Interaction with the N -acetylgalactosamine moiety facilitates pore formation by Cry4Aa toxin, thereby increasing its toxicity

ABSTRACT Cry4Aa is a mosquito-larvicidal toxin produced by Bacillus thuringiensis serovar israelensis . Cry4Aa exhibits a characteristic architecture comprising three domains (I, II, and III) and functions as a pore-forming toxin, consistent with many previously reported insecticidal Cry toxins. However, the mechanism preceding pore formation by Cry4Aa, including receptor recognition, remains to be elucidated. The present study examined the potential involvement of sugar moieties in the Cry4Aa mode of action, with a particular focus on the process of pore formation. Cry4Aa and the sugar N -acetylgalactosamine exhibited considerably increased binding affinity. Furthermore, N -acetylgalactosamine treatment significantly enhanced the toxicity of Cry4Aa against Culex pipiens mosquito larvae. In the presence of N -acetylgalactosamine, the binding of Cry4Aa to phosphatidylcholine liposomes increased by 1.4-fold. The presence of this sugar did not affect the single-channel conductance of Cry4Aa pores formed in artificial lipid bilayers; however, it did appear to increase the number of Cry4Aa pores per designated area. In silico molecular docking analysis revealed promising binding sites within the cavity, surrounded by the Cry4Aa domains. These results suggest that the interaction of Cry4Aa with N -acetylgalactosamine moieties is a vital step in the process of pore formation. This interaction may induce a conformational change in the Cry4Aa molecule that facilitates its insertion into the target membrane. IMPORTANCE Cry toxins produced by the soil bacterium Bacillus thuringiensis are used as an environmentally friendly biopesticide. Cry toxins are generally considered pore-forming toxins; however, the mechanism preceding pore formation appears to be complex and is not yet fully elucidated. This study examined the interaction between mosquito-larvicidal Cry4Aa toxin and sugar moieties, determining considerably higher binding affinity with N -acetylgalactosamine. Furthermore, treatment with this sugar significantly increased the toxicity of Cry4Aa toward Culex pipiens mosquito larvae. Subsequent analyses revealed that treatment with this sugar increases the binding of Cry4Aa to phosphatidylcholine bilayers, thereby facilitating pore formation. The findings of this study enhance our understanding of the mechanism underlying the insecticidal activity of Cry toxins.