RESUMEN
Iron-bound cyclic tetrapyrroles (hemes) are redox-active cofactors in bioenergetic enzymes. However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here, we used cellular, biochemical, structural and computational methods to characterize the structure and function of the heterodimeric bacterial ABC transporter CydDC. We provide multi-level evidence that CydDC is a heme transporter required for functional maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryogenic-electron microscopy approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and occlusion. Our simulations reveal that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to rotate 180°.
Asunto(s)
Proteínas de Escherichia coli , Hemo , Hemo/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Oxidación-Reducción , Conformación ProteicaRESUMEN
Functional reintegration into lipid environments represents a major challenge for in vitro investigation of integral membrane proteins (IMPs). Here, we report a new approach, termed LMNG Auto-insertion Reintegration (LAiR), for reintegration of IMPs into lipid bilayers within minutes. The resulting proteoliposomes displayed an unprecedented capability to maintain proton gradients and long-term stability. LAiR allowed for monitoring catalysis of a membrane-bound, physiologically relevant polyisoprenoid quinone substrate by Escherichia coli cytochromes bo 3 (cbo 3) and bd (cbd) under control of the proton motive force. LAiR also facilitated bulk-phase detection and physiological assessment of the "proton leak" in cbo 3, a controversial catalytic state that previously was only approachable at the single-molecule level. LAiR maintained the multisubunit integrity and higher-order oligomeric states of the delicate mammalian F-ATP synthase. Given that LAiR can be applied to both liposomes and planar membrane bilayers and is compatible with IMPs and lipids from prokaryotic and eukaryotic sources, we anticipate LAiR to be applied broadly across basic research, pharmaceutical applications, and biotechnology.
RESUMEN
Cytochrome bd-I from Escherichia coli is a terminal oxidase in the respiratory chain that plays an important role under stress conditions. Cytochrome bd-I was thought to consist of the major subunits CydA and CydB plus the small CydX subunit. Recent high-resolution structures of cytochrome bd-I demonstrated the presence of an additional subunit, CydH/CydY (called CydH here), the function of which is unclear. In this report, we show that in the absence of CydH, cytochrome bd-I is catalytically active, can sustain bacterial growth and displays haem spectra and susceptibility for haem-binding inhibitors comparable to the wild-type enzyme. Removal of CydH did not elicit catalase activity of cytochrome bd-I in our experimental system. Taken together, in the absence of the CydH subunit cytochrome bd-I retained key enzymatic properties.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Grupo Citocromo b/genética , Grupo Citocromo b/química , Citocromos/genética , Citocromos/química , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Proteínas del Complejo de Cadena de Transporte de Electrón/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , HemoRESUMEN
For the design of next-generation tuberculosis chemotherapy, insight into bacterial defence against drugs is required. Currently, targeting respiration has attracted strong attention for combatting drug-resistant mycobacteria. Q203 (telacebec), an inhibitor of the cytochrome bcc complex in the mycobacterial respiratory chain, is currently evaluated in phase-2 clinical trials. Q203 has bacteriostatic activity against M. tuberculosis, which can be converted to bactericidal activity by concurrently inhibiting an alternative branch of the mycobacterial respiratory chain, cytochrome bd. In contrast, non-tuberculous mycobacteria, such as Mycobacterium smegmatis, show only very little sensitivity to Q203. In this report, we investigated factors that M. smegmatis employs to adapt to Q203 in the presence or absence of a functional cytochrome bd, especially regarding its terminal oxidases. In the presence of a functional cytochrome bd, M. smegmatis responds to Q203 by increasing the expression of cytochrome bcc as well as of cytochrome bd, whereas a M. smegmatisbd-KO strain adapted to Q203 by increasing the expression of cytochrome bcc. Interestingly, single-cell studies revealed cell-to-cell variability in drug adaptation. We also investigated the role of a putative second cytochrome bd isoform postulated for M. smegmatis. Although this putative isoform showed differential expression in response to Q203 in the M. smegmatisbd-KO strain, it did not display functional features similar to the characterised cytochrome bd variant.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Citocromos/metabolismo , Humanos , Imidazoles , Mycobacterium smegmatis , Mycobacterium tuberculosis/metabolismo , Oxidorreductasas/metabolismo , Piperidinas , Piridinas , Tuberculosis/tratamiento farmacológicoRESUMEN
