Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

BACKGROUND: To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori) midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. RESULTS: The Bombyx mori (silkworm) aminopeptidase N (APN) and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM), and unusually tight binding to the cadherin-like receptor (2.6 nM), which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac) was identical to Cry1Aa. CONCLUSIONS: These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research

Biological Pesticides: Biotechnology\u27s Answer to Silent Spring

In the 25 years since the publication of Rachel Carson’s Silent Spring the public has come to realize the environmental impact of heavy use of chemical pesticides. To add insult to injury, many insects, including the disease vector, the mosquito, are now virtually resistant to standard chemical pesticides. Biotechnology is now providing a positive response to these dilemmas through the production and development of improved forms of microbial pest control agents: biological pesticides. Biological pesticides are pathogens, or predators, of insects such as bacteria, fungi, and viruses, and predatory insects or vertebrates such as mosquito fish which reduce the population of the pest. Several biological insecticides are now registered with the EPA and are used as safe alternatives to chemicals. These agents are highly specific to the insect pests which they attack and are extensively tested and shown not to be toxic to man or animals or indeed to non-target insects. Biological pesticides are excellent models for release of genetically-engineered microorganisms into the environment because literally millions of tons of certain of these biological pesticides have been released and extensive studies have been done on their persistence. The goals of genetic engineering are to decrease the costs of production by increasing yield and specific activity of biological pesticides. Through the use of genetic engineering, plants with the genes of biological pesticides incorporated into their own genomes are now in field trials. These new developments further reduce the environmental impact of the pesticide by removing it from the environment niches of insects which do not attack the specific crop. This presentation will review the persistence of non-genetically-engineered microorganisms. The release of newly-engineered organisms into the environment will be critically discussed, considering the ethics and economic impact of genetically-engineered biological pesticides

Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus

BACKGROUND: Aminopeptidase N (APN) type proteins isolated from several species of lepidopteran insects have been implicated as Bacillus thuringiensis (Bt) toxin-binding proteins (receptors) for Cry toxins. We examined brush border membrane vesicle (BBMV) proteins from the mosquito Anopheles quadrimaculatus to determine if APNs from this organism would bind mosquitocidal Cry toxins that are active to it. RESULTS: A 100-kDa protein with APN activity (APN(Anq )100) was isolated from the brush border membrane of Anopheles quadrimaculatus. Native state binding analysis by surface plasmon resonance shows that APN(Anq )100 forms tight binding to a mosquitocidal Bt toxin, Cry11Ba, but not to Cry2Aa, Cry4Ba or Cry11Aa. CONCLUSION: An aminopeptidase from Anopheles quadrimaculatus mosquitoes is a specific binding protein for Bacillus thuringiensis Cry11Ba

Estimation of juvenile striped bass relative abundance in the Virginia portion of Chesapeake Bay, January 2003-December 2003 : annual progress report

The Virginia Institute of Marine Science (VIMS) has conducted a juvenile striped bass seine survey from 1967 through 1973 and from 1980 through the present. The primary objective has been the monitoring of the relative annual recruitment success ofjuvenile stripedbass in the spawning and nursery areas of Lower Chesapeake Bay. Initially (1967-1973), the survey was funded by the U.S. Fish and Wildlife Service and when reinstated in 1980 with funding from the National Marine Fisheries Service under the Emergency Striped Bass Study program. Commencing with the 1988 annual survey, support of the program has been jointly made through the Sportfish Restoration Program (Wallop-Breaux Act), administered through the U.S. Fish and Wildlife Service and the Virginia Marine Resources Commission. This report summarizes the results of the 2003 sampling period and compares these results with the previous work

