A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

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Fluorescence High-Throughput Screening for Inhibitors of TonB Action

Journal of Bacteriology ◽

10.1128/jb.00889-16 ◽

2017 ◽

Vol 199 (10) ◽

Cited By ~ 11

Author(s):

Brittany L. Nairn ◽

Olivia S. Eliasson ◽

Dallas R. Hyder ◽

Noah J. Long ◽

Aritri Majumdar ◽

...

Keyword(s):

Escherichia Coli ◽

Acinetobacter Baumannii ◽

High Throughput ◽

High Throughput Screening ◽

Resistant Bacteria ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Compound Libraries

ABSTRACT Gram-negative bacteria acquire ferric siderophores through TonB-dependent outer membrane transporters (TBDT). By fluorescence spectroscopic hgh-throughput screening (FLHTS), we identified inhibitors of TonB-dependent ferric enterobactin (FeEnt) uptake through Escherichia coli FepA (EcoFepA). Among 165 inhibitors found in a primary screen of 17,441 compounds, we evaluated 20 in secondary tests: TonB-dependent ferric siderophore uptake and colicin killing and proton motive force-dependent lactose transport. Six of 20 primary hits inhibited TonB-dependent activity in all tests. Comparison of their effects on [59Fe]Ent and [14C]lactose accumulation suggested several as proton ionophores, but two chemicals, ebselen and ST0082990, are likely not proton ionophores and may inhibit TonB-ExbBD. The facility of FLHTS against E. coli led us to adapt it to Acinetobacter baumannii. We identified its FepA ortholog (AbaFepA), deleted and cloned its structural gene, genetically engineered 8 Cys substitutions in its surface loops, labeled them with fluorescein, and made fluorescence spectroscopic observations of FeEnt uptake in A. baumannii. Several Cys substitutions in AbaFepA (S279C, T562C, and S665C) were readily fluoresceinated and then suitable as sensors of FeEnt transport. As in E. coli, the test monitored TonB-dependent FeEnt uptake by AbaFepA. In microtiter format with A. baumannii, FLHTS produced Z′ factors 0.6 to 0.8. These data validated the FLHTS strategy against even distantly related Gram-negative bacterial pathogens. Overall, it discovered agents that block TonB-dependent transport and showed the potential to find compounds that act against Gram-negative CRE (carbapenem-resistant Enterobacteriaceae)/ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. Our results suggest that hundreds of such chemicals may exist in larger compound libraries. IMPORTANCE Antibiotic resistance in Gram-negative bacteria has spurred efforts to find novel compounds against new targets. The CRE/ESKAPE pathogens are resistant bacteria that include Acinetobacter baumannii, a common cause of ventilator-associated pneumonia and sepsis. We performed fluorescence high-throughput screening (FLHTS) against Escherichia coli to find inhibitors of TonB-dependent iron transport, tested them against A. baumannii, and then adapted the FLHTS technology to allow direct screening against A. baumannii. This methodology is expandable to other drug-resistant Gram-negative pathogens. Compounds that block TonB action may interfere with iron acquisition from eukaryotic hosts and thereby constitute bacteriostatic antibiotics that prevent microbial colonization of human and animals. The FLHTS method may identify both species-specific and broad-spectrum agents against Gram-negative bacteria.

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The Target of Daptomycin Is Absent from Escherichia coli and Other Gram-Negative Pathogens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02005-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 637-639 ◽

Cited By ~ 62

Author(s):

Christopher P. Randall ◽

Katherine R. Mariner ◽

Ian Chopra ◽

Alex J. O'Neill

Keyword(s):

Escherichia Coli ◽

Antibacterial Activity ◽

Resistance Mechanisms ◽

Efflux Transporters ◽

Permeability Barrier ◽

Gram Negative Bacteria ◽

Intrinsic Resistance ◽

Gram Negative ◽

Content Type ◽

E Coli

ABSTRACTAntistaphylococcal agents commonly lack activity against Gram-negative bacteria likeEscherichia coliowing to the permeability barrier presented by the outer membrane and/or the action of efflux transporters. When these intrinsic resistance mechanisms are artificially compromised, such agents almost invariably demonstrate antibacterial activity against Gram negatives. Here we show that this is not the case for the antibiotic daptomycin, whose target appears to be absent fromE. coliand other Gram-negative pathogens.

