# Mutagenesis Targeting the S153 Residue Within the Transmembrane β-Hairpin of Mosquito-Larvicidal Mpp46Ab Affects Its Toxicity and the Synergistic Toxicity with Cry4Aa

## TL;DR

Scientists studied how changing a specific amino acid in a mosquito-killing toxin affects its toxicity and how it works with another toxin.

## Contribution

The study reveals how S153 mutations in Mpp46Ab influence toxicity and synergy with Cry4Aa through changes in ion permeability of toxin pores.

## Key findings

- The S153I mutant showed significantly increased toxicity against mosquito larvae.
- S153F, S153L, and S153I mutants exhibited reduced synergistic toxicity with Cry4Aa.
- Electrophysiologic analysis showed altered ion permeability in mutant toxin pores.

## Abstract

The mosquito-larvicidal activity of Mpp46Ab, a pore-forming toxin derived from Bacillus thuringiensis TK-E6, has been shown to correlate with the cation selectivity of the toxin pores. In particular, K155 within the transmembrane β-hairpin is considered a good target for the improvement of Mpp46Ab, as increased cation selectivity of the toxin pores and consequently increased mosquito-larvicidal activity were observed in K155E and K155I mutants. In this study, to investigate the importance of S153 adjacent to K155, we constructed a mutant library, in which S153 was randomly replaced by other residues. After mutagenesis and subsequent primary screening using Culex pipiens mosquito larvae, we obtained 10 different Mpp46Ab mutants in addition to the wild type. Interestingly, the S153I mutant exhibited significantly increased toxicity. In addition, the S153F, S153L, and S153I mutants exhibited significantly reduced synergistic toxicity with mosquito-larvicidal Cry4Aa. Electrophysiologic analysis of the mutants revealed an apparent change in the ion-permeability of the toxin pores, which correlated with toxicity. Our results suggest that pore formation is central to the insecticidal activity of Mpp46Ab and that the ion permeability of toxin pores is a potential indicator correlated with both toxicity and synergistic toxicity with other toxins.

We constructed a library of Mpp46Ab mutants, in which S153 within the transmembrane β-hairpin was randomly replaced by other amino acids. Mutagenesis and subsequent primary screening yielded 10 different Mpp46Ab mutants in addition to the wild type. Remarkably, S153 was replaced with a more hydrophobic amino acid in most of the mutants, and the S153I mutant in particular exhibited significantly increased toxicity. Electrophysiologic analysis using artificial lipid bilayers revealed that the single-channel conductance and PK/PCl permeability ratio were significantly increased for S153I pores. This suggests that the formation of highly ion-permeable and highly cation-selective toxin pores increases the influx of cations and water into cells, thereby facilitating osmotic shock. In addition, the S153F, S153L, and S153I mutants exhibited significantly reduced synergistic toxicity with Cry4Aa. Electrophysiologic analysis showed that the S153F, S153L, and S153I mutants form toxin pores with a significantly reduced PK/PNa permeability ratio and a significantly increased PK/PCa permeability ratio compared to wild-type pores. Thus, our results suggest that pore formation is central to the insecticidal activity of Mpp46Ab and that the ion permeability of toxin pores is a potential indicator correlated with both toxicity and synergistic toxicity with other toxins.