RESUMEN
Lateral partitioning of proteins and lipids shapes membrane function. In model membranes, partitioning can be influenced both by bilayer-intrinsic factors like molecular composition and by bilayer-extrinsic factors such as interactions with other membranes and solid supports. While cellular membranes can departition in response to bilayer-intrinsic or -extrinsic disruptions, the mechanisms by which they partition de novo are largely unknown. The plasma membrane of Mycobacterium smegmatis spatially and biochemically departitions in response to the fluidizing agent benzyl alcohol, then repartitions upon fluidizer washout. By screening for mutants that are sensitive to benzyl alcohol, we show that the bifunctional cell wall synthase PonA2 promotes membrane partitioning and cell growth during recovery from benzyl alcohol exposure. PonA2's role in membrane repartitioning and regrowth depends solely on its conserved transglycosylase domain. Active cell wall polymerization promotes de novo membrane partitioning and the completed cell wall polymer helps to maintain membrane partitioning. Our work highlights the complexity of membrane-cell wall interactions and establishes a facile model system for departitioning and repartitioning cellular membranes.
Asunto(s)
Alcohol Bencilo , Pared Celular , Membrana Celular , Mycobacterium smegmatisRESUMEN
BACKGROUND: Physical functioning is a crucial factor for independence and quality of life in old age. The aim of the "bestform-Best function of range of motion" trial is to investigate the effects of a 6 months multimodal machine-based strength, coordination and endurance training on physical function, risk of falls and health parameters in older adults. METHODS: Bestform is a cluster-randomised trial including older adults ≥ 65 years living in senior care facilities in Southern Germany. Senior care facilities are randomly allocated to the control group with usual care (n ≥ 10 care facilities) and to the intervention group (n ≥ 10 care facilities), overall including ≥ 400 seniors. Residents belonging to the intervention group are offered a supervised machine-based exercise training programme twice weekly over 45-60 min over six months in small groups, while those in the usual care facilities will not receive active intervention. The primary outcome is the change in Short Physical Performance Battery over six months between groups. Secondary outcomes are change in risk of falling, fear of falling, number of falls and fall-related injuries, physical exercise capacity, handgrip strength, body composition, cardiac function, blood parameters, quality of life, risk of sarcopenia, activities of daily living, and cognition over three and six months. DISCUSSION: The bestform study investigates the change in physical function between seniors performing exercise intervention versus usual care over six months. The results of the study will contribute to the development of effective physical activity concepts in senior care facilities. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04207307. Registered December 2019.
Asunto(s)
Accidentes por Caídas , Actividades Cotidianas , Humanos , Anciano , Accidentes por Caídas/prevención & control , Miedo , Fuerza de la Mano , Calidad de Vida , Rango del Movimiento Articular , Ejercicio Físico , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
When bacteria adhere to surfaces, the chemical and mechanical character of the cell-substrate interface guides cell function and the development of microcolonies and biofilms. Alternately on bactericidal surfaces, intimate contact is critical to biofilm prevention. The direct study of the buried cell-substrate interfaces at the heart of these behaviors is hindered by the small bacterial cell size and inaccessibility of the contact region. Here, we present a total internal reflectance fluorescence depletion approach to measure the size of the cell-substrate contact region and quantify the gap separation and curvature near the contact zone, providing an assessment of the shapes of the near-surface undersides of adhered bacterial cells. Resolution of the gap height is about 10%, down to a few nanometers at contact. Using 1 and 2 µm silica spheres as calibration standards we report that, for flagella-free Escherichia coli (E. coli) adhering on a cationic poly-l-lysine layer, the cell-surface contact and apparent cell deformation vary with adsorbed cell configuration. Most cells adhere by their ends, achieving small contact areas of 0.15 µm2, corresponding to about 1-2% of the cell's surface. The altered Gaussian curvatures of end-adhered cells suggest the flattening of the envelope within the small contact region. When cells adhere by their sides, the contact area is larger, in the range 0.3-1.1 µm2 and comprising up to â¼12% of the cell's total surface. A region of sharper curvature, greater than that of the cells' original spherocylindrical shape, borders the flat contact region in cases of side-on or end-on cell adhesion, suggesting envelope stress. From the measured curvatures, precise stress distributions over the cell surface could be calculated in future studies that incorporate knowledge of envelope moduli. Overall the small contact areas of end-adhered cells may be a limiting factor for antimicrobial surfaces that kill on contact rather than releasing bactericide.
