Permeabilization of Fungal Membranes by Plant Defensins Inhibits Fungal Growth

ABSTRACT We used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi. Membrane permeabilization induced by plant defensins in Neurospora crassa was biphasic, depending on the plant defensin dose. At high defensin levels (10 to 40 μM), strong permeabilization was detected that could be strongly suppressed by cations in the medium. This permeabilization appears to rely on direct peptide-phospholipid interactions. At lower defensin levels (0.1 to 1 μM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the inhibition of fungal growth. Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from Raphanus sativus, failed to induce cation-resistant permeabilization in N. crassa. Dm-AMP1, a plant defensin from Dahlia merckii, induced cation-resistant membrane permeabilization in yeast (Saccharomyces cerevisiae) which correlated with its antifungal activity. However, Dm-AMP1 could not induce cation-resistant permeabilization in the Dm-AMP1-resistantS. cerevisiae mutant DM1, which has a drastically reduced capacity for binding Dm-AMP1. We think that cation-resistant permeabilization is binding site mediated and linked to the primary cause of fungal growth inhibition induced by plant defensins.

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Specific Binding Sites for an Antifungal Plant Defensin from Dahlia (Dahlia merckii) on Fungal Cells Are Required for Antifungal Activity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.1.54 ◽

2000 ◽

Vol 13 (1) ◽

pp. 54-61 ◽

Cited By ~ 79

Author(s):

Karin Thevissen ◽

Rupert W. Osborn ◽

David P. Acland ◽

Willem F. Broekaert

Keyword(s):

Antifungal Activity ◽

Plasma Membranes ◽

Specific Binding ◽

Plant Defensin ◽

Wild Type ◽

Whole Cells ◽

Specific Binding Sites ◽

Plant Defensins ◽

Related Plant ◽

Wild Type Yeast

Dm-AMP1, an antifungal plant defensin from seeds of dahlia (Dahlia merckii), was radioactively labeled with t-butoxycarbonyl-[35S]-L-methionine N-hydroxy-succinimi-dylester. This procedure yielded a 35S-labeled peptide with unaltered antifungal activity. [35S]Dm-AMP1 was used to assess binding on living cells of the filamentous fungus Neurospora crassa and the unicellular fungus Saccharomyces cerevisiae. Binding of [35S]Dm-AMP1 to fungal cells was saturable and could be competed for by preincubation with excess, unlabeled Dm-AMP1 as well as with Ah-AMP1 and Ct-AMP1, two plant defensins that are highly homologous to Dm-AMP1. In contrast, binding could not be competed for by more distantly related plant defensins or structurally unrelated antimicrobial peptides. Binding of [35S]Dm-AMP1 to either N. crassa or S. cerevisiae cells was apparently irreversible. In addition, whole cells and microsomal membrane fractions from two independently obtained S. cerevisiae mutants selected for resistance to Dm-AMP1 exhibited severely reduced binding affinity for [35S]Dm-AMP1, compared with wild-type yeast. This finding suggests that binding of Dm-AMP1 to S. cerevisiae plasma membranes is required for antifungal activity of this protein.

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In vitro and in vivo antifungal activity of buparvaquone against Sporothrix brasiliensis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00699-21 ◽

2021 ◽

Author(s):

Luana Pereira Borba-Santos ◽

Thayná Lopes Barreto ◽

Taissa Vila ◽

Kung Darh Chi ◽

Fabiana dos Santos Monti ◽

...

Keyword(s):

