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

Abstract

RsAFP2 (Raphanus sativus antifungal peptide 2), an antifungal plant defensin isolated from seed of R. sativus, interacts with glucosylceramides (GlcCer) in membranes of susceptible yeast and fungi and induces membrane permeabilization and fungal cell death. However, using carboxyfluorescein-containing small unilamellar vesicles containing purified GlcCer, we could not observe permeabilization as a consequence of insertion of RsAFP2 in such vesicles. Therefore, we focused on a putative RsAFP2-induced signaling cascade downstream of RsAFP2-binding to GlcCer in fungal membranes. We show that RsAFP2 induces reactive oxygen species (ROS) in Candida albicans wild type in a dose-dependent manner, but not at all in an RsAFP2-resistant ΔgcsC. albicans mutant that lacks the RsAFP2-binding site in its membranes. These findings indicate that upstream binding of RsAFP2 to GlcCer is needed for ROS production leading to yeast cell death. Moreover, the antioxidant ascorbic acid blocks RsAFP2-induced ROS generation, as well as RsAFP2 antifungal activity. These data point to the presence of an intracellular plant defensin-induced signaling cascade, which involves ROS generation and leads to fungal cell growth arrest.

Permeabilization of Fungal Hyphae by the Plant Defensin NaD1 Occurs through a Cell Wall-dependent Process

“…This supports the idea that NaD1-induced permeabilization requires the presence of a receptor. This finding is also consistent with the finding that the plant defensin RsAFP2 is unable to permeabilize liposomes containing glucosylceramide, a known binding partner of the peptide (29). In contrast, LL-37, CP-29, and BMAP-28 caused rapid calcein release due to disruption of the SUVs.…”

Antimicrobial peptides

“…This suggestion is supported by elevated expression of PR-6 in metchnikowin plants compared with that in wild type individuals. 99 The higher level of reactive oxygen species down to expression of antimicrobial peptides 24,82,99,100 supports the notion that these peptides play some part of their roles by modulating the redox milieu, which might ultimately lead to cell death.…”

“…Furthermore, Psd1 regulates interkinetic nuclear migration from Synthesis (S) to Mitosis (M) phase of cell cycle in rat retinal neuroblasts. 81 Additionally, some defensins such as RsAFP2 82 and NaD1 24 modulate the intracellular signaling cascades, specifically, induction of reactive oxygen species (ROS) that upon accumulation may cause membrane damage, organelle breakdown and eventually cell death. Pertinent support for this modulatory activity was found in certain mutants of F. graminearum disrupted in some MAPKKK(s) genes that were hypersensitive to alfalfa MsDef1, barrel clover MtDef2, and radish RsAFP2.…”

“…Though it has been reported that the degree of antimicrobial activity is not dependent on the AMP production level in transgenic plants, 106 most of the publications furnish clear evidence for peptides' doseeffect activity. 82,100,107,108 Some investigators emphasize the possibility of synergism among different AMPs. [109][110][111] In vivo data for different antibacterial peptides show that defensins and linear peptides work in-synergy, 112,113 as exemplified by synergism between LL-37 and human b-defensin HBD-2.…”

Purification, biochemical characterization, and antimicrobial activity of a new lipid transfer protein from Coffea canephora seeds

“…Induction of the endogenous production of ROS in various fungi treated with 200 µg/mL Cc-LTP2 after the growth inhibition assay was evaluated using the fluorescent dye 2',7'-dichlorofluorescein diacetate (Calbiochem; EMD), as described by Aerts et al (2007) with some modifications. Incubation was performed as described in the SYTOX Green uptake assay section.…”