First-year Experience Course: Problem Solving, Inquiry, and Integration

In an effort to address academic deficiencies outlined in recent studies, Virginia Tech’s College of Natural Resources and Environment redesigned its first-year experience class to fit their activities into three components of activities: problem solving, inquiry, and integrative learning. The problem solving component required students to define a problem, identify problem-solving strategies, and propose solutions and hypotheses. The inquiry component of the first-year experience included selecting a research topic, learning how to access information about the topic, learning how to evaluate existing information about the topic, and deciding which information to use to achieve desired results about the topic. The final component of the program, integration of learning, connected different programs of study with in-class learning and outside experience. This component also stressed exploring the relationship between student’s self and their learning experiences. To evaluate these three categories, the Motivated Strategies for Learning Questionnaire and the Information Literacy Test surveys were administered to the students at the beginning of the semester and the end of the semester in order to evaluate student growth in each category, as well as students’ own self-awareness. Quantitative analysis of these two surveys illustrates the effectiveness of the assignments associated with each component. Knowledge gained from the redevelopment of the class, quantitative analysis of the surveys, and plans for additional amendments to the class will be shared during conference proceedings

Ser170 of Bacillus thuringiensis Cry1Ab δ-endotoxin becomes anchored in a hydrophobic moiety upon insertion of this protein into Manduca sexta brush border membranes

<p>Abstract</p> <p>Background</p> <p>Three spin-labeled mutant proteins, mutated at the beginning, middle, and end of α-helix 5 of the <it>Bacillus thuringiensis </it>Cry1Ab δ-endotoxin, were used to study the involvement of these specific amino acid residues in ion transport and to determine conformational changes in the vicinity of these residues when the protein was translocated into a biological membrane.</p> <p>Results</p> <p>Amino acid residue leucine 157, located in the N-terminal portion of α-helix 5, showed no involvement in ion transport, and the environment that surrounds the residue did not show any change when transferred into the biological membrane. Serine 170, located in the middle of the α-helix, showed no involvement in ion transport, but our findings indicate that in the membrane-bound state this residue faces an environment that makes the spin less mobile, as opposed to the mobility observed in an aqueous environment. Serine 176, located in the C-terminal end of the α-helix 5 is shown to be involved in ion transport activity.</p> <p>Conclusion</p> <p>Ion transport data for L157, S170, and S176, along with the mobility of the spin-labels, structural characterization of the resulting proteins, and toxicity assays against a target insect, suggest that the toxin undergoes conformational changes upon protein translocation into the midgut membrane. These conformational changes result in the midregion of the α-helix 5 being exposed to a hydrophobic-like environment. The location of these three residues in the toxin suggests that the entire α-helix becomes inserted in the insect midgut membrane.</p

Blocking binding of Bacillus thuringiensis Cry1Aa to Bombyx mori cadherin receptor results in only a minor reduction of toxicity

<p>Abstract</p> <p>Background</p> <p><it>Bacillus thuringiensis </it>Cry1Aa insecticidal protein is the most active known <it>B. thuringiensis </it>toxin against the forest insect pest <it>Lymantria dispar </it>(gypsy moth), unfortunately it is also highly toxic against the non-target insect <it>Bombyx mori </it>(silk worm).</p> <p>Results</p> <p>Surface exposed hydrophobic residues over domains II and III were targeted for site-directed mutagenesis. Substitution of a phenylalanine residue (F328) by alanine reduced binding to the <it>Bombyx mori </it>cadherin by 23-fold, reduced biological activity against <it>B. mori </it>by 4-fold, while retaining activity against <it>Lymantria dispar</it>.</p> <p>Conclusion</p> <p>The results identify a novel receptor-binding epitope and demonstrate that virtual elimination of binding to cadherin BR-175 does not completely remove toxicity in the case of <it>B. mori</it>.</p