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Heterogeneous and Flexible Transmission ofmcr-1in Hospital-AssociatedEscherichia coli

mBio ◽

10.1128/mbio.00943-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 23

Author(s):

Yingbo Shen ◽

Zuowei Wu ◽

Yang Wang ◽

Rong Zhang ◽

Hong-Wei Zhou ◽

...

Keyword(s):

Escherichia Coli ◽

Genomic Analysis ◽

Multidrug Resistant ◽

Fluoroquinolone Resistance ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Genes Encoding

ABSTRACTThe recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route ofmcr-1amongEnterobacteriaceaespecies in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis ofEscherichia coliisolates collected in a hospital in Hangzhou, China. We found thatmcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread ofmcr-1. Themcr-1gene was found on either chromosomes or plasmids, but in most of theE. coliisolates,mcr-1was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission ofmcr-1and the coexistence ofmcr-1with other genes encoding β-lactamases and fluoroquinolone resistance in theE. coliisolates. These findings indicate thatmcr-1is heterogeneously disseminated in both commensal and pathogenic strains ofE. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology ofmcr-1among hospital-associatedE. colistrains.IMPORTANCEColistin represents one of the very few available drugs for treating infections caused by extensively multidrug-resistant Gram-negative bacteria. The recently emergentmcr-1colistin resistance gene threatens the clinical utility of colistin and has gained global attention. Howmcr-1spreads in hospital settings remains unknown and was investigated by whole-genome sequencing ofmcr-1-carryingEscherichia coliin this study. The findings revealed extraordinary flexibility ofmcr-1in its spread among genetically diverseE. colihosts and plasmids, nosocomial transmission ofmcr-1-carryingE. coli, and the continuous emergence of novel Inc types of plasmids carryingmcr-1and newmcr-1variants. Additionally,mcr-1was found to be frequently associated with other genes encoding β-lactams and fluoroquinolone resistance. These findings provide important information on the transmission and epidemiology ofmcr-1and are of significant public health importance as the information is expected to facilitate the control of this significant antibiotic resistance threat.

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National Surveillance of Antimicrobial Susceptibility of Bacteremic Gram-Negative Bacteria with Emphasis on Community-Acquired Resistant Isolates: Report from the 2019 Surveillance of Multicenter Antimicrobial Resistance in Taiwan (SMART)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01089-20 ◽

2020 ◽

Vol 64 (10) ◽

Author(s):

Po-Yu Liu ◽

Yu-Lin Lee ◽

Min-Chi Lu ◽

Pei-Lan Shao ◽

Po-Liang Lu ◽

...

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Resistance ◽

Klebsiella Pneumoniae ◽

Gram Negative Bacteria ◽

National Surveillance ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Carbapenem Resistant

ABSTRACT A multicenter collection of bacteremic isolates of Escherichia coli (n = 423), Klebsiella pneumoniae (n = 372), Pseudomonas aeruginosa (n = 300), and Acinetobacter baumannii complex (n = 199) was analyzed for susceptibility. Xpert Carba-R assay and sequencing for mcr genes were performed for carbapenem- or colistin-resistant isolates. Nineteen (67.8%) carbapenem-resistant K. pneumoniae (n = 28) and one (20%) carbapenem-resistant E. coli (n = 5) isolate harbored blaKPC (n = 17), blaOXA-48 (n = 2), and blaVIM (n = 1) genes.

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Nonclonal Emergence of Colistin Resistance Associated with Mutations in the BasRS Two-Component System in Escherichia coli Bloodstream Isolates

mSphere ◽

10.1128/msphere.00143-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 2

Author(s):

Axel B. Janssen ◽

Toby L. Bartholomew ◽

Natalia P. Marciszewska ◽

Marc J. M. Bonten ◽

Rob J. L. Willems ◽

...

