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Toxicity, membrane binding and uptake of the Sclerotinia sclerotiorum agglutinin (SSA) in different insect cell lines

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Abstract

The fungal lectin purified from Sclerotinia sclerotiorum, further referred to as Sclerotinia sclerotiorum agglutinin or SSA, possesses insecticidal activity against important pest insects such as pea aphids (Acyrthosiphon pisum). This paper aims at a better understanding of its activity at cellular level. Therefore, different insect cell lines were treated with SSA. These cell lines were derived from different tissues and represent the three major orders of insects important in agriculture: CF-203 (midgut Choristoneura fumiferana, Lepidoptera), GUTAW1 (midgut, Helicoverpa zea, Lepidoptera), High5 cells (ovary, Trichoplusia ni, Lepidoptera), Sf9 (ovary cells from Spodoptera frugiperda, Lepidoptera), S2 (hemocyte, Drosophila melanogaster, Diptera), and TcA (whole body, Tribolium castaneum, Coleoptera). Although the sensitivity to SSA differs between the cell lines, SSA clearly showed toxicity in all six cell lines with median effect concentrations (EC50) ranging between 9 and 42 μg/ml. An in-depth analysis of the mechanism of uptake in the cells revealed superior amounts of FITC-SSA at the membrane of CF-203 cells compared to Sf9 cells, while a similar small amount of SSA was internalized in both cell lines. Pre-incubation with the clathrin-mediated endocytosis inhibitor phenylarsine oxide inhibited the internalization of SSA into the CF-203 and Sf9 cells with a respective reduction of 6- and 1.7-fold. The data are discussed in relation to the importance of cellular uptake mechanism for SSA binding and cytotoxicity.

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Acknowledgements

The authors acknowledge support of the Research Council of Ghent University (BOF-UGent) and the Fund for Scientific Research-Flanders (FWO-Vlaanderen, Belgium). Ying Shen is a recipient of a doctoral grant from the China Scholarship Council (CSC).

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Authors and Affiliations

  1. Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, 9000, Ghent, Belgium

    Ying Shen, Kristof De Schutter, Tomasz Walski & Guy Smagghe

  2. Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, 9000, Ghent, Belgium

    Ying Shen, Kristof De Schutter, Tomasz Walski & Els J. M. Van Damme

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  1. Ying Shen
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  2. Kristof De Schutter
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  4. Els J. M. Van Damme
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  5. Guy Smagghe
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Correspondence to Guy Smagghe.

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Editor: Tetsuji Okamoto

Electronic supplementary material

Supplementary Fig. 1

Effect of different concentrations of SSA on the six insect cell lines. GUTAW1 (A), CF-203 (B), High5 (C), Sf9 (D), S2 (D) and TcA (F) cells were incubated with different concentrations of SSA for 4 days at 27 °C. Data are presented as mean % total cell viability ± SE, based on 2 biological repeats with each 5 technical replicates. (GIF 33 kb)

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Supplementary Fig. 2

Confocal imaging of CF-203 (A-C) and Sf9 cells (D-F), untreated (A, D) or after a 10-min-incubation with 0.1 μg/ml FITC (B, E) or 10.2 μg/ml of SSA (C, F). (GIF 151 kb)

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Shen, Y., De Schutter, K., Walski, T. et al. Toxicity, membrane binding and uptake of the Sclerotinia sclerotiorum agglutinin (SSA) in different insect cell lines. In Vitro Cell.Dev.Biol.-Animal 53, 691–698 (2017). https://doi.org/10.1007/s11626-017-0176-8

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  • DOI: https://doi.org/10.1007/s11626-017-0176-8

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