Phage-encoded cationic antimicrobial peptide used for outer membrane disruption in lysis

AbstractSpanins are required for the last step in bacteriophage lysis: the disruption of the outer membrane. Bioinformatic analysis has shown that ~15% of phages lack a spanin gene, which suggests an alternate mechanism of outer membrane disruption. To address this, we selected virulent podophage ϕKT as a spaninless exemplar and tested ϕKT genes for outer membrane disruption during lysis. Hypothetical novel gene 28 causes outer membrane disruption when co-expressed with ϕKT lysis genes and complements the lysis defect of a λ spanin mutant. Gp28 is a 56 aa cationic peptide with predicted amphipathic helical structure and is associated with the particulate fraction after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic interaction with the membrane. Super high-resolution microscopy supports a primarily outer membrane localization for the peptide. Additionally, holin function is not required for gp28-mediated lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. In standard assays to measure bactericidal and inhibitory effects of antimicrobial peptides on bacterial cells, synthesized gp28 performed equivalently to LL-37. The studies presented here suggest that ϕKT Gp28 disrupts bacterial outer membranes during lysis in a manner akin to antimicrobial peptides.SignificanceHere we provide evidence that ϕKT produces an antimicrobial peptide for outer membrane disruption during lysis. The disruptin is a new paradigm for phage lysis, and has no similarities to other known lysis genes. Many mechanisms have been proposed for the function of antimicrobial peptides, however there is not a consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic selection system to support such studies. Therefore, the ϕKT disruptin may represent the first genetically tractable antimicrobial peptide.

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Phage-encoded cationic antimicrobial peptide required for lysis

Journal of Bacteriology ◽

10.1128/jb.00214-21 ◽

2021 ◽

Author(s):

Ashley Holt ◽

Jesse Cahill ◽

Jolene Ramsey ◽

Cody Martin ◽

Chandler O’Leary ◽

...

Keyword(s):

Antimicrobial Peptide ◽

Outer Membrane ◽

Genetic System ◽

Membrane Disruption ◽

New Paradigm ◽

Hydrophobic Membrane ◽

Cationic Protein ◽

Cationic Antimicrobial Peptide ◽

Host Lysis ◽

K 12

Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in E. coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp 28 in OM disruption. Additionally, gp 28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp 28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp 28 protein prior to lysis. Gp 28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp 28 behaved similar to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp 28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and thus establishes a new class of phage lysis proteins, the disruptins. Significance We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses.

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Spermicidal efficacy of VRP, a synthetic cationic antimicrobial peptide, inducing apoptosis and membrane disruption

Journal of Cellular Physiology ◽

10.1002/jcp.25958 ◽

2017 ◽

Vol 233 (2) ◽

pp. 1041-1050 ◽

Cited By ~ 5

Author(s):

Prasanta Ghosh ◽

Arpita Bhoumik ◽

Sudipta Saha ◽

Sandipan Mukherjee ◽

Sarfuddin Azmi ◽

...

Keyword(s):

Antimicrobial Peptide ◽

Membrane Disruption ◽

Cationic Antimicrobial Peptide

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Mode-of-Action of Antimicrobial Peptides: Membrane Disruption vs. Intracellular Mechanisms

Frontiers in Medical Technology ◽

10.3389/fmedt.2020.610997 ◽

2020 ◽

Vol 2 ◽

Author(s):

Aurélie H. Benfield ◽

Sónia Troeira Henriques

Keyword(s):

Antimicrobial Peptides ◽

Physicochemical Properties ◽

Broad Spectrum ◽

Mode Of Action ◽

Bacterial Pathogens ◽

Model Membranes ◽

Membrane Disruption ◽

Attractive Alternative ◽

Bacterial Cells

Antimicrobial peptides are an attractive alternative to traditional antibiotics, due to their physicochemical properties, activity toward a broad spectrum of bacteria, and mode-of-actions distinct from those used by current antibiotics. In general, antimicrobial peptides kill bacteria by either disrupting their membrane, or by entering inside bacterial cells to interact with intracellular components. Characterization of their mode-of-action is essential to improve their activity, avoid resistance in bacterial pathogens, and accelerate their use as therapeutics. Here we review experimental biophysical tools that can be employed with model membranes and bacterial cells to characterize the mode-of-action of antimicrobial peptides.

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Acyl carrier protein is a bacterial cytoplasmic target of cationic antimicrobial peptide LL-37

Biochemical Journal ◽

10.1042/bj20150432 ◽

2015 ◽

Vol 470 (2) ◽

pp. 243-253 ◽

Cited By ~ 10

Author(s):

Myung-Chul Chung ◽

Scott N. Dean ◽

Monique L. van Hoek

Keyword(s):

Antimicrobial Peptide ◽

Acyl Carrier Protein ◽

Acid Synthesis ◽

Membrane Disruption ◽

Carrier Protein ◽

Cytoplasmic Protein ◽

Bacterial Killing ◽

Cationic Antimicrobial Peptide ◽

Antibiotic Resistant ◽

Future Drug

In addition to membrane disruption, the cathelicidin antimicrobial peptide LL-37 translocates through the bacterial inner membrane to target intracellular molecules. Our data suggest that the CAMP LL-37 is able can specifically bind to the cytoplasmic protein AcpP resulting in the inhibition of fatty acid synthesis and bacterial killing. Our studies introduce a novel mechanism for cationic antimicrobial peptides, which may be useful in future drug development for the treatment of antibiotic-resistant bacterial infection.

