RESUMEN
Oxidation of malate to oxaloacetate, catalyzed by either malate dehydrogenase (Mdh) or malate quinone oxidoreductase (Mqo), is a critical step of the tricarboxylic acid cycle. Both Mqo and Mdh are found in most bacterial genomes, but the level of functional redundancy between these enzymes remains unclear. A bioinformatic survey revealed that Mqo was not as widespread as Mdh in bacteria but that it was highly conserved in mycobacteria. We therefore used mycobacteria as a model genera to study the functional role(s) of Mqo and its redundancy with Mdh. We deleted mqo from the environmental saprophyte Mycobacterium smegmatis, which lacks Mdh, and found that Mqo was essential for growth on nonfermentable carbon sources. On fermentable carbon sources, the Δmqo mutant exhibited delayed growth and lowered oxygen consumption and secreted malate and fumarate as terminal end products. Furthermore, heterologous expression of Mdh from the pathogenic species Mycobacterium tuberculosis shortened the delayed growth on fermentable carbon sources and restored growth on nonfermentable carbon sources at a reduced growth rate. In M. tuberculosis, CRISPR interference of either mdh or mqo expression resulted in a slower growth rate compared to controls, which was further inhibited when both genes were knocked down simultaneously. These data reveal that exergonic Mqo activity powers mycobacterial growth under nonenergy limiting conditions and that endergonic Mdh activity complements Mqo activity, but at an energetic cost for mycobacterial growth. We propose Mdh is maintained in slow-growing mycobacterial pathogens for use under conditions such as hypoxia that require reductive tricarboxylic acid cycle activity.
Asunto(s)
Malato Deshidrogenasa , Malatos , Oxidorreductasas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Ciclo del Ácido Cítrico , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Malatos/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Ácido Oxaloacético/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismoRESUMEN
Our inability to predict which mutations could result in antibiotic resistance has made it difficult to rapidly identify the emergence of resistance, identify pre-existing resistant populations, and manage our use of antibiotics to effectively treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F420H2-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is essential for emergence from hypoxia, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid and delamanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, before being exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas , Farmacorresistencia Bacteriana , Mutación , Mycobacterium tuberculosis , Nitroimidazoles/farmacología , Nitrorreductasas , Oxazoles/farmacología , Ingeniería de Proteínas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Nitrorreductasas/genética , Nitrorreductasas/metabolismo , Polimorfismo GenéticoRESUMEN
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
Exoelectrogens are able to transfer electrons extracellularly, enabling them to respire on insoluble terminal electron acceptors. Extensively studied exoelectrogens, such as Geobacter sulfurreducens and Shewanella oneidensis, are Gram negative. More recently, it has been reported that Gram-positive bacteria, such as Listeria monocytogenes and Enterococcus faecalis, also exhibit the ability to transfer electrons extracellularly, although it is still unclear whether this has a function in respiration or in redox control of the environment, for instance, by reducing ferric iron for iron uptake. In this issue of Journal of Bacteriology, Hederstedt and colleagues report on experiments that directly compare extracellular electron transfer (EET) pathways for ferric iron reduction and respiration and find a clear difference (L. Hederstedt, L. Gorton, and G. Pankratova, J Bacteriol 202:e00725-19, 2020, https://doi.org/10.1128/JB.00725-19), providing further insights and new questions into the function and metabolic pathways of EET in Gram-positive bacteria.
Asunto(s)
Enterococcus faecalis , Shewanella , Transporte de Electrón , Electrones , Geobacter , Homeostasis , Nutrientes , Oxidación-ReducciónRESUMEN
To persist when nutrient sources are limited, aerobic soil bacteria metabolize atmospheric hydrogen (H2). This process is the primary sink in the global H2 cycle and supports the productivity of microbes in oligotrophic environments. H2-metabolizing bacteria possess [NiFe] hydrogenases that oxidize H2 to subatmospheric concentrations. The soil saprophyte Mycobacterium smegmatis has two such [NiFe] hydrogenases, designated Huc and Hhy, that belong to different phylogenetic subgroups. Both Huc and Hhy are oxygen-tolerant, oxidize H2 to subatmospheric concentrations, and enhance bacterial survival during hypoxia and carbon limitation. Why does M. smegmatis require two hydrogenases with a seemingly similar function? In this work, we resolved this question by showing that Huc and Hhy are differentially expressed, localized, and integrated into the respiratory chain. Huc is active in late exponential and early stationary phases, supporting energy conservation during mixotrophic growth and transition into dormancy. In contrast, Hhy is most active during long-term persistence, providing energy for maintenance processes following carbon exhaustion. We also show that Huc and Hhy are obligately linked to the aerobic respiratory chain via the menaquinone pool and are differentially affected by respiratory uncouplers. Consistently, these two enzymes interacted differentially with the respiratory terminal oxidases. Huc exclusively donated electrons to, and possibly physically associated with, the proton-pumping cytochrome bcc-aa3 supercomplex. In contrast the more promiscuous Hhy also provided electrons to the cytochrome bd oxidase complex. These results indicate that, despite their similar characteristics, Huc and Hhy perform distinct functions during mycobacterial growth and survival.
