Biochemical features of the Cry4B toxin of Bacillus thuringiensis subsp. israelensis and its interaction with BT-R3, a bitopic cadherin G-protein coupled receptor in Anopheles gambiae.

Introduction: The cadherin G-protein coupled receptor BT-R3 in the mosquito Anopheles gambiae 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 Bacillus thuringiensis subsp. israelensis (Bti) to BT-R3 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. Described in this study is the behaviour of the Cry4B purified active protein toxin in solution relative to its protoxin predecessor produced by Bti as well as identification of the region within BT-R3 of An. gambiae to which the toxin binds. Materials and Methods: The relationship and behaviour of protoxin and toxin were ascertained in vitro by solubility studies in an alkaline environment like that of the mosquito larval midgut. To identify the specific toxin-binding site within BT-R3, the full-length coding sequence of the bt-r3 gene was amplified and cloned in pENTR/D-TOTO and subcloned in pXINSECT-DEST38 resulting in recombinant pXINSECT-DEST38-bt-r3. Cytotoxicity was analysed using Trichoplusia ni High Five™ insect cells transfected with the pXINSECT-DEST38-bt-r3 plasmid rendering them susceptible to the Cry4B toxin. Truncation mutational analyses, receptor-toxin binding studies and live cell experiments were used to elucidate the toxin-binding site in BT-R3. Results: The N-terminal half of the Cry4B protoxin was cleaved releasing active Cry4B toxin. The nontoxic C-terminal portion was degraded into small peptide fragments. The receptor BT-R3 contained a single toxin-binding site--a 106-amino acid polypeptide bounded by Ile1359 and Ser1464 (1359IS1464) localized in the 11th cadherin repeat of the receptor. Conclusions: The structural features of the toxin-binding site are critical to the specificity, selectivity and affinity of the active toxin and for the design and development of novel Bti-based biopesticides.

Cell death signaling in Anopheles gambiae initiated by Bacillus thuringiensis 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 Bacillus thuringiensis (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 Anopheles gambiae, the primary mosquito vector of malaria, is the consequence of a variety of signaling activities. We show here that Cry4B, acting as first messenger, binds specifically to the bitopic cadherin BT-R3 G-protein-coupled receptor (GPCR) localized in the midgut of A. gambiae, activating the downstream second messenger cyclic adenosine monophosphate (cAMP). The direct result of the Cry4B-BT-R3 binding is the release of αs from the heterotrimeric αßγ-G-protein complex and its activation of adenylyl cyclase (AC). The upshot is an increased level of cAMP, which activates protein kinase A (PKA). The functional impact of cAMP-PKA signaling is the stimulation of Na+/K+-ATPase (NKA) which serves as an Na+/K+ pump to maintain proper gradients of extracellular Na+ and intracellular K+. Increased level of cAMP amplifies NKA and upsets normal ion concentration gradients. NKA, as a scaffolding protein, accelerates the first messenger signal to the nucleus, generating additional BT-R3 molecules and promoting their exocytotic trafficking to the cell membrane. Accumulation of BT-R3 on the cell surface facilitates recruitment of additional toxin molecules which, in turn, amplify the original signal in a cascade-like manner. This report provides the first evidence of a bacterial toxin using NKA via AC/PKA signaling to execute cell death.

Functional and Structural Analysis of the Toxin-Binding Site of the Cadherin G-Protein-Coupled Receptor, BT-R1, for Cry1A Toxins of Bacillus thuringiensis.

The G-protein-coupled receptor BT-R1 in the moth Manduca sexta represents a class of single-membrane-spanning α-helical proteins within the cadherin family that regulate intercellular adhesion and contribute to important signaling activities that control cellular homeostasis. The Cry1A toxins, Cry1Aa, Cry1Ab, and Cry1Ac, produced by Bacillus thuringiensis bind BT-R1 very tightly (Kd = 1.1 nM) and trigger a Mg2+-dependent signaling pathway that involves the stimulation of G-protein α-subunit, which subsequently launches a coordinated signaling cascade, resulting in insect death. The three Cry1A toxins compete for the same binding site on BT-R1, and the pattern of inhibition of insecticidal activity against M. sexta is strikingly similar for all three toxins. The binding domain is localized in the 12th cadherin repeat (EC12: Asp1349 to Arg1460, 1349DR1460) in BT-R1 and to various truncation fragments derived therefrom. Fine mapping of EC12 revealed that the smallest fragment capable of binding is a highly conserved 94-amino acid polypeptide bounded by Ile1363 and Ser1456 (1363IS1456), designated as the toxin-binding site (TBS). Logistical regression analysis revealed that binding of an EC12 truncation fragment containing the TBS is antagonistic to each of the Cry1A toxins and completely inhibits the insecticidal activity of all three. Elucidation of the EC12 motif of the TBS by X-ray crystallography at a 1.9 Å resolution combined with results of competitive binding analyses, live cell experiments, and whole insect bioassays substantiate the exclusive involvement of BT-R1 in initiating insect cell death and demonstrate that the natural receptor BT-R1 contains a single TBS.

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

The Cry1Ab toxin produced by Bacillus thuringiensis binds to a conserved structural motif in the 12th ectodomain module (EC12) of BT-R1, a cadherin G protein-coupled receptor (GPCR) contained in the membrane of midgut epithelial cells of the tobacco hornworm Manduca sexta. 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. Moreover, we generated a fluorescent EC12 probe by converting the 36th residue to cysteine to enable maleimide-mediated conjugation of Alexa-488 fluorescent dye to EC12 by site-directed mutagenesis. In addition, we changed the 44th residue of EC12 to tryptophan, which greatly improved accuracy of protein quantification and traceability. Using the fluorescently labeled EC12 probe for direct and competitive binding assays, we were able to determine binding specificity in solution. These accomplishments will facilitate identification and characterization of the interface sequences for both the Cry1Ab toxin and BT-R1.

