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Mechanism of Action of Insecticidal Crystal Toxins from Bacillus thuringiensis: Biophysical and Biochemical Analyses of the Insertion of Cry1A Toxins into Insect Midgut Membranes

Nair, Manoj Sadasivan

Abstract Details

2008, Doctor of Philosophy, Ohio State University, Biophysics.

The most controversial step in the study of the mechanism of action of insecticidal crystal toxins is that of insertion of the toxin into insect brush border membranes. Conflicting models of insertion of toxin can be categorized into two groups; ones that propose that only certain alpha helices of domain I insert into insect brush border membrane vesicles (BBMV) (Umbrella, Penknife and Serial Receptor Binding models) and others that propose that most of the toxin inserts into BBMV (Aronson, Buried Dragon and Unchanged Structure models).

Protease protection studies of cysteine mutations from all domains of the toxin showed protection of most of the toxin (a 60 kDa form) similar to the wild type Cry1A toxin, when inserted into insect brush border membranes. Studies on steady state fluorescence measurements of these cysteine residues when bound to artificial vesicles or natural brush border membrane vesicles (BBMV), and fluorescence energy transfer measurements in labeled artificial vesicles suggested that residues from all the domains of the toxin inserted into the membrane.

Residues in the loop 2 of Domain II of the toxin that played a vital role in the insertion of the toxin into insect BBMV were identified. Examination of receptor binding and insertion of mutants of these residues have shown that insertion of Cry1Ab into the membrane is dependent on specific residues at positions in this loop. Absence of phenylalanine or a closely related amino acid such as tryptophan at position 371 allowed initial binding of the toxin to the receptor but compromised the insertion of the toxin into insect membrane, thereby confirming that irreversible binding step of the toxin-BBMV interaction is the critical step in the mode of action of the toxin where Domain II is a major candidate mediating the step. Fluorescence blue shift studies into artificial and natural membranes also indicated a difference in the partitioning of the toxin into artificial and natural membranes; thereby suggesting that physiological mode of action required the presence of receptors.

Analyses of the oligomeric states of Cry1A toxins have shown that oligomerization of the toxin before insertion into insect membranes is not essential for toxicity as both monomeric and oligomeric forms induced similar levels of toxicity. Oligomers (defined as prepores) isolated under specified conditions from artificial membranes included all three domains of the toxin and receptor derived peptides used to elicit the prepore, suggesting that all domains of the toxin are able to insert into the membrane even as they were bound to receptor peptides. We also add further evidence that binding of two receptors in a serial manner is not essential for toxicity of Cry proteins.

Our studies present an alternate model where an entire toxin unit (of 60 kDa) binds to a receptor, based on availability and affinity of binding and inserts into insect brush border membrane either as a monomer or oligomeric form of the unit.

Donald Dean (Advisor)
David Stetson (Committee Member)
Charles Bell (Committee Member)
144 p.

Recommended Citations

Citations

  • Nair, M. S. (2008). Mechanism of Action of Insecticidal Crystal Toxins from Bacillus thuringiensis: Biophysical and Biochemical Analyses of the Insertion of Cry1A Toxins into Insect Midgut Membranes [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1218558470

    APA Style (7th edition)

  • Nair, Manoj. Mechanism of Action of Insecticidal Crystal Toxins from Bacillus thuringiensis: Biophysical and Biochemical Analyses of the Insertion of Cry1A Toxins into Insect Midgut Membranes. 2008. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1218558470.

    MLA Style (8th edition)

  • Nair, Manoj. "Mechanism of Action of Insecticidal Crystal Toxins from Bacillus thuringiensis: Biophysical and Biochemical Analyses of the Insertion of Cry1A Toxins into Insect Midgut Membranes." Doctoral dissertation, Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1218558470

    Chicago Manual of Style (17th edition)