Evaluation of loop-mediated isothermal amplification assay along with conventional and real-time PCR assay for sensitive detection of pathogenic Vibrio parahaemolyticus from seafood sample without enrichment.

The primary reason for foodborne illness is improper seafood safety testing, and hence, an appropriate tool for testing is the key to control the outbreaks. The current study aimed to develop a loop-mediated isothermal amplification (LAMP) assay to detect pathogenic Vibrio parahaemolyticus, important foodborne pathogen, targeting tdh, and trh genes. The specificity of the LAMP assay was good without any false-positive and false-negative results. The assay was highly sensitive and could detect the pathogenic V. parahaemolyticus as low as 1 CFU/reaction in spiked seafood samples and 1 pg of extracted DNA. Out of 62 seafood samples from India's southwest coastal region tested with LAMP assay, eight (12.9%) were positive for trh, and seven (11.29%) samples were positive tdh gene. LAMP-based on tdh and trh was found to be significantly more sensitive (p < 0.05) than conventional PCR and nearly equal sensitive as real-time PCR (RT-PCR) for the detection of pathogenic V. parahaemolyticus. Our study shows that LAMP assay can be a better approach as a point-of-care (POC) diagnostic tool and could detect pathogenic V. parahaemolyticus on seafood samples directly without enrichment and isolation. The high sensitivity and simplicity make LAMP assay a better alternative method than the conventional method and RT-PCR for the detection of pathogens. LAMP assay can be considered as a good alternative to PCR for the routine detection of pathogenic V. parahaemolyticus in seafood.

Unraveling the Composition of Insecticidal Crystal Proteins in Bacillus thuringiensis: a Proteomics Approach.

Bacillus thuringiensis (Bt) is the most widely used active ingredient for biological insecticides. The composition of δ-endotoxins (Cry and Cyt proteins) in the parasporal crystal determines the toxicity profile of each Bt strain. However, a reliable method for their identification and quantification has not been available, due to the high sequence identity of the genes that encode the δ-endotoxins and the toxins themselves. Here, we have developed an accurate and reproducible mass spectrometry-based method (liquid chromatography-tandem mass spectrometry-multiple reaction monitoring [LC-MS/MS-MRM]) using isotopically labeled proteotypic peptides for each protein in a particular mixture to determine the relative proportion of each δ-endotoxin within the crystal. To validate the method, artificial mixtures containing Cry1Aa, Cry2Aa, and Cry6Aa were analyzed. Determination of the relative abundance of proteins (in molarity) with our method was in good agreement with the expected values. This method was then applied to the most common commercial Bt-based products, DiPel DF, XenTari GD, VectoBac 12S, and Novodor, in which between three and six δ-endotoxins were identified and quantified in each product. This novel approach is of great value for the characterization of Bt-based products, not only providing information on host range, but also for monitoring industrial crystal production and quality control and product registration for Bt-based insecticides.IMPORTANCEBacillus thuringiensis (Bt)-based biological insecticides are used extensively to control insect pests and vectors of human diseases. Bt-based products provide greater specificity and biosafety than broad-spectrum synthetic insecticides. The biological activity of this bacterium resides in spores and crystals comprising complex mixtures of toxic proteins. We developed and validated a fast, accurate, and reproducible method for quantitative determination of the crystal components of Bt-based products. This method will find clear applications in the improvement of various aspects of the industrial production process of Bt. An important aspect of the production of Bt-based insecticides is its quality control. By specifically quantifying the relative proportion of each of the toxins that make up the crystal, our method represents the most consistent and repeatable evaluation procedure in the quality control of different batches produced in successive fermentations. This method can also contribute to the design of specific culture media and fermentation conditions that optimize Bt crystal composition across a range of Bt strains that target different pestiferous insects. Quantitative information on crystal composition should also prove valuable to phytosanitary product registration authorities that oversee the safety and efficacy of crop protection products.

Hemolytic and cytotoxic activity from cultures of Aureococcus anophagefferens-a causative species of brown tides in the north-western Bohai Sea, China.

