A Seed-Endophytic Bacillus safensis Strain With Antimicrobial Activity Has Genes for Novel Bacteriocin-Like Antimicrobial Peptides

Bacteriocins are a highly diverse group of antimicrobial peptides that have been identified in a wide range of commensal and probiotic organisms, especially those resident in host microbiomes. Rising antibiotic resistance have fueled renewed research into new drug scaffolds such as antimicrobial peptides for use in therapeutics. In this investigation, we examined mung bean seeds for endophytes possessing activity against human and plant pathogens. We isolated a novel strain of Bacillus safensis, from the contents of surface-sterilized mung bean seed, which we termed B. safensis C3. Genome sequencing of C3 identified three distinct biosynthetic systems that produce bacteriocin-based peptides. C3 exhibited antibacterial activity against Escherichia coli, Xanthomonas axonopodis, and Pseudomonas syringae. Robust antimicrobial activity of B. safensis C3 was observed when C3 was co-cultured with Bacillus subtilis. Using the cell-free supernatant of C3 and cation exchange chromatography, we enriched a product that retained antimicrobial activity against B. subtilis. The peptide was found to be approximately 3.3 kDa in size by mass spectrometry, and resistant to proteolysis by Carboxypeptidase Y and Endoproteinase GluC, suggesting that it is a modified variant of an AS-48 like bacteriocin. Our findings open new avenues into further development of novel bacteriocin-based scaffolds for therapeutic development, as well as further investigations into how our discoveries of bacteriocin-producing plant commensal microorganisms may have the potential for an immediate impact on the safety of food supplies.

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De Novo Design of Antimicrobial Peptides With a Special Charge Pattern and Their Application in Combating Plant Pathogens

Frontiers in Plant Science ◽

10.3389/fpls.2021.753217 ◽

2021 ◽

Vol 12 ◽

Author(s):

Eric H. -L. Chen ◽

Cheng-Wei Weng ◽

Yi-Min Li ◽

Ming-Chin Wu ◽

Chien-Chih Yang ◽

...

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Plant Pathogens ◽

De Novo ◽

Plant Protection ◽

Acyl Chain ◽

Fatty Acyl ◽

Plant Diseases ◽

Basic Residue ◽

Wide Range

Plant diseases are important issues in agriculture, and the development of effective and environment-friendly means of disease control is crucial and highly desired. Antimicrobial peptides (AMPs) are known as potential alternatives to chemical pesticides because of their potent broad-spectrum antimicrobial activity and because they have no risk, or have only a low risk, of developing chemical-resistant pathogens. In this study, we designed a series of amphipathic helical peptides with different spatial distributions of positive charges and found that the peptides that had a special sequence pattern “BBHBBHHBBH” (“B” for basic residue and “H” for hydrophobic residue) displayed excellent bactericidal and fungicidal activities in a wide range of economically important plant pathogens. The peptides with higher helical propensity had lower antimicrobial activity. When we modified the peptides with a long acyl chain at their N-terminus, their plant protection effect improved. Our application of the fatty acyl-modified peptides on the leaves of tomato and Arabidopsis plants lessened the infection caused by Pectobacterium carotovorum subsp. carotovorum and Botrytis cinerea. Our study provides important insights on the development of more potent novel AMPs for plant protection.

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Novel Method for Selection of Antimicrobial Peptides from a Phage Display Library by Use of Bacterial Magnetic Particles

Applied and Environmental Microbiology ◽

10.1128/aem.00162-08 ◽

2008 ◽

Vol 74 (24) ◽

pp. 7600-7606 ◽

Cited By ~ 14

Author(s):

