Targeting bacterial energetics to produce new antimicrobials

Drug Resistance Updates 36 (2018) 1–12

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Drug Resistance Updates journal homepage: www.elsevier.com/locate/drup

Invited review

Targeting bacterial energetics to produce new antimicrobials a

Kiel Hards , Gregory M. Cook a b

a,b,⁎

MARK

Department of Microbiology and Immunology, School of Biomedical Sciences, University of Otago, Dunedin 9054, New Zealand Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1042, New Zealand

A R T I C L E I N F O

A B S T R A C T

Keywords: Bioenergetics Antimicrobials Drug discovery Bacteria Respiration

From the war on drug resistance, through cancer biology, even to agricultural and environmental protection: there is a huge demand for rapid and effective solutions to control infections and diseases. The development of small molecule inhibitors was once an accepted “one-size fits all” approach to these varied problems, but persistence and resistance threaten to return society to a pre-antibiotic era. Only five essential cellular targets in bacteria have been developed for the majority of our clinically-relevant antibiotics. These include: cell wall synthesis, cell membrane function, protein and nucleic acid biosynthesis, and antimetabolites. Many of these targets are now compromised through rapidly spreading antimicrobial resistance and the need to target nonreplicating cells (persisters). Recently, an unprecedented medical breakthrough was achieved by the FDA approval of the drug bedaquiline (BDQ, trade name Sirturo) for the treatment of multidrug-resistant tuberculosis disease. BDQ targets the membrane-bound F1Fo-ATP synthase, validating cellular energy generating machinery as a new target space for drug discovery. Recently, BDQ and several other FDA-approved drugs have been demonstrated to be respiratory “uncouplers” disrupting transmembrane electrochemical gradients, in addition to binding to enzyme targets. In this review, we summarize the role of bioenergetic systems in mycobacterial persistence and discuss the multi-targeting nature of uncouplers and the place these molecules may have in future drug development.

1. Bioenergetic systems in persistent bacteria 1.1. Overview of the respiratory circuit Although the term “bioenergetics” could be broadly applied to any biological reaction involving an energy change, it colloquially applies to the mechanisms that organisms use to store and utilize different energy sources (Nicholls and Ferguson, 2013a). All life has evolved to harness the energy inherent in disequilibria, whether it be in chemical mass:action ratios, abiotic gradients (i.e. salt, pH) or electrical potential differences (Schoepp-Cothenet et al., 2013). The main feature of bioenergetic systems are biological membranes (Nicholls and Ferguson, 2013a): they act as a barrier to allow the establishment of electrochemical gradients that are efficiently transduced into chemical energy, according to cellular demand. The study of bioenergetics is focused on understanding the membrane-bound enzyme complexes that effect these transductions. All organisms require an electrochemical gradient, in the form of either a proton motive force (PMF) or sodium motive force (SMF), to survive and grow (Cook et al., 2014a). The energy in these gradients is consumed for a variety of processes, such as the synthesis of ATP and

⁎

active transport of solutes from the environment. ATP and strong metabolic reductants, like NADH, can in fact be generated by soluble cytoplasmic enzymes, which is the primary mechanism used by many fermentative organisms. However, the PMF or SMF is still required and these organisms will use alternative mechanisms, such as ATP hydrolysis or solute export, to drive their generation (Cook et al., 2014a). Furthermore, the generation of the PMF or SMF by membrane-bound respiratory complexes are evolutionarily conserved and existed in the lowest universal common ancestor of Eubacteria and Archaea (Schoepp-Cothenet et al., 2013). Due to their prevalence and conservation, this review will consider only those organisms that encode the intact respiratory chains that defines the backbone of bioenergetics: henceforth “respiratory organisms”. Respiratory organisms require three components for the generation and maintenance of an electrochemical gradient (Fig. 1A): a physical barrier, enzymes to generate the gradient and enzymes to consume the gradient. The electrochemical gradient itself links these components and can be likened to a simple electrical circuit (Fig. 1B). This analogy holds even when discussing complex systems and most bioenergetic parameters are derived from electrical theory (Nicholls and Ferguson, 2013b). For example, the electrochemical gradient can be expressed in

Corresponding author at: Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin 9054, New Zealand. E-mail address: [email protected] (G.M. Cook).

https://doi.org/10.1016/j.drup.2017.11.001 Received 7 June 2017; Received in revised form 25 October 2017; Accepted 31 October 2017 1368-7646/ © 2017 Elsevier Ltd. All rights reserved.

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Fig. 1. Electric circuit analogy for the respiratory chain. Biological membranes (A) form a circuit of protons that have a potential difference (ΔpH, usually measured in mV) across the membrane. The respiratory chain and ATP synthase are analogous to the battery and lightbulb of a simple electrical circuit (B). The proton permeability of the membrane and activity of proton translocating enzymes (e.g. ATP synthase) determine the resistance and hence proton current. Introducing an uncoupler (C), like CCCP, provides a low resistance pathway; causing current and respiratory activity to increase substantially. This is analogous to introducing a wire (D) across the circuit described in (B). Current and voltage values as described in (Nicholls and Ferguson, 2013c).

millivolts and proton (or sodium ion) flux is measured in amps. Most organisms tend to generate an electrochemical gradient of ∼150 mV (Schoepp-Cothenet et al., 2013), which is comprised by varying amounts of both a charge (Δy) and proton/sodium (ΔpH, ΔpNa+) gradients. It is important that consumption and generation are precisely balanced: overly high magnitudes are susceptible to proton-leaks (Brand et al., 1994) and/or increase of the total system’s resistance (respiratory backpressure), while an overly high proton current decreases the total voltage due to internal resistance in the battery-like components (Nicholls and Ferguson, 2013b). In both cases, the mass:action ratio of reducing equivalents (like the NADH/NAD+ couple) will be disturbed, having a feedback effect on all intracellular reactions. An important proof of this theory was the observation that bioenergetically-active membranes could be short-circuited (Nicholls and Ferguson, 2013b). Chemicals that can disrupt the permeability of membranes, by forming physical pores or specifically binding protons or ions, are equivalent to introducing a small wire across an electrical circuit (Fig. 1C & D). The cellular response is to increase the activity of PMF/SMF-generating complexes to be commensurate with the reduced resistance (Nicholls and Ferguson, 2013c). This is ultimately futile due to internal resistance and the lack of useful work performed in this low resistance circuit. Chemicals achieving this are called “uncouplers” as they uncouple the generation of electrochemical gradients from ATP synthesis by F-type ATP synthases. A summary of canonical uncouplers is provided in Fig. 2, where molecules can either dissipate both components of the electrochemical gradient (e.g. CCCP) or only one component (e.g. valinomycin, nigericin, gramicidin). It is therefore important that the functions of individual components of the respiratory circuit are considered in light of the electrochemical gradients, which link the entire system.

