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.
USDA Forest Service, U.S. Department of Agriculture, 359 Main Road, Delaware, OH 43015, USA. avalaitis@fs.fed.us
Algimantas P
AP
Valaitis
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