Tsuyoshi Tanaka ◽

Yoriko Kokuryu ◽

Tadashi Matsunaga

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Phage Display ◽

Magnetic Particles ◽

Peptide Library ◽

Inner Membrane ◽

Phage Display Peptide Library ◽

Wide Range ◽

Bacterial Magnetic Particles ◽

Selection Of

ABSTRACT Antimicrobial peptides were isolated from a phage display peptide library using bacterial magnetic particles (BacMPs) as a solid support. The BacMPs obtained from “Magnetospirillum magneticum” strain AMB-1 consist of pure magnetite (50 to 100 nm in size) and are covered with a lipid bilayer membrane derived from the invagination of the inner membrane. BacMPs are easily purified from a culture of magnetotactic bacteria by magnetic separation. Approximately 4 × 1010 PFU of the library phage (complexity, 2.7 × 109) was reacted with BacMPs. The elution of bound phages from BacMPs was performed by disrupting its membrane with phospholipase D treatment. Six candidate peptides, which were highly cationic and could bind onto the BacMP membrane, were obtained. They exhibited antimicrobial activity against Bacillus subtilis but not against Escherichia coli and Saccharomyces cerevisiae. The amino acid substitution of the selected peptide, KPQQHNRPLRHK (peptide 6-7), to enhance the hydrophobicity resulted in obvious antimicrobial activity against all test microorganisms. The present study shows for the first time that a magnetic selection of antimicrobial peptides from the phage display peptide library was successfully achieved by targeting the actual bacterial inner membrane. This BacMP-based method could be a promising approach for a high-throughput screening of antimicrobial peptides targeting a wide range of species.

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Production of an Engineered Killer Peptide in Nicotiana benthamiana by Using a Potato virus X Expression System

Applied and Environmental Microbiology ◽

10.1128/aem.71.10.6360-6367.2005 ◽

2005 ◽

Vol 71 (10) ◽

pp. 6360-6367 ◽

Cited By ~ 27

Author(s):

Marcello Donini ◽

Chiara Lico ◽

Selene Baschieri ◽

Stefania Conti ◽

Walter Magliani ◽

...

Keyword(s):

Antimicrobial Activity ◽

Pseudomonas Syringae ◽

Potato Virus ◽

Broad Spectrum ◽

Plant Pathogens ◽

Expression System ◽

Potato Virus X ◽

Scale Production ◽

Human Pathogens ◽

Single Chain

ABSTRACT The decapeptide killer peptide (KP) derived from the sequence of a single-chain, anti-idiotypic antibody acting as a functional internal image of a microbicidal, broad-spectrum yeast killer toxin (KT) was shown to exert a strong microbicidal activity against human pathogens. With the aim to exploit this peptide to confer resistance to plant pathogens, we assayed its antimicrobial activity against a broad spectrum of phytopathogenic bacteria and fungi. Synthetic KP exhibited antimicrobial activity in vitro towards Pseudomonas syringae, Erwinia carotovora, Botrytis cinerea, and Fusarium oxysporum. KP was also expressed in plants by using a Potato virus X (PVX)-derived vector as a fusion to the viral coat protein, yielding chimeric virus particles (CVPs) displaying the heterologous peptide. Purified CVPs showed enhanced antimicrobial activity against the above-mentioned plant pathogens and human pathogens such as Staphylococcus aureus and Candida albicans. Moreover, in vivo assays designed to challenge KP-expressing plants (as CVPs) with Pseudomonas syringae pv. tabaci showed enhanced resistance to bacterial attack. The results indicate that the PVX-based display system is a high-yield, rapid, and efficient method to produce and evaluate antimicrobial peptides in plants, representing a milestone for the large-scale production of high-added-value peptides through molecular farming. Moreover, KP is a promising molecule to be stably engineered in plants to confer broad-spectrum resistance to phytopathogens.

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A pattern-triggered immunity-related phenolic, acetosyringone, boosts rapid inhibition of a diverse set of plant pathogenic bacteria

BMC Plant Biology ◽

10.1186/s12870-021-02928-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Ágnes Szatmári ◽

Ágnes M. Móricz ◽

Ildikó Schwarczinger ◽

Judit Kolozsváriné Nagy ◽

Ágnes Alberti ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Cell Membrane ◽