The ATP synthase is a multicomponent enzyme that is largely conserved across the kingdoms of life. In many species the ATP synthase is central in the synthesis of ATP by using the electrochemical proton gradient generated via the electron transport chain. Bacteria inhabit very diverse ecological niches; hence their metabolism to extract nutrients and generation of ATP varies from species to species. Some species are obligate aerobes (e.g., Mycobacterium tuberculosis), relying on oxidative phosphorylation for ATP synthesis, whereas others are strict anaerobes (e.g., Clostridioides difficile) relying primarily on substrate-level phosphorylation using various fermentative pathways. Yet other species, such as Staphylococcus aureus and Escherichia coli are facultative anaerobes and can convert energy via both respiratory and fermentative pathways. The metabolic propensity and growth conditions experienced by bacterial species have a great impact on the necessity of a functional ATP synthase for viability. The ATP synthase has been validated as a druggable target with the approval of the ATP synthase inhibitor bedaquiline for treatment of M. tuberculosis, an organism in which the ATP synthase is essential for growth. Currently, no ATP synthase inhibitors are in clinical use against non-mycobacterial pathogens. In this review, the physiological functions of the ATP synthase in various bacterial pathogens are discussed in relation to the metabolic pathways utilized for providing energy. The ATP synthase is essential in important pathogenic species that are obligate aerobes, obligate anaerobes and aerotolerant anaerobes, whereas it is dispensable for growth in most facultative anaerobic pathogens. Interference with the ATP synthase in facultative anaerobes has physiological consequences, such as membrane hyperpolarization, which can be exploited for combination therapies. Collectively, the available data indicate that the ATP synthase is an interesting target for development of new antimicrobials beyond M. tuberculosis.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Adenosina Trifosfato/metabolismo , Inhibidores Enzimáticos/metabolismo , HumanosRESUMEN
Cytochrome bd-type oxidases play a crucial role for survival of pathogenic bacteria during infection and proliferation. This role and the fact that there are no homologues in the mitochondrial respiratory chain qualify cytochrome bd as a potential antimicrobial target. However, few bd oxidase selective inhibitors have been described so far. In this report, inhibitory effects of Aurachin C (AurC-type) and new Aurachin D (AurD-type) derivatives on oxygen reductase activity of isolated terminal bd-I, bd-II and bo3 oxidases from Escherichia coli were potentiometrically measured using a Clark-type electrode. We synthesized long- (C10, decyl or longer) and short-chain (C4, butyl to C8, octyl) AurD-type compounds and tested this set of molecules towards their selectivity and potency. We confirmed strong inhibition of all three terminal oxidases for AurC-type compounds, whereas the 4(1H)-quinolone scaffold of AurD-type compounds mainly inhibits bd-type oxidases. We assessed a direct effect of chain length on inhibition activity with highest potency and selectivity observed for heptyl AurD-type derivatives. While Aurachin C and Aurachin D are widely considered as selective inhibitors for terminal oxidases, their structure-activity relationship is incompletely understood. This work fills this gap and illustrates how structural differences of Aurachin derivatives determine inhibitory potency and selectivity for bd-type oxidases of E. coli.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de la Membrana Bacteriana Externa/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/metabolismo , Unión Proteica , Quinolonas/química , Quinolonas/farmacologíaRESUMEN
The treatment of infectious diseases caused by multidrug-resistant pathogens is a major clinical challenge of the 21st century. The membrane-embedded respiratory cytochrome bd-type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. Escherichia coli encodes for two cytochrome bd isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between cydABX (Ecbd-I) and appCBX (Ecbd-II) operon encoded cytochrome bd variants have remained elusive in the past. Here, we demonstrate that cytochrome bd-II catalyzes oxidation of benzoquinols while possessing additional specificity for naphthoquinones. Our data show that although menaquinol-1 (MK1) is not able to directly transfer electrons onto cytochrome bd-II from E. coli, it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome bd-II to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two bd oxidase isoforms, but two notable differences are apparent upon inspection: (i) Ecbd-II does not contain a CydH-like subunit, thereby exposing heme b595 to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of Ecbd-I Our work completes the structural landscape of terminal respiratory oxygen reductases of E. coli and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in E. coli.
