In vitro analysis of the anticancer activity of Lysinibacillus sphaericus binary toxin in human cancer cell lines.
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SUBMITTER: Chankamngoen W
PROVIDER: S-EPMC7394974 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Lysinibacillus sphaericus harboring Binary (BinA and BinB) toxins is highly toxic against Anopheles and Culex mosquito larvae. The Anopheles Ag55 cell line is a suitable model for investigating the mode of Bin toxin action. Based on the low-levels of α-glycosidase Agm3 mRNA in Ag55 cells and the absence of detectable Agm3 proteins, we hypothesized that a scavenger receptor could be mediating Bin cytotoxicity. Preliminary RNA interference knockdown of the expressed scavenger receptors, combined with Bin cytotoxicity assays, was conducted. The scavenger Receptor C1 (SCRC1) became the focus of this study, as a putative receptor for Bin toxins in Ag55 cells, and SCRBQ2 was selected as a negative control. Open reading frames encoding SCRC1 and SCRBQ2 were cloned and expressed in vitro, and polyclonal antibodies were prepared for immunological analyses. The RNAi silencing of SCRC1 and SCRBQ2 resulted in the successful knockdown of both SCRC1 and SCRBQ2 transcripts and protein levels. The cytolytic toxicity of Bin against Ag55 cells was severely reduced after the SCRC1-RNAi treatment. The phagocytic receptor protein SCRC1 mediates endocytosis of the Bin toxin into Ag55 cells, thereby facilitating its internal cytological activity. The results support a mechanism of the Bin toxin entering Ag55 cells, possibly via SCRC1-mediated endocytosis, and encourage investigations into how Bin is transferred from its bound form on the midgut epithelial cells into the epithelial endocytic system.
| S-EPMC11359365 | biostudies-literature
Project description:BackgroundThe resistance of a Culex quinquefasciatus strain to the binary (Bin) larvicidal toxin from Lysinibacillus sphaericus is due to the lack of expression of the toxin's receptors, the membrane-bound Cqm1 α-glucosidases. A previous transcriptomic profile of the resistant larvae showed differentially expressed genes coding Cqm1, lipases, proteases and other genes involved in lipid and carbohydrate metabolism. This study aimed to investigate the metabolic features of Bin-resistant individuals by comparing the activity of some enzymes, energy reserves, fertility and fecundity to a susceptible strain.MethodsThe activity of specific enzymes was recorded in midgut samples from resistant and susceptible larvae. The amount of lipids and reducing sugars was determined for larvae and adults from both strains. Additionally, the fecundity and fertility parameters of these strains under control and stress conditions were examined.ResultsEnzyme assays showed that the esterase activities in the midgut of resistant larvae were significantly lower than susceptible ones using acetyl-, butyryl- and heptanoyl-methylumbelliferyl esthers as substrates. The α-glucosidase activity was also reduced in resistant larvae using sucrose and a synthetic substrate. No difference in protease activities as trypsins, chymotrypsins and aminopeptidases was detected between resistant and susceptible larvae. In larval and adult stages, the resistant strain showed an altered profile of energy reserves characterized by significantly reduced levels of lipids and a greater amount of reducing sugars. The fertility and fecundity of females were similar for both strains, indicating that those changes in energy reserves did not affect these reproductive parameters.ConclusionsOur dataset showed that Bin-resistant insects display differential metabolic features co-selected with the phenotype of resistance that can potentially have effects on mosquito fitness, in particular, due to the reduced lipid accumulation.
| S-EPMC10413512 | biostudies-literature
Project description: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.
| S-EPMC8073846 | biostudies-literature
Project description:The binary (Bin) toxin from Lysinibacillus sphaericus is effective to mosquito larvae, but its utilization is threatened by the development of insect resistance. Bin toxin is composed of the BinB subunit required for binding to midgut receptors and the BinA subunit that causes toxicity after cell internalization, mediated by BinB. Culex quinquefasciatus resistance to this toxin is caused by mutations that prevent expression of Bin toxin receptors in the midgut. Previously, it was shown that the Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis restores Bin toxicity to Bin-resistant C. quinquefasciatus and to Aedes aegypti larvae, which are naturally devoid of functional Bin receptors. Our goal was to elucidate the mechanism involved in Cyt1Aa synergism with Bin in such larvae. In vivo assays showed that the mixture of Bin toxin, or its BinA subunit, with Cyt1Aa was effective to kill resistant larvae. However, no specific binding interaction between Cyt1Aa and the Bin toxin, or its subunits, was observed. The synergy between Cyt1Aa and Bin toxins is dependent on functional Cyt1Aa, as demonstrated by using the nontoxic Cyt1AaV122E mutant toxin affected in oligomerization and membrane insertion, which was unable to synergize Bin toxicity in resistant larvae. The synergism correlated with the internalization of Bin or BinA into anterior and medium midgut epithelial cells, which occurred only in larvae treated with wild-type Cyt1Aa toxin. This toxin is able to overcome failures in the binding step involving BinB receptor by allowing the internalization of Bin toxin, or its BinA subunit, into the midgut cells.IMPORTANCE One promising management strategy for mosquito control is the utilization of a mixture of L. sphaericus and B. thuringiensis subsp. israelensis insecticidal toxins. From this set, Bin and Cyt1Aa toxins synergize and display toxicity to resistant C. quinquefasciatus and to A. aegypti larvae, whose midgut cells lack Bin toxin receptors. Our data set provides evidence that functional Cyt1Aa is essential for internalization of Bin or its BinA subunit into such cells, but binding interaction between Bin and Cyt1Aa is not observed. Thus, this mechanism contrasts with that for the synergy between Cyt1Aa and the B. thuringiensis subsp. israelensis Cry toxins, where active Cyt1Aa is not necessary but a specific binding between Cry and Cyt1Aa is required. Our study established the initial molecular basis of the synergy between Bin and Cyt1Aa, and these findings enlarge our knowledge of their mode of action, which could help to develop improved strategies to cope with insect resistance.
