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.

Channel Formation in Cry Toxins: An Alphafold-2 Perspective.

Bacillus thuringiensis (Bt) strains produce pore-forming toxins (PFTs) that attack insect pests. Information for pre-pore and pore structures of some of these Bt toxins is available. However, for the three-domain (I-III) crystal (Cry) toxins, the most used Bt toxins in pest control, this crucial information is still missing. In these Cry toxins, biochemical data have shown that 7-helix domain I is involved in insertion in membranes, oligomerization and formation of a channel lined mainly by helix α4, whereas helices α1 to α3 seem to have a dynamic role during insertion. In the case of Cry1Aa, toxic against Manduca sexta larvae, a tetrameric oligomer seems to precede membrane insertion. Given the experimental difficulty in the elucidation of the membrane insertion steps, we used Alphafold-2 (AF2) to shed light on possible oligomeric structural intermediates in the membrane insertion of this toxin. AF2 very accurately (<1 Å RMSD) predicted the crystal monomeric and trimeric structures of Cry1Aa and Cry4Ba. The prediction of a tetramer of Cry1Aa, but not Cry4Ba, produced an 'extended model' where domain I helices α3 and α2b form a continuous helix and where hydrophobic helices α1 and α2 cluster at the tip of the bundle. We hypothesize that this represents an intermediate that binds the membrane and precedes α4/α5 hairpin insertion, together with helices α6 and α7. Another Cry1Aa tetrameric model was predicted after deleting helices α1 to α3, where domain I produced a central cavity consistent with an ion channel, lined by polar and charged residues in helix α4. We propose that this second model corresponds to the 'membrane-inserted' structure. AF2 also predicted larger α4/α5 hairpin n-mers (14 ≤n ≤ 17) with high confidence, which formed even larger (~5 nm) pores. The plausibility of these models is discussed in the context of available experimental data and current paradigms.

Biosynthesized Silver Nanoparticles Using Morus alba (White Mulberry) Leaf Extract as Potential Antibacterial and Anticancer Agents.

In this study, we report the green synthesis of silver nanoparticles (AgNPs) from Morus alba or white mulberry leaf extract (MLE) and assess their antibacterial and anticancer potential. The GC-MS analysis of MLE confirmed the existence of phenolic compounds, serving as reducing, capping, and stabilizing agents in the biosynthesis of AgNPs. The MLE-AgNPs were spherical, with an average particle size of 20-44.5 nm and a face-centered cubic structure. EDX spectra confirmed the formation of AgNPs, and a negative zeta potential value (-14.5 mV) suggested their physicochemical stability. Excellent antibacterial activity was demonstrated by MLE-AgNPs against Acinetobacter baumannii strains with a MIC of 2 µg/mL, while good activity was observed against other Gram-negative (Escherichia coli and Salmonella typhimurium) and Gram-positive (Bacillus subtilis and Staphylococcus aureus) bacteria with a MIC of 32 µg/mL. In vitro cytotoxic effects on MCF-7 (human breast cancer cells) and MCF-10A (normal human mammary epithelial cells) were investigated by the MTT assay. The half-maximal inhibitory concentrations (IC50) against MCF-7 cells were 18 and 33 µg/mL for MLE-AgNPs and MLE, respectively, with no effect on normal MCF-10A cells. Altogether, the results support the high antibacterial and anticancer potential of biosynthesized AgNPs by white mulberry leaf extract.

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.

An aromatic cluster in Lysinibacillus sphaericus BinB involved in toxicity and proper in-membrane folding.

The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. Based on structural alignments with other toxins, an aromatic cluster in the C-terminal domain of BinB (termed here BC) has been proposed to be important for toxicity. We tested this experimentally using BinB mutants bearing single mutations in this aromatic cluster. Consistent with the hypothesis, two of these mutations, F311A and F315A, were not toxic to Culex quinquefasciatus larvae and were unable to permeabilize liposomes or elicit ion channel activity, in contrast to wild-type BinB. Despite these effects, none of these mutations altered significantly the interaction between the activated forms of the two subunits in solution. These results indicate that these aromatic residues on the C-terminal domain of BinB are critical for toxin insertion in membranes. The latter can be by direct contact of these residues with the membrane surface, or by facilitating the formation a membrane-inserting oligomer.

