Pracownie badawcze

Dr hab. Alicja Węgrzyn, Prof. IBB PAN

Pracownia Terapii Fagowej

Zakres badań

Our laboratory is interested in mechanisms of bacteriophage development and their use in therapies for bacterial infections in animals. We investigate various aspects of phage therapy, including safety and efficacy. The long-term goal of our studies is to introduce phages for the prevention and treatment of bacterial infections in animals and the protection of food and medical devices.

Laboratory location: Gdańsk

Badania

Najważniejsze osiągnięcia badawcze

  • We uncovered roles of genes that are included in the exo-xis region in the regulation of phage development.
  • We discovered the first phage-encoded micro-RNA molecule that is functional in bacterial cells.
  • We demonstrated the large biodiversity (both genetic and functional) of bacteriophages that were isolated from a single habitat.
  • We described a newly discovered bacteriophage that exhibits extremely rapid development in host cells.
  • We found that a series of newly isolated and characterized bacteriophages can be potentially used in phage therapy against Salmonella enterica infections in chickens.

Opis badań

Bacteriophages have been used as models in microbiology, genetics, and molecular biology for over 70 years. However, more recent discoveries indicated that phages may be involved in the pathogenicity of different bacterial strains, while also being considered therapeutical agents for bacterial infections (so-called “phage therapy”). To combat the phage-mediated pathogenicity of bacteria that bear prophages that code for various toxins and to utilize bacteriophages in phage therapy, understanding the molecular mechanisms that occur during the development of phages in bacterial cells is crucial. Therefore, our studies focus on regulation of the development of bacteriophages, both those that code for Shiga toxins (so-called Stx phages, used as models of toxin-converting phages) and those that can be potentially toxic.

During this project, led by the head of this laboratory (National Science Center [NCN] project no. 1924/B/P01/2010/39, entitled “Mechanisms of regulation of development of bacteriophages coding for Shiga toxins,” 2010-2014), we have studied principles of the control of DNA replication of Stx phages. We discovered that the DNA replication of these phages can be inhibited by higher levels of guanosine tetraphosphage (ppGpp),1 whereas the synthesis of this unusual nucleotide may be stimulated by phenetyl isothiocyanate, making this compound a potential drug for the treatment of infections that are caused by Shiga toxin-producing Escherichia coli strains, which can cause severe disease in humans.2

Our next experiments were performed during studies that were funded by another grant (NCN project no. 2013/09/B/NZ2/02366, entitled “The role of the exo-xis region in the regulation of development of lambdoid bacetriophages, exemplified by phage lambda and phages carrying Shiga toxin genes, responsible for pathogenicity of enterohemorrhagic strains of Escherichia coli, 2014-2017, PI: Prof. Alicja Węgrzyn). The exo-xis region is an evolutionarily conserved fragment of genomes of lambdoid bacteriophages, but very little was known about the roles of genes that are included in this region. Systematic and complex studies of functions of genes that are included in this region indicated that they are involved in the regulation of bacteriophage development, particularly at stages of the “lysis vs. lysogenization” decision and prophage induction. Such regulatory properties might be crucial for the infectivity of E. coli strains that produce Shiga toxins because their virulence depends on lysogenization with Stx phages and subsequent prophage induction. We demonstrated the differential expression of exo-xis region genes under various conditions that significantly influenced the control of lysogenization and prophage induction.3 Moreover, we discovered the first micro-RNA molecule that is encoded by bacteriophages, which is also the first described micro-RNA type that was shown to be functional in prokaryotic cells.4 This molecule is produced as a 80-nt long transcript, which is then processed to form a 20-nt long functional modulator of gene expression, resembling eukaryotic micro-RNA. Furthermore, we demonstrated that genes from the exo-xis region are crucial for the oxidative stress-mediated induction of Stx prophages, thus underscoring their roles in the expression of Shiga toxin-producing E. coli virulence.5 Finally, we characterized functions of particular genes from the exo-xis region, suggesting the potential unusual properties of their products.6

