Biochemical features of the Cry4B toxin of subsp. and its interaction with BT-R, a bitopic cadherin G-protein coupled receptor in .

Introduction: The cadherin G-protein coupled receptor BT-R in the mosquito is a single membrane-spanning α-helical (bitopic) protein that represents the most abundant and functionally diverse group of membrane proteins. Binding of the Cry4B toxin of subsp. (Bti) to BT-R triggers a Mg2+-dependent signalling pathway in the mosquito that involves stimulation of G protein α-subunit, which subsequently launches a coordinated signalling cascade involving Na/K-ATPase.

Cell death signaling in initiated by Cry4B toxin involves Na/K ATPase.

Identifying the mechanisms by which bacterial pathogens kill host cells is fundamental to understanding how to control and prevent human and animal disease. In the case of (Bt), such knowledge is critical to using the bacterium to kill insect vectors that transmit human and animal disease. For the Cry4B toxin produced by Bt, its capacity to kill , the primary mosquito vector of malaria, is the consequence of a variety of signaling activities.

"The Defined Toxin-binding Region of the Cadherin G-protein Coupled Receptor, BT-R, for the Active Cry1Ab Toxin of ".

The bacterium (Bt) produces protoxin proteins in parasporal crystals. Proteolysis of the protoxin generates an active toxin which is a potent microbial insecticide. Additionally, Bt toxin genes have been introduced into genetically modified crops to produce insecticidal toxins which protect crops from insect invasion.

Interaction of Fluorescently Labeled Cadherin G Protein-coupled Receptor with the Cry1Ab Toxin of Bacillus thuringiensis.

The Cry1Ab toxin produced by binds to a conserved structural motif in the 12 ectodomain module (EC12) of BT-R, a cadherin G protein-coupled receptor (GPCR) contained in the membrane of midgut epithelial cells of the tobacco hornworm . Toxin binding transmits a signal into the cells and turns on a multi-step signal transduction pathway, culminating in cell death. Using chromatographically purified Cry1Ab and EC12 proteins, we demonstrated the direct formation of a stable complex between these two proteins in solution and visualized it on a native polyacrylamide gel.

Cytotoxicity of the Bacillus thuringiensis Cry4B toxin is mediated by the cadherin receptor BT-R₃ of Anopheles gambiae.

We demonstrate for the first time the selective cytotoxicity of Bacillus thuringiensis subsp. israelensis Cry4B toxin mediated by BT-R₃ using a cell-based system, which employs High Five insect cells stably expressing BT-R₃. Discovery and validation of BT-R₃ as the Cry4B receptor was accomplished using a web-based computational pipeline platform that facilitates high-throughput insecticidal target identification utilizing the Anopheles gambiae genome.

The Cry4B toxin of Bacillus thuringiensis subsp. israelensis kills Permethrin-resistant Anopheles gambiae, the principal vector of malaria.

Resurgence of malaria has been attributed, in part, to the development of resistance by Anopheles gambiae, a principal vector of the disease, to various insecticidal compounds such as Permethrin. Permethrin, a neurotoxicant, is widely used to impregnate mosquito nets. An alternative strategy to control mosquitoes is the use of Bacillus thuringiensis subsp.

Bacillus thuringiensis: a genomics and proteomics perspective.

Bacillus thuringiensis (Bt) is a unique bacterium in that it shares a common place with a number of chemical compounds which are used commercially to control insects important to agriculture and public health. Although other bacteria, including B. popilliae and B.

Genomic imprinting: the influence of differential methylation in the two sexes.

Genomic imprinting is an epigenetic form of gene regulation that entails differential sex-specific methylation of the alleles of a gene. Such methylation distinguishes male and female genomes and is inherited in a parent-of-origin-specific manner. Sex-specific imprints are established in the germline during gametogenesis and remain intact throughout embryonic and postnatal development.

Susceptibility of Manduca sexta to Cry1Ab toxin of Bacillus thuringiensis correlates directly to developmental expression of the cadherin receptor BT-R(1).

The cadherin receptor BT-R(1), localized in the midgut epithelium of the tobacco hornworm, Manduca sexta, is coupled to programmed oncotic-like cell death, which is triggered by the univalent binding of the Cry1Ab toxin of Bacillus thuringiensis (Bt) to the receptor. Kinetic analysis of BT-R(1) expression during larval development reveals that the density of BT-R(1) on the midgut surface increases dramatically along with an equivalent rise in the concentration of Cry1Ab toxin molecules needed to kill each of the five larval stages of the insect. The increase in the number of BT-R(1) molecules per midgut surface area requires additional toxin molecules to kill older versus younger larvae, as evidenced by the corresponding LC(50) values.

Enhanced exocytosis of the receptor BT-R(1) induced by the Cry1Ab toxin of Bacillus thuringiensis directly correlates to the execution of cell death.

