Biosynthesis of Cry5B-Loaded Sulfur Nanoparticles using Arthrobotrys oligospora Filtrate: Effects on Nematicidal Activity, Thermal Stability, and Pathogenicity against Caenorhabditis elegans.

Cry5B, a crystal protein produced by Bacillus thuringiensis (Bt), is a bionematicide with potent nematicidal activity against various plant-parasitic and free-living nematodes. This protein, however, is susceptible to destruction by ultraviolet light, proteolytic enzymes, and high temperatures. This study aims to produce Cry5B protein for bionematicidal use and improve its stability and nematicidal efficacy by loading it intoArthrobotrys oligospora-mediated sulfur nanoparticles (AO-SNPs). Based on the mortality assay, the Cry5B protein exhibited dose-dependent nematicidal activity against the model organismCaenorhabditis elegans. The nematicidal activity, thermal stability, and pathogenic effects of Cry5B-loaded AO-SNPs (Cry5B-SNPs) were compared to those of free Cry5B. After 3 h of exposure to heat at 60 °C, Cry5B-SNPs had greater nematicidal activity than free Cry5B protein, indicating the effective formulation of Cry5B-SNPs that could be used as an alternative to current nematicide delivery strategies.

Screening and characterization of Bacillus thuringiensis isolates for high production of Vip3A and Cry proteins and high thermostability to control Spodoptera spp.

Bacillus thuringiensis (Bt) is an entomopathogenic bacterium that produces crystalline (Cry and Cyt) and soluble (vegetative insecticidal proteins or Vips) proteins during the sporulation and vegetative growth phases, respectively. Combining Cry and Vip proteins could delay insect resistance development and exhibit synergistic activity against various insect pests. This study aims to screen Bt isolates collected from Thailand for high Vip3A and Cry protein production levels and high thermostability to control Spodoptera spp. Among the selected Bt isolates with high target protein synthesis, Bt isolate 506 was found to be safe for further biopesticide formulation due to the absence of non-specific metabolite, as determined by the detection of thermo-stable ß-exotoxin I based on biological assays and PCR analysis. Bt isolate 506 showed the presence of Cry1A, Cry2A, and Vip3A-type proteins identified as Cry1Aa45, Cry2Aa22, and Vip3A87, respectively. The insecticidal activity of whole culture extracts containing Vip3A and Cry mixtures and culture supernatants containing secreted Vip3A protein was evaluated against the second-instar larvae of S. exigua and S. frugiperda. The Bt isolate 506 showed high toxicity against both insects, and the insecticidal proteins produced by this isolate retained their activity after heating at 50 °C. This Bt isolate is a promising candidate for further development as a biopesticide against lepidopteran pests.

Vegetative insecticidal protein (Vip3A) production by Bacillus thuringiensis Bt294 and its efficacy against Lepidopteran pests (Spodoptera exigua).

A vegetative insecticidal protein, Vip3A, is highly active against lepidopteran pests, which are the most important pests in most tropical countries. An important aspect of the successful commercial production of this bacterial insecticide is the development of bacterial culture media that maximize the titres of this protein and cost reduction. This study aimed to investigate and optimize Vip3A production by Bacillus thuringiensis Bt294 using statistical methods and 3-step sequential approaches. The experimental design showed that the production of Vip3A was maximized to 300 mg/L when the bacterium was cultivated in medium composed of 5.05 g/L glycerol, 49.17 g/L soytone, 30.05 g/L casein hydrolysate, 1.99 g/L CaCl2.2H2O, 7.5 mg/L CuSO4, 15 mg/L MnSO4.H2O, 9.4 g/L K2HPO4, 2.2 g/L KH2PO4, 0.2 g/L MgSO4.7H2O, 5 g/L yeast extract, 2.5 mg/L NiCl2.6H2O and 3 mL/L vitamin solution. B. thuringiensis Bt294 Vip3A toxin was highly toxic to Spodoptera exigua with LC50 values of 187.1 ng/cm2 at 7 days. This result demonstrated that a high titre of Vip3A produced by B. thuringiensis Bt294 will be useful as a biological control agent. This optimization will allow production to be scaled up for commercial production in the future.

