Somphob Leetachewa, Ph.D.
|Ph.D. (Molecular Genetics and Genetic Engineering), Mahidol University, 2006
Field of Research:Microbial toxinStudies an interaction of mosquito larvicidal toxins and their receptors
Aedes aegypti mosquito is an important vector of dengue fever causing global death. Cry4 and Cry11 toxins produced from the Bacillus thuringiensis bacteria are specifically toxic to mosquito larvae. I am interested in an action mechanism of Cry4Ba, toxin-receptor interaction with its applications through structure-function relationship, cytolytic pore-formation, cellular & molecular biology and immunological techniques.
Previously structure-function relationship studies revealed that critical roles of the positively charged side chains of Arg-158 and Arg-136 in helix 4 of Cry4Ba and Cry11A in the pore forming activity of the Cry4Ba toxin. While, the purified Cry4Ba α4-α5 hairpins were shown to be highly capable of inducing liposome permeability, suggesting this region that is responsible for pore formation of the toxin.
Aminopeptidase N (APN), membrane-bound alkaline phosphatase (mALP) and cadherin-like proteins (CadLP) have been identified from lepidopteran and obviously characterized as specific receptors for Bt d-endotoxins. It is doubtful if these receptors could serve as receptors for dipteran specific Cry toxins. ALPs which are common at epithelial microvilli of insect larval gut. A. aegypti membrane-bound ALP was successfully cloned into Sf9 insect cells and shown to be a good candidate for the Cry4Ba-toxin receptor. GPI anchored APNs were identified using data mining, and utilized for design of long and double stranded RNAs which were able to specific knockdown APN gene from A. aegypti larvae by soaking technique. Interestingly, the knock downed larvae were markedly resistant to the Cry4Ba toxin.
In the mosquito larval gut, peritrophic membrane (PM) lining before the epithelial membrane serves as a physical and protective barrier. In the proposed mechanism, after enzymatic activation of Cry toxin by lumen enzymes, the toxin needs to cross the PM to reach a target on the epithelial membrane. The PM is consisted of one to three laminated layers of chitin fibrils held by chitin binding proteins and glycoproteins. Our previous publication suggested that the Cry4Ba toxin bound to the PM proteins and subsequently altered permeability of A. aegypti larval PM, allowing the toxin to pass through the PM. Therefore, my current research focuses on identification of a Cry4Ba receptor from the PM and manipulation of Cry4Ba-toxin receptor interaction in order to improve the larvicidal efficacy of the Cry4Ba toxin in the near future.