title: Immunology of the protein toxin of bacterial insect pathogens
Creators: name: Pendleton, Ian Roderick
Publisher: name: University of Glasgow
Year: 1967
Subjects: subject: Microbiology, molecular biology.

description: The toxicity for certain insect larvae of the parasporal protein crystal of the bacterium Bacillus thuringiensis is well documented. There is evidence that the crystal is a protoxin which is activated to form a toxin by digestion in the insect gut. It is believed that difference in host-specificity and toxicity of strains of B. thuringiensis may be reflected by differences in the composition of the crystal toxins. A method was developed for the isolation of pure crystals from mature cultures of B. thuringiensis consisting of 50% spores and 50% crystals. The method is essentially a 2-stage process; in the first stage a flotation technique removes.60% of the spores from the mixture and in the second stage a phase separation technique completes the purification. 60-65% of the crystals were recovered from the culture at a purity of 99%. Preparations of crystals from 23 strains of B. thuringiensis were used to prepare antisera. A selection from these antisera enabled an antigenic analysis of solutions of the crystals from 94 strains of B. thuringiensis to be carried out using the Ouchterlony gel diffusion technique. Although crystals stimulated production of only one antibody, solutions of many of the crystals contained several antigens. The 94 strains were divisible into 32 groups on the basis of the antigens of crystal solutions. Altogether 11 different crystal antigens were distinguished in the strains of B. thuringiensis investigated. Crystals from 93 strains of B. thuringiensis were digested by the gut juice of larvae of Pieris brassicae. Ouchterlony gel diffusion indicated that digestion of the crystals resulted in loss of 4 out of the 11 antigens detected in crystal solutions; other antigens were more resistant to digestion. Some antigens not detectable in the crystal solutions appeared in the digests. The antigenic composition of crystal digests was less varied than that of crystal solutions. 2 The soluble products of digestion of crystals of the Berliner strain of B. thuringiensis were investigated by gel filtration. Two fractions were separated: fraction C1 was composed of material of molecular weight 200,000 and fraction C2 was composed of material of molecular weight 5,000-10,000. Fraction C1 was not toxic on injection into the haemocoel of larvae of P. brassicae although fraction C2 was toxic by this route and so is believed to contain the activated toxin. Digestion experiments also indicated that fraction C1 was the precursor of fraction C2 in the digestion process. 3 antigens were detected in fraction C2 of the crystal digest. A gel filtration technique was designed to investigate the association of the antigens present in fraction C2 with particular molecules. Gel filtration of mixtures of monospecific antisera and fraction C2 indicated that the 3 antigens in fraction C2 were present as different antigenic sites on one peptide molecule. These mixtures were fed to larvae of P. brassicae. The results indicated that one of the 3 antigens was necessary for the toxic function of fraction 02. Neutralisation of the other 2 antigens individually had no apparent effect on toxicity; neutralisation of both of these antigens together reduced toxicity. Evidence is presented to suggest that the protoxin crystal is activated by insect gut juices by digestion to yield a single toxic fraction of comparatively low Molecular weight.