| S-EPMC7082581 | biostudies-literature
Project description:Culex quinquefasciatus resistance to the binary (Bin) toxin, the major larvicidal component from Lysinibacillus sphaericus, is associated with mutations in the cqm1 gene, encoding the Bin-toxin receptor. Downregulation of the cqm1 transcript was found in the transcriptome of larvae resistant to the L. sphaericus IAB59 strain, which produces both the Bin toxin and a second binary toxin, Cry48Aa/Cry49Aa. Here, we investigated the transcription profiles of two other mosquito colonies having Bin resistance only. These confirmed the cqm1 downregulation and identified transcripts encoding the enzyme pantetheinase as the most downregulated mRNAs in both resistant colonies. Further quantification of these transcripts reinforced their strong downregulation in Bin-resistant larvae. Multiple genes were found encoding this enzyme in Cx. quinquefasciatus and a recombinant pantetheinase was then expressed in Escherichia coli and Sf9 cells, with its presence assessed in the midgut brush border membrane of susceptible larvae. The pantetheinase was expressed as a ~70 kDa protein, potentially membrane-bound, which does not seem to be significantly targeted by glycosylation. This is the first pantetheinase characterization in mosquitoes, and its remarkable downregulation might reflect features impacted by co-selection with the Bin-resistant phenotype or potential roles in the Bin-toxin mode of action that deserve to be investigated.
| S-EPMC10813629 | biostudies-literature
Project description: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.
| S-EPMC5391067 | biostudies-literature
Project description:Bacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.
| S-EPMC3038859 | biostudies-literature
Project description:Lysinibacillus sphaericus strains belonging the antigenic group H5a5b produce spores with larvicidal activity against larvae of Culex mosquitoes. C7, a new isolated strain, which presents similar biochemical characteristics and Bin toxins in their spores as the reference strain 2362, was, however, more active against larvae of Culex mosquitoes. The contribution of the surface layer protein (S-layer) to this behaviour was envisaged since this envelope protein has been implicated in the pathogenicity of several bacilli, and we had previously reported its association to spores. Microscopic observation by immunofluorescence detection with anti S-layer antibody in the spores confirms their attachment. S-layers and BinA and BinB toxins formed high molecular weight multimers in spores as shown by SDS-PAGE and western blot detection. Purified S-layer from both L. sphaericus C7 and 2362 strain cultures was by itself toxic against Culex sp larvae, however, that from C7 strain was also toxic against Aedes aegypti. Synergistic effect between purified S-layer and spore-crystal preparations was observed against Culex sp. and Aedes aegypti larvae. This effect was more evident with the C7 strain. In silico analyses of the S-layer sequence suggest the presence of chitin-binding and hemolytic domains. Both biochemical characteristics were detected for both S-layers strains that must justify their contribution to pathogenicity.
| S-EPMC4213006 | biostudies-literature
Project description:The Cry48Aa/Cry49Aa binary toxin of Bacillus sphaericus was recently discovered by its ability to kill Culex quinquefasciatus mosquito larvae through a novel interaction between its two components. We have investigated the target specificity of this toxin and show it to be non-toxic to coleopteran, lepidopteran and other dipteran insects, including closely related Aedes and Anopheles mosquitoes. This represents an unusually restricted target range for crystal toxins from either B. sphaericus or Bacillus thuringiensis. Gut extracts from Culex and Aedes larvae show differential processing of the Cry48Aa protein, with the location of cleavage sites in Culex reflecting those previously shown for the activation of Cry4 toxins in mosquitoes. Pre-activation of Cry48Aa/Cry49Aa with Culex extracts, however, fails to induce toxicity to Aedes larvae. Co-administration of Cry49Aa with Cry4Aa gives higher than predicted toxicity, perhaps suggesting weak synergism against Culex larvae between Cry49Aa and other three-domain Cry toxins.
| S-EPMC3638318 | biostudies-literature
Project description: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.
2017-03-22 | GSE96838 | GEO