fig. S2) 40 .

Inhibition of NADH oxidation by 2B8 is restored by CFZ.
To directly test whether 2B8 increased the NADH/NAD + ratio by inhibiting NADH oxidation, NADH decay catalyzed by M. smegmatis IMVs was evaluated by fluorescence at 340 ex /460 em 41 . NADH oxidation was almost completely inhibited by 5 mM KCN (Fig. 5a,b), confirming that NADH oxidation by M. smegmatis IMVs was performed by enzymes of the ETC and tuberculosis mc 2 6206 strain was monitored in real-time using high-resolution respirometry. Upon bacterial inoculation, the oxygen consumption rate (OCR) was measured at steady state (white columns, without compounds). Compounds were added in stepwise increments (depicted under each drug) and the change in OCR reported as fold-increase compared to the steady state without treatment. The lower right panel is a composite of the actual OCR levels achieved after stepwise increments in compound concentration, as depicted for respective compounds (for clarity, statistical significance and compound concentrations were not included for the lower right panel). Treatment with 2B8 and CCCP resulted in a bell-shaped effect: increased OCR was observed at lower concentrations, whereas higher concentrations resulted in OCR levels similar to no treatment. RA13 induced minimal changes in OCR, with only a small increase at 125 µM. BDQ increased OCR until 9 µM and this was maintained (albeit with a decreasing trend), upon treatment with additional BDQ. The OCR increase induced by TRZ was sustained at all the tested concentrations. Experiments were repeated three different times and all biological replicates were analyzed together. *p < 0.05, **p < 0.01, ***p < 0.001 by ANOVA. not cytoplasmic enzymes contaminating the IMVs. As expected, an inhibitor of NDH-2 such as TRZ 21 , significantly slowed down NADH oxidation (Fig. 5a,b). In contrast to KCN, however, NADH oxidation in the presence of TRZ progressed almost to completion when followed for an extended period of time (Fig. 5b), possibly catalyzed by mycobacterial type-1 NADH dehydrogenase 18 . Consistent with the effects on the NADH/NAD + ratio in intact mycobacteria (Fig. 4), NADH oxidation by M. smegmatis IMVs was significantly inhibited at concentrations greater than 62.5 µM 2B8 (Fig. 5a). Inhibition of NADH oxidation by 125 µM 2B8 was similar to KCN in that it persisted over an extended period of time (Fig. 5c). As previously reported 36,39 , NADH oxidation by mycobacterial IMVs was also inhibited by 18 µM BDQ (Fig. 5a), consistent with the increased NADH/NAD + ratio induced by BDQ in live M. tuberculosis (Fig. 4). Finally, it was observed that 125 µM RA13 did not have any effect, while 15 µM CCCP even had the tendency to accelerate NADH oxidation (Fig. 5a), however this was not statistically significant. These results suggested 2B8 could be increasing the NADH/NAD + ratio indirectly by inhibiting the ETC downstream, similar to KCN. Alternatively, 2B8 treatment could be blocking NADH oxidation by inhibiting mycobacterial NADH dehydrogenases 18 .
