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COMMON PART


Project Number18-76-00028

Project titleStudy of factors, which influence the expression and specificity of Bacillus thuringiensis toxins

Project LeadAntonets Kirill

AffiliationFederal State Budget Scientific Institution All-Russia Research Institute for Agricultural Microbiology,

Implementation period 07.2018 - 06.2020 

Research area 06 - AGRICULTURAL SCIENCES, 06-104 - Agricultural biotechnologies

KeywordsBacillus thuringiensis, biological pest-control agents, agrobiotechnology, toxins, plant protection, genomics, molecular dynamics


 

PROJECT CONTENT


Annotation
The producing of ecological safe products is an important goal of modern agriculture and biotechnology. It can be achieved by substituting chemical pesticides in favor of biological plant protection from insects. Many different of organisms including nematodes, fungi and bacteria, are used as such agents. Many biological agents are based on the different strains of the gram-positive spore-forming bacterium Bacillus thuringiensis. This species of bacteria produces a broad spectrum of diverse toxins, because of which a significant amount of economically valuable insecticidal strains was developed on its basis for a long period of studies. The most important and variable group of toxins are endotoxins of the Cry superfamily, which are incorporated in the parospalic bodies during the formation of endospores. Despite the high relevance of research in the field of creating new ecologically safe plant protection methods, the molecular mechanisms of the specificity of the toxins of B. thuringiensis remain insufficiently studied, which prevents the effective molecular design of toxins to create economically significant strains, which are highly selective against given species of insect pests. This Project is dedicated to the identifying of genome features of B. thuringiensis strains and amino acid sequences of protein toxins, which are responsible for the specificity of this bacterium against pests and expression level of the toxins. The study will be carried out using the collection of B. thuringiensis strains in ARRIAM, among which there are both highly effective strains against some groups of insect pests and strains that have lost the ability to produce toxins. Within the Project complete sequences of the genomes of the B. thuringiensis strains will be obtained. The analysis of the obtained sequences will reveal the sequences of genes encoding Cry-toxins. Comparison of the sequences from strains belonging to the three different serotypes, will allow us to determine the variable regions responsible for the specificity of toxins. Also, the comparison of the whole-genome sequences of strains will allow to determine the changes of bacterial chromosome or extrachromosomal replicons (including plasmids) that lead to a decrease in the level of toxins production. An important part of the project is to test the ability to predict the sequence of Cry-toxins acting on insects of a given species. Using the molecular dynamics, we will optimize the surface of the toxin responsible for binding to the receptor protein in the intestine of insects. This will allow the toxin, which initially did not interact with the receptor of the given species of insect pest, to bind to it and cause the death of the intestinal epithelial cells, which leads to the death of the insect. Taking together, within this Project a comprehensive bioinformatics and experimental analysis of the specificity of the insecticidal toxins of three different B. thuringiensis serotypes will be carried out, and a new approach to the molecular design of toxins with specified properties will be assayed. The data obtained within this Project, will create a basis for development of the innovative biological agents, safe for humans and highly selective against specific species of insect pests.

Expected results
The expected results of the Project are of high importance and novelty. As a research object, strains of B. thuringiensis of three different serotypes from the ARRIAM collection will be used. At the first stage of the Project, the genomes of these strains will be sequenced using the whole-genome next generation sequencing. The results of this analysis will reveal the variable regions of Cry-toxins, which determine the specificity of their action. Changes in the genomes of B. thuringiensis strains that lead to a decrease in the level of toxin production will also be analyzed. The results obtained will be the first comprehensive study of genomic peculiarities affecting the specificity and level of production of toxins B. thuringiensis. Using molecular dynamics methods, the toxin sequence will be optimized to provide binding to the receptor of those insect species, to which this toxin initially had no affinity. Thus, a change in the specificity of B. thuringiensis toxins and their acquisition of insecticidal properties against those insect species for which no insecticidal activity has been observed, will be achieved. Since bioinformatic predictions require experimental verification, plasmids will be obtained for the production of appropriate toxin variants in the expressional E. coli systems, and an experimental verification of their efficacy will be performed. Thus, for the first time, the design of the amino acid sequence of the insecticidal toxin of B. thuringiensis with the desired properties will be carried out in silico. These results will be of great importance at the world level, since they will not only allow a deeper exploration of the molecular mechanisms of bacterial toxin action, but will also significantly simplify and accelerate the development of innovative new generation biological agents to control the population of insect pests, which are safe for humans and the environment.