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NOVEL FAMILY OF PROLINE-RICH ANTIMICROBIAL PEPTIDES INHIBITING BACTERIAL TRANSLATION

BIOTECHNOLOGY: STATE OF THE ART AND PERSPECTIVES ◽

10.37747/2312-640x-2021-19-173-174 ◽

2021 ◽

Vol 1 (19) ◽

pp. 173-174

Author(s):

R.N. Kruglikov ◽

T.V. Ovchinnikova ◽

P.V. Panteleev

Keyword(s):

Biological Activity ◽

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Bacterial Cells ◽

Bacterial Translation

A new proline-rich antimicrobial peptide from alpaca (Vicugnia pacos) was obtained, as well as its modified analogs. The biological activity of the peptides was studied, and also the role of the N- and C-terminal fragments, when acting on bacterial cells, was analyzed.

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Structural studies on the antimicrobial peptide Brevinin 1E by spectroscopic methods

Spectroscopy An International Journal ◽

10.1155/2003/650369 ◽

2003 ◽

Vol 17 (2-3) ◽

pp. 127-138 ◽

Cited By ~ 1

Author(s):

Woo-Sung Son ◽

Ji-Sun Kim ◽

Hyung-Eun Kim ◽

Sang-Ho Park ◽

Bong-Jin Lee

Keyword(s):

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Three Dimensional ◽

Helical Structure ◽

Structural Motif ◽

Dimensional Structure ◽

Resonance Spectroscopy ◽

Random Structure ◽

Three Dimensional Structure ◽

Pharmacologically Active

Skin extracts of frogs are a rich source of pharmacologically active peptides such as caeruleins, tachykinins, bradykinins, thyrotropin-releasing hormone, bombesin-like and opioid peptides. A large variety of antimicrobial peptides has been isolated fromRanaspecies. These peptides, grouped in several families on the basis of differing length and distinct activity, were found to have one structural motif in common: an intramolecular disulfide bridge located at the C-terminal end, forming a seven-member ring, which was designated ‘Rana box’. Brevinin 1E is a 24-residue antimicrobial peptide isolated from the skin of a frog,Rana brevipoda. This peptide shows a broad range of antimicrobial activity against prokaryotic cells but shows very much hemolytic activity against human red blood cells. The solution structure of Brevinin 1E was studied by using CD (circular dichroism) and NMR (nuclear magnetic resonance) spectroscopy. CD investigation revealed that Brevinin 1E adopts random structure in aqueous solution but adopts mainlyα-helical structure in TFE/water (6 : 4, v/v) solution. The three-dimensional structure of Brevinin 1E was determined in 60% TFE/water solution using homonuclear NMR spectroscopy. This peptide showed mainly anα-helical structure with amphipathic property. Its three-dimensional structure is similar to those of other peptides such as magainin, nigrocin and ranalexin. Therefore, Brevinin 1E can be classified into the family of antimicrobial peptides containing a single linearα-helix that interact with target microbial membrane, leading to cell death through disruption of membrane integrity.

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Identification of a Genetic Locus Responsible for Antimicrobial Peptide Resistance inClostridium difficile

Infection and Immunity ◽

10.1128/iai.00731-10 ◽

2010 ◽

Vol 79 (1) ◽

pp. 167-176 ◽

Cited By ~ 78

Author(s):

Shonna M. McBride ◽

Abraham L. Sonenshein

Keyword(s):