1.2. Electron transfer and components of bacterial respiratory chains Most reactions involved in the generation of electrochemical gradients are redox driven. High-energy precursors, such as NADH, are oxidized to release high-energy electrons, depending on the reduction potential of the particular redox couple (Table 1). This is transferred through several enzyme complexes and lipid-soluble electron carriers, with increasing reduction potentials, and the released energy is harnessed to generate electrochemical gradients by vectorial proton pumping or charge separation/redox loop mechanisms (Fig. 3). Electrons generally terminate on the reduction of oxygen, due to its high reduction potential, but alternatives such as fumarate and nitrate are also commonly used (Cook et al., 2014a). The total system is referred to as the electron transport chain or respiratory chain. Bacterial respiratory chains are far more diverse than their eukaryotic counterparts (Cook et al., 2014a), but follow the same general structure: oxidative electron liberating complexes are connected to reductive electron terminating complexes via electron carriers (quinones, cytochrome c) and intermediary complexes in certain cases. For electron donating complexes, bacteria most frequently encode from two types of NADH:quinone oxidoreductase (Ndh1 c.f. complex I, Ndh2), succinate dehydrogenases (c.f. complex II), formate dehydrogenases and hydrogenases. While for electron accepting complexes, bacteria most frequently encode two types of terminal cytochrome oxidase (heme-copper oxidases, c.f. complex IV and cytochrome bd), nitrate reductases, nitrite reductases, fumarate reductases, tetrathionate reductases and hydrogenases (Cook et al., 2014a). Menaquinone derivatives are the most frequently utilized electron carriers, as they are the ancestral quinone (Schoepp-Cothenet et al., 2013), while higher potential ubiquinones can be utilized in some Alpha-, Beta- and Gammaproteobacteria. Given that complex IV frequently exists in functional association with quinol:cytochrome c oxidases (c.f. complex III) (Melo and Teixeira, 2016), it is unlikely that cytochrome c exists as an 2

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Fig. 2. Comparison of protonophores and ionophores. (1) Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) is an electrogenic protonophore. CCCP is driven to the periplasm by the Δψ, while CCCPH is driven to the cytoplasm by the ΔpH. It can equilibrate both Δψ and ΔpH. (2) Valinomycin is an electrogenic ionophore, selective for potassium ions, which equilibrates the potassium gradient and dissipates the Δψ. (3) Nigericin is a hydrophobic weak carboxylic acid, which can traverse the membrane as its either protonated acid or neutral salt. It dissipates chemical gradients (i.e. ΔpH) but maintains the charge (one positive charge exchanged for one positive charge: electroneutral). (4) Gramicidin is a channel-forming ionophore, making the membrane more permeable to ions, with all gradients equilibrating according to passive diffusion. The effect of each chemical on pH gradients, alkali metal concentration ([metal]) or the charge gradient (i.e. Δψ) is indicated.

1.3. Bacterial persistence and the role of respiration

Table 1 Redox potential of electron donors and acceptors discussed in this work. Redox couple +

H /H2 NAD+/NADH Cytochrome c3 ox/red FAD/FADH2 Oxaloacetate2−/malate2− Menaquinone ox/red S4O62−/S2O32− Fumarate/succinate Ubiquinone ox/red NO2−/NO NO3−/NO2− O2/H2O a

Eo’

(mV)

Persistence is a physiological phenomenon where a subpopulation of bacterial cells resist antibiotic killing, but without modifying their genetic code (Harms et al., 2016). This is distinguished from true developmental processes like sporulation (Rittershaus et al., 2013). It is well described for infection models of Salmonella, uropathogenic Escherchia coli and Mycobacterium tuberculosis (Blango and Mulvey, 2010; Claudi et al., 2014; Manina et al., 2015). The true extent of clinical persistence is not understood, although notable parallels can be drawn with several different infections and diseases; such as therapeutic-resistant cancer stem cell populations and Candida albicans infection (LaFleur et al., 2006; Zhang et al., 2015). The molecular mechanisms underlying bacterial persistence are poorly understood, although the stringent response and toxin antitoxin systems are heavily implicated in the formation of persistent bacteria (Maisonneuve et al., 2016). A universal feature of persistence is the induction of a slow growing or metabolically dormant state (Rittershaus et al., 2013). This would, by definition, implicate respiration and energy metabolism, however very few studies have directly investigated its role in drug persistence. The SOS-induced TisB peptide has been found to allow the formation of ciprofloxacin persistence in E. coli (Dörr et al., 2010), which is known to reduce intracellular ATP concentrations in E. coli (Unoson and Wagner, 2008). The latter authors proposed this was due to dissipation of the PMF, although this was not directly tested. In support of respiratory effects, and the most direct support for such a hypothesis so far, it was found that energizing cells with respiratory carbon sources enhanced killing by aminoglycosides in E. coli and Staphylococcus aureus (Allison et al., 2011). This was due to the resuscitation of cells through an

a

−414 −320 −290 −220 −172 −74 +24 +33 +113 +350 +433 +818

Values as described in Thauer et al. (1977).

independent electron carrier, unlike quinones. A schematic of the reactions performed in each complex is shown in Fig. 4. There is also evidence from methanogens that ferredoxins may also be involved in respiratory electron transport, while some hydrogenases are co-transcribed with ferredoxins (Berney et al., 2014b; Welte et al., 2010). Compared to the four complexes used in mitochondria, there is a huge diversity in the configuration of electron donors, acceptors and transfer molecules that microorganisms can use (this list being by no means exhaustive). This diversity allows Bacteria and Archaea to occupy a wide variety of habitats and reconfigure their electron transport chain components according to changing habitat conditions.

