ISSN: 2471-9315
Chanan Angsuthanasombat, Somsri Sakdee, Chompounoot Imtong, Chalermpol Kanchanawarin and Gerd Katzenmeier
Mahidol University, Thailand
Prince of Songkla University, Thailand
Kasetsart University, Thailand
Posters & Accepted Abstracts: Appli Micro
We have been devoted to discovering a multitude of toxic mechanisms of bacterial insecticidal proteins, i.e. Cry4Aa & Cry4Ba toxins from Bacillus thuringiensis (Bt), which are highly toxic to larvae of Aedes and Anopheles species, vectors of dengue viruses and malaria, respectively. The insecticidal feature of these Cry mosquito-active toxins is generally attributed to their capability to form oligomeric pores, causing lysis of target mid-gut cells. However, molecular description of their oligomerization process and target specificity has not been clearly defined. In this report, we employed two direct rendering techniques, i.e. single-particle negative-stain electron microscopy and high-speed atomic force microscopy, for visualizing 3D structure and trimeric assembly of the membrane-associated Cry4Ba toxin. We clearly showed that a membrane-induced state of toxin monomers is a critical prerequisite for the formation of a potential pre-pore trimer. Moreover, the polarity of the Cry4Ba �?±4-�?±5 loop residue-Asn166 was found to be important for ion permeation and pore-opening. We further demonstrated that single-reversal charge in the �?²10-�?²11 receptor-binding loops, Cry4Aa-Lys514 or Cry4Ba-Asp454, reflected their different toxicity against target mosquito-larvae. Furthermore, we demonstrated that Cry4Ba utilizes two aromatic loop-residues, Tyr332 and Phe364 which are respectively located in �?²2-�?²3 and �?²4-�?²5 loops, comprising the receptor-binding domain, for synergistic interactions with its alternative receptorâ�?�?Cyt2Aa2 from Bt subsp. darmstadiensis. We further showed that Thr328 and Thr369 could form H-bonding responsible for holding together these two receptor-binding hairpins (i.e., �?²2-�?²3 and �?²4-�?²5) in relevant to toxicity synergism with Cyt2Aa2. Altogether, we now feel able to tackle the key steps viz., their insecticidal mechanism, particularly on toxin-receptor interactions and the events following insertion of part of the toxin into membrane phase to form a trans-membrane leakage pore. Comprehensive understandings of the actual underlying toxic mechanism would bolster the future development of a better engineered bio-pesticide for control of such disease-carrying vectors.