المؤلفون: Anaya, Paulina, Onofre, Janette, Torres-Quintero, Mary Carmen, Sánchez, Jorge, Gill, Sarjeet S, Bravo, Alejandra, Soberón, Mario

مصطلحات موضوعية: Zoology, Biochemistry and Cell Biology, Biological Sciences, Biodefense, Vaccine Related, Prevention, Aedes, Animals, Bacillus thuringiensis, Bacillus thuringiensis Toxins, Bacterial Proteins, Endotoxins, Erythrocytes, Hemolysin Proteins, Insecticides, Larva, Polymerization, Rabbits, Cyt1Aa toxin, Hemolysis, Aedes aegypti, Red blood cells, Oligomerization, Medicinal and Biomolecular Chemistry, Entomology, Biochemistry and cell biology

الوصف: Bacillus thuringiensis (Bt) Cyt1Aa toxin shows toxicity to mosquitoes, to certain coleopteran pests and also to red blood cells (RBC). However, its mode of action in the different target cells is not well defined. This protein is a single α-β domain pore-forming toxin, where a β sheet is wrapped by two α-helices layers. The Cyt1Aa α-helix hairpin in the N-terminal has been proposed to be involved in initial membrane binding and oligomerization, while the β sheet inserts into the membrane to form a pore that lyze the cells. To determine the role of the N-terminal α-helix hairpin region of Cyt1Aa in its mode of action, we characterized different single point mutations located in helices α-1 and α-2. Eight cysteine substitutions in different residues were produced in Bt, and we found that three of them: Cyt1AaA65C, Cyt1AaL85C and Cyt1AaN89C, lost insecticidal toxicity against Aedes aegypti larvae but retained similar or increased hemolytic activity towards rabbit RBC. Analysis of toxin binding and oligomerization using Ae. aegypti midgut brush border membrane vesicles showed that the three Cyt1Aa mutants non-toxic to Ae. aegypti were affected in oligomerization. However, these mutants were still hemolytic. Our data shows that oligomerization of Cyt1Aa toxin is essential for its toxicity to Ae. aegypti but not for its toxicity against RBC indicating that the mode of action of Cyt1Aa is different in these distinct target membranes.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/6xr5s0rjTest

المؤلفون: Tetreau, Guillaume, Banneville, Anne-Sophie, Andreeva, Elena A, Brewster, Aaron S, Hunter, Mark S, Sierra, Raymond G, Teulon, Jean-Marie, Young, Iris D, Burke, Niamh, Grünewald, Tilman A, Beaudouin, Joël, Snigireva, Irina, Fernandez-Luna, Maria Teresa, Burt, Alister, Park, Hyun-Woo, Signor, Luca, Bafna, Jayesh A, Sadir, Rabia, Fenel, Daphna, Boeri-Erba, Elisabetta, Bacia, Maria, Zala, Ninon, Laporte, Frédéric, Després, Laurence, Weik, Martin, Boutet, Sébastien, Rosenthal, Martin, Coquelle, Nicolas, Burghammer, Manfred, Cascio, Duilio, Sawaya, Michael R, Winterhalter, Mathias, Gratton, Enrico, Gutsche, Irina, Federici, Brian, Pellequer, Jean-Luc, Sauter, Nicholas K, Colletier, Jacques-Philippe

المصدر: Nature Communications. 11(1)

مصطلحات موضوعية: Agricultural Biotechnology, Agricultural, Veterinary and Food Sciences, Chemical Sciences, Animals, Bacillus thuringiensis Toxins, Bacterial Proteins, Cell Membrane, Crystallography, X-Ray, Disulfides, Endotoxins, HEK293 Cells, Hemolysin Proteins, Humans, Hydrogen-Ion Concentration, Insecticides, Mice, Microscopy, Atomic Force, NIH 3T3 Cells, Protein Conformation, Sf9 Cells

الوصف: Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.

