RESUMEN
Bacterial cytoplasmic membrane vesicles provide a unique experimental system for studying active transport. Vesicles are prepared by lysis of osmotically sensitized cells (i.e., protoplasts or spheroplasts) and comprise osmotically intact, unit-membrane-bound sacs that are approximately 0.5-1.0 µm in diameter and devoid of internal structure. Their metabolic activities are restricted to those provided by the enzymes of the membrane itself, and each vesicle is functional. The energy source for accumulation of a particular substrate can be determined by studying which compounds or experimental conditions drive solute accumulation, and metabolic conversion of the transported substrate or the energy source is minimal. These properties of the vesicle system constitute a considerable advantage over intact cells, as the system provides clear definition of the reactions involved in the transport process. This discussion is not intended as a general review but is concerned with respiration-dependent active transport in membrane vesicles from Escherichia coli. Emphasis is placed on experimental observations demonstrating that respiratory energy is converted primarily into work in the form of a solute concentration gradient that is driven by a proton electrochemical gradient, as postulated by the chemiosmotic theory of Peter Mitchell.
Asunto(s)
Vesículas Citoplasmáticas/metabolismo , Escherichia coli/metabolismo , Biología Molecular/historia , Transporte Biológico , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Membrana Celular/efectos de los fármacos , Escherichia coli/citología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Ácido Láctico/metabolismo , Masculino , Estados UnidosRESUMEN
All cells fold their genomes, including bacterial cells, where the chromosome is compacted into a domain-organized meshwork called the nucleoid. How compaction and domain organization arise is not fully understood. Here, we describe a method to estimate the average mesh size of the nucleoid in Escherichia coli. Using nucleoid mesh size and DNA concentration estimates, we find that the cytoplasm behaves as a poor solvent for the chromosome when the cell is considered as a simple semidilute polymer solution. Monte Carlo simulations suggest that a poor solvent leads to chromosome compaction and DNA density heterogeneity (i.e., domain formation) at physiological DNA concentration. Fluorescence microscopy reveals that the heterogeneous DNA density negatively correlates with ribosome density within the nucleoid, consistent with cryoelectron tomography data. Drug experiments, together with past observations, suggest the hypothesis that RNAs contribute to the poor solvent effects, connecting chromosome compaction and domain formation to transcription and intracellular organization.
Asunto(s)
Cromosomas Bacterianos/química , Escherichia coli/metabolismo , Conformación de Ácido Nucleico , Solventes/química , Transcripción Genética , Aminoglicósidos/farmacología , Simulación por Computador , ADN Bacteriano/química , Difusión , Escherichia coli/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Tamaño de la Partícula , ARN Bacteriano/metabolismo , Ribosomas/metabolismo , Ribosomas/ultraestructura , Transcripción Genética/efectos de los fármacosRESUMEN
Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.
Asunto(s)
Antibacterianos/química , Antineoplásicos/química , Infecciones por Escherichia coli/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Nucleótidos/metabolismo , Ribonucleótido Reductasas/química , Antibacterianos/uso terapéutico , Antineoplásicos/uso terapéutico , Biocatálisis , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Infecciones por Escherichia coli/enzimología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Humanos , Simulación del Acoplamiento Molecular , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Nucleótidos/química , Oxidación-Reducción , Estructura Secundaria de Proteína , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Relación Estructura-ActividadRESUMEN
Metformin is the first-line therapy for treating type 2 diabetes and a promising anti-aging drug. We set out to address the fundamental question of how gut microbes and nutrition, key regulators of host physiology, affect the effects of metformin. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we developed a high-throughput four-way screen to define the underlying host-microbe-drug-nutrient interactions. We show that microbes integrate cues from metformin and the diet through the phosphotransferase signaling pathway that converges on the transcriptional regulator Crp. A detailed experimental characterization of metformin effects downstream of Crp in combination with metabolic modeling of the microbiota in metformin-treated type 2 diabetic patients predicts the production of microbial agmatine, a regulator of metformin effects on host lipid metabolism and lifespan. Our high-throughput screening platform paves the way for identifying exploitable drug-nutrient-microbiome interactions to improve host health and longevity through targeted microbiome therapies. VIDEO ABSTRACT.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Microbioma Gastrointestinal/efectos de los fármacos , Interacciones Microbiota-Huesped/efectos de los fármacos , Hipoglucemiantes/uso terapéutico , Metformina/uso terapéutico , Agmatina/metabolismo , Animales , Caenorhabditis elegans/microbiología , Proteína Receptora de AMP Cíclico , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Humanos , Hipoglucemiantes/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Longevidad/efectos de los fármacos , Metformina/farmacología , Nutrientes/metabolismoRESUMEN