Mutation of a Threonine Residue in αD-ß4 Loop of Cyt2Aa2 Protein Influences Binding on Fluid Lipid Membranes.

Cyt proteins are insecticidal proteins originally from Bacillus thuringiensis. The lipid binding of the Cyt2Aa2 protein depends on the phase of the lipid bilayer. In this work, the importance of the conserved T144 residue in the αD-ß4 loop for lipid binding on fluid lipid membranes was investigated via atomic force microscopy (AFM). Lipid membrane fluidity could be monitored for the following lipid mixture systems: POPC/DPPC, POPC/SM, and DOPC/SM. AFM results revealed that the T144A mutant was unable to bind to pure POPC bilayers. Similar topography between the wildtype and T144A mutant was seen for the POPC/Chol system. Small aggregates of T144A mutant were observed in the POPC and DOPC domains of the lipid mixture systems. In addition, the T144A mutant had no cytotoxic effect against human colon cancer cells. These results suggest that alanine replacement into threonine 144 hinders the binding of Cyt2Aa2 on liquid lipid membranes. These observations provide a possibility to modify the Cyt2Aa2 protein to specific cells via lipid phase selection.

Proteomic Analysis and Promoter Modification of Bacillus thuringiensis to Improve Insecticidal Vip3A Protein Production.

Insecticidal protein Vip3A secreted from B. thuringiensis is a potential biocontrol agent for control of lepidopteran pests. Under laboratory conditions, high albeit variable Vip3A production from the local isolate Bt294 was only obtained from a much enriched TB culture medium. Proteomic analysis and strain improvement were therefore performed to improve Vip3A production. Studies indicated that the buffer capacity, carbon source, and nitrogen source are critical to efficiently produce Vip3A. Medium with lower amounts of peptone and yeast extract (compared to TB), with an additional carbon source and phosphate buffer (LB*G medium) was found to give reasonable yields of Vip3A. Proteomic analysis revealed higher expression of proteins involved in glutamate and histidine biosynthesis in cells cultured in TB compared to LB about 58 and 33 times, respectively. Experiments confirmed that glutamate supplementation could increase Vip3A production. In addition, promoter substitution with that of cry3A increased Vip3A yields by about 20-30%. Overall, very high yields of Vip3A could be obtained by culturing Bt294 (Pcry3A-vip3Aa64) in LB*G medium with glutamate supplementation.

Production of Lysinibacillus sphaericus Mosquitocidal Protein Mtx2 from Bacillus subtilis as a Secretory Protein.

BACKGROUND: Mtx2 is a mosquitocidal toxin produced during the vegetative growth of Lysinibacillus sphaericus. The protein shows synergism with other toxins against mosquito larvae; hence it could be used in mosquito control formulations. The protein expression system is needed for Mtx2 development as a biocontrol agent. OBJECTIVE: This study aimed to set up a Bacillus subtilis system to produce Mtx2 as a secreted protein since the protein contains a putative signal peptide. METHODS: Initially, four different promoters (P43, Pspac, PxylA, and PyxiE) were compared for their strength using GFP as a reporter in B. subtilis. Subsequently, six different signal peptides (SacB, Epr, AmyE, AprE, LipA, and Vip3A) were tested in conjunction with the selected promoter and mtx2 to evaluate levels of Mtx2 secreted by B. subtilis WB800, an extracellular protease-deficient strain. RESULTS: The promoter PyxiE showed the highest GFP intensity and was selected for further study. Mtx2 was successfully produced as a secreted protein from signal peptides LipA and AmyE, and it exhibited larvicidal activity against Aedes aegypti. CONCLUSION: B. subtilis was successfully developed as a host for the production of secreted Mtx2, and the protein retained its larvicidal activity. Although the Mtx2 production level still needs improvement, the constructed plasmids could be used to produce other soluble proteins.

The Fungus Metarhizium sp. BCC 4849 Is an Effective and Safe Mycoinsecticide for the Management of Spider Mites and Other Insect Pests.