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الوصول الحر: https://escholarship.org/uc/item/1wd1x011Test

المؤلفون: Chen, Jianwu, Aimanova, Karly G, Gill, Sarjeet S

المصدر: PLOS Neglected Tropical Diseases. 14(2)

مصطلحات موضوعية: Biochemistry and Cell Biology, Biological Sciences, Infectious Diseases, Vector-Borne Diseases, Biotechnology, Genetics, Emerging Infectious Diseases, 1.1 Normal biological development and functioning, Underpinning research, Aedes, Animals, Bacillus thuringiensis Toxins, Bacterial Proteins, Cadherins, Digestive System, Endotoxins, Hemolysin Proteins, Insect Proteins, Larva, Protein Binding, Medical and Health Sciences, Tropical Medicine, Biological sciences, Biomedical and clinical sciences, Health sciences

الوصف: Aedes cadherin (AaeCad, AAEL024535) has been characterized as a receptor for Bacillus thuringiensis subsp. israelensis (Bti) Cry11A toxins. However, its role in development is still unknown. In this study, we modified the cadherin gene using ZFN and TALEN. Even though we obtained heterozygous deletions, no homozygous mutants were viable. Because ZFN and TALEN have lower off-targets than CRISPR/Cas9, we conclude the cadherin gene is essential for Aedes development. In contrast, in lepidopteran insects loss of a homologous cadherin does not appear to be lethal, since homozygous mutants are viable. To analyze the role of AaeCad in vivo, we tagged this protein with EGFP using CRISPR-Cas9-mediated homologous recombination and obtained a homozygous AaeCad-EGFP line. Addition of Aedes Rad51 mRNA enhanced the rate of recombination. We then examined AaeCad protein expression in most tissues and protein dynamics during mosquito development. We observe that AaeCad is expressed in larval and adult midgut-specific manner and its expression pattern changed during the mosquito development. Confocal images showed AaeCad has high expression in larval caecae and posterior midgut, and also in adult midgut. Expression of AaeCad is observed primarily in the apical membranes of epithelial cells, and not in cell-cell junctions. The expression pattern observed suggests AaeCad does not appear to play a role in these junctions. However, we cannot exclude its role beyond cell-cell adhesion in the midgut. We also observed that Cry11A bound to the apical side of larval gastric caecae and posterior midgut cells exactly where AaeCad-EGFP was expressed. Their co-localization suggests that AaeCad is indeed a receptor for the Cry11A toxin. Using this mosquito line we also observed that low doses of Cry11A toxin caused the cells to slough off membranes, which likely represents a defense mechanism, to limit cell damage from Cry11A toxin pores formed in the cell membrane.

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الوصول الحر: https://escholarship.org/uc/item/0cb8p1w2Test

المؤلفون: Qiu, Yulou, Li, Pan, Dong, Sa, Zhang, Xiaoshuai, Yang, Qianru, Wang, Yulong, Ge, Jing, Hammock, Bruce D, Zhang, Cunzheng, Liu, Xianjin

المصدر: Journal of Agricultural and Food Chemistry. 66(4)

مصطلحات موضوعية: Agricultural, Veterinary and Food Sciences, Food Sciences, Biotechnology, Immunization, Animals, Antibodies, Anti-Idiotypic, Antibodies, Monoclonal, Bacillus thuringiensis Toxins, Bacterial Proteins, Bacteriophages, Camelus, Edible Grain, Endotoxins, Food Contamination, Hemolysin Proteins, Luminescent Measurements, Lymphocytes, Peptide Library, RNA, Single-Domain Antibodies, Cry1Ab toxin, anti-idiotypic camel nanobody, competitive chemiluminescent immunoassay, Chemical Sciences, Agricultural and Veterinary Sciences, Engineering, Food Science, Agricultural, veterinary and food sciences, Chemical sciences

