RESUMEN
There is a need to discover and develop non-toxic antibiotics that are effective against metabolically dormant bacteria, which underlie chronic infections and promote antibiotic resistance. Traditional antibiotic discovery has historically favored compounds effective against actively metabolizing cells, a property that is not predictive of efficacy in metabolically inactive contexts. Here, we combine a stationary-phase screening method with deep learning-powered virtual screens and toxicity filtering to discover compounds with lethality against metabolically dormant bacteria and favorable toxicity profiles. The most potent and structurally distinct compound without any obvious mechanistic liability was semapimod, an anti-inflammatory drug effective against stationary-phase E. coli and A. baumannii. Integrating microbiological assays, biochemical measurements, and single-cell microscopy, we show that semapimod selectively disrupts and permeabilizes the bacterial outer membrane by binding lipopolysaccharide. This work illustrates the value of harnessing non-traditional screening methods and deep learning models to identify non-toxic antibacterial compounds that are effective in infection-relevant contexts.
RESUMEN
The discovery of novel structural classes of antibiotics is urgently needed to address the ongoing antibiotic resistance crisis1-9. Deep learning approaches have aided in exploring chemical spaces1,10-15; these typically use black box models and do not provide chemical insights. Here we reasoned that the chemical substructures associated with antibiotic activity learned by neural network models can be identified and used to predict structural classes of antibiotics. We tested this hypothesis by developing an explainable, substructure-based approach for the efficient, deep learning-guided exploration of chemical spaces. We determined the antibiotic activities and human cell cytotoxicity profiles of 39,312 compounds and applied ensembles of graph neural networks to predict antibiotic activity and cytotoxicity for 12,076,365 compounds. Using explainable graph algorithms, we identified substructure-based rationales for compounds with high predicted antibiotic activity and low predicted cytotoxicity. We empirically tested 283 compounds and found that compounds exhibiting antibiotic activity against Staphylococcus aureus were enriched in putative structural classes arising from rationales. Of these structural classes of compounds, one is selective against methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci, evades substantial resistance, and reduces bacterial titres in mouse models of MRSA skin and systemic thigh infection. Our approach enables the deep learning-guided discovery of structural classes of antibiotics and demonstrates that machine learning models in drug discovery can be explainable, providing insights into the chemical substructures that underlie selective antibiotic activity.
Asunto(s)
Antibacterianos , Aprendizaje Profundo , Descubrimiento de Drogas , Animales , Humanos , Ratones , Antibacterianos/química , Antibacterianos/clasificación , Antibacterianos/farmacología , Antibacterianos/toxicidad , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Redes Neurales de la Computación , Algoritmos , Enterococos Resistentes a la Vancomicina/efectos de los fármacos , Modelos Animales de Enfermedad , Piel/efectos de los fármacos , Piel/microbiología , Descubrimiento de Drogas/métodos , Descubrimiento de Drogas/tendenciasRESUMEN
In vivo, bacterial actin MreB assembles into dynamic membrane-associated filamentous structures that exhibit circumferential motion around the cell. Current knowledge of MreB biochemical and polymerization properties in vitro remains limited and is mostly based on MreB proteins from Gram-negative species. In this study, we report the first observation of organized protofilaments by electron microscopy and the first 3D-structure of MreB from a Gram-positive bacterium. We show that Geobacillus stearothermophilus MreB forms straight pairs of protofilaments on lipid surfaces in the presence of ATP or GTP, but not in the presence of ADP, GDP or non-hydrolysable ATP analogs. We demonstrate that membrane anchoring is mediated by two spatially close short hydrophobic sequences while electrostatic interactions also contribute to lipid binding, and show that the population of membrane-bound protofilament doublets is in steady-state. In solution, protofilament doublets were not detected in any condition tested. Instead, MreB formed large sheets regardless of the bound nucleotide, albeit at a higher critical concentration. Altogether, our results indicate that both lipids and ATP are facilitators of MreB polymerization, and are consistent with a dual effect of ATP hydrolysis, in promoting both membrane binding and filaments assembly/disassembly.