Asunto(s)
Adhesión Bacteriana , Escherichia coli , Escherichia coli/fisiología , Adhesión Bacteriana/fisiología , Biopelículas , Bacterias , Membrana Celular , Antibacterianos , Cationes , Propiedades de SuperficieRESUMEN
The intracellular membrane domain (IMD) is a laterally discrete region of the mycobacterial plasma membrane, enriched in the subpolar region of the rod-shaped cell. Here, we report genome-wide transposon sequencing to discover the controllers of membrane compartmentalization in Mycobacterium smegmatis. The putative gene cfa showed the most significant effect on recovery from membrane compartment disruption by dibucaine. Enzymatic analysis of Cfa and lipidomic analysis of a cfa deletion mutant (Δcfa) demonstrated that Cfa is an essential methyltransferase for the synthesis of major membrane phospholipids containing a C19:0 monomethyl-branched stearic acid, also known as tuberculostearic acid (TBSA). TBSA has been intensively studied due to its abundant and genus-specific production in mycobacteria, but its biosynthetic enzymes had remained elusive. Cfa catalyzed the S-adenosyl-l-methionine-dependent methyltransferase reaction using oleic acid-containing lipid as a substrate, and Δcfa accumulated C18:1 oleic acid, suggesting that Cfa commits oleic acid to TBSA biosynthesis, likely contributing directly to lateral membrane partitioning. Consistent with this model, Δcfa displayed delayed restoration of subpolar IMD and delayed outgrowth after bacteriostatic dibucaine treatment. These results reveal the physiological significance of TBSA in controlling lateral membrane partitioning in mycobacteria. IMPORTANCE As its common name implies, tuberculostearic acid is an abundant and genus-specific branched-chain fatty acid in mycobacterial membranes. This fatty acid, 10-methyl octadecanoic acid, has been an intense focus of research, particularly as a diagnostic marker for tuberculosis. It was discovered in 1934, and yet the enzymes that mediate the biosynthesis of this fatty acid and the functions of this unusual fatty acid in cells have remained elusive. Through a genome-wide transposon sequencing screen, enzyme assay, and global lipidomic analysis, we show that Cfa is the long-sought enzyme that is specifically involved in the first step of generating tuberculostearic acid. By characterizing a cfa deletion mutant, we further demonstrate that tuberculostearic acid actively regulates lateral membrane heterogeneity in mycobacteria. These findings indicate the role of branched fatty acids in controlling the functions of the plasma membrane, a critical barrier for the pathogen to survive in its human host.
Asunto(s)
Dibucaína , Mycobacterium , Humanos , Mycobacterium/metabolismo , Ácidos Esteáricos/metabolismo , Ácidos Grasos , Ácido Oléico , Metiltransferasas/metabolismoRESUMEN
The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. We describe a novel assay that combines metabolic tagging of the peptidoglycan, which sits directly beneath the mycomembrane, click chemistry of test molecules, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in mycobacteria by testing a small panel of azide-tagged molecules. The general trend is similar across the two types of mycobacteria with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria.
