Antifungal Mechanism of Vip3Aa, a Vegetative Insecticidal Protein, against Pathogenic Fungal Strains.

Seong-Cheol Park,Jin-Young Kim,Su-Hyang Yoo,Hyosuk Son,Mi-Kyeong Jang,Seong-Eun Mun,Hye Song Lim,Jong-Kook Lee,Jung Ro Lee

doi:10.3390/antibiotics10121558

Abstract

Discovering new antifungal agents is difficult, since, unlike bacteria, mammalian and fungal cells are both eukaryotes. An efficient strategy is to consider new antimicrobial proteins that have variety of action mechanisms. In this study, a cDNA encoding Bacillus thuringiensis Vip3Aa protein, a vegetative insecticidal protein, was obtained at the vegetative growth stage; its antifungal activity and mechanism were evaluated using a bacterially expressed recombinant Vip3Aa protein. The Vip3Aa protein demonstrated various concentration- and time-dependent antifungal activities, with inhibitory concentrations against yeast and filamentous fungi ranging from 62.5 to 125 µg/mL and 250 to 500 µg/mL, respectively. The uptake of propidium iodide and cellular distributions of rhodamine-labeled Vip3Aa into fungal cells indicate that its growth inhibition mechanism involves its penetration within cells and subsequent intracellular damage. Furthermore, we discovered that the death of Candida albicans cells was caused by the induction of apoptosis via the generation of mitochondrial reactive oxygen species and binding to nucleic acids. The presence of significantly enlarged Vip3Aa-treated fungal cells indicates that this protein causes intracellular damage. Our findings suggest that Vip3Aa protein has potential applications in the development of natural antimicrobial agents.

Highlights

Bacillus thuringiensis is the most widely used biological insecticide for controlling insect pests, primarily Lepidoptera and Coleoptera species [1]
While studying the insecticidal activity of the Vip3Aa protein, we developed an interest in the characteristics and novel functions of the Vip3Aa protein, which are similar to those of common antimicrobial peptides (AMPs) [22]
The full-length cDNA of Vip3Aa was cloned into the pET28a vector and expressed in E. coli BL21(DE3)

Summary

Introduction

Bacillus thuringiensis is the most widely used biological insecticide for controlling insect pests, primarily Lepidoptera and Coleoptera species [1]. Among the insecticidal proteins secreted by B. thuringiensis, the parasporal inclusion crystal (Cry) toxins are the most wellknown and widely used. Cry toxins accumulate during sporulation in the B. thuringiensis strain, resulting in a crystalline inclusion with a variety of morphologies. When pests consume Cry toxins, the alkaline digestive tract of the insects denatures the insoluble crystals, making them soluble and susceptible to digestion by proteases found in the pest gut, which releases the toxin from the crystal [2]. The Cry toxin penetrates the cell membrane of the pest digestive tract, paralyzing the gut and forming a pore. The pest eventually stops eating and starves to death [2,3]

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