Cry1Ab toxin produced by Bacillus thuringiensis exerts insecticidal action upon binding to BT-R(1), a cadherin receptor localized in the midgut epithelium of the tobacco hornworm Manduca sexta. The univalent binding of toxin to receptor transmits a death signal into the cell and turns on a multi-step signal transduction pathway involving adenylyl cyclase (AC) and protein kinase A (PKA), which drives the biochemical events that culminate in oncotic cell death. Here, we report that cell killing by the Cry1Ab toxin is a dynamic episode in which the toxin promotes exocytotic transport of BT-R(1) from intracellular membrane vesicles to the plasma membrane.

Univalent binding of the Cry1Ab toxin of Bacillus thuringiensis to a conserved structural motif in the cadherin receptor BT-R1.

The Cry1Ab toxin produced by Bacillus thuringiensis (Bt) exerts insecticidal action upon binding to BT-R1, a cadherin receptor localized in the midgut epithelium of the tobacco hornworm Manduca sexta [Dorsch, J. A., Candas, M.

A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis.

Many pathogenic organisms and their toxins target host cell receptors, the consequence of which is altered signaling events that lead to aberrant activity or cell death. A significant body of literature describes various molecular and cellular aspects of toxins associated with bacterial invasion, colonization, and host cell disruption. However, there is little information on the molecular and cellular mechanisms associated with the insecticidal action of Bacillus thuringiensis (Bt) Cry toxins.

The proteomics of sickle cell disease: profiling of erythrocyte membrane proteins by 2D-DIGE and tandem mass spectrometry.

Quantitative changes in the red blood cell membrane proteome in sickle cell disease were analyzed using the two-dimensional fluorescence difference gel electrophoresis 2D-DIGE technique. From over 500 analyzed two-dimensional gel spots, we found 49 protein gel spots whose content in sickle cell membranes were changed by at least 2.5-fold as compared to control cells.

The human erythrocyte proteome: analysis by ion trap mass spectrometry.

This report describes an analysis of the red blood cell proteome by ion trap tandem mass spectrometry in line with liquid chromatography. Mature red blood cells lack all internal cell structures and consist of cytoplasm within a plasma membrane envelope. To maximize outcome, total red blood cell protein was divided into two fractions of membrane-associated proteins and cytoplasmic proteins.

Selective antagonism to the cadherin BT-R1 interferes with calcium-induced adhesion of epithelial membrane vesicles.

BT-R(1) is a member of the cadherin superfamily of proteins and is expressed in the midgut epithelium of Manduca sexta during larval development. Previously, we showed that calcium ions influence the structure and stability of BT-R(1) on brush border membrane vesicles (BBMVs) prepared from M. sexta midgut epithelium.

Expression of a midgut-specific cadherin BT-R1 during the development of Manduca sexta larva.

The btr-1 gene of Manduca sexta (GenBank AF319973) encodes a cadherin, BT-R(1) (210-kDa), which contains 12 ectodomain modules in association with a number of motifs potentially involved in interactions with cadherin and integrin. The molecule is a target receptor for Bacillus thuringiensis Cry1A toxins that bind to BT-R(1) with high affinity and specificity. BT-R(1) is localized exclusively in the midgut epithelium.

Insect resistance to Bacillus thuringiensis: alterations in the indianmeal moth larval gut proteome.

Insect resistance to the Cry toxins of Bacillus thuringiensis (Bt) has been examined previously using a number of traditional biochemical and molecular techniques. In this study, we utilized a proteomic approach involving two-dimensional differential gel electrophoresis, mass spectrometry, and function-based activity profiling to examine changes in the gut proteins from the larvae of an Indianmeal moth (IMM, Plodia interpunctella) colony exhibiting resistance to Bt. We found a number of changes in the levels of certain specific midgut proteins that indicate increased glutathione utilization, elevation in oxidative metabolism, and differential maintenance of energy balance within the midgut epithelial cells of the Bt-resistant IMM larva.

Proteolytic cleavage of the developmentally important cadherin BT-R1 in the midgut epithelium of Manduca sexta.

BT-R1 (M(r) = 210 kDa) represents a new type of insect cadherin that is expressed specifically in the midgut epithelium during growth and development of Manduca sexta larvae. It also is a target receptor for the Cry1A toxins of the entomopathogenic bacterium Bacillus thuringiensis. Expression of BT-R1, which varies during larval development, correlates with the abundance of the protein and with the differential cleavage of the molecule at each developmental stage.

Changes in protease activity and Cry3Aa toxin binding in the Colorado potato beetle: implications for insect resistance to Bacillus thuringiensis toxins.

Widespread commercial use of Bacillus thuringiensis Cry toxins to control pest insects has increased the likelihood for development of insect resistance to this entomopathogen. In this study, we investigated protease activity profiles and toxin-binding capacities in the midgut of a strain of Colorado potato beetle (CPB) that has developed resistance to the Cry3Aa toxin of B. thuringiensis subsp.

Structural characterization of the hemocytes of Plodia interpunctella.

Six types of hemocytes were identified in fifth instars of the Indian meal moth, Plodia interpunctella. The morphology of these cells was characterized by phase contrast and electron microscopy, with Sudan black B, Giemsa, Janus green B, and periodic acid-Schiff staining. Reaction of the hemocytes with seven fluorescing lectin conjugates revealed distinctive binding patterns by their plasma and nuclear membranes and cytoplasmic inclusions.