scholarly journals Identification of EnvC and Its Cognate Amidases as Novel Determinants of Intrinsic Resistance to Cationic Antimicrobial Peptides

2016 ◽  
Vol 60 (4) ◽  
pp. 2222-2231 ◽  
Author(s):  
Tamiko Oguri ◽  
Won-Sik Yeo ◽  
Taeok Bae ◽  
Hyunwoo Lee
Keyword(s):  
Antimicrobial Peptides ◽  
Common Factor ◽  
Animal Experiments ◽  
Enteric Bacteria ◽  
Polymyxin B ◽  
Intrinsic Resistance ◽  
Cationic Antimicrobial Peptides ◽  
Gram Negative ◽  
Content Type ◽  
E Coli

ABSTRACTCationic antimicrobial peptides (CAMPs) are an essential part of the innate immune system. Some Gram-negative enteric pathogens, such asSalmonella enterica, show intrinsic resistance to CAMPs. However, the molecular basis of intrinsic resistance is poorly understood, largely due to a lack of information about the genes involved. In this study, using a microarray-based genomic technique, we screened the Keio collection of 3,985Escherichia colimutants for altered susceptibility to human neutrophil peptide 1 (HNP-1) and identifiedenvCandzapBas novel genetic determinants of intrinsic CAMP resistance. In CAMP killing assays, anE. coliΔenvCEcor ΔzapBEcmutant displayed a distinct profile of increased susceptibility to both LL-37 and HNP-1. Both mutants, however, displayed wild-type resistance to polymyxin B and human β-defensin 3 (HBD3), suggesting that the intrinsic resistance mediated by EnvC or ZapB is specific to certain CAMPs. A correspondingSalmonellaΔenvCSemutant showed similarly increased CAMP susceptibility. TheenvCmutants of bothE. coliandS. entericadisplayed increased surface negativity and hydrophobicity, which partly explained the increased CAMP susceptibility. However, the ΔenvCEcmutant, but not the ΔenvCSemutant, was defective in outer membrane permeability, excluding this defect as a common factor contributing to the increased CAMP susceptibility. Animal experiments showed that theSalmonellaΔenvCSemutant had attenuated virulence. Taken together, our results indicate that the role ofenvCin intrinsic CAMP resistance is likely conserved among Gram-negative enteric bacteria, demonstrate the importance of intrinsic CAMP resistance for full virulence ofS. enterica, and provide insight into distinct mechanisms of action of CAMPs.

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

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

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.

scholarly journals Group of peptides that act synergistically with hydrophobic antibiotics against gram-negative enteric bacteria.

1996 ◽  
Vol 40 (8) ◽  
pp. 1801-1805 ◽  
Author(s):  
M Vaara ◽  
M Porro
Keyword(s):  
Synthetic Peptide ◽  
Bacterial Species ◽  
Cetylpyridinium Chloride ◽  
Enteric Bacteria ◽  
Polymyxin B ◽  
Disulfide Bridge ◽  
Gram Negative ◽  
E Coli ◽  
Cationic Detergents ◽  
Lysine Residues

A synthetic peptide, KFFKFFKFFK [corrected], consisting of cationic lysine residues and hydrophobic phenylalanine residues was found to sensitize gram-negative bacteria to hydrophobic and amphipathic antibiotics. At a concentration of 3 micrograms/ml, it decreased the MIC of rifampin for smooth, encapsulated Escherichia coli by a factor of 300. Other susceptible bacterial species included Enterobacter cloacae, Klebsiella pneumoniae, and Salmonella typhimurium, but Pseudomonas aeruginosa was resistant. Similar results were obtained with another synthetic peptide, IKFLKFLKFLK [corrected]. The fractional inhibitory concentration indices for the synergism of these peptides with rifampin, erythromycin, fusidic acid, and novobiocin were very close to those determined for the previously characterized potent outer-membrane-disorganizing agents polymyxin B nonapeptide and deacylpolymyxin B. KFFKFFKFFK [corrected] had direct activity against the gram-positive organism Micrococcus strain ML36, was strongly hemolytic, and was as active on polymyxin-resistant E. coli mutants as on their parent. These three attributes made KFFKFFKFFK [corrected] different from polymyxin derivatives and similar to cationic detergents, such as cetylpyridinium chloride. However, whereas the MIC of cetylpyridinium chloride for E. coli is low (0.5 to 4 micrograms/ml), that of KFFKFFKFFK [corrected] is much higher (30 to 100 micrograms/ml). Other groups of synthetic peptides studied included polymyxin-like peptides with an intrachain disulfide bridge. Their synergism with antibiotics was less marked. Still other peptides, including KEKEKEKEKE and KKKKKKFLFL, lacked any synergism with the probe antibiotics.