Five isolates of Metarhizium sp. were evaluated for their pathogenicity against the spider mite (Tetranychus truncatus Ehara) (Acari: Tetranychidae) and Metarhizium sp. BCC 4849 resulted in the highest mortality (82%) on the 5th day post-inoculation (DPI). Subsequent insect bioassay data indicated similar high virulence against five other insects: African red mites (Eutetranychus africanus Tucker) (Acari: Tetranychidae), bean aphid (Aphis craccivora Koch) (Hemiptera: Aphididae), cassava mealybug (Phenacoccus manihoti Matile-Ferrero) (Hemiptera: Pseudococcidae), sweet potato weevil (Cylas formicarius Fabricius) (Coleoptera: Brentidae), and oriental fruit fly (Bactrocera dorsalis Hendel) (Diptera: Tephritidae), at mortalities of 92-99%, on 3rd-6th DPI, and in laboratory conditions. The pathogenicity assay against E. africanus in hemp plants under greenhouse conditions indicated 85-100% insect mortality on 10th DPI using the fungus alone or in combination with synthetic acaricide. Genome sequencing of Metarhizium sp. BCC 4849 revealed the high abundance of proteins associated with zinc-, heme-, and iron-binding; oxidation-reduction; and transmembrane transport, implicating its versatile mode of interaction with the environment and adaptation to various ion homeostasis. The light and scanning electron microscopy indicated that at 24 h post inoculation (PI), adhesion and appressorial formation occurred, notably near the setae. Most infected mites had stopped moving and started dying by 48-72 h PI. Elongated hyphal bodies and oval blastospores were detected in the legs. At 96-120 h PI or longer, dense mycelia and conidial mass had colonized the interior and exterior of dead mites, primarily at the bottom than the upper part. The shelf-life study also indicated that conidial formulation combined with an oxygen-moisture absorber markedly enhanced the viability and germination after storage at 35 °C for four months. The fungus was tested as safe for humans and animals, according to our toxicological assays.

Antagonistic Effect of Truncated Fragments of Bacillus thuringiensis Vip3Aa on the Larvicidal Activity of its Full-length Protein.

BACKGROUND: Vip3Aa is a vegetative insecticidal protein produced by Bacillus thuringiensis. The protein is produced as an 88-kDa protoxin that could be processed by insect gut proteases into a 22-kDa N-terminal and a 66-kDa C-terminal fragments. The C-terminal part could bind to a specific receptor while the N-terminal part is required for toxicity and structural stability. OBJECTIVE: To demonstrate the antagonistic effect of truncated fragments on the insecticidal activity of the full-length Vip3Aa. METHODS: The full-length protein (Vip3Aa), a 66-kDa C-terminal fragment (Vip3Aa-D199) and a predicted carbohydrate binding module (CBM) were produced in Escherichia coli. Purified proteins were mixed at different ratios and fed to Spodoptera litura and Spodoptera exigua larvae. Mortality was recorded and compared between larvae fed with individual toxin and mixtures of the full-length and truncated toxins. RESULTS: Production level of the Vip3Aa-D199 was significantly decreased comparing to that of the full-length protein. Vip3Aa-D199 and CBM fragment were not toxic to insect larvae whereas Vip3Aa showed high toxicity with LC50 about 200 ng/cm2. Feeding the larvae with mixtures of the Vip3Aa and Vip3Aa-D199 at different ratios revealed antagonistic effect of the Vip3Aa-D199 on the toxicity of Vip3Aa. Results showed that the lethal time (LT 50 and LT 95) of larvae fed the mixture toxins was longer than those fed the Vip3Aa alone. In addition, a CBM fragment could inhibit toxicity of the full-length Vip3Aa. CONCLUSION: Our results demonstrated that the Vip3Aa-D199 and a CBM fragment could complete for the membrane binding thus rendering activity of the full-length Vip3Aa.

In vitro analysis of the anticancer activity of Lysinibacillus sphaericus binary toxin in human cancer cell lines.