Brown tides were first observed in 2009 in the north-western Bohai Sea (Qinhuangdao sea area), China, and blooms have occurred at different scales in late spring every year since then. Although the detrimental effects on marine organisms of the causative phytoplankton species Aureococcus anophagefferens have been extensively studied, the mechanism remains poorly understood. We used erythrocytes and adrenal gland chromaffin tumor cells (PC12) to explore the hemolytic activity and cytotoxicity, respectively, of chloroform and methanol extracts of cultured A. anophagefferens isolated from the north-western Bohai Sea area. The methanol extracts showed no hemolytic or cytotoxic activity. Chloroform extracts had a potent hemolytic effect on rabbit erythrocytes; thin layer chromatography (TLC) indicated that the hemolysin was a kind of glycolipid compound. Erythrocyte lysis assay showed that erythrocytes of sea bream were sensitive to the hemolysin, whereas those of human and chicken erythrocytes were insensitive. The hemolytic effects were elevated as temperatures rose from 4 °C to 37 °C. Hemolytic blocking experiments showed that sphingomyelin and d-xylose can inhibit hemolysis significantly, while osmotic protectants with different hydrated molecular diameters had no inhibition, and the hemolysins had no obvious phospholipase activity. The chloroform extracts of A. anophagefferens had significant inhibitory effects on the viability of PC12 cells, and can induce efflux of lactic dehydrogenase (LDH) of PC12 cells and lead to their necrosis.

Cry1A Proteins are Cytotoxic to HeLa but not to SiHa Cervical Cancer Cells.

BACKGROUND: Bacillus thuringiensis toxins are effective against multiple biological targets such as insects, nematodes, mites, protozoa, and importantly, human cancer cells. One of the main mechanisms by which Cry toxins to trigger cell death is the specific recognition of cadherin-like membrane cell receptors. OBJECTIVE: This work aimed to assess the cytotoxicity of the Cry1Ab and Cry1Ac toxins from Bacillus thuringiensis in HeLa, cervical cancer cell line, as well as their antitumor activity in mouse models. METHODS: We analyzed several biological targets of Cry1Ab and Cry1Ac including erythrocytes, insect larvae, as well as cancer and non-cancer cell lines. The viability of HeLa, SiHa, MCF7 and HaCat cells was assessed by MTT 24 h after the administration of Cry toxins. We also studied apoptosis as a possible cytotoxicity mechanism in HeLa. The capacity of Cry toxins to eliminate tumors in xenograft mouse models was also analyzed. RESULTS: Both toxins, Cry1Ab and Cry1Ac, showed specific cytotoxic activity in HeLa (HPV18+) cervical cancer cell line, with a Cry1Ab LC50 of 2.5 µg/ml, and of 0.5 µg/ml for Cry1Ac. Apoptosis was differentially induced in HeLa cells using the same concentration of Cry1Ab and Cry1Ac toxins. Cry1Ac eliminated 50% of the tumors at 10 µg/ml, and eliminate 100% of the tumors at 30 and 50 µg/ml. CONCLUSION: Bacillus thuringiensis Cry1A toxins show dual cytotoxic activity, in insects as well as in HeLa cancer cell line.

Rapid Purification of Endotoxin-Free RTX Toxins.

Cytolytic leukotoxins of the repeat in toxin (RTX) family are large proteins excreted by gram-negative bacterial pathogens through the type 1 secretion system (T1SS). Due to low yields and poor stability in cultures of the original pathogens, it is useful to purify recombinant fatty-acylated RTX cytolysins from inclusion bodies produced in E. coli. Such preparations are, however, typically contaminated by high amounts of E. coli lipopolysaccharide (LPS or endotoxin). We report a simple procedure for purification of large amounts of biologically active and endotoxin-free RTX toxins. It is based on the common feature of RTX cytolysins that are T1SS-excreted as unfolded polypeptides and fold into a biologically active toxin only upon binding of calcium ions outside of the bacterial cell. Mimicking this process, the RTX proteins are solubilized from inclusion bodies with buffered 8 M urea, bound onto a suitable chromatographic medium under denaturing conditions and the contaminating LPS is removed through extensive on-column washes with buffers containing 6 to 8 M urea and 1% Triton X-100 or Triton X-114. Extensive on-column rinsing with 8 M urea buffer removes residual detergent and the eluted highly active RTX protein preparations then contain only trace amounts of LPS. The procedure is exemplified using four prototypic RTX cytolysins, the Bordetella pertussis CyaA and the hemolysins of Escherichia coli (HlyA), Kingella kingae (RtxA), and Actinobacillus pleuropneumoniae (ApxIA).

