N-terminal Activation Is an Essential Early Step in the Mechanism of Action of the Bacillus thuringiensis Cry1Ac Insecticidal Toxin

Resistance to Bacillus thuringiensis Cry1Ac toxin was characterized in a population of Helicoverpa zea larvae previously shown not to have an alteration in toxin binding as the primary resistance mechanism to this toxin. Cry1Ac-selected larvae (AR1) were resistant to protoxins and toxins of Cry1Ab, Cry1Ac, and the corresponding modified proteins lacking helix ␣-1 (Cry1AbMod and Cry1AcMod). When comparing brush border membrane vesicles (BBMVs) prepared from susceptible (LC) and AR1 larval midguts, there were only negligible differences in overall Cry1Ac toxin binding, though AR1 had 18% reversible binding, in contrast to LC, in which all binding was irreversible. However, no differences were detected in Cry1Ac-induced pore formation activity in BBMVs from both strains. Enzymatic activities of two putative Cry1Ac receptors (aminopeptidase N [APN] and alkaline phosphatase [ALP]) were significantly reduced (2-fold and 3-fold, respectively) in BBMVs from AR1 compared to LC larvae. These reductions corresponded to reduced protein levels in midgut luminal contents only in the case of ALP, with an almost 10-fold increase in specific ALP activity in midgut fluids from AR1 compared to LC larvae. Partially purified H. zea ALP bound Cry1Ac toxin in ligand blots and competed with Cry1Ac toxin for BBMV binding. Based on these results, we suggest the existence of at least one mechanism of resistance to Cry1A toxins in H. zea involving binding of Cry1Ac toxin to an ALP receptor in the larval midgut lumen of resistant larvae.

Section: Fig 4 Effect Of Cry1ac Toxin On Ksupporting

confidence: 73%

Association of Cry1Ac Toxin Resistance in Helicoverpa zea (Boddie) with Increased Alkaline Phosphatase Levels in the Midgut Lumen

Caccia

Moar

Chandrashekhar

et al. 2012

“…There were reports that the N terminus may prevent the interaction of toxin with target membrane, and the removal of the N-terminal peptide is a necessary step in toxin activation. In addition, the C terminus is believed to be important for crystallization and may by extension apply to the activity of Cry toxin, but it is not required for insecticidal activity (6). Therefore, the cleavages of the N terminus and C terminus may enhance the insecticidal activity of Cry protein.…”

Section: Discussionmentioning

confidence: 99%

Novel Bacillus thuringiensis δ-Endotoxin Active against Locusta migratoria manilensis

Lei

Dan

et al. 2011

A novel ␦-endotoxin gene was cloned from a Bacillus thuringiensis strain with activity against Locusta migratoria manilensis by PCR-based genome walking. The sequence of the cry gene was 3,432 bp long, and it encoded a Cry protein of 1,144 amino acid residues with a molecular mass of 129,196.5 kDa, which exhibited 62% homology with Cry7Ba1 in the amino acid sequence. The ␦-endotoxin with five conserved sequence blocks in the amino-terminal region was designated Cry7Ca1 (GenBank accession no. EF486523). Protein structure analysis suggested that the activated toxin of Cry7Ca1 has three domains: 227 residues forming 7 ␣-helices (domain I); 213 residues forming three antiparallel ␤-sheets (domain II); and 134 residues forming a ␤-sandwich (domain III). The three domains, respectively, exhibited 47, 44, and 34% sequence identity with corresponding domains of known Cry toxins. SDS-PAGE and Western blot analysis showed that Cry7Ca1, encoded by the full-length open reading frame of the cry gene, the activated toxin 1, which included three domains but without the N-terminal 54 amino acid residues and the C terminus, and the activated toxin 2, which included three domains and N-terminal 54 amino acid residues but without the C terminus, could be expressed in Escherichia coli. Bioassay results indicated that the expressed proteins of Cry7Ca1 and the activated toxins (toxins 1 and 2) showed significant activity against 2nd instar locusts, and after 7 days of infection, the estimated 50% lethal concentrations (LC 50 s) were 8.98 g/ml for the expressed Cry7Ca1, 0.87 g/ml for the activated toxin 1, and 4.43 g/ml for the activated toxin 2. The ␦-endotoxin also induced histopathological changes in midgut epithelial cells of adult L. migratoria manilensis.Bacillus thuringiensis is a spore-forming entomopathogenic bacterium that produces parasporal crystals composed of proteins called ␦-endotoxins during sporulation (8,19,36,45). It is reported that B. thuringiensis exhibits specific activity against Lepidoptera, Diptera, Coleoptera, Homoptera, Hymenoptera, Mallophaga, nematodes, and other invertebrates (2,7,9,24,44). B. thuringiensis is a uniquely specific, safe, and effective biological insecticide that has been used for more than 50 years (3,30). The increased use of B. thuringiensis in biological control and its potential application in medicine have encouraged researchers to find novel strains with different toxic spectra or high specific activity and new functional genes of B. thuringiensis (13,22,26,28).There are two known classes of ␦-endotoxins in B. thuringiensis: the insect-specific insecticidal crystal (Cry) proteins and the Diptera-specific cytolytic (Cyt) proteins. The Cry proteins are encoded by cry genes, include more than 480 different genes belonging to 124 subgroups of 60 groups, and show variable degrees of sequence homology (see the B. thuringiensis [Bt] toxin nomenclature website at http://www.lifesci.sussex.ac .uk/home/Neil_Crickmore/Bt/). Most of the Cry proteins contain five conserved sequence blocks acco...

