Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens.
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. "
ABSTRACT: The mechanisms used by baculoviruses to exit the midgut and cause systemic infection of their insect hosts have been debated for decades. After being ingested, baculoviruses reach the midgut, where several host barriers need to be overcome in order to establish successful infection. One of these barriers is the basal lamina, a presumably virus-impermeable extracellular layer secreted by the epithelial cells lining the midgut and trachea. This review discusses new evidence that demonstrates how these viruses breach the basal lamina and establish efficient systemic infections. The biochemical mechanisms involved in dismantling basal lamina during baculovirus infection may also provide new insights into the process of basal lamina remodeling in invertebrate and vertebrate animals.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). "
ABSTRACT: Parasites have evolved different virulence strategies to manipulate host physiological functions. The parasitoid wasp Cotesia congregata induces developmental arrest and immune suppression of its Lepidopteran host Manduca sexta. In this interaction, a symbiotic virus (C. congregata Bracovirus, CcBV) associated with the wasp is essential for parasitism success. The virus is injected into the host with wasp eggs and virus genes are expressed in host tissues. Among potential CcBV virulence genes, cystatins, which are tight binding inhibitors of C1A cysteine proteases, are suspected to play an important role in the interaction owing to their high level of expression. So far, however, potential in vivo targets in M. sexta are unknown. Here, we characterized for the first time four M. sexta C1A cysteine proteases corresponding to cathepsin L and cathepsin B and two different '26-29 kDa' cysteine proteases (MsCath1 and MsCath2). Our analyses revealed that MsCath1 and MsCath2 are transcriptionally downregulated in the course of parasitism. Moreover, viral Cystatin1 and MsCath1 co-localize in the plasma following parasitism, strongly suggesting that they interact. We also show that parasitism induces a general increase of cysteine protease activity which is later controlled. The potential involvement of cysteine proteases in defense against parasitoids is discussed.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). "
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.Journal of Insect Physiology 06/2008; 54(5):777-89. DOI:10.1016/j.jinsphys.2008.02.008 · 2.50 Impact Factor