Progress 10/01/10 to 09/30/15
Outputs
Target Audience:For this research project the primary audience is the scientific community in academia, industry and the government. In additional there is an international audience that views this research with a great deal of interest. The target audience is reached via scientific publications and talks presented at national and international meetings. Sarjeet Gill and Jianwu Chen presented results at international and national meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Assistant Specialists, Karly Aimanova and Jianwu Chen, and the postdoc Estefania Contreras Navarro were each given independent projects. Jianwu Chen was allowed to initiate a new project for gene knockout using Crisper methodology. In addition he was allowed to mentor both a graduate student and an undergraduate student. Further he presented a poster at the Entomological Society of America Annual Meeting. Karly Aimanova was given a project to transform mosquito species that expresses dsRNA in vivo. Maria Ramirez is minority undergraduate students who were given an opportunity to work in the laboratory to learn insect dissection and insect maintenance techniques. How have the results been disseminated to communities of interest?Since the primary audience isthe scientific community in academia, industry and the government we published all our results in international peer reviewed journals. A large number of papers and book chapters were published during the five year project. Sarjeet Gill and Jianwu Chen presented results at international and national meetings. What do you plan to do during the next reporting period to accomplish the goals?This is a final report
Impacts
What was accomplished under these goals?
We accomplished all the goals that we set out to. The five year project was very successful. Importantly we were able to get a five year grant from NIH to fund the research we proposed. In the first objective we showed specific cadherin domains are indeed involved in toxicity; in particular cadherin repeats (CR) 8-11. We showed by expressing AeCad in Aedes C6/36 cells, that this cadherin is important because cells become Cry11Aa-sensitive. In the second sub-aim we showed the Cry11Ba loops α8, 1 and 3 are involved in toxicity. Loops 1 and 3 are of greater significance in toxicity to Aedes and Culex larvae, than to Anopheles larvae. Further, the Cry4Ba loops - α8, loop 1, and loop 3 compete with Cry11Ba binding to brush border membrane vesicles (BBMV); suggesting Cry11Ba and Cry4Ba have common binding sites in Ae. aegypti BBMV. But these BBMV binding sites differ from that of Cry11Aa. In the third sub-aim we proved that cadherin plays an important role in Cry11Aa oligomerization further supporting our model of toxin action. In the fourth sub-aim we showed that suppression of cadherin expression by in vivo silencing (Fig. 1B, C) leads to mosquito larvae being 13X less sensitive to Cry11Aa toxin, but not to Cry11Ba or Cry4Ba. The second objective was to characterize the role of alkaline phosphatases in toxicity. Our proposed focus was only on ALP09077. Our first sub-aim determined that loop II of Cry4Ba is important for toxicity and binding to larval BBMV, and that the toxicity is correlated with ALP09077 binding. In a second sub-aim, we showed using dsRNA feeding that ALP09077 plays a functional role in Cry11A toxicity, but less so with Cry4Ba. However, we noted that other ALPs may be involved in toxicity. Thus rather than focusing only on a few ALPs, we changed the approach used. Since field strains are not resistant to Bti, we lab-selected a Cry11Aa-resistant line. This strain, named G30, showed high level Cry11A resistance (124X), cross-resistance to Cry4Aa (66X), low resistance to Cry4Ba (13X) and negligible resistance to Cry11Ba (2X). Using a cross reacting ALP antibody we noted that ALP, but not AeCad, protein levels were decreased by ~40% in this strain. We then compared the midgut transcriptome of this resistant strain with that of wild-type using RNA seq. We observed that three ALP transcripts were statistically significantly altered. In addition to these two objective we also elucidated the characteristics of Cyt1A in pore-formation and synergism. We showed that as with Cry11Aa, Cry4Ba activity is synergized by Cyt1Aa, and that Cyt1Aa loop β6-α5 K198A, E204A and β7 K225A mutants affected binding and synergism with Cry4Ba. In addition, domain II loop α-8 of Cry4Ba is involved in binding and in synergism with Cyt1Aa, since the Cry4Ba mutants in this domain showed decreased binding and synergism with Cyt1Aa. These data suggest that as with the synergism between Cry11Aa and Cyt1Aa toxins, Cyt1Aa also functions as a Cry4Ba receptor. We also characterized Cyt1Aa domains involved in oligomerization and membrane insertion. The Cyt1A N-terminal region is involved in toxin aggregation, while the C-terminal domain is involved in the toxin's interaction with the lipid membrane. Also helix α3 mutants were not hemolytic and insecticidal, because they were affected in oligomerization and unable to insert into the membrane. However, these mutants were still able to synergize Cry11Aa toxicity indicating the oligomerization and membrane insertion steps, are distinct from Cyt1Aa's ability to synergize.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Canton, P.E., Cancino-Rodezno, A., Gill S.S., Soberon M., and Bravo A. 2015. Transcriptional cellular responses in midgut tissue of Aedes aegypti larvae following intoxication with Cry11Aa toxin from Bacillus
thuringiensis. BMC Genomics Dec 9;16:1042. doi: 10.1186/s12864-015-2240-7
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: For this research project the primary audience is the scientific community in academia, industry and the government. In additional there is an international audience that views this research with a great deal of interest. Two approaches were used to disseminate our funding this year. The first is to publish the results in international peer reviewed journals. Five papers were published. The second was to present results at meetings. Sarjeet Gill and Jianwu Chen presented results at international and national meetings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Assistant Specialists, Karly Aimanova and Jianwu Chen, and the postdoc Estefania Contreras Navarro were each given independent projects. Jianwu Chen was allowed to initiate a new project for gene knockout using Crisper methodology. In addition he was allowed to mentor both a graduate student and an undergraduate student. Further he presented a poster at the Entomological Society of America Annual Meeting. Karly Aimanova was given a project to transform mosquito species that expresses dsRNA in vivo. Maria Ramirez and Jacqueline Ledezma are minority undergraduate students who were given an opportunity to work in the laboratory to learn insect dissection and insect maintenance techniques. How have the results been disseminated to communities of interest? For this research project the primary audience is the scientific community in academia, industry and the government. In additional there is an international audience that views this research with a great deal of interest. Two approaches were used to disseminate our funding this year. The first is to publish the results in international peer reviewed journals. Five papers were published. The second was to present results at meetings. Sarjeet Gill and Jianwu Chen presented results at international and national meetings. What do you plan to do during the next reporting period to accomplish the goals? Last year we initiated a project to identify mosquitocidal toxins in an anaerobic strain of CLOSTRIDIUM BIFERMANTANS. We plan to identify the toxins that are toxic to ANOPHELES mosquitoes. We also plan to continue this project in the coming year and plan to submit a new grant proposal for extramural funding. In addition we plan to continue our existing work on BACILLUS THURINGIENSIS ISRAELENSIS. In particular we plan to use Crisper/Cas9 to disrupt key proteins involved in toxicity. We also will submit a grant proposal to continue this line of investigation
Impacts
What was accomplished under these goals?