Fig. 3. Vectorial and redox loop mechanisms of PMF generation. (1) Vectorial proton transfer involves the direct enzymatic transfer of protons from the cytoplasm to the periplasm. This is driven by the energy released in the particular redox reaction (NADH oxidation pictured). (2) Redox loop transfer (a.k.a. charge separation or scalar proton translocation) is an indirect transfer of protons across the membrane. This is achieved by oxidization and reduction of quinones at opposite sides of the membrane, dependent on the enzyme’s location of the quinone catalytic site. The reaction inherently releases protons into the periplasm, but is scalar due to the bulk distribution of quinones in the membrane (i.e. the oxidized quinone may be different from the one reduced). Nuo, type I NADH:quinone oxidoreductase; Ndh, type II NADH:quinone oxidoreductase; Nar, nitrate reductase.

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Fig. 4. Common components of bacterial respiratory chains. (A) Reactions performed by common electron donating complexes of bacterial respiratory chains. Pictured from left to right: type I NADH:quinone oxidoreductase (Nuo), type II NADH:quinone oxidoreductase (Ndh), succinate dehydrogenase (SDH), formate dehydrogenase (Fdh), hydrogenase (Hyd). (B) Reactions performed by common electron accepting complexes of bacterial respiratory chains. Pictured from left to right: cytochrome bc1-aa3 supercomplex (Qcr-Cta, complex III/IV), cytochrome bd oxidase (Cyd), nitrate reductase (Nar), fumarate reductase (Frd), tetrathionite reductase (Ttr), hydrogenase (Hyd, a distinct class from the previous hydrogenase).

genus. Fermentative hydrogen production through [NiFe]-hydrogenases allow M. smegmatis to survive extended periods of microaerobic conditions (Berney et al., 2014a). Encoding [NiFe]-hydrogenases itself would have seemed paradoxical until recently, as canonical complexes are inactivated by molecular oxygen (Vincent et al., 2005), but mycobacteria encode the newly discovered oxygen-insensitive enzymes (Berney et al., 2014b; Greening et al., 2015). Cells do not truly ferment to drive growth through ATP derived from substrate level phosphorylation, as the F1Fo-ATP synthase is essential on fermentable and nonfermentable carbon sources (Tran and Cook, 2005). However, adaptation of an anaerobic-type metabolism to aerobic lifestyles appears to give mycobacteria their trademark hardiness and may reflect on the mechanisms of persistence as a whole. Pathogenic mycobacteria notably do not encode homologous hydrogenases, although appear to have adopted the same general principle. Export of succinate as a fermentation-like end product enables M. tuberculosis to maintain the membrane potential (Δψ) during equivalent microaerobic conditions (Eoh and Rhee, 2013), while the pathogen can use fumarate and nitrate for alternative terminal electron acceptors (Malm et al., 2009; Sohaskey, 2008; Watanabe et al., 2011). This does not support anaerobic growth and is again the adaptation of these enzymes to maintain redox balance under hypoxia (Cook et al., 2014b). Individual mycobacterial respiratory complexes are discussed in the following section. Metabolism in the absence of growth, at first glance, appears to be a bioenergetic paradox. The demand for ATP will be reduced to that solely required for cell maintenance (non-growth) processes. We have previously calculated that the maintenance ATP requirements for M. smegmatis (non-growth functions) are ∼88-fold less than that for growth (Tran and Cook, unpublished data). Therefore, the F1Fo-ATP synthase, the primary mechanism of clearing protons from the periplasmic space and preventing PMF hyperpolarization, needs to be 88fold less active to avoid ATP accumulation in the absence of growth. The observed decrease in intracellular ATP during non-replicating persistence suggests that the F1Fo-ATP synthase may in actuality be even less active (Gengenbacher et al., 2010). Cells would theoretically need to reduce their respiratory chain activity to compensate (or risk

NADH-driven respiratory chain, which was confirmed genetically, by uncoupling cells through cyanide treatment. The authors proposed that the generation of a PMF facilitated uptake of aminoglycosides, as quinolones and β-lactams were not responsive to these treatments. One might reconsider this proposal in the light of recent data that proposes a correlation between antibiotic cidalitiy (including aminoglycosides) and respiratory activity (Lobritz et al., 2015), although as this study was performed against growing cells the results cannot be unambiguously compared. Regardless, it is known that a PMF is required for the viability of persistent M. tuberculosis (Rao et al., 2008). These findings suggest that low efficiency or non-proton pumping respiratory chain configurations are used to maintain flux through the respiratory chain (sometimes referred to as redox balance). Flux, rather than the absence of respiration, is necessary for the reasons discussed above. Understanding the bioenergetic adaptations of persistent cells is the key to understanding their physiology. This has been well studied in the Mycobacterium genus and will be discussed in the following section. 2. Respiration and energy generation in mycobacteria 2.1. Respiratory flexibility and metabolism in the absence of growth Mycobacteria are a genus of organisms that have the ability to metabolize various energy sources in the absence of growth (Cook et al., 2009, 2014b). This is distinguished from drug-persistence as mycobacteria have been shown to adopt this lifestyle in response to environmental cues, rather than stochastically in response to drug treatment (Berney and Cook, 2010; Gomez and McKinney, 2004), although the states appear physiologically equivalent. Mycobacteria have adapted to inhabit a wide range of intracellular and extracellular environments; including notable human pathogens such as M. tuberculosis and soil saphrophytes such as Mycobacterium smegmatis (Cook et al., 2009). Key to this is the proficiency of mycobacteria at switching their electron donor and acceptor utilization according to availability. Originally thought to be obligately aerobic non-fermentative organisms, several studies are redefining the metabolic breadth of this 4

