Destabilization of α-Helical Structure in Solution Improves Bactericidal Activity of Antimicrobial Peptides: Opposite Effects on Bacterial and Viral Targets

ABSTRACTWe have previously examined the mechanism of antimicrobial peptides on the outer membrane of vaccinia virus. We show here that the formulation of peptides LL37 and magainin-2B amide in polysorbate 20 (Tween 20) results in greater reductions in virus titer than formulation without detergent, and the effect is replicated by substitution of polysorbate 20 with high-ionic-strength buffer. In contrast, formulation with polysorbate 20 or high-ionic-strength buffer has the opposite effect on bactericidal activity of both peptides, resulting in lesser reductions in titer for both Gram-positive and Gram-negative bacteria. Circular dichroism spectroscopy shows that the differential action of polysorbate 20 and salt on the virucidal and bactericidal activities correlates with the α-helical content of peptide secondary structure in solution, suggesting that the virucidal and bactericidal activities are mediated through distinct mechanisms. The correlation of a defined structural feature with differential activity against a host-derived viral membrane and the membranes of both Gram-positive and Gram-negative bacteria suggests that the overall helical content in solution under physiological conditions is an important feature for consideration in the design and development of candidate peptide-based antimicrobial compounds.

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Cathelicidin Antimicrobial Peptides with Reduced Activation of Toll-Like Receptor Signaling Have Potent Bactericidal Activity against Colistin-Resistant Bacteria

mBio ◽

10.1128/mbio.01418-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 7

Author(s):

Cheng Kao ◽

Xiaoyan Lin ◽

Guanghui Yi ◽

Yunliang Zhang ◽

Dean A. Rowe-Magnus ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Bactericidal Activity ◽

Bacterial Infections ◽

Inflammatory Responses ◽

Human Cells ◽

Resistant Bacteria ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Human Red Blood Cells ◽

Minor Injuries

ABSTRACT The world is at the precipice of a postantibiotic era in which medical procedures and minor injuries can result in bacterial infections that are no longer effectively treated by antibiotics. Cathelicidins are peptides produced by animals to combat bacterial infections and to regulate innate immune responses. However, cathelicidins are potent activators of the inflammatory response. Cathelicidins with reduced proinflammatory activity and potent bactericidal activity in the low micromolar range against Gram-negative bacteria have been identified. Motifs in cathelicidins that impact bactericidal activity and cytotoxicity to human cells have been elucidated and used to generate peptides that have reduced activation of proinflammatory cytokine production and reduced cytotoxicity to human cells. The resultant peptides have bactericidal activities comparable to that of colistin and can kill colistin-resistant bacteria. IMPORTANCE Cathelicidins are antimicrobial peptides that can also increase inflammatory responses. This combination of activities can cause complications in the treatment of bacterial infections despite the pressing need for new antimicrobials. We have identified cathelicidins with decreased activation of inflammatory responses. The peptides kill Gram-negative bacteria at low micromolar concentrations by binding to and perturbing the integrity of the bacterial membrane. The peptides were also engineered to further decrease lysis of human red blood cells. The peptides have activities comparable to those of the polymyxins, a class of antibiotics to which plasmid-borne resistance is rapidly spreading and can kill colistin-resistant bacteria. These peptides are promising candidates for the development of novel antibacterial agents.

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In Vitro Antibacterial Activities of AF 3013, the Active Metabolite of Prulifloxacin, against Nosocomial and Community Italian Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.12.3616-3622.2001 ◽

2001 ◽

Vol 45 (12) ◽

pp. 3616-3622 ◽

Cited By ~ 28

Author(s):

Maria Pia Montanari ◽

Marina Mingoia ◽

Pietro Emanuele Varaldo

Keyword(s):

Bactericidal Activity ◽

Active Metabolite ◽

Antibacterial Activities ◽

Minimal Bactericidal Concentration ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Gram Positive Bacteria ◽

Bactericidal Activities ◽

Time Kill

ABSTRACT AF 3013, the active metabolite of prulifloxacin, was tested to determine its inhibitory and bactericidal activities against 396 nosocomial and 258 community Italian isolates. Compared with that of ciprofloxacin, its activity (assessed in MIC and minimal bactericidal concentration tests) was generally similar or greater against gram-positive bacteria and greater against gram-negative bacteria. In time-kill assays using selected isolates, its bactericidal activity was comparable to that of ciprofloxacin.

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

BMC Microbiology ◽

10.1186/s12866-021-02176-4 ◽

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.