Keyword(s):

Escherichia Coli ◽

Lipid A ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Anionic Lipid ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Two Component ◽

Resistant Strains

ABSTRACT Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one Escherichia albertii strain. These were the only colistin-resistant strains of 1,140 bloodstream Escherichia isolates collected in a tertiary hospital over a 10-year period (2006 to 2015). Core-genome phylogenetic analysis showed that each patient was colonized by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen. IMPORTANCE Multidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole-genome sequence analysis and experimental validation to characterize the mechanisms through which Escherichia coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.

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Bacterial Metal Resistance: Coping with Copper without Cooperativity?

mBio ◽

10.1128/mbio.00653-21 ◽

2021 ◽

Author(s):

Nicholas P. Greene ◽

Vassilis Koronakis

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Cell Envelope ◽

Metal Resistance ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Rnd Transporter ◽

Entire Cell

In Escherichia coli and other Gram-negative bacteria, tripartite efflux pumps (TEPs) span the entire cell envelope and serve to remove noxious molecules from the cell. CusBCA is a TEP responsible for copper and silver detoxification in E. coli powered by the resistance-nodulation-cell division (RND) transporter, CusA.

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Synergistic Interactions of Vancomycin with Different Antibiotics against Escherichia coli: Trimethoprim and Nitrofurantoin Display Strong Synergies with Vancomycin against Wild-Type E. coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03502-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 276-281 ◽

Cited By ~ 38

Author(s):

Alice Zhou ◽

Tina Manzhu Kang ◽

Jessica Yuan ◽

Casey Beppler ◽

Caroline Nguyen ◽

...

Keyword(s):

Escherichia Coli ◽

The Other ◽

Gram Negative Bacteria ◽

Functional Categories ◽

Wild Type ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Synergistic Interactions ◽

Pairwise Interactions

ABSTRACTGram-negative bacteria are normally resistant to the antibiotic vancomycin (VAN), which cannot significantly penetrate the outer membrane. We usedEscherichia colimutants that are partially sensitive to VAN to study synergies between VAN and 10 other antibiotics representing six different functional categories. We detected strong synergies with VAN and nitrofurantoin (NTR) and with VAN and trimethoprim (TMP) and moderate synergies with other drugs, such as aminoglycosides. These synergies are powerful enough to show the activity of VAN against wild-typeE. coliat concentrations of VAN as low as 6.25 μg/ml. This suggests that a very small percentage of exogenous VAN does enterE. colibut normally has insignificant effects on growth inhibition or cell killing. We used the results of pairwise interactions with VAN and the other 10 antibiotics tested to place VAN into a functional category of its own, as previously defined by Yeh et al. (P. Yeh, A. I. Tschumi, and R. Kishony, Nat Genet 28:489–494, 2006,http://dx.doi.org/10.1038/ng1755).

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Screening for Antibacterial Inhibitors of the UDP-3-O-(R-3-Hydroxymyristoyl)- N-Acetylglucosamine Deacetylase (LpxC) Using a High-Throughput Mass Spectrometry Assay

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109355319 ◽

2009 ◽

Vol 15 (1) ◽

pp. 52-61 ◽

Cited By ~ 33

Author(s):

Erik F. Langsdorf ◽

Asra Malikzay ◽

William A. Lamarr ◽

Dayna Daubaras ◽

Cynthia Kravec ◽

...

Keyword(s):

Mass Spectrometry ◽

Escherichia Coli ◽

Thermal Stability ◽

High Throughput ◽

High Throughput Screening ◽

Antibacterial Agents ◽

Screening Tool ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Mass Spectrometry Assay

A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)- Nacetylglucosamine deacetylase, LpxC, is described. This reaction is essential in the biosynthesis of lipopolysaccharide (LPS) of gram-negative bacteria and is an attractive target for the development of new antibacterial agents. The assay uses the RapidFire™ mass spectrometry platform to measure the native LpxC substrate and the reaction product and thereby generates a ratiometric readout with minimal artifacts due to detection interference. The assay was robust in a high-throughput screen of a library of more than 700,000 compounds arrayed as orthogonal mixtures, with a median Z' factor of 0.74. Selected novel inhibitors from the screening campaign were confirmed as binding to LpxC by biophysical measurements using a thermal stability shift assay. Some inhibitors showed whole-cell antimicrobial activity against a sensitive strain of Escherichia coli with reduced LpxC activity (strain D22; minimum inhibitory concentrations ranging from 0.625-20 µg/mL). The results show that mass spectrometry—based screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions.