Asunto(s)
Mycobacterium tuberculosis , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Pared Celular/química , Pared Celular/metabolismo , Transporte Biológico , Antibacterianos/química , Antibacterianos/metabolismoRESUMEN
Some of the most dangerous bacterial pathogens (Gram-negative and mycobacterial) deploy a formidable secondary membrane barrier to reduce the influx of exogenous molecules. For Gram-negative bacteria, this second exterior membrane is known as the outer membrane (OM), while for the Gram-indeterminate Mycobacteria, it is known as the "myco" membrane. Although different in composition, both the OM and mycomembrane are key structures that restrict the passive permeation of small molecules into bacterial cells. Although it is well-appreciated that such structures are principal determinants of small molecule permeation, it has proven to be challenging to assess this feature in a robust and quantitative way or in complex, infection-relevant settings. Herein, we describe the development of the bacterial chloro-alkane penetration assay (BaCAPA), which employs the use of a genetically encoded protein called HaloTag, to measure the uptake and accumulation of molecules into model Gram-negative and mycobacterial species, Escherichia coli and Mycobacterium smegmatis, respectively, and into the human pathogen Mycobacterium tuberculosis. The HaloTag protein can be directed to either the cytoplasm or the periplasm of bacteria. This offers the possibility of compartmental analysis of permeation across individual cell membranes. Significantly, we also showed that BaCAPA can be used to analyze the permeation of molecules into host cell-internalized E. coli and M. tuberculosis, a critical capability for analyzing intracellular pathogens. Together, our results show that BaCAPA affords facile measurement of permeability across four barriers: the host plasma and phagosomal membranes and the diderm bacterial cell envelope.
Asunto(s)
Escherichia coli , Mycobacterium tuberculosis , Humanos , Escherichia coli/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Pared Celular/metabolismo , Mycobacterium tuberculosis/genéticaRESUMEN
Increasing the speed, specificity, sensitivity, and accessibility of mycobacteria detection tools are important challenges for tuberculosis (TB) research and diagnosis. In this regard, previously reported fluorogenic trehalose analogues have shown potential, but their green-emitting dyes may limit sensitivity and applications in complex settings. Here, we describe a trehalose-based fluorogenic probe featuring a molecular rotor turn-on fluorophore with bright far-red emission (RMR-Tre). RMR-Tre, which exploits the unique biosynthetic enzymes and environment of the mycobacterial outer membrane to achieve fluorescence activation, enables fast, no-wash, low-background fluorescence detection of live mycobacteria. Aided by the red-shifted molecular rotor fluorophore, RMR-Tre exhibited up to a 100-fold enhancement in M.â tuberculosis labeling compared to existing fluorogenic trehalose probes. We show that RMR-Tre reports on M.â tuberculosis drug resistance in a facile assay, demonstrating its potential as a TB diagnostic tool.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Sondas Moleculares , Trehalosa , Colorantes FluorescentesRESUMEN
In mycobacteria, the glucose-based disaccharide trehalose cycles between the cytoplasm, where it is a stress protectant and carbon source, and the cell envelope, where it is released as a byproduct of outer mycomembrane glycan biosynthesis and turnover. Trehalose recycling via the LpqY-SugABC transporter promotes virulence, antibiotic recalcitrance, and efficient adaptation to nutrient deprivation. The source(s) of trehalose and the regulation of recycling under these and other stressors are unclear. A key technical gap in addressing these questions has been the inability to trace trehalose recycling in situ, directly from its site of liberation from the cell envelope. Here we describe a bifunctional chemical reporter that simultaneously marks mycomembrane biosynthesis and subsequent trehalose recycling with alkyne and azide groups. Using this probe, we discovered that the recycling efficiency for trehalose increases upon carbon starvation, concomitant with an increase in LpqY-SugABC expression. The ability of the bifunctional reporter to probe multiple, linked steps provides a more nuanced understanding of mycobacterial cell envelope metabolism and its plasticity under stress.