Pseudomonas Syringae ◽

Bacterial Cell ◽

Plant Pathogens ◽

Bacterial Attachment ◽

Bacterial Metabolism ◽

Free Form ◽

Wide Range ◽

Bacterial Cell Membrane

Abstract Background Acetosyringone (3,5-dimethoxy-4-hydroxyacetophenone, AS) is a syringyl-type phenolic compound rarely found in plants in free form. It has been shown earlier to inhibit the growth of Pseudomonas bacteria in the presence of hydrogen peroxide and peroxidase (AS mix). Results We detected elevated levels of free AS in Nicotiana tabacum and N. benthamiana plants after inducing pattern-triggered immunity (PTI) by injecting bacterial elicitor flg22, or pathogenicity-mutant Pseudomonas syringae pv. syringae 61 hrcC- bacteria; but not after inoculations with compatible or incompatible pathogens at the time of PTI onset. In this study, we demonstrate that the antibacterial effect of the AS mix is general, as growth of several Gram-negative and -positive phytopathogenic bacteria was characteristically inhibited. The inhibition of bacterial metabolism by the AS mix was rapid, shown by the immediate drop of luminescence intensity of P. syringae pv. tomato DC3000 lx strain after addition of AS mix. The mechanism of the bacteriostatic effect was investigated using fluorescent reporter dye assays. SYTOX Green experiments supported others’ previous findings that the AS mix does not result in membrane permeabilization. Moreover, we observed that the mode of action could be depolarization of the bacterial cell membrane, as shown by assays carried out with the voltage sensitive dye DIBAC4(3). Conclusions Level of free acetosyringone is elevated during plant PTI responses in tobacco leaves (N. tabacum and N. benthamiana). When combined with hydrogen peroxide and peroxidase (AS mix), components of the mix act synergistically to inhibit bacterial metabolism and proliferation rapidly in a wide range of plant pathogens. This effect is related to depolarization rather than to permeabilization of the bacterial cell membrane. Similar AS mixture to the in vivo model might form locally at sites of invading bacterial attachment to the plant cells and the presence of acetosyringone might have an important role in the inhibition of bacterial proliferation during PTI.

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Evaluation of Antibacterial Properties of Chimeric Bovine Lactoferrin Peptide for Inhibition of Food and Plant Pathogens

International Journal of Infection ◽

10.5812/iji.104594 ◽

2020 ◽

Vol 7 (2) ◽

Author(s):

Hamid Sarhadi ◽

Mohammad Hasan Jahandar ◽

Abbas Tanhaeian

Keyword(s):

Listeria Monocytogenes ◽

Antimicrobial Peptides ◽

Salmonella Typhimurium ◽

Pseudomonas Syringae ◽

Plant Pathogens ◽

Antibacterial Properties ◽

Antimicrobial Activities ◽

Inhibitory Effect ◽

Bovine Lactoferrin ◽

Pathogenic Microorganisms

: The resistance of microorganisms to conventional antibiotics has prompted researchers to produce new antimicrobial compounds. Antimicrobial peptides can be alternatives to chemical antibiotics. Antimicrobial peptides are produced approximately by all living organisms to fight infection. Lactoferrin is an iron glycoprotein that plays an important role in the immune system. Lactoferricin and lactoferrampine have stronger antimicrobial activities than lactoferrin. In this study, we investigated the inhibitory effects of a combination of chimeric bovine lactoferricin and lactoferrampine on microorganisms including Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Enterococcus faecalis, and Listeria monocytogenes, as well as plant pathogens including Pseudomonas syringae pv. syringae, Pseudomonas viridiflava, Xanthomonas translucens, Xanthomonas perforans, Erwinia amylovora, Pectobacterium carotovorum, and Agrobacterium tumefaciens. The results showed that chimeric bovine lactoferrin had a good inhibitory effect against pathogenic microorganisms and plant pathogens. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) for pathogenic microorganisms Salmonella typhimurium and Listeria monocytogenes were 7.562 µg/mL and 15.125 µg/mL, respectively, which showed the highest sensitivity to chimeric bovine lactoferrin. The MIC and MBC for plant pathogens were 0.497 µg/mL and 0.997 µg/mL, respectively, which were related to Pseudomonas syringae pv. syringae and it showed the highest sensitivity to chimeric bovine lactoferrin.

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Limacus flavus yellow slug: bioactive molecules in the mucus

10.1101/2021.05.06.442857 ◽

2021 ◽

Author(s):