To explore these possibilities, it was evaluated whether CFZ could restore NADH oxidation when inhibited by 2B8. It was previously shown that in the presence of a reduced quinone pool (i.e. KCN treatment), CFZ restores NADH oxidation by serving as a surrogate electron acceptor in an NDH-2 catalyzed reaction 20 . However, this would not occur in the presence of an NDH-2 inhibitor such as TRZ 20 . As reported 20 , NADH oxidation by IMVs exposed to KCN was restored upon addition of CFZ, with NADH oxidation essentially reaching completion were quantified by RLUs at 2 and 24 h after treatment. Number of CFUs was also determined in parallel and data normalized to RLUs/CFUs. After 2 h treatment, only BDQ and TRZ significantly reduced M. tuberculosis intracellular ATP levels. After 24 h, in addition to BDQ and TRZ, intracellular ATP was also reduced with 62.5 and 125 µM 2B8, and CCCP. In contrast, RA13 had no effect. Experiments were repeated three separate times and all replicates were pooled together for analysis. **p < 0.01, ***p < 0.001 by ANOVA. Figure 4. 2B8 increases M. tuberculosis NADH/NAD + ratio. After 2 or 24 h treatment, intracellular NADH and NAD + concentrations were determined and the NADH/NAD + ratio was calculated. Treatment with 62.5 and 125 µM 2B8 for 2 h resulted in a significant increase of the NADH/NAD + ratio. Treatment with BDQ and TRZ also increased the ratio significantly. In contrast, 125 µM RA13 did not. After 24 h treatment, 62.5 and 125 µM 2B8, BDQ, and TRZ resulted in a significantly elevated NADH/NAD + ratio. However, RA13 and CCCP were ineffective at changing the NADH/NAD + ratio. Experiments were repeated three separate times and all replicates were pooled for analysis. *p < 0.05, **p < 0.01, ***p < 0.001 by ANOVA.
Scientific RepoRts | (2019) 9:1513 | https://doi.org/10.1038/s41598-018-38064-7 ( Fig. 5b). In contrast, the rate of NADH oxidation in the presence of TRZ was not altered by CFZ, with both curves superimposing (Fig. 5b). NADH oxidation in the presence of 2B8 was also restored by CFZ (Fig. 5c), albeit to a lower extent than observed for KCN ( Fig. 5b), as evidenced by the fact that oxidation did not progress to completion during the assayed time (Fig. 5c). This result with CFZ indicated that 2B8's effect on NADH oxidation is not through direct inhibition of mycobacterial NDH-2.

2B8 inhibits the ETC in M. smegmatis IMVs.
To specifically evaluate 2B8's effects on the mycobacterial ETC, M. smegmatis IMVs were energized with NADH, succinate or both, and INT reduction was monitored over time. Since INT is reduced by the ETC prior to cytochrome c oxidase 42 , KCN treatment did not affect INT reduction by IMVs energized with either single or combined substrates (Fig. 6). As expected, TRZ activity depended on the specific substrate(s) used to energize the IMVs: none, potent or intermediate inhibition was observed in the presence of succinate (Fig. 6a), NADH (Fig. 6b), or both substrates (Fig. 6c), respectively. In stark contrast, 78.125-125 µM 2B8 potently inhibited the ETC activity when initiated by succinate (Fig. 6a), NADH ( Fig. 6b) or both (Fig. 6c). In fact, out of all the tested drugs, 2B8 was the only one to potently inhibit ETC activity initiated by succinate. Again, CCCP had a tendency to increase ETC activity ( Fig. 6, but was not significant), whereas 125 µM RA13 only affected INT reduction in the presence of both substrates (Fig. 6c). Supplementary Table S2 summarizes the effects of different compounds on mycobacterial bioenergetics. In sum, high concentrations of 2B8 inhibited the ETC regardless of the substrate(s) energizing M. smegmatis IMVs, hence its effect is not due to a selective inhibition of NADH dehydrogenases. Moreover, 2B8 compromises mycobacterial bioenergetics differently than a classical uncoupler such as CCCP.   β-lactam potentiation by drugs targeting mycobacterial bioenergetics. Collectively, these studies confirmed the initial hypothesis that 2B8 targeted mycobacterial bioenergetics. Therefore, it was important to determine whether other compounds targeting mycobacterial bioenergetics, could recapitulate our initial observation that 2B8 potentiates β-lactams 29 . As shown in Table 2, β-lactam potentiation was stronger for 2B8, followed by TRZ and CCCP. In contrast, the effect was lower for BDQ and negligible for RA13.

