Synergistic Activity between Two Antifungal Proteins, the Plant Defensin NaD1 and the Bovine Pancreatic Trypsin Inhibitor

Bleackley, Mark R.; Dawson, Charlotte S.; McKenna, James A.; Quimbar, Pedro; Hayes, Brigitte M. E.; Weerden, Nicole L. van der; Anderson, Marilyn A.

doi:10.1128/msphere.00390-17

Cited by 15 publications

(12 citation statements)

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“…The peptides were tested for antifungal activity toward the filamentous fungi F. graminearum by performing hyphal growth inhibition assays according to the method of Bleackley et al (2017) with slight modifications. Fungal isolates were grown under constant agitation on a carboxymethyl cellulose (CMC) sporulation medium ( Cappellini and Peterson, 1965 ) at 170rpm and 25°C for 5–7days until spores were abundantly produced.…”

Section: Methodsmentioning

confidence: 99%

Peptides Derived From the α-Core and γ-Core Regions of a Putative Silybum marianum Flower Defensin Show Antifungal Activity Against Fusarium graminearum

et al. 2021

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Fusarium graminearum is the etiological agent of Fusarium head blight (FHB), a disease that produces a significant decrease in wheat crop yield and it is further aggravated by the presence of mycotoxins in the affected grains that may cause health problems to humans and animals. Plant defensins and defensin-like proteins are antimicrobial peptides (AMPs); they are small basic, cysteine-rich peptides (CRPs) ubiquitously expressed in the plant kingdom and mostly involved in host defence. They present a highly variable sequence but a conserved structure. The γ-core located in the C-terminal region of plant defensins has a conserved β-hairpin structure and is a well-known determinant of the antimicrobial activity among disulphide-containing AMPs. Another conserved motif of plant defensins is the α-core located in the N-terminal region, not conserved among the disulphide-containing AMPs, it has not been yet extensively studied. In this report, we have cloned the putative antimicrobial protein DefSm2, expressed in flowers of the wild plant Silybum marianum. The cDNA encodes a protein with two fused basic domains of an N-terminal defensin domain (DefSm2-D) and a C-terminal Arg-rich and Lys-rich domain. To further characterize the DefSm2-D domain, we built a 3D template-based model that will serve to support the design of novel antifungal peptides. We have designed four potential antifungal peptides: two from the DefSm2-D α-core region (SmAPα1-21 and SmAPα10-21) and two from the γ-core region (SmAPγ27-44 and SmAPγ29-35). We have chemically synthesized and purified the peptides and further characterized them by electrospray ionization mass spectrometry (ESI-MS) and Circular dichroism (CD) spectroscopy. SmAPα1-21, SmAPα10-21, and SmAPγ27-44 inhibited the growth of the phytopathogen F. graminearum at low micromolar concentrations. Conidia exposure to the fungicidal concentration of the peptides caused membrane permeabilization to the fluorescent probe propidium iodide (PI), suggesting that this is one of the main contributing factors in fungal cell killing. Furthermore, conidia treated for 0.5h showed cytoplasmic disorganization as observed by transmission electron microscopy (TEM). Remarkably, the peptides derived from the α-core induced morphological changes on the conidia cell wall, which is a promising target since its distinctive biochemical and structural organization is absent in plant and mammalian cells.

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Section: Methodsmentioning

confidence: 99%

Peptides Derived From the α-Core and γ-Core Regions of a Putative Silybum marianum Flower Defensin Show Antifungal Activity Against Fusarium graminearum

et al. 2021

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“…15 N labelled NaD1 was produced in Pichia pastoris as described in ( Chen et al, 2006 ) and purified by cation-exchange chromatography and RP-HPLC as described in ( Hayes et al, 2014 ). HXP4 ( Bleackley et al, 2017 ), DmAMP1 ( Osborn et al, 1995 ) and TsD10 were expressed in Pichia pastoris and purified by cation-exchange chromatography and RP-HPLC as described in ( Hayes et al, 2013 , Lay et al, 2012 ).…”