Binary or Bin toxin produced by Lysinibacillus sphaericus is composed of BinA (42 kDa) and BinB (51 kDa) subunits. These work together to exert maximal toxicity against mosquito larvae via pore formation and induction of apoptosis. The C-terminal domains in both subunits are homologous to those of aerolysin-type ß pore-forming toxins, including parasporin-2 (PS2). The latter is one of the Bacillus thuringiensis toxins that exhibits specific cytotoxicity against human cancer cells. The present study investigates the possible anticancer activity of Bin toxin using PS2 as a control. We demonstrate that treatment with a high concentration of trypsin-activated Bin inhibits cell proliferation in human cancer cells A549, Caco-2, HepG2, HK-1 and KKU-M055. In the most susceptible cells, HK-1, Bin toxin exposure led to morphological alterations, decreased migration, decreased adhesion activity and apoptosis induction. Although these effects necessitated high concentrations, they suggest that Bin toxin may be optimized as a novel potential cancer-therapeutic agent.

Protein-Lipid Interaction of Cytolytic Toxin Cyt2Aa2 on Model Lipid Bilayers of Erythrocyte Cell Membrane.

Cytolytic toxin (Cyt) is a toxin among Bacillus thuringiensis insecticidal proteins. Cyt toxin directly interacts with membrane lipids for cytolytic action. However, low hemolytic activity is desired to avoid non-specific effects in mammals. In this work, the interaction between Cyt2Aa2 toxin and model lipid bilayers mimicking the erythrocyte membrane was investigated for Cyt2Aa2 wild type (WT) and the T144A mutant, a variant with lower hemolytic activity. Quartz crystal microbalance with dissipation (QCM-D) results revealed a smaller lipid binding capacity for the T144A mutant than for the WT. In particular, the T144A mutant was unable to bind to the phosphatidylcholine lipid (POPC) bilayer. However, the addition of cholesterol (Chol) or sphingomyelin (SM) to the POPC bilayer promoted binding of the T144 mutant. Moreover, atomic force microscopy (AFM) images unveiled small aggregates of the T144A mutant on the 1:1 sphingomyelin/POPC bilayers. In contrast, the lipid binding trend for WT and T144A mutant was comparable for the 1:0.4 POPC/cholesterol and the 1:1:1 sphingomyelin/POPC/cholesterol bilayers. Furthermore, the binding of WT and T144A mutant onto erythrocyte cells was investigated. The experiments showed that the T144A mutant and the WT bind onto different areas of the erythrocyte membrane. Overall the results suggest that the T144 residue plays an important role for lipid binding.

Intracellular localization and cytotoxicity of Bacillus thuringiensis Vip3Aa against Spodoptera frugiperda (Sf9) cells.

Vip3Aa protein is produced by Bacillus thuringiensis during vegetative growth and displays high toxicity against a wide range of lepidopteran insect larvae such as Spodoptera exigua and Spodoptera frugiperda, both important insect pests worldwide. Vip3Aa protein is synthesized as a protoxin (proVip3Aa) and becomes activated by digestion with either trypsin or insect gut proteases. The activated Vip3Aa protein (actVip3Aa) binds to a specific receptor in the brush border epithelial midgut cells, causing cell death via apoptosis, possibly induced by its pore-forming activity. Here we investigated the actVip3Aa intracellular localization to explain the molecular mechanism leading to the cytotoxicity of Vip3Aa toxin. The Spodoptera frugiperda (Sf9) cell line was incubated with fluorescently labeled Vip3Aa, namely Alexa488-actVip3Aa, and the intracellular localization was analyzed through a laser scanning confocal microscope. The Alexa488-actVip3Aa was internalized into the Sf9 cells. Immunofluorescence detection demonstrated that Alexa488-actVip3Aa did not colocalize with early endosomes which is usually implicated in clathrin-mediated endocytosis, suggesting that the actVip3Aa does not use clathrin-dependent endocytosis to transport into the cytosol. Intracellular visualization also shows that actVip3Aa does not directly target to mitochondria upon entry into the cytosol. Following internalization, actVip3Aa causes cell division disruption that subsequently could trigger cell death via apoptosis.

Modulation of Cas9 level for efficient CRISPR-Cas9-mediated chromosomal and plasmid gene deletion in Bacillus thuringiensis.