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Actinas , Nucleótidos , Actinas/metabolismo , Nucleótidos/metabolismo , Polimerizacion , Adenosina Trifosfato/metabolismo , Lípidos , Proteínas Bacterianas/metabolismoRESUMEN
Device-associated bloodstream infections can cause serious medical problems and cost-intensive postinfection management, defining a need for more effective antimicrobial coatings. Newly developed coatings often show reduced bacterial colonization and high hemocompatibility in established in vitro tests, but fail in animal studies or clinical trials. The poor predictive power of these models is attributed to inadequate representation of in vivo conditions. Herein, a new single-pass blood flow model, with simultaneous incubation of the test surface with bacteria and freshly-drawn human blood, is presented. The flow model is validated by comparative analysis of a recently developed set of antiadhesive and contact-killing polymer coatings, and the corresponding uncoated polycarbonate surfaces. The results confirm the model's ability to differentiate the antimicrobial activities of the studied surfaces. Blood activation data correlate with bacterial surface coverage: low bacterial adhesion is associated with low inflammation and hemostasis. Shear stress correlates inversely with bacterial colonization, especially on antiadhesive surfaces. The introduced model is concluded to enable the evaluation of novel antimicrobial materials under in vivo-like conditions, capturing interactions between bacteria and biomaterials surfaces in the presence of key components of the ex vivo host response.
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Antiinfecciosos , Animales , Humanos , Antiinfecciosos/farmacología , Materiales Biocompatibles , Adhesión Bacteriana , Polímeros , Bacterias , Materiales Biocompatibles Revestidos/farmacología , AntibacterianosRESUMEN
All bacterial cells must expand their envelopes during growth. The main load-bearing and shape-determining component of the bacterial envelope is the peptidoglycan cell wall. Bacterial envelope growth and shape changes are often thought to be controlled through enzymatic cell wall insertion. We investigated the role of cell wall insertion for cell shape changes during cell elongation in Gram-negative bacteria. We found that both global and local rates of envelope growth of Escherichia coli remain nearly unperturbed upon arrest of cell wall insertion-up to the point of sudden cell lysis. Specifically, cells continue to expand their surface areas in proportion to biomass growth rate, even if the rate of mass growth changes. Other Gram-negative bacteria behave similarly. Furthermore, cells plastically change cell shape in response to differential mechanical forces. Overall, we conclude that cell wall-cleaving enzymes can control envelope growth independently of synthesis. Accordingly, the strong overexpression of an endopeptidase leads to transiently accelerated bacterial cell elongation. Our study demonstrates that biomass growth and envelope forces can guide cell envelope expansion through mechanisms that are independent of cell wall insertion.
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Pared Celular , Escherichia coli , Pared Celular/metabolismo , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Ciclo Celular , Bacterias Gramnegativas/metabolismo , Peptidoglicano/metabolismoRESUMEN
Surface curvature both emerges from, and influences the behavior of, living objects at length scales ranging from cell membranes to single cells to tissues and organs. The relevance of surface curvature in biology is supported by numerous experimental and theoretical investigations in recent years. In this review, first, a brief introduction to the key ideas of surface curvature in the context of biological systems is given and the challenges that arise when measuring surface curvature are discussed. Giving an overview of the emergence of curvature in biological systems, its significance at different length scales becomes apparent. On the other hand, summarizing current findings also shows that both single cells and entire cell sheets, tissues or organisms respond to curvature by modulating their shape and their migration behavior. Finally, the interplay between the distribution of morphogens or micro-organisms and the emergence of curvature across length scales is addressed with examples demonstrating these key mechanistic principles of morphogenesis. Overall, this review highlights that curved interfaces are not merely a passive by-product of the chemical, biological, and mechanical processes but that curvature acts also as a signal that co-determines these processes.
