Random Mutational Analysis Targeting Residue K155 within the Transmembrane β-Hairpin of the Mosquitocidal Mpp46Ab Toxin

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

Characterization of the channel-pores formed by Bacillus thuringiensis Cry46Ab toxin in planar lipid bilayers

Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with aerolysin-type architecture, and has been shown that co-administration of Cry46Ab with other mosquitocidal Cry toxins results in synergistic toxicity against Culex pipiens Coquillett (Diptera: Culicidae) mosquito larvae. Cry46Ab, therefore, is expected to find use in improving the insecticidal activity of B. thuringiensis-based bioinsecticides. In the present study, the mode of action of Cry46Ab was explored by single-channel measurements of Cry46Ab channel-pores. The single-channel conductances of channel-pores formed in planar lipid bilayers by Cry46Ab were determined to be 31.8 +/- 2.7 pS in 150 mM NaCl and 24.2 +/- 0.7 pS in 150 mM CaCl2. Ion-selectivity measurements revealed that the channel-pores formed by Cry46Ab were cation selective. The permeability ratio of K+ to Cl-was approximately 4, and the preferences for cations were K+ > Na+, K+ > Ca2+, and Ca2+ > Na+. A calcein release assay using liposomes suggested that Cry46Ab influences the integrity of membrane vesicles. Formation of cation-selective channel-pores has been observed with other insecticidal Cry toxins that have structures distinct from those of Cry46Ab; the capability of forming such pores may be a property required of insecticidal toxins

Mutational analysis of the transmembrane α4-helix of Bacillus thuringiensis mosquito-larvicidal Cry4Aa toxin

Cry4Aa, produced by Bacillus thuringiensis subsp. israelensis, exhibits specific toxicity to larvae of medically important mosquito genera. Cry4Aa functions as a pore-forming toxin, and a helical hairpin (α4-loop-α5) of domain I is believed to be the transmembrane domain that forms toxin pores. Pore formation is considered to be a central mode of Cry4Aa action, but the relationship between pore formation and toxicity is poorly understood. In the present study, we constructed Cry4Aa mutants in which each polar amino acid residues within the transmembrane α4 helix was replaced with glutamic acid. Bioassays using Culex pipiens mosquito larvae and subsequent ion permeability measurements using symmetric KCl solution revealed an apparent correlation between toxicity and toxin pore conductance for most of the Cry4Aa mutants. In contrast, the Cry4Aa mutant H178E was a clear exception, almost losing its toxicity but still exhibiting a moderately high conductivity of about 60% of the wild-type. Furthermore, the conductance of the pore formed by the N190E mutant (about 50% of the wild-type) was close to that of H178E, but the toxicity was significantly higher than that of H178E. Ion selectivity measurements using asymmetric KCl solution revealed a significant decrease in cation selectivity of toxin pores formed by H178E compared to N190E. Our data suggest that the toxicity of Cry4Aa is primarily pore related. The formation of toxin pores that are highly ion-permeable and also highly cation-selective may enhance the influx of cations and water into the target cell, thereby facilitating the eventual death of mosquito larvae

Molecular Basis on Nitrogen Utilization in Rice(Recent Topics of the Agricultunal Biological Science in Tohoku University)

Rice (Oryza sativa L.) is the major provision for half of the world population and is the important model crop in terms of synteny. Nitrogen is a massive prerequisite element for rice during its life span. During evolutionary processes, rice has acquired strategic systems of nitrogen metabolism for the survival, i.e., the highly efficient ammonium assimilation in roots and nitrogen remobilization (nitrogen recycling). In our laboratory, research is underway to elucidate molecular mechanisms, cellular functions and the communication mechanisms in nitrogen metabolisms, especially ammonium assimilation in roots and nitrogen recycling, in rice. In this article, aim and overview of our research projects, and some recent research topics are shown

Channel-pore cation selectivity is a major determinant of Bacillus thuringiensis Cry46Ab mosquitocidal activity

Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type architecture, and it is expected to be used as a novel bioinsecticide. Cry46Ab acts as a functional pore-forming toxin, and characteristics of the resulting channel pores, including ion selectivity, have been analyzed. However, the relationship between channel-pore ion selectivity and insecticidal activity remains to be elucidated. To clarify the effects of charged amino acid residues on the ion permeability of channel-pores and the resulting insecticidal activity, in the present study, we constructed Cry46Ab mutants in which a charged amino acid residue within a putative transmembrane β-hairpin region was replaced with an oppositely charged residue. Bioassays using Culex pipiens mosquito larvae revealed that the mosquitocidal activity was altered by the mutation. A K155E Cry46Ab mutant exhibited toxicity apparently higher than that of wild-type Cry46Ab, but the E159K and E163K mutants exhibited decreased toxicity. Ions selectivity measurements demonstrated that the channel pores formed by both wild-type and mutant Cry46Abs were cation selective, and their cation preference was also similar. However, the degree of cation selectivity was apparently higher in channel pores formed by the K155E mutant, and reduced selectivity was observed with the E159K and E163K mutants. Our data suggest that channel-pore cation selectivity is a major determinant of Cry46Ab mosquitocidal activity and that cation selectivity can be controlled via mutagenesis targeting the transmembrane β-hairpin region

Mammalian enzymes for preventing transcriptional errors caused by oxidative damage

8-Oxo-7,8-dihydroguanine (8-oxoGua) is produced in cells by reactive oxygen species normally formed during cellular metabolic processes. This oxidized base can pair with both adenine and cytosine, and thus the existence of this base in messenger RNA would cause translational errors. The MutT protein of Escherichia coli degrades 8-oxoGua-containing ribonucleoside di- and triphosphates to the monophosphate, thereby preventing the misincorporation of 8-oxoGua into RNA. Here, we show that for human the MutT-related proteins, NUDT5 and MTH1 have the ability to prevent translational errors caused by oxidative damage. The increase in the production of erroneous proteins by oxidative damage is 28-fold over the wild-type cells in E.coli mutT deficient cells. By the expression of NUDT5 or MTH1 in the cells, it is reduced to 1.4- or 1.2-fold, respectively. NUDT5 and MTH1 hydrolyze 8-oxoGDP to 8-oxoGMP with V(max)/K(m) values of 1.3 × 10(−3) and 1.7 × 10(−3), respectively, values which are considerably higher than those for its normal counterpart, GDP (0.1–0.5 × 10(−3)). MTH1, but not NUDT5, possesses an additional activity to degrade 8-oxoGTP to the monophosphate. These results indicate that the elimination of 8-oxoGua-containing ribonucleotides from the precursor pool is important to ensure accurate protein synthesis and that both NUDT5 and MTH1 are involved in this process in human cells

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

A 2,175-bp modified gene (cry11Ba-S1) encoding Cry11Ba from Bacillus thuringiensis subsp. jegathesan was designed and the recombinant protein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The recombinant Cry11Ba was highly toxic against Culex pipiens mosquito larvae, being nine and 17 times more toxic than mosquitocidal Cry4Aa and Cry11Aa from Bacillus thuringiensis subsp. israelensis, respectively. Interestingly, a further increase in the toxicity of the recombinant Cry11Ba was achieved by mixing with Cry4Aa, but not with Cry11Aa. These findings suggested that Cry11Ba worked synergistically with Cry4Aa, but not with Cry11Aa, in exhibiting toxicity against C. pipiens larvae. On the other hand, the amount of Cry toxin bound to brush border membrane vesicles (BBMVs) did not significantly change between individual toxins and the toxin mixtures, suggesting that the increase in toxins binding to BBMVs was not a reason for the observed synergistic effect. It is generally accepted that synergism of toxins is a potentially powerful tool for enhancing insecticidal activity and managing Cry toxin resistance in mosquitoes. The mixture of Cry4Aa and Cry11Ba in order to increase toxicity would be very valuable in terms of mosquito control