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.

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).

The crystal structure of JNK from Drosophila melanogaster reveals an evolutionarily conserved topology with that of mammalian JNK proteins.

BACKGROUND: The c-Jun N-terminal kinases (JNKs), members of the mitogen-activated protein kinase (MAPK) family, engage in diverse cellular responses to signals produced under normal development and stress conditions. In Drosophila, only one JNK member is present, whereas ten isoforms from three JNK genes (JNK1, 2, and 3) are present in mammalian cells. To date, several mammalian JNK structures have been determined, however, there has been no report of any insect JNK structure. RESULTS: We report the first structure of JNK from Drosophila melanogaster (DJNK). The crystal structure of the unphosphorylated form of DJNK complexed with adenylyl imidodiphosphate (AMP-PNP) has been solved at 1.79 Å resolution. The fold and topology of DJNK are similar to those of mammalian JNK isoforms, demonstrating their evolutionarily conserved structures and functions. Structural comparisons of DJNK and the closely related mammalian JNKs also allow identification of putative catalytic residues, substrate-binding sites and conformational alterations upon docking interaction with Drosophila scaffold proteins. CONCLUSIONS: The DJNK structure reveals common features with those of the mammalian JNK isoforms, thereby allowing the mapping of putative catalytic and substrate binding sites. Additionally, structural changes upon peptide binding could be predicted based on the comparison with the closely-related JNK3 structure in complex with pepJIP1. This is the first structure of insect JNK reported to date, and will provide a platform for future mutational studies in Drosophila to ascertain the functional role of insect JNK.

Interaction of Lysinibacillus sphaericus binary toxin with mosquito larval gut cells: Binding and internalization.

The binary toxin produced by Lysinibacillus sphaericus is composed of BinA and BinB subunits. Together, but not separately, the two subunits are highly toxic to Culex quinquefasciatus larvae, but show no toxicity to Aedes aegypti. The molecular mechanism underlying intoxication has not been clearly elucidated. The present study compares the binding and the internalization of binary toxin into the midgut epithelial cells of susceptible C. quinquefasciatus mosquito larvae with those of Bin-refractory A. aegypti. The guts from larvae fed with fluorescently labeled toxin were dissected and analyzed using a confocal laser scanning microscope. When fed with a mixture of both components, co-localization of BinA and BinB was detected both on the cell surface and in the cytoplasm of Culex larval gut cells. However, administration of BinA alone resulted in localization only on the cell membrane, whereas BinB alone was detected both on the cell membrane and inside the cytoplasm. In contrast, when a mixture of both components, or each individual component, was fed to Aedes larvae, BinA and BinB were unable to reach the cytoplasm and were localized only on the cell membrane. These results are consistent with the suggestion that the internalization of BinA is essential for toxicity, and that BinB is required for this internalization into susceptible larval gut cells.

Lysinibacillus sphaericus binary toxin induces apoptosis in susceptible Culex quinquefasciatus larvae.

During sporulation, a Gram-positive bacterium Lysinibacillus sphaericus (Ls) produces the mosquito larvicidal binary toxin composed of 2 subunits, BinA and BinB. Full toxicity against Culex and Anopheles mosquito larvae is achieved when both subunits are administered together at equimolar amounts. Although cellular responses to Bin toxin have been reported in previous studies, it remains essential to extensively examine the cytopathic effects in vivo to define the underlying mechanism of larval death. In this study, 4th instar Culex quinquefasciatus larvae fed with different doses of Bin toxin were analyzed both for ultrastructural as well as biochemical effects. Typical morphological changes consistent with apoptosis were observed in mosquito larvae exposed to Bin toxin, including mitochondrial swelling, chromatin condensation, cytoplasmic vacuolization and apoptotic cell formation. Bin toxin also induced the activation of caspase-9 and caspase-3 in larval midgut cells. Our current observations thus suggest that Bin toxin triggers apoptosis via an intrinsic or mitochondrial pathway in vivo, possibly contributing to larval death.

Crystal structure of BinB: a receptor binding component of the binary toxin from Lysinibacillus sphaericus.

