Mistletoe viscotoxins induce membrane permeabilization and spore death in phytopathogenic fungi

Abstract

Viscotoxins (Vts) are basic peptides expressed in mistletoe leaves, seeds and stems which have been shown to be cytotoxic to mammalian cells. The aim of this study was to analyse whether Vts were able to control and/or inhibit the growth of phytopathogenic fungi to obtain a clue to their biological function. Incubation of two Vt isoforms, VtA(3) and VtB, at a final concentration of 10 micro M resulted in a complete blockage of the germination of spores from three different pathogenic fungi. It was also shown that lower concentrations than 10 micro M of VtA(3) and VtB inhibit their mycelial growth in a dose-dependent manner. The protein dose required to inhibit the growth of Fusarium solani and Sclerotinia sclerotiorum to a 50% was between 1.5 and 3.75 micro M, which represents a potent activity. No significant differences in the antifungal potency for each Vt isoform, either VtA(3) and VtB, were observed, although they have been shown to exert differential cytotoxicity on mammalian cells. It was also demonstrated that Vts act as fungicidal compounds. To explore the basis of the antifungal activity the ability of VtA(3) to induce changes in membrane permeability and on the oxidative status of F. solani spores was analysed. By using a specific fluorescent probe on intact spores, it was demonstrated that VtA(3) produces rapid changes in fungal membrane permeability. It also induces H(2)O(2) production verified by a histochemical staining. The data presented in this study support a direct role of Vts in the plant defence determined by their lethal effect on fungal pathogens.

Antimicrobial activity and mechanism of action of a thionin‐like peptide from Capsicum annuum fruits and combinatorial treatment with fluconazole against Fusarium solani

“…The viscotoxin VtA 3 from Viscum album and other thionins were also able to inhibit germination of F. solani conidia at 50 μg mL −1 , although this inhibition was analyzed for 16 h of incubation. As for mycelial growth, 7.5 μg mL −1 of VtA 3 was necessary to achieve 50% inhibition, and this potent activity was also verified against Sclerotinia sclerotiorum cells . Studies have shown that the inhibitory concentration of AMPs varies widely and is dependent on the tested fungus.…”

“…The same research group showed that ∼50 μg mL −1 of VtA 3 is able to modify the permeability of membranes of F. solani conidia using the probe Sytox green. These authors also observed that with only 5 min of incubation, fungal conidia were permeabilized and prolonged exposure of conidia to VtA3 progressively increased the percentage of permeabilized cells, reaching 70% in 30 min . Taveira et al demonstrated that Ca Thi was able to cause permeabilization in all six important Candida species tested, suggesting that permeabilization may be the first in a sequence of cellular changes caused by Ca Thi that lead to the death of the microorganism.…”

Sensitization of K562 Leukemia Cells to Doxorubicin by the Viscum album Extract

“…Today, mistletoe extract [V. album extract (VAE)] therapy is among the most commonly used and thoroughly studied complementary treatments in Europe (Beuth and Schierholz, 2007;Fasching et al, 2007;Molassiotis et al, 2005Molassiotis et al, , 2006aMolassiotis et al, , 2006b. Many kinds of metabolites have been isolated from the European mistletoe, and some are synthesized by the host and incorporated into V. album (Cordero et al, 1993;Giudici et al, 2004). Viscotoxins and amphipathic and basic polypeptides (Schaller et al, 1998); tyramine, phenylethylamine, choline, and acetylcholine (Hegnauer, 1966); aminoalkaloids (Amer et al, 2012); phenolic acids and flavonoids (Deliorman et al, 2002;Fukunaga et al, 1987;Luczkiewicz et al, 2001;Vicas et al, 2011); phenylpropanoids (Panossian et al, 1998;Wagner et al, 1986); terpenoids (Fukunaga et al, 1987); long-chain fatty acids and hydrocarbons as well as trace amounts of volatile components including trans-abergamotene, trans-b-farnesene, loliolide, and vomifoliol (Cebović et al, 2008); and oligosaccharides and polysaccharides (Arda et al, 2003;Jordan and Wagner, 1986) belong to active metabolites isolated from the mistletoe.…”

New Antimicrobial Agents of Plant Origin

“…Most of those with proven antimicrobial activity were obtained from monocot species of the grass (Poaceae) family (Oard et al 2004;Fujimura et al 2005) or dicot plants of the mistletoe (Viscaceae) family (Giudici et al 2004). However, related sequences are present in many other plants suggesting that many more thionins with antimicrobial properties remain to be discovered (Silverstein et al 2007).…”

“…This structure is stabilized by three conserved disulfide bridges, which are supplemented by a fourth one in some groups. The toxicity of thionins is associated with an increase in membrane permeability that causes depolarization and cell death (Giudici et al 2004;BerrocalLobo et al 2009). Their positive charges allow them to bind negatively charged phospholipid heads while a hydrophobic region could interact with the lipid tails.…”