Mosquitoes transmit some of the world's most serious diseases, including malaria, filariasis, yellow fever, and encephalitis. With the emergence of pesticide- and drug-resistant mosquitoes, and the increased concern over chemical pesticide toxicity, the use of environmentally-safe biological control agents, such as Bacillus thuringiensis (Bt) is more important than ever. Bt subsp. israelensis (Bti) produce mosquitocidal crystal proteins, also call delta- endotoxins, within crystalline inclusions during sporulation. Recent studies suggest that optimal mosquitocidal activity requires synergistic interactions between the delta-endotoxins. In addition, optimal mosquitocidal activity is dependent on the production of these delta-endotoxins as crystalline inclusions. Our long term research objectives are to define the mechanism of synergistic interactions between the delta-enotoxins and characterize the process for in vivo proteins crystallization. In this proposal, our studies will focus on several delta-endotoxins, including CytA and CryIVD, and the cryIVD operon-encoded 20-kDa protein insolated from Bti. The following are the specific aims for this 5-year proposal: 1) Isolate and characterize the active 20-kDa protein; 2) Determine the binding specificity between the 20-kDa protein and CytA; 3) Determine the role of the 20-kDa protein in Bt crystal protein synthesis and degradation; 4) Determine the mechanism of synergy between CytA and CryIVD; 5) Determine the mechanism of CytA's crystal- dependent mosquitocidal activity; and 5) Determine the CytA structure(s) responsible for synergism through site-directed mutagenesis. A more comprehensive understanding of the synergistic interaction between these mosquitocidal proteins will provide new strategies to design more effective Bt strains for insect control. Understanding the mechanism of in vivo protein crystallization is important not only for improving the effectiveness and expanding the target range of Bt biopesticides, but also for consideration of Bt as a novel gene expression system for the over- production of proteins as crystals.