Asunto(s)
Mycobacterium , Trehalosa , Trehalosa/metabolismo , Pared Celular/metabolismo , Membrana Celular/metabolismo , Carbono/metabolismoRESUMEN
Mycobacteria share an unusually complex, multilayered cell envelope, which contributes to adaptation to changing environments. The plasma membrane is the deepest layer of the cell envelope and acts as the final permeability barrier against outside molecules. There is an obvious need to maintain the plasma membrane integrity, but the adaptive responses of the plasma membrane to stress exposure remain poorly understood. Using chemical treatment and heat stress to fluidize the membrane, we show here that phosphatidylinositol (PI)-anchored plasma membrane glycolipids known as PI mannosides (PIMs) are rapidly remodeled upon membrane fluidization in Mycobacterium smegmatis. Without membrane stress, PIMs are predominantly in a triacylated form: two acyl chains of the PI moiety plus one acyl chain modified at one of the mannose residues. Upon membrane fluidization, we determined the fourth fatty acid is added to the inositol moiety of PIMs, making them tetra-acylated variants. Additionally, we show that PIM inositol acylation is a rapid response independent of de novo protein synthesis, representing one of the fastest mass conversions of lipid molecules found in nature. Strikingly, we found that M. smegmatis is more resistant to the bactericidal effect of a cationic detergent after benzyl alcohol pre-exposure. We further demonstrate that fluidization-induced PIM inositol acylation is conserved in pathogens such as Mycobacterium tuberculosis and Mycobacterium abscessus. Our results demonstrate that mycobacteria possess a mechanism to sense plasma membrane fluidity change. We suggest that inositol acylation of PIMs is a novel membrane stress response that enables mycobacterial cells to resist membrane fluidization.
Asunto(s)
Inositol , Mycobacterium tuberculosis , Acilación , Alcoholes Bencílicos , Detergentes , Ácidos Grasos , Glucolípidos , Inositol/metabolismo , Manosa/química , Manosa/metabolismo , Manósidos/química , Mycobacterium tuberculosis/metabolismo , Fosfatidilinositoles/metabolismoRESUMEN
A primary component of all known bacterial cell walls is the peptidoglycan (PG) layer, which is composed of repeating units of sugars connected to short and unusual peptides. The various steps within PG biosynthesis are targets of potent antibiotics as proper assembly of the PG is essential for cellular growth and survival. Synthetic mimics of PG have proven to be indispensable tools to study the bacterial cell structure, growth, and remodeling. Yet, a common component of PG, meso-diaminopimelic acid (m-DAP) at the third position of the stem peptide, remains challenging to access synthetically and is not commercially available. Here, we describe the synthesis and metabolic processing of a selenium-based bioisostere of m-DAP (selenolanthionine) and show that it is installed within the PG of live bacteria by the native cell wall crosslinking machinery in mycobacterial species. This PG probe has an orthogonal release mechanism that could be important for downstream proteomics studies. Finally, we describe a bead-based assay that is compatible with high-throughput screening of cell wall enzymes. We envision that this probe will supplement the current methods available for investigating PG crosslinking in m-DAP-containing organisms.
Asunto(s)
Mycobacterium , Selenio , Pared Celular/química , Ácido Diaminopimélico/metabolismo , Mycobacterium/metabolismo , Peptidoglicano/químicaRESUMEN
Cell wall peptidoglycan is a heteropolymeric mesh that protects the bacterium from internal turgor and external insults. In many rod-shaped bacteria, peptidoglycan synthesis for normal growth is achieved by two distinct pathways: the Rod complex, comprised of MreB, RodA, and a cognate class B penicillin-binding protein (PBP), and the class A PBPs (aPBPs). In contrast to laterally growing bacteria, pole-growing mycobacteria do not encode an MreB homolog and do not require SEDS protein RodA for in vitro growth. However, RodA contributes to the survival of Mycobacterium tuberculosis in some infection models, suggesting that the protein could have a stress-dependent role in maintaining cell wall integrity. Under basal conditions, we find here that the subcellular distribution of RodA largely overlaps that of the aPBP PonA1 and that both RodA and the aPBPs promote polar peptidoglycan assembly. Upon cell wall damage, RodA fortifies Mycobacterium smegmatis against lysis and, unlike aPBPs, contributes to a shift in peptidoglycan assembly from the poles to the sidewall. Neither RodA nor PonA1 relocalize; instead, the redistribution of nascent cell wall parallels that of peptidoglycan precursor synthase MurG. Our results support a model in which mycobacteria balance polar growth and cell-wide repair via spatial flexibility in precursor synthesis and extracellular insertion. IMPORTANCE Peptidoglycan synthesis is a highly successful target for antibiotics. The pathway has been extensively studied in model organisms under laboratory-optimized conditions. In natural environments, bacteria are frequently under attack. Moreover, the vast majority of bacterial species are unlikely to fit a single paradigm of cell wall assembly because of differences in growth mode and/or envelope structure. Studying cell wall synthesis under nonoptimal conditions and in nonstandard species may improve our understanding of pathway function and suggest new inhibition strategies. Mycobacterium smegmatis, a relative of several notorious human and animal pathogens, has an unusual polar growth mode and multilayered envelope. In this work, we challenged M. smegmatis with cell wall-damaging enzymes to characterize the roles of cell wall-building enzymes when the bacterium is under attack.