Section: Methodsmentioning

confidence: 99%

“…S. cerevisiae cells were prepared by diluting to an OD 600 = 0.01 and C. albicans cells were diluted to OD 600 = 0.0002 in ½ strength potato dextrose broth (1/2 PDB) (Becton Dickinson). Assays investigating the combined effect of chemical cell wall synthesis inhibitors with defensins were performed as described in ( Bleackley et al, 2017 ). Synergy was assessed using the fractional inhibitory concentration (FIC) equation.…”

Section: Methodsmentioning

confidence: 99%

“…To assess the effect of exogenous 1,3-β-glucan on the activity of NaD1, a survival assay was employed essentially as described in ( Bleackley et al, 2017 ). A two-fold dilution series of laminarin (Sigma) from 50 mg/mL was prepared in MilliQ H 2 O and 10 µL of each concentration plus a no laminarin control were transferred to the wells of a U-bottom 96 well microtitre plate (Greiner Bio-one).…”

Section: Methodsmentioning

confidence: 99%

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The interaction with fungal cell wall polysaccharides determines the salt tolerance of antifungal plant defensins

et al. 2019

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The fungal cell wall is the first point of contact between fungal pathogens and host organisms. It serves as a protective barrier against biotic and abiotic stresses and as a signal to the host that a fungal pathogen is present. The fungal cell wall is made predominantly of carbohydrates and glycoproteins, many of which serve as binding receptors for host defence molecules or activate host immune responses through interactions with membrane-bound receptors. Plant defensins are a large family of cationic antifungal peptides that protect plants against fungal disease. Binding of the plant defensin NaD1 to the fungal cell wall has been described but the specific component of the cell wall with which this interaction occurred was unknown. The effect of binding was also unclear, that is whether the plant defensin used fungal cell wall components as a recognition motif for the plant to identify potential pathogens or if the cell wall acted to protect the fungus against the defensin. Here we describe the interaction between the fungal cell wall polysaccharides chitin and β-glucan with NaD1 and other plant defensins. We discovered that the β-glucan layer protects the fungus against plant defensins and the loss of activity experienced by many cationic antifungal peptides at elevated salt concentrations is due to sequestration by fungal cell wall polysaccharides. This has limited the development of cationic antifungal peptides for the treatment of systemic fungal diseases in humans as the level of salt in serum is enough to inactivate most cationic peptides.

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“…C. parapsilosis (D). Letras diferentes representam diferença significativa entre médias por meio do teste de Tukey (P<0,05).Na literatura há relatos de inibidores de proteases com atividade inibitória frente espécies de Candida(BLEACKLEY et al, 2017) e também especificamente, inibidores de tripsina frente a células planctônicas (DOKKA e DAVULURI, 2014) e inibidores de tripsina com efeito antibiofilme de Candida spp (PATRIOTA et al, 2016)…”

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Morbidade Em Mulheres Por Câncer Colorretal No Estado Do Ceará (2002 a 2013)

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Sales²,

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et al. 2019

As Ciências Da Vida Frente Ao Contexto Contemporâneo 2

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Ciência. 2. Ciências da vida-Pesquisa-Brasil. I. Pereira, Denise. II. Série. CDD 570.9 Elaborado por Maurício Amormino Júnior-CRB6/2422 O conteúdo dos artigos e seus dados em sua forma, correção e confiabilidade são de responsabilidade exclusiva dos autores. 2019 Permitido o download da obra e o compartilhamento desde que sejam atribuídos créditos aos autores, mas sem a possibilidade de alterá-la de nenhuma forma ou utilizá-la para fins comerciais.

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Synergistic Activity between Two Antifungal Proteins, the Plant Defensin NaD1 and the Bovine Pancreatic Trypsin Inhibitor

Cited by 15 publications

References 43 publications

Peptides Derived From the α-Core and γ-Core Regions of a Putative Silybum marianum Flower Defensin Show Antifungal Activity Against Fusarium graminearum

Peptides Derived From the α-Core and γ-Core Regions of a Putative Silybum marianum Flower Defensin Show Antifungal Activity Against Fusarium graminearum

The interaction with fungal cell wall polysaccharides determines the salt tolerance of antifungal plant defensins

Morbidade Em Mulheres Por Câncer Colorretal No Estado Do Ceará (2002 a 2013)

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