Patricia Yumi Hayashida ◽

Pedro Ismael Silva Junior

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Water Solubility ◽

Micrococcus Luteus ◽

Chemical Characterization ◽

New Drugs ◽

Bioactive Molecules ◽

Resistant Bacteria ◽

Drug Resistant Bacteria ◽

Wide Range

Background: Snails and slugs were used as a treatment for many health problems therefore ancient times. Since the antimicrobial resistance became a major global thread, antimicrobial peptides have been considered as a potential source for development of new drugs, especially for drug-resistant bacteria. Nowadays reports confirm that the mucous secretions have antimicrobial, antiviral and antifungal properties. Methods: The present study has the objective to characterize and evaluate antimicrobial peptides of Limacus flavus mucus. The mucus was obtained by thermal shock and submitted to RP-HPLC. Fractions were used to perform the antimicrobial activity and hemolytic assays, electrophoresis (SDS-Page Gel) and submitted to mass spectrometry (LC-MS / MS). Identification and characterization was performed by PeaksX+ software. The physicochemical parameters were evaluated with bioinformatics tools, which predicted water solubility, iso-electric point, charge net and its primary structure. Results: Three fractions were isolated from the mucus of L. flavus and presented antifungal and antibacterial activity. The mucus showed greater inhibition for filamentous fungi (Aspergillus niger), yeast (Cryptococcus neoformans), Gram positive bacteria (Bacillus subtilis, Micrococcus luteus) and Gram negative bacteria (Enterobacter cloacae). These fractions also did not show hemolytic activity for human blood cells (erythrocytes). Fractions sequences were identified and presents Mw <3kDa, WLGH, DLQW, YLRW, respectively. Conclusion: This study revealed three antimicrobial peptides of L. flavus mucus with a wide range of antimicrobial activity and its physic-chemical characterization. Keywords: Limacus flavus, mucus, slug, antimicrobial peptide, bioactive molecules, resistance, microorganisms.

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Discovery of novel antimicrobial peptides: A transcriptomic study of the sea anemone Cnidopus japonicus

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720018400061 ◽

2018 ◽

Vol 16 (02) ◽

pp. 1840006 ◽

Cited By ~ 4

Author(s):

Ekaterina N. Grafskaia ◽

Nadezhda F. Polina ◽

Vladislav V. Babenko ◽

Daria D. Kharlampieva ◽

Pavel A. Bobrovsky ◽

...

Keyword(s):

Antimicrobial Activity ◽

Antimicrobial Peptides ◽

Search Algorithm ◽

Sea Anemone ◽

Amino Acid Sequences ◽

Innate Immune ◽

Bacterial Strains ◽

Wide Range ◽

Structural Peculiarities ◽

Living Organisms

As essential conservative component of the innate immune systems of living organisms, antimicrobial peptides (AMPs) could complement pharmaceuticals that increasingly fail to combat various pathogens exhibiting increased resistance to microbial antibiotics. Among the properties of AMPs that suggest their potential as therapeutic agents, diverse peptides in the venoms of various predators demonstrate antimicrobial activity and kill a wide range of microorganisms. To identify potent AMPs, the study reported here involved a transcriptomic profiling of the tentacle secretion of the sea anemone Cnidopus japonicus. An in silico search algorithm designed to discover toxin-like proteins containing AMPs was developed based on the evaluation of the properties and structural peculiarities of amino acid sequences. The algorithm revealed new proteins of the anemone containing antimicrobial candidate sequences, and 10 AMPs verified using high-throughput proteomics were synthesized. The antimicrobial activity of the candidate molecules was experimentally estimated against Gram-positive and -negative bacteria. Ultimately, three peptides exhibited antimicrobial activity against bacterial strains, which suggests that the method can be applied to reveal new AMPs in the venoms of other predators as well.

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Morphological and molecular characterisation of Streptomyces spp. which suppress pathogenic fungi

African Crop Science Journal ◽

10.4314/acsj.v28i4.6 ◽

2020 ◽

Vol 28 (4) ◽

pp. 555-566

Author(s):

N. Goredema ◽

T. Ndowora ◽

R. Shoko ◽

E. Ngadze

Keyword(s):

Antimicrobial Activity ◽

Fusarium Oxysporum ◽

Aspergillus Flavus ◽

Plant Pathogens ◽

Morphological Characteristics ◽

Molecular Characteristics ◽

Penicillium Italicum ◽

Streptomyces Species ◽

Genus Streptomyces ◽

Wide Range

Streptomyces species are aerobes and chemoorganotrophic bacteria. These microorganisms produce a wide range of industrially significant compounds, specifically antibiotics and anti fungal substances. The objective of this study was to characterise soil-borne Streptomyces isolates using morphological and molecular traits in order to identify them to species level, and leverage from their potential to suppress the growth of Aspergillus flavus, Fusarium oxysporum and Penicillium italicum. Twenty-seven soil-borne putative Streptomyces, which elicited comprehensive antimicrobial activity against Aspergillus flavus, Fusarium oxysporum and Penicillium italicum, in a previous study, were evaluated. On the basis of morphology, the bacteria resembled the genus Streptomyces. Initially, colonies phenotypically appeared to have a relatively smooth surface but as growth progressed the bacteria developed a weft of aerial mycelium granular, powdery or velvety in appearance. Bacteria produced a wide variety of pigments which in turn were responsible for the colour of the vegetative and aerial mycelia, colour ranged from white to cream or buff shades and yellow to orange or brown. Microscopic analyses and morphological characteristics generated sub-groups of the isolates and clustered them according to their similarities. One bacterial strain was randomly selected from each cluster and investigated using molecular characteristics. Partial 16S rDNAs from the selected representative isolates from each subgroup, were sequenced and phylogenetic analysis performed. The 16S rDNA sequences of the isolates indicated that they were related to Streptomyces species: S. bungoensis, S. thermocarboxydus, S. corchorusii and S. lasaliensis, that are known secondary metabolite producers possessing antimicrobial activity against plant pathogens.