Microbial populations can maximize fitness in dynamic environments through bet hedging, a process wherein a subpopulation assumes a phenotype not optimally adapted to the present environment but well adapted to an environment likely to be encountered. Here, we show that oxygen induces fluctuating expression of the trimethylamine oxide (TMAO) respiratory system of Escherichia coli, diversifying the cell population and enabling a bet-hedging strategy that permits growth following oxygen loss. This regulation by oxygen affects the variance in gene expression but leaves the mean unchanged. We show that the oxygen-sensitive transcription factor IscR is the key regulator of variability. Oxygen causes IscR to repress expression of a TMAO-responsive signaling system, allowing stochastic effects to have a strong effect on the output of the system and resulting in heterogeneous expression of the TMAO reduction machinery. This work reveals a mechanism through which cells regulate molecular noise to enhance fitness.
Asunto(s)
Escherichia coli/metabolismo , Transducción de Señal , Aerobiosis , Anaerobiosis , Secuencia de Bases , Sitios de Unión , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Metilaminas/metabolismo , Metilaminas/farmacología , Oxígeno/metabolismo , Proteínas Periplasmáticas/química , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Fosfotransferasas/química , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Regulación hacia ArribaRESUMEN
Multidrug resistance is a global threat as the clinically available potent antibiotic drugs are becoming exceedingly scarce. For example, increasing drug resistance among gram-positive bacteria is responsible for approximately one-third of nosocomial infections. As ribosomes are a major target for these drugs, they may serve as suitable objects for novel development of next-generation antibiotics. Three-dimensional structures of ribosomal particles from Staphylococcus aureus obtained by X-ray crystallography have shed light on fine details of drug binding sites and have revealed unique structural motifs specific for this pathogenic strain, which may be used for the design of novel degradable pathogen-specific, and hence, environmentally friendly drugs.
Asunto(s)
Antibacterianos/síntesis química , Proteínas Bacterianas/química , Diseño de Fármacos , Ribosomas/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/microbiología , Cristalografía por Rayos X , Deinococcus/efectos de los fármacos , Deinococcus/genética , Deinococcus/metabolismo , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Modelos Moleculares , Ribosomas/metabolismo , Ribosomas/ultraestructura , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Thermus thermophilus/efectos de los fármacos , Thermus thermophilus/genética , Thermus thermophilus/metabolismoRESUMEN
Evolvabilitythe capacity to generate beneficial heritable variationis a central property of biological systems. However, its origins and modulation by environmental factors have not been examined systematically. Here, we analyze the fitness effects of all single mutations in TEM-1 ß-lactamase (4,997 variants) under selection for the wild-type function (ampicillin resistance) and for a new function (cefotaxime resistance). Tolerance to mutation in this enzyme is bimodal and dependent on the strength of purifying selection in vivo, a result that derives from a steep non-linear ampicillin-dependent relationship between biochemical activity and fitness. Interestingly, cefotaxime resistance emerges from mutations that are neutral at low levels of ampicillin but deleterious at high levels; thus the capacity to evolve new function also depends on the strength of selection. The key property controlling evolvability is an excess of enzymatic activity relative to the strength of selection, suggesting that fluctuating environments might select for high-activity enzymes.
Asunto(s)
Resistencia a la Ampicilina , Cefotaxima/farmacología , Evolución Molecular Dirigida , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , beta-Lactamasas/genética , Ampicilina/farmacología , Escherichia coli/enzimología , Aptitud Genética , Mutación , Resistencia betalactámica , beta-Lactamasas/químicaRESUMEN
All bacteria form persisters, cells that are multidrug tolerant and therefore able to survive antibiotic treatment. Due to the low frequencies of persisters in growing bacterial cultures and the complex underlying molecular mechanisms, the phenomenon has been challenging to study. However, recent technological advances in microfluidics and reporter genes have improved this scenario. Here, we summarize recent progress in the field, revealing the ubiquitous bacterial stress alarmone ppGpp as an emerging central regulator of multidrug tolerance and persistence, both in stochastically and environmentally induced persistence. In several different organisms, toxin-antitoxin modules function as effectors of ppGpp-induced persistence.