 

REPORTS


Annotation of the results obtained in 2019
The scope of the Project in 2019 was devoted to the experimental study of Cry toxins identified in the genomes of Bacillus thuringiensis during the first year of work. Several different methods have been tested for the extraction and purification of recombinant Cry toxins, and one has been chosen that provides sufficient protein for experimentation. An optimal E. coli producer strain was also selected. At the same time, although the method of isolation and purification of recombinant proteins provided the necessary amount of recombinant Cry toxins, it was shown that during the extraction of the proteins, their spatial structure is disturbed, which leads to the loss of their activity. To optimize the action of the Cry toxin against a new insect species, the sequence of Cry1Ab12, a toxin that according to the literature is specific against insects of the order Lepidoptera, but not Diptera, was chosen. The spatial structures of the toxins Cry1Ab12 and Cry4Ba1 (the latter was selected as the toxin active against Diptera) and the potential receptor for aminopeptidase N APN1 A. aegypti were predicted. Then, the structures of the Cry1Ab12-APN1 and Cry4Ba1-APN1 complexes were predicted. It was found that the complexes of the Cry1Ab12 and Cry4Ba1 toxins with the APN1 receptor showed high structural similarity, despite the significant differences in the amino acid sequence of these Cry toxins. Based on the structure of the Cry1Ab12-APN1 complex, positions of the Cry1Ab12 protein were selected, which are located on the surface of interaction with the APN1 receptor. Using enumeration of amino acids at these positions, a number of substitutions were predicted in the Cry1Ab12 protein, which led to the stabilization of the complex, and thus allowed the Cry1Ab12 toxin sequence to be obtained, optimized for interaction with A. aegypti aminopeptidase N. The high similarity of the structures of the Cry1Ab12-APN1 complex and the comples of Diptera-specific Cry4Ba1 toxin and APN1 suggests that a number of stabilizing substitutions on the surface of interaction of the toxin with the receptor should be sufficient to increase the activity of the corresponding Cry toxin with respect to the desired group of insects. The designed toxin has shown low activity against the larvae of the A. aegypti mosquito, which allows us to conclude that the host spectrum changes after a modification of the primary structure of this protein. In general, the results obtained during the second year of the Project implementation allow us to conclude that it is possible to use the software tools to optimize the sequences of Cry toxins in order to increase their activity against specific groups of insects. It gives opportunities for agriculture and biotechnology in the context of creating new biological pest control agents with a given range of specificity for specific groups of insects. Publications of the second year of the Project: 1. Malovichko Y.V., Nizhnikov A.A., Antonets K.S. Repertoire of the Bacillus thuringiensis virulence factors unrelated to major classes of protein toxins and its role in specificity of host-pathogen interactions // Toxins, 2019, V.11, e347. https://doi.org/10.3390/toxins11060347 (impact-factor 3,895, Q1) 2. Shikov A.E., Malovichko Yu.V., Skitchenko R.K., Nizhnikov A.A., Antonets K.S. No More Tears: Mining Sequencing Data for Novel Bt Cry Toxins with CryProcessor // Toxins, 2020, V12(3), e204, https://doi.org/10.3390/toxins12030204 (impact-factor 3,895, Q1) 3. Belousova M.E., Grishechkina S.D., Ermolova V.P., Antonets K.S., Mardanov A.V., Rakitin A.L., Beletsky A.V., Ravin N.V., Nizhnikov A.A. Whole genome sequencing of Bacillus thuringiensis var. darmstadiensis 56 strain and the study of insecticidal activity of the biological preparation on its basis // Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2020, V.55(1), p. 87-96, https://doi.org/10.15389/agrobiology.2020.1.87eng (imapct-factor 0,47) 4. Malovichko Yu.V., Shikov A.E., Skitchenko R.K., Nizhnikov A.A., Antonets K.S. Revealing mechanisms of Bacillus thuringiensis host specificity via molecular modelling of the Cry toxin-receptor interactions // BMC Bioinformatics, 2019, V.20 (Suppl 17), 516, https://doi.org/10.1186/s12859-019-3122-9 (impact factor 2,511, Q1) 5. Malovichko Yu.V., Afonin A.M., Belousova M.E., Nizhnikov A.A., Antonets K.S. Long-read sequencing of Bacillus thuringiensis strains reveals genome rearrangements affecting their virulence effectiveness // FEBS Open Bio, 2019, 9 (Suppl. 1), pp. 65-431, https://doi.org/10.1002/2211-5463.12675 (impact-factor 2,021).

 

Publications

1. Malovichko Yu.V., Nizhnikov A.A., Antonets K.S. Repertoire of the Bacillus thuringiensis Virulence Factors Unrelated to Major Classes of Protein Toxins and Its Role in Specificity of Host-Pathogen Interactions Toxins (MDPI), 11(6), 347 (year - 2019).

2. Malovichko Yu.V., Shikov A.E., Skitchenko R.K., Nizhnikov A.A., Antonets K.S. Revealing mechanisms of Bacillus thuringiensis host specificity via molecular modelling of the Cry toxin-receptor interactions BMC Bioinformatics, 20 (Suppl 17):516 (year - 2019).