BACILLUS THURINGIENSIS ISRAELENSIS (BTI) is highly toxic to different mosquito and black fly species that are vectors of human disease pathogens. Due to the emergence of insect-resistance to chemical insecticides, BTI formulations are being used worldwide to control mosquitoes. Hence these formulations continue to be used worldwide for the control of the most important pests of agriculture and human health. The continued use of these formulations is likely to enhance the selection pressure to develop resistance to the toxins that form the active ingredients in these formulations. Consequently it is anticipated that insect resistance to BACILLUS THURINGIENSIS toxins will become more frequent in the coming years. Such an occurrence could limit the utility of BACILLUS THURINGIENSIS-crops that form a large part of US agriculture. Similarly the continued use of bacterial formulations for the control of insect vectors of human diseases also enhances the selection pressure for resistance. This bacterium produces a crystal inclusion composed of at least four toxins: Cry4A, Cry4B, Cry11A and Cyt1A. The specificity of this bacterial strain to its target insects is dependent on receptor binding of the individual toxins to specific midgut proteins. In previous years we showed that the Cry11A toxin binds a variety of different proteins in AEDES AEGYPTI larval midgut. Our previous data indicated that a number of proteins bind Cry11A. We previously cloned a full-length cadherin from Aedes aegypti larvae and reported this protein binds Cry11Aa toxin from of BACILLUS THURINGIENSIS ISRAELENSIS with high affinity. Based on these results, we investigated if the cadherin is involved in the in vivo toxicity of Cry11Aa toxin to AEDES AEGYPTI. We established a mosquito cell line stably expressing the full-length the cadherin and transgenic mosquitoes with silenced cadherin expression. Cells expressing the cadherin showed increased sensitivity to Cry11Aa toxin. Cry11Aa toxin at 400 nM killed approximately 37% of the cells in 3 h. Otherwise, transgenic mosquitoes with silenced cadherin expression showed increased tolerance to Cry11Aa toxin. Furthermore, cells expressing this cadherin triggered Cry11Aa oligomerization. These results show this cadherin plays a pivotal role in Cry11Aa toxicity to AEDES AEGYPTI larvae by mediating Cry11Aa oligomerization. However, since high toxicity was not obtained in cadherin-expressing cells, an additional receptor may be needed for manifestation of full toxicity. Moreover, cells expressing this cadherin were sensitive to Cry4Aa and Cry11Ba, but not Cry4Ba. However transgenic mosquitoes with silenced cadherin expression showed no tolerance to Cry4Aa, Cry4Ba, and Cry11Ba toxins. These results suggest that while Aedes cadherin may mediate Cry4Aa and Cry11Ba toxicity, this cadherin but is not the main receptor of Cry4Aa, Cry4Ba and Cry11Ba toxin in AEDES AEGYPTI. To further elucidate the mechanism of this toxin we developed an AEDES AEGYPTI resistant strain that shows high-level resistance to Cry11Aa toxin. After 27 selections with Cry11Aa toxin, the larvae showed a 124-fold resistance ratio for Cry11Aa. These resistant larvae larvae showed cross-resistance to Cry4Aa (66-fold resistance), less to Cry4Ba (13-fold), but not to Cry11Ba (2-fold). Midguts from these resistant larvae did not show detectable difference in the processing of the Cry11Aa toxin compared to that in susceptible larvae. Brush border membrane vesicles (BBMV) from resistant larvae bound slightly less Cry11Aa compared to susceptible larvae BBMV. To identify potential proteins associated with Cry11A resistance, not only transcript changes in the larval midgut were analyzed using NextGen sequencing and qPCR, but alterations of previously identified receptor proteins were investigated using immunoblots. The transcripts of 375 genes were significantly increased and those of 208 genes were down regulated in the resistant larvae midgut compared to the susceptible larvae. None of the transcripts for previously identified receptors of Cry11Aa (AEDES cadherin, ALP1, APN1, and APN2) were altered in these analyses. The genes for the identified functional receptors in resistant larvae midgut did not contain any mutation in their sequences nor was there any change in their transcript expression levels compared to susceptible larvae. However, ALP proteins were expressed at reduced levels (~40%) in the resistant strain BBMV. APN proteins and their activity were also slightly reduced in resistance strain. The transcript levels of ALPs (AAEL013330 and AAEL015070) and APNs (AAEL008158, AAEL008162) were significantly reduced. These results strongly suggest that ALPs and APNs could be associated with Cry11Aa resistance in AEDES AEGYPTI. Continued analysis of the molecular basis of toxin action including studies of insect specificity, of insect resistance to Cry toxins and of the role of receptor molecules in toxicity will provide new ways for a rational design of Cry toxins to control insect pests important in agriculture or in human health. Such developments will extend the useful life span of this technology. The management and control of mosquito vectors of human disease currently rely primarily on chemical insecticides. However, larvicidal treatments can be effective, and if based on biological insecticides, they can also ameliorate the risk posed to human health by chemical insecticides. The aerobic BACILLUS and LYSINIBACILLUS have been used for vector control for a number of decades. But a more cost-effective use would be an anaerobic bacterium because of the ease with which these can be cultured. More recently, the anaerobic bacterium CLOSTRIDIUM BIFERMANTANS MALAYSIA was reported to have high mosquitocidal activity. We cloned four toxins encoded by the Cry operon, Cry16A, Cry17A, Cbm17.1, and Cbm17.2, and show that are all required for toxicity, and these toxins collectively show remarkable selectivity for AEDES rather than ANOPHELES mosquitoes, even though CLOSTRIDIUM BIFERMANTANS MALAYSIA is more toxic to ANOPHELES. Hence, toxins that target ANOPHELES are different from those expressed by the Cry operon.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Cant�n PE, L�pez-D�az JA,Gill SS, Bravo A, Sober�n M. 2014. Membrane binding and oligomer membrane insertion are necessary but insufficient for Bacillus thuringiensis Cyt1Aa toxicity. Peptides. 53:286-291.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
G�mez I, S�nchez J, Mu�oz-Garay C, Matus V, Gill SS, Sober�n M, Bravo A. 2014 Bacillus thuringiensis Cry1A toxins are versatile proteins with multiple modes of action: two distinct pre-pores are involved in toxicity. Biochem J. 459:383-296.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Lee SB, Chen J, Aimanova KG, Gill SS. 2014. Aedes cadherin mediates the in vivo toxicity of the Cry11Aa toxin to Aedes aegypti. Peptides. S0196-9781(14)00215-0. doi: 10.1016/j.peptides.2014.07.015.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Lee SB, Aimanova KG, Gill SS. 2014. Alkaline phosphatases and aminopeptidases are associated with Cry11Aa resistance in Aedes aegypti. Insect Biochem. Mol Biol. 54:112-21 p.doi:10.1016/j.ibmb
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Qureshi N, Chawla S, Likitvivatanavong S, Lee HL, Gill SS. 2014. The Cry toxin operon from Clostridium bifermantans malaysia is highly toxic to Aedes larval mosquitoes. Appl Environ Microbiol. 80:5689-5697
- Type:
Books
Status:
Published
Year Published:
2014
Citation:
Dhadialla TS. and Gill SS. 2014. Advances in Insect Physiology Vol.17: Insect Midgut and Insecticidal Proteins. Elsevier Academic Press, 423p.
|
Progress 01/01/13 to 09/30/13
Outputs
Target Audience: For this research project the primary audience is the scientific community in academia, industry and the government. In additional there is an international audience that views this research with a great deal of interest. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Assistant Specialists, Karly Aimanova and Jianwu Chen were each given independent projects. Jianwu Chen was allowed to initiate a new project for gene knockout using Talen methodology. In addition he was allowed to mentor both a graduate student and an undergraduate student. Further he presented a talk at an international symposium organized by the Center for Disease Vector Research and a poster at the Entomological Society of America Annual Meeting. Karly Aimanova was given a project to transform mosquito species that expresses dsRNA in vivo. Subum Lee graduated with his PhD this year. He presented his work in a poster at the Entomological Society of America Annual Meeting. Amanda Ortega and Maria Ramirez are minority undergraduate students who were given an opportunity to work in the laboratory to learn insect dissection and insect maintenance techniques. How have the results been disseminated to communities of interest? For this research project the primary audience is the scientific community in academia, industry and the government. In additional there is an international audience that views this research with a great deal of interest. Two approaches were used to disseminate our funding this year. The first is to publish the results in international peer reviewed journals. Two papers were published. The second was to present results at meetings. Sarjeet Gill, Jianwu Chen and Subum Lee presented results at international and national meetings. What do you plan to do during the next reporting period to accomplish the goals? While cadherin is a critical receptor for the Cry11A toxin, for other proteins like Cry4Ba, Cry4A other receptors, including the alkaline phosphatase appears to be more critical. As noted above we continue to believe that toxicity binding changes depending on whether the toxin is monomeric or oligomeric. Hence we plan to analyze the role of specific receptors in the toxicity of the three toxins in BTI, and how these modulate oligomerization of the toxins. In addition we are also initiating a project to identify mosquitocidal toxins in an anaerobic strain of CLOSTRIDIUM BIFERMANTANS. This effort will allow us to be competitive for other extramural funding.
Impacts
What was accomplished under these goals?