scholarly journals Induction of the Cpx Envelope Stress Pathway Contributes to Escherichia coli Tolerance to Antimicrobial Peptides

2013 ◽  
Vol 79 (24) ◽  
pp. 7770-7779 ◽  
Author(s):  
Bianca Audrain ◽  
Lionel Ferrières ◽  
Amira Zairi ◽  
Guillaume Soubigou ◽  
Curtis Dobson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptides ◽  
Antimicrobial Peptide ◽  
Polymyxin B ◽  
Genetic Response ◽  
Content Type ◽  
E Coli ◽  
Multiresistant Bacteria ◽  
Envelope Stress ◽  
Stress Pathway

ABSTRACTAntimicrobial peptides produced by multicellular organisms as part of their innate system of defense against microorganisms are currently considered potential alternatives to conventional antibiotics in case of infection by multiresistant bacteria. However, while the mode of action of antimicrobial peptides is relatively well described, resistance mechanisms potentially induced or selected by these peptides are still poorly understood. In this work, we studied the mechanisms of action and resistance potentially induced by ApoEdpL-W, a new antimicrobial peptide derived from human apolipoprotein E. Investigation of the genetic response ofEscherichia coliupon exposure to sublethal concentrations of ApoEdpL-W revealed that this antimicrobial peptide triggers activation of RcsCDB, CpxAR, and σEenvelope stress pathways. This genetic response is not restricted to ApoEdpL-W, since several other antimicrobial peptides, including polymyxin B, melittin, LL-37, and modified S4dermaseptin, also activate severalE. colienvelope stress pathways. Finally, we demonstrate that induction of the CpxAR two-component system directly contributes toE. colitolerance toward ApoEdpL-W, polymyxin B, and melittin. These results therefore show thatE. colisenses and responds to different antimicrobial peptides by activation of the CpxAR pathway. While this study further extends the understanding of the array of peptide-induced stress signaling systems, it also provides insight into the contribution of Cpx envelope stress pathway toE. colitolerance to antimicrobial peptides.

scholarly journals Pharmacodynamic Evaluation of MRX-8, a Novel Polymyxin, in the Neutropenic Mouse Thigh and Lung Infection Models against Gram-Negative Pathogens

2020 ◽  
Vol 64 (11) ◽  
Author(s):  
Alexander J. Lepak ◽  
Wen Wang ◽  
David R. Andes
Keyword(s):  
Polymyxin B ◽  
Lung Model ◽  
Dose Fractionation ◽  
Time Curve ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Infection Models ◽  
Dose Ranging

ABSTRACT MRX-8 is a novel polymyxin analogue in development for the treatment of infections caused by Gram-negative pathogens, including those resistant to other antibiotic classes. In the present study, we examined the pharmacodynamic activity of MRX-8 against a variety of common Gram-negative pathogens in the neutropenic mouse thigh and lung models. Additionally, we examined polymyxin B (PMB) as a comparator. Plasma pharmacokinetics of MRX-8 and PMB were linear over a broad dosing range of 0.156 to 10 mg/kg of body weight and had similar AUC0–∞ (area under the drug concentration-time curve from 0 h to infinity) exposures of MRX-8, 0.22 to 12.64 mg · h/liter, and PMB, 0.12 to 13.22 mg · h/liter. Dose fractionation was performed for MRX-8 using a single Escherichia coli isolate, and the results demonstrated that both Cmax (maximum concentration of drug in serum)/MIC and AUC/MIC ratios were strongly associated with efficacy. In the thigh model, dose-ranging studies included strains of E. coli (n = 3), Pseudomonas aeruginosa (n = 2), Klebsiella pneumoniae (n = 3), and Acinetobacter baumannii (n = 1). Both MRX-8 and PMB exhibited increased effects with increasing doses. MRX-8 and PMB free AUC/MIC exposures for net stasis were similar for E. coli and K. pneumoniae at 20 to 30. Notably, for P. aeruginosa and A. baumannii, the free AUC/MIC ratio for stasis was numerically much smaller for MRX-8 at 6 to 8 than for PMB at 16 to 37. In the lung model, MRX-8 was also more effective than PMB when dosed to achieve similar free-drug AUC exposures over the study period. MRX-8 is a promising novel polymyxin analogue with in vivo activity against many different clinically relevant species in both the mouse thigh and lung models.