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Shi et al., 2010). This suggests that succinate dehydrogenases and malate:quinone oxidoreductases are key members of the non-proton pumping respiratory chain, used during mycobacterial persistence.

backpressure and wasteful energy loss through passive proton leaks), consume the PMF through an alternative mechanism, or generate a smaller PMF from the same input of reductant. In reality, the mycobacterial membrane is extremely impermeable to protons, with a three-fold lower proton permeability compared to B. subtilis (Tran et al., 2005). This suggests that passive proton leakage is a comparatively small problem for this genus. Mycobacteria do not appear to maintain a high rate of respiration in response, however, as slow growing chemostat M. smegmatis cells maintain a PMF ∼50 mV lower than during unregulated batch growth (Berney and Cook, 2010; Rao et al., 2001). Cells in fact appear to adopt lower efficiency, non-proton pumping, respiratory chain compositions during persistence (Shi et al., 2005). It is also known that mycobacteria are proficient at interconversion of the Δψ and ΔpH components of the PMF (Rao et al., 2001), implying cation transporters are a major mechanism for modulating the cell’s overall PMF. Taken together, this would suggest that mycobacteria tightly regulate the generation of a PMF to be commensurate with the need to recycle reducing equivalents, in non-replicating conditions. Tightly linked to this are the reactions of central carbon metabolism, which can both produce and utilize reducing equivalents and ATP. Two specific reactions are directly linked to respiratory chain activity. The succinate to fumarate and malate to oxaloacetate steps of the citric acid cycle are catalyzed by the respiratory-linked succinate dehydrogenases and malate:quinone oxidoreductases, respectively (Cook et al., 2014b). During hypoxia, M. tuberculosis favors the accumulation of the succinate through the glyoxylate shunt and methylcitrate cycle (Eoh and Rhee, 2014, 2013), two alternative anabolic pathways for the citric acid cycle that produce less NADH than the standard route (Fig. 5). This overall reaction appears to form a cycle sustaining succinate dehydrogenase and malate:quinone oxidoreductase activity, while avoiding the generation of NADH. This cycle must be sustained in the long term by continued propionyl-CoA and acetyl-CoA input. These precursors can be derived from fatty acid metabolism of triacyl glycerides, known to accumulate in response to non-growth conditions (Daniel et al., 2011;

2.2. Diversity of respiratory complexes in mycobacteria Mycobacteria encode a diverse respiratory chain with multiple primary dehydrogenases and terminal acceptor reductases (Fig. 6). In this section, focus is given to respiratory complexes identified as particularly important to persistence or drug resistance of the genus. Mycobacteria utilize menaquinone/menaquinol (MQ/MQH2) as the primary conduit between electron donating and electron accepting reactions (Cook et al., 2014b). MQ has a stronger reduction potential compared to ubiquinone (Em = −74 mV compared to +113 mV for ubiquinone) (Thauer et al., 1977), therefore some primary dehydrogenase reactions will not occur spontaneously in mycobacteria. As the primary means of transferring electrons from complex to complex, the MQ biosynthesis pathway is understandably important for both growth and non-replicating persistence (Dhiman et al., 2009). Mycobacteria synthesize menaquinone MK-9(II-H2) from chorismate and polyisoprenyldiphosphate precursors from two converging pathways that generally resemble the E. coli synthesis pathway (Debnath et al., 2012). More recently, it has been reported that a second pool of polyketide quinones may be synthesized in oxygen limited biofilms (Anand et al., 2015). The reduction potential of these polyketide quinones have not yet been solved and the complexes capable of switching between MQ and polyketide quinone utilization are not known. NADH is the primary electron donor for growth in mycobacteria and is consumed by two different dehydrogenases: the type I NADH dehydrogenase (Nuo), equivalent to the mitochondrial complex I, and a microorganism-specific type II NADH dehydrogenase (Ndh2). Ndh2 appears to be the preferred NADH dehydrogenase: Nuo is not essential for M. tuberculosis growth or persistence (Rao et al., 2008), is only 5% as active as Ndh2 in M. smegmatis (Vilcheze et al., 2005) and is downregulated in M. tuberculosis infection models (Schnappinger et al.,

Fig. 5. Citric acid cycle in mycobacteria and its metabolic rerouting. Selected pathways of central carbon metabolism in mycobacteria are shown. Coloured arrows, according to the key, indicate the production or consumption of particular cofactors. An alternative cycle through the glyoxylate shunt and methylcitrate cycle is indicated by bold arrows and is discussed in text.

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Fig. 6. Organization of electron transport chain components in mycobacteria. Mycobacteria connect electron donating and accepting complexes using a menaquinone pool, other classes of quinone are not encoded in mycobacteria. Only primary dehydrogenases, those that directly connect to quinone reduction, are shown. Several other cytoplasmic enzymes are capable of contributing to the NADH/NAD+ pool and are not shown. DH – dehydrogenase, G-3-P – glycerol-3phosphate, CO – carbon monoxide, MQ – menaquinone, OR – oxidoreductase.