BACILLUS THURINGIENSIS ISRAELENSIS (BTI) is highly toxic to different mosquito and black fly species that are vectors of human disease pathogens. Due to the emergence of insect-resistance to chemical insecticides, BTI formulations are being used worldwide to control mosquitoes. Hence these formulations continue to be used worldwide for the control of the most important pests of agriculture and human health. The continued use of these formulations is likely to enhance the selection pressure to develop resistance to the toxins that form the active ingredients in these formulations. Consequently it is anticipated that insect resistance to BACILLUS THURINGIENSIS toxins will become more frequent in the coming years. Such an occurrence could limit the utility of BACILLUS THURINGIENSIS-crops that form a large part of US agriculture. Similarly the continued use of bacterial formulations for the control of insect vectors of human diseases also enhances the selection pressure for resistance. This bacterium produces a crystal inclusion composed of at least four toxins: Cry4A, Cry4B, Cry11A and Cyt1A. The specificity of this bacterial strain to its target insects is dependent on receptor binding of the individual toxins to specific midgut proteins. In previous years we showed that the Cry11A toxin binds a variety of different proteins in AEDES AEGYPTI larval midgut. Our previous data indicated that a number of proteins bind Cry11A. In the present work we continue to focus on this objective. We showed that this toxin binds midgut brush border membrane vesicles (BBMV) with an affinity of 30 nM. Previously an aminopeptidase N (APN), named AaeAPN2, was identified as a putative Cry11A toxin binding protein by pull-down assays using biotinylated Cry11A toxin. Here we show this protein localizes to the apical membrane of epithelial cells in the proximal and distal regions of larval caeca. The AaeAPN2 protein binds Cry11A with high affinity, 8.6 nM. Further, full-length AaeAPN2 and its fragments were cloned and expressed. The toxin-binding region was identified and further competitive assays demonstrated that Cry11A binding to BBMV was efficiently competed by the full-length AaeAPN2 and the two AaeAPN2 fragments. In bioassays against AEDES AEGYPTI larvae, the presence of the full-length and a partial fragment of AaeAPN2 enhanced Cry11A larval mortality. Taken together, we conclude that this APN is a binding protein and plays a role in Cry11A toxicity. BTI also produces a hemolytic Cyt1A toxin that interacts with Cry11A to synergize the activity of the latter toxin. It was previously proposed that Cyt toxins do not interact with protein receptors but directly interact with specific midgut cell lipids. Here, we analyzed if oligomerization and membrane insertion of Cyt1A are necessary steps to synergize Cry11A toxicity. We characterized Cyt1A alpha helix C mutants that were affected in oligomerization, in membrane insertion and also in hemolytic and insecticidal activities. However, these mutants were still able to synergize Cry11A toxicity indicating these steps are independent events of Cyt1A synergistic activity. Furthermore, the data indicate that formation of stable Cyt1A-oligomeric structure is a key step for membrane insertion, hemolysis and insecticidal activity. We also analyzed the oligomerization of the mutant Cry1AbMod protein, which kills insects resistant to Cry toxins, but has less potency against susceptible insects. We found that the Cry1AbMod-protoxin efficiently induces oligomerization, but not the activated Cry1AbMod-toxin, explaining the loss of potency of Cry1AbMod against susceptible insects. Our data support the pore formation model involving sequential interaction with different midgut proteins, leading to pore formation in the target-membrane. We also show that the Cry1A toxins can form two different pre-pores. Continued analysis of the molecular basis of toxin action including studies of insect specificity, of insect resistance to Cry toxins and of the role of receptor molecules in toxicity will provide new ways for a rational design of Cry toxins to control insect pests important in agriculture or in human health. Such developments will extend the useful life span of this technology.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Chen, J., Likitvivatanavong, S., Aimanova, K. G, and Gill, S. S. 2013. A 104 kDa Aedes aegypti aminopeptidase N is a putative receptor for the Cry11A toxin from Bacillus thuringiensis subsp. israelensis Insect Biochem Mol Biol. 43:1201-1208.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
L�pez-Diaz, J. A., Cant�n, P.E., Gill, S.S., Soberon, M. and Bravo, A. 2013
Oligomerization is a key step in Cyt1Aa membrane insertion and toxicity but not necessary to synergize Cry11A toxicity in Aedes aegypti larvae. Environ. Microbiol. 15, 30303039.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: BACILLUS THURINGIENSIS ISRAELENSIS (BTI) is highly toxic to different mosquito and black fly species that are vectors of human disease pathogens. Due to the emergence of insect-resistance to chemical insecticides, BTI formulations are being used worldwide to control mosquitoes. This bacterium produces a crystal inclusion composed of at least four toxins: Cry4A, Cry4B, Cry11A and Cyt1A. Receptor binding is a key factor in determining the specificity of Cry toxins. In previous years we showed that the Cry11A and Cry4Ba toxins bind variety of different proteins in the midgut of AEDES AEGYPTI larvae. In our last report we summarize data showing that silencing alkaline phosphatase (ALP1) expression in AEDES AEGYPTI affected Cry11Aa and Cry4Ba toxicity indicating that this protein was involved in Cry4Ba and Cry11Aa toxicity. However, Cry11Aa relied more on this binding protein than Cry4Ba as shown by the effect of silencing ALP1 expression in the toxicity of these toxins. These data shows that this alkaline phosphatase is a functional receptor for the Cry4Ba and Cry11Aa toxins. The data suggested that Cry4Ba relied in more than one GPI-anchored protein. Recently another ALP isoform (mALP) was shown to be involved in Cry4Ba toxicity. In order to determine the role of both ALP1 and mALP in the toxicity of Cry11Aa and Cry4Ba we cloned mALP and expressed it in E. COLI. Preliminary data shows that both Cry11Aa and Cry4Ba bind mALP with similar binding affinities. We will characterize Cry11Aa and Cry4Ba mutants that were shown to be affected in toxicity and binding to ALP1. Also we cloned both mALP and ALP1 in Drosophila melanogaster using a Gal4-UAS system using a driver GAL fly that express these proteins in the gut of larvae. We are still in the process of cloning flies that express these AEDES AEGYPTI ALP isoforms to determine the toxicity of both Cry4Ba and Cry11Aa. BTI also produces Cyt proteins. Like the Cry proteins the Cyt proteins are mosquitocidal. The Cyt toxins play an important role on the toxicity against mosquitoes since they synergize the insecticidal effect of Cry toxins and are able to overcome resistance to Cry toxins. Cry and Cyt toxins interact by specific epitopes and this interaction is important to induce synergistic activity. It was proposed that Cyt toxins do not interact with protein-receptors, and that they have a direct interaction with the specific lipids present in the insect midgut cells. Hence we analyzed if oligomerization of Cyt1Aa and membrane insertion are necessary steps to induce synergism with Cry11Aa toxin. We isolate mutants in helix C of Cyt1Aa and show that they were severely affected in oligomerization, in membrane insertion and also in hemolytic and insecticidal activity. However these mutants were still able to synergize Cry11Aa toxicity indicating that oligomerization, membrane insertion and toxicity of Cyt1Aa are independent events of its synergistic activity. Furthermore, the data indicates that oligomerization is a key step in membrane insertion and toxicity of Cyt1Aa. This data was been put together into a manuscript for publication. PARTICIPANTS: PI, Sarjeet S. Gill, Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521. Others who have worked on the project at UC Riverside are: The following worked on the project this year: Amy Evans, Jianwu Chen, Karly Aimanova and Subum Lee. Nancy Mora a minority undergraduate student was given an opportunity to work in the laboratory to learn mosquito culture techniques. Subum Lee a graduate student initiated work on the mode of action of the Cry11A toxin. Jianwu Chen an Assistant Specialist was given an independent project that involving molecular biology research. Karly Aimanova an Assistant Specialist was involved in doing some immunohistochemistry while Amy Evans was involved in molecular biology research. Partner Organization and Collaborator: Alejandra Bravo and Mario Soberon, Co-PIs on the project from Departamento de Microbiologıa Molecular, Instituto de Biotecnologıa, Universidad Nacional Autonoma de Mexico, Apdo. Postal 510-3, Cuernavaca 62250, Morelos, Mexico. Others in Mexico who worked on the project include: Claudia Rodriguez-Almazan, Carlos Munoz-Garay, and Isabel Gomez. TARGET AUDIENCES: For this research project the primary audience is the scientific community in academia, industry and the government. In additional there is an international audience that views this research with a great deal of interest. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
BACILLUS THURINGIENSIS formulations continue to be used worldwide for the control of the most important pests of agriculture and human health. The continued use of these formulations is likely to enhance the selection pressure to develop resistance to the toxins that form the active ingredients in these formulations. Consequently it is anticipated that insect resistance to BACILLUS THURINGIENSIS toxins will become more frequent in the coming years. Such an occurrence could limit the utility of BACILLUS THURINGIENSIS-crops that form a large part of US agriculture. Similarly the continued use of bacterial formulations for the control of insect vectors of human diseases also enhances the selection pressure for resistance. Our research using the Cry toxins is aimed at elucidating the mechanism of action. These studies have enabled us to show how these toxins act in the different insect orders. In a recent publication we proposed a more dynamic model of how a toxin can bind to two different receptors in the midgut of insects. As noted last year this model shows that proteolytic action that leads to intramolecular cleavage of the toxin is an essential for toxin insertion. In previous years we identified four different proteins act as receptors for the mosquitocidal Cry11Aa and Cry4Ba proteins - an alkaline phosphatase, a cadherin, an aminopeptidase, and a glycosidase protein. This year we further analyzed differences in the interaction of the Cry11A and Cry4Ba toxins with not only the cadherin protein but also alkaline phosphatases. This data reinforces the concept that we developed last year that the binding of toxins from BTI to the mosquito midgut is even more complex than originally anticipated. Hence, while a number of receptors mediate toxicity to the Cry toxins, the manner by which each of the mosquitocidal Cry toxins interact with the receptor proteins varies. For example, the Cry11A uses the cadherin and ALP1 as a receptor the Cry4Ba toxin uses alkaline phosphatases and a yet unidentified receptor for binding. It is likely that the third toxin in BTI, Cry4A, will likely using other proteins for interaction. Further, the interaction of these toxins with the synergistic Cyt1A toxin is also complex. This year we found that the interaction sites that are involved in Cyt1A membrane insertion and toxicity are independent events of its synergistic activity. Continued analysis of the molecular basis of toxin action including studies of insect specificity, of insect resistance to Cry toxins and of the role of receptor molecules in toxicity will provide new ways for a rational design of Cry toxins to control insect pests important in agriculture or in human health. Such developments will extend the useful life span of this technology.