Discussion
PMF and bioenergetic homeostasis provide electrochemical gradients and high-energy bonds required for bacterial physiology and survival 10 . Based on our previous observation that 2B8, a cationic amphiphile, affected energy-dependent processes such as protein secretion and lipid export (but not lipid biosynthesis) 29 , it was hypothesized this anti-biofilm compound potentiated β-lactams by collapsing components of the mycobacterial PMF. Using a series of mechanistic studies with intact mycobacteria or IMVs, it is shown herein that 2B8 collapses both components of the PMF and inhibits the ETC, collectively disturbing mycobacterial bioenergetics. Indeed, 2B8 affected multiple bioenergetics parameters including redox homeostasis, both components of the PMF (Δψ and ΔpH), oxygen consumption, ATP generation, ETC activity, NADH/NAD + ratio and NADH oxidation (Supplementary Table S2). These results plausibly explain the diverse, yet related effects induced by 2B8 such as biofilm disruption 28,43 , reversal of antibiotic tolerance 28 , and antibiotic potentiation 29 . Furthermore, it provides a tool to develop compounds with improved adjunct activity that could shorten antimicrobial therapy in TB and other infectious diseases. 2B8 differentially affected mycobacterial bioenergetics parameters in a concentration-dependent manner (Supplementary Table S3). Specifically, whereas uncoupling activity was observed below 62.5 µM, higher concentrations predominantly inhibited the ETC. Similar to other weak cationic amphiphiles 32 , the uncoupler activity is probably due to the ionizable group in 2B8's aminoimidazole moiety (pKa~8.5). Even though this headgroup is also present in RA13 and other inactive 2-AI compounds 28 , their overall potency is additionally dictated by the structure of different alkyl chains, covalently attached to the linker unit 43 . For instance, in the inactive RA13 it consists of a single chain, thirteen carbons in length ( Supplementary Fig. S1). Meanwhile, a shorter and branched alkyl chain is present in 2B8 (Supplementary Fig. S1). These modifications do not significantly impact their hydrophobicity (logD value at pH 7.4 for 2B8 and RA13 is 3.6 and 3.65, respectively), previously shown to correlate with uncoupling activity in cationic amphiphiles 32 . Instead, the shorter and/or branched alkyl chains in 2B8 could confer an enhanced ability to either traverse the mycobacterial cell envelope, partition in and/or flip-flop across the cell membrane to collapse the mycobacterial PMF and interfere with the ETC.
Compelling evidence indicate 2B8's mechanism of action is not limited to uncoupling activity. In fact, 2B8's MIC against M. tuberculosis (Table 1), correlates with the higher dose required to inhibit mycobacterial ETC, rather than the lower dose inducing uncoupling effects. Taking into consideration the importance of PMF on bacterial physiology, this finding was rather surprising and needs further investigation. Nevertheless, 2B8's effect on mycobacterial bioenergetics (PMF collapse and inhibition of the ETC), conforms to the strategy "uncoupler + additional target" proposed for cationic amphiphiles 32,44 , and currently being unveiled in both traditional and novel drugs. This is the case for pyrazinamide 45,46 , a first-line antibiotic currently used to treat TB. Moreover, TRZ was shown to collapse Staphylococcus aureus PMF 47 , in addition to the well-characterized inhibition of NDH-2. Herein, a similar result was obtained when evaluating TRZ effects on mycobacterial bioenergetics. Importantly, significant mycobacterial uncoupling activity was recently described for the novel compounds BDQ 35,36 and SQ109 48,49 , previously shown to target the mycobacterial ATP synthase 12 and the mycolate transporter MmpL3 50,51 , respectively. Inhibition of menaquinone biosynthesis was actually identified as a third target for SQ109 49 . Hitting numerous targets has several repercussions in drug research and development. It broadens the antimicrobial spectrum, as reported for SQ109 52 , nitazoxanide 53 and 2-AI compounds 54 . Furthermore, it decreases the likelihood of selecting for resistant mutants. Indeed, in the order of >10 12 bacteria were required to isolate mutants resistant to nitazoxanide 53 , whereas mycobacterial mutants to SQ109 have not been isolated 50 .
