|Harrison, Robert - Bob|
Submitted to: Molecular Insect Science International Symposium Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 5/19/2006
Publication Date: 5/20/2006
Citation: Harrison, R.L., Huarong, L., Tang, H., Bonning, B.C. 2006. Mechanism of insecticidal action of a basement membrane-degrading protease. Molecular Insect Science International Symposium Proceedings. Tuscon,AZ May 20,2006- May 24, 2006.
Technical Abstract: ScathL is a cathepsin-L like cysteine protease that digests components of the basement membrane during insect metamorphosis. On the basis that basement membranes constitute a barrier to dissemination of baculoviruses within the insect host, a recombinant baculovirus (AcMLF9.ScathL) that expresses ScathL was constructed. AcMLF9.ScathL kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild type virus and triggers melanization of larvae shortly before death. To elucidate the mechanism of insecticidal action of ScathL, we are testing five hypotheses; (1) ScathL results in death of the host insect independent of cysteine protease activity: By testing a baculovirus expressing a catalytic site mutant of ScathL, we have shown that cysteine protease activity is required for the insecticidal activity of ScathL. (2) ScathL causes damage to tissues other than the basement membrane: At high concentrations, ScathL results in damage to the gut. However, this damage may be a direct effect of ScathL proteolytic activity, or an indirect effect resulting from lysis of cells underlying disrupted basement membrane. Fragmentation of internal tissues occurs in melanized AcMLF9.ScathL-infected larvae. (3) ScathL damages the basement membrane barrier to virus dissemination allowing more rapid spread or altered tissue tropism of the virus: Damage to the basement membrane that overlies the gut, allows for more rapid movement of budded virus into the hemocoel. (4) ScathL activates the immune response in an unregulated manner by acting directly on prophenoloxidase (PPO): Although ScathL activity was significantly higher in melanized larvae, there were no differences in phenoloxidase activity between AcMLF9.ScathL and control treatments. ScathL does not activate PPO directly in vitro. (5) ScathL degrades components of the basement membrane that results in death independent of the immune system: We are using polydnavirus-derived immunosuppressive genes to separate the effects of melanization and the associated production of toxic free radicals, from the potentially lethal impact of basement membrane damage on physiological processes.