Another line of our research focused on characterizing the biodiversity of bacteriophages. Such studies are crucial for the development of phage therapy. The vast majority of bacteriophages are specific to their hosts, and this specificity is restricted to bacterial species and strains. Thus, effective phage therapy requires the isolation and characterization of many different phages and establishment of a relatively large collection of these viruses. We obtained a grant (NCN no. 2015/17/B/NZ9/01724, entitled “Functional biodiversity of bacteriophages – studies at molecular level and potential biotechnological importance,” 2016-2020, PI: Prof. Alicja Węgrzyn) that is devoted to the isolation and characterization of environmental bacteriophages that infect different bacterial species. Analyzing samples from a single environment (i.e., urban sewage), we isolated and characterized the largest group of bacteriophages that derived from a single habitat. Importantly, these phages were characterized in a complex way, including genome analysis, morphological characterization, the determination of development type and kinetics of propagation in bacterial cells, and sensitivity to various environmental conditions and chemical and physical agents.7 These studies demonstrated the enormous biodiversity of bacteriophages, even when they occur in a single habitat. This work has been highly evaluated and frequently cited in the scientific literature. Further studies led to the isolation and characterization of very interesting bacteriophages, including one that exhibits extremely rapid intracellular development8 and one that infects specifically Shiga toxin-producing E. coli strains.

Another objective of our laboratory was to determine the link between DNA replication and central carbon metabolism in E. coli cells. Results of these studies proved the existence of this relationship. Moreover, such a relationship also exists between DNA replication and a process called the stringent response, which is activated after nutrient limitations.10 These studies are a result of the implementation of the following NCN projects: 2011/02/A/NZ1/00009, 2012/05/N/NZ1/00535, and 2016/23/D/NZ1/02601.

A current project (NCN project no. 2017/27/B/NZ9/00393, entitled “Biological studies on efficacy and safety of phage therapy on the model of chicken infection with Salmonella,” 2018-2021, PI: Prof. Alicja Węgrzyn) is a large-scale study of the isolation and characterization of Salmonella-specific phages in experiments with chickens that are infected with S. enterica. The safety and efficacy of phage therapy is compared with antibiotic treatment. The characterization of bacteriophages was published,9 and experiments with animals have been performed, with detailed data analysis ongoing.

Our further studies will focus on two aspects: (i) further detailed studies of structures and functions of unusual proteins that are encoded by genes from the exo-xis region of lambdoid bacteriophages (a relevant grant application has been submitted to the NCN) and (ii) the optimization of phage therapy to combat bacterial infections in poultry, fish, and other animals.

  • Bibliography
  1. Nowicki et al. J Bacteriol. 2013; 195: 5007-5015. doi: 10.1128/JB.00592-13.
  2. Nowicki et al. Antimicrob Agents Chemother. 2014; 58: 2304-2315. doi: 10.1128/AAC.02515-13.
  3. Bloch et al. PLoS ONE. 2014; 9: doi: 10.1371/journal.pone.0108233.
  4. Nejman-Faleńczyk et al. Sci Rep. 2015; 5: doi: 10.1038/srep10080.
  5. Licznerska et al. Oxid Med Cell Longev. 2016; 2016: 8453135. doi: 10.1155/2016/8453135.
  6. Dydecka et al. Front Microbiol. 2017; 8: 1618. doi: 10.3389/fmicb.2017.01618.
  7. Jurczak-Kurek et al. Sci Rep. 2016; 6: 34338. doi: 10.1038/srep34338.
  8. Topka et al. Front Microbiol. 2019; 9: 3326. doi: 10.3389/fmicb.2018.03326.
  9. Kosznik-Kwaśnicka et al. Int J Mol Sci. 2020; 21: 6152. doi: 10.3390/ijms21176152.
  10. Fernandez-Coll et al. mBio. 2020; 11:e03223-19. doi: 10.1128/mBio.03223-19.

