Laboratory of molecular bioengineering
Head: Konstantin Miroshnikov
The Laboratory conducts a complex molecular biological study of bacterial viruses (bacteriophages). The researchers conduct a major project to study and systemize bacteriophages that infect agricultural organisms, identify the roles of proteins and peptides encoded by bacteriophage genes, and also participate in the development of phage cocktail formulations and technologies for their production and control.
Bacteriophages are the most numerous creatures on Earth, their diversity is estimated as 1015 taxonomic species. Many of the processes that comprise the classics of molecular biology today have been studied in detail using bacteriophages as investigation objects. The Laboratory's capabilities allow conducting a full cycle of experiments in the area of genetic engineering: from the strategy choice of cloning an individual gene and its chemical enzymatic synthesis to the development of a method of isolation, purification, complete physical and chemical identification and studies of biological activity, scaling of expression and the stage of fermentation.
The Laboratory has been developed an algorithm for studying bacteriophages with the use of modern methods of genomics, structural and functional proteomics, and physico-chemical methods for determining the structures of proteins and protein complexes. The Laboratory was founded by Professor in 1985 at the Ivanovskiy Institute of Virology of the USSR Academy of Medical Sciences. In 1994-1997 the Laboratory was based in the Bakh Institute of Biochemistry of the Russian Academy of Sciences, since 1997 it is a member of the IBCh RAS team. Since 2013, the Laboratory is headed by Konstantin Miroshnikov.
Genomics of bacteriophages
Since 1999, the Laboratory has been participating in the international program to study Pseudomonas aeruginosa bacteriophages.
Pseudomonas aeruginosa is an extremely important bacterium in the soil and freshwater reservoirs, an important component in ecosystems. Bacteriophages that regulate the balance of pseudomonas populations play an important ecological role. Pseudomonades are a conditional pathogen that can cause nosocomial infections. Due to the natural and acquired antibiotic resistance P. aeruginosa from these infections is quite difficult to get rid of. Therefore, bacteriophages of pseudomonads are often considered as an additional and even an alternative method for the treatment of pseudomonas infections. Today about 20 taxonomic genera of bacteriophages infecting pseudomonas are known, and their number is constantly growing. Depending on the habitat conditions, the diversity of P. aeruginosa strains is very large, respectively, the specific variety of bacteriophages infecting these strains also opens up great opportunities for research. P. aeruginosa serves as one of the model objects for studying the microbiome of Lake Baikal. The Laboratory participated in the description and approval of the genera phiKZ-like, KMV-like, PB1-like, M6-like Pseudomonas bacteriophages.
Currently the Laboratory is conducting a major project to study and systematize bacteriophages that infect agricultural pathogens. Enterobacteria belonging to genera Pectobacterium and Dickeya cause black leg and soft-rot bacterioses of potato, which bring enormous damage to potato production. To develop the concept of biological crop protection and control of pathogens, an in-depth understanding of the genetic organization of the relevant bacteriophages is needed.
Sequencing of bacteriophage genomes isproceeded in cooperation with the "Genome" Center of ICBFM SB RAS (Novosibirsk), the Limnological Institute of the SB RAS (Irkutsk), Immanuel Kant Baltic Federal University (Kaliningrad), Katholieke Universiteit Leuven (Belgium).
Proteomics of bacteriophages
Viruses are the smallest representatives of wildlife, they are distinguished by a small and compactly organized genome. However, until now the exact function of at least a third of the genes of viruses is still unknown. The identification of the role of proteins and peptides encoded by bacteriophage genes is the subject of the Laboratory's interest.