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Abc Transporter ◽

Genetic Locus ◽

Polymyxin B ◽

Transcriptional Analysis ◽

Cationic Antimicrobial Peptide ◽

Low Levels ◽

Antimicrobial Peptide Resistance ◽

Abc Transporter Genes

ABSTRACTClostridium difficilecauses chronic intestinal disease, yet little is understood about how the bacterium interacts with and survives in the host. To colonize the intestine and cause persistent disease, the bacterium must circumvent killing by host innate immune factors, such as cationic antimicrobial peptides (CAMPs). In this study, we investigated the effect of model CAMPs on growth and found thatC. difficileis not only sensitive to these compounds but also responds to low levels of CAMPs by expressing genes that lead to CAMP resistance. By plating the bacterium on medium containing the CAMP nisin, we isolated a mutant capable of growing in three times the inhibitory concentration of CAMPs. This mutant also showed increased resistance to the CAMPs gallidermin and polymyxin B, demonstrating tolerance to different types of antimicrobial peptides. We identified the mutated gene responsible for the resistance phenotype as CD1352. This gene encodes a putative orphan histidine kinase that lies adjacent to a predicted ABC transporter operon (CD1349 to CD1351). Transcriptional analysis of the ABC transporter genes revealed that this operon was upregulated in the presence of nisin in wild-type cells and was more highly expressed in the CD1352 mutant. The insertional disruption of the CD1349 gene resulted in significant decreases in resistance to the CAMPs nisin and gallidermin but not polymyxin B. Because of their role in cationic antimicrobial peptide resistance, we propose the designationcprABCfor genes CD1349 to CD1351 andcprKfor the CD1352 gene. These results provide the first evidence of aC. difficilegene associated with antimicrobial peptide resistance.

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Pse-T2, an Antimicrobial Peptide with High-Level, Broad-Spectrum Antimicrobial Potency and Skin Biocompatibility against Multidrug-ResistantPseudomonas aeruginosaInfection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01493-18 ◽

2018 ◽

Vol 62 (12) ◽

Cited By ~ 7

Author(s):

Hee Kyoung Kang ◽

Chang Ho Seo ◽

Tudor Luchian ◽

Yoonkyung Park

Keyword(s):

Antimicrobial Peptide ◽

Membrane Integrity ◽

Helical Structure ◽

Multidrug Resistant ◽

Bacterial Cells ◽

Bacterial Strains ◽

Necrosis Factor Alpha ◽

Content Type ◽

Bacterial Membranes

ABSTRACTPseudin-2, isolated from the frogPseudis paradoxa, exhibits potent antibacterial activity but also cytotoxicity. In an effort to develop clinically applicable antimicrobial peptides (AMPs), we designed pseudin-2 analogs with Lys substitutions, resulting in elevated amphipathic α-helical structure and cationicity. In addition, truncated analogs of pseudin-2 and Lys-substituted peptides were synthesized to produce linear 18-residue amphipathic α-helices, which were further investigated for their mechanism and functions. These truncated analogs exhibited higher antimicrobial activity and lower cytotoxicity than pseudin-2. In particular, Pse-T2 showed marked pore formation, permeabilization of the outer/inner bacterial membranes, and DNA binding. Fluorescence spectroscopy and scanning electron microscopy showed that Pse-T2 kills bacterial cells by disrupting membrane integrity.In vivo, wounds infected with multidrug-resistant (MDR)Pseudomonas aeruginosahealed significantly faster when treated with Pse-T2 than did untreated wounds or wounds treated with ciprofloxacin. Moreover, Pse-T2 facilitated infected-wound closure by reducing inflammation through suppression of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α). These data suggest that the small antimicrobial peptide Pse-T2 could be useful for future development of therapeutic agents effective against MDR bacterial strains.

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Induction by Cationic Antimicrobial Peptides and Involvement in Intrinsic Polymyxin and Antimicrobial Peptide Resistance, Biofilm Formation, and Swarming Motility of PsrA in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00594-08 ◽

2008 ◽

Vol 190 (16) ◽

pp. 5624-5634 ◽

Cited By ~ 47

Author(s):

W. James Gooderham ◽

Manjeet Bains ◽

Joseph B. McPhee ◽

Irith Wiegand ◽

Robert E. W. Hancock

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Peptides ◽

Antimicrobial Peptide ◽

Outer Membrane ◽

Biofilm Formation ◽

Metabolic Energy ◽

Wild Type ◽

Swarming Motility ◽

Cationic Antimicrobial Peptides ◽

Antimicrobial Peptide Resistance

ABSTRACT Pseudomonas aeruginosa is an important opportunistic pathogen that causes infections that can be extremely difficult to treat due to its high intrinsic antibiotic resistance and broad repertoire of virulence factors, both of which are highly regulated. It is demonstrated here that the psrA gene, encoding a transcriptional regulator, was upregulated in response to subinhibitory concentrations of cationic antimicrobial peptides. Compared to the wild type and the complemented mutant, a P. aeruginosa PAO1 psrA::Tn5 mutant displayed intrinsic supersusceptibility to polymyxin B, a last-resort antimicrobial used against multidrug-resistant infections, and the bovine neutrophil antimicrobial peptide indolicidin; this supersusceptibility phenotype correlated with increased outer membrane permeabilization by these agents. The psrA mutant was also defective in simple biofilm formation, rapid attachment, and swarming motility, all of which could be complemented by the cloned psrA gene. The role of PsrA in global gene regulation was studied by comparing the psrA mutant to the wild type by microarray analysis, demonstrating that 178 genes were up- or downregulated ≥2-fold (P ≤ 0.05). Dysregulated genes included those encoding certain known PsrA targets, those encoding the type III secretion apparatus and effectors, adhesion and motility genes, and a variety of metabolic, energy metabolism, and outer membrane permeability genes. This suggests that PsrA might be a key regulator of antimicrobial peptide resistance and virulence.

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