Publications
- Jimenez AI, Reyes EZ, Cancino-Rodezno A, Bedoya-Perez LP, Caballero-Flores GG, Muriel-Millan LF, Likitvivatanavong S, Gill SS, Bravo A, Soberon M. 2012 Aedes aegypti alkaline phosphatase ALP1 is a functional receptor of Bacillus thuringiensis Cry4Ba and Cry11Aa toxins. Insect Biochem Mol Biol. 42:683-9.
- Cancino-Rodezno A, Lozano L, Oppert C, Castro JI, Lanz-Mendoza H, Encarnacion S, Evans AE, Gill SS, Soberon M, Jurat-Fuentes JL, Bravo A. 2012. Comparative proteomic analysis of Aedes aegypti larval midgut after intoxication with Cry11Aa toxin from Bacillus thuringiensis. PLoS One. 7:e37034.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: BACILLUS THURINGIENSIS ISRAELENSIS (BTI) is highly toxic to different mosquito and black fly species that are vectors of human disease pathogens. Due to the emergence of insect-resistance to chemical insecticides, BTI formulations are being used worldwide to control mosquitoes. This bacterium produces a crystal inclusion composed of at least four toxins: Cry4A, Cry4B, Cry11A and Cyt1A. Receptor binding is a key factor in determining the specificity of Cry toxins. In previous years we showed that the Cry11A and Cry4Ba toxins bind variety of different proteins in the midgut of AEDES AEGYPTI larvae. Our previous data indicated that there are differences in the way Cry11Aa and Cry4Ba appear to bind to midgut membrane proteins. We had shown last year that the Cry11Aa toxin binds the AE. AEGYPTI cadherin with high affinity (16.7 nM KD) to a fragment containing the membrane proximal part of the ectodomain of this receptor molecule (cadherin repeats CR7-11). Binding competition experiments showed that Cry4Aa and the Cry11Ba, produced by another bacterial strain, B. T. JEGATHESAN, competed with binding of Cry11Aa to this cadherin CR7-11 fragment showing that these toxins share the same cadherin binding sites. Further this binding to cadherin facilitates Cry toxin oligomer formation. In contrast we showed, the Cry4Ba toxin did not compete with Cry11Aa binding to CR7-11, indicating that Cry4Ba may not rely on binding to cadherin for toxicity. This year we followed this line of investigation and showed that indeed there are differences in the binding of Cry11Aa and Cry4Ba. Using plasmon resonance we showed the Cry4Ba toxin binds the CR7-11 cadherin fragment with a nine-fold lower binding affinity compared to that of Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated IN VITRO in the absence of the CR7-11 fragment. In contrast the Cry11Aa toxin only formed oligomers in the presence of the CR7-11 fragment. Pore formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA showed that silenced larvae were more tolerant to Cry11Aa in contrast to Cry4Ba that showed similar toxic levels to that of control larvae. Our data indeed shows that cadherin binding does not appear to be a rate limiting step for Cry4Ba toxicity to AE. AEGYPTI larvae. What then does the Cry4Ba toxin bind to In previous years we showed that the Cry toxins also bind to alkaline phosphatases and aminopeptidase-Ns. Indeed our preliminary evidence suggests that the Cry4Ba toxin binds to one of the alkaline phosphatases, ALP1. Thus our data suggest that three different midgut proteins, i.e., cadherin, AaeALP1, and AaeAPN1, are involved in Cry11Aa and Cry4Ba binding to midgut brush border membranes in this mosquito. PARTICIPANTS: PI, Sarjeet S. Gill, Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521. Others who have worked on the project at UC Riverside are: The following worked on the project this year: Amy Evans, Supaporn Likitvivatanavong, Karly Aimanova and Subum Lee. Amanda Ortega a minority undergraduate student was given an opportunity to work in the laboratory to learn insect dissection techniques. Subum Lee a graduate student initiated work on the mode of action of the Cry11A toxin. Supaporn Likitvivatanavong is a postdoctoral who was given an independent project that involved molecular biology research. Karly Aimanova an Assistant Specialist was involved in doing some immunohistochemistry while Amy Evans was involved in molecular biology research. TARGET AUDIENCES: For this research project the primary audience is the scientific community in academia, industry and the government. In additional there is an international audience that views this research with a great deal of interest. PROJECT MODIFICATIONS: There was no major change the last year.
Impacts
BACILLUS THURINGIENSIS formulations continue to be used worldwide for the control of the most important pests of agriculture and human health. The continued use of these formulations is likely to enhance the selection pressure to develop resistance to the toxins that form the active ingredients in these formulations. Consequently it is anticipated that insect resistance to BACILLUS THURINGIENSIS toxins will become more frequent in the coming years. Such an occurrence could limit the utility of BACILLUS THURINGIENSIS-crops that form a large part of US agriculture. Similarly the continued use of bacterial formulations for the control of insect vectors of human diseases also enhances the selection pressure for resistance. Our research using the Cry toxins is aimed at elucidating the mechanism of action. These studies have enabled us to show how these toxins act in the different insect orders. In a recent publication we proposed a more dynamic model of how a toxin can bind to two different receptors in the midgut of insects. As noted last year this model shows that proteolytic action that leads to intramolecular cleavage of the toxin is an essential for toxin insertion. In previous years we identified four different proteins act as receptors for the mosquitocidal Cry11Aa and Cry4Ba proteins - an alkaline phosphatase, a cadherin, an aminopeptidase, and a glycosidase protein. This year we showed that there are differences in the manner in which the Cry11Aa and Cry4Ba proteins interact with cadherin protein. Hence our data shows there is even more complexity to toxin binding to the mosquito midgut than we anticipated last year. Hence, while a number of receptors mediate toxicity to the Cry toxins, the manner by which each of the mosquitocidal Cry toxins interact with the receptor proteins varies. Thus while cadherin is a critical receptor for the Cry11A toxin, for other proteins like Cry4Ba the other receptors, including the alkaline phosphatase appears to be more critical. Further, as noted last year we continue to believe that toxicity binding changes depending on whether the toxin is monomeric or oligomeric. Continued analysis of the molecular basis of toxin action including studies of insect specificity, of insect resistance to Cry toxins and of the role of receptor molecules in toxicity will provide new ways for a rational design of Cry toxins to control insect pests important in agriculture or in human health. Such developments will extend the useful life span of this technology.
Publications
- Rodriguez-Almazan, C., Ruiz de Escudero, I., Canton, P.C., Munoz-Garay, C, Perez, C., Gill, S.S., Soberon, M. and Bravo, A. 2011. The Amino- and Carboxyl-Terminal Fragments of the Bacillus thuringiensis Cyt1Aa Toxin Have Differential Roles in Toxin Oligomerization and Pore Formation. Biochem. 50, 388-396.
- Likitvivatanavong, S., Chen, J., Bravo, A., Soberon, M., and Gill, S. S. 2011. Multiple Receptors as Targets of Cry Toxins in Mosquitoes, J. Agric. Fd. Chem, 59:2829-38.
- Bravo, A., Likitvivatanavong, S., Gill, S. S., and Soberon, M. 2011. Bacillus thuringiensis: A story of a successful bioinsecticide. Insect Biochem Mol Biol. 41:423-31.