How 2B8 blocks mycobacterial ETC remains to be fully defined. Despite several attempts, resistant mutants have not been successfully obtained. However, this might require evaluating higher mycobacterial numbers as discussed above. The probability of isolating resistant mutants is also determined by the nature of the target(s), being less feasible if membrane function rather than enzymatic activity is compromised 10 . As reported for other cationic amphiphiles 32 , the hydrophobic moiety in 2B8 could mediate drug-membrane interactions affecting membrane packaging and/or lipid diffusion. These more subtle and acute effects on membrane physiology could affect the ETC and precede any evidence of the catastrophic mycobacterial cell membrane disruption observed only after prolonged incubation (24 h) with 2B8 ≥ 125 µM 29 . That 2B8 but not TRZ-mediated inhibition of NADH oxidation was partially restored by CFZ, ruled out NDH-2 as the target for 2B8. Furthermore, the fact that 2B8 inhibited INT reduction when the mycobacterial ETC was energized with either succinate, NADH (despite not directly inhibiting NDH-2), or both substrates suggests at least two distinct scenarios (Fig. 7): (a) Upstream of  Experiments are currently underway to determine how mycobacteria withstand 2B8's collapse of the PMF. Transcriptomics analysis indicated that in response to 2B8 29 , M. tuberculosis upregulated genes previously shown to be involved in drug efflux such as mmpL5 55 , rv1218c and rv1258c 56 , or detoxification such as rv3161c 57 . However, taking into consideration the fact that efflux pumps require PMF 58 , it is unlikely this constitutes a major mechanism counteracting 2B8's effects. Instead, mycobacteria could inactivate 2B8 by modifying the ionizable headgroup and/or the alkyl chains, jointly required for its activity. This is currently being evaluated to determine if 2B8's stability and/or efficacy is amenable to improvement via medicinal chemistry. Transcriptomics analysis also provided evidence that 2B8 induced remodeling of mycobacterial metabolism 29 . Mycobacterial carbon and nitrogen metabolism was altered by 2B8 treatment, as suggested by the upregulation of icl1, prpC and prpD (involved in the methylcitrate cycle 59 ), and downregulation of glnA1 and pknG (involved in the biosynthesis and regulation of glutamine and glutamate 60,61 ), respectively. Interestingly, it was previously shown that metabolic remodeling endowed M. tuberculosis the ability to survive for four days in the presence of BDQ at 300 × MIC 39 , a concentration determined herein to uncouple mycobacteria, in agreement with the initial results from Cook 35 and later confirmed by Steyn 36 . The metabolic adaptations induced by BDQ and 2B8 are very different and it remains to be determined if they play a role to counteract 2B8. However, this highlights the fact that mycobacteria are able to temporarily persist in the presence of uncoupling concentrations of an excellent drug such as BDQ (or for that matter, 2B8). Moreover, it emphasizes that uncoupling might not be sufficient against TB and when considering the strategy "uncoupler + additional target", the second component could be just as important or even more.
Lastly, the ability to potentiate β-lactam antibiotics against M. tuberculosis was compared between several compounds targeting mycobacterial bioenergetics at different sites (Table 2). Not surprisingly, carbenicillin and meropenem were particularly potentiated by TRZ, a compound affecting both mycobacterial bioenergetics and cell envelope permeability 29,62 , similar to 2B8. Meanwhile, β-lactam potentiation by CCCP was intermediate. This could be envisioned as the maximum β-lactam potentiation achieved by a compound principally acting as an uncoupler. Conversely, the weak β-lactam potentiation observed with BDQ at 0.5 × MIC could be attributed to the fact that BDQ does not have uncoupling activity in mycobacteria at this lower concentration 36 . In sum, at the tested drug concentrations, the magnitude of β-lactam potentiation (2B8 > TRZ > CCCP) could be collectively determined by: a) effects on mycobacterial bioenergetics (CCCP > TRZ~2B8), plus b) enhanced mycobacterial cell envelope permeability (only induced by 2B8 and TRZ 29,62 ), conducive to increased β-lactam penetration in 2B8-treated M. tuberculosis 29 . Ongoing experiments are being performed to determine if this concerted effect on mycobacterial bioenergetics and cell envelope permeability, is the mechanism behind 2B8's anti-biofilm properties.