Asunto(s)
Grupo Citocromo b/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Oxidorreductasas/metabolismo , Grupo Citocromo b/genética , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Oxidorreductasas/genética , Conformación Proteica , Isoformas de ProteínasRESUMEN
Cardiolipin (CL) is a lipid that is found in the membranes of bacteria and the inner membranes of mitochondria. CL can increase the activity of integral membrane proteins, in particular components of respiratory pathways. We here report that CL activated detergent-solubilized cytochrome bd, a terminal oxidase from Escherichia coli. CL enhanced the oxygen consumption activity ~ twofold and decreased the apparent KM value for ubiquinol-1 as substrate from 95 µM to 35 µM. Activation by CL was also observed for cytochrome bd from two Gram-positive species, Geobacillus thermodenitrificans and Corynebacterium glutamicum, and for cytochrome bo3 from E. coli. Taken together, CL can enhance the activity of detergent-solubilized cytochrome bd and cytochrome bo3.
Asunto(s)
Grupo Citocromo b , Geobacillus , Consumo de OxígenoRESUMEN
The goal of the present study was to select a Gram-positive (Gram+) and Gram-negative (Gram-) strain to measure antimicrobial activity in environmental samples, allowing high-throughput environmental screening. The sensitivity of eight pre-selected bacterial strains were tested to a training set of ten antibiotics, i.e. three Gram+ Bacillus subtilis strains with different read-outs, and five Gram- strains. The latter group consisted of a bioluminescent Allivibrio fischeri strain and four Escherichia coli strains, i.e. a wild type (WT) and three strains with a modified cell envelope to increase their sensitivity. The WT B. subtilis and an E. coli strain newly developed in this study, were most sensitive to the training set. This E. coli strain carries an open variant of an outer membrane protein combined with an inactivated multidrug efflux transport system. The assay conditions of these two strains were optimized and validated by exposure to a validation set of thirteen antibiotics with clinical and environmental relevance. The assay sensitivity ranged from the ng/mL to µg/mL range. The applicability of the assays for toxicological characterization of aquatic environmental samples was demonstrated for hospital effluent extract. A future application includes effect-directed analysis to identify yet unknown antibiotic contaminants or their transformation products.
Asunto(s)
Bioensayo , Antibacterianos , Bacillus subtilis , Escherichia coli , Ensayos Analíticos de Alto Rendimiento , Pruebas de Sensibilidad MicrobianaRESUMEN
Cytochrome bd, a component of the prokaryotic respiratory chain, is important under physiological stress and during pathogenicity. Electrons from quinol substrates are passed on via heme groups in the CydA subunit and used to reduce molecular oxygen. Close to the quinol binding site, CydA displays a periplasmic hydrophilic loop called Q-loop that is essential for quinol oxidation. In the carboxy-terminal part of this loop, CydA from Escherichia coli and other proteobacteria harbors an insert of ~60 residues with unknown function. In the current work, we demonstrate that growth of the multiple-deletion strain E. coli MB43∆cydA (∆cydA∆cydB∆appB∆cyoB∆nuoB) can be enhanced by transformation with E. coli cytochrome bd-I and we utilize this system for assessment of Q-loop mutants. Deletion of the cytochrome bd-I Q-loop insert abolished MB43∆cydA growth recovery. Swapping the cytochrome bd-I Q-loop for the Q-loop from Geobacillus thermodenitrificans or Mycobacterium tuberculosis CydA, which lack the insert, did not enhance the growth of MB43∆cydA, whereas swapping for the Q-loop from E. coli cytochrome bd-II recovered growth. Alanine scanning experiments identified the cytochrome bd-I Q-loop insert regions Ile318-Met322, Gln338-Asp342, Tyr353-Leu357, and Thr368-Ile372 as important for enzyme functionality. Those mutants that completely failed to recover growth of MB43∆cydA also lacked oxygen consumption activity and heme absorption peaks. Moreover, we were not able to isolate cytochrome bd-I from these inactive mutants. The results indicate that the cytochrome bd Q-loop exhibits low plasticity and that the Q-loop insert in E. coli is needed for complete, stable, assembly of cytochrome bd-I.
