A target engagement assay to assess uptake, potency and retention of antibiotics in living bacteria

Abstract A major challenge in antibiotics drug discovery is to turn potent biochemical inhibitors of essential bacterial components into effective antimicrobials. This difficulty is underpinned by a lack of methods to investigate the physicochemical properties needed for candidate antibiotics to permeate the bacterial cell envelope and avoid clearance by the action of bacterial efflux pumps. To address these issues, here we used a target engagement assay to measure the equilibrium and kinetics binding parameters of antibiotics to their molecular targets in live bacteria. We validated this approach for a known antibiotic target, dihydrofolate reductase, using the Gram-negative bacteria Escherichia coli and the emerging human pathogen Mycobacterium abscessus. We expect the use of similar target engagement assays to expedite the discovery and progression of novel, cell-permeable antibiotics with on-target activity.

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Packaging Covered with Antiviral and Antibacterial Coatings Based on ZnO Nanoparticles Supplemented with Geraniol and Carvacrol

International Journal of Molecular Sciences ◽

10.3390/ijms22041717 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1717

Author(s):

Małgorzata Mizielińska ◽

Paweł Nawrotek ◽

Xymena Stachurska ◽

Magdalena Ordon ◽

Artur Bartkowiak

Keyword(s):

Synergistic Effect ◽

Moderate Activity ◽

Gram Negative Bacteria ◽

Human Pathogen ◽

Safety Measures ◽

Gram Negative ◽

Airborne Viruses ◽

Respiratory Droplets ◽

Active Substances ◽

External Coating

The purpose of the study was to obtain an external coating based on nanoparticles of ZnO, carvacrol, and geraniol that could be active against viruses such as SARS-Co-V2. Additionally, the synergistic effect of the chosen substances in coatings was analyzed. The goal of the study was to measure the possible antibacterial activity of the coatings obtained. Testing antiviral activity with human pathogen viruses, such as SARS-Co-V2, requires immense safety measures. Bacteriophages such as phi 6 phage represent good surrogates for the study of airborne viruses. The results of the study indicated that the ZC1 and ZG1 coatings containing an increased amount of geraniol or carvacrol and a very small amount of nanoZnO were found to be active against Gram-positive and Gram-negative bacteria. It is also important that a synergistic effect between these active substances was noted. This explains why polyethylene (PE) films covered with the ZC1 or ZG1 coatings (as internal coatings) were found to be the best packaging materials to extend the quality and freshness of food products. The same coatings may be used as the external coatings with antiviral properties. The ZC1 and ZG1 coatings showed moderate activity against the phi 6 phage that has been selected as a surrogate for viruses such as coronaviruses. It can be assumed that coatings ZG1 and ZC1 will also be active against SARS-CoV-2 that is transmitted via respiratory droplets.

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Interaction of Gram-Negative Bacteria with the Lysosomal Fraction of Polymorphonuclear Leukocytes II. Changes in the Cell Envelope of Escherichia coli

Infection and Immunity ◽

10.1128/iai.1.3.311-318.1970 ◽

1970 ◽

Vol 1 (3) ◽

pp. 311-318

Author(s):

D. Friedberg ◽

I. Friedberg ◽

M. Shilo

Keyword(s):

Escherichia Coli ◽

Polymorphonuclear Leukocytes ◽

Cytoplasmic Membrane ◽

Morphological Changes ◽

Cell Envelope ◽

Gram Negative Bacteria ◽

Intact Cells ◽

Lysosomal Fraction ◽

E Coli ◽

Absorbing Material

Interaction of lysosomal fraction with Escherichia coli caused damage to the cell envelope of these intact cells and to the cytoplasmic membrane of E. coli spheroplasts. The damage to the cytoplasmic membrane was manifested in the release of 260-nm absorbing material and β-galactosidase from the spheroplasts, and by increased permeability of cryptic cells to O -nitrophenyl-β- d -galactopyranoside; damage to the cell wall was measured by release of alkaline phosphatase. Microscope observation showed morphological changes in the cell envelope.

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Fine structural observations of Desulfovibrio desulfuricans

Canadian Journal of Microbiology ◽

10.1139/m73-121 ◽

1973 ◽

Vol 19 (6) ◽

pp. 753-756

Author(s):

Terrence M. Hammill ◽

Geno J. Germano

Keyword(s):

Electron Microscopy ◽

Cell Division ◽

Cell Envelope ◽

Desulfovibrio Desulfuricans ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Ultrathin Sections ◽

Basal Apparatus ◽

Specialized Region ◽

Whole Mounts

Glutaraldehyde-fixed, platinum-carbon-shadowed whole mounts, and ultrathin sections of glutaraldehyde-OsO4-fixed cells of Desulfovibrio desulfuricans were observed by electron microscopy. The preparations demonstrated a typical Vibrio form with a single polar flagellum. The cell envelope and the formation of external blebs were shown to be similar to other gram-negative bacteria. The protoplast, apparently devoid of mesosomes or other membranous structures, was densely packed with ribosomes and contained a fibrous nucleoid. A specialized region near the flagellar end of the cell was commonly observed and termed the basal apparatus. Cell division appeared to be by constriction.

