Delta endotoxin inhibits Rb+ uptake, lowers cytoplasmic pH and inhibits a K+-ATPase inManduca sexta CHE cells

Abstract

Delta endotoxin, a 68 kilodalton protein isolated from Bacillus thuringiensis spp. Kurstaki, is a potent entomocidal agent that alters a K+ current across midgut tissue of many phytophagous insects. This toxin completely inhibited the vanadate-sensitive 86Rb+ uptake and mimicked the vanadate-induced decrease in cytosolic pH in a cell line (CHE) originating from Manduca sexta embryonic tissue. The toxin also inhibited a K+-sensitive-ATPase in the plasma membranes isolated from these cells. Using the K+-sensitive-ATPase substrate p-nitrophenyl phosphate, delta endotoxin was found to have a Ki of 0.4 microM. These data suggest that the toxin inhibits a K+-ATPase responsible for 86Rb+ uptake in the CHE cells. The relationship between the toxin inhibition of K+-ATPase and toxin-altered K+ current is discussed.

Analysis of Cry8Ka5-binding proteins from Anthonomus grandis (Coleoptera: Curculionidae) midgut

“…Although Cry1Ac is nontoxic to vertebrates, inhibition of V-ATPases from mammals has been described in dogs and rats (English and Cantley, 1986;Rani and Balaraman, 1997). In insects, where they are located in goblet cells, English and Cantley (1985) reported inhibition of a K + sensitive ATPase in Manduca sexta by a d-endotoxin.…”

The mode of action of Bacillus thuringiensis crystal proteins

“…Such an inhibition would explain the decreased K + concentration in the goblet cell cavity from isolated midguts of M. sexta fed dissolved crystal proteins (Gupta et al, 1985). Although toxic fragments were found to inhibit in vitro a membrane bound K + -ATPase isolated from cells of the embryonic M. sexta cell line CHE (English & Cantley, 1985) and a purified (Na + -K+)-ATPase from dog kidney (English & Cantley, 1986), these results were obtained with cells and enzymes not occurring in the larval midgut and the crystal protein fragments used in these studies appeared to be trypsin sensitive. These facts render the observed inhibition of ATPases as an unlikely step in the mechanism of toxic action.…”

Mechanisms of inducible resistance against Bacillus thuringiensis endotoxins in invertebrates

“…After insertion into the membrane bilayer, the toxin interferes with the activity of K + / amino acid transport, inhibiting K + and amino acid assimilation in the gut lumen, causing imbalance in pH, ion and other macromolecules in the gut, and finally leads to the lysis of the cell and insect death (Harvey & Wolfersberger, 1979;English & Cantley, 1985;Knowles & Ellar, 1987;Reuveni & Dunn, 1991;Wolfersberger, 1991;Leonardi et al, 1997;Tran et al, 2001).…”