- Rodriguez-Almazan C, Reyes EZ, Zuniga-Navarrete F, Munoz-Garay C, Gomez I, Evans AM, Likitvivatavanong S, Bravo A, Gill SS, Soberon M. 2012. Cadherin binding is not a limiting step for Bacillus thuringiensis subs. israelensis Cry4Ba toxicity to Aedes aegypti larvae. Biochem J. Published on 14 Feb 2012 as manuscript BJ20111579.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: BACILLUS THURINGIENSIS ISRAELENSIS (BTI) is highly toxic to different mosquito and black fly species that are vectors of human disease pathogens. Due to the emergence of insect-resistance to chemical insecticides, BTI formulations are being used worldwide to control mosquitoes. This bacterium produces a crystal inclusion composed of at least four toxins: Cry4A, Cry4B, Cry11A and Cyt1A. Receptor binding is a key factor in determining the specificity of Cry toxins. Last year we showed that the Cry11A and Cry4Ba toxins use three different proteins to bind to in the midgut of AEDES AEGYPTI larvae. This year we evaluated the binding capacity of Cry11Ba toxin, which is one of the most toxic proteins to mosquito larvae produced by BACILLUS THURINGIENSIS. We show it binds the brush border membrane vesicles (BBMV) with high affinity, showing an apparent dissociation constant of 8.2nM. We previously reported that an anti-cadherin antibody competes with Cry11Ba binding to BBMV, suggesting a possible role of cadherin as a toxin receptor. We provide evidence that specific cadherin repeat regions are involved in this interaction. Using cadherin fragments as competitors, a C-terminal fragment, which contains cadherin repeat 7 (CR7) to CR11 competed with Cry11Ba binding to BBMV. This binding was also efficiently competed by the CR9, CR10, and CR11 peptide fragments. Moreover, we show CR11 to be an important region of interaction with Cry11Ba toxin. An alkaline phosphatase (AaeALP1) and an aminopeptidase-N (AaeAPN1) also competed with Cry11Ba binding to AE. AEGYPTI BBMV. Finally, we found that Cry11Ba and Cry4Ba share some common binding sites. Synthetic peptides corresponding to some of the loop regions of Cry4Ba can compete with Cry11Ba binding to BBMV, suggesting Cry11Ba and Cry4Ba have common sites involved in binding midgut membranes. The data suggest that three different midgut proteins, i.e., cadherin, AaeALP1, and AaeAPN1, are involved in Cry11Ba binding to midgut brush border membranes in this mosquito. In a separate study we analyze the response of Cry toxins to the Lepidopteran MANDUCA SEXTA and the Dipteran AE. AEGYPTI using toxins specifically toxic to each, namely the Cry1Ab and Cry11Aa, respectively. We show that one of the pathways activated in response to the toxins is the mitogen-activated protein kinase p38 pathway (MAPK p38) that activates a complex defense response. We show that MAPK p38 is activated at the posttranslational level after Cry toxin intoxication in both insect orders. Further gene silencing of MAPK p38 in vivo, resulted in both insect species becoming hypersensitive to Cry toxin action, suggesting that the MAPK p38 pathway is involved in insect defense against Bt Cry toxins. This finding may have biotechnological applications for enhancing the activity of some Bt Cry toxins against specific insect pests. PARTICIPANTS: PI, Sarjeet S. Gill, Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521. Others who have worked on the project at UC Riverside are: Amy Evans, Supaporn Likitvivatanavong, Karly Aimanova and Subum Lee. Amanda Ortega a minority undergraduate student was given an opportunity to work in the laboratory to learn insect dissection techniques. Subum Lee a graduate student initiated work on the mode of action of the Cry11A toxin. Supaporn Likitvivatanavong is a postdoctoral who was given an independent project that involved molecular biology research. Karly Aimanova an Assistant Specialist was involved in doing some immunohistochemistry while Amy Evans was involved in molecular biology research. Partner Organization and Collaborator: Alejandra Bravo and Mario Soberon, Co-PIs on the project from Departamento de Microbiologıa Molecular, Instituto de Biotecnologıa, Universidad Nacional Autonoma de Mexico, Apdo. Postal 510-3, Cuernavaca 62250, Morelos, Mexico. Others in Mexico who worked on the project include: Angeles Cancino-Rodezno, Sabino Pacheco and Claudia Rodriguez-Almazan. TARGET AUDIENCES: For this research project the primary audience is the scientific community in academia, industry and the government. In additional there is an international audience that views this research with a great deal of interest. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
BACILLUS THURINGIENSIS formulations continue to be used worldwide for the control of the most important pests of agriculture and human health. The continued use of these formulations is likely to enhance the selection pressure to develop resistance to the toxins that form the active ingredients in these formulations. Consequently it is anticipated that insect resistance to BACILLUS THURINGIENSIS toxins will become more frequent in the coming years. Such an occurrence could limit the utility of BACILLUS THURINGIENSIS-crops that form a large part of US agriculture. Similarly the continued use of bacterial formulations for the control of insect vectors of human diseases also enhances the selection pressure for resistance. Our research using the Cry toxins is aimed at elucidating the mechanism of action. These studies have enabled us to show how these toxins act in the different insect orders. In a recent publication we proposed a more dynamic model of how a toxin can bind to two different receptors in the midgut of insects. As noted last year this model shows that proteolytic action that leads to intramolecular cleavage of the toxin is an essential for toxin insertion. In previous years we identified four different proteins act as receptors for the mosquitocidal Cry11A and Cry4B proteins - an alkaline phosphatase, a cadherin, an aminopeptidase, and a glycosidase protein. This year we showed that the first three of these proteins also act as receptors for the Cry11Ba toxin. This data shows there is significant complexity to toxin binding to the mosquito midgut. There are a variety of receptors that could mediate toxicity. A critical receptor for the Cry11A toxin is the cadherin protein. However, for other proteins like Cry4Ba the other receptors appear to be more critical. As noted last year we continue to believe that toxicity binding changes depending on whether the toxin is monomeric or oligomeric. Continued analysis of the molecular basis of toxin action including studies of insect specificity, of insect resistance to Cry toxins and of the role of receptor molecules in toxicity will provide new ways for a rational design of Cry toxins to control insect pests important in agriculture or in human health. Such developments will extend the useful life span of this technology.
Publications
- Pacheco, S., Gomez, I., Arenas, I., Saab-Rincon, G., Rodriquez-Almazan, C., Gill, S. S., Bravo, A. and Soberon, M. 2009. Domain II Loop 3 of BACILLUS THURINGIENSIS Cry1Ab Toxin Is Involved in a "Ping Pong" Binding Mechanism with MANDUCA SEXTA Aminopeptidase-N and Cadherin Receptors. J. Biol. Chem 284:32759-32757.
- Cancino-Rodezno., A., Alexander, C., Villasenor, R., Pacheco, S., Porta, H., Pauchet, Y., Soberon, M., Gill, S.S. and Bravo, A. 2010. The mitogen-activated protein kinase p38 is involved in insect defense against Cry toxins from BACILLUS THURINGIENSIS. Insect Biochem. Mol. Biol. 40:58-63.
- Likitvivatanavong, S., Chen, J., Bravo, A., Soberon, M., Gill, S. S. 2011. Cadherin, Alkaline Phosphatase, and Aminopeptidase N as Receptors of Cry11Ba Toxin from BACILLUS THURINGIENSIS subsp. JEGATHESAN in AEDES AEGYPTI. Appl. Environ. Microbiol 77: 24-31.
