Synergism of Bacillus thuringiensis toxins by a fragment of a toxin-binding cadherin

• Published in: Proceedings of the National Academy of Sciences - PNAS 2007-08, Vol.104 (35), p.13901-13906
• Main Authors: Chen, Jiang, Hua, Gang, Jurat-Fuentes, Juan Luis, Abdullah, Mohd Amir, Adang, Michael J
• Format: Article
• Language: English
• Subjects: Animals; Bacillus thuringiensis; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Bacterial Proteins - pharmacology; bacterial toxins; Bacterial Toxins - chemistry; Bacterial Toxins - metabolism; Bacterial Toxins - pharmacology; binding capacity; binding proteins; Bioassay; Biochemistry; Biological Sciences; Cadherins; crystal proteins; E coli; Endotoxins - chemistry; Endotoxins - metabolism; Endotoxins - pharmacology; Escherichia coli; Escherichia coli - genetics; Hemolysin Proteins - chemistry; Hemolysin Proteins - metabolism; Hemolysin Proteins - pharmacology; Insect control; Insect larvae; insect proteins; insecticidal properties; insecticidal proteins; intestinal mucosa; Kinetics; Larva - drug effects; Lepidoptera; Manduca - drug effects; Manduca - growth & development; Manduca sexta; Microvilli; Midgut; Mortality; Peptide Fragments - chemistry; Peptide Fragments - pharmacology; Peptides; Pesticides; Protein Denaturation; Protein Folding; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Spectroscopy; Spectrum analysis; synergism; Toxicity; Toxins; transmembrane proteins
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0706011104

The insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) are broadly used to control insect pests with agricultural importance. The cadherin Bt-R₁ is a binding protein for Bt Cry1A toxins in midgut epithelia of tobacco hornworm (Manduca sexta). We previously identified the Bt-R₁ region most proximal to the cell membrane (CR12-MPED) as the essential binding region required for Cry1Ab-mediated cytotoxicity. Here, we report that a peptide containing this region expressed in Escherichia coli functions as a synergist of Cry1A toxicity against lepidopteran larvae. Far-UV circular dichroism and ¹H-NMR spectroscopy confirmed that our purified CR12-MPED peptide mainly consisted of β-strands and random coils with unfolded structure. CR12-MPED peptide bound brush border membrane vesicles with high affinity (Kd = 32 nM) and insect midgut microvilli but did not alter Cry1Ab or Cry1Ac binding localization in the midgut. By BIAcore analysis we demonstrate that Cry1Ab binds CR12-MPED at high (9 nM)- and low (1 μM)-affinity sites. CR12-MPED-mediated Cry1A toxicity enhancement was significantly reduced when the high-affinity Cry1A-binding epitope (¹⁴¹⁶GVLTLNIQ¹⁴²³) within the peptide was altered. Because the mixtures of low Bt toxin dose and CR12-MPED peptide effectively control target insect pests, our discovery has important implications related to the use of this peptide to enhance insecticidal activity of Bt toxin-based biopesticides and transgenic Bt crops.