mycobacteria can vary based on the growth conditions (Berney and Cook, 2010; Rao et al., 2001), this could plausibly explain the regulatory differences. Malate:quinone oxidoreductases (MQO) have only recently become appreciated for their roles in bacterial metabolism. They catalyze essentially the same reaction as malate dehydrogenases (MDH), but are instead coupled to quinones (Molenaar et al., 2000; van der Rest et al., 2000). Structurally, they seem very similar to type II NADH dehydrogenases and are proposed to have a common ancestor (Mogi et al., 2009). M. tuberculosis encodes both MQO (rv2852c) and MDH (rv1240). While MQO does not appear to be essential for M. tuberculosis (Griffin et al., 2011; Zhang et al., 2012), it has been proposed the concert action of MQO and MDH is functionally redundant with the reaction catalyzed by Ndh2 (Miesel et al., 1998; Molenaar et al., 2000; Vilcheze et al., 2005). Roles for MQO may therefore exist when NADH is not utilized as an electron donor. The uncontested terminal electron acceptor for mycobacterial respiration is oxygen. While studies have reported supplementary utilization of other alternative electron acceptors (Berney et al., 2014a; Sohaskey and Modesti, 2009; Tan et al., 2010; Watanabe et al., 2011); no study has yet reported the growth, survival or persistence of mycobacteria under strictly anaerobic conditions. Oxygen reduction is performed by two distinct complexes. The first is a proton-pumping supercomplex (Kim et al., 2015) of menaquinol:cytochrome c oxidoreductase (cytochrome bcc/Qcr) and an aa3-type cytochrome c oxidase (Cta), analogous to mitochondrial complexes III and IV. Second is a cytochrome bd-type oxidase, which does not pump protons and is proposed to have a high affinity for oxygen as described for the E. coli enzyme (D’Mello et al., 1996). The Qcr-Cta supercomplex is the preferred pathway under routine culture conditions, is dispensable in M. smegmatis and is essential in M. tuberculosis (Matsoso et al., 2005). Cytochrome bd, on the other hand, has a variety of roles in both persistence and general drug resistance. This complex is the predominant terminal complex in the respiratory chain of oxygen-limited mycobacteria (Berney and Cook, 2010; Shi et al., 2005), suggesting a role in mycobacterial persistence, and in the fast-growing saprophyte M. smegmatis, cytochrome bd can compensate for the disruption of the QcrCta complex (Matsoso et al., 2005). The complex has recently been

2003). Ndh2 is encoded by either one or two homologs, depending on the species (Cook et al., 2014b), and is suggested to be essential for the growth of M. tuberculosis (Griffin et al., 2011; Sassetti et al., 2003; Weinstein et al., 2005). Unlike the multi-subunit, proton-pumping, Nuo; Ndh2 is a single small (50–60 kDa) membrane-bound protein that catalyzes the same reaction, but without proton pumping. It is only partially embedded in the membrane (monotopic) and contains a noncovalently bound FAD co-factor (Heikal et al., 2014). Mutations in the M. smegmatis Ndh2 result in pleiotropic phenotypes, but most notably resulted in resistance to isoniazid (INH) and ethionamide (ETH) (Miesel et al., 1998; Vilcheze et al., 2005). These phenotypes have been attributed to altered NADH/NAD+ ratios perturbing intracellular redox homeostasis. While NADH may not be a primary electron donor during standard mycobacterial persistence (Eoh and Rhee, 2014, 2013), its contribution to drug induced persistence should not be overlooked. The importance of alternative electron donors, succinate and malate, from the citric acid cycle were discussed in the previous section. Succinate dehydrogenases (SDHs), equivalent to mitochondrial complex II, are multi-subunit integral membrane proteins that utilize succinate as reductant for electron transport (Hägerhäll, 1997). They contain FAD, iron sulfur clusters and varying amounts of b-type heme (Lancaster, 2013). They have homology with fumarate reductases (FRDs), which perform the reverse reaction (discussed below), and the reaction performed in vivo cannot be predicted based solely on amino acid sequence (Cook et al., 2014b). Mycobacteria can encode either 2 or 3 annotated SDHs/FRDs based on the species (Hartman et al., 2014; Pecsi et al., 2014; Watanabe et al., 2011). It is not clear why it is necessary to encode 3 highly similar enzymes, but Sdh1 and Sdh2 have been found to be differentially upregulated between energy- and oxygen-limiting conditions (Berney and Cook, 2010). This suggests that each enzyme may be responsible for coping with the different environmental pressures that induce non-growing states. The succinate dehydrogenase reaction does not occur spontaneously in mycobacteria, as MQ is a better reducing agent than succinate (Em = +33 mV; ΔG° = +21 kJ/mol). Some organisms, like Bacillus species, consume the PMF to overcome this challenge (Lancaster et al., 2006; Schirawski and Unden, 1998). It is not known if Sdh1 or Sdh2 can interact with the PMF, or if they do so differently. Given that the PMF compositions of 6

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largely attributed to insufficient molecular diversity of the compound libraries screened. A switch to empirical non-target based phenotypic screening that interrogates all essential targets simultaneously under physiological conditions produced the first drug discovered in four decades to treat TB disease. However, other phenotypic screens have met with mixed success and many of the hits are non-specific membrane-active inhibitors such as detergents and uncouplers (Farha et al., 2013; Payne et al., 2007). Non-growth is key feature of all drug persistence and growth therefore appears to be an improper parameter to assess the efficacy of a drug, so parameters related to persistence and survival should be made the primary screening focus. This can be achieved by infection model screening, as used to identify the preclinical tuberculosis candidate Q203 (Pethe et al., 2013), or through the measurement of intracellular parameters like ATP (this is further discussed in section 3.3) (Mak et al., 2012). Therefore, while the rise of drug resistance is an important consideration, the true pitfall of current antibiotics is their focus on targeting actively growing cells. The thematic scenario of M. tuberculosis infection is an appropriate case study in this regard. Current first-line regimens for drug-susceptible TB includes isoniazid, rifampicin, pyrazinamide, and ethambutol (World Health Organization, 2010). Rifampicin targets RNA polymerase, pyrazinamide has multiple targets and the remaining drugs interfere with cell wall synthesis (Lewis, 2013). Treatment timeframes with these compounds ranges in the order of 6 months (World Health Organization, 2010) due to the capacity for M. tuberculosis to form persister cells, especially in response to isoniazid (Wallis et al., 2007, 1999; Wayne and Hayes, 1996). Furthermore, drug resistance is reported for all front- and second-line therapeutics (Almeida Da Silva and Palomino, 2011), with multi- and extensively-drug resistant tuberculosis (MDR- and XDR- TB) being classified by the WHO as a public health crisis (World Health Organization, 2016). In these cases, treatment is recommended for a minimum of 18 months after culture conversion (World Health Organization, 2010), with a global treatment success rate of ∼50% for rifampicin-resistant infection (World Health Organization, 2016). Compounds that hit multiple targets in nongrowing cells are needed to both reduce the treatment timeframe and generate new tools for treating MDR- and XDR-TB.