- Rodriguez-Almazan, C., Ruiz de Escudero, I., Canton, P.C., Munoz-Garay, C, Perez, C., Gill, S.S., Soberon, M. and Bravo, A. 2011. The Amino- and Carboxyl-Terminal Fragments of the Bacillus thuringensis Cyt1Aa Toxin Have Differential Roles in Toxin Oligomerization and Pore Formation. Biochem. 50, 388-396.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: BACILLUS THURINGIENSIS ISRAELENSIS (BTI) is highly toxic to different mosquito and black fly species that are vectors of human disease pathogens. Due to the emergence of insect-resistance to chemical insecticides, BTI formulations are being used worldwide to control mosquitoes. This bacterium produces a crystal inclusion composed of at least four toxins: Cry4A, Cry4B, Cry11A and Cyt1A. Cry11A toxin is the most active toxin in BTI against AEDES AEGYPTI larvae. Receptor binding is a key factor in determining the specificity of Cry toxins. Last year we identified a 65 kDa protein GPI-anchored alkaline phosphatase (ALP) as a Cry11Aa receptor in A. AEGYPTI larvae. This year we showed this toxins binds to additional proteins. Using biotinylated Cry11Aa toxin and pull down assays of solubilized brush border membrane vesicles prepared from midguts of mosquito larvae we eluted three proteins. These were identified by mass spectrometry as aminopeptidases N (APN), one of which was a 140 kDa protein, named AaeAPN1 (AAEL 012778 in VectorBase). The cDNA for this protein was cloned and used for expression of the protein E. COLI and in Sf21 insect cells. AaeAPN1 protein expressed in Sf21 cells was enzymatically active, had a GPI-anchor but did not bind Cry11Aa. A truncated AaeAPN1, however, binds Cry11Aa with high affinity, and also Cry11Ba but with lower affinity. Using antibodies that were developed to the protein, we showed the protein localizes to the apical side of posterior midgut epithelial cells of larva. AaeAPN1 in BBMV prepared from mosquito larval midgut but not Sf21 expressed AaeAPN1 can be detected by wheat germ agglutinin suggesting the native but not the Sf21 cell expressed APN1 contains N-acetylglucosamine moieties. As indicated last year we reported that in addition to a 65 kDa GPI-anchored alkaline phosphatase (ALP) the toxin also binds a 250 kDa membrane protein. Our cloning indicated the protein was an AEDES cadherin (AaeCAD). Using an antibody to this protein we showed it detects a 250-kDa protein in immunoblots of larval brush border membrane vesicles (BBMV). The antibody inhibits Cry11Aa toxin binding to BBMV and immunolocalizes the cadherin protein to apical membranes of distal and proximal caecae and posterior midgut epithelial cells. This localization is consistent with areas to which Cry11Aa toxin binds and causes pathogenicity. Using toxin overlay assays we showed that one cadherin fragment that contains CR 7-11 bound Cry11Aa and this binding was primarily through toxin domain II loop alpha8 and loop 2. Cadherin repeats CR8-11 but not CR7 bound Cry11Aa under non-denaturing conditions. Cry11Aa bound the cadherin fragment with high affinity with an apparent KD of 16.7 nM. Finally we showed this Cry11Aa binding site could also be competed by Cry11Ba and Cry4Aa but not Cry4Ba. These results indicate that AEDES cadherin is possibly a receptor for Cry11A, and together with its ability to bind an ALP suggest a similar mechanism of toxin action as previously proposed for lepidopteran insects. PARTICIPANTS: PI, Sarjeet S. Gill, Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521. Others who have worked on the project at UC Riverside are: The following worked on the project this year: Amy Evans, Supaporn Likitvivatanavong, Jianwu Chen and Karly Aimanova. Amanda Ortega a minority undergraduate student was given an opportunity to work in the laboratory to learn insect dissection techniques. Both Jianwu Chen and Supaporn Likitvivatanavong are postdoctorals who were given independent projects that involved molecular biology research. Karly Aimanova a postdoctoral was involved in doing some immunohistochemistry while Amy Evans was involved in molecular biology research. Partner Organization and Collaborator: Alejandra Bravo and Mario Soberon, Co-PIs on the project from Departamento de Microbiologıa Molecular, Instituto de Biotecnologıa, Universidad Nacional Autonoma de Mexico, Apdo. Postal 510-3, Cuernavaca 62250, Morelos, Mexico. Others in Mexico who worked on the project include: Claudia Martinez Anaya and Sabino Pacheco. TARGET AUDIENCES: For this research project the primary audience is the scientific community in academia, industry and the government. In additional there is an international audience that views this research with a great deal of interest. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
BACILLUS THURINGIENSIS toxins have been employed successfully in recent years to control the most important pests of agriculture and human health, but not all insect pests are targets for Cry toxins and it is anticipated that insect resistance to BACILLUS THURINGIENSIS toxins will become more frequent in the coming years and could limit the utility of BACILLUS THURINGIENSIS-crops in the near future. In our publications we have presented different experimental strategies that have been used to improve Cry toxin action. In most cases, the improvement of toxins came as a direct result of understanding their mode of action in the different insect orders. In a recent publication we proposed a modified model of the one we have earlier. In this model the activation process can be modified by addition of protease inhibitors or by introduction of intramolecular cleavage sites in the toxin. In previous years we identified three different proteins that act as receptors - an alkaline phosphatase for the Cry11A toxin, a glycosidase for the Cry4A toxin, and also to a high affinity to a cadherin like protein. This year we showed that this toxin also binds with to an aminopeptidase. This data shows there is significant complexity to toxin binding to the mosquito midgut. There are a variety of receptors that could mediate toxicity. A critical receptor is the cadherin protein. However, other receptors can also act to as sites of toxin binding. We believe that toxicity binding changes depending on whether the toxin is monomeric or oligomeric. Continued analysis of the molecular basis of toxin action including studies of insect specificity, of insect resistance to Cry toxins and of the role of receptor molecules in toxicity will provide new ways for a rational design of Cry toxins to control insect pests important in agriculture or in human health. Such developments will extend the useful life span of this technology.
Publications
- Pacheco, S., Gomez, I., Gill, S. S., Bravo, A. and Soberon, M. 2009. Enhancement of insecticidal activity of Bacillus thuringiensis Cry1A toxins by fragments of a toxin-binding cadherin correlates with oligomer formation. Peptides 30:583-588.
- Soberon, M., Gill, S. S. and Bravo, A. 2009. Signaling versus punching hole: How do Bacillus thuringiensis toxins kill insect midgut cells Cell Mol Life Sci. 66:1337-1349.
- Fernandez, L., Martinez-Anaya C., Lira E., Chen, JW., Evans, A., Hernandez-Martinez, S., Lanz-Mendoza H., Bravo, A., Gill, S. S., Soberon, M. 2009. Cloning and epitope mapping of Cry11Aa-binding sites in the Cry11Aa-receptor alkaline phosphatase from Aedes aegypti. Biochemistry. 48, 8899-8907.
- Chen, J.W., Aimanova, K., Martinez, C., Bravo, A., Soberon, M., Gill, S. S. 2009. Aedes aegypti cadherin serves as a putative receptor of the Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis. Biochemical J. 424, 191-200.
- Chen, J. W., Aimanova, K., Pan, S. Q, Gill, S. S. 2009. Characterization and identification of Aedes aegypti aminopeptidase (APN) receptors of Bacillus thuringiensis subsp. israelensis Cry11A toxin. Ins Biochem. Mol. Biol. 39, 688-696.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: BACILLUS THURINGIENSIS ISRAELENSIS (BTI) is highly toxic to different mosquito and black fly species that are vectors of human disease pathogens. Due to the emergence of insect-resistance to chemical insecticides, BTI formulations are being used worldwide to control mosquitoes. This bacterium produces a crystal inclusion composed of at least four toxins: Cry4A, Cry4B, Cry11A and Cyt1A. Cry11A toxin is the most active toxin in BTI against AEDES AEGYPTI larvae. Receptor binding is a key factor in determining the specificity of Cry toxins. Last year we identified a 65 kDa protein GPI-anchored alkaline phosphatase (ALP) as a Cry11Aa receptor in A. AEGYPTI larvae. This year by using a pepSpot arrays of Cry11Aa domain II-III we identified two ALP regions (TDGSIKFAR and RVQSQNSGNRM) that competes binding of Cry11Aa to BBMV and toxicity. Further at least one Cry11Aa mutant, a triple alanine substitution (RVQSQAAANRM) attenuates Cry11Aa toxicity. We also previously reported the Cry11A also bound an unidentified 200 kDa membrane protein. We speculated that this protein was a cadherin like protein. Using sequences obtained from the AEDES genome, we amplified a portion of the C-terminal of AEDES cadherin. This partial cadherin fragment was expressed as a His-tag protein, and the purified protein used for production of rabbit anti-AaeCad antibody. To show AaeCad plays a role in binding of the Cry11A toxin to A. AEGYPTI midgut epithelia, we performed a competition assay using larval midgut epithelial membranes. Using biotinylated Cry11A we showed the anti-AaeCad antibody competed readily with the toxin. We then developed antibodies to this cadherin fragment, and the antibody shows intense immunofluorescence on the apical side of the distal and proximal caeca and on the apical membrane of posterior midgut epithelial cells. These are the same tissues, which bind Cry toxins from BTI and show subsequent pathological responses. These results indicate the cadherin molecule is a critical molecule that mediates toxicity. The full-length cadherin molecule was then cloned, and our data shows the gene is incorrectly annotated in the Aedes genome. Three partial cadherin fragments covering all the cadherin repeats were cloned, expressed, purified by nickel-affinity chromatography and then analyzed for Cry11A toxin binding. One of these fragments that contains cadherin repeats (CR) 7-11 bound Cry11A toxin strongly, whereas fragments covering cadherin repeats 1-7 did not bind the toxin. To further determine the toxin binding regions on the large fragment, each cadherin repeat (CR7-11) were amplified, cloned, expressed and used for toxin binding. Only CR9 and CR10 bound the toxin, and these fragments can compete with Cry11A binding to the midgut membranes. To determine which of the predicted Cry11 loop regions are involved in binding to cadherin and the cadherin repeats, synthetic peptides corresponding to the four Cry11A loop regions, loop α-8, loop 1, loop 2 and loop 3 were synthesized and analyzed. Both loop α-8 and 2 could compete with Cry11A binding to the cadherin and repeats CR9 and CR10. The affinity of toxin binding to cadherin was quite high. PARTICIPANTS: PI, Sarjeet S. Gill, Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521. Others who have worked on the project at UC Riverside are: The following worked on the project this year: Amy Evans, Monica Ramirez-Rivas, Jianwu Chen and Karly Aimanova. Monica Ramirez-Rivas a minority undergraduate student was given an opportunity to work in the laboratory to learn insect culture techniques. Both Jianwu Chen and Karly Aimanova are postdoctorals who were given independent projects that involved molecular biology research. Partner Organization and Collaborator: Alejandra Bravo and Mario Soberon, Co-PIs on the project from Departamento de Microbiologıa Molecular, Instituto de Biotecnologıa, Universidad Nacional Autonoma de Mexico, Apdo. Postal 510-3, Cuernavaca 62250, Morelos, Mexico. Others in Mexico who worked on the project include: Claudia Martinez Anaya and Sabino Pacheco. TARGET AUDIENCES: For this research project the primary audience is the scientific community in academia, industry and the government. The research done in the last year has led to contact between Alejandra Bravo and Mario Soberon and Monsanto Inc. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
BACILLUS THURINGIENSIS toxins have been employed successfully in recent years to control the most important pests of agriculture and human health, but not all insect pests are targets for Cry toxins and it is anticipated that insect resistance to BACILLUS THURINGIENSIS toxins will become more frequent in the coming years and could limit the utility of BACILLUS THURINGIENSIS-crops in the near future. In our publications we have presented different experimental strategies that have been used to improve Cry toxin action. In most cases, the improvement of toxins came as a direct result of understanding their mode of action in the different insect orders. In a recent publication we proposed a modified model of the one we have earlier. In this model the activation process can be modified by addition of protease inhibitors or by introduction of intramolecular cleavage sites in the toxin. In previous years we identified two different proteins that act as receptors - an alkaline phosphatase for the Cry11A toxin and a glycosidase for the Cry4A toxin. This year we showed the Cry11A toxin binds with high affinity to a cadherin like protein. The toxin binding site was mapped to cadherin repeats 9 and 10. In addition this Cry11A toxin minds to two different aminopeptidases. In the Cry11A and 11B toxins the domains that are important for toxicity are mapped to loop regions. These were identified as loop -8, loop 2 and loop 3. It appears that the numerous toxins present in mosquitocidal BACILLUS THURINGIENSIS can bind to different receptors. Consequently, it is likely that this diversity in receptor targets will help delay resistance development. Continued analysis of the molecular basis of toxin action including studies of insect specificity, of insect resistance to Cry toxins and of the role of receptor molecules in toxicity will provide new ways for a rational design of Cry toxins to control insect pests important in agriculture or in human health. Such developments will extend the useful life span of this technology.