Asunto(s)
Proteínas Bacterianas , Peptidoglicano , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Peptidoglicano/metabolismoRESUMEN
The muscle disease sarcopenia, which is characterised by a loss of muscle strength, muscle quantity, and physical performance, restricts mobility and independence in an ageing society. The aim of this systematic review and meta-analysis is to analyse the effects that long-term progressive resistance training interventions performed on weight machines have on sarcopenia (European Working Group on Sarcopenia in Older People) and how the interventions are composed. In total, 779 articles published between 2000 and 2020 were scanned (PubMed, Web of Science, CINAHL) and 14 randomised controlled trials were included within the review. Populations, interventions, control groups and outcomes were analysed. Subsequent meta-analysis (10 studies, 902 participants) revealed that the time needed in a chair-stand-test, as an indicator for leg strength, was predominantly reduced, whereas grip strength remained unchanged after the interventions. Data concerning the effects of machine-based progressive resistance training on muscle quantity were insufficient for meta-analysis. Physical performance measured by undergoing the Timed-Up-and-Go-test, gait speed test, Short Physical Performance Battery and 6 min-walk-test improved significantly as well. The quality of evidence (GRADE) in the analysed studies was low or moderate. In summary, machine-based progressive resistance training has the potential to reverse sarcopenia in the oldest old, as reflected by enhanced muscle strength and physical performance. The systematic review revealed promising initial results for muscle quantity.
Asunto(s)
Entrenamiento de Fuerza , Sarcopenia , Anciano , Anciano de 80 o más Años , Fuerza de la Mano , Humanos , Fuerza Muscular/fisiología , Músculo Esquelético/fisiología , Sarcopenia/prevención & controlRESUMEN
Because bacterial adhesion to surfaces is associated with infections and biofilm growth, it has been a longstanding goal to develop coatings that minimize biomolecular adsorption and eliminate bacteria adhesion. We demonstrate that, even on carefully-engineered non-bioadhesive coatings such as polyethylene glycol (PEG) layers that prevent biomolecule adsorption and cell adhesion, depletion interactions from non-adsorbing polymer in solution (such as 10 K PEG or 100 K PEO) can cause adhesion and retention of Escherichia coli cells, defeating the antifouling functionality of the coating. The cells are immobilized and remain viable on the timescale of the study, at least up to 45 minutes. When the polymer solution is replaced by buffer, cells rapidly escape from the surface, consistent with expectations for the reversibility of depletion attractions. The dissolved polymer additionally causes cells to aggregate in solution and aggregates rapidly dissociate to singlets upon tenfold dilution in buffer, also consistent with depletion. Hydrodynamic forces can substantially reduce the adhesion of aggregates on surfaces in conditions where single cells adhere via depletion. The findings reported here suggest that because bacteria thrive in polymer-rich environments both in vivo and in situ, depletion interactions may make it impossible to avoid bacterial retention on surfaces.