الوصف: Cry toxins have been widely used in genetically modified organisms for pest control, raising public concern regarding their effects on the natural environment and food safety. In this work, a phage-mediated competitive chemiluminescent immunoassay (c-CLIA) was developed for determination of Cry1Ab toxin using anti-idiotypic camel nanobodies. By extracting RNA from camels' peripheral blood lymphocytes, a naive phage-displayed nanobody library was established. Using anti-Cry1Ab toxin monoclonal antibodies (mAbs) against the library for anti-idiotypic antibody screening, four anti-idiotypic nanobodies were selected and confirmed to be specific for anti-Cry1Ab mAb binding. Thereafter, a c-CLIA was developed for detection of Cry1Ab toxin based on anti-idiotypic camel nanobodies and employed for sample testing. The results revealed a half-inhibition concentration of developed assay to be 42.68 ± 2.54 ng/mL, in the linear range of 10.49-307.1 ng/mL. The established method is highly specific for Cry1Ab recognition, with negligible cross-reactivity for other Cry toxins. For spiked cereal samples, the recoveries of Cry1Ab toxin ranged from 77.4% to 127%, with coefficient of variation of less than 9%. This study demonstrated that the competitive format based on phage-displayed anti-idiotypic nanobodies can provide an alternative strategy for Cry toxin detection.

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الوصول الحر: https://escholarship.org/uc/item/8r03f07vTest
https://escholarship.org/content/qt8r03f07v/qt8r03f07v.pdfTest

المؤلفون: Chen, Jianwu, Aimanova, Karly, Gill, Sarjeet S

مصطلحات موضوعية: Biochemistry and Cell Biology, Biomedical and Clinical Sciences, Medicinal and Biomolecular Chemistry, Biological Sciences, Chemical Sciences, Clinical Sciences, Vector-Borne Diseases, Emerging Infectious Diseases, Infectious Diseases, Aedes, Alkaline Phosphatase, Animals, Animals, Genetically Modified, Bacillus thuringiensis Toxins, Bacterial Proteins, Cadherins, Digestive System, Endotoxins, Gene Knockdown Techniques, HSP70 Heat-Shock Proteins, Hemolysin Proteins, Insect Proteins, Larva, Models, Animal, Receptors, Cell Surface, Sf9 Cells, Aedesaegypti, Alkaline phosphatase, Bacillus thuringiensis, Receptor, Function, Toxin, Medical and Health Sciences, Endocrinology & Metabolism, Medical biochemistry and metabolomics, Medicinal and biomolecular chemistry

الوصف: Presently three major groups of proteins from Aedes aegypti, cadherin, alkaline phosphatases (ALP) and aminopeptidases N (APN), have been identified as Cry11Aa toxin receptors. To further characterize their role on toxicity, transgenic mosquitoes with silenced Aedes cadherin expression were previously generated and the role of cadherin in mediating the toxicity of four different mosquitocidal toxins (Cry11Aa, Cry11Ba, Cry4Aa and Cry4Ba) was demonstrated. Here, we investigated the role of another reported Cry11Aa receptor, ALP1. As with Aedes cadherin, this protein is localized in the apical cell membrane of distal and proximal gastric caecae and the posterior midgut. We also successfully generated transgenic mosquitoes that knockdowned ALP1 transcript levels using an inducible Aedes heat shock promoter, Hsp70A driving dsALP1RNA. Four different mosquitocidal toxins were used for larval bioassays against this transgenic mosquito. Bioassay results show thatCry11Aa toxicity to these transgenic larvae following a heat shock decreased (4.4 fold) and Cry11Ba toxicity is slightly attenuated. But Cry4Aa and Cry4Ba toxicity to ALP1 silenced larvae is unchanged. Without heat shock, toxicity of all four toxins does not change, suggesting this heat shock promoter is heat-inducible. Notably, transgenic mosquitoes with ALP1 knockdown are about 3.7 times less resistant to Cry11Aa toxin than those with Aedes cadherin knockdown. These results demonstrate that the ALP1 is an important secondary receptor for Cry11Aa and Cry11Ba, but it might not be involved in Cry4Aa and Cry4Ba toxicity.