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Fenómenos Mecánicos , Membrana Celular , MorfogénesisRESUMEN
Trypanosoma brucei, the causative agent of human African trypanosomiasis, is highly motile and must be able to move in all three dimensions for reliable cell division. These characteristics make long-term microscopic imaging of live T. brucei cells challenging, which has limited our understanding of important cellular events. To address this issue, we devised an imaging approach that confines cells in small volumes within cast agarose microwells that can be imaged continuously for up to 24 h. Individual T. brucei cells were imaged through multiple rounds of cell division with high spatial and temporal resolution. We developed a strategy that employs in-well "sentinel" cells to monitor potential imaging toxicity during loss-of-function experiments such as small-molecule inhibition and RNAi. Using our approach, we show that the asymmetric daughter cells produced during T. brucei division subsequently divide at different rates, with the old-flagellum daughter cell dividing first. The flagellar detachment phenotype that appears during inhibition of the Polo-like kinase homolog TbPLK occurs in a stepwise fashion, with the new flagellum initially linked by its tip to the old, attached flagellum. We probe the feasibility of a previously proposed "back-up" cytokinetic mechanism and show that cells that initiate this process do not appear to complete cell division. This live-cell imaging method will provide a novel avenue for studying a wide variety of cellular events in trypanosomatids that have previously been inaccessible.
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División Celular/fisiología , Microscopía Intravital/métodos , Trypanosoma brucei brucei/fisiologíaRESUMEN
Mechanical rupture, or lysis, of the cytoplasmic membrane is a common cell death pathway in bacteria occurring in response to ß-lactam antibiotics. A better understanding of the cellular design principles governing the susceptibility and response of individual cells to lysis could indicate methods of potentiating ß-lactam antibiotics and clarify relevant aspects of cellular physiology. Here, we take a single-cell approach to bacterial cell lysis to examine three cellular features-turgor pressure, mechanosensitive channels, and cell shape changes-that are expected to modulate lysis. We develop a mechanical model of bacterial cell lysis and experimentally analyze the dynamics of lysis in hundreds of single Escherichia coli cells. We find that turgor pressure is the only factor, of these three cellular features, which robustly modulates lysis. We show that mechanosensitive channels do not modulate lysis due to insufficiently fast solute outflow, and that cell shape changes result in more severe cellular lesions but do not influence the dynamics of lysis. These results inform a single-cell view of bacterial cell lysis and underscore approaches of combatting antibiotic tolerance to ß-lactams aimed at targeting cellular turgor.
RESUMEN
Precision surface engineering is key to advanced biomaterials. A new platform of PEGylated styrene-maleic acid copolymers for adsorptive surface biofunctionalization is reported. Balanced amphiphilicity renders the copolymers water-soluble but strongly affine for surfaces. Fine-tuning of their molecular architecture provides control over adsorptive anchorage onto specific materials-which is why they are referred to as "anchor polymers" (APs)-and over structural characteristics of the adsorbed layers. Conjugatable with an array of bioactives-including cytokine-complexing glycosaminoglycans, cell-adhesion-mediating peptides and antimicrobials-APs can be applied to customize materials for demanding biotechnologies in uniquely versatile, simple, and robust ways. Moreover, homo- and heterodisplacement of adsorbed APs provide unprecedented means of in situ alteration and renewal of the functionalized surfaces. The related options are exemplified with proof-of-concept experiments of controlled bacterial adhesion, human umbilical vein endothelial cell, and induced pluripotent cell growth on AP-functionalized surfaces.
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Materiales Biocompatibles/química , Polímeros/química , Antiinfecciosos/química , Antiinfecciosos/farmacología , Materiales Biocompatibles/farmacología , Adhesión Celular/efectos de los fármacos , Citocinas/química , Glicosaminoglicanos/química , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Maleatos/química , Oligopéptidos/química , Polietilenglicoles/química , Polímeros/farmacología , Estireno/química , Propiedades de SuperficieRESUMEN
Bactericidal antibiotics kill bacteria by perturbing various cellular targets and processes. Disruption of the primary antibiotic-binding partner induces a cascade of molecular events, leading to overproduction of reactive metabolic by-products. It remains unclear, however, how these molecular events contribute to bacterial cell death. Here, we take a single-cell physical biology approach to probe antibiotic function. We show that aminoglycosides and fluoroquinolones induce cytoplasmic condensation through membrane damage and subsequent outflow of cytoplasmic contents as part of their lethality. A quantitative model of membrane damage and cytoplasmic leakage indicates that a small number of nanometer-scale membrane defects in a single bacterium can give rise to the cellular-scale phenotype of cytoplasmic condensation. Furthermore, cytoplasmic condensation is associated with the accumulation of reactive metabolic by-products and lipid peroxidation, and pretreatment of cells with the antioxidant glutathione attenuates cytoplasmic condensation and cell death. Our work expands our understanding of the downstream molecular events that are associated with antibiotic lethality, revealing cytoplasmic condensation as a phenotypic feature of antibiotic-induced bacterial cell death.