Functional Reconstitution of HlyB, a Type I Secretion ABC Transporter, in Saposin-A Nanoparticles.

Type I secretion systems (T1SS) are ubiquitous transport machineries in Gram-negative bacteria. They comprise a relatively simple assembly of three membrane-localised proteins: an inner-membrane complex composed of an ABC transporter and a membrane fusion protein, and a TolC-like outer membrane component. T1SS transport a wide variety of substrates with broad functional diversity. The ABC transporter hemolysin B (HlyB), for example, is part of the hemolysin A-T1SS in Escherichia coli. In contrast to canonical ABC transporters, an accessory domain, a C39 peptidase-like domain (CLD), is located at the N-terminus of HlyB and is essential for secretion. In this study, we have established an optimised purification protocol for HlyB and the subsequent reconstitution employing the saposin-nanoparticle system. We point out the negative influence of free detergent on the basal ATPase activity of HlyB, studied the influence of a lysolipid or lipid matrix on activity and present functional studies with the full-length substrate proHlyA in its folded and unfolded states, which both have a stimulatory effect on the ATPase activity.

Efficient production of endotoxin depleted bioactive α-hemolysin of uropathogenic Escherichia coli.

Uropathogenic E. coli (UPEC), especially associated with severe urinary tract infections (UTI) pathologies, harbors an important virulence factor known as α-hemolysin (110 kDa). Hemolytic activity of α-hemolysin (HlyA) requires modification (acylation) of two lysine residues of HlyA by HlyC, part of operon hlyCABD. Most of the previous studies had used whole operon hlyCABD and gene tolC cloning for the production of active α-hemolysin. Studies involving α-hemolysin are limited due to the cumbersome and manual method of purification for this toxin. Here, we report a simple method for production of both active and inactive recombinant α-hemolysin by cloning only hlyA and hlyC genes of operon hlyCABD. Presence of both active and inactive α-hemolysin would be advantageous for functional characterization. After translation, the yield of the purified α-hemolysin was 1 mg/200 ml. Functionality of the recombinant α-hemolysin protein was confirmed using hemolytic assay. This is the first report of the production of active and inactive recombinant α-hemolysin for functional studies.

Erythroliposomes: Integrated Hybrid Nanovesicles Composed of Erythrocyte Membranes and Artificial Lipid Membranes for Pore-Forming Toxin Clearance.

Pore-forming toxins (PFTs) are the most common bacterial virulence proteins and play a significant role in the pathogenesis of bacterial infections; thus, PFTs are an attractive therapeutic target in bacterial infections. Inspired by the pore-forming process and mechanism of PFTs, we designed an integrated hybrid nanovesicle-the erythroliposome (called the RM-PL)-for PFT detoxification by fusing natural red blood cell (RBC) membranes with artificial lipid membranes. The lipid and RBC membranes were mutually beneficial when integrated into a hybrid nanovesicle structure. The RBC membrane endowed RM-PLs with the capacity for detoxification, while the PEGylated lipid membrane stabilized the RM-PLs and greatly improved the detoxification capacity of the RBC membrane. With α-hemolysin (Hlα) as a model PFT, we demonstrated that RM-PLs could not only significantly reduce the toxicity of Hlα to erythrocytes in vitro but also effectively sponge Hlα in vivo and rescue mice from Hlα-induced damage. Moreover, the high detoxification capacity of RM-PLs was shown to be partly related to the expression of the Hlα receptor protein, a disintegrin and metalloproteinase domain-containing protein 10 on the RBC membrane. Consequently, as a component integrating natural and artificial materials, the erythroliposome nanoplatform inspires potential strategies for antivirulence therapy.

Identification of an insecticidal toxin produced by Enterobacter sp. strain 532 isolated from diseased Bombyx mori silkworms.

