| Biopesticides based on the bacterium Bacillus thuringiensis (BT) have attracted wide attention as safe alternatives to chemical pesticides. There is a family of protein toxins synthesized by BT that are insecticidal specifically to lepidopteran, dipteran and coleopteran insects which include moths, mosquitoes and beetles, respectively. Despite broad interest in these toxins, little is known about the molecular mechanism(s) of their toxicity and the insect receptor molecules that bind these toxins.; This dissertation is focused on the identification and cloning of a receptor for cryIA(b) toxin of BT. Ligand blotting with {dollar}sp{lcub}125{rcub}{dollar}IcryIA(b) toxin and immunoprecipitation of the toxin-binding protein complex with anti-cryIA(b) antiserum identified a 210-kDa protein present in Manduca sexta midgut brush border membrane vesicles (BBMV) as a specific receptor for this particular toxin. The radiolabeled toxin bound to the 210-kDa protein on blots with a {dollar}rm Ksb{lcub}d{rcub}{dollar} value of 0.708 nM and could be specifically blocked by unlabelled cryIA(b) toxin but not by unlabelled toxins from other subspecies of BT such as cryIIIA that kills beetles and cryIVD that kills mosquitoes. The 210-kDa protein is present only in the cryIA(b)-susceptible insect M. sexta and is absent in the beetle Leptinotarsa decemlineata and the mosquito Aedes aegypti,both of which are not susceptible to the cryIA(b) toxin. The BT cryIA(b) toxin receptor has been named BT-R{dollar}sb1.{dollar}; Natural BT-R{dollar}sb1{dollar} was purified and partial amino acid sequences of five internal peptides were determined. A BT-R{dollar}sb1{dollar} cDNA clone was identified from a M. sexta cDNA library using oligonucleotide probes based on the internal peptide sequences. The deduced amino acid sequence from the BT-R{dollar}sb1{dollar} clone revealed that this protein has a single transmembrane region, a large extra-cellular domain and a small 101-amino acid cytoplasmic domain. The BT-R{dollar}sb1{dollar} clone was expressed as a 210-kDa membrane protein in transfected COS-7 and human embryonic 293 cells. The 293 cells expressing BT-R{dollar}sb1{dollar} specifically bound the cryIA(b) toxin of BT. Interestingly, BT-R{dollar}sb1{dollar} shares 20-40% sequence similarity with the cadherin superfamily of proteins.; Results of the studies presented in this dissertation point out that toxin binding is an essential first step in the mode of action of BT toxins. Insect host range specificity of BT toxins is predicated on receptor mediated differences in the midgut of insects as well as on the individual toxins themselves. The receptor molecule BT-R{dollar}sb1,{dollar} present in M. sexta, is responsible for the high affinity binding of the cryIA(b) toxin. However, toxin binding to the receptor alone is not sufficient for cytotoxicity. The mode of action appears to be a complex, multi-step process. |
