Alanine Scanning Analyses of the Three Major Loops in Domain II of Bacillus thuringiensis Mosquitocidal Toxin Cry4Aa

Abstract

Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.

Bacillus thuringiensis Cry11Ba works synergistically with Cry4Aa but not with Cry11Aa for toxicity against mosquito Culex pipiens (Diptera: Culicidae) larvae

“…GST-Cry4Aa also showed relatively high toxicity, with an LC50 value of 0.97 ± 0.34 µg/ml (Fig. 2), which was similar to that for Cry4Aa reported previously (Howlader et al 2009(Howlader et al , 2010. The toxicity of GST-Cry11Aa, which had an LC50 value of 1.91 ± 0.37 µg/ml was slightly lower than that for GST-Cry4Aa (Fig.…”

“…Brush border membrane vesicles (BBMVs) were prepared from 7-to 10-day-old C. pipiens larvae (4 th instar) as described previously (Howlader et al 2010).…”

Isolation and characterization of entomopathogenic bacteria from soil samples from the western region of Cuba

“…Protein profiles by SDS‐PAGE from bacterial cell lysates have been used routinely for the differentiation and characterization of isolates of B. thuringiensis in order to determine the main entomopathogenic factors (Sreshty et al 2011, Ibarra et al 2003). Cry4 protein (127–135 kDa) has great pathogenic variability with activity against dipterans, nematodes, mites, and Lepidoptera (Mena et al 2003, Howlader et al 2010). The 28 kDa band corresponding to Cyt 1A has detergent activity (Manceva et al 2005).…”

The Relationship between Glycan Binding and Direct Membrane Interactions in Vibrio cholerae Cytolysin, a Channel-forming Toxin

“…This study builds upon the previous findings by offering a systematic scan of the entire VCC rim domain, an analysis of how membrane and glycan interactions are related, and additional insights into the mechanism of pore assembly. Similar methods have been instrumental in uncovering membrane interactions for other toxins, most notably the cholesterol-dependent cytolysin perfringolysin O (PFO) (23) and the Cry4Aa mosquitocidal toxin from Bacillus thuringiensis (24). This approach was instrumental in identifying a dipeptide (threonine-leucine) TL sequence responsible for cholesterol recognition in PFO (23).…”