RESULTS

Table 1

Solubilization of Bacillus thuringiensis subsp. medellin Cyt1Ab1 toxin in buffers of different pH.

Buffer

pH

Obtained concentration (g/l)

Na2HPO4 50 mM, KH2PO4 50 mM

5.0

0.28

Na2HPO4 50 mM, KH2PO4 50 mM

6.0

0.30

Na2HPO4 50 mM, KH2PO4 50 mM

7.0

0.34

Tris 50 mM

8.0

0.38

Na2CO3 50 mM, NaHCO3 50 mM

9.0

0.50

Na2CO3 50 mM, NaHCO3 50 mM

10.0

0.67

Na2CO3 50 mM, NaHCO3 50 mM

10.6

1.16

Na2CO3 50 mM, NaHCO3 50 mM

11.0

1.51

NaOH 50 mM, EDTA 10 mM

12.0

2.33

 

Solubility of the Cyt1Ab1 toxin was higher at the alkaline buffers. The maximal solubilization was obtained in the buffer of pH 12.0 which produced 2.33 g/ml of solubilized toxin. This toxin is preferentially solubilized at higher pH values.

 

Figure 1

Effect of pH in proteolytic processing of Cyt1Ab1 toxin

The proteolysis of the Cyt1Ab1 toxin was tested on buffers of different pH. The toxin showed a little activation at pH 10.6 and 11 with C. quinquefasciatus gut extract, trypsin and chymotrypsin, a complete activation of toxin at defined fragments were seen at pH 12.

 

Figure 2

Time course assay for activation of Cyt1Ab1 toxin.

 

The action of C. quinquefasciatus gut extracts in the processing of Cyt1Ab1 was observed after 1 min of initiated the digestion, being most evident 10 min later and finally converted to 25 kDa fragment. For trypsin, the fragment of 26 kDa appeared at 14 h of activation. The processing by chymotrypsin generated intermediaries ending with formation of 25 kDa which appeared at 24 h.

 

Figure 3

In vivo processing of Cyt1Ab1 crystal toxin by third instar C. Quinquefasciatus larvae

 

As observed in the Western blot of the mosquito C. quinquefasciatus guts intoxicated with the Cyt1Ab1 toxin, the processing of the parasporal inclusion generated a 25 kDa fragment, similar in size to those observed during the in vitro digestion.

 

Table 2

Mosquitocidal and hemolytic activities of Cyt1Ab1 inclusions, solubilized and proteolytically processed against third instar C. quinquefasciatus larvae and sheep red blood cells.

Toxin

Half Lethal Concentration

(LC50) g /ml

Half Hemolytic Dose

(HD50) g/ml

Cyt1Aa1crystal inclusions

Cyt1Ab1crystal inclusions

0.74 (0.5-0.9)*

1.3 (0.9 -1.7)

------

Not hemolytic

Cyt1Ab1 Solubilized

Not toxic

4.5 (3.4-6.1)

Cyt1Ab1 activated by C. quinquefasciatus gut extracts

Not toxic

1.4 (0.4-2.0)

Cyt1Ab1 activated by trypsin

Not toxic

2.2 (1.6-3.3)

Cyt1Ab1 activated by chymotrypsin

Not toxic

1.9 (1.5-2.6)

*Values represent the 95% confidence limits, determined by Probit analysis.

The alkali-solubilized toxin showed an HD50 of 4.5 g/ml in sheep red blood cells, this activity was potenciated when the toxin was in vitro processed by mosquito larvae gut extracts, trypsin and chymotrypsin. Mosquitocidal activity assay of the Cyt1Ab1 against third instar C. quinquefasciatus larvae showed a LC50 of 1.3 g/ml. In contrast, the soluble and processed toxin did not show any toxicity in the bioassays evaluated, even at the concentration of 25 g/ml.