Although Enterobacter sp. 532 shows pathogenicity in Bombyx mori, the insecticidal mechanisms are unclear. Here, we identified and characterised an insecticidal protein from Enterobacter. The insecticidal protein was purified from the strain and inoculated into B. mori larvae. Intracellular proteins were prepared, purified and separated by preparative native polyacrylamide gel electrophoresis (PAGE); one protein band had insecticidal activity. Sodium dodecyl sulfate-PAGE showed the presence of several bands, indicating that the insecticidal protein formed a complex. Peptide mass fingerprinting of a prominent 255.3-kDa band revealed 64 peptides that matched one protein with 33.0% sequence coverage. This protein was a homologue of the A component of the toxin complex (Tc), and the VRP1 domain was conserved; thus, the gene was named itcA (insecticidal toxin complex A). In the itcA downstream region, B and C component gene homologues were found, and these genes were located on an 86.2-kb contig sequence. Two repA genes and 27 genes related to conjugation transfer of plasmids were located on the contig, suggesting that the contig originated from a mobilisable plasmid. Therefore, these findings suggested that the strain may have acquired the Tc genes by horizontal transfer. This is the first description of Tc produced by the genus Enterobacter.

Sialic acid facilitates binding and cytotoxic activity of the pore-forming Clostridium perfringens NetF toxin to host cells.

NetF-producing type A Clostridium perfringens is an important cause of canine and foal necrotizing enteritis. NetF, related to the ß-sheet pore-forming Leukocidin/Hemolysin superfamily, is considered a major virulence factor for this disease. The main purpose of this work is to demonstrate the pore-forming activity of NetF and characterize the chemical nature of its binding site. Electron microscopy using recombinant NetF (rNetF) confirmed that NetF is able to oligomerize and form large pores in equine ovarian (EO) cell membranes and sheep red blood cells. These oligomeric pores appear to be about 4-6 nm in diameter, and the number of oligomer subunits to vary from 6 to 9. Sodium periodate treatment rendered EO cells non-susceptible to NetF, suggesting that NetF binding requires cell surface carbohydrates. NetF cytotoxicity was also inhibited by a lectin that binds sialic acid, by sialidase, and by free sialic acid in excess, all of which clearly implicate sialic acid-containing membrane carbohydrates in NetF binding and/or toxicity for EO cells. Binding of NetF to sheep red blood cells was not inhibited by the gangliosides GM1, GM2 and GM3, nor did the latter promote membrane permeabilization in liposomes, suggesting that they do not constitute the cellular receptors. In contrast, treatment of EO cells with different proteases reduced their susceptibility to NetF, suggesting that the NetF receptor is a sialic acid-containing glycoprotein.

Membrane localization of the Repeats-in-Toxin (RTX) Leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans.

The oral bacterium, Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis, as well as systemic infections including endocarditis, produces numerous virulence factors, including a repeats-in-toxin (RTX) protein called leukotoxin (LtxA), which kills human immune cells. The strains of A. actinomycetemcomitans most closely associated with disease have been shown to produce the most LtxA, suggesting that LtxA plays a significant role in the virulence of this organism. LtxA, like many of the RTX toxins, can be divided into four functional domains: an N-terminal hydrophobic domain, which contains a significant fraction of hydrophobic residues and has been proposed to play a role in the membrane interaction of the toxin; the central domain, which contains two lysine residues that are the sites of post-translational acylation; the repeat domain that is characteristic of the RTX toxins, and a C-terminal domain thought to be involved in secretion. In its initial interaction with the host cell, LtxA must bind to both cholesterol and an integrin receptor, lymphocyte function-associated antigen-1 (LFA-1). While both interactions are essential for toxicity, the domains of LtxA involved remain unknown. We therefore undertook a series of experiments, including tryptophan quenching and trypsin digestion, to characterize the structure of LtxA upon interaction with membranes of various lipid compositions. Our results demonstrate that LtxA adopts a U-shaped conformation in the membrane, with the N- and C-terminal domains residing outside of the membrane.

Mapping an Equilibrium Folding Intermediate of the Cytolytic Pore Toxin ClyA with Single-Molecule FRET.