Asunto(s)
Adhesión Bacteriana , Biopelículas , Adsorción , Bacterias , Polietilenglicoles , Propiedades de SuperficieRESUMEN
Motivated by observations of cell orientation at biofilm-substrate interfaces and reports that cell orientation and adhesion play important roles in biofilm evolution and function, we investigated the influence of surface chemistry on the orientation of Escherichia coli cells captured from flow onto surfaces that were cationic, hydrophobic, or anionic. We characterized the initial orientations of nonmotile cells captured from gentle shear relative to the surface and flow directions. The broad distribution of captured cell orientations observed on cationic surfaces suggests that rapid electrostatic attractions of cells to oppositely charged surfaces preserve the instantaneous orientations of cells as they rotate in the near-surface shearing flow. By contrast, on hydrophobic and anionic surfaces, cells were oriented slightly more in the plane of the surface and in the flow direction compared with that on the cationic surface. This suggests slower development of adhesion at hydrophobic and anionic surfaces, allowing cells to tip toward the surface as they adhere. Once cells were captured, the flow was increased by 20-fold. Cells did not reorient substantially on the cationic surface, suggesting a strong cell-surface bonding. By contrast, on hydrophobic and anionic surfaces, increased shear forced cells to tip toward the surface and align in the flow direction, a process that was reversible upon reducing the shear. These findings suggest mechanisms by which surface chemistry may play a role in the evolving structure and function of microbial communities.
Asunto(s)
Adhesión Bacteriana , Escherichia coli , Biopelículas , Interacciones Hidrofóbicas e Hidrofílicas , Propiedades de SuperficieRESUMEN
Many antibiotics target the assembly of cell wall peptidoglycan, an essential, heteropolymeric mesh that encases most bacteria. In rod-shaped bacteria, cell wall elongation is spatially precise yet relies on limited pools of lipid-linked precursors that generate and are attracted to membrane disorder. By tracking enzymes, substrates, and products of peptidoglycan biosynthesis in Mycobacterium smegmatis, we show that precursors are made in plasma membrane domains that are laterally and biochemically distinct from sites of cell wall assembly. Membrane partitioning likely contributes to robust, orderly peptidoglycan synthesis, suggesting that these domains help template peptidoglycan synthesis. The cell wall-organizing protein DivIVA and the cell wall itself promote domain homeostasis. These data support a model in which the peptidoglycan polymer feeds back on its membrane template to maintain an environment conducive to directional synthesis. Our findings are applicable to rod-shaped bacteria that are phylogenetically distant from M. smegmatis, indicating that horizontal compartmentalization of precursors may be a general feature of bacillary cell wall biogenesis.
Asunto(s)
Pared Celular/metabolismo , Mycobacterium smegmatis/metabolismo , Peptidoglicano/metabolismo , Ciclo Celular , Membrana Celular/metabolismoRESUMEN
The mycomembrane layer of the mycobacterial cell envelope is a barrier to environmental, immune, and antibiotic insults. There is considerable evidence of mycomembrane plasticity during infection and in response to host-mimicking stresses. Since mycobacteria are resource and energy limited under these conditions, it is likely that remodeling has distinct requirements from those of the well-characterized biosynthetic program that operates during unrestricted growth. Unexpectedly, we found that mycomembrane remodeling in nutrient-starved, nonreplicating mycobacteria includes synthesis in addition to turnover. Mycomembrane synthesis under these conditions occurs along the cell periphery, in contrast to the polar assembly of actively growing cells, and both liberates and relies on the nonmammalian disaccharide trehalose. In the absence of trehalose recycling, de novo trehalose synthesis fuels mycomembrane remodeling. However, mycobacteria experience ATP depletion, enhanced respiration, and redox stress, hallmarks of futile cycling and the collateral dysfunction elicited by some bactericidal antibiotics. Inefficient energy metabolism compromises the survival of trehalose recycling mutants in macrophages. Our data suggest that trehalose recycling alleviates the energetic burden of mycomembrane remodeling under stress. Cell envelope recycling pathways are emerging targets for sensitizing resource-limited bacterial pathogens to host and antibiotic pressure.IMPORTANCE The glucose-based disaccharide trehalose is a stress protectant and carbon source in many nonmammalian cells. Mycobacteria are relatively unique in that they use trehalose for an additional, extracytoplasmic purpose: to build their outer "myco" membrane. In these organisms, trehalose connects mycomembrane biosynthesis and turnover to central carbon metabolism. Key to this connection is the retrograde transporter LpqY-SugABC. Unexpectedly, we found that nongrowing mycobacteria synthesize mycomembrane under carbon limitation but do not require LpqY-SugABC. In the absence of trehalose recycling, compensatory anabolism allows mycomembrane biosynthesis to continue. However, this workaround comes at a cost, namely, ATP consumption, increased respiration, and oxidative stress. Strikingly, these phenotypes resemble those elicited by futile cycles and some bactericidal antibiotics. We demonstrate that inefficient energy metabolism attenuates trehalose recycling mutant Mycobacterium tuberculosis in macrophages. Energy-expensive macromolecule biosynthesis triggered in the absence of recycling may be a new paradigm for boosting host activity against bacterial pathogens.