Asunto(s)
Grupo Citocromo b/química , Grupo Citocromo b/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/química , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Alanina/genética , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Grupo Citocromo b/aislamiento & purificación , Proteínas del Complejo de Cadena de Transporte de Electrón/aislamiento & purificación , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/aislamiento & purificación , Hemo/metabolismo , Mutagénesis/genética , Proteínas Mutantes/química , Proteínas Mutantes/aislamiento & purificación , Proteínas Mutantes/metabolismo , Oxidorreductasas/aislamiento & purificación , Consumo de Oxígeno , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
The branched respiratory chain of Mycobacterium tuberculosis has attracted attention as a highly promising target for next-generation antibacterials. This system includes two terminal oxidases of which the exclusively bacterial cytochrome bd represents the less energy-efficient one. Albeit dispensable for growth under standard laboratory conditions, cytochrome bd is important during environmental stress. In this review, we discuss the role of cytochrome bd during infection of the mammalian host and in the defense against antibacterials. Deeper insight into the biochemistry of mycobacterial cytochrome bd is needed to understand the physiological role of this bacteria-specific defense factor. Conversely, cytochrome bd may be utilized to gain information on mycobacterial physiology in vitro and during host infection. Knowledge-based manipulation of cytochrome bd function may assist in designing the next-generation tuberculosis combination chemotherapy.
Asunto(s)
Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Grupo Citocromo b/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Antituberculosos/metabolismo , Membrana Celular/metabolismo , Descubrimiento de Drogas , Quimioterapia Combinada , Transporte de Electrón/efectos de los fármacos , Humanos , Inhibidores de la Síntesis de la Proteína/metabolismo , Transducción de Señal , Tuberculosis/tratamiento farmacológicoRESUMEN
Accumulating evidence suggests that the bactericidal activity of some antibiotics may not be directly initiated by target inhibition. The activity of isoniazid (INH), a key first-line bactericidal antituberculosis drug currently known to inhibit mycolic acid synthesis, becomes extremely poor under stress conditions, such as hypoxia and starvation. This suggests that the target inhibition may not fully explain the bactericidal activity of the drug. Here, we report that INH rapidly increased Mycobacterium bovis BCG cellular ATP levels and enhanced oxygen consumption. The INH-triggered ATP increase and bactericidal activity were strongly compromised by Q203 and bedaquiline, which inhibit mycobacterial cytochrome bc1 and FoF1 ATP synthase, respectively. Moreover, the antioxidant N-acetylcysteine (NAC) but not 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) abrogated the INH-triggered ATP increase and killing. These results reveal a link between the energetic (ATP) perturbation and INH's killing. Furthermore, the INH-induced energetic perturbation and killing were also abrogated by chemical inhibition of NADH dehydrogenases (NDHs) and succinate dehydrogenases (SDHs), linking INH's bactericidal activity further to the electron transport chain (ETC) perturbation. This notion was also supported by the observation that INH dissipated mycobacterial membrane potential. Importantly, inhibition of cytochrome bd oxidase significantly reduced cell recovery during INH challenge in a culture settling model, suggesting that the respiratory reprogramming to the cytochrome bd oxidase contributes to the escape of INH killing. This study implicates mycobacterial ETC perturbation through NDHs, SDHs, cytochrome bc1, and FoF1 ATP synthase in INH's bactericidal activity and pinpoints the participation of the cytochrome bd oxidase in protection against this drug under stress conditions.