Asunto(s)
Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium smegmatis/metabolismo , Oxidorreductasas/metabolismo , Aerobiosis , Mycobacterium smegmatis/enzimologíaRESUMEN
The ability to persist in the absence of growth triggered by low oxygen levels is a critical process for the survival of mycobacterial species in many environmental niches. MSMEG_5243 (fsq), a gene of unknown function in Mycobacterium smegmatis, is up-regulated in response to hypoxia and regulated by DosRDosS/DosT, an oxygen- and redox-sensing two-component system that is highly conserved in mycobacteria. In this communication, we demonstrate that MSMEG_5243 is a flavin-sequestering protein and henceforth refer to it as Fsq. Using an array of biochemical and structural analyses, we show that Fsq is a member of the diverse superfamily of flavin- and deazaflavin-dependent oxidoreductases (FDORs) and is widely distributed in mycobacterial species. We created a markerless deletion mutant of fsq and demonstrate that fsq is required for cell survival during hypoxia. Using fsq deletion and overexpression, we found that fsq enhances cellular resistance to hydrogen peroxide treatment. The X-ray crystal structure of Fsq, solved to 2.7 Å, revealed a homodimeric organization with FAD bound noncovalently. The Fsq structure also uncovered no potential substrate-binding cavities, as the FAD is fully enclosed, and electrochemical studies indicated that the Fsq:FAD complex is relatively inert and does not share common properties with electron-transfer proteins. Taken together, our results suggest that Fsq reduces the formation of reactive oxygen species (ROS) by sequestering free FAD during recovery from hypoxia, thereby protecting the cofactor from undergoing autoxidation to produce ROS. This finding represents a new paradigm in mycobacterial adaptation to hypoxia.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Hipoxia , Mycobacterium/crecimiento & desarrollo , Estrés Oxidativo , Oxígeno/metabolismo , Sustancias Protectoras/metabolismo , Proteínas Bacterianas/genética , Catálisis , Cristalografía por Rayos X , Transporte de Electrón , Modelos Moleculares , Mycobacterium/metabolismo , Oxidación-Reducción , Conformación ProteicaRESUMEN
The recent discovery of small molecules targeting the cytochrome bc1 :aa3 in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc1 :aa3 consists of a menaquinone:cytochrome c reductase (bc1 ) and a cytochrome aa3 -type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc1 :aa3 complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc1 :aa3 , as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis.