The binary toxin (Bin), produced by Lysinibacillus sphaericus, is composed of BinA (42 kDa) and BinB (51 kDa) proteins, which are both required for full toxicity against Culex and Anopheles mosquito larvae. Specificity of Bin toxin is determined by the binding of BinB component to a receptor present on the midgut epithelial membranes, while BinA is proposed to be a toxic component. Here, we determined the first crystal structure of the active form of BinB at a resolution of 1.75 Å. BinB possesses two distinct structural domains in its N- and C-termini. The globular N-terminal domain has a ß-trefoil scaffold which is a highly conserved architecture of some sugar binding proteins or lectins, suggesting a role of this domain in receptor-binding. The BinB ß-rich C-terminal domain shares similar three-dimensional folding with aerolysin type ß-pore forming toxins, despite a low sequence identity. The BinB structure, therefore, is a new member of the aerolysin-like toxin family, with probably similarities in the cytolytic mechanism that takes place via pore formation.

Anticancer efficacy of biosynthesized silver nanoparticles loaded with recombinant truncated parasporin-2 protein.

Bacterial toxins have received a great deal of attention in the development of cancer treatments. Parasporin-2 (PS2Aa1 or Mpp46Aa1) is a Bacillus thuringiensis parasporal protein that preferentially destroys human cancer cells while not harming normal cells, making it a promising anticancer treatment. With the efficient development and sustainable silver nanoparticles (AgNPs) synthesis technology, the biomedical use of AgNPs has expanded. This study presents the development of a novel nanotoxin composed of biosynthesized silver nanoparticles loaded with the N-terminal truncated PS2Aa1 toxin. MOEAgNPs were synthesized using a biological method, with Moringa oleifera leaf extract and maltose serving as reducing and capping agents. The phytochemicals present in M. oleifera leaf extract were identified by GC-MS analysis. MOEAgNPs were loaded with N-terminal truncated PS2Aa1 fused with maltose-binding protein (MBP-tPS2) to formulate PS2-MOEAgNPs. The PS2-MOEAgNPs were evaluated for size, stability, toxin loading efficacy, and cytotoxicity. PS2-MOEAgNPs demonstrated dose-dependent cytotoxicity against the T-cell leukemia MOLT-4 and Jurkat cell lines but had little effect on the Hs68 fibroblast or normal cell line. Altogether, the current study provides robust evidence that PS2-MOEAgNPs can efficiently inhibit the proliferation of T-cell leukemia cells, thereby suggesting their potential as an alternative to traditional anticancer treatments.

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.

Crystallization and preliminary X-ray crystallographic analysis of the functional form of BinB binary toxin from Bacillus sphaericus.

The binary toxin from Bacillus sphaericus consists of two proteins, BinA and BinB, which work together to exert toxicity against mosquito larvae. BinB is proposed to be a receptor-binding domain and internalizes BinA into the midgut cells, resulting in toxicity via an unknown mechanism. The functional form of BinB has been successfully crystallized. The crystals of BinB diffracted to a resolution of 1.75 Å and belong to space group P6(2)22, with unit-cell parameters a = b = 95.2, c = 154.9 Å. Selenomethionine-substituted BinB (SeMetBinB) was prepared and crystallized for experimental phasing. The SeMetBinB crystal data were collected at a wavelength of 0.979 Å and diffracted to a resolution of 1.85 Å.

Amino acid residues in the N-terminal region of the BinB subunit of Lysinibacillus sphaericus binary toxin play a critical role during receptor binding and membrane insertion.

