title: PEGylation Enhances Mosquito-Larvicidal Activity of Lysinibacillus sphaericus Binary Toxin.
abstract: Toxic strains of Lysinibacillus sphaericus have been used in the field for larval control of mosquito vector diseases. The high toxicity of L. sphaericus is attributed to the binary (BinAB) toxin produced as parasporal crystalline inclusions during the early stages of sporulation. BinA and BinB, the primary components of these spore-crystals, exert high toxicity when administered together. However, instability, short half-lives, and rapid proteolytic digestion can limit their use as an effective insecticide. BinA alone displays larvicidal toxicity, in the absence of BinB, albeit with much reduced activity. Here for the first time, we demonstrate the beneficial effect of PEGylation (covalent attachment of polyethylene glycol) on mosquito-larvicidal activity of BinA. Polymer conjugation was achieved using 750 Da polyethylene glycol (PEG) at two different pH values (pH 7.2 and 8.5). Two different isoforms of the biopolymers, purified to homogeneity, were highly water-soluble and resistant to trypsin and proteinase K. The mono-PEGylated BinA isoforms also displayed preservation of the toxin structure with improved thermal stability by about 3-5 °C, as evident from thermal denaturation studies by circular dichroism and differential scanning fluorimetry. Notably, PEGylation enhanced BinA toxicity by nearly 6-fold. The PEGylated BinA isoforms alone displayed high larvicidal activity (LC50value of ∼3.4 ng/mL) against the third instar Culex larvae, which compares favorably against LC50reported for the combination of BinA and BinB proteins. Since BinA can be synthesized easily through recombinant technology and easily PEGylated, the conjugated biopolymers offer a promising opportunity for mosquito control programs.
authors.affiliation: Homi Bhabha National Institute, Training School Complex , Anushaktinagar, Mumbai 400094, India.
authors.forename: Mahima
authors.initials: M
authors.lastname: Sharma
authors.forename: Ramesh S
authors.initials: RS
authors.lastname: Hire
authors.forename: Ashok B
authors.initials: AB
authors.lastname: Hadapad
authors.forename: Gagan D
authors.initials: GD
authors.lastname: Gupta
authors.affiliation: Homi Bhabha National Institute, Training School Complex , Anushaktinagar, Mumbai 400094, India.
authors.forename: Vinay
authors.identifier: 0000-0002-7619-6236
authors.initials: V
authors.lastname: Kumar
mesh_terms: D000818:Animals; D001406:Bacillaceae / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D009033:Culicidae; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007306:Insecticides / Q000737:chemistry*; D007814:Larva; D008958:Models, Molecular; D011092:Polyethylene Glycols / Q000737:chemistry*; D055550:Protein Stability; D059748:Proteolysis; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D001427:Bacterial Toxins; D007306:Insecticides; C046134:binB protein, Bacillus sphaericus; D011092:Polyethylene Glycols