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Discovery and characterisation of novel circular bacteriocin plantacyclin B21AG from Lactobacillus plantarum B21

10.1101/2020.04.10.035774 ◽

2020 ◽

Cited By ~ 3

Author(s):

Aida Golneshin ◽

Mian Chee Gor ◽

Ben Vezina ◽

Nicholas Williamson ◽

Thi Thu Hao Van ◽

...

Keyword(s):

Antimicrobial Activity ◽

Amino Acid ◽

Lactobacillus Plantarum ◽

De Novo ◽

Peptide Sequencing ◽

Exchange Chromatography ◽

Leader Peptide ◽

Proteolytic Resistance ◽

Wide Range ◽

Circular Bacteriocin

AbstractLactobacillus plantarum B21 isolated from Vietnamese sausage (nem chua) has previously displayed broad antimicrobial activity against gram positive bacteria including foodborne pathogens Listeria monocytogenes and Clostridium perfringens. This study successfully identified the antimicrobial agent as plantacyclin B21AG, a 5668 Da circular bacteriocin demonstrating high thermostability, resistance to a wide range of pH, proteolytic resistance and temporal stability. We report a reverse genetics approach used to identify and characterise plantacyclin B21AG. The bacteriocin was purified from culture supernatant by a short process consisting of concentration, n-butanol extraction and cation exchange chromatography. A de novo peptide sequencing using LC-MS/MS techniques identified two putative peptide fragments which were mapped to the genome of Lactobacillus plantarum B21. This revealed an ORF corresponding to a putative circular bacteriocin with a 33-amino acid leader peptide and 58-amino acid mature peptide found on native plasmid pB21AG01. The corresponding gene cluster, consisted of seven genes associated with post-translational circularisation, immunity and secretion. The robust nature of plantacyclin B21AG, its antimicrobial activity and associated machinery for cyclisation make it an interesting biotechnological target for further development, and application as a food-safe antimicrobial.

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Apoptotic Cell Death is a Common Response to Pathogen Attack in Oats

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.10.1000 ◽

2002 ◽

Vol 15 (10) ◽

pp. 1000-1007 ◽

Cited By ~ 51

Author(s):

Nan Yao ◽

Satoshi Imai ◽

Yasuomi Tada ◽

Hitoshi Nakayashiki ◽

Yukio Tosa ◽

...

Keyword(s):

Cell Death ◽

Pseudomonas Syringae ◽

Pathogenic Bacteria ◽

Plant Pathogens ◽

Magnaporthe Grisea ◽

Apoptotic Cell ◽

Chromatin Condensation ◽

Apoptotic Cell Death ◽

Wide Range ◽

Pathogen Attack

We have examined the characteristics of cell death induced by pathogen infection in oats with respect to following hallmark apoptotic features: DNA laddering, chromatin condensation, and electron microscopic-bterminal deoxynucleotidyl transferase-mediated UTP end labeling positive response. A wide range of plant pathogens representing different levels of parasitism in susceptible and resistant interactions were used for the inocula, which include (i) an obligate parasite, Puccinia coronata f. sp. avenae (the crown rust fungus); (ii) a facultative biotroph parasite, Magnaporthe grisea (the blast fungus); (iii) pathogenic bacteria, Pseudomonas syringae pv. atropurpurea and P. syringae pv. coronafaciens (the halo or stripe blights of oats); and (iv) Ryegrass mottle virus. Surprisingly, any of the pathogens used induced most of the apoptotic features in oat cells at and around the infection sites, indicating that apoptotic cell death is a common phenomenon in oats during pathogen attack. The localization and the timing of apoptotic cell death during a course of infection were, however, quite different depending on the interactions (compatible or incompatible) and the pathogens (fungi, bacteria, or viruses). Possible roles of apoptotic cell death in the susceptible and resistant interactions are discussed.

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