The 303-residue cytolytic toxin ClyA forms a stable α-helical monomer. In the presence of detergents or membranes, however, the protein makes a large conformational transition to the protomer state, which is competent for assembly into a dodecameric cytolytic pore. In this study, we map the structure of the ClyA monomer during denaturant-induced unfolding with single-molecule Förster resonance energy transfer (FRET) spectroscopy. To this end, we probe intramolecular distances of six different segments of ClyA by placing donor and acceptor fluorophores at corresponding positions along the chain. We identify an intermediate state that contains the folded core consisting of three of the α-helices that make up the helical bundle present in the structure of both the monomer and the protomer, but with the C- and N-terminal helices unfolded, in accord with the secondary structure content estimated from circular dichroism (CD) spectroscopy. The existence of this intermediate is likely to be a consequence of the structural bistability underlying the biological function of ClyA: The terminal helices are part of the largest rearrangements during protomer formation, and the local differences in stability we detect may prime the protein for the required conformational transition.

pH-Dependent exhibition of hemolytic activity by an extract of Hypsizygus marmoreus fruiting bodies.

The current study found that an extract from the fruiting bodies of the edible mushroom Hypsizygus marmoreus exhibited hemolytic activity against sheep red blood cells when its pH was lowered. Although hemolytic activity was not detected when an extract had a neutral pH, an extract with a low pH exhibited potent hemolytic activity. The maximal hemolytic activity was exhibited by an extract with a pH of 5.5. A heat-treated extract did not exhibit hemolytic activity before its pH was lowered, and that activity was inhibited in the presence of PMSF and EDTA. The turbidity of the extract increased during lowering of its pH, and the precipitate fraction exhibited hemolytic activity. Fractionation by a modified Bligh and Dyer method and TLC analyses suggested that a hemolytic compound in the extract might be a type of lipid. These results suggest that a hemolytic lipid-like compound in an extract of H. marmoreus fruiting bodies may be released by a non-active precursor substance(s) through metalloenzyme(s) while the extract has a low pH.

Essential oil of Siparuna guianensis as an alternative tool for improved lepidopteran control and resistance management practices.

Although the cultivation of transgenic plants expressing toxins of Bacillus thuringiensis (Bt) represents a successful pest management strategy, the rapid evolution of resistance to Bt plants in several lepidopteran pests has threatened the sustainability of this practice. By exhibiting a favorable safety profile and allowing integration with pest management initiatives, plant essential oils have become relevant pest control alternatives. Here, we assessed the potential of essential oils extracted from a Neotropical plant, Siparuna guianensis Aublet, for improving the control and resistance management of key lepidopteran pests (i.e., Spodoptera frugiperda and Anticarsia gemmatalis). The essential oil exhibited high toxicity against both lepidopteran pest species (including an S. frugiperda strain resistant to Cry1A.105 and Cry2Ab Bt toxins). This high insecticidal activity was associated with necrotic and apoptotic effects revealed by in vitro assays with lepidopteran (but not human) cell lines. Furthermore, deficits in reproduction (e.g., egg-laying deterrence and decreased egg viability), larval development (e.g., feeding inhibition) and locomotion (e.g., individual and grouped larvae walking activities) were recorded for lepidopterans sublethally exposed to the essential oil. Thus, by similarly and efficiently controlling lepidopteran strains susceptible and resistant to Bt toxins, the S. guianensis essential oil represents a promising management tool against key lepidopteran pests.

Confocal micrographs: automated segmentation and quantitative shape analysis of neuronal cells treated with ostreolysin A/pleurotolysin B pore-forming complex.

Detailed shape analysis of cells is important to better understand the physiological mechanisms of toxins and determine their effects on cell morphology. This study aimed to develop a procedure for accurate morphological analysis of cell shape and use it as a tool to estimate toxin activity. With the aim of optimizing the method of cell morphology analysis, we determined the influence of ostreolysin A and pleurotolysin B complex (OlyA/PlyB) on the morphology of murine neuronal NG108-15 cells. A computational method was introduced and successfully applied to quantify morphological attributes of the NG108-15 cell line before and after 30 and 60 min exposure to OlyA/PlyB using confocal microscopy. The modified circularity measure [Formula: see text] for shape analysis was applied, which defines the degree to which the shape of the neuron differs from a perfect circle. It enables better detection of small changes in the shape of cells, making the outcome easily detectable numerically. Additionally, we analyzed the influence of OlyA/PlyB on the cell area, allowing us to detect the cells with blebs. This is important because the formation of plasma membrane protrusions such as blebs often reflects cell injury that leads to necrotic cell death. In summary, we offer a novel analytical method of neuronal cell shape analysis and its correlation with the toxic effects of the pore-forming OlyA/PlyB toxin in situ.