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Antibacterianos/farmacología , Membrana Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Aminoglicósidos/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Citoplasma/metabolismo , Escherichia coli/citología , Escherichia coli/metabolismo , Fluoroquinolonas/farmacología , Pruebas de Sensibilidad Microbiana/métodos , Viabilidad Microbiana/efectos de los fármacos , Microscopía de Fuerza Atómica/métodos , Microscopía Fluorescente/métodos , Análisis de la Célula Individual/métodosRESUMEN
Performance and safety of materials in contact with living matter are determined by sequential and competitive protein adsorption. However, cause and consequences of these processes remain hard to be generalized and predicted. In a new attempt to address that challenge, the authors compared and analyzed the protein adsorption and displacement on various thoroughly characterized polymer substrates using a combination of surface-sensitive techniques. A multiple linear regression approach was applied to model the dependence of protein adsorption, desorption, and exchange dynamics on protein and surface characteristics. While the analysis confirmed that protein properties primarily govern the observed adsorption and retention phenomena and hydrophobicity as well as surface charge are the most relevant polymer surface properties, the authors have identified several protein-surface combinations that deviate from these patterns and deserve further investigation.
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Interacciones Hidrofóbicas e Hidrofílicas , Polímeros/química , Proteínas/química , AdsorciónRESUMEN
Cell shape and cell volume are important for many bacterial functions. In recent years, we have seen a range of experimental and theoretical work that led to a better understanding of the determinants of cell shape and size. The roles of different molecular machineries for cell-wall expansion have been detailed and partially redefined, mechanical forces have been shown to influence cell shape, and new connections between metabolism and cell shape have been proposed. Yet the fundamental determinants of the different cellular dimensions remain to be identified. Here, we highlight some of the recent developments and focus on the determinants of rod-like cell shape and size in the well-studied model organisms Escherichia coli and Bacillus subtilis.
RESUMEN
Advanced blood contacting biomaterials are designed to combine antiseptic and anticoagulant functionalities. Here, we present a new in vitro methodology for the analysis of bacterial adhesion and growth after the preceding human whole blood incubation of the tested materials. Poly(styrene) surfaces as well as thrombin-responsive and non-responsive poly(ethylene glycol)-heparin hydrogel coatings, with and without silver functionalization, were analyzed with this approach using freshly drawn human whole blood and various human pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli). Adsorbed blood proteins and adherent immune cells were observed to suppress bacterial colonization on poly(styrene) surfaces. Silver functionalization of responsive and non-responsive poly(ethylene glycol)-heparin hydrogels had no influence on microbial attachment but decreased bacterial proliferation and viability. Whole blood pre-incubation did not affect the antimicrobial properties of the tested silver-modified hydrogels. In sum, our introduced multistage incubation test revealed the antibacterial effects as well as antiseptic-permissive characteristics of blood-borne interfacial layers on polymeric biomaterials.
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Antibacterianos/farmacología , Anticoagulantes/farmacología , Sangre/efectos de los fármacos , Heparina/análogos & derivados , Hidrogeles/farmacología , Plata/química , Antibacterianos/química , Anticoagulantes/química , Células Cultivadas , Heparina/farmacología , Humanos , Hidrogeles/química , Polietilenglicoles/química , Plata/farmacologíaRESUMEN
MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.
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Bacillus subtilis/citología , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Pared Celular/metabolismo , Peptidoglicano/metabolismo , Transporte de ProteínasRESUMEN
In nature, microorganisms exhibit different volumes spanning six orders of magnitude 1 . Despite their capability to create different sizes, a clonal population in a given environment maintains a uniform size across individual cells. Recent studies in eukaryotic and bacterial organisms showed that this homogeneity in cell size can be accomplished by growing a constant size between two cell cycle events (that is, the adder model 2-6 ). Demonstration of the adder model led to the hypothesis that this phenomenon is a consequence of convergent evolution. Given that archaeal cells share characteristics with both bacteria and eukaryotes, we investigated whether and how archaeal cells exhibit control over cell size. To this end, we developed a soft-lithography method of growing the archaeal cells to enable quantitative time-lapse imaging and single-cell analysis, which would be useful for other microorganisms. Using this method, we demonstrated that Halobacterium salinarum, a hypersaline-adapted archaeal organism, grows exponentially at the single-cell level and maintains a narrow-size distribution by adding a constant length between cell division events. Interestingly, the archaeal cells exhibited greater variability in cell division placement and exponential growth rate across individual cells in a population relative to those observed in Escherichia coli 6-9 . Here, we present a theoretical framework that explains how these larger fluctuations in archaeal cell cycle events contribute to cell size variability and control.