Asunto(s)
Membrana Celular/metabolismo , Pared Celular/metabolismo , Metabolismo Energético/efectos de los fármacos , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/metabolismo , Trehalosa/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/biosíntesis , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/efectos de los fármacos , Pared Celular/efectos de los fármacos , Factores Cordón/metabolismo , Factores Cordón/farmacología , Diarilquinolinas/farmacología , Metabolismo Energético/genética , Galactanos/metabolismo , Galactanos/farmacología , Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Glucosa/farmacología , Maltosa/metabolismo , Maltosa/farmacología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Ácidos Micólicos/metabolismo , Ácidos Micólicos/farmacología , Rifampin/farmacología , Trehalosa/farmacologíaRESUMEN
Transpeptidation reinforces the structure of cell-wall peptidoglycan, an extracellular heteropolymer that protects bacteria from osmotic lysis. The clinical success of transpeptidase-inhibiting ß-lactam antibiotics illustrates the essentiality of these cross-linkages for cell-wall integrity, but the presence of multiple, seemingly redundant transpeptidases in many species makes it challenging to determine cross-link function. Here, we present a technique to link peptide strands by chemical rather than enzymatic reaction. We employ biocompatible click chemistry to induce triazole formation between azido- and alkynyl-d-alanine residues that are metabolically installed in the peptidoglycan of Gram-positive or Gram-negative bacteria. Synthetic triazole cross-links can be visualized using azidocoumarin-d-alanine, an amino acid derivative that undergoes fluorescent enhancement upon reaction with terminal alkynes. Cell-wall stapling protects Escherichia coli from treatment with the broad-spectrum ß-lactams ampicillin and carbenicillin. Chemical control of cell-wall structure in live bacteria can provide functional insights that are orthogonal to those obtained by genetics.
Asunto(s)
Bacterias/química , Pared Celular/química , Reactivos de Enlaces Cruzados/química , Péptidos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Pared Celular/efectos de los fármacos , Escherichia coli/química , Escherichia coli/efectos de los fármacos , Humanos , beta-Lactamas/farmacologíaRESUMEN
The propensity of broad-spectrum antibiotics to indiscriminately kill both pathogenic and beneficial bacteria has a profound impact on the spread of resistance across multiple bacterial species. Alternative approaches that narrow antibacterial specificity towards desired pathogenic bacterial population are of great interest. Here, we report an enzyme-responsive antibiotic-loaded nanoassembly strategy for narrow delivery of otherwise broad-spectrum antibiotics. We specifically target Staphylococcus aureus (S. aureus), an important blood pathogen that secretes PC1 ß-lactamases. Our nanoassemblies selectively eradicate S. aureus grown in vitro with other bacteria, highlighting its potential capability in targeting the desired pathogenic bacterial population.
Asunto(s)
Infecciones Estafilocócicas , Staphylococcus aureus , Antibacterianos/farmacología , Bacterias , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/tratamiento farmacológicoRESUMEN
Bacteria surround themselves with cell walls to maintain cell rigidity and protect against environmental insults. Here we review chemical and biochemical techniques employed to study bacterial cell wall biogenesis. Recent advances including the ability to isolate critical intermediates, metabolic approaches for probe incorporation, and isotopic labeling techniques have provided critical insight into the biochemistry of cell walls. Fundamental manuscripts that have used these techniques to discover cell wall-interacting proteins, flippases, and cell wall stoichiometry are discussed in detail. The review highlights that these powerful methods and techniques have exciting potential to identify and characterize new targets for antibiotic development.