OBJECTIVES: To set up an efficient gene editing system in Bacillus thuringiensis (Bt) using CRISPR-Cas9 by demonstrating deletion of chromosomal and plasmid genes. RESULTS: CRISPR-Cas9 from Streptococcus pyogenes was found to function in Bt cells, resulting in DNA cleavage that is lethal to the cells. The system was assessed for its ability to mediate gene editing by knock-out of the protease genes nprA (neutral protease A) and aprA (alkaline protease A). Gene editing was not detected when the Bacillus-derived pBCX was used to carry CRISPR-Cas9 elements and a DNA repair template. When the Cas9 promoter was replaced with the sporulation-specific promoter cyt2A, a Bt ∆nprA clone was obtained, but this plasmid construct did not give reproducible results. Bt ∆nprA ∆aprA and Bt ∆aprA deletion mutants were finally generated when the Lactobacillus plantarum-derived plasmid pLPPR9 was used, likely due to its lower copy number reducing Cas9 toxicity. Only three to four clones each needed to be screened to identify the desired gene-modified mutants. Conversely, efficient editing of the plasmid vip3A gene required the use of pBCX and longer homology sequences for the repair template. CONCLUSIONS: Capitalizing on the differential impact of plasmid copy number and homology arm length, we devised distinct yet simple and efficient approaches to chromosomal and plasmid gene deletion for Bt that condense the screening process, minimize screening, and facilitate multiple consecutive gene editing steps.

Lipid phase influences the binding of Bacillus thuringiensis Cyt2Aa2 toxin on model lipid membranes.

Bacillus thuringiensis is a bacterium that produces many insecticidal proteins including cytolytic proteins or Cyt toxins. Although the Cyt toxin shows specific toxicity against Dipteran insect species, the toxin binds directly to the lipid membrane without a specific protein receptor requirement. In this work, we have investigated the interaction of Cyt2Aa2 toxin with lipid bilayers composed of different lipid phases. By means of atomic force microscopy (AFM), lipid phase separation was observed for 1:1 and 4:1 M mixtures of DPPC/POPC bilayers. The exposure of Cyt2Aa2 to these lipid bilayers revealed that the toxin selectively bound to Ld lipid bilayer (corresponding to POPC). In turn, it did not bind to the Lo and So phases (corresponding to DPPC). Interestingly, for the bilayer of 4:1 DPPC/POPC mixture, the binding of Cyt2Aa2 was localized at the lipid phase boundary instead of Ld domain as occurred for the 1:1 DPPC/POPC bilayer. In addition, quartz crystal microbalance with dissipation experiments confirmed AFM results. In particular, the measurements showed that amount of protein bound to 1:1 DPPC/POPC (with phase separation) was half of the binding quantified for the Ld phase lipid bilayer (pure POPC and 1:4 DPPC/POPC mixture). These results indicate that the lipid phase (lipid acyl chain) influences the Cyt2Aa2-lipid interaction.

Tyrosine-776 of Vip3Aa64 from Bacillus thuringiensis is Important for Retained Larvicidal Activity During High-Temperature Storage.

Vip3Aa (vegetative insecticidal protein) secreted by Bacillus thuringiensis (Bt) is highly toxic to lepidopteran insects. The Bt isolate M190 produces Vip3Aa35 at high concentrations, and Vip3Aa35 was found to be very effective against Spodoptera exigua. Unfortunately, the use of Vip3Aa35 in pest control is limited by its short shelf life when stored at high temperatures, retaining activity for only 1 month at 37 °C. To find a more stable alternative, we screened 500 isolates of Bt collected from various locations in Thailand and discovered Bt isolate 294 which produced large amounts of Vip3Aa64 that exhibited high toxicity against S. exigua but could be stored at 37 °C for up to 3 months. Vip3Aa35 and Vip3Aa64 have only nine amino acid differences between them, with six of those residues being located at the C terminus. Vip3Aa35 and Vip3Aa64 chimeras revealed that the C-terminal sequence is important for the retained larvicidal activity observed with Vip3Aa64. Various single amino acid substitutions were created to identify the key amino acids responsible for this stability. A single residue, Tyr776, was found to be solely responsible, with the Vip3Aa35:N776Y acquiring thermostability similar to Vip3Aa64 while the Vip3Aa64:Y776N exhibited Vip3Aa35-like thermostability.

Cholesterol Increases Lipid Binding Rate and Changes Binding Behavior of Bacillus thuringiensis Cytolytic Protein.