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Correct Sorting of Lipoproteins into the Inner and Outer Membranes ofPseudomonas aeruginosaby theEscherichia coliLolCDE Transport System

mBio ◽

10.1128/mbio.00194-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 4

Author(s):

Christian Lorenz ◽

Thomas J. Dougherty ◽

Stephen Lory

Keyword(s):

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Transport Systems ◽

Gram Negative Bacteria ◽

Inner Membrane ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Outer Membranes

ABSTRACTBiogenesis of the outer membrane of Gram-negative bacteria depends on dedicated macromolecular transport systems. The LolABCDE proteins make up the machinery for lipoprotein trafficking from the inner membrane (IM) across the periplasm to the outer membrane (OM). The Lol apparatus is additionally responsible for differentiating OM lipoproteins from those for the IM. InEnterobacteriaceae, a default sorting mechanism has been proposed whereby an aspartic acid at position +2 of the mature lipoproteins prevents Lol recognition and leads to their IM retention. In other bacteria, the conservation of sequences immediately following the acylated cysteine is variable. Here we show that inPseudomonas aeruginosa, the three essential Lol proteins (LolCDE) can be replaced with those fromEscherichia coli. TheP. aeruginosalipoproteins MexA, OprM, PscJ, and FlgH, with different sequences at their N termini, were correctly sorted by either theE. coliorP. aeruginosaLolCDE. We further demonstrate that an inhibitor ofE. coliLolCDE is active againstP. aeruginosaonly when expressing theE. coliorthologues. Our work shows that Lol proteins recognize a wide range of signals, consisting of an acylated cysteine and a specific conformation of the adjacent domain, determining IM retention or transport to the OM.IMPORTANCEGram-negative bacteria build their outer membranes (OM) from components that are initially located in the inner membrane (IM). A fraction of lipoproteins is transferred to the OM by the transport machinery consisting of LolABCDE proteins. Our work demonstrates that the LolCDE complexes of the transport pathways ofEscherichia coliandPseudomonas aeruginosaare interchangeable, with theE. coliorthologues correctly sorting theP. aeruginosalipoproteins while retaining their sensitivity to a small-molecule inhibitor. These findings question the nature of IM retention signals, identified inE. colias aspartate at position +2 of mature lipoproteins. We propose an alternative model for the sorting of IM and OM lipoproteins based on their relative affinities for the IM and the ability of the promiscuous sorting machinery to deliver lipoproteins to their functional sites in the OM.

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Cues from the Membrane: Bacterial Glycerophospholipids

Journal of Bacteriology ◽

10.1128/jb.00136-17 ◽

2017 ◽

Vol 199 (13) ◽

Cited By ~ 3

Author(s):

Zachary D. Dalebroux

Keyword(s):

Escherichia Coli ◽

Survival Strategies ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Adaptation Mechanisms ◽

Membrane Envelope ◽

Critical Components ◽

New Evidence

ABSTRACT In this issue of the Journal of Bacteriology, V. W. Rowlett et al. unveil new Escherichia coli circuitry linking membrane glycerophospholipid (GPL) homeostasis to bacterial stress response and adaptation mechanisms (J Bacteriol 199:e00849-16, 2017, https://doi.org/10.1128/JB.00849-16 ). Glycerophospholipids comprise critical components of the dual-membrane envelope of Gram-negative bacteria and participate in many processes. The new evidence suggests that, in some instances, distinct E. coli GPL molecules function for distinct biochemistry and bacteria sense perturbations in membrane GPL concentrations to coordinate survival strategies. Understanding GPL sensing and remodeling mechanisms will be important moving forward, given the breadth of function for these molecules in bacteriology.

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