Highly Effective Broad Spectrum Chimeric Larvicide That Targets Vector Mosquitoes Using a Lipophilic Protein

Abstract

Two mosquitocidal bacteria, Bacillus thuringiensis subsp. israelensis (Bti) and Lysinibacillus sphaericus (Ls) are the active ingredients of commercial larvicides used widely to control vector mosquitoes. Bti's efficacy is due to synergistic interactions among four proteins, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa, whereas Ls's activity is caused by Bin, a heterodimer consisting of BinA, the toxin, and BinB, a midgut-binding protein. Cyt1Aa is lipophilic and synergizes Bti Cry proteins by increasing midgut binding. We fused Bti's Cyt1Aa to Ls's BinA yielding a broad-spectrum chimeric protein highly mosquitocidal to important vector species including Anopheles gambiae, Culex quinquefasciatus, and Aedes aegypti, the latter an important Zika and Dengue virus vector insensitive to Ls Bin. Aside from its vector control potential, our bioassay data, in contrast to numerous other reports, provide strong evidence that BinA does not require conformational interactions with BinB or microvillar membrane lipids to bind to its intracellular target and kill mosquitoes.

Figure 1

Parasporal inclusions of chimeric Cyt1Aa-BinA synthesized using the 4Q7 acrystalliferous strain of Bacillus thuringiensis subsp. israelensis. (A) Schematic of the cyt1Aa-binA gene fusion. A 0.84-kbp fragment containing the cyt1Aa gene BtIII promoter (PBtIII) and cyt1Aa open reading frame (ORF) was cloned in frame with a 1.4 kbp fragment harboring the binA ORF flanked by its native transcription terminator (ter). The nucleotide sequences at the fusion site (underlined) and the coded amino acids (KLGA, lysine-leucine-glycine-alanine) are shown above the HindIII site, as are the positions of applicable restriction sites and the 20-kDa like chaperone-like protein gene under control of the cry1Ac gene promoter (Pcry1Ac) used for cloning in pBU4 to generate the expression vector pBU-cyt1Aa-binA. The Cyt1Aa-BinA protoxin is composed of 623 amino acids and has a molecular mass of 69.6 kDa; the predicted proteolytically active forms of Cyt1Aa (22.7 kDa) and BinA (38.8 kDa) are shown. (B) Micrograph (x1000) of 4Q7/pBU-cyt1Aa-binA grown for 48 hr showing sporulated cells with endospores (s) and parasporal inclusions (c); free spores and inclusions are also present, which is typical after lysis of B. thuringiensis cells.

Figure 2

Protein profile and antigenicity of the Cyt1Aa-BinA chimera. Inclusion bodies were purified from Bacillus thuringiensis subsp. israelensis 4Q7 strains producing (A) Cyt1Aa (4Q7/pWF45; lanes 1, 2), Cyt1A-BinA (4Q7/pBU-cyt1Aa-binA; lanes 3, 4), (B) Cyt1Aa and BinAB (4Q7/45S1; lanes 1, 2) or Cyt1A-BinA (4Q7/pBU-cyt1A-binA; lanes 3, 4). Inclusions were solubilized and fractionated by SDS-PAGE in a 10% gel and electroblotted for Western analysis using rabbit anti-Cyt1Aa and anti-BinA antibodies (Ab). Lanes 1, 2, and 3, 4, respectively, 0.75 μg and 1.5 μg of protein; molecular masses: Cyt1Aa, 27.2 kDa; BinA, 42 kDa; BinB, 52 kDa; and Cyt1Aa-BinA 69.6 kDa. (C) SDS-PAGE demonstrating proteolytic cleavage of Cyt1Aa-BinA by trypsin, with Cyt1Aa as a control. Purified parasporal inclusions were solubilized in 50 mM NaOH, supernatants collected and neutralized with HCl, and digested with the enzyme at 28 °C. Untreated samples, 1.5 hr (lane 1), and trypsin-treated samples, 0.5 hr (lane 2) and 1.5 hr (lane 3). (D) Midgut histopathology caused by Cyt1Aa-BinA chimera in fourth instars of Culex quinquefasciatus 8 hours post-treatment at the LC₉₅ concentration; Control midgut epithelium, (i) and (ii), respectively, 100x and 400x magnification. Midgut epithelium of a treated larva (iii) and (iv), respectively 100x and 600x magnification. Note the vacuoles in cells designated by arrows in D (iv) that have sloughed from the midgut basement membrane (C, 100x; D, 600x). The central circular area in A is the food column surrounded by the peritrophic membrane. MW, protein molecular mass standards; kDa, kilodaltons.