Cytolytic protein (Cyt) is a member of insecticidal proteins produced by Bacillus thuringiensis. Cyt protein has activity against insect cells and mammalian cells, which differ in lipid and cholesterol composition. This study presents the lipid binding behavior of Cyt2Aa2 protein on model membranes containing different levels of cholesterol content by combining Quartz Crystal Microbalance with Dissipation (QCM-D) and Atomic Force Microscopy (AFM). QCM-D results revealed that cholesterol enhances the binding rate of Cyt2Aa2 protein onto lipid bilayers. In addition, the thicker lipid bilayer was observed for the highest cholesterol content. These results were confirmed by AFM. The analysis of protein surface coverage as a function of time showed a slower process for 5:0 and 5:0.2 (POPC:Chol) ratios than for 5:1 and 5:2 (POPC:Chol) ratios. Significantly, the Cyt2Aa2-lipid binding behavior and the protein⁻lipid layer were different for the 5:3 (POPC:Chol) ratio. Furthermore, AFM images revealed a transformation of Cyt2Aa2/lipid layer structure from strip pattern to ring shape structures (which showed a strong repulsion with AFM tip). In summary, cholesterol increases the binding rate and alters the lipid binding behavior of Cyt2Aa2 protein, although it is not required for Cyt2Aa2 protein binding onto lipid bilayers.

pH regulates pore formation of a protease activated Vip3Aa from Bacillus thuringiensis.

Vip3Aa insecticidal protein is produced from Bacillus thuringiensis and exerts a broad spectrum of toxicity against lepidopteran insect species. Although Vip3Aa has been effectively used as part of integrated pest management strategies, the mechanism of the toxin remains unclear. Here, we investigated the effect of pH in a range from 5.0 to 10.0 on the pore-forming activity of the trypsin activated Vip3Aa (actVip3Aa) by in vitro pore-forming assays. Based on calcein release assay, actVip3Aa could permeabilize the artificial neutral liposomes under all the pH tested, except pH10.0. The maximum membrane permeability of actVip3Aa was detected at pH8.0 and the permeability decreased and abolished when exposing to acidic and alkaline conditions, respectively. The planar lipid bilayer experiment revealed that actVip3Aa formed ion channels at pH5.0-8.0 but no current signals were detected at pH10.0, consistent with the observation from calcein release assay. The toxin formed ion channels with a diameter of 1.4nm at pH8.0 and pore size was gradually decreased when reducing the pH. This study provided a view of the molecular mechanism of Vip3Aa by which the pore-forming activity is regulated by pH.

Bacillus thuringiensis Cyt2Aa2 binding on lipid/cholesterol bilayer depends on protein concentration and time.

Bacillus thuringiensis produces cytolytic proteins (Cyt) that show toxicity against dipteran insect larvae acting directly on the cell membrane. Up to now, two different models have been proposed to explain the interaction mechanism of the cytolytic protein Cyt2Aa2 on lipid membranes: pore-forming and detergent-like action. Here we report on the interaction of Cyt2Aa2 with lipid/cholesterol bilayers at early stage (far from equilibrium) as a function of protein concentration. Quartz crystal microbalance with dissipation (QCM-D) measurements showed that the rate of protein adsorption increased with concentration, although the mass of the final protein-lipid was similar after two hours. In addition, the dissipation (compliance of the hybrid lipid/protein layer) increased with decreasing protein concentration. Furthermore, atomic force microscopy (AFM) revealed that the structure of the protein-lipid layer was concentration and time dependent. A rigid hybrid homogeneous layer was observed at protein concentrations of 50 µg/ml and 100 µg/ml after 30 min. At lower concentrations, 10 µg/ml and 17.5 µg/ml, protein adsorption on the lipid layer led to the formation of small aggregates. Interestingly, at 25 µg/ml a transition of a hole-like structure into a homogeneous layer was observed. This suggests that 25 µg/ml is a threshold concentration for the binding mechanism of Cyt2Aa2 on to lipid membranes.

Molecular analysis of Culex quinquefasciatus larvae responses to Lysinibacillus sphaericus Bin toxin.