Publications
- Jimenez-Juarez, N., Munoz-Garay, C., Gomez, I., Gill, S. S., Soberon, M., and Bravo, A. (2007) The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta. Peptides. 2008 29:318-23.
- Fernandez, L. E., Gomez, I., Pacheco, S., Arenas, I., Gill, S. S., Bravo, A., and Soberon, M. (2007) Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins. Peptides. 2008 29:324-9.
- Bravo A and Soberon M. (2008) How to cope with insect resistance to Bt toxins Trends Biotechnol. 26:573-9.
- Pardo-Lopez L, Munoz-Garay C, Porta H, Rodriguez-Almazan C, Soberon M, Bravo A. (2008) Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis. Peptides. 2008 Aug 19. [Epub ahead of print]
- Pacheco S, Gomez I, Gill SS, Bravo A, and Soberon M. (2008) Enhancement of insecticidal activity of Bacillus thuringiensis Cry1A toxins by fragments of a toxin-binding cadherin correlates with oligomer formation. Peptides. 2008 Aug 20. [Epub ahead of print]
- Soberon M, Gill SS, and Bravo A. (2008) Signaling versus punching hole: How do Bacillus thuringiensis toxins kill insect midgut cells Cell Mol Life Sci. 2009 Jan 13. [Epub ahead of print]
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Progress 01/01/07 to 12/31/07
Outputs
Receptor binding is a key factor in determining the specificity of Cry toxins from Bacillus thuringiensis (Bti). Last year we identified a 65 kDa protein as a GPI-anchored alkaline phosphatase (ALP) as Cry11Aa receptor in Ae. aegypti larvae. This year we identified three cDNA ALP clones from Ae. aegypti (ALPAR93, ALP2 and ALP3). The three ALP clones were expressed in E. coli. ELISA binding assays showed recombinant ALPAR93 bound Cry11Aa and Cry4Ba while recombinant ALP2 and ALP3 showed low Cry toxin binding. These results show that ALPAR93 is the Cry11Aa receptor identified on Ae. aegypti. Homologous and heterologous binding competitions showed that Cry11Aa and Cry4B share binding sites on ALPAR93 since Cry11Aa and Cry4Ba competed the binding of biotinylated-Cry11Aa to ALPAR93. We analyzed the role of the Cry11Aa domain III in interacting with ALP93. An array of overlapping synthetic peptides corresponding to the Cry11Aa domain III was synthesized and spotted into
cellulose membranes. Binding analysis of ALP93 to this peptide array indicated that specific Cry11Aa domain III bound ALP93 protein. To determine the ALPAR93 domains involved in Cry11Aa binding nine overlapping fragments of 150 residues of ALPAR93 were produced in E. coli. Ligand blots using Cry11Aa toxin showed that the toxin bound to two regions in the amino-terminal end of ALPAR93 and in the middle part of the protein since the toxin bound to fragments 1, 2, 4, 5 and 6. As representatives, we showed that fragments 2 and 5 competed the binding of labeled Cry11Aa to Ae. aegypti BBMV's as ALPAR93. Interestingly, Cry11Aa mutant E266A in loop -8, that was previously shown to be affected in BBMV binding and toxicity, did not bind to ALPAR93 nor to ALPAR93 fragments 1 and 2 but retained binding to fragments 5 and 6 indicating that loop -8 binds the amino terminal end of ALPAR93. In addition to the ALPs, we identified two proteins of 95 and 140 kDa that bound the Cry11Aa
toxins. These proteins, isolated by pull down assays using Cry11Aa as a ligand, have aminopeptidase but no alkaline phosphatase activity. In the coming year we will isolate and characterize the cDNAs that encode for these proteins. Finally, in previous work we showed that Cyt1Aa synergizes or suppresses resistance to Cry11Aa toxin by functioning as a membrane-bound receptor. Thus Bti not only produces a toxin but also its functional receptor, thereby promoting toxin binding to the target membrane and causing toxicity. This results in Bti being a highly effective pathogenic bacterium. Also, we previously reported that in the presence of Cyt1Aa toxin a 250 kDa Cry11Aa oligomer is formed after proteolytic activation, in contrast to the proteolytic activation in the absence of Cyt1Aa. This year we demonstrated that the Cry11Aa oligomer induced pore-formation in liposomes and, that Cry11Aa and Cyt1Aa mutants affected the interaction between these toxins. These changes in the toxin
interaction also affect synergism and on Cry11Aa oligomer formation, indicating that Cyt1Aa synergizes Cry11Aa activity by facilitating the formation of a pre-pore oligomer structure that is membrane insertion competent.
Impacts
In previous years we showed the Cyt1Aa toxin acts as a surrogate receptor for the Cry11Aa and Cry4B toxins. This year we showed that the Cyt1Aa toxin acts by facilitating formation of Cry11Aa oligomers that are membrane insertion competent. Thus the toxin is acting in a mechanism that is very similar to that of other Cry toxin receptors. Our investigation this year also defined the interaction of the Cry11Aa toxin with its receptor. Both the toxin and receptor domains involved in this interaction were mapped. We were also showed that toxins from the Bacillus thuringiensis subsp. israeliensis bind to a variety of proteins in the midgut of mosquitoes. This work will enable us and other investigators to predict the possible mechanism by which resistance occurs when it is detected in the field.
Publications
- Gomez, I., Pardo-Lopez, L., Munoz-Garay, C., Fernandez, L. E., Perez, C., Sanchez, J., Soberon, M., and Bravo, A. (2007) Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis. Peptides. 28:169-73
- Jimenez-Juarez, A., Munoz-Garay, C., Gomez, I., Saab-Rincon, G., Damian-Alamazo, J. Y., Gill, S. S., Soberon, M., and Bravo, A. (2007) Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae. Journal of Biological Chemistry. 282: 21222-21229.
- Perez, C., Munoz-Garay, C., C. Portugal, L., Sanchez, J., Gill, S. S., Soberon, M., and Bravo, A. (2007) Bacillus thuringiensis subsp. israelensis Cyt1Aa enhances activity of Cry11Aa toxin by facilitating the formation of a pre-pore oligomeric structure. Cellular Microbiology. 9: 2931-2937.
- Soberon, M., Pardo-Lopez, L., Lopez, I., Gomez, I., Tabashnik, B., and Bravo A. (2007) Engineering Modified Bt Toxins to Counter Insect Resistance. Science. 318: 1640-1642.
