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. Once the Cry4B toxin binds to the BT-R3 receptor, a cell death pathway is manifested by sequential cytological changes that include membrane blebbing, cell swelling and lysis. Cry4B toxin associates with cell membrane in both oligomeric and monomeric forms. Monomeric toxin binds specifically to BT-R3 whereas oligomer interacts with cell membrane non-specifically. Cytotoxicity and cell death are the direct result of binding of toxin monomer to BT-R3. The oligomeric form of Cry4B toxin is not involved in cell death. Both the location of the toxin-binding region within BT-R3 and its structural motif are critical to the binding affinity and specificity of the toxin. The toxin-binding region of BT-R3 appears to be located in EC11, the most membrane proximal EC module within the extracellular domain. It is characterized by the presence of two highly conserved amino acid sequences within their N- and C-termini that flank EC11. These sequences represent signature motifs that mark the toxin-binding function in BT-R3. The two sequences form two adjacent ß-strands within the ß-barrel of EC11, the positioning of which is a hallmark of all Cry toxin receptors thus far reported.

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. israelensis (Bti) because there is no observable resistance in the field to the bacterium. Bti kills mosquitoes by targeting cadherin molecules residing in the midgut epithelium of larvae of the insect. Cry proteins (Cry4A, Cry4B, Cry10A and Cry11A) produced by the bacterium during the sporulation phase of its life cycle bind to the cadherin molecules, which serve as receptors for the proteins. These Cry proteins have variable specificity to a variety of mosquitoes, including Culex and Aedes as well as Anopheles. Importantly, selective mosquitocidal action is occasioned by binding of the respective Cry toxins to cadherins distinctive to individual mosquito species. Differential fractionation of the four Cry proteins from a novel Bti isolate (M1) and cloning and expression of their genes in Escherichia coli revealed that Cry4B is the only Cry protein that exerts insecticidal action against An. gambiae. Indeed, it does so against a Permethrin-resistant strain of the mosquito. The other three Cry proteins are ineffective. Multiple sequence alignments of the four Cry proteins revealed a divergent sequence motif in the Cry4B toxin, which most likely determines binding of the toxin to its cognate receptor, BT-R3, in An. gambiae and to its specific toxicity. A model showing Cry4B toxin binding to BT-R3 is presented.

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. The resultant dramatic increase in BT-R(1) displayed on the surface of toxin-treated cells effects the recruitment and concomitant binding of additional toxin monomers which, in turn, amplifies the original signal in a cascade-like manner. Blocking the activation of AC/PKA signal transduction by either EDTA or PKAi inhibits exocytotic trafficking of BT-R(1) and prevents cell death. Moreover, the exocytosis inhibitor Exo1 blocks translocation of receptor and progression of cell death alike. Obviously, movement of BT-R(1) is mediated by toxin-induced signal transduction and amplification of this signaling apparently is critical to the execution of cell death.

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., Griko, N. B., Maaty, W. S., Midboe, E. G., Vadlamudi, R. K., and Bulla, L. A., Jr. (2002) Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R1 in Manduca sexta: involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis, Insect Biochem. Mol. Biol. 32, 1025-1036]. BT-R1 represents a family of invertebrate cadherins whose ectodomains (ECs) are composed of multiple cadherin repeats (EC1 through EC12). In the present work, we determined the Cry1Ab toxin binding site in BT-R1 in the context of cadherin structural determinants. Our studies revealed a conserved structural motif for toxin binding that includes two distinct regions within the N- and C-termini of EC12. These regions are characterized by unique sequence signatures that mark the toxin-binding function in BT-R1 as well as in homologous lepidopteran cadherins. Structure modeling of EC12 discloses the conserved motif as a single broad interface that holds the N- and C-termini in close proximity. Binding of toxin to BT-R1, which is univalent, and the subsequent downstream molecular events responsible for cell death depend on the conserved motif in EC12.

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. Recently, we reported that the Cry1Ab toxin produced by Bt kills insect cells by activating a Mg(2+)-dependent cytotoxic event upon binding of the toxin to its receptor BT-R(1). Here we show that binding of Cry toxin to BT-R(1) provokes cell death by activating a previously undescribed signaling pathway involving stimulation of G protein (G(alphas)) and adenylyl cyclase, increased cAMP levels, and activation of protein kinase A. Induction of the adenylyl cyclase/protein kinase A pathway is manifested by sequential cytological changes that include membrane blebbing, appearance of ghost nuclei, cell swelling, and lysis. The discovery of a toxin-induced cell death pathway specifically linked to BT-R(1) in insect cells should provide insights into how insects evolve resistance to Bt and into the development of new, safer insecticides.

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. In 38 cases we observed an increase and in 11 cases a decrease in content in the sickle cell membranes. The proteins of interest were identified by in-gel tryptic digestion followed by liquid chromatography in line with tandem mass spectrometry. From 38 analyzed gel spots, we identified 44 protein forms representing different modifications of 22 original protein sequences. The majority of the identified proteins fall into small groups of related proteins of the following five categories: actin accessory proteins--four proteins, components of lipid rafts--two proteins, scavengers of oxygen radicals--two proteins, protein repair participants--six proteins, and protein turnover components--three proteins. The number of proteins whose content in sickle RBC membrane is decreased is noticeably smaller, and most are either components of lipid rafts or actin accessory proteins. Elevated content of protein repair participants as well as oxygen radical scavengers may reflect the increased oxidative stress observed in sickle cells.

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. The cleavage of BT-R1 is calcium dependent, and consequently, Ca2+ directly influences the structural integrity of BT-R1. Indeed, removal of calcium ions by chelating agents promotes cleavage of the BT-R1 ectodomain, resulting in formation of fragments that are similar to those observed during larval development. Partial purification of proteins from brush border membrane vesicles (BBMVs) by gel filtration chromatography hinders the cleavage of BT-R1 in the presence of EDTA and EGTA, indicating that there is specific proteolytic activity associated with the BBMV. This specific proteolytic cleavage of BT-R1 not only alters the integrity of BT-R1 but it most likely is implicated in cell adhesion events during differentiation and development of M. sexta midgut epithelium. We propose a model for calcium-dependent protection of BT-R1 as well as a cleavage pattern that may modulate the molecular interactions and adhesive properties of its ectodomain. Molecular characterization of such a protection mechanism should lead to a better understanding of how the function of specific cadherins is modulated during tissue differentiation and insect development.