found to mediate the cellular response to several antibiotics and reactive oxygen species (Arora et al., 2014; Berney et al., 2014c; Hards et al., 2015; Lu et al., 2015), although it is unknown if this is due to promoting persistence or a direct effect. The E. coli cytochrome bd homolog has been reported to possess both catalase (Borisov et al., 2013) and quinol:peroxidase activity (Al-Attar et al., 2016), allowing it to directly detoxify hydrogen peroxide. The biochemical role of this complex in persistent mycobacteria is poorly understood. Other electron acceptors, for M. tuberculosis specifically, include fumarate and nitrate. The pathogen encodes a fumarate reductase, in addition to its two succinate dehydrogenases. Metabolomic analysis suggests it is a bona fide fumarate reductase (Watanabe et al., 2011), although this has not been directly tested through biochemical methods. The authors proposed that Sdh1 and Sdh2 are functionally redundant with the fumarate reductase, in a similar fashion to the E. coli enzymes (Maklashina et al., 1998). The nitrate reductase appears to have similarly modest effects, supplementing rapid switches to hypoxia (Sohaskey, 2008). Fumarate reductase is absent and nitrate reductase has a very low activity in M. smegmatis (Pecsi et al., 2014; Cook et al., 2014b, 2017), suggesting roles in virulence rather than environmental mycobacterial persistence. ATP synthesis in mycobacteria is driven by proton-coupled F1FoATP synthases (F1Fo). While mycobacteria encode all the glycolytic enzymes necessary for ATP synthesis by substrate-level phosphorylation, F1Fo is essential for growth on fermentable or non-fermentable carbon sources (Tran and Cook, 2005), suggesting that ATP is obligately produced through F1Fo for growth. While ATP synthases can usually operate in reverse ATP hydrolysis mode, to generate a PMF, the mycobacterial enzyme is incapable of hydrolyzing ATP (Haagsma et al., 2010). Given the relative impermeability of the mycobacterial membrane to protons (Tran et al., 2005), it could be hypothesized that ATP synthesis is the sole mechanism by which mycobacteria can truly consume their PMF (rather than exchange it, in the case of cation transporters). Specializations for that role may have rendered this particular enzyme incapable of operating in the reverse (ATP hydrolysis) direction. It has not been ruled out that sheer cellular demand for ATP to power mycobacterial growth underpins the enzyme’s essentiality. Overall, a hypothetical schematic for a non-proton pumping respiratory chain, signifying persistence, would most likely involve type II NADH dehydrogenases, succinate dehydrogenases and malate:quinone oxidoreductases as the electron inputs. Cytochrome bd is the best candidate for the electron terminating respiratory complex. Redox balance of NADH, if not maintained by type II NADH dehydrogenases, could also be maintained by central carbon metabolism enzymes, like malate dehydrogenases. Understanding how these components generate a PMF under non-growing conditions is of particular interest for advancing our knowledge of persistence as a whole.

3.2. Recent developments in the inhibition of energy generation A cautious-but-positive outlook has developed in the scientific community since the FDA approval of bedaquiline (BDQ), for treating MDR-TB, in December 2012 (Jones, 2013). This was the first drug approved for tuberculosis treatment in four decades. BDQ does not affect traditional drug-target spaces, but instead inhibits the mycobacterial F1Fo-ATP synthase (Andries et al., 2005; Haagsma et al., 2011) and in doing so defined energy generation as a new drug-target space. BDQ binds to the c-subunit rotor in the membrane embedded part of the F1Fo-ATP synthase (Koul et al., 2007; Preiss et al., 2015), inhibiting the enzyme, and results in a decrease in intracellular ATP levels (Koul et al., 2014, 2008). Whether the decrease in intracellular ATP directly results in cell death is a subject open to debate. Mycobacteria can be routinely depleted of ATP (Frampton et al., 2012) and yet remain viable. However, there is no consensus on a lethal intracellular ATP concentration and the lack of standardized reporting precludes any real comparison of data. Rather, the lack of temporal correlation between intracellular ATP and BDQ’s killing kinetics (Koul et al., 2014) is a telling result. The cellular consequences of directly inhibiting energy-generating complexes are undergoing active investigation. Lewis and colleagues have recently reported that intracellular ATP depletion induces persister cell formation in both Staphylococcus aureus and E. coli implicating ATP generation in persistence (Conlon et al., 2016; Gavrish et al., 2014; Shan et al., 2017). S. aureus is intrinsically resistant to polymyxins (polymyxin B and colistin), an important class of antimicrobial peptides used to treat infections caused by gram-negative bacteria. Inhibition of ATP synthase in S. aureus eliminates

3. Energetics as a new drug target space 3.1. Current drug targets and their pitfalls There are approximately 200 conserved essential proteins in bacteria, yet the majority of clinically relevant broad spectrum antimicrobials affect only 5 major targets or pathways (Lewis, 2013) (Fig. 7). For example, β-lactams cause osmotic defects through cell wall ultrastructural changes, quinolones prevent DNA synthesis by arresting the DNA replication fork and aminoglycosides promote tRNA mismatching at the ribosome (Kohanski et al., 2010). Most new compounds are simply derivatives of chemical classes for which there are already underlying resistance mechanisms. It has been difficult to discover new antimicrobials and there is a very long time to market making this an unattractive commercial proposition for pharmaceutical companies. Of the 70 HTS campaigns run between 1995 and 2001 (67 target based, 3 whole cell) by GlaxoSmithKline, only five leads were discovered (Payne et al., 2007). This lack of success was due to a number of factors, but 7

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Fig. 7. Cellular targets for the majority of clinically relevant antibiotics. The traditional targets for antibiotics are cell wall synthesis, protein synthesis, nucleic acid biosynthesis and antimetabolites. Inhibitors that target cell membrane function are not shown (e.g. polymyxins). Examples of common inhibitors/inhibitor classes are indicated in blue and are reviewed in (Lewis, 2013). Bedaquiline targets the F1Fo-ATP synthase, the enzyme responsible for the majority of ATP synthesis in many bacterial pathogens.