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Synergy and remarkable specificity of antimicrobial peptides in vivo using a systematic knockout approach

eLife ◽

10.7554/elife.44341 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 29

Author(s):

Mark Austin Hanson ◽

Anna Dostálová ◽

Camilla Ceroni ◽

Mickael Poidevin ◽

Shu Kondo ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Fungal Pathogens ◽

Gene Editing ◽

Cationic Peptides ◽

Gram Negative Bacteria ◽

Biological Processes ◽

Gram Negative ◽

Bacteria And Fungi

Antimicrobial peptides (AMPs) are host-encoded antibiotics that combat invading microorganisms. These short, cationic peptides have been implicated in many biological processes, primarily involving innate immunity. In vitro studies have shown AMPs kill bacteria and fungi at physiological concentrations, but little validation has been done in vivo. We utilized CRISPR gene editing to delete most known immune-inducible AMPs of Drosophila, namely: 4 Attacins, 2 Diptericins, Drosocin, Drosomycin, Metchnikowin and Defensin. Using individual and multiple knockouts, including flies lacking these ten AMP genes, we characterize the in vivo function of individual and groups of AMPs against diverse bacterial and fungal pathogens. We found that Drosophila AMPs act primarily against Gram-negative bacteria and fungi, contributing either additively or synergistically. We also describe remarkable specificity wherein certain AMPs contribute the bulk of microbicidal activity against specific pathogens, providing functional demonstrations of highly specific AMP-pathogen interactions in an in vivo setting.

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SPS-neutralization in tissue samples for efficacy testing of antimicrobial peptides

BMC Infectious Diseases ◽

10.1186/s12879-019-4700-1 ◽

2019 ◽

Vol 19 (1) ◽

Author(s):

Gabrielle Sherella Dijksteel ◽

Peter H. Nibbering ◽

Magda M. W. Ulrich ◽

Esther Middelkoop ◽

Bouke K. H. L. Boekema

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Antimicrobial Agents ◽

Ex Vivo ◽

Residual Activity ◽

Gram Negative Bacteria ◽

Tissue Samples ◽

Gram Negative ◽

Viable Bacteria

Abstract Background Accurate determination of the efficacy of antimicrobial agents requires neutralization of residual antimicrobial activity in the samples before microbiological assessment of the number of surviving bacteria. Sodium polyanethol sulfonate (SPS) is a known neutralizer for the antimicrobial activity of aminoglycosides and polymyxins. In this study, we evaluated the ability of SPS to neutralize residual antimicrobial activity of antimicrobial peptides [SAAP-148 and pexiganan; 1% (wt/v) in PBS], antibiotics [mupirocin (Bactroban) and fusidic acid (Fucidin) in ointments; 2% (wt/wt))] and disinfectants [2% (wt/wt) silver sulfadiazine cream (SSD) and 0.5% (v/v) chlorhexidine in 70% alcohol]. Methods Homogenates of human skin models that had been exposed to various antimicrobial agents for 1 h were pipetted on top of Methicillin-resistant Staphylococcus aureus (MRSA) on agar plates to determine whether the antimicrobial agents display residual activity. To determine the optimal concentration of SPS for neutralization, antimicrobial agents were mixed with PBS or increasing doses of SPS in PBS (0.05–1% wt/v) and then 105 colony forming units (CFU)/mL MRSA were added. After 30 min incubation, the number of viable bacteria was assessed. Next, the in vitro efficacy of SAAP-148 against various gram-positive and gram-negative bacteria was determined using PBS or 0.05% (wt/v) SPS immediately after 30 min incubation of the mixture. Additionally, ex vivo excision wound models were inoculated with 105 CFU MRSA for 1 h and exposed to SAAP-148, pexiganan, chlorhexidine or PBS for 1 h. Subsequently, samples were homogenized in PBS or 0.05% (wt/v) SPS and the number of viable bacteria was assessed. Results All tested antimicrobials displayed residual activity in tissue samples, resulting in a lower recovery of surviving bacteria on agar. SPS concentrations at ≥0.05% (wt/v) were able to neutralize the antimicrobial activity of SAAP-148, pexiganan and chlorhexidine, but not of SSD, Bactroban and Fucidin. Finally, SPS-neutralization in in vitro and ex vivo efficacy tests of SAAP-148, pexiganan and chlorhexidine against gram-positive and gram-negative bacteria resulted in significantly higher numbers of CFU compared to control samples without SPS-neutralization. Conclusions SPS was successfully used to neutralize residual activity of SAAP-148, pexiganan and chlorhexidine and this prevented an overestimation of their efficacy.

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Cathelicidin Peptides Restrict Bacterial Growth via Membrane Perturbation and Induction of Reactive Oxygen Species

mBio ◽

10.1128/mbio.02021-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 5

Author(s):

Dean A. Rowe-Magnus ◽

Adenine Y. Kao ◽

Antonio Cembellin Prieto ◽

Meng Pu ◽

Cheng Kao

Keyword(s):