The binary toxin produced by Lysinibacillus sphaericus is composed of BinA and BinB subunits that work together in governing toxicity against mosquito larvae. BinA is proposed to be important for toxicity, whereas BinB has been shown to act as a specific receptor-binding component. The precise function of both subunits, however, is not well established. Here, we investigated the function of the N-terminal region of BinB subunit initially by introducing triple alanine substitutions at positions 35PEI37 and 41FYN43. Both block mutations abolished the larvicidal activity. Single point mutations (P35A, E36A, I37A, F41A, Y42A, N43A) were generated in order to identify amino acids that are critical for the toxin activity. Mosquito-larvicidal activity was significantly reduced in P35A, E36A, F41A and Y42A mutants. However, these mutants retained ability to form in vitro interaction with the BinA counterpart. Immunohistochemistry analysis revealed that P35A, F41A and N43A bind to the larval midgut membrane at comparable levels to that of the wild type BinB. In contrast, greatly reduced binding activity was observed in the Y42A, suggesting an important role of this residue in receptor binding. Alanine substitution at P35 resulted in a marked decrease in membrane penetration, indicating its functional importance for the membrane insertion. These results suggest the important roles of the N-terminal region of BinB in both the receptor recognition and the membrane interaction.

Study on Cellular Localization of Bin Toxin and its Apoptosis-inducing Effect on Human Nasopharyngeal Carcinoma Cells.

BACKGROUND: Bacterial pore-forming toxins, BinA and BinB together known as the binary toxin are potent insecticidal proteins, that share structural homology with antitumor bacterial parasporin-2 protein. The underlying molecular mechanism of Bin toxin-induced cancer cell cytotoxicity requires more knowledge to understand whether the toxin induced human cytotoxic effects occur in the same way as that of parasporin-2 or not. METHODS: In this study, anticancer properties of Lysinibacillus sphaericus derived Bin toxin on HK1 were evaluated through MTT assay, morphological analysis and lactate dehydrogenase efflux assay. Induction of apoptosis was determined from RT-qPCR, caspase activity and cytochrome c release assay. Internalization pattern of Bin toxin in HK1 cells was studied by confocal laser-scanning microscopic analysis. RESULTS: Activated Bin toxin had strong cytocidal activity to HK1 cancer cell line at 24 h postinoculation. Both BinA and BinB treated HK1 cells showed significant inhibition of cell viability at 12 µM. Induction of apoptotic mediators from RT-qPCR and caspase activity analyses indicated the activation of programmed cell death in HK1 cells in response to Bin toxin treatment. Internalization pattern of Bin toxin studied by using confocal microscopy indicated the localization of BinA on cell surface and internalization of BinB in the cytoplasm of cancer cells as well as colocalization of BinA with BinB. Evaluation of cytochrome c release also showed the association of BinB and BinA+BinB with mitochondria. CONCLUSION: Bin toxin is a cytotoxic protein that induces cytotoxic and apoptotic events in HK1 cells, and may have high therapeutic potential as an anti-cancer agent.

Engineering BinB Pore-Forming Toxin for Selective Killing of Breast Cancer Cells.

Breast cancer is one of the most common cancers in women worldwide. Conventional cancer chemotherapy always has adverse side effects on the patient's healthy tissues. Consequently, combining pore-forming toxins with cell-targeting peptides (CTPs) is a promising anticancer strategy for selectively destroying cancer cells. Here, we aim to improve the target specificity of the BinB toxin produced from Lysinibacillus sphaericus (Ls) by fusing a luteinizing hormone-releasing hormone (LHRH) peptide to its pore-forming domain (BinBC) to target MCF-7 breast cancer cells as opposed to human fibroblast cells (Hs68). The results showed that LHRH-BinBC inhibited MCF-7 cell proliferation in a dose-dependent manner while leaving Hs68 cells unaffected. BinBC, at any concentration tested, did not affect the proliferation of MCF-7 or Hs68 cells. In addition, the LHRH-BinBC toxin caused the efflux of the cytoplasmic enzyme lactate dehydrogenase (LDH), demonstrating the efficacy of the LHRH peptide in directing the BinBC toxin to damage the plasma membranes of MCF-7 cancer cells. LHRH-BinBC also caused MCF-7 cell apoptosis by activating caspase-8. In addition, LHRH-BinBC was predominantly observed on the cell surface of MCF-7 and Hs68 cells, without colocalization with mitochondria. Overall, our findings suggest that LHRH-BinBC could be investigated further as a potential cancer therapeutic agent.

Expression and purification of the active soluble form of Bacillus sphaericus binary toxin for structural analysis.