interface in the Fo component created an unregulated pore for equilibrating the pH gradient (Hards et al., 2015). The mechanism of maintaining the charge gradient was unresolved. Others have reported a consistent effect of BDQ on oxygen consumption in M. tuberculosis (Lamprecht et al., 2016), but found no change in the PMF. The authors used NADH to initiate respiration prior to BDQ treatment (Lamprecht et al., 2016), compared to our experiment which used succinate (Hards et al., 2015). We have subsequently found that NADH-energized membranes of M. smegmatis are indeed resistant to BDQ uncoupling (Hards and Cook, unpublished data), suggesting that the electron donors used for respiration are critical in determining the uncoupling efficacy of BDQ. Whether this is due to the differing respiratory components used to generate a PMF from these electron donors, or the activation of NADH-dependent drug efflux pumps is unknown. Not all uncouplers appear to act through this mechanism, however, as SQ109 (N-adamantan-2-yl-N'-((E)-3,7-dimethyl-octa-2,6-dienyl)ethane-1,2-diamine) and the leprosy drug clofazimine are proposed to uncouple in an analogous way to CCCP (Feng et al., 2015) (Fig. 2). An E. coli oxidase null mutant (i.e. deleted for all three oxidases) was resistant to killing by aminoglycosides, β-lactams and DNA gyrase inhibitors, which was correlated to the inability to stimulate oxygen consumption (Lobritz et al., 2015). This suggests that aerobic respiration is necessary for antibiotic killing, providing an explanation for the growth rate dependency of persistence (Rittershaus et al., 2013). However, as the experiments were performed in minimal media, lacking alternative electron acceptors − like fumarate or nitrate, the role of anaerobic respiratory chains in this regard is unknown. It has previously proposed that the production of reactive oxygen species is a common mechanism underlying all bactericidal antibiotics (Kohanski et al., 2007). Respiratory dysregulation, by uncoupling, provides a mechanism to produce such reactive oxygen species. Whether uncoupling is indeed related to a universal mechanism of antibiotic killing is a discussion muddied by the many apparent mechanisms of drug-

intrinsic resistance to polymyxins opening a new avenue for repurposing these exclusively Gram-negative agents against Gram-positive bacteria by targeting ATP synthesis (Vestergaard et al., 2017). These recent studies demonstrate the untapped opportunity to not only combat persister cells that are refractory to eradication by antimicrobials, but also broaden the spectrum of our current antimicrobial armoury. Since BDQ, several new inhibitors are being discovered or rediscovered for their activities against all respiratory chain components, not just the ATP synthase. For example, a number of groups have identified a series of imidazopyridine amide (IPA) compounds that interfere with energy metabolism and are active in the low nanomolar range in vitro (Kang et al., 2014; Moraski et al., 2013; Pethe et al., 2013). The leading drug candidate in this series Q203 recently progressed to clinical development phase I under a U.S. FDA investigational new drug application. Q203 targets the cytochrome bcc component of the mycobacterial III/IV supercomplex (Pethe et al., 2013). It’s potent in vivo activity suggests this complex is required in both growing and quiescent cells, a notable counterpoint to the hypothetical persistence respiratory chain proposed in this review; although mouse models do not produce encapsulated granulomas (Guirado and Schlesinger, 2013) and so the environmental cues, promoting persistence, may not be present. At the time of writing, only genetic evidence has been found to suggest cytochrome bcc inhibition and no biochemical confirmation (indicative of target engagement) or deeper understanding has been garnered for the consequences of inhibiting the mycobacterial enzyme. More recently it has been demonstrated that several existing drugs, antitubercular or otherwise, are uncouplers of respiration (Feng et al., 2015; Hards et al., 2015; Lobritz et al., 2015). BDQ was found to dissipate the ΔpH component of the PMF, but not the Δψ, in M. smegmatis (Hards et al., 2015). This would be analogous to the action of nigericin (Fig. 2), but a point mutation in the F1Fo-ATP synthase (AtpED32V) nullified the effect. It was proposed that disruption of the a-c subunit 8

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Fig. 8. Strategies for targeting bacterial bioenergetics. (A) Structures of selected antituberculosis drugs and other relevant inhibitors and (B) The respiratory chain, proton motive force and F-type ATP synthase (c.f. Fig. 1) connection can be disrupted by 5 conceivable strategies, as discussed in text. (1) Inhibition of respiratory chain components, as exemplified by Q203. (2) Activation of respiratory chain components, as exemplified by QQ8c and clofazimine (CFZ, under defined conditions). (3) Dissipation of the PMF through protonophoric or ionophoric mechanisms, SQ109 notably also inhibits menaquinone biosynthesis, preventing further respiratory activation. (4) Dissipation of the PMF through protein-based mechanisms, such as the creation of a proton pore in the mycobacterial F1Fo-ATP synthase. It is currently unknown if BDQ also collapses the PMF by chemical mechanisms. (5) Inhibition of the F1Fo-ATP synthase, the original strategy that highlighted bioenergetics as a viable target space. N,N-dicyclohexylcarbodiimide (DCCD) is a classical covalent inhibitor of the F1Fo-ATP synthase.

death (Frampton et al., 2012). If it is demonstrated that ATP depletion does not define the action of BDQ then new inhibitors discovered on this basis may fail to have the intended in vivo activity. Regardless of its contribution of cidality, uncoupling seems to be a key parameter underpinning respiratory-targeted therapeutics and should be routinely documented in future drug-discovery programs. It is notable that PMF dissipation has not been demonstrated at the whole cell level, for antimycobacterials like BDQ, likely due to compensatory stimulation of respiration. Oxygen consumption may therefore be a more appropriate surrogate parameter for the PMF. It has precedent linking its deregulation to uncoupling and cidality (Hards et al., 2015; Lamprecht et al., 2016; Lobritz et al., 2015), while the state of several respiratory chain components can easily be inferred from properly conducted experiments (Nicholls and Ferguson, 2013c). One obvious flaw is that oxygen consumption will not suit the analysis of compounds that inhibit alternative electron acceptor complexes. However, given bacteriostatic and bactericidal antibiotics antagonize each other, due to their inhibition and stimulation of oxygen consumption respectively (Lobritz et al., 2015), inferences on antibiotic synergy could be proposed before the fact. The safety of this target space, given humans generate energy by broadly similar mechanisms and with several conserved proteins, is a valid concern. Dismissing concerns based on a 20,000-fold selectivity difference (Haagsma et al., 2009) of BDQ may be inappropriate as conflicting reports exist on the effect of BDQ on eukaryotic oxygen consumption: while there is no effect on bovine heart mitochondria, HepG2 and RAW264.7 cells (Haagsma et al., 2009; Lamprecht et al.,