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الوصول الحر: https://escholarship.org/uc/item/6969z3nmTest

المؤلفون: Dan Sun, Liuhong Zhu, Le Guo, Shaoli Wang, Qingjun Wu, Neil Crickmore, Xuguo Zhou, Alejandra Bravo, Mario Soberón, Zhaojiang Guo, Youjun Zhang

مصطلحات موضوعية: ABC transporters, Bacillus thuringiensis, CRISPR/Cas9, GPI-anchored proteins, Plutella xylostella, Receptor redundancy, Toxin action, ATP-Binding Cassette Transporters, Animals, Bacillus thuringiensis Toxins, Bacterial Proteins, CD13 Antigens, Endotoxins, Hemolysin Proteins, Insect Proteins, Insecticide Resistance, Larva, Moths

الوصف: Background Biopesticides and transgenic crops based on Bacillus thuringiensis (Bt) toxins are extensively used to control insect pests, but the rapid evolution of insect resistance seriously threatens their effectiveness. Bt resistance is often polygenic and complex. Mutations that confer resistance occur in midgut proteins that act as cell surface receptors for the toxin, and it is thought they facilitate its assembly as a membrane-damaging pore. However, the mechanistic details of the action of Bt toxins remain controversial. Results We have examined the contribution of two paralogous ABC transporters and two aminopeptidases N to Bt Cry1Ac toxicity in the diamondback moth, Plutella xylostella, using CRISPR/Cas9 to generate a series of homozygous polygenic knockout strains. A double-gene knockout strain, in which the two paralogous ABC transporters ABCC2 and ABCC3 were deleted, exhibited 4482-fold resistance to Cry1A toxin, significantly greater than that previously reported for single-gene knockouts and confirming the mutual functional redundancy of these ABC transporters in acting as toxin receptors in P. xylostella. A double-gene knockout strain in which APN1 and APN3a were deleted exhibited 1425-fold resistance to Cry1Ac toxin, providing the most direct evidence to date for these APN proteins acting as Cry1Ac toxin receptors, while also indicating their functional redundancy. Genetic crosses of the two double-gene knockouts yielded a hybrid strain in which all four receptor genes were deleted and this resulted in a >?34,000-fold resistance, indicating that while both types of receptor need to be present for the toxin to be fully effective, there is a level of functional redundancy between them. The highly resistant quadruple knockout strain was less fit than wild-type moths, but no fitness cost was detected in the double knockout strains. Conclusion Our results provide direct evidence that APN1 and APN3a are important for Cry1Ac toxicity. They support our overarching hypothesis of a versatile mode of ...

العلاقة: 10779/uos.23488697.v1; https://figshare.com/articles/journal_contribution/A_versatile_contribution_of_both_aminopeptidases_N_and_ABC_transporters_to_Bt_Cry1Ac_toxicity_in_the_diamondback_moth/23488697Test

الإتاحة: https://figshare.com/articles/journal_contribution/A_versatile_contribution_of_both_aminopeptidases_N_and_ABC_transporters_to_Bt_Cry1Ac_toxicity_in_the_diamondback_moth/23488697Test

المؤلفون: Le Guo, Zhouqiang Cheng, Jianying Qin, Dan Sun, Shaoli Wang, Qingjun Wu, Neil Crickmore, Xuguo Zhou, Alejandra Bravo, Mario Soberón, Zhaojiang Guo, Youjun Zhang

مصطلحات موضوعية: Animals, Bacillus thuringiensis, Bacillus thuringiensis Toxins, Bacterial Proteins, Endotoxins, Granulovirus, Hemolysin Proteins, Insect Proteins, Insecticide Resistance, Insecticides, Larva, MAP Kinase Signaling System, Moths, Transcription Factors

