Specificity of Bacillus thuringiensis var. colmeri insecticidal delta-endotoxin is determined by differential proteolytic processing of the protoxin by larval gut proteases

Area of Science:

• Microbiology
• Insect Toxicology
• Biochemistry

Background:

• Bacillus thuringiensis (Bt) produces crystal delta-endotoxins toxic to insects.
• The Bt var. colmeri serotype 21 delta-endotoxin exhibits toxicity against both lepidopteran and dipteran larvae.
• Understanding the activation and specificity of these toxins is crucial for developing targeted biopesticides.

Purpose of the Study:

• To investigate the differential activation and insect specificity of the delta-endotoxin from Bacillus thuringiensis var. colmeri.
• To elucidate the molecular basis for the selective toxicity against lepidopteran and dipteran insects.
• To characterize the activated toxin fragments and their origins.

Main Methods:

• Solubilization of native crystal delta-endotoxin under alkaline reducing conditions.
• Activation of the protoxin using trypsin and gut extracts from Pieris brassicae (lepidopteran) and Aedes aegypti (dipteran) larvae.
• In vitro toxicity assays using insect cell lines and in vivo bioassays.
• Gel electrophoresis and peptide mapping to analyze protoxin conversion and toxin structure.

Main Results:

• Activation with Aedes aegypti gut extract yielded a dipteran-specific toxin (52-kDa), while trypsin or P. brassicae enzymes produced a lepidopteran-specific toxin (55-kDa).
• In vitro and in vivo bioassays confirmed the insect-specific toxicity of the activated toxins.
• Peptide mapping and two-step activation suggested the dipteran toxin is derived from the lepidopteran toxin via mosquito-specific enzymes.
• All toxins cross-reacted with antiserum to Bt var. kurstaki P1 toxin, and this antiserum neutralized their cytotoxicity.

Conclusions:

• The 130-kDa protoxin of Bt var. colmeri is processed into distinct insect-specific toxins by specific insect gut proteases.
• Aedes aegypti gut enzymes convert the protoxin to a dipteran-specific toxin, potentially via modification of a lepidopteran-specific intermediate.
• These findings highlight the potential for developing targeted Bacillus thuringiensis-based insecticides by exploiting differential toxin activation mechanisms.