pH regulates pore formation of a protease activated Vip3Aa from Bacillus thuringiensis

Thittaya Kunthic; Hirokazu Watanabe; Ryuji Kawano; Yoshikazu Tanaka; Boonhiang Promdonkoy; Min Yao; Panadda Boonserm

doi:10.1016/j.bbamem.2017.08.018

pH regulates pore formation of a protease activated Vip3Aa from Bacillus thuringiensis

Biochim Biophys Acta Biomembr. 2017 Nov;1859(11):2234-2241. doi: 10.1016/j.bbamem.2017.08.018. Epub 2017 Sep 1.

Authors

Thittaya Kunthic¹, Hirokazu Watanabe², Ryuji Kawano², Yoshikazu Tanaka³, Boonhiang Promdonkoy⁴, Min Yao⁵, Panadda Boonserm⁶

Affiliations

¹ Institute of Molecular Biosciences, Mahidol University, Salaya, Phuttamonthon, Nakhon Pathom 73170, Thailand.
² Department of Life Science and Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
³ Laboratory of X-ray Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan; Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan; Japan Science and Technology Agency, PRESTO, Sendai 980-8577, Japan.
⁴ National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Khlong Luang, Pathumthani 12120, Thailand.
⁵ Laboratory of X-ray Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan.
⁶ Institute of Molecular Biosciences, Mahidol University, Salaya, Phuttamonthon, Nakhon Pathom 73170, Thailand. Electronic address: panadda.boo@mahidol.ac.th.

PMID: 28865796
DOI: 10.1016/j.bbamem.2017.08.018

Abstract

Vip3Aa insecticidal protein is produced from Bacillus thuringiensis and exerts a broad spectrum of toxicity against lepidopteran insect species. Although Vip3Aa has been effectively used as part of integrated pest management strategies, the mechanism of the toxin remains unclear. Here, we investigated the effect of pH in a range from 5.0 to 10.0 on the pore-forming activity of the trypsin activated Vip3Aa (actVip3Aa) by in vitro pore-forming assays. Based on calcein release assay, actVip3Aa could permeabilize the artificial neutral liposomes under all the pH tested, except pH10.0. The maximum membrane permeability of actVip3Aa was detected at pH8.0 and the permeability decreased and abolished when exposing to acidic and alkaline conditions, respectively. The planar lipid bilayer experiment revealed that actVip3Aa formed ion channels at pH5.0-8.0 but no current signals were detected at pH10.0, consistent with the observation from calcein release assay. The toxin formed ion channels with a diameter of 1.4nm at pH8.0 and pore size was gradually decreased when reducing the pH. This study provided a view of the molecular mechanism of Vip3Aa by which the pore-forming activity is regulated by pH.

Keywords: Bacillus thuringiensis; Ion channels; Membrane insertion; Planar lipid bilayers; Pore formation; Vip3Aa.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacillus thuringiensis / metabolism
Bacterial Proteins / chemistry
Bacterial Proteins / metabolism
Bacterial Proteins / pharmacology
Bacterial Proteins / physiology*
Cell Membrane Permeability* / drug effects
Hydrogen-Ion Concentration
In Vitro Techniques
Lipid Bilayers / chemistry
Lipid Bilayers / metabolism
Pest Control, Biological
Proteolysis
Trypsin / metabolism

Substances

Bacterial Proteins
Lipid Bilayers
Vip3A protein, Bacillus thuringiensis
Trypsin