- Jimenez-Juarez, N., Munoz-Garay, C., Gomez, I., Gill, S. S., Soberon, M., and Bravo, A. (2007) The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta. Peptides. Accepted. Eprint
- Fernandez, L. E., Gomez, I., Pacheco, S., Arenas, I., Gill, S. S., Bravo, A., and Soberon, M. (2007) Employing phage display to study the mode of action of Bacillus thuringiensis Cry toxins. Peptides. Accepted Eprint
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Progress 01/01/06 to 12/31/06
Outputs
Bacillus thuringiensis subsp israelensis (Bti) produces a number of crystalline (Cry) toxins, of which Cry11A is the most active toxin against Aedes aegypti. For the toxic activity of this Cry toxin, receptor binding is a key determinant for specificity as observed with other three domain Cry toxins. The Cry11Aa toxin regions involved in the interaction with its receptor include exposed loop alpha-8 of Cry11Aa toxin, located in domain II, since certain single point mutants in this region affected binding and toxicity. Also synthetic peptides corresponding to exposed regions in domain II (loop a8, b4 and loop 3) competed with the binding of Cry11Aa to membrane vesicles from mosquito midgut microvilli suggesting that beta-4 and loop 3 might also be involved in Cry11Aa-receptor interaction. Work reported last year led to the identification of a GPI-anchored alkaline phosphatase as a receptor of Cry11Aa in Aedes aegypti larval midgut cells. This year we identified three
alkaline phosphatase cDNA clones from the midgut of this mosquito species. Sequence analyses suggest that all three of the ALP were GPI anchored. Two of these ALP cDNA's were cloned in E. coli expression vectors and purified. Ligand blot binding analyses showed that the two proteins bound Cry11Aa. ELISA binding assays showed loop alpha-8 affected toxicity and also toxin binding, suggesting that one of the clones we isolated is the GPI-ALP receptor of Cry11Aa we characterized last year. As observed in the literature domain III is an important domain of lepidopteran Cry toxins involved in receptor interaction. To determine if domain III is also involved in ALP binding an array of overlapping synthetic peptide corresponding to Cry11Aa domain III was synthesized and spotted into cellulose membranes. Binding analysis of ALP to this peptide array indicated that Cry11Aa domain III sequences rqvsqnsgnrm and tdgsikfar bound ALP protein. The lack of resistance in mosquitoes to Bti is due to the
presence of the Cyt1Aa protein in the crystal. In addition, synergism between Cyt1Aa and the Cry proteins of Bti has been observed. Work reported last year indicated that Cyt1Aa synergizes or suppresses resistance to Cry11Aa toxin by functioning as a membrane-bound receptor. During this grant period we characterized the mechanism of synergism of cyt1Aa with the mosquitocidal Cry4B toxin also produced by Bti. As shown previously with Cry11Aa, Cry4B domain II mutants were affected in synergism with Cyt1Aa. Also synthetic peptide arrays of Cyt1Aa identified the same loop beta6-alphaE is involved in binding to Cry4B. Finally, in this period we also analyzed the effect of Cyt1Aa binding on the proteolytic activation of Cry11Aa. To determine if the interaction of Cry11Aa protoxin with Cyt1Aa could induce oligomer formation we performed proteolytic activation of Cry11Aa protoxin in the presence of cyt1Aa protoxin or activated toxin and analyzed the Cry11Aa protein by Western blot. Our
results showed that in the presence of Cyt1Aa toxin a 250 kDa Cry11Aa oligomer is formed in contrast to the proteolytic activation in the absence of Cyt1Aa.
Impacts
Bacillus thuringiensis subsp. israelensis is a highly effective pathogenic bacterium that has been used world-wide for the control of disease vectors. In spite of it use for more than two decades no resistance has developed to date, primarily because of the presence of toxins with different mechanisms of action. However, the mechanism of action of these toxins is not known. Our investigation last year showed the Cyt1Aa toxin acts as a surrogate receptor for the Cry11Aa toxin. This year we showed that the Cyt1Aa toxin also acts as a receptor for the Cry4B toxin. It thus appears that this Bacillus strain makes a protein, which is not only by itself cytolytic, but it also acts as a receptor for other toxins produced in this strain. These results suggest that the same mechanism of synergism operates in the synergism of Cyt1Aa and Cry4B as we previously observed with Cry11Aa and Cyt1Aa, that is Cyt1Aa is a membrane bound receptor of Cry4B. However, additional data is
needed to demonstrate that this is indeed the case and to prove this hypothesis. As indicated last year these are indeed novel findings. We also cloned three alkaline phosphatases, one of which interacts with the Cry11A toxin. In addition, we also identified specific domains involved in binding to Cry11A toxin binding.
Publications
- Soberon, M., Fernandez, L. E., Perez, C., Gill, S. S., and Bravo, A. (2007) Mode of action of mosquitocidal Bacillus thuringiensis toxins. Toxicon. Accepted.
- Bravo, A., Gill, S. S., and Soberon, M. (2007) Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon. 49:423-35.
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Progress 01/01/05 to 12/31/05
Outputs
Bacillus thuringiensis subsp. israelensis produces a number of crystalline (Cry) toxins, of which Cry11A is the most active toxin against Aedes aegypti. The Cry11Aa toxin regions involved in the interaction with its receptor remain uncharacterized. Using phage-display, a powerful methodology that we have previously used, we identified the receptor-binding epitopes of Cry11Aa toxin. We found that exposed loop apha8 of Cry11Aa toxin, located in domain II, is an important epitope that is involved in receptor interaction. Synthetic peptides corresponding to exposed regions in domain II (loop apha8, beta4 and loop 3) competed with the binding of Cry11Aa to membrane vesicles from Aedes aegypti midgut microvilli. To demonstrate the role of this loop α-8 in receptor interaction we isolated a peptide-displaying phage (P5.tox), which recognizes loop apha8 in Cry11Aa, that interferes the interaction of the Cry11A toxin with the midgut receptor. This phage also attenuates
the toxicity of Cry11A in larval bioassays. Finally site directed mutagenesis of loop apha8 residues affected toxicity and receptor binding, indicating an important role for this domain in toxicity to larval mosquitoes. Using biotinylated Cry11Aa toxin we also identified a 65 kDa glycosylphosphatidyl-inositol (GPI)-anchored alkaline phosphatase as a functional receptor of the Cry11Aa toxin in Aedes aegypti midgut cells. Two (a 100 kDa and a 65 kDa) GPI-anchored proteins that bound Cry11Aa toxin were preferentially extracted after treatment of brush border membrane vesicles (BBMV) from Ae. aegypti midgut epithelia with phospholipase C. The 65 kDa protein was further purified by toxin affinity chromatography. The 65 kDa protein had high alkaline phosphatase activity. Peptide-displaying phages that were selected by their ability to bind BBMV of larval gut by biopanning (P1.BBMV and P8.BBMV) were shown to bind the 65 kDa GPI-anchored alkaline phosphatase. These phages also competed with
the Cry11Aa toxin binding to BBMV. Using confocal microscopy of phage P1.BBMV binding to fixed midgut tissue sections we showed that the GPI-anchored alkaline phosphatase was preferentially distributed in the posterior part of the midgut and in the caeca of Ae. aegypti. We also showed that the Cry11Aa binds to the same regions of the midgut, and that the Cry11Aa toxin competed with P1.BBMV and P8.BBMV binding to BBMV. Since these phages bound to the same site as the Cry11Aa toxin we then reasoned that these phages should also attenuate Cry11Aa toxicity. Indeed in larval bioassays we observed in vivo attenuation of Cry11Aa toxicity in the presence of these phages. Our results shows that the GPI-anchored alkaline phosphatase is an important receptor molecule involved in Cry11Aa interaction with midgut cells and its toxicity to Ae. aegypti larvae.
Impacts
Bacillus thuringiensis subsp. israelensis is a highly effective pathogenic bacterium that has been used world-wide for the control of disease vectors. In spite of it use for more than two decades no resistance has developed to date, primarily because of the presence of toxins with different mechanisms of action. However, the mechanism of action of these toxins is not known. In this past year we established two key findings. The first is the identification of a receptor for one of the toxin in this strain, Cry11Aa. This receptor is a lipid anchored alkaline phosphatase. We also identified the domains through which the Cry11Aa toxin interacts with its receptor. The second key finding during this first year of the grant is that the cytolytic toxin in this strain, Cyt1Aa, can act as a functional receptor for the Cry11Aa. Interestingly, the Cry11Aa domain that interacts with the cytolytic toxin is the same as that which interacts with the alkaline phosphatase. Hence this
mosquitocidal bacterial strain produces a toxin that can act as a receptor for another toxin in the same strain. This indeed is a very novel finding.
Publications
- Fernandez LE, Perez C, Segovia L, Rodriguez MH, Gill SS, Bravo A, Soberon M. 2005. Cry11Aa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop alpha-8 of domain II. FEBS Lett. 579:3508-3514.
- Fernandez LE, Aimanova KG, Gill SS, Bravo A, Soberon M. 2006. A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae. Biochem. J. 394:77-84.
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