Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens.

Department of Entomology, Iowa State University, 418 Science II, Ames, IA 50011-3222, USA.
Tissue and Cell (Impact Factor: 1.05). 01/2008; 39(6):431-43. DOI: 10.1016/j.tice.2007.08.003
Source: PubMed

ABSTRACT ScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, which digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus (AcMLF9.ScathL) expressing ScathL kills larvae of the tobacco budworm Heliothis virescens significantly faster than the wild type virus and triggers melanization and tissue fragmentation shortly before death. The tissue fragmentation was assumed to be a direct consequence of basement membrane degradation by ScathL. The goal of this study was to investigate the tissue specificity of ScathL when expressed by AcMLF9.ScathL using light, transmission and scanning electron microscopy. Baculovirus expression of ScathL resulted in damage to the basement membrane overlying the midgut, fat body and muscle fibers in larvae infected with AcMLF9.ScathL, but not in larvae infected with the control virus AcMLF9.ScathL.C146A or wild type virus AcMNPV C6. Injection of recombinant ScathL and high levels of baculovirus-mediated expression of ScathL resulted in complete loss of the gut. Extensive damage to the basement membrane mediated by ScathL likely resulted in loss of viability of the underlying tissue and subsequent death of the insect. These results confirm the conclusion of an earlier study (Philip, J.M.D., Fitches, E., Harrison, R.L., Bonning, B.C., Gatehouse, J.A., 2007. Characterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs. Insect Biochem. Mol. Biol. 37, 589-600) of the remarkable specificity of this protease.

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    • "Second, baculoviruses may exit the midgut using tracheal elements; thus, basal lamina surrounding midgut-associated tracheae in orally infected T. ni with viruses expressing AcFGF was examined by transmission electron microscopy. In the presence of AcFGF, the basal lamina of tracheal cells associated with the midgut was disorganized; instead of a thin uniform sheath surrounding the cells, it appeared fragmented (Means and Passarelli, 2010) and reminiscent of that observed when larvae were infected with viruses expressing basal laminae degrading enzymes (Tang et al., 2007). Analysis with a virus lacking Acfgf showed some rearrangement of tracheal cell basal lamina but not as drastically as with the virus carrying Acfgf. "
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    Virology 02/2011; 411(2):383-92. DOI:10.1016/j.virol.2011.01.009 · 3.28 Impact Factor
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    • "Interestingly, it has been shown that a recombinant baculovirus expressing the S. peregrina cathepsin L (SCathL) induces melanization of larvae (Harrison and Bonning, 2001). In this case, melanization was not directly activated by SCathL but was postulated to be induced by the SCathL-dependent degradation of the basement membrane and recruitment of hemocytes at the sites of injury, because damaged tissues are most probably recognized as non-self (Tang et al., 2007; Li et al., 2008). "
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    Biological Chemistry 05/2009; 390(5-6):493-502. DOI:10.1515/BC.2009.061 · 2.69 Impact Factor
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    • "In preliminary experiments, it was noted that the pericardial cells, dorsal aorta and alary muscles appeared to be melanized in some larvae killed by injection of ScathL. We examined these tissues in the larvae killed by ScathL injection using a light microscope (Nikon SMZ1500) and by scanning electron microscopy (SEM) as described previously (Tang et al., 2007). "
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    ABSTRACT: ScathL is a cathepsin L-like cysteine protease derived from the flesh fly Sarcophaga peregrina that functions in basement membrane (BM) remodeling during insect development. A recombinant baculovirus expressing ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus. Here, we show that the occurrence of larval melanization prior to death was closely associated with the onset of high cysteine protease activity of ScathL in the hemolymph of fifth instars infected with AcMLF9.ScathL, but not with AcMLF9.ScathL.C146A, a recombinant baculovirus expressing a catalytic site mutant of ScathL. Fragmented fat body, ruptured gut and malpighian tubules, and melanized tracheae were observed in AcMLF9.ScathL-infected larvae. Phenoloxidase activity in hemolymph was unchanged, but the pool of prophenoloxidase was significantly reduced in virus-infected larvae and further reduced in AcMLF9.ScathL-infected larvae. The median lethal dose (LD(50)) for purified ScathL injected into fifth-instar H. virescens was 11.0 microg/larva. ScathL was also lethal to adult pea aphids, Acyrthosiphon pisum with a similar loss of integrity of the gut and fat body. Injection with purified ScathL.C146A or bovine trypsin at 20 microg/larva did not produce any effect in either insect. These results illustrate the potent insecticidal effects of ScathL cysteine protease activity and the potential for use of ScathL in development of insect resistant transgenic plants when combined with an appropriate delivery system.
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