3. Malovichko Yu.V., Afonin A.M., Belousova M.E., Nizhnikov A.A., Antonets K.S. Long-read sequencing of Bacillus thuringiensis strains reveals genome rearrangements affecting their virulence effectiveness FEBS Open Bio, 9 (Suppl. 1), pp. 65-431 (year - 2019).

4. Shikov A.E., Malovichko Yu.V., Skitchenko R.K., Nizhnikov A.A., Antonets K.S. No More Tears: Mining Sequencing Data for Novel Bt Cry Toxins with CryProcessor Toxins, V.12(3), 204 (year - 2020).

5. - В Петербурге ускорили поиск веществ для борьбы с сельхозвредителями ТАСС Наука, 08.04.2020 (year - ).

6. - Микробиологи разработали программу для поиска новых инсектицидов газета.ru, 08.04.2020 (year - ).

7. - Компьютерная программа поможет искать новые средства борьбы с насекомыми-вредителями полит.ру, 08.04.2020 (year - ).

8. - Микробиологи разработали программу для поиска новых инсектицидов Indicator.ru, 11.04.2020 (year - ).

9. Belousova M.E., Grishechkina S.D., Ermolova V.P., Antonets K.S., Mardanov A.V., Rakitin A.L., Beletsky A.V., Ravin N.V., Nizhnikov A.A. Секвенирование генома штамма Bacillus thuringiensis var. darmstadiensis 56 и изучение инсектицитдной активности биологического препарата на его основе Сельскохозяйственная биология, Т.55(1), с. 87-96 (year - 2020).


Annotation of the results obtained in 2018
In the scope of the Project in 2018, we carried out whole-genome sequencing of twelve Bacillus thuringiensis strains belonging to three different serotypes: var. thuringiensis, var. darmstadiensis and var. israeliensis. Sequencing was carried out using two different technologies: Illumina HiSeqX, which produces many short accurate reads, and Oxford Nanopore, which produces long reads, but more error-prone than Illumina. Long reads were used to obtain de novo preliminary assemblies of genomes. After several stages of error correction using both long reads and short reads, assemblies of genomes with a low error rate were obtained. For some strains, we obtained a complete assembly of the genome, in which contig corresponds to a replicon. The assembly of the genome of B. thuringiensis var. darmstadiensis obtained in this study, is the best and most complete among publicly available assemblies for this serotype. Protein-encoding sequences in the genomes of the studied strains were annotated, and we performed a search for genes encoding Cry toxins. Since toxins are highly variable, a special computer program based on the use of Hidden Markov Model (HMM) was developed to search for genes encoding for them. This program allows to search for Cry toxins in an array of protein sequences and to annotate their domain structure. The accuracy of the prediction and the performance of this program exceeds the available analogues. Using developed program, the repertoire of Cry toxins, which can be produced by the strains B. thuringiensis used in the study, was identified. It was found that the strains belonging to serotypes var. thuringiensis and var. israeliensis contained several genes encoding Cry toxins in their genome, while strains var. darmstadiensis bore only one gene that encodes a Cry toxin, which according to the literature data possesses mainly nematocidal activity. Thus, we can conclude that the insecticidal activity of B. thuringiensis var. darmstadiensis is associated with other non-Cry toxins. Sequences of toxins were compared to each other and it was found that they are completely identical between strains of the same serotype, even at the nucleotide level, which may indicate that these strains have the same origin. At the same time, toxins from different serotypes differed by more than 30%, which may indicate that adaptation to different hosts affects a large number of positions in the amino acid sequence of toxins. We also found that the inability of some strains of serotype var. israeliensis to form crystals during sporulation was associated with the loss of the plasmid, on which all three genes encoding the Cry toxins in these strains are located. Thus, in 2018, we carried out full genome sequencing of 12 strains of B. thuringiensis, de novo assembled their genomes, and determined the repertoire of Cry toxins, which determine their host specificity. A new program for the search and annotation of Cry toxins was also developed, which exceeds the existing analogues in the prediction accuracy and the performance. The identification of genes encoding Cry toxins in the strains under study made it possible to proceed to an experimental study of the species specificity of their action. Publication of the Project in 2018: 1. Malovichko Y.V., Afonin A.A., Belousova M.E., Ermolova V.P., Grishechkina S.D., Nizhnikov A.A., Antonetz K.S. Comparative genomics of the insecticidal bacterium Bacillus thuringiensis using Oxford Nanopore sequencing // Mol. Biol. Cell, 2018, V.28, P611 (Abstract P3470) (Abstracts of ASCB/EMBO Meeting 2018, San Diego, USA)

 

Publications

1. Malovichko Y.V., Afonin A.A., Belousova M.E., Ermolova V.P., Grishechkina S.D., Nizhnikov A.A., Antonetz K.S. Comparative genomics of the insecticidal bacterium Bacillus thuringiensis using Oxford Nanopore sequencing Molecular Biology of the Cell, V. 29(26), 3063-63, P3470 (year - 2018).