BTI 6-endotoxin

Figure 1. SDS-PAGG analysis of alkaline-dissolved Bacillus thuringiensis subspecies kurstaki (BTK) and israelensis (BTI) -endotoxin at 25 yg per track (1) BTK -endotoxin from Biochem Products - US Division (Salsbury Labs., Inc.), (2) BTI 6-endotoxin from Sandoz Inc., (3) BTI (Sandoz) -endotoxin purified by DEAE-anion exchange chromatography, (4) percipitate formed after dialysis of BTI (Sandoz) -endotoxin into pH 4.5 sodium acetate buffer, (5) soluble fraction after dialysis of BTI (Sandoz) -endotoxin into pH 4.5 sodium acetate buffer, (6) BTI (Sandoz) -endotoxin purified by Sephadex G-75 gel filtration chromatography at Rf 1.35, (7) at Rf 1.58, and (8) BTI strain IFC-1 -endotoxin from Biochem Products - US Division (Salsbury Labs., Inc.). S, molecular weights as indicated X1000 for bovine serum albumin (BSA), ovalbumin (OA), trypsin, and myoglobin. Reproduced with permission from Ref. 29. Copyright 1984, Academic Press, Inc.

Table III. Toxicity of partially purified alkaline-solubilized BTI 6-endotoxin fed and injected into A. aegypti and T. n, respectively.

Ug/ml + 1 S.D. with slope of probit analysis in parenthesis. Larvae were incubated in water with BTI 6-endotoxin added. 

The injection of alkaline-dissolved BTI 6-endotoxin led to a number of immediate neuromuscular effects (Table IV) including,... 

Table IV. Symptoms elicited after the injection of alkaline-dissolved BTI 6-endotoxin (Track 2) into mice and insects...

A number of other lines of evidence also suggested that there may be another mode-of-action for BTI poisoning by injection other than its known, general cytolytic activity (3,22-24). Using the appearance of cytosolic lactate dehydrogenase (LDH) in insect hemolymph post-injection as a marker for cytotoxicity (36-37), we found that dissolved BTI 6-endotoxin was a potent cytotoxin. When T. ni, however, were injected with dissolved BTI 5-endotoxin and then incubated at 28, 15, and 9°, there was an increase in the LD50 with a decrease in temperature (Figure 2) but the LDH levels at 3.5 PPM BTI were unaffected by temperature. 

A 25K dalton component was isolated at relatively high purity as determined by SDS-PAGE (Figure 5, Track 3) from alkaline-dissolved BTI 6-endotoxin (Figure 5, Track 1). The... 

Figure 4. Time dependency of nervous activity in the ventral nerve cord of Trichoplusia ni injected with 3.7 PPM alkaline-dissolved BTI 6-endotoxin (Sandoz), with 10 PPM methamidophos (MMP) and with 35 PPM phospholipase-A2 (P-A2). Mechanical sensory stimulation is given at arrow S. The control response was the same as the recording for P-A2. BTI and P-A2 were injected into T ni at their respective LDjq.

Figure 5. SDS-Page analysis of alkaline-dissolved Bacillus thuringiensis israelensis (BTI) 6-endotoxin from Sandoz Inc. at 25 yg per track (1) BTI 6-endotoxin as prepared in Figure 1 (Track 2), (2) soluble fraction after dialysis of BTI 6-endotoxin into pH 4.5 sodium acetate buffer, and (3) 25K component from BTI 6-endotoxin after pH 4.5 percipitation and DEAE-anion exchange chromatography. S, molecular weight markers from top to bottom bovine serum albumin (68K daltons), ovalbumin (43K), and myoglobin (16K).

A partially purified Bacillus thurlnglensis var. israelensls (Bti) 6-endotoxin was used to Immunize rabbits. The antisera obtained have an improved specificity towards the mosquito larvacidal activity of the toxin, as opposed to antiserum raised when the whole crystal was used as immunogen. Using a two step/indirect ELISA (enzyme linked immunosorbent assay) procedure developed in our laboratory, fourteen experimental formulations were tested, and the results were compared with bioassays. An average of 69.1 international units 20% c.v. was found to associate with each ug of toxin detected by the ELISA. Our data indicate that when toxin specific antisera are available, Immunoassays can be used to predict the biological activity of Bti samples with reasonable accuracy. 

C. BTI (Sandoz) was further purified by DEAE-Cellulose (DE-52, Whatman, 30 ml) in 0.025 M Tris-HCL (pH 8.00), eluted with a 10 h, 100 ml, 0.0-0.5 M NaCl linear gradient by acid precipitation where the DEAE elutant was dialyzed into 0.05 H sodium acetate (pH 4.5) and the percipitate removed by centrifugation and by Sephadex 6-75 super fine (Pharmacia) gel permeation chromatography (95 x 1.3 cm i.d. column) in 0.025 M sodium phosphate (pH 8.0). Alkaline-dissolved and partially purified 6-endotoxin was analyzed by 12.5% SDS-PAGE (.28), stained with Coomassie brilliant blue (Figure 1). 

The alkaline-dissolved 6-endotoxin of BTI (Sandoz, Track 2) was toxic by injection to all animals tested except Tenebrio molitor (Coleoptera Tenebrionidae) (Table I). Mice,... 

Table I. Injected toxicity of aikaline-solubilized BTI (Sandoz, Track 2) and BTK (Salsbury, Track 1) 6-endotoxin.

The basis for this difference is unknown but these species differences could be useful in the elucidation of the mechanism for toxicity. BTI toxicity by injection also was not peculiar to the Sandoz strain but was also noted for the IFC-1 strain from Salsbury (Table III). By contrast the alkaline-dissolved 6-endotoxin of BTK (Track 1) showed no toxicity when injected into the same species (Table I). Obvious fundamental differences exist between the dissolved 6-endotoxin of BTI and BTK. 

The 6-endotoxin of B. . israelensis (H-14) is not as clearly understood as that of BTk. The parasporal crystal of BTi contains several proteins with molecular weights ranging from 26,000 to over 100,000d (9). Several authors have reported that the 26,000d protein has insecticidal activity (j , 11, 12). However, other reports suggest that the toxin is 66,000d, (13, 1 ). Because BTi has hemolytic and cytolytic activities in addition to insecticidal activities (15) the possibility exists that multiple toxin proteins exist in the crystal. However, evidence from genetics... 

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