Lysinibacillus sphaericus produces the mosquito larvicidal binary toxin consisting of BinA and BinB, which are both required for toxicity against Culex and Anopheles larvae. The molecular mechanisms behind Bin toxin-induced damage remain unexplored. We used whole-genome microarray-based transcriptome analysis to better understand how Culex larvae respond to Bin toxin treatment at the molecular level. Our analyses of Culex quinquefasciatus larvae transcriptome changes at 6, 12, and 18 h after Bin toxin treatment revealed a wide range of transcript signatures, including genes linked to the cytoskeleton, metabolism, immunity, and cellular stress, with a greater number of down-regulated genes than up-regulated genes. Bin toxin appears to mainly repress the expression of genes involved in metabolism, the mitochondrial electron transport chain, and the protein transporter of the outer/inner mitochondrial membrane. The induced genes encode proteins linked to mitochondrial-mediated apoptosis and cellular detoxification including autophagic processes and lysosomal compartments. This study is, to our knowledge, the first microarray analysis of Bin toxin-induced transcriptional responses in Culex larvae, providing a basis for an in-depth understanding of the molecular nature of Bin toxin-induced damage.

Conditions for homogeneous preparation of stable monomeric and oligomeric forms of activated Vip3A toxin from Bacillus thuringiensis.

Bacillus thuringiensis vegetative insecticidal proteins like Vip3A have been used for crop protection and to delay resistance to existing insecticidal Cry toxins. However, little is known about Vip3A's behavior or its mechanism of action, and a structural model is required. Herein, in an effort to facilitate future crystallization and functional studies, we have used the orthogonal biophysical techniques of light scattering and sedimentation to analyze the aggregation behavior and stability of trypsin-activated Vip3A toxin in solution. Both scattering and sedimentation data suggest that at pH 10 the toxin is monomeric and adopts an elongated shape, but after overnight incubation aggregation was observed at all pH values tested (5-12). The narrowest size distribution was observed at pH 7, but it was consistent with large oligomers of ~50 nm on average. The addition of ß-D-glucopyranoside (OG) helped in achieving preparations that were stable and with a narrower particle size distribution. In this case, scattering was consistent with a 4-nm monomeric globular Vip3A form. After OG dialysis, 40-nm particles were detected, with a molecular weight consistent with homotetramers. Therefore, OG is proposed as the detergent of choice to obtain a Vip3A crystal for structural studies, either before (monomers) or after dialysis (tetramers).

Bacillus thuringiensis Cyt2Aa2 toxin disrupts cell membranes by forming large protein aggregates.

Bacillus thuringiensis (Bt) Cyt2Aa2 showed toxicity against Dipteran insect larvae and in vitro lysis activity on several cells. It has potential applications in the biological control of insect larvae. Although pore-forming and/or detergent-like mechanisms were proposed, the mechanism underlying cytolytic activity remains unclear. Analysis of the haemolytic activity of Cyt2Aa2 with osmotic stabilizers revealed partial toxin inhibition, suggesting a distinctive mechanism from the putative pore formation model. Membrane permeability was studied using fluorescent dye entrapped in large unilamellar vesicles (LUVs) at various protein/lipid molar ratios. Binding of Cyt2Aa2 monomer to the lipid membrane did not disturb membrane integrity until the critical protein/lipid molar ratio was reached, when Cyt2Aa2 complexes and cytolytic activity were detected. The complexes are large aggregates that appeared as a ladder when separated by agarose gel electrophoresis. Interaction of Cyt2Aa2 with Aedes albopictus cells was investigated by confocal microscopy and total internal reflection fluorescent microscopy (TIRF). The results showed that Cyt2Aa2 binds on the cell membrane at an early stage without cell membrane disruption. Protein aggregation on the cell membrane was detected later which coincided with cell swelling. Cyt2Aa2 aggregations on supported lipid bilayers (SLBs) were visualized by AFM. The AFM topographic images revealed Cyt2Aa2 aggregates on the lipid bilayer at low protein concentration and subsequently disrupts the lipid bilayer by forming a lesion as the protein concentration increased. These results supported the mechanism whereby Cyt2Aa2 binds and aggregates on the lipid membrane leading to the formation of non-specific hole and disruption of the cell membrane.