الوصف: The benefits of biopesticides and transgenic crops based on the insecticidal Cry-toxins from Bacillus thuringiensis (Bt) are considerably threatened by insect resistance evolution, thus, deciphering the molecular mechanisms underlying insect resistance to Bt products is of great significance to their sustainable utilization. Previously, we have demonstrated that the down-regulation of PxmALP in a strain of Plutella xylostella (L.) highly resistant to the Bt Cry1Ac toxin was due to a hormone-activated MAPK signaling pathway and contributed to the resistance phenotype. However, the underlying transcriptional regulatory mechanism remains enigmatic. Here, we report that the PxGATAd transcription factor (TF) is responsible for the differential expression of PxmALP observed between the Cry1Ac susceptible and resistant strains. We identified that PxGATAd directly activates PxmALP expression via interacting with a non-canonical but specific GATA-like cis-response element (CRE) located in the PxmALP promoter region. A six-nucleotide insertion mutation in this cis-acting element of the PxmALP promoter from the resistant strain resulted in repression of transcriptional activity, affecting the regulatory performance of PxGATAd. Furthermore, silencing of PxGATAd in susceptible larvae reduced the expression of PxmALP and susceptibility to Cry1Ac toxin. Suppressing PxMAP4K4 expression in the resistant larvae transiently recovered both the expression of PxGATAd and PxmALP, indicating that the PxGATAd is a positive responsive factor involved in the activation of PxmALP promoter and negatively regulated by the MAPK signaling pathway. Overall, this study deciphers an intricate regulatory mechanism of PxmALP gene expression and highlights the concurrent involvement of both trans-regulatory factors and cis-acting elements in Cry1Ac resistance development in lepidopteran insects.

العلاقة: 10779/uos.23489762.v1; https://figshare.com/articles/journal_contribution/MAPK-mediated_transcription_factor_GATAd_contributes_to_Cry1Ac_resistance_in_diamondback_moth_by_reducing_PxmALP_expression/23489762Test

الإتاحة: https://figshare.com/articles/journal_contribution/MAPK-mediated_transcription_factor_GATAd_contributes_to_Cry1Ac_resistance_in_diamondback_moth_by_reducing_PxmALP_expression/23489762Test

المؤلفون: Canton, Pablo Emiliano, Cancino-Rodezno, Angeles, Gill, Sarjeet S, Soberón, Mario, Bravo, Alejandra

المصدر: BMC genomics. 16(1)

مصطلحات موضوعية: Gastrointestinal Tract, Animals, Aedes, Bacterial Proteins, Endotoxins, Insecticides, Cluster Analysis, Reproducibility of Results, Gene Expression Profiling, Inhibitory Concentration 50, Computational Biology, Gene Expression Regulation, Larva, Drug Resistance, Hemolysin Proteins, Molecular Sequence Annotation, High-Throughput Nucleotide Sequencing, Transcriptome, Bacillus thuringiensis Toxins, Aedes aegypti, Bacillus thuringiensis subsp israelensis, RNA-seq, biological control, defense response, Cry toxins, Biological Sciences, Information and Computing Sciences, Medical and Health Sciences, Bioinformatics

الوصف: BackgroundAlthough much is known about the mechanism of action of Bacillus thuringiensis Cry toxins, the target tissue cellular responses to toxin activity is less understood. Previous transcriptomic studies indicated that significant changes in gene expression occurred during intoxication. However, most of these studies were done in organisms without a sequenced and annotated reference genome. A reference genome and transcriptome is available for the mosquito Aedes aegypti, and its importance as a disease vector has positioned its biological control as a primary health concern. Through RNA sequencing we sought to determine the transcriptional changes observed during intoxication by Cry11Aa in A. aegypti and to analyze possible defense and recovery mechanisms engaged after toxin ingestion.ResultsIn this work the changes in the transcriptome of 4(th) instar A. aegypti larvae exposed to Cry11Aa toxin for 0, 3, 6, 9, and 12 h were analyzed. A total of 1060 differentially expressed genes after toxin ingestion were identified with two bioconductoR packages: DESeq2 and EdgeR. The most important transcriptional changes were observed after 9 or 12 h of toxin exposure. GO enrichment analysis of molecular function and biological process were performed as well as Interpro protein functional domains and pBLAST analyses. Up regulated processes include vesicular trafficking, small GTPase signaling, MAPK pathways, and lipid metabolism. In contrast, down regulated functions are related to transmembrane transport, detoxification mechanisms, cell proliferation and metabolism enzymes. Validation with RT-qPCR showed large agreement with Cry11Aa intoxication since these changes were not observed with untreated larvae or larvae treated with non-toxic Cry11Aa mutants, indicating that a fully functional pore forming Cry toxin is required for the observed transcriptional responses.ConclusionsThis study presents the first transcriptome of Cry intoxication response in a fully sequenced insect, and reveals possible conserved cellular processes that enable larvae to contend with Cry intoxication in the disease vector A. aegypti. We found some similarities of the mosquito responses to Cry11Aa toxin with previously observed responses to other Cry toxins in different insect orders and in nematodes suggesting a conserved response to pore forming toxins. Surprisingly some of these responses also correlate with transcriptional changes observed in Bti-resistant and Cry11Aa-resistant mosquito larvae.