Metodologia

In our studies, we have used various modern microbiological and molecular methods. Bacteriophages are isolated and purified to a high degree using ultracentrifugation in CsCl gradients. Morphological characterization is performed using electron microscopy. Analyses of cell cycle parameters (initiation age, C period duration, and number of origins at birth time) and DNA profiles are investigated using flow cytometry. The regulation of gene expression is studied using modern molecular methods, including real-time polymerase chain reaction, automatic Western blot (WES system), various hybridization methods, and techniques for protein-nucleic acid interactions.

Wybrane publikacje

  • Different expression patterns of genes from the exo-xis region of bacteriophage l and Shiga toxin-converting bacteriophage F24B following infection or prophage induction in Escherichia coli. Bloch S, Nejman-Faleńczyk B, Dydecka A, Łoś JM, Felczykowska A, Węgrzyn A, Węgrzyn G. PLOS ONE. 2014; 9: doi: 10.1371/journal.pone.0108233.
  • A small, microRNA-size, ribonucleic acid regulating gene expression and development of Shiga toxin-converting bacteriophage F24B. Nejman-Faleńczyk B, Bloch S, Licznerska K, Dydecka A, Felczykowska A, Topka G, Węgrzyn A, Węgrzyn G. Scientific Reports. 2015; 5: doi: 10.1038/srep10080.
  • Biodiversity of bacteriophages: morphological and biological properties of a large group of phages isolated from urban sewage. Jurczak-Kurek A, Gąsior T, Nejman-Faleńczyk B, Bloch S, Dydecka A, Topka G, Necel A, Jakubowska-Deredas M, Narajczyk M, Richert M, Mieszkowska A, Wróbel B, Węgrzyn G, Węgrzyn A. Scientific Reports. 2016; 6: doi: 10.1038/srep34338.
  • Characterization of bacteriophage vB-EcoS-95, isolated from urban sewage and revealing extremely rapid lytic development. Topka G, Bloch S, Nejman-Faleńczyk B, Gąsior T, Jurczak-Kurek A, Necel A, Dydecka A, Richert M, Węgrzyn G, Węgrzyn A. Frontiers in Microbiology. 2019; 9: doi: 10.3389/fmicb.2018.03326.
  • Characteristics of a series of three bacteriophages infecting Salmonella enterica strains. Kosznik-Kwaśnicka K, Ciemińska K, Grabski M, Grabowski Ł, Górniak M, Jurczak-Kurek A, Węgrzyn G, Węgrzyn A. International Journal of Molecular Sciences. 2020; 21: 6152. doi: 10.3390/ijms21176152.

Współpraca

  • Michael Cashel, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, USA
  • Logan W. Donaldson, Department of Biology, York University, Toronto, ON, Canada.
  • Marc Le Borgne, Department of Bioactive Molecules and Medicinal Biochemistry, Claude Bernard University, Lyon, France
  • Laurent Janniere, Genoscope – Centre National de Séquençage, François Jacob Institute of Biology, Evry, France
  • Magdalena Narajczyk, Laboratory of Electron Microscopy, Faculty of Biology, University of Gdansk, Gdansk, Poland
  • Sylwia Rodziewicz-Motowidło, Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Gdansk, Poland
  • Lidia Piechowicz, Department of Microbiology, Medical University of Gdansk, Gdansk, Poland.
  • Luciano Saso, Department of Physiology and Pharmacology „Vittorio Erspamer”, Sapienza University, Rome, Italy
  • Tomasz Wołkowicz, Department of Bacteriology and Biocontamination Control, National Institute of Public Health-National Institute of Hygiene, Warsaw, Poland

Nagrody i wyróżnienia

  • Sylwia Bloch. Prize of Minister of Science and Higher Education for outstanding PhD thesis. 2017. Warsaw, Poland.
  • Alicja Węgrzyn. Prize of Polish Genetic Society for the best series of original publications from years 2007-2010. 2010. Warsaw, Poland.
  • Alicja Węgrzyn. Prize of Minister of Science and Higher Education for a cycle of publications on mechanisms of expression of genes coding for Shiga toxins in Escherichia coli 2010. Warsaw, Poland.

Publikacje