Antifungal Activity ◽

Mammalian Cells ◽

Plasma Membranes ◽

Galleria Mellonella ◽

Fungal Growth ◽

Sporothrix Brasiliensis ◽

First Choice ◽

Altered Cell

Sporotrichosis has become an important zoonosis in Brazil and Sporothrix brasiliensis is the primary species transmitted by cats. Improvement of animal treatment will help control and limit the spread and geographic expansion of sporotrichosis. Accordingly, buparvaquone, an antiprotozoal hydroxynaphthoquinone agent marketed as Butalex®, was evaluated in vitro and in vivo against feline-borne isolates of S. brasiliensis . Buparvaquone inhibited in vitro fungal growth at concentrations 4-fold lower than itraconazole (the first-choice antifungal used for sporotrichosis) and was 408 times more selective for S. brasiliensis than mammalian cells. Yeasts treated with a subinhibitory concentration of buparvaquone exhibited mitochondrial dysfunction, ROS and neutral lipid accumulation, and impaired plasma membranes. Also, scanning electron microscopy images revealed buparvaquone altered cell wall integrity and induced cell disruption. I n vivo experiments in a Galleria mellonella model revealed that buparvaquone (single dose of 5 mg/kg) is more effective than itraconazole against infections with S. brasiliensis yeasts. Combined, our results indicate that buparvaquone has a great in vitro and in vivo antifungal activity against S. brasiliensis , revealing the potential application of this drug as an alternative treatment for feline sporotrichosis.

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The Antifungal Activity of RsAFP2, a Plant Defensin from Raphanus sativus, Involves the Induction of Reactive Oxygen Species in Candida albicans

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000104753 ◽

2007 ◽

Vol 13 (4) ◽

pp. 243-247 ◽

Cited By ~ 110

Author(s):

An M. Aerts ◽

Isabelle E.J.A. François ◽

Els M.K. Meert ◽

Qiu-Tian Li ◽

Bruno P.A. Cammue ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Candida Albicans ◽

Antifungal Activity ◽

Raphanus Sativus ◽

Reactive Oxygen ◽

Oxygen Species ◽

Plant Defensin

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Characterization and antifungal activity of a plant peptide expressed in the interaction between Capsicum annuum fruits and the anthracnose fungus

Bioscience Reports ◽

10.1042/bsr20192803 ◽

2019 ◽

Vol 39 (12) ◽

Author(s):

Álan Chrisleyr Maracahipes ◽

Gabriel Bonan Taveira ◽

Lorran Yves Sousa-Machado ◽

Olga Lima Tavares Machado ◽

Rosana Rodrigues ◽

...

Keyword(s):

Antifungal Activity ◽

Capsicum Annuum ◽

Protein Profile ◽

Membrane Permeabilization ◽

Ros Production ◽

Growth Inhibition Assay ◽

Plant Defensins ◽

Related Plant ◽

Basic Peptides

Abstract Plant defensins are low molecular weight basic peptides ranging from 5 to 7 kDa, with capacity of inhibiting various pathogens, including fungi. They are present in different tissues of plants, including floral parts and fruits of Capsicum sp. The IIF48 extract, present in immature fruits of Capsicum annuum inoculated with C. gloeosporioides, was able to inhibit up to 100% growth ‘in vitro’ of the fungus Colletotrichum gloeosporioides. The main objective of this work was the purification and antifungal activity characterization of a defense-related plant defensin-like isolated of the IIF48 immature fruits extract. The IIF48 extract was subjected to HPLC purification and 13 fractions were obtained, followed by a tricine gel electrophoresis to obtain the protein profile. The different fractions were submitted to a growth inhibition assay against C. gloeosporioides fungus. Fraction 7 (F7) was the most active causing 73% inhibition. Because of the higher F7 activity and the presence of only a peptide of approximately 5 kDa this fraction was subjected to N-terminal sequencing. F7 fraction was carried out plasma membrane permeabilization assays, induction of intracellular ROS production analysis and investigated mitochondrial membrane potential. The F7 fraction showed significant inhibitory activity on the tested fungus, besides promoting membrane permeabilization, induction of endogenous ROS production in Colletotrichum cells and impairing mitochondrial functionality. The first 18 amino acid sequence of the F7 fraction peptide suggests homology to plant-like defensin and was named IIFF7Ca. We also concluded that IIFF7Ca peptide has an effective antimicrobial action against the fungus C. gloeosporioides.

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Antifungal Activity of Lactobacillus plantarum ZZUA493 and Its Application to Extend the Shelf Life of Chinese Steamed Buns

Foods ◽

10.3390/foods11020195 ◽

2022 ◽

Vol 11 (2) ◽

pp. 195

Author(s):

Shanshan Zhao ◽

Xiangmei Hao ◽

Fengyuan Yang ◽

Yuan Wang ◽

Xiaomiao Fan ◽

...