Engineering Bacillus thuringiensis Cyt1Aa toxin specificity from dipteran to lepidopteran toxicity.

The Cyt and Cry toxins are different pore-forming proteins produced by Bacillus thuringiensis bacteria, and used in insect-pests control. Cry-toxins have a complex mechanism involving interaction with several proteins in the insect gut such as aminopeptidase N (APN), alkaline phosphatase (ALP) and cadherin (CAD). It was shown that the loop regions of domain II of Cry toxins participate in receptor binding. Cyt-toxins are dipteran specific and interact with membrane lipids. We show that Cry1Ab domain II loop3 is involved in binding to APN, ALP and CAD receptors since point mutation Cry1Ab-G439D affected binding to these proteins. We hypothesized that construction of Cyt1A-hybrid proteins providing a binding site that recognizes gut proteins in lepidopteran larvae could result in improved Cyt1Aa toxin toward lepidopteran larvae. We constructed hybrid Cyt1Aa-loop3 proteins with increased binding interaction to Manduca sexta receptors and increased toxicity against two Lepidopteran pests, M. sexta and Plutella xylostella. The hybrid Cyt1Aa-loop3 proteins were severely affected in mosquitocidal activity and showed partial hemolytic activity but retained their capacity to synergize Cry11Aa toxicity against mosquitos. Our data show that insect specificity of Cyt1Aa toxin can be modified by introduction of loop regions from another non-related toxin with different insect specificity.

Cry64Ba and Cry64Ca, Two ETX/MTX2-Type Bacillus thuringiensis Insecticidal Proteins Active against Hemipteran Pests.

Genetically modified crops that express insecticidal Bacillus thuringiensis (Bt) proteins have become a primary approach for control of lepidopteran (moth) and coleopteran (beetle) pests that feed by chewing the plants. However, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. In this study, we describe two Cry toxins (Cry64Ba and Cry64Ca) from Bt strain 1012 that showed toxicity against two important hemipteran rice pests, Laodelphax striatellus and Sogatella furcifera Both of these proteins contain an ETX/MTX2 domain and share common sequence features with the ß-pore-forming toxins. Coexpression of cry64Ba and cry64Ca genes in the acrystalliferous Bt strain HD73- resulted in high insecticidal activity against both hemipteran pests. No toxicity was observed on other pests such as Ostrinia furnacalis, Plutella xylostella, or Colaphellus bowringi Also, no hemolytic activity or toxicity against cancer cells was detected. Binding assays showed specific binding of the Cry64Ba/Cry64Ca toxin complex to brush border membrane vesicles isolated from L. striatellus Cry64Ba and Cry64Ca are Bt Cry toxins highly effective against hemipteran pests and could provide a novel strategy for the environmentally friendly biological control of rice planthoppers in transgenic plants.IMPORTANCE In Asia, rice is an important staple food, whose production is threatened by rice planthoppers. To date, no effective Bacillus thuringiensis (Bt) protein has been shown to have activity against rice planthoppers. We cloned two Bt toxin genes from Bt strain 1012 that showed toxicity against small brown planthoppers (Laodelphax striatellus) and white-backed planthoppers (Sogatella furcifera). To our knowledge, the proteins encoded by the cry64Ba and cry64Ca genes are the most efficient insecticidal Bt Cry proteins with activity against hemipteran insects reported so far. Cry64Ba and Cry64Ca showed no toxicity against some lepidopteran or coleopteran pests. These two proteins should be able to be used for integrated hemipteran pest management.

Broad specificity immunoassay for detection of Bacillus thuringiensis Cry toxins through engineering of a single chain variable fragment with mutagenesis and screening.

The cultivation of genetically modified crops (GMCs) has greatly increased worldwide. Given the fact that over 10 Bacillus thuringiensis Cry toxins have been applied in GMCs, there is a need to develop an efficient and economically affordable detection method that simultaneously screen such compounds with similar structures in crops and foodstuff. Here we described an approach using a site-directed mutagenesis that enhances the generic specificity of single chain variable fragment (scFv). After three rounds of panning against mixed antigen (Cry1Ab, Cry1Ac, Cry1B, Cry1C and Cry1F) from the constructed mutagenesis library, one mutant showed greater ability to bind to a range of Cry toxins. The phage mutant, named D9, was cloned into pET26b expression vector, and then induced and purified. The mutant D9 was used to develop an indirect competitive immunoassay that sucessfully recognizes five targeted Cry1 toxins with a working range from 0.20 to 2.22µgmL-1. The recoveries of five Cry toxins from spiked rice samples ranged from 86.67% to 96.67%, with a coefficient of variation less than 8%. The results suggest that the immunoassay based on a scFv is a promising approach for detection of Cry toxins with a broad specificity.