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الوصول الحر: https://escholarship.org/uc/item/9px1b4sdTest

المؤلفون: Lee, Su-Bum, Chen, Jianwu, Aimanova, Karlygash G, Gill, Sarjeet S

مصطلحات موضوعية: Biodefense, Vaccine Related, Vector-Borne Diseases, Prevention, Emerging Infectious Diseases, Infectious Diseases, Aedes, Animals, Animals, Genetically Modified, Bacillus thuringiensis Toxins, Bacterial Proteins, Cadherins, Cell Line, Endotoxins, Female, Gene Expression, Hemolysin Proteins, Insect Proteins, Insecticides, Male, Protein Multimerization, Bacillus thuringiensis, Cry11Aa toxin, Cadherin, Transgenic mosquito, Oligomerization, Cytotoxicity, Medical and Health Sciences, Endocrinology & Metabolism

الوصف: Cadherin plays an important role in the toxicity of Bacillus thuringiensis Cry proteins. We previously cloned a full-length cadherin from Aedes aegypti larvae and reported this protein binds Cry11Aa toxin from B. thuringiensis subsp. israelensis with high affinity, ≈16.7nM. Based on these results, we investigated if Aedes cadherin is involved in the in vivo toxicity of Cry11Aa toxin to Ae. aegypti. We established a mosquito cell line stably expressing the full-length Aedes cadherin and transgenic mosquitoes with silenced Aedes cadherin expression. Cells expressing the Aedes cadherin showed increased sensitivity to Cry11Aa toxin. Cry11Aa toxin at 400nM killed approximately 37% of the cells in 3h. Otherwise, transgenic mosquitoes with silenced Aedes cadherin expression showed increased tolerance to Cry11Aa toxin. Furthermore, cells expressing Aedes cadherin triggered Cry11Aa oligomerization. These results show the Aedes cadherin plays a pivotal role in Cry11Aa toxicity to Ae. 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 Aedes cadherin were sensitive to Cry4Aa and Cry11Ba, but not Cry4Ba. However transgenic mosquitoes with silenced Aedes 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 Ae. aegypti.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/8wd3b5gxTest

المؤلفون: Sawaya, Michael R, Cascio, Duilio, Gingery, Mari, Rodriguez, Jose, Goldschmidt, Lukasz, Colletier, Jacques-Philippe, Messerschmidt, Marc M, Boutet, Sébastien, Koglin, Jason E, Williams, Garth J, Brewster, Aaron S, Nass, Karol, Hattne, Johan, Botha, Sabine, Doak, R Bruce, Shoeman, Robert L, DePonte, Daniel P, Park, Hyun-Woo, Federici, Brian A, Sauter, Nicholas K, Schlichting, Ilme, Eisenberg, David S

المصدر: Proceedings of the National Academy of Sciences of the United States of America. 111(35)

مصطلحات موضوعية: Generic health relevance, Bacillus thuringiensis, Bacillus thuringiensis Toxins, Bacterial Proteins, Crystallization, Crystallography, X-Ray, Endotoxins, Hemolysin Proteins, Lasers, Spores, Bacterial, Synchrotrons, X-Ray Diffraction, XFEL, Cry3A insecticidal toxin, serial femtosecond crystallography

الوصف: It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼ 5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.

وصف الملف: application/pdf

الوصول الحر: https://escholarship.org/uc/item/8g8793xzTest