Isolation and characterization of the insecticidal toxin binding site from the receptor BT-R(1) of Manduca sexta

Crystal protein inclusion bodies produced by the gram-positive bacterium Bacillus thuringiensis (BT) are attractive biopesticide alternatives to chemical-based pesticides. These proteins are produced at the time the bacterium sporulates and exhibit insecticidal activity toward three orders of insects, Lepidoptera (moths and butterflies), Diptera (mosquitoes and blackflies) and Coleoptera (beetles). Parasitic worms, from the class Nematoda, also are susceptible to these proteins. Insect mortality is mediated through the binding of toxin to a high affinity receptor, BT-R{dollar}sb1{dollar} in Manduca sexta, causing disruption in the natural function of the midgut cells and death. Despite the current attention being directed toward this pathology, little is known about the natural function of the receptors involved in toxicity.; The purpose of this dissertation is to define and characterize the region of the BT-R{dollar}sb1{dollar} receptor protein to which the Cry1A toxins (Cry1Aa, Cry1Ab and Cry1Ac) bind. In vitro translation of the 172-kDa receptor, from the cDNA clone, in rabbit reticulocyte lysates (RRL) or E. coli produces a protein that binds these Cry1A radiolabeled toxins on ligand blots. A 169-amino acid peptide fragment, called BHBT-R{dollar}sb1,{dollar} excised from the 233-amino acid C-terminus of BT-R{dollar}sb1{dollar} and translated in either expression system also binds the radiolabeled Cry1A toxins on ligand blots. Binding of Cry1Ab to this fragment can be specifically blocked with addition of unlabeled Cry1Aa or Cry1Ab giving a Kd value of 39 nM for this receptor-ligand interaction. The 169-amino acid fragment is the only segment of BT-R{dollar}sb1{dollar} to which Cry1Aa, Cry1Ab or Cry1Ac binds, indicating that this fragment is the single toxin binding site in the receptor BT-R{dollar}sb1.{dollar}; BHBT-R{dollar}sb1{dollar} protein expressed in E. coli was purified using a nickel affinity column specific for a his-tag attached to the N-terminus of the protein. The pure protein was used in a bioassay to demonstrate that this fragment of the natural receptor, BT-R{dollar}sb1,{dollar} could block the insecticidal activity of Cry1Ab in the model insect M. sexta. An LC{dollar}sb{lcub}50{rcub}{dollar} dose of Cry1Ab, 7.5 ng/cm{dollar}sp2,{dollar} fed to the insects in the presence of a 1500-nM excess of BHBT-R{dollar}sb1{dollar} reduced insect mortality to zero. Further, at a dose of 9ng/cm{dollar}sp2{dollar} in the presence of a 1600-nM excess of BHBT-R{dollar}sb1,{dollar} insect mortality was reduced from 90% to 50%. A logistical regression analysis of the bioassay data reveals that the chance for insect survival increases 0.1-0.3% for each additional 1 nM of BHBT-R{dollar}sb1{dollar} that is available to the feeding insect.; The experiments presented in this dissertation establish that the natural receptor BT-R{dollar}sb1{dollar} in M. sexta for the Cry1A toxins examined family contains a single toxin binding site. This binding site has extremely high affinity for these toxins and is located in the last 233 amino acids of the C-terminus of BT-R{dollar}sb1.{dollar} Moreover, the purified peptide fragment, BHBT-R{dollar}sb1,{dollar} can function as an antagonist to Cry1Ab toxin. These types of receptor studies should lead to better understanding of the nature of insecticidal toxins as well as the development of superior biopesticides based on BT toxins.