Asunto(s)
Bacterias/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple , Guanosina Tetrafosfato/metabolismo , Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Hongos/efectos de los fármacos , Transducción de SeñalRESUMEN
Penicillin and related beta-lactams comprise one of our oldest and most widely used antibiotic therapies. These drugs have long been known to target enzymes called penicillin-binding proteins (PBPs) that build the bacterial cell wall. Investigating the downstream consequences of target inhibition and how they contribute to the lethal action of these important drugs, we demonstrate that beta-lactams do more than just inhibit the PBPs as is commonly believed. Rather, they induce a toxic malfunctioning of their target biosynthetic machinery involving a futile cycle of cell wall synthesis and degradation, thereby depleting cellular resources and bolstering their killing activity. Characterization of this mode of action additionally revealed a quality control function for enzymes that cleave bonds in the cell wall matrix. The results thus provide insight into the mechanism of cell wall assembly and suggest how best to interfere with the process for future antibiotic development.
Asunto(s)
Amdinocilina/farmacología , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , beta-Lactamas/farmacología , Pared Celular/efectos de los fármacos , Pared Celular/enzimología , Escherichia coli/citología , Escherichia coli/enzimología , Glicósido Hidrolasas/antagonistas & inhibidores , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano/metabolismoRESUMEN
Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering1 to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription-replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.
Asunto(s)
Antibacterianos , Sitios de Unión , ARN Polimerasas Dirigidas por ADN , Escherichia coli , Mutación , Rifampin , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Roturas del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Farmacorresistencia Bacteriana/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Nucleótidos/deficiencia , Nucleótidos/metabolismo , Regiones Promotoras Genéticas , Rifampin/química , Rifampin/metabolismo , Rifampin/farmacología , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
Persistence refers to the phenomenon in which isogenic populations of antibiotic-sensitive bacteria produce rare cells that transiently become multidrug tolerant. Whether slow growth in a rare subset of cells underlies the persistence phenotype has not be examined in wild-type bacteria. Here, we show that an exponentially growing population of wild-type Escherichia coli cells produces rare cells that stochastically switch into slow growth, that the slow-growing cells are multidrug tolerant, and that they are able to resuscitate. The persistence phenotype depends hierarchically on the signaling nucleotide (p)ppGpp, Lon protease, inorganic polyphosphate, and toxin-antitoxins. We show that the level of (p)ppGpp varies stochastically in a population of exponentially growing cells and that the high (p)ppGpp level in rare cells induces slow growth and persistence. (p)ppGpp triggers slow growth by activating toxin-antitoxin loci through a regulatory cascade depending on inorganic polyphosphate and Lon protease.
Asunto(s)
Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Guanosina Pentafosfato/metabolismo , Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/efectos de los fármacos , Polifosfatos/metabolismo , Proteasa La/metabolismo , Transcripción GenéticaRESUMEN
Mutagenesis is responsive to many environmental factors. Evolution therefore depends on the environment not only for selection but also in determining the variation available in a population. One such environmental dependency is the inverse relationship between mutation rates and population density in many microbial species. Here, we determine the mechanism responsible for this mutation rate plasticity. Using dynamical computational modelling and in culture mutation rate estimation, we show that the negative relationship between mutation rate and population density arises from the collective ability of microbial populations to control concentrations of hydrogen peroxide. We demonstrate a loss of this density-associated mutation rate plasticity (DAMP) when Escherichia coli populations are deficient in the degradation of hydrogen peroxide. We further show that the reduction in mutation rate in denser populations is restored in peroxide degradation-deficient cells by the presence of wild-type cells in a mixed population. Together, these model-guided experiments provide a mechanistic explanation for DAMP, applicable across all domains of life, and frames mutation rate as a dynamic trait shaped by microbial community composition.