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

2012 ◽  
Vol 57 (1) ◽  
pp. 637-639 ◽  
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.

scholarly journals Different effects of transcriptional regulators MarA, SoxS and Rob on susceptibility of Escherichia coli to cationic antimicrobial peptides (CAMPs): Rob-dependent CAMP induction of the marRAB operon

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 570-578 ◽  
Author(s):  
Douglas M. Warner ◽  
Stuart B. Levy
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptides ◽  
Bacterial Infections ◽  
Transcriptional Regulators ◽  
Polymyxin B ◽  
Cationic Antimicrobial Peptides ◽  
Antibiotic Resistant ◽  
E Coli ◽  
Camp Induction ◽  
Increased Susceptibility

Cationic antimicrobial peptides (CAMPs), a component of the mammalian immune system, protect the host from bacterial infections. The roles of the Escherichia coli transcriptional regulators MarA, SoxS and Rob in susceptibility to these peptides were examined. Overexpression of marA, either in an antibiotic-resistant marR mutant or from a plasmid, decreased bacterial susceptibility to CAMPs. Overexpression of the soxS gene from a plasmid, which decreased susceptibility to antibiotics, unexpectedly caused no decrease in CAMP susceptibility; instead it produced increased susceptibility to different CAMPs. Deletion or overexpression of rob had little effect on CAMP susceptibility. The marRAB operon was upregulated when E. coli was incubated in sublethal amounts of CAMPs polymyxin B, LL-37 or human β-defensin-1; however, this upregulation required Rob. Deletion of acrAB increased bacterial susceptibility to polymyxin B, LL-37 and human β-defensin-1 peptides. Deletion of tolC yielded an even greater increase in susceptibility to these peptides and also led to increased susceptibility to human α-defensin-2. Inhibition of cellular proton-motive force increased peptide susceptibility for wild-type and acrAB deletion strains; however, it decreased susceptibility of tolC mutants. These findings demonstrate that CAMPs are both inducers of marA-mediated drug resistance through interaction with Rob and also substrates for efflux in E. coli. The three related transcriptional regulators show different effects on bacterial cell susceptibility to CAMPs.

scholarly journals pH Dependence of Microbe Sterilization by Cationic Antimicrobial Peptides

2013 ◽  
Vol 57 (7) ◽  
pp. 3312-3320 ◽  
Author(s):  
William F. Walkenhorst ◽  
J. Wolfgang Klein ◽  
Phuong Vo ◽  
William C. Wimley
Keyword(s):  
Ionic Strength ◽  
Antimicrobial Peptides ◽  
Ph Dependence ◽  
Gram Positive ◽  
Cationic Antimicrobial Peptides ◽  
Gram Negative ◽  
Content Type ◽  
Ionic Strength Effect ◽  
Effects Of Ph ◽  
Species Specific

ABSTRACTWe recently described a family of cationic antimicrobial peptides (CAMPs) selected from a combinatorial library that exhibited potent, broad-spectrum activity at neutral pH and low ionic strength. To further delimit the utility and activity profiles of these peptides, we investigated the effects of solution conditions, such as pH and ionic strength, on the efficacy of the peptide antimicrobials against a panel of microorganisms. Peptide minimum sterilizing concentrations (MSCs) varied linearly with pH for each subtype within our family of CAMPs for all organisms tested. The peptides were much less effective against Gram-negative bacteria at high pH, consistent with a decrease in net positive charge on the peptides. A similar trend was observed for the fungusCandida albicans. Surprisingly, the opposite pH trend was observed with the Gram-positiveStaphylococcus aureus. In addition, an additive ionic strength effect was observed with increasing buffer strengths at identical pH values. The extreme difference in the observed pH behavior between Gram-negative and Gram-positive organisms is attributed to the presence of native charged molecules in the much thicker peptidoglycan layer of the Gram-positive organism. The novel species-specific effects of pH observed here have important implications for applications using CAMPs and for the design of novel CAMPs.