The binary toxin produced from Bacillus sphaericus is highly toxic against larvae of Culex and Anopheles mosquitoes. The two major components of the binary toxin are 42-kDa BinA and 51-kDa BinB, which are produced as crystalline inclusions during sporulation. Currently, there is no detailed knowledge of the molecular mechanism of the binary toxin, mainly due to the lack of structural information. Herein, we describe an expression protocol with modified conditions allowing production of soluble, biologically active BinA and BinB for further structural analysis. The binA and binB genes from B. sphaericus 2297 strain were independently cloned and fused with a polyhistidine tag at their N-termini. Both (His)(6)-tagged BinA and (His)(6)-tagged BinB were expressed as soluble forms at low temperature. Highly pure proteins were obtained after two-step purification by Ni-NTA affinity and size exclusion chromatography. In vitro activation by trypsin digestion generated a resistant fragment, of 40kDa for BinA, and of 45kDa for BinB, and an oligomeric complex of BinA and BinB in solution was observed after proteolytic activation. Their functional and structural properties were confirmed by a biological assay and far-UV circular dichroism, respectively. The mixture of BinA and BinB, either as a protoxin or as a trypsin-activated form, exhibited high mosquito-larvicidal activity against Culex quinquefasciatus larvae with LC(50) of about 10ng/ml, while no toxicity was observed from the single binary toxin component. Results from far-UV circular dichroism of BinA and BinB suggest the presence of mainly ß-structure. The expression and purification protocols reported here will be useful for the production of the active and homogeneous binary toxin to allow further detailed structural investigation.

Functional characterization of truncated fragments of Bacillus sphaericus binary toxin BinB.

Bacillus sphaericus produces a mosquitocidal binary toxin composed of two subunits, BinA (42 kDa) and BinB (51 kDa). Both components are required for maximum toxicity against mosquito larvae. BinB has been proposed to provide specificity by binding to the epithelial gut cell membrane, while BinA may be responsible for toxicity. To identify regions in BinB responsible for receptor binding and for interaction to BinA, we used six BinB shorter constructs derived from both the N-terminal and the C-terminal halves of the protein. All constructs expressed as inclusion bodies in Escherichia coli, similarly to the wild-type protein. A marked decrease in larvicidal activity was observed when BinA was used in combination with these BinB constructs, used either individually or in pairs from both N and C-halves of BinB. Nevertheless, immunohistochemistry analyses demonstrate that these constructs are able to bind to the epithelium gut cell membrane, and in vitro protein-protein interaction assays revealed that these constructs can bind to BinA. These results show that fragments corresponding to both halves of BinB are able to bind the receptor and to interact with BinA, but both halves are required by the toxin to exhibit full larvicidal activity.

Cytotoxic Effects and Intracellular Localization of Bin Toxin from Lysinibacillus sphaericus in Human Liver Cancer Cell Line.

Binary toxin (Bin toxin), BinA and BinB, produced by Lysinibacillus sphaericus has been used as a mosquito-control agent due to its high toxicity against the mosquito larvae. The crystal structures of Bin toxin and non-insecticidal but cytotoxic parasporin-2 toxin share some common structural features with those of the aerolysin-like toxin family, thus suggesting a common mechanism of pore formation of these toxins. Here we explored the possible cytotoxicity of Bin proteins (BinA, BinB and BinA + BinB) against Hs68 and HepG2 cell lines. The cytotoxicity of Bin proteins was evaluated using the trypan blue exclusion assay, MTT assay, morphological analysis and LDH efflux assay. The intracellular localization of Bin toxin in HepG2 cells was assessed by confocal laser scanning microscope. HepG2 cells treated with BinA and BinB (50 µg/mL) showed modified cell morphological features and reduced cell viability. Bin toxin showed no toxicity against Hs68 cells. The EC50 values against HepG2 at 24 h were 24 ng/mL for PS2 and 46.56 and 39.72 µg/mL for BinA and BinB, respectively. The induction of apoptosis in treated HepG2 cells was confirmed by upregulation of caspase levels. The results indicated that BinB mediates the translocation of BinA in HepG2 cells and subsequently associates with mitochondria. The study supports the possible development of Bin toxin as either an anticancer agent or a selective delivery vehicle of anticancer agents to target mitochondria of human cancer cells in the future.