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Archaea/citología , Archaea/crecimiento & desarrollo , Bacterias/citología , Bacterias/crecimiento & desarrollo , División Celular , Archaea/fisiología , Bacterias/patogenicidad , Ciclo Celular , Escherichia coli/citología , Escherichia coli/crecimiento & desarrollo , Escherichia coli/fisiología , Halobacterium salinarum/citología , Modelos Biológicos , Análisis de la Célula Individual/métodos , Factores de TiempoRESUMEN
The shapes of most bacteria are imparted by the structures of their peptidoglycan cell walls, which are determined by many dynamic processes that can be described on various length scales ranging from short-range glycan insertions to cellular-scale elasticity1-11. Understanding the mechanisms that maintain stable, rod-like morphologies in certain bacteria has proved to be challenging due to an incomplete understanding of the feedback between growth and the elastic and geometric properties of the cell wall3,4,12-14. Here, we probe the effects of mechanical strain on cell shape by modelling the mechanical strains caused by bending and differential growth of the cell wall. We show that the spatial coupling of growth to regions of high mechanical strain can explain the plastic response of cells to bending4 and quantitatively predict the rate at which bent cells straighten. By growing filamentous Escherichia coli cells in doughnut-shaped microchambers, we find that the cells recovered their straight, native rod-shaped morphologies when released from captivity at a rate consistent with the theoretical prediction. We then measure the localization of MreB, an actin homologue crucial to cell wall synthesis, inside confinement and during the straightening process, and find that it cannot explain the plastic response to bending or the observed straightening rate. Our results implicate mechanical strain sensing, implemented by components of the elongasome yet to be fully characterized, as an important component of robust shape regulation in E. coli.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/citología , Escherichia coli/fisiología , Pared Celular/química , Pared Celular/genética , Pared Celular/fisiología , Escherichia coli/química , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Microscopía , Microscopía Fluorescente , Modelos Biológicos , Peptidoglicano/química , Peptidoglicano/metabolismo , Estrés FisiológicoRESUMEN
An estimated 1.5 billion microbial infections occur globally each year and result in â¼4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridge-based system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuum-degassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of â¼10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting. IMPORTANCE: This paper describes a portable system for rapidly identifying bacteria in resource-limited environments; we highlight the capabilities of the technology by detecting different pathogens within the ESKAPE collection, which cause nosocomial infections. The system is designed around isothermal DNA-based assays housed within an autonomous plastic cartridge that are designed with the end user in mind, who may have limited technological training. Displaying excellent sensitivity and specificity, the assay systems that we demonstrate may enable future diagnoses of bacterial infection to guide the development of effective chemotherapies and may have a role in areas beyond health where rapid detection is valuable, including in industrial processing and manufacturing, food security, agriculture, and water quality testing.