scholarly journals Proteomic response of Escherichia coli to a membrane lytic and iron chelating truncated Amaranthus tricolor defensin

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Tessa B. Moyer ◽  
Ashleigh L. Purvis ◽  
Andrew J. Wommack ◽  
Leslie M. Hicks
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Antimicrobial Peptides ◽  
Mechanism Of Action ◽  
Gram Negative Bacteria ◽  
Amaranthus Tricolor ◽  
Core Motif ◽  
Gram Negative ◽  
E Coli ◽  
Membrane Lysis

Abstract Background Plant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays. Results Atr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae. Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe3+ and chelate Fe2+ at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors. Conclusions This study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.

scholarly journals Endotoxemia as a Diagnostic Tool for Patients with Suspected Bacteremia Caused by Gram-Negative Organisms: a Meta-Analysis of 4 Decades of Studies

2015 ◽  
Vol 53 (4) ◽  
pp. 1183-1191 ◽  
Author(s):  
James C. Hurley ◽  
Piotr Nowak ◽  
Lars Öhrmalm ◽  
Charalambos Gogos ◽  
Apostolos Armaganidis ◽  
...  
Keyword(s):  
Confidence Interval ◽  
Meta Analysis ◽  
Inclusion Criteria ◽  
Summary Receiver Operating Characteristic ◽  
Detection Rates ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Patient Groups ◽  
Gram Negative Organisms

The clinical significance of endotoxin detection in blood has been evaluated for a broad range of patient groups in over 40 studies published over 4 decades. The influences of Gram-negative (GN) bacteremia species type and patient inclusion criteria on endotoxemia detection rates in published studies remain unclear. Studies were identified after a literature search and manual reviews of article bibliographies, together with a direct approach to authors of potentially eligible studies for data clarifications. The concordance between GN bacteremia and endotoxemia expressed as the summary diagnostic odds ratios (DORs) was derived for three GN bacteremia categories across eligible studies by using a hierarchical summary receiver operating characteristic (HSROC) method. Forty-two studies met broad inclusion criteria, with between 2 and 173 GN bacteremias in each study. Among all 42 studies, the DORs (95% confidence interval) were 3.2 (1.7 to 6.0) and 5.8 (2.4 to 13.7) in association with GN bacteremias withEscherichia coliand those withPseudomonas aeruginosa, respectively. Among 12 studies of patients with sepsis, the proportion of endotoxemia positivity (95% confidence interval) among patients withP. aeruginosabacteremia (69% [57 to 79%];P= 0.004) or withProteusbacteremia (76% [51 to 91%];P= 0.04) was significantly higher than that among patients without GN bacteremia (49% [33 to 64%]), but this was not so for patients bacteremic withE. coli(57% [40 to 73%];P= 0.55). Among studies of the sepsis patient group, the concordance of endotoxemia with GN bacteremia was surprisingly weak, especially forE. coliGN bacteremia.

scholarly journals Bordetella pertussis Lipid A Glucosamine Modification Confers Resistance to Cationic Antimicrobial Peptides and Increases Resistance to Outer Membrane Perturbation

2014 ◽  
Vol 58 (8) ◽  
pp. 4931-4934 ◽  
Author(s):  
Nita R. Shah ◽  
Robert E. W. Hancock ◽  
Rachel C. Fernandez
Keyword(s):  
Immune System ◽  
Antimicrobial Peptides ◽  
Outer Membrane ◽  
Bordetella Pertussis ◽  
Lipid A ◽  
Whooping Cough ◽  
Human Immune System ◽  
Cationic Antimicrobial Peptides ◽  
Content Type ◽  
Membrane Perturbation

ABSTRACTBordetella pertussis, the causative agent of whooping cough, has many strategies for evading the human immune system. Lipopolysaccharide (LPS) is an important Gram-negative bacterial surface structure that activates the immune system via Toll-like receptor 4 and enables susceptibility to cationic antimicrobial peptides (CAMPs). We show modification of the lipid A region of LPS with glucosamine increased resistance to numerous CAMPs, including LL-37. Furthermore, we demonstrate that this glucosamine modification increased resistance to outer membrane perturbation.

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