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.

"The Defined Toxin-binding Region of the Cadherin G-protein Coupled Receptor, BT-R1, for the Active Cry1Ab Toxin of Bacillus thuringiensis".

The bacterium Bacillus thuringiensis (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. The insecticidal activity of Cry toxins is mediated by specific interaction between toxins and their respective cellular receptors. One such toxin (Cry1Ab) exerts toxicity by first targeting the 12th ectodomain region (EC12) of the moth cadherin receptor BT-R1. Binding promotes a highly regulated signaling cascade event that concludes in oncotic-like cell death. We previously determined that conserved sequence motifs near the N- and C-termini of EC12 are critical for toxin binding in insect cells. Here, we have established that Cry1Ab specifically binds to EC12 as a soluble heterodimeric complex with extremely high affinity (Kd = 19.5 ± 1.6 nM). Binding assays using Cry1Ab toxin and a fluorescently labeled EC12 revealed that the heterodimeric complex is highly specific in that no such formation occurs between EC12 and other Cry toxins active against beetle and mosquito. Disruption of one or both terminal sequence motifs in EC12 eliminates complex formation. Until now, comprehensive biophysical characterization of Cry1Ab recognition and binding by the BT-R1 receptor was unresolved. The findings presented here provide insight on the molecular determinants in the Cry family of toxins and should facilitate the assessment and advancement of their use as pesticidal agents.

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.

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.

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. sphaericus, are used as microbial insecticides, their spectrum of insecticidal activity is quite limited compared to Bt. Importantly, Bt is safe for humans and is the most widely used environmentally compatible biopesticide worldwide. Furthermore, insecticidal Bt genes have been incorporated into several major crops, rendering them insect resistant, and thus providing a model for genetic engineering in agriculture.This review highlights what the authors consider the most relevant issues and topics pertaining to the genomics and proteomics of Bt. At least one of the authors (L.A.B.) has spent most of his professional life studying different aspects of this bacterium with the goal in mind of determining the mechanism(s) by which it kills insects. The other authors have a much shorter experience with Bt but their intellect and personal insight have greatly enriched our understanding of what makes Bt distinctive in the microbial world. Obviously, there is personal interest and bias reflected in this article notwithstanding oversight of a number of published studies. This review contains some material not published elsewhere although several ideas and concepts were developed from a broad base of scientific literature up to 2010.

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.

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.

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.

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. Both fractions were divided into subfractions, and proteins were identified in each fraction separately through tryptic digestion. Membrane protein digests were collected from externally exposed proteins, internally exposed proteins, "spectrin extract" mainly consisting of membrane skeleton proteins, and membrane proteins minus spectrin extract. Cytoplasmic proteins were divided into 21 fractions based on molecular mass by size exclusion chromatography. The tryptic peptides were separated by reverse-phase high-performance liquid chromatography and identified by ion trap tandem mass spectrometry. A total of 181 unique protein sequences were identified: 91 in the membrane fractions and 91 in the cytoplasmic fractions. Glyceraldehyde-3-phosphate dehydrogenase was identified with high sequence coverage in both membrane and cytoplasmic fractions. Identified proteins include membrane skeletal proteins, metabolic enzymes, transporters and channel proteins, adhesion proteins, hemoglobins, cellular defense proteins, proteins of the ubiquitin-proteasome system, G-proteins of the Ras family, kinases, chaperone proteins, proteases, translation initiation factors, and others. In addition to the known proteins, there were 43 proteins whose identification was not determined.

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.