Asunto(s)
Mycobacterium tuberculosis/metabolismo , Oxidorreductasas/química , Mutaciones Letales Sintéticas , Adenosina Trifosfato/química , Animales , Antineoplásicos/farmacología , Antituberculosos/farmacología , Reductasas del Citocromo/metabolismo , Diarilquinolinas/farmacología , Transporte de Electrón , Complejo IV de Transporte de Electrones/metabolismo , Eliminación de Gen , Humanos , Inflamación , Ratones , Ratones Endogámicos BALB C , Proteínas Mitocondriales , Infecciones por Mycobacterium/microbiología , Mycobacterium bovis , Mycobacterium tuberculosis/genética , Fosforilación Oxidativa , Oxidorreductasas/genética , Oxígeno/química , Proteínas de Plantas , Células THP-1RESUMEN
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 non-replicating 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.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Metabolismo Energético/efectos de los fármacos , Mycobacterium tuberculosis/fisiología , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Antituberculosos/uso terapéutico , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Diarilquinolinas/farmacología , Diarilquinolinas/uso terapéutico , Diseño de Fármacos , Descubrimiento de Drogas/métodos , Farmacorresistencia Bacteriana Múltiple , Complejo I de Transporte de Electrón , Humanos , Potenciales de la Membrana/efectos de los fármacos , Terapia Molecular Dirigida/métodos , Mycobacterium tuberculosis/efectos de los fármacos , ATPasas de Translocación de Protón/antagonistas & inhibidores , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Desacopladores/farmacología , Desacopladores/uso terapéuticoRESUMEN
The majority of microbial cells in global soils exist in a spectrum of dormant states. However, the metabolic processes that enable them to survive environmental challenges, such as nutrient-limitation, remain to be elucidated. In this work, we demonstrate that energy-starved cultures of Pyrinomonas methylaliphatogenes, an aerobic heterotrophic acidobacterium isolated from New Zealand volcanic soils, persist by scavenging the picomolar concentrations of H2 distributed throughout the atmosphere. Following the transition from exponential to stationary phase due to glucose limitation, the bacterium up-regulates by fourfold the expression of an eight-gene operon encoding an actinobacteria-type H2-uptake [NiFe]-hydrogenase. Whole-cells of the organism consume atmospheric H2 in a first-order kinetic process. Hydrogen oxidation occurred most rapidly under oxic conditions and was weakly associated with the cell membrane. We propose that atmospheric H2 scavenging serves as a mechanism to sustain the respiratory chain of P. methylaliphatogenes when organic electron donors are scarce. As the first observation of H2 oxidation to our knowledge in the Acidobacteria, the second most dominant soil phylum, this work identifies new sinks in the biogeochemical H2 cycle and suggests that trace gas oxidation may be a general mechanism for microbial persistence.
Asunto(s)
Acidobacteria/metabolismo , Gases , Microbiología del Suelo , Secuencia de Aminoácidos , Atmósfera , Carbono/química , Cromatografía de Gases , Transporte de Electrón , Electrones , Regulación Bacteriana de la Expresión Génica , Hidrógeno/química , Hidrogenasas/metabolismo , Cinética , Datos de Secuencia Molecular , Oxidación-Reducción , Oxígeno/química , Filogenia , Reproducibilidad de los Resultados , Homología de Secuencia de Aminoácido , Suelo/químicaRESUMEN
In the Earth's lower atmosphere, H2 is maintained at trace concentrations (0.53 ppmv/0.40 nM) and rapidly turned over (lifetime ≤ 2.1 y(-1)). It is thought that soil microbes, likely actinomycetes, serve as the main global sink for tropospheric H2. However, no study has ever unambiguously proven that a hydrogenase can oxidize this trace gas. In this work, we demonstrate, by using genetic dissection and sensitive GC measurements, that the soil actinomycete Mycobacterium smegmatis mc(2)155 constitutively oxidizes subtropospheric concentrations of H2. We show that two membrane-associated, oxygen-dependent [NiFe] hydrogenases mediate this process. Hydrogenase-1 (Hyd1) (MSMEG_2262-2263) is well-adapted to rapidly oxidize H2 at a range of concentrations [Vmax(app) = 12 nmolâ gâ dw(-1)â min(-1); Km(app) = 180 nM; threshold = 130 pM in the Δhyd23 (Hyd1 only) strain], whereas Hyd2 (MSMEG_2719-2720) catalyzes a slower-acting, higher-affinity process [Vmax(app) = 2.5 nmolâ gâ dw(-1)â min(-1); Km(app) = 50 nM; threshold = 50 pM in the Δhyd13 (Hyd2 only) strain]. These observations strongly support previous studies that have linked group 5 [NiFe] hydrogenases (e.g., Hyd2) to the oxidation of tropospheric H2 in soil ecosystems. We further reveal that group 2a [NiFe] hydrogenases (e.g., Hyd1) can contribute to this process. Hydrogenase expression and activity increases in carbon-limited cells, suggesting that scavenging of trace H2 helps to sustain dormancy. Distinct physiological roles for Hyd1 and Hyd2 during the adaptation to this condition are proposed. Soil organisms harboring high-affinity hydrogenases may be especially competitive, given that they harness a highly dependable fuel source in otherwise unstable environments.
