Investigation of the pore-forming mechanism of a cytolytic delta-endotoxin from Bacillus thuringiensis.

Cyt2Aa1 is a cytolytic protein produced by Bacillus thuringiensis subsp. kyushuensis. Penetration of the toxin into membranes has been studied to learn more about membrane-insertion mechanisms and transmembrane-pore formation. The haemolysis assay of Cyt2Aa1 showed a steep and sigmoidal dose-response curve, indicating that toxin aggregation or oligomerization is required for pore formation. Studies of the effect of temperature on pore formation and fluorimetric studies of acrylodan-labelled toxin suggest that toxin inserts into the membrane before oligomerizing to form a pore. Low temperature neither inhibited membrane binding nor closed pores that have been formed, but markedly inhibited oligomerization of the toxin molecules. When toxin-treated red blood cells at 4 degrees C were transferred to a toxin-free solution at 37 degrees C, no significant increase in haemolysis was observed. This result suggests that membrane-bound toxin could not diffuse laterally and interact with other molecules to form a pore. From these results, we propose that Cyt2Aa1 binds and inserts into the membrane as a monomer. Oligomerization occurs when toxin molecules have bound in close proximity to each other and pores are formed from large oligomers

Cholesterol Increases Lipid Binding Rate and Changes Binding Behavior of <i>Bacillus thuringiensis</i> 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&#8315;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

Isoleucine at position 150 of Cyt2Aa toxin from Bacillus thuringiensis plays an important role during membrane binding and oligomerization

Cyt2Aa2 is a mosquito larvicidal and cytolytic toxin producedby Bacillus thuringiensis subsp. darmstadiensis. The toxin becomesinactive when isoleucine at position 150 was replacedby alanine. To investigate the functional role of this position,Ile150 was substituted with Leu, Phe, Glu and Lys. All mutantproteins were produced at high level, solubilized in carbonatebuffer and yielded protease activated product similar to thoseof the wild type. Intrinsic fluorescence spectra analysis suggestedthat these mutants retain similar folding to the wildtype. However, mosquito larvicidal and hemolytic activitiesdramatically decreased for the I150K and were completelyabolished for I150A and I150F mutants. Membrane bindingand oligomerization assays demonstrated that only I150E andI150L could bind and form oligomers on lipid membrane similarto that of the wild type. Our results suggest that amino acidat position 150 plays an important role during membranebinding and oligomerization of Cyt2Aa2 toxin. [BMB Reports2013; 46(3): 175-180

Real-time qPCR confirmation of cell death-related genes in <i>Culex</i> treated with Bin toxin at 6, 12, or 18 h.

<p>Bar charts represent -fold changes in caspase-1, caspase-3, and cytochrome c gene expression levels relative to levels in the non-treated <i>C</i>. <i>quinquefasciatus</i> larval gut. Error bars indicate the standard error of the mean from four biological replicates by Student’s <i>t</i>-test. Numeric data is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175473#pone.0175473.s003" target="_blank">S3 Table</a>.</p

Molecular analysis of <i>Culex quinquefasciatus</i> larvae responses to <i>Lysinibacillus sphaericus</i> Bin toxin

<div><p><i>Lysinibacillus sphaericus</i> produces the mosquito larvicidal binary toxin consisting of BinA and BinB, which are both required for toxicity against <i>Culex</i> and <i>Anopheles</i> larvae. The molecular mechanisms behind Bin toxin-induced damage remain unexplored. We used whole-genome microarray-based transcriptome analysis to better understand how <i>Culex</i> larvae respond to Bin toxin treatment at the molecular level. Our analyses of <i>Culex quinquefasciatus</i> 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 <i>Culex</i> larvae, providing a basis for an in-depth understanding of the molecular nature of Bin toxin-induced damage.</p></div

Functional classification of the Bin toxin intoxication transcriptome.

<p>The significantly changed transcripts in <i>Culex</i> after Bin toxin treatment for 6, 12, or 18 h were subsequently classified into 11 functional groups: 1) cytoskeletal and structural function (CST); 2) chemosensory reception (CSR); 3) blood and sugar food digestive (DIG); 4) diverse functions (DIV); 5) immunity (IMM); 6) metabolism (MET); 7) proteolysis (PRT); 8) redox, stress, and mitochondrion (RSM); 9) replication, transcription, and translation (RTT); 10) transport (TRP); and 11) unknown function (UNK). Bin toxin treatment responsive gene expression data is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175473#pone.0175473.s002" target="_blank">S2 Table</a>.</p

Physical Factors Affecting the Scale-Up of Vegetative Insecticidal Protein (Vip3A) Production by <i>Bacillus thuringiensis</i> Bt294

Vip3A (vegetative insecticidal protein) is a representative member of the Vip3 family, which is widely used for lepidopteran pest control. This Vip3A protein, a non-growth-associated protein, is an effective bioinsecticide against insect pests, but there is relatively little information about its production processes at large scales. Hence, the effects of environmental factors on Vip3A production by Bacillus thuringiensis Bt294 (antifoam agents, shaking speeds, agitation and aeration rates), as well as controlling physical conditions such as the lowest point of dissolved oxygen and controlling of culture pH, were observed in shaking flasks and bioreactors. The results showed that antifoam agents, flask types and shaking speeds had significant effects on Vip3A and biomass production. Cultivation without pH control and DO control in 5 L bioreactors at lower agitation and aeration rates, which was not favorable for biomass production, resulted in a high Vip3A protein production of 5645.67 mg/L. The scale-up studies of the Vip3A protein production in a pilot-scale 750 L bioreactor gave 3750.0 mg/L. Therefore, this study demonstrated the significant effects of agitation, aeration rates and culture pH on Vip3A production by B. thuringiensis Bt294. Balancing of physical conditions was necessary for obtaining the highest yield of Vip3A by slowing down the production rate of biomass. Moreover, this Vip3A protein has high potential as a bioinsecticide for lepidopteran pest control in organic crops. This information will be important for significantly increasing the Vip3A protein concentration by the bacterium and will be useful for field application at a lower cost