“…Hence, an in-depth study on the synthesis mechanisms of B. thuringiensis parasporal crystals and spores under different physiologically relevant conditions at the proteomic level is highly significant. To date, most B. thuringiensis proteomic studies have mainly focused on parasporal crystal purification and identification (7,52), spore composition and function (13,22), classification of Bacillus strains (16), and so on. There has been no report on the large-scale characterization of the B. thuringiensis proteome, which is becoming one of the international hot spots of research.…”

mentioning

confidence: 99%

Proteomic Analysis of Bacillus thuringiensis at Different Growth Phases by Using an Automated Online Two-Dimensional Liquid Chromatography-Tandem Mass Spectrometry Strategy

Huang

Ding

Sun

et al. 2012

The proteome of a new Bacillus thuringiensis subsp. kurstaki strain, 4.0718, from the middle vegetative (T 1 ), early sporulation (T 2 ), and late sporulation (T 3 ) phases was analyzed using an integrated liquid chromatography (LC)-based protein identification system. The system comprised two-dimensional (2D) LC coupled with nanoscale electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. After deletion of redundant proteins from the different batches and B. thuringiensis subspecies, 918, 703, and 778 proteins were identified in the respective three phases. Their molecular masses ranged from 4.6 Da to 477.4 Da, and their isoelectric points ranged from 4.01 to 11.84. Function clustering revealed that most of the proteins in the three phases were functional metabolic proteins, followed by proteins participating in cell processes. Small molecular and macromolecular metabolic proteins were further classified according to the Kyoto Encyclopedia of Genes and Genome and BioCyc metabolic pathway database. Three protoxins (Cry2Aa, Cry1Aa, and Cry1Ac) as well as a series of potential intracellular active factors were detected. Many significant proteins related to spore and crystal formation, including sporulation proteins, help proteins, chaperones, and so on, were identified. The expression patterns of two identified proteins, CotJc and glutamine synthetase, were validated by Western blot analysis, which further confirmed the MS results. This study is the first to use shotgun technology to research the proteome of B. thuringiensis. Valuable experimental data are provided regarding the methodology of analyzing the B. thuringiensis proteome (which can be used to produce insecticidal crystal proteins) and have been added to the related protein database. Bacillus thuringiensis is characterized by the production of a parasporal body during sporulation, which contains one or more Cry and/or Cyt proteins (also known as ␦-endotoxins) in crystalline form (17, 42). Many B. thuringiensis subspecies are used as bacterial insecticides and sources of genes for the recombinant bacteria and B. thuringiensis strains applied for insecticidal products (5, 31). Since the 1980s, substantial progress has been made in the development and production of new genetically engineered B. thuringiensis insecticides, especially in the transformation of insecticidal crystal proteins (ICPs), due to the ever-increasing research on the B. thuringiensis protein production mechanisms and genetic structure. For B. thuringiensis, many researchers have found great difficulty in achieving marked improvements in ICP only by traditional fermentation and mutagenesis treatment, which hinders the practical applications of B. thuringiensis. The challenge is probably rooted in the fact that except for the closely synergistic effect with spore formation (2), ICPs are synthesized via multiple intracellular reactions, which are further complicated by various factors, including c...