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Antibiotic transport kinetics in Gram-negative bacteria revealed via single-cell uptake analysis and mathematical modelling

10.1101/645507 ◽

2019 ◽

Author(s):

Jehangir Cama ◽

Margaritis Voliotis ◽

Jeremy Metz ◽

Ashley Smith ◽

Jari Iannucci ◽

...

Keyword(s):

Rational Design ◽

Cell Envelope ◽

Time Lapse ◽

Drug Accumulation ◽

Cell Uptake ◽

Gram Negative Bacteria ◽

Label Free ◽

Transport Pathways ◽

Gram Negative ◽

Accumulation Kinetics

AbstractThe double-membrane cell envelope of Gram-negative bacteria is a formidable barrier to intracellular antibiotic accumulation. A quantitative understanding of antibiotic transport in these cells is crucial for drug development, but this has proved elusive due to the complexity of the problem and a dearth of suitable investigative techniques. Here we combine microfluidics and time-lapse auto-fluorescence microscopy to quantify antibiotic uptake label-free in hundreds of individual Escherichia coli cells. By manipulating the microenvironment, we showed that drug (ofloxacin) accumulation is higher in growing versus non-growing cells. Using genetic knockouts, we provide the first direct evidence that growth phase is more important for drug accumulation than the presence or absence of individual transport pathways. We use our experimental results to inform a mathematical model that predicts drug accumulation kinetics in subcellular compartments. These novel experimental and theoretical results pave the way for the rational design of new Gram-negative antibiotics.

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Binding From both sides: TolR and full-length OmpA bind and maintain the local structure of the E. coli cell wall

10.1101/409466 ◽

2018 ◽

Cited By ~ 1

Author(s):

Alister T. Boags ◽

Firdaus Samsudin ◽

Syma Khalid

Keyword(s):

Cell Wall ◽

Electrostatic Interactions ◽

Cell Envelope ◽

Binding Domain ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

E Coli ◽

Non Covalent Interactions ◽

Covalent Interactions

SUMMARYWe present a molecular modeling and simulation study of the of the E. coli cell envelope, with a particular focus on the role of TolR, a native protein of the E. coli inner membrane in interactions with the cell wall. TolR has been proposed to bind to peptidoglycan, but the only structure of this protein thus far is in a conformation in which the putative peptidoglycan binding domain is not accessible. We show that a model of the extended conformation of the protein in which this domain is exposed, binds peptidoglycan largely through electrostatic interactions. We show that non-covalent interactions of TolR and OmpA with the cell wall, from the inner membrane and outer membrane sides respectively, maintain the position of the cell wall even in the absence of Braun’s lipoprotein. When OmpA is truncated to remove the peptidoglycan binding domain, TolR is able to pull the cell wall down towards the inner membrane. The charged residues that mediate the cell-wall interactions of TolR in our simulations, are conserved across a number of species of Gram-negative bacteria.

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An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump

eLife ◽

10.7554/elife.24905 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 70

Author(s):

Zhao Wang ◽

Guizhen Fan ◽

Corey F Hryc ◽

James N Blaza ◽

Irina I Serysheva ◽

...

Keyword(s):

Transport Mechanism ◽

Drug Transport ◽

Efflux Pump ◽

Cell Envelope ◽

Gram Negative Bacteria ◽

Multidrug Efflux ◽

Protein Assemblies ◽

Multidrug Efflux Pump ◽

Channel Opening ◽

Activated State

Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the Escherichia coli AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump.

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Diacylglycerol kinase A is essential for polymyxin resistance provided by EptA, MCR-1 and other lipid A phosphoethanolamine transferases.

Journal of Bacteriology ◽

10.1128/jb.00498-21 ◽

2021 ◽

Author(s):

Alexandria B. Purcell ◽

Bradley J. Voss ◽

M. Stephen Trent

Keyword(s):

Lipid A ◽

Cell Envelope ◽

Diacylglycerol Kinase ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

Severe Reduction ◽

Bacterial Fitness ◽

Phosphoethanolamine Transferases ◽

Kinase A

Gram-negative bacteria utilize glycerophospholipids (GPLs) as phospho-form donors to modify various surface structures. These modifications play important roles in bacterial fitness in diverse environments influencing cell motility, recognition by the host during infection, and antimicrobial resistance. A well-known example is the modification of the lipid A component of lipopolysaccharide by the phosphoethanolamine (pEtN) transferase EptA that utilizes phosphatidyethanoalmine (PE) as the phospho-form donor. Addition of pEtN to lipid A promotes resistance to cationic antimicrobial peptides (CAMPs), including the polymyxin antibiotics like colistin. A consequence of pEtN modification is the production of diacylglycerol (DAG) that must be recycled back into GPL synthesis via the diacylglycerol kinase A (DgkA). DgkA phosphorylates DAG forming phosphatidic acid, the precursor for GPL synthesis. Here we report that deletion of dgkA in polymyxin-resistant E. coli results in a severe reduction of pEtN modification and loss of antibiotic resistance. We demonstrate that inhibition of EptA is regulated post-transcriptionally and is not due to EptA degradation during DAG accumulation. We also show that the inhibition of lipid A modification by DAG is a conserved feature of different Gram-negative pEtN transferases. Altogether, our data suggests that inhibition of EptA activity during DAG accumulation likely prevents disruption of GPL synthesis helping to maintain cell envelope homeostasis.

