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

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

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

A Lipid Bilayer Formed on a Hydrogel Bead for Single Ion Channel Recordings

Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, alpha-hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion

A Report on Overseas Teaching Practicum by Graduate Students in Elementary/Secondary Schools in the United States (Ⅶ)

The present reports is on the 7th overseas teaching practicum in the United States by 15 graduate students of Hiroshima University, Japan, partly organized by Hiroshima University Global Partnership School Center since 2007. The group was comprised of 13 elementary school and 2 secondary school education major graduate students. They planned and conducted lessons in English in three local public schools in North Carolina. The expected outcomes of this project were: 1) to self-develop practical instructional competence by teaching pupils with diverse backgrounds in the U.S.; 2) to enhance the abilities in developing teaching materials through hands-on teaching experiences in English; and 3) to acquire the abilities to design, implement and evaluate programs for promoting global partnership. In addition, the teaching experience was followed by cross-cultural study visits to Raleigh, NC and Washington, D.C. It helped to boost our group motivation that the local media, newspaper and TV, and the city Board of Education covered our visit. It is hoped that this project will enhance the students’ teaching competence in designing quality materials/lessons and classroom communication skills in English

Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 variant

SARS-CoV-2オミクロンBA.2.75株（通称ケンタウロス）のウイルス学的性状の解明. 京都大学プレスリリース. 2022-10-12.The SARS-CoV-2 Omicron BA.2.75 variant emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically distinct from BA.5, the currently predominant BA.2 descendant. Here, we show that BA.2.75 has a greater effective reproduction number and different immunogenicity profile than BA.5. We determined the sensitivity of BA.2.75 to vaccinee and convalescent sera as well as a panel of clinically available antiviral drugs and antibodies. Antiviral drugs largely retained potency but antibody sensitivity varied depending on several key BA.2.75-specific substitutions. The BA.2.75 spike exhibited a profoundly higher affinity for its human receptor, ACE2. Additionally, the fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were greater than those of BA.2. Our multilevel investigations suggest that BA.2.75 acquired virological properties independent of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5

Convergent evolution of SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant

In late 2022, various Omicron subvariants emerged and cocirculated worldwide. These variants convergently acquired amino acid substitutions at critical residues in the spike protein, including residues R346, K444, L452, N460, and F486. Here, we characterize the convergent evolution of Omicron subvariants and the properties of one recent lineage of concern, BQ.1.1. Our phylogenetic analysis suggests that these five substitutions are recurrently acquired, particularly in younger Omicron lineages. Epidemic dynamics modelling suggests that the five substitutions increase viral fitness, and a large proportion of the fitness variation within Omicron lineages can be explained by these substitutions. Compared to BA.5, BQ.1.1 evades breakthrough BA.2 and BA.5 infection sera more efficiently, as demonstrated by neutralization assays. The pathogenicity of BQ.1.1 in hamsters is lower than that of BA.5. Our multiscale investigations illuminate the evolutionary rules governing the convergent evolution for known Omicron lineages as of 2022

Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants

In late 2022, SARS-CoV-2 Omicron subvariants have become highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged through the recombination of two cocirculating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022. XBB.1 is the variant most profoundly resistant to BA.2/5 breakthrough infection sera to date and is more fusogenic than BA.2.75. The recombination breakpoint is located in the receptor-binding domain of spike, and each region of the recombinant spike confers immune evasion and increases fusogenicity. We further provide the structural basis for the interaction between XBB.1 spike and human ACE2. Finally, the intrinsic pathogenicity of XBB.1 in male hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provides evidence suggesting that XBB is the first observed SARS-CoV-2 variant to increase its fitness through recombination rather than substitutions

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

Abstract: The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era