Asunto(s)
Escherichia coli , Tasa de Mutación , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Peróxido de Hidrógeno/metabolismo , Mutación , Inactivación Metabólica/genéticaRESUMEN
The polypeptide exit tunnel is an important functional compartment of the ribosome where the newly synthesized proteins are surveyed. The tunnel is the target of clinically important macrolide antibiotics. Macrolides plug the tunnel and are believed to stop production of all proteins. Contrary to this view, we show that drug-bound ribosomes can synthesize a distinct subset of cellular polypeptides. The structure of a protein defines its ability to thread through the antibiotic-obstructed tunnel. Synthesis of certain polypeptides that initially bypass translational arrest can be stopped at later stages of elongation while translation of some proteins goes to completion. Our findings reveal that small-molecule effectors can accentuate the discriminatory properties of the ribosomal exit tunnel and that macrolide antibiotics reshape the cellular proteome rather than block global protein synthesis.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Macrólidos/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Ribosomas/efectos de los fármacos , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Extensión de la Cadena Peptídica de Translación , Ribosomas/metabolismoRESUMEN
Stress responses allow cells to adapt to changes in external conditions by activating specific pathways1. Here we investigate the dynamics of single cells that were subjected to acute stress that is too strong for a regulated response but not lethal. We show that when the growth of bacteria is arrested by acute transient exposure to strong inhibitors, the statistics of their regrowth dynamics can be predicted by a model for the cellular network that ignores most of the details of the underlying molecular interactions. We observed that the same stress, applied either abruptly or gradually, can lead to totally different recovery dynamics. By measuring the regrowth dynamics after stress exposure on thousands of cells, we show that the model can predict the outcome of antibiotic persistence measurements. Our results may account for the ubiquitous antibiotic persistence phenotype2, as well as for the difficulty in attempts to link it to specific genes3. More generally, our approach suggests that two different cellular states can be observed under stress: a regulated state, which prepares cells for fast recovery, and a disrupted cellular state due to acute stress, with slow and heterogeneous recovery dynamics. The disrupted state may be described by general properties of large random networks rather than by specific pathway activation. Better understanding of the disrupted state could shed new light on the survival and evolution of cells under stress.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Viabilidad Microbiana/efectos de los fármacos , Estrés Fisiológico/fisiología , Escherichia coli/citología , Privación de Alimentos , Análisis de la Célula Individual , Factores de TiempoRESUMEN
Antibiotics that target Gram-negative bacteria in new ways are needed to resolve the antimicrobial resistance crisis1-3. Gram-negative bacteria are protected by an additional outer membrane, rendering proteins on the cell surface attractive drug targets4,5. The natural compound darobactin targets the bacterial insertase BamA6-the central unit of the essential BAM complex, which facilitates the folding and insertion of outer membrane proteins7-13. BamA lacks a typical catalytic centre, and it is not obvious how a small molecule such as darobactin might inhibit its function. Here we resolve the mode of action of darobactin at the atomic level using a combination of cryo-electron microscopy, X-ray crystallography, native mass spectrometry, in vivo experiments and molecular dynamics simulations. Two cyclizations pre-organize the darobactin peptide in a rigid ß-strand conformation. This creates a mimic of the recognition signal of native substrates with a superior ability to bind to the lateral gate of BamA. Upon binding, darobactin replaces a lipid molecule from the lateral gate to use the membrane environment as an extended binding pocket. Because the interaction between darobactin and BamA is largely mediated by backbone contacts, it is particularly robust against potential resistance mutations. Our results identify the lateral gate as a functional hotspot in BamA and will allow the rational design of antibiotics that target this bacterial Achilles heel.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Fenilpropionatos/química , Fenilpropionatos/farmacología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Cristalografía por Rayos X , Diseño de Fármacos , Escherichia coli/citología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Espectrometría de Masas , Simulación de Dinámica Molecular , Estructura Secundaria de ProteínaRESUMEN
The use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding the cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes, but strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at internal in-frame and out-of-frame start sites, can be functionally important and contribute to the "alternative" bacterial proteome. The internal start sites may also play regulatory roles in gene expression.
