Activation of Bacillus thuringiensis delta-endotoxin.

Title: Activation of Bacillus thuringiensis delta-endotoxin.
Authors: Milne, Ross.
Date: 1996
Abstract: Bacillus thuringiensis (Bt) produces a proteinaceous parasporal crystalline inclusion which is pathogenic to insect larvae. The insecticidal activities of crystals from different subspecies of Bt are highly specific for larvae within a particular insect order and in some cases for specific larvae within a family. Lepidopteran-active Bt crystals are composed of 130 kDa $\delta$-endotoxin (protoxin molecules, covalently linked by disulfide bridges). On ingestion by susceptible larvae, the $\delta$-endotoxin is converted to a 58-68 kDa toxin by the action of the high pH gut juice (pH $\sim$ 10-11) containing proteolytic enzymes and other proteins which associate with the toxin. One of the objectives of the research was to determine the role of enzymes/proteins in the spruce budworm (Choristoneura fumiferana) gut in determining the activity/specificity of a toxin. The gut juice of the spruce budworm larvae was found to contain a single trypsin-like serine protease (CFT-1) which was responsible for the activation of the $\delta$-endotoxin. CFT-1 was purified from gut juice by a combination of size exclusion and ion exchange chromatography. Some characteristics, such as molecular mass, N-terminal and active site sequences, charge and substrate specificity were similar to mammalian pancreatic trypsins. However, differences were observed in the burst kinetics and pH dependence of the catalysis where the apparent ionization constant of the active site histidine was over 1 pH unit higher than in the mammalian serine proteases. Another significant difference was that CFT-1 was stable for long periods of time at pH values as high as 11 whereas mammalian trypsins rapidly autolyse at pH values greater than 8. Inhibition of CFT-1 in gut juice prevented activation of the $\delta$-endotoxin. However, gut juice activation produced a slightly smaller toxin than the CFT-1 alone. It was observed during the digestion of $\delta$-endotoxins with gut juices that toxin was precipitated. This precipitate was shown to be caused by a protein in the gut juice. This protein has a molecular mass of 75 kDa and anionic character based on ion-exchange chromatography. A weak elastase-like activity was also shown to be associated with the precipitating protein. When the aggregate of toxin and gut juice protein was dissociated, it was observed that smaller toxin fragments were generated indicating that the toxin had been further proteolysed. The precipitation and degradation of toxin by gut juice may explain the observation that, when gut juice is used to activate the $\delta$-endotoxin, the in vitro recovery of toxin is much lower than expected. Therefore precipitation may play a role in determining the toxicity towards various insect larvae. Evidence had been obtained that DNA was associated with the $\delta$-endotoxin and toxin in vitro after the usual purification procedures and this DNA appeared to play an important role in the activation of the $\delta$-endotoxin. A study was undertaken to determine whether DNA found in purified Bt crystal preparations was also associated with crystals in vivo. The results obtained from photomicrography of Bt during its stages of development showed that DNA condenses in the region where the parasporal crystal forms. These results provided further evidence that the DNA found to be associated with purified crystals in vitro was not artifactual.
CollectionTh├Ęses, 1910 - 2010 // Theses, 1910 - 2010
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