It should be noted that whole-cell experiments were performed mainly in M. tuberculosis, whereas assays with IMVs were exclusively performed using M. smegmatis-derived membranes. Even though using an avirulent mycobacteria could be a limitation to our study, for the following reasons we consider M. smegmatis a valid source of mycobacterial membranes not significantly impacting the conclusions: a) whole-cell assays using M. smegmatis or M. tuberculosis gave similar results. This was the case for the membrane potential assay presented herein using M. smegmatis and DisC 3 (5) (Fig. 1), additionally evaluated by flow cytometry and DiOC 2 (3) staining in M. tuberculosis (not shown). The former is presented because it has the benefit of being a real-time assay; b) the overall potency of the tested compounds did not differ significantly between assays using M. tuberculosis whole-cells and M. smegmatis IMVs; c) there was a good correlation between both types of assays. For instance, the in vivo M. tuberculosis NADH/NAD + ratio was increased by TRZ, 2B8 and BDQ, but tended to decrease in the presence of CCCP (Fig. 4). These results paralleled the effects these compounds had on NADH oxidation (Fig. 5) and electron transport (Fig. 6) in M. smegmatis IMVs; d) our results with M. smegmatis IMVs evaluating restoration of NADH oxidation by CFZ (Fig. 5) and BDQ's effect on NADH oxidation (Fig. 5), are similar to those reported for M. tuberculosis IMVs 20,36 ; and e) due to the inside out membrane topology in IMVs, the mycobacterial inner membrane would be external and readily accessible to the compounds. Thus, the difference in M. smegmatis and M. tuberculosis cell envelope composition [3][4][5] , known to impact drug diffusion and probably contributing to 2B8's higher MIC against M. tuberculosis 29 , is bypassed in IMVs. Indeed, this could explain why 2B8's inhibitory effect on the ETC of M. smegmatis IMVs was observed at concentrations ≥78 μM (Fig. 6), whereas higher concentrations (≥93 μM) were required to decrease OCR (Fig. 2). It is however, possible that subtle differences reported in the ETC of these related organisms 18 , could have some repercussions on the experimental outcome.
In conclusion, it was determined that 2B8 collapses both components of the mycobacterial PMF and interferes with the ETC. Further investigation into the mechanism of action of 2B8 could lead to the elucidation of novel drug targets in M. tuberculosis and increase our knowledge in the field of mycobacterial bioenergetics.

Methods
All materials were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless indicated.
Bacterial strains, media, and culture conditions. M. smegmatis mc 2 155 and the BSL2 strain M. tuberculosis H37Rv mc 2 6206 were grown as before 29

Real-time measurement of oxygen consumption rate by high-resolution respirometry.
Mycobacterial oxygen consumption in the presence or absence of 2-AI compounds or other drugs was monitored in real-time using Oroboros Oxygraph-2k (http://www.oroboros.at, Oroboros Instruments, Innsbruck, Austria). The chambers of Oroboros Oxygraph-2k were filled with 2.5 mL of 7H9 supplemented with 0.2% dextrose and glycerol. Basal oxygen level (nmol/mL) and oxygen consumption rate (OCR) (pmol/s × mL) without bacteria was measured and calibrated using Datlab4 software (Oroboros Instruments). Thereafter, mid-log phase M. smegmatis or M. tuberculosis cultures were adjusted to an OD 600 of 0.5 in the same media used to fill the chambers. Finally, 100 or 400 µL of M. smegmatis or M. tuberculosis respectively, was injected into the Oroboros Oxygraph-2k chambers with a Hamilton Microliter Syringe (Hamilton Company, Reno, NV, USA) and the OCR level of non-treated bacteria was recorded. Continuous OCR monitoring was performed upon addition of increasing concentrations of 2-AI compounds, CCCP, BDQ or TRZ, as indicated in each experiment. Fold change in OCR levels were β-lactam potentiation assay. β-lactam potentiation assay was performed as previously described 29 .
