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Random Mutational Analysis Targeting Residue K155 within the Transmembrane β-Hairpin of the Mosquitocidal Mpp46Ab
Midoka Miyazaki1, Mami Asakura1, Toru Ide1
1Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University, 3-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan.
Abstract:
Mpp46Ab is a mosquito-larvicidal pore-forming toxin derived from Bacillus thuringiensis TK-E6. Pore formation is believed to be a central mode of Mpp46Ab action, and the cation selectivity of the channel pores, in particular, is closely related to its mosquito-larvicidal activity. In the present study, we constructed a mutant library in which residue K155 within the transmembrane β-hairpin was randomly replaced with other amino acid residues. Upon mutagenesis and following primary screening using Culex pipiens mosquito larvae, we obtained 15 mutants in addition to the wild-type toxin. Bioassays using purified proteins revealed that two mutants, K155E and K155I, exhibited toxicity significantly higher than that of the wild-type toxin. Although increased cation selectivity was previously reported for K155E channel pores, we demonstrated in the present study that the cation selectivity of K155I channel pores was also significantly increased. Considering the characteristics of the amino acids, the charge of residue 155 may not directly affect the cation selectivity of Mpp46Ab channel pores. Replacement of K155 with glutamic acid or isoleucine may induce a similar conformational change in the region associated with the ion selectivity of the Mpp46Ab channel pores. Mutagenesis targeting the transmembrane β-hairpin may be an effective strategy for enhancing the ion permeability of the channel pores and the resulting mosquito-larvicidal activity of Mpp46Ab.
Insights
Mutating a key residue in the mosquito-larvicidal toxin Mpp46Ab significantly enhanced its toxicity and cation selectivity. This suggests targeting the transmembrane β-hairpin is a promising strategy for developing new larvicides.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- Mpp46Ab is a pore-forming toxin from Bacillus thuringiensis with mosquito-larvicidal activity.
- Channel pore cation selectivity is crucial for Mpp46Ab's larvicidal efficacy.
Purpose of the Study:
- To investigate the role of residue K155 in Mpp46Ab's transmembrane β-hairpin on its function.
- To enhance Mpp46Ab's mosquito-larvicidal activity through targeted mutagenesis.
Main Methods:
- Random mutagenesis of residue K155 in the Mpp46Ab transmembrane β-hairpin.
- Screening of mutants using Culex pipiens mosquito larvae.
- Bioassays with purified mutant proteins to assess toxicity and cation selectivity.
Main Results:
- Fifteen mutants of Mpp46Ab were generated, with K155E and K155I showing significantly higher toxicity than wild-type.
- Both K155E and K155I mutants exhibited increased cation selectivity in their channel pores.
- The charge of residue 155 may not directly influence cation selectivity, suggesting conformational changes are involved.
Conclusions:
- Mutagenesis of the transmembrane β-hairpin is an effective strategy to improve Mpp46Ab's larvicidal properties.
- Modifying residue K155 can enhance ion permeability and Mpp46Ab's efficacy against mosquitoes.
- Further research into conformational changes could optimize Mpp46Ab-based larvicides.