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. Reprogramming of methylation patterns in gametes is essential to sex-specific inheritance of imprinted genes and assures exclusive harboring of female- and male-specific imprinted patterns in maternal and paternal gametes, respectively. The consequences of genomic imprinting are manifested by its loss, which can lead to a variety of disorders, the most prominent ones being Prader-Willi and Angelman syndromes. Although a great deal of research has been carried out to examine various imprinting mechanisms, little is known about the establishment and regulation of imprinted genes. In the present paper, we describe several epigenetic mechanisms that have relevance in imprinting and that may have impact on embryonic development, fetal growth and animal cloning.

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. Based on these observations, we developed a mathematical model to quantify both the expression of BT-R(1) and the susceptibility of M. sexta larvae to the Cry1Ab toxin. Interestingly, the toxin-receptor ratio remains constant during larval development regardless of larval size and mass. This ratio apparently is critical for insecticidal activity and the decrease in toxin effectiveness during larval development is due primarily to the number of effective toxins and available receptors in the larval midgut. Evidently, susceptibility of M. sexta to the Cry1Ab toxin of Bt correlates directly to the developmental expression of BT-R(1) in this insect.

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.

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. In the present study, the effects of calcium and Cry1Ab toxin, produced by Bacillus thuringiensis, on the adhesive properties of BBMVs were investigated. Addition of calcium to a suspension of BBMVs promoted adhesion and aggregation of the vesicles. Treatment of BBMVs with trypsin or lowering the pH (pH 4.0) of the BBMV suspension abolished calcium-induced vesicle aggregation, whereas treatment with deglycosylating enzymes did not affect the aggregation of vesicles, indicating that adhesion and clustering of BBMVs involves protein-protein interactions. Preincubation of BBMVs with Cry1Ab toxin, which specifically binds to BT-R(1) with high affinity and disrupts the midgut epithelium of M. sexta, caused a 50% decrease in calcium-induced vesicle aggregation. The inhibitory effects of the Cry1Ab toxin on BBMV aggregation was blocked completely when the toxin was preincubated with a peptide containing the toxin-binding site of BT-R(1). Cry3A toxin, which is similar in molecular structure to Cry1Ab but does not bind to BT-R(1) and is not toxic to M. sexta larvae, did not affect BBMV aggregation. The results of this study demonstrate that the adhesive function of BT-R(1) is compromised by the Cry1Ab toxin, which acts as a selective antagonist, and supports the notion that BT-R(1) is critical in preserving the integrity of larval midgut epithelium in M. sexta.

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. The amount of BT-R(1) protein increases dramatically during larval development, paralleling accumulation of its mRNA. The 5'-UTR of the btr-1 gene contains sequence motifs that most likely recruit specific transcription factors, particularly, those that determine posterior patterning and that control intestinal cell proliferation, differentiation and identity during development. The increase in abundance of BT-R(1) may be required to support not only the differentiation of the epithelial cells but also the establishment of physiological function and structural integrity of the midgut during larval development in M. sexta. We believe that BT-R(1) is essential to larval midgut epithelial organization during rapid cell proliferation and tissue growth in M. sexta because disruption of such organization and functionality occasioned by the binding of the Cry1A toxins of B. thuringiensis to BT-R(1) causes death to the insect.

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. Additionally, the electrophoretic migration pattern of a low molecular mass acidic protein, which apparently is an ortholog of F(1)F(0)-ATPase, was considerably altered in the Bt-resistant insect indicating that variations in amino acid content or modifications of certain proteins also are important components of the resistance phenomenon in the IMM. Furthermore, there was a dramatic decrease in the level of chymotrypsin-like proteinase in the midgut of the Bt-resistant larva, signifying that reduction of chymotrypsin activity, and subsequently decreased activation of Cry toxin in the insect midgut, is an important factor in the resistant state of the IMM. The proteomic analysis of larval gut proteins utilized in this study provides a useful approach for consolidating protein changes and physiological events associated with insect resistance to Bt. Our results support the hypothesis that physiological adaptation of insects and resistance to Bt is multifaceted, including protein modification and changes in the synthesis of specific larval gut proteins. We believe that increased oxidative metabolism may be an adaptive response of insects that undergo survival challenge and that it could mediate detoxification as well as higher rates of generalized and localized mutations that enhance their resistance and provide survival advantage.

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.

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. tenebrionis. Histological examination revealed that the structural integrity of the midgut tissue in the toxin-resistant (R) insect was retained whereas the same tissue was devastated by toxin action in the susceptible (S) strain. Function-based activity profiling using zymographic gels showed specific proteolytic bands present in midgut extracts and brush border membrane vesicles (BBMV) of the R strain not apparent in the S strain. Aminopeptidase activity associated with insect midgut was higher in the R strain than in the S strain. Enzymatic processing of toxin did not differ in either strain and, apparently, is not a factor in resistance. BBMV from the R strain bound approximately 60% less toxin than BBMV from the S strain, whereas the kinetics of toxin saturation of BBMV was 30 times less in the R strain than in the S strain. However, homologous competition inhibition binding of (125)I-Cry3Aa to BBMV did not reveal any differences in binding affinity (K(d) approximately 0.1 microM) between the S and R strains. The results indicate that resistance by the CPB to the Cry3Aa toxin correlates with specific alterations in protease activity in the midgut as well as with decreased toxin binding. We believe that these features reflect adaptive responses that render the insect refractory to toxin action, making this insect an ideal model to study host innate responses and adaptive changes brought on by bacterial toxin interaction.