Asunto(s)
Atmósfera/química , Hidrógeno/metabolismo , Hidrogenasas/metabolismo , Mycobacterium smegmatis/enzimología , Microbiología del Suelo , Cromatografía de Gases , Hidrógeno/análisis , Oxidación-Reducción , Oxígeno/metabolismoRESUMEN
OBJECTIVES: Quinolinequinones (QQ) have been shown to inhibit the growth of mycobacterial species, but their mode(s) of action and molecular target(s) remain unknown. To facilitate further development of QQ as antimycobacterial drugs, we investigated the molecular mechanism and target of QQ in mycobacteria. METHODS: Cell viability of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin was determined in the presence of QQ8c, a representative QQ compound, and isoniazid, a frontline antitubercular drug. The effect of QQ8c on mycobacterial energetics was studied using inverted membrane vesicles. NADH oxidation and formation of reactive oxygen species (ROS) were measured in the presence and absence of KCN. Generation of ROS was measured via oxygen consumption in an oxygen electrode. The effects of QQ8c were compared with the antimycobacterial drug clofazimine in side-by-side experiments. RESULTS: QQ8c challenge resulted in complete sterilization of cultures with no refractory resistant population observed. QQ8c stimulated NADH oxidation by type II NADH dehydrogenase (NDH-2) and oxygen consumption in inverted membrane vesicles. Large quantities of ROS were produced in the presence of QQ8. Even when oxygen consumption was blocked with KCN, activation of NDH-2 by QQ8c occurred suggesting QQ8c was redox cycling. CONCLUSIONS: QQ8c targets NDH-2 of the mycobacterial respiratory chain leading to activation of NADH oxidation and generating bactericidal levels of ROS in a manner similar to, but more effectively than, the antimycobacterial drug clofazimine. Our results validate respiratory-generated ROS as an avenue for antimycobacterial drug development.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , NADH Deshidrogenasa/metabolismo , Quinolonas/farmacología , Quinonas/farmacología , Proteínas Bacterianas/metabolismo , Clofazimina/farmacología , Descubrimiento de Drogas , Transporte de Electrón/efectos de los fármacos , Activación Enzimática , Viabilidad Microbiana/efectos de los fármacos , Mycobacterium bovis/efectos de los fármacos , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/fisiología , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The 2014 report from the World Health Organization (WHO) on antimicrobial resistance revealed an alarming rise in antibiotic resistance all around the world. Unlike classical antibiotics, with the exception of a few species, no acquired resistance towards antimicrobial peptides (AMPs) has been reported. Therefore, AMPs represent leads for the development of novel antibiotics. Caenopore-5 is constitutively expressed in the intestine of the nematode Caenorhabditis elegans and is a pore-forming AMP. The protein (82 amino acids) was successfully synthesised by using Boc solid-phase peptide synthesis and native chemical ligation. No γ-linked by-product was observed despite the use of a C-terminal Glu-thioester. The folding of the synthetic protein was confirmed by (1) H NMR spectroscopy and circular dichroism and compared with data recorded for recombinant caenopore-5. The permeabilisation activities of the protein and of shortened analogues were evaluated.
Asunto(s)
Antiinfecciosos/síntesis química , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Secuencia de Aminoácidos , Animales , Antiinfecciosos/química , Proteínas de Caenorhabditis elegans/síntesis química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/farmacología , Membrana Celular/efectos de los fármacos , Técnicas de Química Sintética , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Permeabilidad , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Técnicas de Síntesis en Fase SólidaRESUMEN
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.
Asunto(s)
Antituberculosos/farmacología , Diarilquinolinas/farmacología , Viabilidad Microbiana/efectos de los fármacos , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/fisiología , Desacopladores/farmacología , Medios de Cultivo/química , Perfilación de la Expresión Génica , Humanos , Análisis por Micromatrices , Técnicas MicrobiológicasRESUMEN
We have known for 40 years that soils can consume the trace amounts of molecular hydrogen (H2) found in the Earth's atmosphere.This process is predicted to be the most significant term in the global hydrogen cycle. However, the organisms and enzymes responsible for this process were only recently identified. Pure culture experiments demonstrated that several species of Actinobacteria, including streptomycetes and mycobacteria, can couple the oxidation of atmospheric H2 to the reduction of ambient O2. A combination of genetic, biochemical, and phenotypic studies suggest that these organisms primarily use this fuel source to sustain electron input into the respiratory chain during energy starvation. This process is mediated by a specialized enzyme, the group 5 [NiFe]-hydrogenase, which is unusual for its high affinity, oxygen insensitivity, and thermostability. Atmospheric hydrogen scavenging is a particularly dependable mode of energy generation, given both the ubiquity of the substrate and the stress tolerance of its catalyst. This minireview summarizes the recent progress in understanding how and why certain organisms scavenge atmospheric H2. In addition, it provides insight into the wider significance of hydrogen scavenging in global H2 cycling and soil microbial ecology.