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Infecciones Bacterianas/diagnóstico , Infección Hospitalaria/diagnóstico , ADN Bacteriano/análisis , Dispositivos Laboratorio en un Chip , Microfluídica/métodos , Sistemas de Atención de Punto , Acinetobacter baumannii/clasificación , Acinetobacter baumannii/genética , Infecciones Bacterianas/microbiología , Infección Hospitalaria/microbiología , Cartilla de ADN/genética , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple , Enterobacter/clasificación , Enterobacter/genética , Enterococcus faecium/clasificación , Enterococcus faecium/genética , Humanos , Klebsiella pneumoniae/clasificación , Klebsiella pneumoniae/genética , Microfluídica/instrumentación , Pseudomonas aeruginosa/clasificación , Pseudomonas aeruginosa/genética , Staphylococcus aureus/clasificación , Staphylococcus aureus/genéticaRESUMEN
In this article we describe two techniques for exploring the relationship between bacterial cell shape and the intracellular organization of proteins. First, we created microchannels in a layer of agarose to reshape live bacterial cells and predictably control their mean cell wall curvature, and quantified the influence of curvature on the localization and distribution of proteins in vivo. Second, we used agarose microchambers to reshape bacteria whose cell wall had been chemically and enzymatically removed. By combining microstructures with different geometries and fluorescence microscopy, we determined the relationship between bacterial shape and the localization for two different membrane-associated proteins: i) the cell-shape related protein MreB of Escherichia coli, which is positioned along the long axis of the rod-shaped cell; and ii) the negative curvature-sensing cell division protein DivIVA of Bacillus subtilis, which is positioned primarily at cell division sites. Our studies of intracellular organization in live cells of E. coli and B. subtilis demonstrate that MreB is largely excluded from areas of high negative curvature, whereas DivIVA localizes preferentially to regions of high negative curvature. These studies highlight a unique approach for studying the relationship between cell shape and intracellular organization in intact, live bacteria.
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Bacillus subtilis/citología , Bioingeniería/métodos , Pared Celular/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citología , Esferoplastos/química , Bacillus subtilis/metabolismo , Escherichia coli/metabolismo , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente , Sefarosa/químicaRESUMEN
The Min proteins (MinC, MinD, and MinE) form a pole-to-pole oscillator that controls the spatial assembly of the division machinery in Escherichia coli cells. Previous studies identified that interactions of MinD with phospholipids positioned the Min machinery at the membrane. We extend these studies by measuring the affinity, kinetics, and ATPase activity of E. coli MinD, MinE, and MinDE binding to supported lipid bilayers containing varying compositions of anionic phospholipids. Using quartz crystal microbalance measurements, we found that the binding affinity (K(d)) for the interaction of recombinant E. coli MinD and MinE with lipid bilayers increased with increasing concentration of the anionic phospholipids phosphatidylglycerol and cardiolipin. The K(d) for MinD (1.8 µM) in the presence of ATP was smaller than for MinE (12.1 µM) binding to membranes consisting of 95:5 phosphatidylcholine/cardiolipin. The simultaneous binding of MinD and MinE to membranes revealed that increasing the concentration of anionic phospholipid stimulates the initial rate of adsorption (k(on)). The ATPase activity of MinD decreased in the presence of anionic phospholipids. These results indicate that anionic lipids, which are concentrated at the poles, increase the retention of MinD and MinE and explain its dwell time at this region of bacterial cells. These studies provide insight into interactions between MinD and MinE and between these proteins and membranes that are relevant to understanding the process of bacterial cell division, in which the interaction of proteins and membranes is essential.
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Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Adenosina Trifosfatasas/química , Adsorción , Antibacterianos/farmacología , Sitios de Unión , Cardiolipinas/química , División Celular , Relación Dosis-Respuesta a Droga , Cinética , Membrana Dobles de Lípidos/metabolismo , Liposomas/metabolismo , Fosfolípidos/química , Proteínas Recombinantes/químicaRESUMEN
Although bacterial cells are known to experience large forces from osmotic pressure differences and their local microenvironment, quantitative measurements of the mechanical properties of growing bacterial cells have been limited. We provide an experimental approach and theoretical framework for measuring the mechanical properties of live bacteria. We encapsulated bacteria in agarose with a user-defined stiffness, measured the growth rate of individual cells and fit data to a thin-shell mechanical model to extract the effective longitudinal Young's modulus of the cell envelope of Escherichia coli (50-150 MPa), Bacillus subtilis (100-200 MPa) and Pseudomonas aeruginosa (100-200 MPa). Our data provide estimates of cell wall stiffness similar to values obtained via the more labour-intensive technique of atomic force microscopy. To address physiological perturbations that produce changes in cellular mechanical properties, we tested the effect of A22-induced MreB depolymerization on the stiffness of E. coli. The effective longitudinal Young's modulus was not significantly affected by A22 treatment at short time scales, supporting a model in which the interactions between MreB and the cell wall persist on the same time scale as growth. Our technique therefore enables the rapid determination of how changes in genotype and biochemistry affect the mechanical properties of the bacterial envelope.