induced uncoupling and insufficient studies correlating drug-induced PMF dissipation and cell death. 3.3. Outlook on respiratory inhibition and the role of uncouplers Targeting energy generation in pathogenic bacteria has only been seriously considered at the molecular level in the past decade, and at the clinical level for less than half that time (Jones, 2013; Cook et al., 2017). The success of this relatively new area of research depends on our understanding of the compounds and targets that are forming high profile leads of investigation. Concerns have already been raised towards the use of bedaquiline, due to QT interval prolongation and unexplained deaths in clinical trials (Diacon et al., 2014; Fox and Menzies, 2013; Kakkar and Dahiya, 2014). Resistance, in the form of efflux pumps, has already been reported (Andries et al., 2014; Hartkoorn et al., 2014) and antagonistic effects with other new leads, like pretomanid (Tasneen et al., 2011; Wallis et al., 2012), are poorly understood. Accelerated FDA approval programs, such as that used for bedaquiline, behooves researchers to pursue a greater fundamental understanding than what might normally be expected for new antibiotics, lest ineffective compounds be released to the medical community. The question of efficacy should naturally be considered first. ATP depletion is starting to be used as a “gold-standard” measure for bioenergetic inhibitors (Koul et al., 2008; Li et al., 2014; Lu et al., 2011; Pethe et al., 2013), but observations have been made that ATP depletion is not temporally correlated (Koul et al., 2014) or indicative of cell 9

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pathogenic respiratory chains; at genetic, biochemical and physiological levels, is still poorly resolved and restricts our ability to target mitochondrially-conserved complexes. The mechanisms of drug-induced uncoupling are not unambiguously proven, and the role of drugs dissipating only the charge (membrane potential) or pH (transmembrane pH gradient) component of the PMF is not understood. Finally, the ability of organisms to compensate for respiratory inhibition at a substrate-level (fermentation-like responses) and oxidative-level (respiratory chain reconfiguration) level is not understood. One important conclusion, however, is that all respiratory chain components are intrinsically linked by PMFs and so respiration should be considered as a pathway, as opposed to separate protein targets. The continued investigation of respiratory complexes from persisters and pathogens, to this end, will no doubt continue to challenge and refine our concepts of bioenergetic systems.

2016), others have found that BDQ stimulates oxygen consumption in MCF7 human fibroblasts (Fiorillo et al., 2016). This result, although not discussed as such by the authors, would be consistent with uncoupling. This should not be considered critical evidence, as uncoupling is not necessarily lethal and could be managed by a clinician; assuming they are appropriately aware of the potential for weight loss, overheating and malnutrition. The black-market weight-loss drug 2,4-dinitrophenol (Grundlingh et al., 2011), an admittedly far more acute scenario of uncoupling, may serve as an appropriate case study. Identifying and improving the management of adverse effects may be more beneficial then fanciful attempts to avoid them. As for new targets within bioenergetics, there appears to be five broad strategies for future drug-development (Fig. 8). The two most obvious are inhibition of individual respiratory chain components and inhibition of the ATP synthesis (Cook et al., 2017). However, as demonstrated for SQ109 (Feng et al., 2015; Li et al., 2014), the additional ability to act as a protonophore can enhance potency and seems to be a desirable property. While it is tough to envision classical protonophores and ionophores being developed as a therapeutics, it is worth noting that mycobacteria are highly susceptible to valinomycin and nigericin (Rao et al., 2001, 2008). As a fourth, although somewhat related, strategy: being able to uncouple by creating dysregulated proton pores in mycobacterial membrane proteins, as proposed for BDQ (Hards et al., 2015), may be an acceptable compromise between selectivity and activity. Finally, activating respiratory complexes may be just as effective as inhibiting them (Heikal et al., 2016; Vilchèze et al., 2017). Inappropriately consuming reductant may forcefully resuscitate persistent mycobacteria, perhaps in a similar manner to the aminoglycoside sensitization of persister E. coli and S. aureus cells (Allison et al., 2011). It could also emulate the metabolite depletion and ROS production inferred to occur for uncouplers, without the selectivity concerns. This has been proposed to occur for a recently identified activator of respiration, QQ8c (Heikal et al., 2016) and may serve as a model compound in this regard. It also seems plausible that several respiratory chain components should be targeted despite their existence in humans. The mycobacterial F1Fo has a unique blockage in ATP hydrolysis (Haagsma et al., 2010), while the mycobacterial succinate dehydrogenase are proposed to couple the menaquinone (Pecsi et al., 2014) (rather than ubiquinone). While several approaches in drug-development are focusing on the human-absent complexes, these unique mycobacterial adaptations may give validity to targeting proteins that have mitochondrial homologs. As an interesting compromise, consider how poorly effective compounds may benefit this target space: cytochrome bd deletion hypersensitizes mycobacteria to BDQ treatment (Berney et al., 2014c; Hards et al., 2015; Kalia et al., 2017) and Q203 so perhaps weak inhibitors of cytochrome bd may enhance combination therapies, if a strong one cannot be found. If this logic stands for other mycobacterial complexes, a weak inhibitor against complexes with human homologs could have minimized toxicity while still being effective, if used correctly. This is not considering the fact that the adaptations of mycobacterial respiratory proteins may yet allow selective targeting. It may be time to revise the notion that each inhibitor needs to be effective in its own right, as the field’s increased understanding may allow for the repurposing of discarded compounds. Taken together, there appears to be ample opportunity to refine and expand both our understanding of pathogenic respiration and our capacity to prevent it.

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