Asunto(s)
Antituberculosos/farmacología , Proteínas del Complejo de Cadena de Transporte de Electrón/antagonistas & inhibidores , Transporte de Electrón/efectos de los fármacos , Isoniazida/farmacología , Mycobacterium bovis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Grupo Citocromo b/antagonistas & inhibidores , Diarilquinolinas/farmacología , Complejo IV de Transporte de Electrones/metabolismo , Imidazoles/farmacología , Potenciales de la Membrana/efectos de los fármacos , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo , Oxidación-Reducción/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Piperidinas/farmacología , Piridinas/farmacologíaRESUMEN
Bedaquiline (BDQ), an inhibitor of the mycobacterial F1Fo-ATP synthase, has revolutionized the antitubercular drug discovery program by defining energy metabolism as a potent new target space. Several studies have recently suggested that BDQ ultimately causes mycobacterial cell death through a phenomenon known as uncoupling. The biochemical basis underlying this, in BDQ, is unresolved and may represent a new pathway to the development of effective therapeutics. In this communication, we demonstrate that BDQ can inhibit ATP synthesis in Escherichia coli by functioning as a H+/K+ ionophore, causing transmembrane pH and potassium gradients to be equilibrated. Despite the apparent lack of a BDQ-binding site, incorporating the E. coli Fo subunit into liposomes enhanced the ionophoric activity of BDQ. We discuss the possibility that localization of BDQ at F1Fo-ATP synthases enables BDQ to create an uncoupled microenvironment, by antiporting H+/K+ Ionophoric properties may be desirable in high-affinity antimicrobials targeting integral membrane proteins.
Asunto(s)
Adenosina Trifosfato/biosíntesis , Antituberculosos/farmacología , Diarilquinolinas/farmacología , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Ionóforos/farmacología , ATPasas de Translocación de Protón/metabolismo , Concentración de Iones de HidrógenoRESUMEN
Mycobacterial energy metabolism currently attracts strong attention as new target space for development of anti-tuberculosis drugs. The imidazopyridine Q203 targets the cytochrome bcc complex of the respiratory chain, a key component in energy metabolism. Q203 blocks growth of Mycobacterium tuberculosis at nanomolar concentrations, however, it fails to actually kill the bacteria, which may limit the clinical applicability of this candidate drug. In this report we show that inhibition of cytochrome bd, a parallel branch of the mycobacterial respiratory chain, by aurachin D invoked bactericidal activity of Q203. In biochemical assays using inverted membrane vesicles from Mycobacterium tuberculosis and Mycobacterium smegmatis we found that inhibition of respiratory chain activity by Q203 was incomplete, but could be enhanced by inactivation of cytochrome bd, either by genetic knock-out or by inhibition with aurachin D. These results indicate that simultaneously targeting the cytochrome bcc and the cytochrome bd branch of the mycobacterial respiratory chain may turn out as effective strategy for combating M. tuberculosis.
Asunto(s)
Antituberculosos/farmacología , Citocromos/antagonistas & inhibidores , Imidazoles/farmacología , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Piperidinas/farmacología , Piridinas/farmacología , Transporte de Electrón/efectos de los fármacos , Quinolonas/farmacologíaRESUMEN
Cytochrome bd is a component of the oxidative phosphorylation pathway in many Gram-positive and Gram-negative bacteria. Next to its role as a terminal oxidase in the respiratory chain this enzyme plays an important role as a survival factor in the bacterial stress response. In Mycobacterium tuberculosis and related mycobacterial strains, cytochrome bd is an important component of the defense system against antibacterial drugs. In this report we describe and evaluate an mCherry-based fluorescent reporter for detection of cytochrome bd expression in Mycobacterium marinum. Cytochrome bd was induced by mycolic acid biosynthesis inhibitors such as isoniazid and most prominently by drugs targeting oxidative phosphorylation. We observed no induction by inhibitors of protein-, DNA- or RNA-synthesis. The constructed expression reporter was suitable for monitoring mycobacterial cytochrome bd expression during mouse macrophage infection and in a zebrafish embryo infection model when using Mycobacterium marinum. Interestingly, in both these infection models cytochrome bd levels were considerably higher than during in vitro culturing of M. marinum. The expression reporter described here can be a valuable tool for elucidating the role of cytochrome bd as a survival factor.