Keyword(s):

Lactic Acid ◽

Antifungal Activity ◽

Shelf Life ◽

Lactobacillus Plantarum ◽

Fungal Growth ◽

Microtiter Plate ◽

Food Preservation ◽

Phenyllactic Acid ◽

Strong Activity ◽

Fungal Growth Inhibition

Lactic acid bacteria (LAB) can produce many kinds of antifungal substances, which have been widely proven to have antifungal activity. In this study, 359 strains of LAB were screened for antifungal activity against Aspergillus niger (A. niger) using the 96-well microtiter plate method, and three showed strong activity. Of these, ZZUA493 showed a broad-spectrum antifungal ability against A. niger, Aspergillus oryzae, Trichoderma longibrachiatum, Aspergillus flavus and Fusarium graminearum. ZZUA493 was identified as Lactobacillus plantarum. Protease treatment, the removal of hydrogen peroxide with catalase and heat treatment had no effect on the antifungal activity of the cell-free supernatant (CFS) of ZZUA493; organic acids produced by ZZUA493 appeared to have an important role in fungal growth inhibition. The contents of lactic acid, acetic acid and phenyllactic acid in the CFS tended to be stable at 48 h, and amounted to 28.5, 15.5 and 0.075 mg/mL, respectively. In addition, adding ZZUA493, as an ingredient during their preparation, prolonged the shelf life of Chinese steamed buns. Overall, ZZUA493 appears to have good potential as a fungal inhibitor for food preservation.

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Dual Effects of Plant Steroidal Alkaloids on Saccharomyces cerevisiae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00289-06 ◽

2006 ◽

Vol 50 (8) ◽

pp. 2732-2740 ◽

Cited By ~ 67

Author(s):

Veronika Simons ◽

John P. Morrissey ◽

Maita Latijnhouwers ◽

Michael Csukai ◽

Adam Cleaver ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Antifungal Activity ◽

Secondary Metabolites ◽

Induced Defense ◽

Substantial Reduction ◽

Plant Secondary Metabolites ◽

Defense Responses ◽

Membrane Permeabilization ◽

Ergosterol Biosynthesis ◽

Induced Defense Responses

ABSTRACT Many plant species accumulate sterols and triterpenes as antimicrobial glycosides. These secondary metabolites (saponins) provide built-in chemical protection against pest and pathogen attack and can also influence induced defense responses. In addition, they have a variety of important pharmacological properties, including anticancer activity. The biological mechanisms underpinning the varied and diverse effects of saponins on microbes, plants, and animals are only poorly understood despite the ecological and pharmaceutical importance of this major class of plant secondary metabolites. Here we have exploited budding yeast (Saccharomyces cerevisiae) to investigate the effects of saponins on eukaryotic cells. The tomato steroidal glycoalkaloidα -tomatine has antifungal activity towards yeast, and this activity is associated with membrane permeabilization. Removal of a single sugar from the tetrasaccharide chain of α-tomatine results in a substantial reduction in antimicrobial activity. Surprisingly, the complete loss of sugars leads to enhanced antifungal activity. Experiments with α-tomatine and its aglycone tomatidine indicate that the mode of action of tomatidine towards yeast is distinct from that of α-tomatine and does not involve membrane permeabilization. Investigation of the effects of tomatidine on yeast by gene expression and sterol analysis indicate that tomatidine inhibits ergosterol biosynthesis. Tomatidine-treated cells accumulate zymosterol rather than ergosterol, which is consistent with inhibition of the sterol C24 methyltransferase Erg6p. However, erg6 and erg3 mutants (but not erg2 mutants) have enhanced resistance to tomatidine, suggesting a complex interaction of erg mutations, sterol content, and tomatidine resistance.

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Antifungal activity of plant essential oils and selected pseudomonas strains against Phomopsis theicola

Pesticidi i fitomedicina ◽

10.2298/pif1702121s ◽

2017 ◽

Vol 32 (2) ◽

pp. 121-127

Author(s):

Mira Starovic ◽

Danijela Ristic ◽

Goran Aleksic ◽

Snezana Pavlovic ◽

Mehmet Özcan ◽

...