Robust Network Stability of Mosquitoes and Human Pathogens of Medical Importance

Background: The exact number of mosquito species relevant to human health is unknown, posing challenges in understanding the scope and breadth of vector–pathogen relationships, and how resilient mosquito vector–pathogen networks are to targeted eradication of vectors. Methods: We performed an extensive literature survey to determine the associations between mosquito species and their associated pathogens of human medical importance. For each vector–pathogen association, we then determined the strength of the associations (i.e., natural infection, lab infection, lab dissemination, lab transmission, known vector). A network analysis was used to identify relationships among all pathogens and vectors. Finally, we examined how elimination of either random or targeted species affected the extinction of pathogens. Results: We found that 88 of 3578 mosquito species (2.5%) are known vectors for 78 human disease-causing pathogens; however, an additional 243 species (6.8%) were identified as potential or likely vectors, bringing the total of all mosquitos implicated in human disease to 331 (9.3%). Network analysis revealed that known vectors and pathogens were compartmentalized, with the removal of six vectors being enough to break the network (i.e., cause a pathogen to have no vector). However, the presence of potential or likely vectors greatly increased redundancies in the network, requiring more than 41 vectors to be eliminated before breaking the network. Conclusion: Although \u3c 10% of mosquitoes are involved in transmitting pathogens that cause human disease, our findings point to inherent robustness in global mosquito vector–pathogen networks

Preferential protection of domains ii and iii of bacillus thuringiensis cry1aa toxin by brush border membrane vesicles

Título español: Protección preferencial de los dominios II y III de la toxina Cry1Aa de Bacillus thuringiensis en Vesículas de Membrana de Borde de Cepillo Abstract The surface exposed Leucine 371 on loop 2 of domain II, in Cry1Aa toxin, was mutated to Lysine to generate the trypsin-sensitive mutant, L371K. Upon trypsin digestion L371K is cleaved into approximately 37 and 26 kDa fragments. These are separable on SDS-PAGE, but remain as a single molecule of 65 kDa upon purification by liquid chromatography. The larger fragment is domain I and a portion of domain II (amino acid residues 1 to 371). The smaller 26-kDa polypeptide is the remainder of domain II and domain III (amino acids 372 to 609). When the mutant toxin was treated with high dose of M. sexta gut juice both fragments were degraded. However, when incubated with M. sexta BBMV, the 26 kDa fragment (domains II and III) was preferentially protected from gut juice proteases. As previously reported, wild type Cry1Aa toxin was also protected against degradation by gut juice proteases when incubated with M. sexta BBMV. On the contrary, when mouse BBMV was added to the reaction mixture neither Cry1Aa nor L371K toxins showed resistance to M. sexta gut juice proteases and were degraded. Since the whole Cry1Aa toxin and most of the domain II and domain III of L371K are protected from proteases in the presence of BBMV of the target insect, we suggest that the insertion of the toxin into the membrane is complex and involves all three domains. Key words: Bacillus thuringiensis, site directed mutagenesis, -endotoxin. Resumen La superficie de la toxina Cry1Aa, en el asa 2 del dominio II contiene expuesta la leucina 371, la cual fue modificada a lisina produciendo una mutante sensible a la tripsina, L371K. Esta mutante produce dos fragmentos de 37 y 26 kDa por acción de la tripsina que son separables por SDS-PAGE, pero que a la purificación por cromatografía líquida se mantienen como una sola molécula de 65 kDa. El fragmento grande contiene al dominio I y una parte del dominio II (aminoácidos 1 al 371). El polipéptido de 26 kDa contiene la parte restante del dominio II y dominio III (aminoácidos 372 al 609). Cuando la toxina mutante fue tratada con dosis altas de jugo intestinal de Manduca sexta, ambos fragmentos fueron degradados. Sin embargo, cuando fueron incubados en VMBC de M. sexta, el fragmento de 26 kDa fue protegido preferencialmente de las proteasas intestinales. Como se ha reportado, la toxina silvestre Cry1Aa también es protegida de la degradación de las proteasas cuando es incubada en VMBC de M. sexta. Sin embargo, cuando se adicionó VMBC de ratón a la mezcla de reacción, ni la toxina Cry1Aa ni la mutante L371K mostraron resistencia a las proteasas y fueron degradadas. Dado que la toxina completa de Cry1Aa y casi todo de los dominios II y III de L371K están protegidos de proteasas en presencia de VMBC del insecto, este estudio sugiere que la inserción de la toxina en la membrana involucra los tres dominios. Palabras clave: Bacillus thuringiensis, mutagénesis sitio dirigida, - endotoxin