Bacillus thuringiensis pore-forming toxins trigger massive shedding of GPI-anchored aminopeptidase N from gypsy moth midgut epithelial cells.
The insecticidal Cry proteins produced by Bacillus thuringiensis strains are pore-forming toxins (PFTs) that bind to the midgut brush border membrane and cause extensive damage to the midgut epithelial cells of susceptible insect larvae. Force-feeding B. thuringiensis PFTs to Lymantria dispar larvae elicited rapid and massive shedding of a glycosylphosphatidylinositol (GPI)-anchored aminopeptidase N (APN) from midgut epithelial cells into the luminal fluid, and depletion of the membrane-anchored enzyme on the midgut epithelial cells. The amount of APN released into the luminal fluid of intoxicated larvae was dose- and time-dependent, and directly related to insecticidal potency of the PFTs. The induction of toxin-induced shedding of APN was inhibited by cyclic AMP and MAPK kinase (MEK) inhibitors PD98059 and U0126, indicating that signal transduction in the MEK/ERK pathway is involved in the regulation of the shedding process. APN released from epithelial cells appears to be generated by the action of a phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of the GPI anchor based upon detection of a cross-reacting determinant (CRD) on the protein shed into the luminal fluid. Alkaline phosphatase was also released from the gut epithelial cells, supporting the conclusion that other GPI-anchored proteins are released as a consequence of the activation PI-PLC. These observations are the basis of a novel and highly sensitive tool for evaluating the insecticidal activity of new Cry proteins obtained though discovery or protein engineering.
Valaitis
Algimantas P
AP
USDA Forest Service, U.S. Department of Agriculture, 359 Main Road, Delaware, OH 43015, USA. avalaitis@fs.fed.us
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000494:pharmacology*; D018826:CD13 Antigens / Q000378:metabolism*; D004731:Endotoxins / Q000494:pharmacology*; D004847:Epithelial Cells / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000187:drug effects / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000494:pharmacology*; D007814:Larva / Q000187:drug effects / Q000378:metabolism; D009036:Moths / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction; D018675:Toxicity Tests; D010738:Type C Phospholipases / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D017261:Glycosylphosphatidylinositols; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases; D018826:CD13 Antigens