Keyword(s):

Antifungal Activity ◽

Essential Oils ◽

Plant Protection ◽

Fungal Growth ◽

Plant Growth Promoting Rhizobacteria ◽

Plant Protection Products ◽

Phytopathogenic Fungus ◽

Plant Essential Oils ◽

Fungal Growth Inhibition

Development of natural plant protection products as an alternative to synthetic fungicides is of significant importance regarding the environment. This study was carried out with an objective to investigate in vitro antifungal activities of several essential oils extracted from oregano, basil, myrtle and Turkish pickling herb, and the plant growth-promoting rhizobacteria in the genus Pseudomonas, against the phytopathogenic fungus Phomopsis theicola. Microdilution methods were used to determine the minimum inhibitory concentrations (MIC) of selected antimicrobial essential oils (EOs). All EOs exhibited significant levels of antifungal activity against the tested fungal isolates. The oregano EO was found the most potent one (MIC - 5.5 ?g/mL), followed by basil (MIC - 75.0?g/mL), myrtle (MIC - 775 ?g/mL) and Turkish pickling herb (MIC - 7750 ?g/mL). Inhibition of Ph. theicola mycelial growth was observed for all tested Pseudomonas spp. strains. K113 and L1 strains were highly effective and achieved more than 60% of fungal growth inhibition using the overnight culture and more than 57% inhibition by applying cell-free supernatants of both strains. A future field trial with K113 and L1 cultures and cell-free supernatants, containing extracellular metabolites toward Ph. theicola, will estimate their effectiveness and applicability as an alternative to chemical protection of apple trees.

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A defensin with highly potent antipathogenic activities from the seeds of purple pole bean

Bioscience Reports ◽

10.1042/bsr20090004 ◽

2009 ◽

Vol 30 (2) ◽

pp. 101-109 ◽

Cited By ~ 30

Author(s):

Peng Lin ◽

Jack Ho Wong ◽

Tzi Bun Ng

Keyword(s):

Gel Filtration ◽

Antifungal Drugs ◽

Fungal Resistance ◽

Plant Defensin ◽

Sytox Green ◽

Plant Defensins ◽

Congo Red Staining ◽

Chromatographic Media ◽

Erythrocyte Membrane Permeability ◽

Hepatoma Hepg2

A 5443 Da peptide with sequence homology to defensins was purified from purple pole beans (Phaseolus vulgaris cv. ‘Extra-long Purple Pole bean’). This peptide was isolated by adsorption on an affinity chromatographic medium Affi-Gel Blue gel and ion-exchange chromatographic media SP-Sepharose (sulfopropyl-Sepharose) and Mono S and by gel filtration on Superdex peptide. The peptide inhibited mycelial growth in Mycosphaerella arachidicola, Helminthosporium maydis, Fusarium oxysporum, Verticillium dahliae, Rhizoctonia solani, Candida albicans and Setosphaeria turcica with an IC50 of 0.8, 0.9, 2.3, 3.2, 4.3, 4.8 and 9.8 μM respectively. Its antifungal potency was higher than that of the plant defensin coccinin (IC50>50 μM). It induced membrane permeabilization in C. albicans as evidenced by SYTOX Green uptake, but did not affect erythrocyte membrane permeability. It inhibited growth in M. arachidicola by inducing chitin accumulation at hyphal tips as was shown by Congo Red staining. The antifungal activity was pH stable and thermostable. The peptide inhibited the proliferation of hepatoma (HepG2), breast cancer (MCF7), colon cancer (HT29) and cervical cancer (SiHa) cells but not that of human embryonic liver (WRL68) cells. Its anti-HepG2 activity (IC50=4.1±0.8 μM, n=3) was higher than that of another plant defensin, gymnin (IC50>50 μM). Its anti-MCF7 activity (IC50=8.3±0.3 μM, n=3) was similar to that of other plant defensins. It reduced the activity of HIV-1 reverse transcriptase with an IC50 of 0.5±0.1 μM, n=3, much more potently than other plant defensins (IC50>40 μM). There is the possibility of using the purple pole bean defensin for producing antifungal drugs and/or transgenic plants with fungal resistance.

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