Asunto(s)
Antibacterianos/farmacología , Citocromos/genética , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Infecciones por Mycobacterium/microbiología , Mycobacterium/efectos de los fármacos , Mycobacterium/genética , Animales , Macrófagos , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Pez CebraRESUMEN
Drug-resistant mycobacterial infections are a serious global health challenge, leading to high mortality and socioeconomic burdens in developing countries worldwide. New innovative approaches, from identification of new targets to discovery of novel chemical scaffolds, are urgently needed. Recently, energy metabolism in mycobacteria, in particular the oxidative phosphorylation pathway, has emerged as an object of intense microbiological investigation and as a novel target pathway in drug discovery. New classes of antibacterials interfering with elements of the oxidative phosphorylation pathway are highly active in combating dormant or latent mycobacterial infections, with a promise of shortening tuberculosis chemotherapy. The regulatory approval of the ATP synthase inhibitor bedaquiline and the discovery of Q203, a candidate drug targeting the cytochrome bc1 complex, have highlighted the central importance of this new target pathway. In this review, we discuss key features and potential applications of inhibiting energy metabolism in our quest for discovering potent novel and sterilizing drug combinations for combating tuberculosis. We believe that the combination of drugs targeting elements of the oxidative phosphorylation pathway can lead to a completely new regimen for drug-susceptible and multidrug-resistant tuberculosis.
Asunto(s)
Antituberculosos/farmacología , Descubrimiento de Drogas/tendencias , Metabolismo Energético/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Fosforilación Oxidativa/efectos de los fármacosRESUMEN
Cytochrome bd is a prokaryotic terminal oxidase that catalyses the electrogenic reduction of oxygen to water using ubiquinol as electron donor. Cytochrome bd is a tri-haem integral membrane enzyme carrying a low-spin haem b558, and two high-spin haems: b595 and d. Here we show that besides its oxidase activity, cytochrome bd from Escherichia coli is a genuine quinol peroxidase (QPO) that reduces hydrogen peroxide to water. The highly active and pure enzyme preparation used in this study did not display the catalase activity recently reported for E. coli cytochrome bd. To our knowledge, cytochrome bd is the first membrane-bound quinol peroxidase detected in E. coli. The observation that cytochrome bd is a quinol peroxidase, can provide a biochemical basis for its role in detoxification of hydrogen peroxide and may explain the frequent findings reported in the literature that indicate increased sensitivity to hydrogen peroxide and decreased virulence in mutants that lack the enzyme.