Reactive Oxygen Species ◽

Antimicrobial Peptides ◽

Single Cell ◽

Reactive Oxygen ◽

Cellular Damage ◽

Primary Effect ◽

Gram Negative Bacteria ◽

Oxygen Species ◽

Gram Positive ◽

Gram Negative

ABSTRACT All metazoans produce antimicrobial peptides (AMPs) that have both broad antimicrobial and immunomodulatory activity. Cathelicidins are AMPs that preferentially kill Gram-negative bacteria in vitro, purportedly by assembling into higher-order structures that perforate the membrane. We utilized high-resolution, single-cell fluorescence microscopy to examine their mechanism of action in real time. Engineered cathelicidins rapidly bound to Gram-negative and Gram-positive cells and penetrated the cytoplasmic membrane. Rapid failure of the peptidoglycan superstructure in regions of active turnover caused leakage of cytoplasmic contents and the formation of membrane-bound blebs. A mutation anticipated to destabilize interactions between cathelicidin subunits had no effect on bactericidal activity, suggesting that cathelicidins have activities beyond perforating the membrane. Nanomolar concentrations of cathelicidins, although not bactericidal, reduced the growth rate of Gram-negative and Gram-positive bacteria. The cells exhibited expression changes in multiple essential processes, including protein synthesis, peptidoglycan biosynthesis, respiration, and the detoxification of reactive oxygen species (ROS). Time-lapse imaging revealed that ROS accumulation preceded bleb formation, and treatments that reduced cellular ROS levels overcame these bactericidal effects. We propose that that the primary effect of cathelicidins is to induce the production of ROS that damage bacterial molecules, leading to slowed growth or cell death. Given their low circulating levels in vivo, AMPs may serve to slow bacterial population expansion so that cellular immunity systems can respond to and battle the infection. IMPORTANCE Antimicrobial peptides (AMPs) are an important part of the mammalian innate immune system in the battle against microbial infection. How AMPs function to control bacteria is not clear, as nearly all activity studies use nonphysiological levels of AMPs. We monitored peptide action in live bacterial cells over short time frames with single-cell resolution and found that the primary effect of cathelicidin peptides is to increase the production of oxidative molecules that cause cellular damage in Gram-positive and Gram-negative bacteria.

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Proteomic Analysis of Resistance of Gram-Negative Bacteria to Chlorhexidine and Impacts on Susceptibility to Colistin, Antimicrobial Peptides, and Ceragenins

Frontiers in Microbiology ◽

10.3389/fmicb.2019.00210 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 10

Author(s):

Marjan M. Hashemi ◽

Brett S. Holden ◽

Jordan Coburn ◽

Maddison F. Taylor ◽

Scott Weber ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Proteomic Analysis ◽

Gram Negative Bacteria ◽

Gram Negative

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In Vitro Anti-Helicobacter pyloriActivities of New Rifamycin Derivatives, KRM-1648 and KRM-1657

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.5.1072 ◽

1999 ◽

Vol 43 (5) ◽

pp. 1072-1076 ◽

Cited By ~ 45

Author(s):

Junko K. Akada ◽

Mutsunori Shirai ◽

Kenji Fujii ◽

Kiwamu Okita ◽

Teruko Nakazawa

Keyword(s):

Helicobacter Pylori ◽

Cell Lysis ◽

Antimicrobial Activities ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Bactericidal Activities ◽

H Pylori ◽

Time Kill

ABSTRACT The new rifamycin derivatives KRM-1657 and KRM-1648 were evaluated for their in vitro antimicrobial activities against 44 strains ofHelicobacter pylori. Although the drugs were not very active against other gram-negative bacteria, the MICs at which 90% of isolates are inhibited for these drugs were lower (0.002 and 0.008 μg/ml, respectively) than those of amoxicillin and rifampin forH. pylori. Time-kill studies revealed that the bactericidal activities of these agents were due to cell lysis. The results presented here indicate that these new rifamycin derivatives may be useful for the eradication of H. pylori infections.

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Antimicrobial Peptides from Rat-Tailed Maggots of the Drone Fly Eristalis tenax Show Potent Activity against Multidrug-Resistant Gram-Negative Bacteria

Microorganisms ◽

10.3390/microorganisms8050626 ◽

2020 ◽

Vol 8 (5) ◽

pp. 626 ◽

Cited By ~ 1

Author(s):

Rolf Hirsch ◽

Jochen Wiesner ◽

Armin Bauer ◽

Alexander Marker ◽

Heiko Vogel ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Multidrug Resistant ◽

Therapeutic Window ◽

Gram Negative Bacteria ◽

Strong Activity ◽

Gram Negative ◽

Physiological Conditions ◽

Manure Storage ◽

Defense Molecules ◽

Generation Sequencing

The spread of multidrug-resistant Gram-negative bacteria is an increasing threat to human health, because novel compound classes for the development of antibiotics have not been discovered for decades. Antimicrobial peptides (AMPs) may provide a much-needed breakthrough because these immunity-related defense molecules protect many eukaryotes against Gram-negative pathogens. Recent concepts in evolutionary immunology predict the presence of potent AMPs in insects that have adapted to survive in habitats with extreme microbial contamination. For example, the saprophagous and coprophagous maggots of the drone fly Eristalis tenax (Diptera) can flourish in polluted aquatic habitats, such as sewage tanks and farmyard liquid manure storage pits. We used next-generation sequencing to screen the E. tenax immunity-related transcriptome for AMPs that are synthesized in response to the injection of bacterial lipopolysaccharide. We identified 22 AMPs and selected nine for larger-scale synthesis to test their activity against a broad spectrum of pathogens, including multidrug-resistant Gram-negative bacteria. Two cecropin-like peptides (EtCec1-a and EtCec2-a) and a diptericin-like peptide (EtDip) displayed strong activity against the pathogens, even under simulated physiological conditions, and also achieved a good therapeutic window. Therefore, these AMPs could be used as leads for the development of novel antibiotics.

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