Asunto(s)
Actinobacteria/metabolismo , Proteínas Bacterianas/metabolismo , Hidrógeno/metabolismo , Hidrogenasas/metabolismo , Actinobacteria/enzimología , Actinobacteria/genética , Microbiología del Aire , Atmósfera/química , Proteínas Bacterianas/genética , Ecosistema , Hidrogenasas/genéticaRESUMEN
Mycobacterium smegmatis is an obligate aerobe that harbours three predicted [NiFe] hydrogenases, Hyd1 (MSMEG_22622263), Hyd2 (MSMEG_2720-2719) and Hyd3 (MSMEG_3931-3928). We show here that these three enzymes differ in their phylogeny, regulation and catalytic activity. Phylogenetic analysis revealed that Hyd1 groups with hydrogenases that oxidize H2 produced by metabolic processes, and Hyd2 is homologous to a novel group of putative high-affinity hydrogenases. Hyd1 and Hyd2 respond to carbon and oxygen limitation, and, in the case of Hyd1, hydrogen supplementation. Hydrogen consumption measurements confirmed that both enzymes can oxidize hydrogen. In contrast, the phylogenetic analysis and activity measurements of Hyd3 are consistent with the enzyme evolving hydrogen. Hyd3 is controlled by DosR, a regulator that responds to hypoxic conditions. The strict dependence of hydrogen oxidation of Hyd1 and Hyd2 on oxygen suggests that the enzymes are oxygen tolerant and linked to the respiratory chain. This unique combination of hydrogenases allows M. smegmatis to oxidize hydrogen at high (Hyd1) and potentially tropospheric (Hyd2) concentrations, as well as recycle reduced equivalents by evolving hydrogen (Hyd3). The distribution of these hydrogenases throughout numerous soil and marine species of actinomycetes suggests that oxic hydrogen metabolism provides metabolic flexibility in environments with changing nutrient fluxes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Hidrogenasas/metabolismo , Mycobacterium smegmatis/enzimología , Aerobiosis , Proteínas Bacterianas/genética , Hidrógeno/metabolismo , Hidrogenasas/genética , Familia de Multigenes , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Operón , Oxidación-Reducción , Oxígeno/metabolismo , FilogeniaRESUMEN
Mycobacterial bioenergetics is a validated target space for antitubercular drug development. Here, we identify BB2-50F, a 6-substituted 5-(N,N-hexamethylene)amiloride derivative as a potent, multi-targeting bioenergetic inhibitor of Mycobacterium tuberculosis. We show that BB2-50F rapidly sterilizes both replicating and non-replicating cultures of M. tuberculosis and synergizes with several tuberculosis drugs. Target identification experiments, supported by docking studies, showed that BB2-50F targets the membrane-embedded c-ring of the F1Fo-ATP synthase and the catalytic subunit (substrate-binding site) of succinate dehydrogenase. Biochemical assays and metabolomic profiling showed that BB2-50F inhibits succinate oxidation, decreases the activity of the tricarboxylic acid (TCA) cycle, and results in succinate secretion from M. tuberculosis. Moreover, we show that the lethality of BB2-50F under aerobic conditions involves the accumulation of reactive oxygen species. Overall, this study identifies BB2-50F as an effective inhibitor of M. tuberculosis and highlights that targeting multiple components of the mycobacterial respiratory chain can produce fast-acting antimicrobials.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Succinato Deshidrogenasa/metabolismo , Succinato Deshidrogenasa/farmacología , Antituberculosos/química , Tuberculosis/tratamiento farmacológico , Adenosina Trifosfato , Inhibidores Enzimáticos/farmacología , SuccinatosRESUMEN
The bioenergetic mechanisms by which Mycobacterium tuberculosis survives hypoxia are poorly understood. Current models assume that the bacterium shifts to an alternate electron acceptor or fermentation to maintain membrane potential and ATP synthesis. Counterintuitively, we find here that oxygen itself is the principal terminal electron acceptor during hypoxic dormancy. M. tuberculosis can metabolize oxygen efficiently at least two orders of magnitude below the concentration predicted to occur in hypoxic lung granulomas. Despite a difference in apparent affinity for oxygen, both the cytochrome bcc:aa3 and cytochrome bd oxidase respiratory branches are required for hypoxic respiration. Simultaneous inhibition of both oxidases blocks oxygen consumption, reduces ATP levels, and kills M. tuberculosis under hypoxia. The capacity of mycobacteria to scavenge trace levels of oxygen, coupled with the absence of complex regulatory mechanisms to achieve hierarchal control of the terminal oxidases, may be a key determinant of long-term M. tuberculosis survival in hypoxic lung granulomas.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Mycobacterium tuberculosis/metabolismo , Oxígeno/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Oxidorreductasas/metabolismo , Homeostasis , Tuberculosis/microbiología , Hipoxia , Adenosina Trifosfato/metabolismo , Citocromos/metabolismoRESUMEN