Asunto(s)
Citocromos/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Proteínas de Escherichia coli/metabolismo , Hidroquinonas/metabolismo , Oxidorreductasas/metabolismo , Grupo Citocromo b , Citocromos/química , Proteínas del Complejo de Cadena de Transporte de Electrón/química , Proteínas de Escherichia coli/química , Hidroquinonas/química , Cinética , Oxidorreductasas/química , Especificidad por SustratoRESUMEN
Ca(2+)-sensor proteins control the secretion of many neuroendocrine substances. Calcium-secretion coupling may involve several mechanisms. First, Ca(2+)-dependent association of their tandem C2 domains with phosphatidylserine may induce membrane curvature and thereby enhance fusion. Second, their association with SNARE complexes may inhibit membrane fusion in the absence of a Ca(2+) trigger. Here we present a method using two optically trapped beads coated with SNARE-free synthetic membranes to elucidate the direct role of the C2AB domain of the soluble Ca(2+)-sensor Doc2b. Contacting membranes are often coupled by a Doc2b-coated membrane stalk that resists forces up to 600 pN upon bead separation. Stalk formation depends strictly on Ca(2+) and phosphatidylserine. Real-time fluorescence imaging shows phospholipid but not content mixing, indicating membrane hemifusion. Thus, Doc2b acts directly on membranes and stabilizes the hemifusion intermediate in this cell-free system. In living cells, this mechanism may co-occur with progressive SNARE complex assembly, together defining Ca(2+)-secretion coupling.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas del Tejido Nervioso/química , Calcio/química , Membranas Artificiales , Fosfatidilserinas/química , Fosfolípidos/químicaRESUMEN
Targeting respiration and ATP synthesis has received strong interest as a new strategy for combatting drug-resistant Mycobacterium tuberculosis. Mycobacteria employ a respiratory chain terminating with two branches. One of the branches includes a cytochrome bc1 complex and an aa3-type cytochrome c oxidase while the other branch terminates with a cytochrome bd-type quinol oxidase. In this communication we show that genetic inactivation of cytochrome bd, but not of cytochrome bc1, enhances the susceptibility of Mycobacterium smegmatis to hydrogen peroxide and antibiotic-induced stress. The type-II NADH dehydrogenase effector clofazimine and the ATP synthase inhibitor bedaquiline were bacteriostatic against wild-type M. smegmatis, but strongly bactericidal against a cytochrome bd mutant. We also demonstrated that the quinone-analog aurachin D inhibited mycobacterial cytochrome bd at sub-micromolar concentrations. Our results identify cytochrome bd as a key survival factor in M. smegmatis during antibiotic stress. Targeting the cytochrome bd respiratory branch therefore appears to be a promising strategy that may enhance the bactericidal activity of existing tuberculosis drugs.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Peróxido de Hidrógeno/toxicidad , Mycobacterium smegmatis/efectos de los fármacos , Complejos de ATP Sintetasa/antagonistas & inhibidores , Complejos de ATP Sintetasa/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Clofazimina/farmacología , Diarilquinolinas/farmacología , Complejo IV de Transporte de Electrones/genética , Técnicas de Inactivación de Genes , Mutación , Mycobacterium smegmatis/enzimología , NADH Deshidrogenasa/química , NADH Deshidrogenasa/metabolismo , Quinolonas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Estrés FisiológicoRESUMEN
OBJECTIVES: It is not fully understood why inhibiting ATP synthesis in Mycobacterium species leads to death in non-replicating cells. We investigated the bactericidal mode of action of the anti-tubercular F1Fo-ATP synthase inhibitor bedaquiline (Sirturo™) in order to further understand the lethality of ATP synthase inhibition. METHODS: Mycobacterium smegmatis strains were used for all the experiments. Growth and survival during a bedaquiline challenge were performed in multiple media types. A time-course microarray was performed during initial bedaquiline challenge in minimal medium. Oxygen consumption and proton-motive force measurements were performed on whole cells and inverted membrane vesicles, respectively. RESULTS: A killing of 3 log10 cfu/mL was achieved 4-fold more quickly in minimal medium (a glycerol carbon source) versus rich medium (LB with Tween 80) during bedaquiline challenge. Assessing the accelerated killing condition, we identified a transcriptional remodelling of metabolism that was consistent with respiratory dysfunction but inconsistent with ATP depletion. In glycerol-energized cell suspensions, bedaquiline caused an immediate 2.3-fold increase in oxygen consumption. Bedaquiline collapsed the transmembrane pH gradient, but not the membrane potential, in a dose-dependent manner. Both these effects were dependent on binding to the F1Fo-ATP synthase. CONCLUSIONS: Challenge with bedaquiline results in an electroneutral uncoupling of respiration-driven ATP synthesis. This may be a determinant of the bactericidal effects of bedaquiline, while ATP depletion may be a determinant of its delayed onset of killing. We propose that bedaquiline binds to and perturbs the a-c subunit interface of the Fo, leading to futile proton cycling, which is known to be lethal to mycobacteria.