Nematicidal Activity of Cry1Ea11 from Bacillus thuringiensis BRC-XQ12 Against the Pine Wood Nematode (Bursaphelenchus xylophilus).

The nematicidal activity of 92 Bacillus thuringiensis strains against the pine wood nematode Bursaphelenchus xylophilus, one of the world's top 10 plant-parasitic nematodes, was determined. The insecticidal crystal proteins (ICPs) from Bacillus thuringiensis BRC-XQ12 were the most toxic to Bursaphelenchus xylophilus, with a lethal concentration 50 (LC50) of 32.13 µg/ml. Because the ICPs expressed by Bacillus thuringiensis BRC-XQ12 were closest to Cry1Ea6 and B. thuringiensis BRC-XQ12 contained four kinds of cry1 subgenes (cry1Aa, cry1Cb, cry1Ea, and cry1Ia), Cry1Ea was most likely to be the key active component against the nematode. The 3,516-bp cry1Ea11 gene from BRC-XQ12, as designated by the B. thuringiensis δ-endotoxin nomenclature committee, was expressed in Escherichia coli. Purified Cry1Ea11 showed an LC50 of 32.53 and 23.23 µg/ml at 24 and 48 h, with corresponding virulence equations of Y = 32.15X + 1.38 (R2 = 0.9951) and Y = 34.29X + 3.16 (R2 = 0.9792), respectively. In order to detect the pathway of B. thuringiensis Cry1Ea11 into Bursaphelenchus xylophilus, the nematode was fed with NHS-rhodamine-labeled GST-Cry1Ea11. The results of confocal laser-scanning microscopy showed that the 159-kDa GST-Cry1Ea11 could be detected in the stylet and the esophageal lumen of the pine wood nematode, indicating that GST-Cry1Ea11 could enter into the nematode through the stylet. As far as we know, no Cry1 proteins have been shown to have activity against plant-parasitic nematodes before. These results demonstrate that Cry1Ea11 is a promising nematicidal protein for controlling pine wilt disease rendered by B. xylophilus, further dramatically broadening the spectrum of Bacillus thuringiensis ICPs.

The First Cry2Ac-Type Protein Toxic to Helicoverpa armigera: Cloning and Overexpression of Cry2ac7 Gene from SBS-BT1 Strain of Bacillus thuringiensis.

The Cry (crystal) proteins from Bacillus thuringiensis are known to have toxicity against a variety of insects and have been exploited to control insect pests through transgenic plants and biopesticides. B. thuringiensis SBS BT-1 carrying the cry2 genes was isolated from soil samples in Pakistan. The 2-kb full length cry2Ac gene was cloned, sequenced, and submitted to the EMBL DNA database (Accession No. AM292031). For expression analysis, Escherichia coli DH5α was transformed with the fragment sub-cloned in pET22b expression vector using NdeI and HindIII restriction sites, and later confirmed by restriction endonuclease analysis. To assess the toxicity of Cry2Ac7 protein against lepidopteran and dipteran insects, BL21 (codon plus) strain of E. coli was further transformed with the recombinant plasmid. The 65-kDa protein was expressed in the form of inclusion bodies up to 180 OD units per liter of the medium. Inclusions were washed with a buffer containing 1.5% Triton-X 100 and >90% pure Cry2Ac7 was obtained. The inclusion bodies were dissolved in 50 mM K2CO3 (pH 11.5), dialyzed, and freeze-dried. This freeze-dried protein as well as inclusion bodies were used in bioassays against larvae of Helicoverpa armigera and Musca domestica. The freeze-dried protein was toxic to H. armigera larvae with an LC50 value of 131 ng/mL. However, Cry2Ac7 produced in E. coli did not show any mortality to M. domestica larvae. This is the first report of Cry2Ac protein toxic to H. armigera.