Transport of proteins across and into membranes is a fundamental biological process with the vast majority being conducted by the ubiquitous Sec machinery. In bacteria, this is usually achieved when the SecY-complex engages the cytosolic ATPase SecA (secretion) or translating ribosomes (insertion). Great strides have been made towards understanding the mechanism of protein translocation. Yet, important questions remain - notably, the nature of the individual steps that constitute transport, and how the proton-motive force (PMF) across the plasma membrane contributes. Here, we apply a recently developed high-resolution protein transport assay to explore these questions. We find that pre-protein transport is limited primarily by the diffusion of arginine residues across the membrane, particularly in the context of bulky hydrophobic sequences. This specific effect of arginine, caused by its positive charge, is mitigated for lysine which can be deprotonated and transported across the membrane in its neutral form. These observations have interesting implications for the mechanism of protein secretion, suggesting a simple mechanism through which the PMF can aid transport by enabling a 'proton ratchet', wherein re-protonation of exiting lysine residues prevents channel re-entry, biasing transport in the outward direction.
Asunto(s)
Proteínas de Escherichia coli , Arginina/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lisina/metabolismo , Transporte de Proteínas , Canales de Translocación SEC/metabolismoRESUMEN
Due to its central role in energy generation and bacterial viability, mycobacterial bioenergetics is an attractive therapeutic target for anti-tuberculosis drug discovery. Building upon our work on antimycobacterial dioxonaphthoimidazoliums that were activated by a proximal positive charge and generated reactive oxygen species upon reduction by Type II NADH dehydrogenase, we herein studied the effect of a distal positive charge on the antimycobacterial activity of naphthoquinoneimidazoles by incorporating a trialkylphosphonium cation. The potency-enhancing properties of the linker length were affirmed by structure-activity relationship studies. The most active compound against M. tb H37Rv displayed good selectivity index (SI = 34) and strong bactericidal activity in the low micromolar range, which occurred through rapid bacterial membrane depolarization that resulted in depletion of intracellular ATP. Through this work, we demonstrated a switch of the scaffold's mode-of-action via relocation of positive charge while retaining its excellent antibacterial activity and selectivity.
RESUMEN
A revised total synthesis of aurachin D (1a), an isoprenoid quinolone alkaloid that targets Mycobacterium tuberculosis (Mtb) cytochrome bd (cyt-bd) oxidase, was accomplished using an oxazoline ring-opening reaction. The ring opening enabled access to a range of electron-poor analogues, while electron-rich analogues could be prepared using the Conrad-Limpach reaction. The aryl-substituted and side-chain-modified aurachin D analogues were screened for inhibition of Mtb cyt-bd oxidase and growth inhibition of Mtb. Nanomolar inhibition of Mtb cyt-bd oxidase was observed for the shorter-chain analogue 1d (citronellyl side chain) and the aryl-substituted analogues 1g/1k (fluoro substituent at C6/C7), 1t/1v (hydroxy substituent at C5/C6) and 1u/1w/1x (methoxy substituent at C5/C6/C7). Aurachin D and the analogues did not inhibit growth of nonpathogenic Mycobacterium smegmatis, but the citronellyl (1d) and 6-fluoro-substituted (1g) inhibitors from the Mtb cyt-bd oxidase assay displayed moderate growth inhibition against pathogenic Mtb (MIC = 4-8 µM).