Changes in protease activity and Cry3Aa toxin binding in the Colorado potato beetle: implications for insect resistance to Bacillus thuringiensis toxins.
Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increased the likelihood for development of insect resistance to this entomopathogen. In this study, we investigated protease activity profiles and toxin-binding capacities in the midgut of a strain of Colorado potato beetle (CPB) that has developed resistance to the Cry3Aa toxin of B. thuringiensis subsp. tenebrionis. Histological examination revealed that the structural integrity of the midgut tissue in the toxin-resistant (R) insect was retained whereas the same tissue was devastated by toxin action in the susceptible (S) strain. Function-based activity profiling using zymographic gels showed specific proteolytic bands present in midgut extracts and brush border membrane vesicles (BBMV) of the R strain not apparent in the S strain. Aminopeptidase activity associated with insect midgut was higher in the R strain than in the S strain. Enzymatic processing of toxin did not differ in either strain and, apparently, is not a factor in resistance. BBMV from the R strain bound approximately 60% less toxin than BBMV from the S strain, whereas the kinetics of toxin saturation of BBMV was 30 times less in the R strain than in the S strain. However, homologous competition inhibition binding of (125)I-Cry3Aa to BBMV did not reveal any differences in binding affinity (K(d) approximately 0.1 microM) between the S and R strains. The results indicate that resistance by the CPB to the Cry3Aa toxin correlates with specific alterations in protease activity in the midgut as well as with decreased toxin binding. We believe that these features reflect adaptive responses that render the insect refractory to toxin action, making this insect an ideal model to study host innate responses and adaptive changes brought on by bacterial toxin interaction.
Loseva
Olga
O
Center for Biotechnology and Bioinformatics and the Department of Molecular and Cell Biology, The University of Texas at Dallas, PO Box 830688, FO3.1, Richardson, TX 75083-0688, USA.
Ibrahim
Mohamed
M
Candas
Mehmet
M
Koller
C Noah
CN
Bauer
Leah S
LS
Bulla
Lee A
LA
D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D001517:Coleoptera / Q000187:drug effects / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004064:Digestive System / Q000201:enzymology; D010450:Endopeptidases / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism*; D006460:Hemolysin Proteins; D007305:Insecticide Resistance; D007306:Insecticides / Q000378:metabolism*; D007814:Larva; D008024:Ligands; D010572:Pest Control, Biological; D014020:Tissue Extracts
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; D008024:Ligands; D014020:Tissue Extracts; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D010450:Endopeptidases; D000626:Aminopeptidases; D003546:Cysteine Endopeptidases