Analyses of toxin-receptor interaction in Manduca sexta using Bacillus thuringiensis Cry1A mutant toxins
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The mechanism of action of Bacillus thuringiensis Cry1A toxin in susceptible larvae involves activation of toxin, binding to apical membrane proteins of larval midgut, insertion of toxins into membrane, and pore formation. I studied two aspects of the mechanism of action of Cry1A toxins in this research using Cry1A mutant toxins. The first aspect analyzed pore-forming properties of Cry1A domain I and domain II mutant toxins with Manduca sexta brush border membrane vesicles (BBMV) using a light scattering assay. The second aspect focused on identifying alternate M. sexta Cry1Acbinding protein(s) and studying effect of protein denaturation on toxin-receptor binding specificity. In the light scattering assay, wild-type toxins Cry1Ac and Cry1Ab and mutant toxins A92D and A92E were able to form pores in BBMV in the presence of KCl. However, in the presence of sucrose, mutants A92D and A92E did not form pores, unlike Cry1Ac and Cry1Ab which was probably due to a charge-masking effect by KCl. Our results with the light scattering assay indicated that toxin-structures in membrane resembled pores. We studied binding of Cry1Ac and Cry1Ac mutant 509QNR511-AAA with M. sexta BBMV and MsAPN-1 under denaturing and non-denaturing conditions. Toxin-binding specificity was altered under denaturing conditions indicating that binding epitopes absent on native protein surface were exposed under denaturing conditions. Finally, the presence of M. sexta BBMV protein(s) that could bind Cry1Ac independent of N-acetylgalactosamine (GalNAc) was investigated. Cry1Ac mutant 509QNR511-AAA with a disrupted GalNAc-interaction pocket is toxic to M. sexta even though binding to MsAPN-1 is abolished suggesting that protein(s) other than MsAPN-1 mediates GalNAc-independent Cry1Ac toxicity. We isolated a 110 kDa protein (Ms110- APN) by toxin-affinity chromatography and cloned the full-length Ms110-APN cDNA and expressed the protein in E. coli. Cry1Ac and 509QNR511-AAA bound E. coliexpressed Ms110-APN indicating that Ms110-APN is a GalNAc-independent Cry1Acbinding protein in M. sexta. The overall conclusions of my studies with Cry1A mutant toxins are that Cry1A toxins utilize different brush border proteins to interact with midgut membrane. Toxins also increase the chance of membrane insertion by utilizing its positively charged residues to interact with negatively charged midgut membrane.