Asunto(s)
Genoma Bacteriano/genética , Iniciación de la Cadena Peptídica Traduccional , Proteoma/genética , Proteómica , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Codón Iniciador/genética , Diterpenos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genoma Bacteriano/efectos de los fármacos , ARN Mensajero/genética , Ribosomas/efectos de los fármacos , Ribosomas/genéticaRESUMEN
Antibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the fluoroquinolone antibiotic ciprofloxacin (cipro) induces mutations by triggering transient differentiation of a mutant-generating cell subpopulation, using reactive oxygen species (ROS). Cipro-induced DNA breaks activate the Escherichia coli SOS DNA-damage response and error-prone DNA polymerases in all cells. However, mutagenesis is limited to a cell subpopulation in which electron transfer together with SOS induce ROS, which activate the sigma-S (σS) general-stress response, which allows mutagenic DNA-break repair. When sorted, this small σS-response-"on" subpopulation produces most antibiotic cross-resistant mutants. A U.S. Food and Drug Administration (FDA)-approved drug prevents σS induction, specifically inhibiting antibiotic-promoted mutagenesis. Further, SOS-inhibited cell division, which causes multi-chromosome cells, promotes mutagenesis. The data support a model in which within-cell chromosome cooperation together with development of a "gambler" cell subpopulation promote resistance evolution without risking most cells.
Asunto(s)
Antibacterianos/efectos adversos , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Mutagénesis/genética , División Celular/efectos de los fármacos , Ciprofloxacina/efectos adversos , Daño del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/genética , Farmacorresistencia Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/patogenicidad , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Mutagénesis/efectos de los fármacos , Mutación , Especies Reactivas de Oxígeno/metabolismo , Respuesta SOS en Genética/efectos de los fármacos , Factor sigma/genéticaRESUMEN
Cell dormancy is a widespread mechanism used by bacteria to evade environmental threats, including antibiotics. Here we monitored bacterial antibiotic tolerance and regrowth at the single-cell level and found that each individual survival cell shows different "dormancy depth," which in return regulates the lag time for cell resuscitation after removal of antibiotic. We further established that protein aggresome-a collection of endogenous protein aggregates-is an important indicator of bacterial dormancy depth, whose formation is promoted by decreased cellular ATP level. For cells to leave the dormant state and resuscitate, clearance of protein aggresome and recovery of proteostasis are required. We revealed that the ability to recruit functional DnaK-ClpB machineries, which facilitate protein disaggregation in an ATP-dependent manner, determines the lag time for bacterial regrowth. Better understanding of the key factors regulating bacterial regrowth after surviving antibiotic attack could lead to new therapeutic strategies for combating bacterial antibiotic tolerance.
Asunto(s)
Adenosina Trifosfato/metabolismo , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Metabolismo Energético/efectos de los fármacos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Agregado de Proteínas , Endopeptidasa Clp/genética , Endopeptidasa Clp/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Concentración de Iones de Hidrógeno , Viabilidad Microbiana/efectos de los fármacos , Análisis de la Célula Individual , Factores de TiempoRESUMEN
The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Bacteriocinas/biosíntesis , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Complejos Multienzimáticos/metabolismo , Ribosomas/enzimología , Inhibidores de Topoisomerasa II/metabolismo , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bacteriocinas/química , Bacteriocinas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Ribosomas/efectos de los fármacos , Ribosomas/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacología , Difracción de Rayos XRESUMEN
Alternative to the conventional search for single-target, single-compound treatments, combination therapies can open entirely new opportunities to fight antibiotic resistance. However, combinatorial complexity prohibits experimental testing of drug combinations on a large scale, and methods to rationally design combination therapies are lagging behind. Here, we developed a combined experimental-computational approach to predict drug-drug interactions using high-throughput metabolomics. The approach was tested on 1,279 pharmacologically diverse drugs applied to the gram-negative bacterium Escherichia coli. Combining our metabolic profiling of drug response with previously generated metabolic and chemogenomic profiles of 3,807 single-gene deletion strains revealed an unexpectedly large space of inhibited gene functions and enabled rational design of drug combinations. This approach is applicable to other therapeutic areas and can unveil unprecedented insights into drug tolerance, side effects, and repurposing. The compendium of drug-associated metabolome profiles is available at https://zampierigroup.shinyapps.io/EcoPrestMet, providing a valuable resource for the microbiological and pharmacological communities.