Briefly, the minimum inhibitory concentration (MIC) of nigericin, valinomycin, CCCP, BDQ and TRZ against M. tuberculosis was determined. Thereafter, the MIC of carbenicillin or meropenem against M. tuberculosis was determined in the presence or absence of nigericin, valinomycin, CCCP, BDQ and TRZ at a concentration equivalent to 50% of their respective MIC. β-lactam potentiation was calculated by dividing the MIC of each β-lactam by the MIC of each β-lactam plus drug.
Generation of IMVs. Preparation of IMVs from M. smegmatis cells was done as previously described with minor modifications 14 . Briefly, 5-10 g wet weight of M. smegmatis were resuspended at a 1:2 ratio (wt/vol) in breaking buffer (50 mM MOPS pH 7.5, 2 mM MgCl 2 ) and protease inhibitor cocktail (Roche, Basel, Switzerland). The suspension was stirred for 1 h at room temperature in the presence of 1.2 mg/mL lysozyme. The MgCl 2 concentration was adjusted to 15 mM and 0.2 mg/mL DNase I was added. Bacteria were lysed by passing six times through a pre-chilled French Press at 20,000 psi (Thermo Electron, Waltham, MA, USA). The lysate was centrifuged at 3,000 × g for 30 min to pellet unbroken cells and the supernatant was further centrifuged at 27,000 × g for 30 min to pellet cell wall. The supernatant was harvested and centrifuged for 1 h at 100,000 × g using an ultracentrifuge (Beckman Coulter, Brea, CA, USA), to pellet IMVs. After removing the supernatant, pelleted IMVs were resuspended in breaking buffer and the protein concentration was measured using Pierce BCA protein assay (Thermo Scientific, Waltham, MA, USA). Glycerol was added to a final concentration of 10% and aliquots of IMVs stored at −80 °C until further use.
Determination of ΔpH collapse with IMVs. Collapse of ΔpH was determined in M. smegmatis IMVs as previously described with minor modifications 35 . The pH-sensitive, fluorescent dye 9-amino-6-chloro-2-methoxyacridine (ACMA, Life Technologies) was used instead of acridine orange. IMVs were diluted to 0.1125 mg/mL in 10 mM HEPES (pH 7.5), 100 mM KCl, 5 mM MgCl 2 and added to a black-wall, 96-well plate (Corning). IMVs were pre-incubated with 2 µM ACMA at 37 °C for 30 minutes and the baseline 410 ex /460 em fluorescence was measured using a Biotek Synergy HT multi-mode plate reader. IMVs were energized with 5 mM NADH and incubated until ACMA fluorescence was quenched due to generation of ΔpH. ETC activity assay with IMVs. The ETC activity was measured as previously described with minor modifications 64  15 µM CCCP, 18 µM BDQ, 80 µM TRZ, and 5 mM potassium cyanide (KCN). ETC activity was initiated by adding 75 µL of 0.2% Triton X-100 in PBS supplemented with either 1 mM NADH, 130 mM sodium succinate or both. Absorbance at 490 nm was immediately monitored at 37 °C for 10 minutes using a SpectraMax M series multi-mode plate reader (Molecular Devices, Sunnyvale, CA, USA).
Determination of NADH oxidation by IMVs. NADH oxidation was measured using fluorescence emission at 460 nm when excited at 340 nm as previously described 41  Restoring NADH oxidation with CFZ. NADH oxidation assay was performed as described above. M.
smegmatis IMVs were incubated with 1 mM NADH in the presence of 125 µM 2B8, 80 µM TRZ or 5 mM KCN and NADH oxidation was monitored for 2 min. Thereafter, to determine if CFZ could restore NADH oxidation by acting as an alternative electron acceptor for NDH-2 20 , 42 µM CFZ or DMSO was added and NADH oxidation was monitored for an additional 10 minutes. CFZ was added a second time and NADH oxidation monitored as above.
Statistical analysis. Statistical analyses were carried out using Student t-test or one way ANOVA with Tukey's post hoc test using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). P-values less than 0.05 were considered significant.

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.