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How the assembly and protection of the bacterial cell envelope depend on cysteine residues

Journal of Biological Chemistry ◽

10.1074/jbc.rev120.011201 ◽

2020 ◽

Vol 295 (34) ◽

pp. 11984-11994 ◽

Cited By ~ 1

Author(s):

Jean-François Collet ◽

Seung-Hyun Cho ◽

Bogdan I. Iorga ◽

Camille V. Goemans

Keyword(s):

Amino Acid ◽

Cell Envelope ◽

Multilayered Structure ◽

Cysteine Residues ◽

Permeability Barrier ◽

Gram Negative Bacteria ◽

Amino Acid Residues ◽

Gram Negative ◽

Oxidative Inactivation ◽

Peptidoglycan Cell Wall

The cell envelope of Gram-negative bacteria is a multilayered structure essential for bacterial viability; the peptidoglycan cell wall provides shape and osmotic protection to the cell, and the outer membrane serves as a permeability barrier against noxious compounds in the external environment. Assembling the envelope properly and maintaining its integrity are matters of life and death for bacteria. Our understanding of the mechanisms of envelope assembly and maintenance has increased tremendously over the past two decades. Here, we review the major achievements made during this time, giving central stage to the amino acid cysteine, one of the least abundant amino acid residues in proteins, whose unique chemical and physical properties often critically support biological processes. First, we review how cysteines contribute to envelope homeostasis by forming stabilizing disulfides in crucial bacterial assembly factors (LptD, BamA, and FtsN) and stress sensors (RcsF and NlpE). Second, we highlight the emerging role of enzymes that use cysteine residues to catalyze reactions that are necessary for proper envelope assembly, and we also explain how these enzymes are protected from oxidative inactivation. Finally, we suggest future areas of investigation, including a discussion of how cysteine residues could contribute to envelope homeostasis by functioning as redox switches. By highlighting the redox pathways that are active in the envelope of Escherichia coli, we provide a timely overview of the assembly of a cellular compartment that is the hallmark of Gram-negative bacteria.

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Analysis of lipoprotein transport depletion in Vibrio cholerae using CRISPRi

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906158116 ◽

2019 ◽

Vol 116 (34) ◽

pp. 17013-17022 ◽

Cited By ~ 8

Author(s):

Florence Caro ◽

Nicole M. Place ◽

John J. Mekalanos

Keyword(s):

Outer Membrane ◽

Vibrio Cholerae ◽

Drug Target ◽

Membrane Vesicle ◽

Essential Genes ◽

Gram Negative Bacteria ◽

Human Pathogen ◽

Membrane Reorganization ◽

A Cell ◽

Classical Genetics

Genes necessary for the survival or reproduction of a cell are an attractive class of antibiotic targets. Studying essential genes by classical genetics, however, is inherently problematic because it is impossible to knock them out. Here, we screened a set of predicted essential genes for growth inhibition using CRISPR-interference (CRISPRi) knockdown in the human pathogen Vibrio cholerae. We demonstrate that CRISPRi knockdown of 37 predicted essential genes inhibits V. cholerae viability, thus validating the products of these genes as potential drug target candidates. V. cholerae was particularly vulnerable to lethal inhibition of the system for lipoprotein transport (Lol), a central hub for directing lipoproteins from the inner to the outer membrane (OM), with many of these lipoproteins coordinating their own essential processes. Lol depletion makes cells prone to plasmolysis and elaborate membrane reorganization, during which the periplasm extrudes into a mega outer membrane vesicle or “MOMV” encased by OM which dynamically emerges specifically at plasmolysis sites. Our work identifies the Lol system as an ideal drug target, whose inhibition could deplete gram-negative bacteria of numerous proteins that reside in the periplasm.

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The Rcs Phosphorelay Is a Cell Envelope Stress Response Activated by Peptidoglycan Stress and Contributes to Intrinsic Antibiotic Resistance

Journal of Bacteriology ◽

10.1128/jb.01740-07 ◽

2008 ◽

Vol 190 (6) ◽

pp. 2065-2074 ◽

Cited By ~ 153

Author(s):

Mary E. Laubacher ◽

Sarah E. Ades

Keyword(s):

Outer Membrane ◽

Single Molecule ◽

Stress Responses ◽

Structural Integrity ◽

Cell Envelope ◽

Gram Negative Bacteria ◽

Intrinsic Resistance ◽

Gram Negative ◽

Envelope Stress ◽

Peptidoglycan Layer

ABSTRACTGram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identifyEscherichia colistress responses activated following inhibition of specific PBPs by the β-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance ofE. colito β-lactam antibiotics.

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