The main objects of proteomic studies are the key structural and functional proteins that provide the bacteriophage infectious cycle:
- Receptor tail proteins. The infectious cycle of the bacteriophage starts with the specific attachment of the virus to the bacterium surface and the active introduction of the viral DNA into the cytoplasm. Attachment is provided by the proteins of the tail spikes and fibers of the bacteriophage. Tail spike proteins have very rigid trimeric structures with original protein folds, and often possess an additional enzymatic activity for the destruction of protective bacterial exopolysaccharides. The study of enzymatic mechanisms, the substrate specificity and structure of such proteins is important both for a fundamental understanding of bacteriophage biology, and for the application of recombinant proteins in protein engineering and for the control of bacterial biofilms. Studies are carried out in cooperation with The Zelinsky Organic Chemistry RAS, The Vinogradsky Institute of Microbiology RAS, The Bach Institute of Biochemistry RAS, The University of Texas Galveston (USA), Universidade de Santiago de Compostela (Spain).
- Cytolytic enzymes (endolysins). At the final stage of the infectious cycle, bacteriophage enzymes the destroy the host cell, enabling an exit of progeny viruses. Such enzymes are very diverse in their properties, structure and substrate specificity. Recombinant endolysins are often regarded as "enzybiotics", antimicrobials for the treatment of antibiotic-resistant varieties of pathogenic bacteria. Research is carried out in cooperation with the Chemical and Biological Departments of Moscow State University, the Shubnikov Institute of Crystallography RAS, The University of Texas at El Paso, The Purdue University (USA), Katholieke Universiteit Leuven (Belgium).
- Molecular chaperones. One of the most important problems of recombinant technologies is the difficulty to obtain a target protein with a natural spatial structure. Folding a polypeptide chain into a biologically active form is often not a spontaneous process, and requires the participation of additional factors. Folding assistants, according to the first described representative of this class, are called chaperones. Although bacteriophages, like all viruses, are dependent on the metabolic systems of the host cell, genes encoding chaperones have been found in a number of phage genomes. Probably, these genes were acquired by bacteriophages as a result of horizontal transfer and adapted to their needs. The study of such proteins is useful for understanding the mechanisms of folding, which is often called the "third component of the genetic code". Also, the bacteriophages proteins often possess "internal chaperones" – energetically excessive domains. In addition, to rapid and spontaneous folding into the target structure, such domains ("folds") direct the folding of a part of the polypeptide chain attached to them. The use of chaperones and folds is an attractive tool for protein engineering. With its help, a number of proteins, previously incapable to form an active structure during synthesis in a bacterial cell, was obtained and studied. Research is carried out in cooperation with the Biological Department of Moscow State University, Institute of Crystallography RAS, The University of Texas at El Paso (USA).
3. Practical application of bacteriophages (medicine, agriculture)
Russia is one of the few countries in the world where bacteriophage preparations are manufactured industrially and are used in practical medicine as antimicrobial agents. However, production standards were developed more than 30 years ago, and manufacturers are faced with the task of bringing the drugs in line with modern requirements formulated by the scientific and medical communities. The Laboratory of Molecular Bioengineering interacts in the development of phage cocktail formulations and production and control technology with the manufacturer of medical phage preparations "Microgen". An attractive strategy is also the use of bacteriophages in agriculture to combat bacterial plant diseases. Interaction in the areas of pathogen diagnostics, the selection of bacteriophages and the production of agricultural preparations occurs with the large scientific and production agricultural holding (Moscow region).
|Konstantin Miroshnikov, C-MAS, D.Sc||pr. r. firstname.lastname@example.org|
|Dorofeeva L.V.||s. r. f.|
|Alexander Ignatov, D.Sc||l. r. f.|
|Mihail Shnejder, Ph.D.||s. r. f.|
|Sykilinda N.N., Ph.D.||s. r. f.|
|Evseev P.V.||r. f.|
|Olga Timoshina||j. r. f.|
|Golovin D.L.||t. q. - lab. as.|
|Komarevtsev S.K.||j. r. f.|
|Landishev N.N.||t. q. - lab. as.|
|Tokmakova A.D.||j. r. f.|
|Kurochkina L.P., Ph.D.|
|Anna Lukyanova, Ph.D.|
|Mesyandjinov V.V., D.Sc|