Laboratories

Małgorzata Łobocka, PhD, DSc, Prof.

Laboratory of Bacteriophage Biology

Research Scope

The long-term goal of our studies is to elucidate unknown aspects of bacteriophage biology that underlie the wide host range of certain phages, their survival strategies among hosts in various environments, and their evasion of bacterial phage-defense mechanisms. We are especially interested is phage-encoded mechanisms that prevent the bacterial response to stress that is associated with phage infection.

Research

Main Scientific Achievements

  • Contributions to the genomic characteristic of obligatory lytic staphylococcal bacteriophages that represent the Rosenblumvirus and Kayvirus genera and uncovering the main mechanism that protects Rosenblumvirus phages from the action of staphylococcal type I restriction-modification systems.
  • Demonstration of the ability of obligatorily lytic staphylococcal phages to form PCSPs with their bacterial hosts.
  • The development of a new method to evaluate and compare the therapeutic efficacy of bacteriophages in vivo.
  • Contribution to uncovering the physiological function of SELO family ampylase that is conserved in all forms of life.

Research Description

Our interests include bacteriophages of a wide host range, especially those that infect pathogenic bacteria. Some of them can develop only lytically, killing their bacterial host upon the release of progeny phages, and can be potentially used as antibacterial agents to treat bacterial infections. Others can alternatively remain in cells in the form of DNA that is either integrated with a chromosome or maintained as a plasmid. They can encode features that are adaptive for bacteria and are main factors of horizontal gene transfer. All of these phages are composed of a double-stranded DNA-filled head and a tail and represent the most ancient virus group that evolved before the diversification of bacteria and archea. The sizes of their genomes vary between a dozen to over 300 kbs. The biology of these phages and their interactions with bacteria in natural environments that are inhabited by their hosts have started to be uncovered only recently. Functions of the majority of their genes are unknown. Homologies between proteins, if any, are mostly structural. Little is known about the factors that contribute to their wide host range. We characterized at the level of the genomic sequence host specificity and certain other properties over 30 obligatorily lytic phages of a wide host range that infect Staphylococcus, Pseudomonas, Enterococcus, or enterobacteria. Despite genomic similarities between phages that represent the same genera, many of them differ in their strain specificity, lytic efficacy, and other types of interactions with their hosts. Our attempt to identify these differences in detail and understand the mechanisms that are responsible for them is based on comparative studies of the infectivity of similar phages to bacterial strains of genomically diversified panels and on comparative studies of phage-bacteria interactions. An external environment for these interactions in the case of human or animal pathogens is the body of infected organisms. Thus, some comparative studies are performed under in vivo conditions. One of our main goals is to correlate the observed differences between phage physiology and phage-bacteria interactions with genetic differences between phages and between their various hosts. This will allow future phage modifications in response to therapeutic needs and the prediction of bacterial susceptibility to particular phages. Among major objects of our interest are staphylococcal obligatorily lytic phages of a wide strain range within the Staphylococcus aureus species. Those of large (~140 kb) genomes belong to the Kayvirus genus, and those of small (~17 kb) genomes belong to the Rosenblumvirus genus. We demonstrated that the strategy of these phages to protect their bacterial hosts from eradication that is caused by phage-mediated lysis is the formation of so-called phage carrier state populations (PCSPs). In PCSPs, phages coexist with unstable phage-resistant bacteria and at least a small fraction of phage-sensitive bacteria that serve as a constant source of newly released phages. We showed that the ability of staphylococcal phages to form PCSPs does not preclude their therapeutic efficacy. Moreover, major factors that determine the resistance of bacteria to phages of Kayvirus and Rosenblumviruse genera differ, providing a rationale for the use of combinations of these phages in antistaphylococcal therapies. The correlation of Rosenblumvirus infectivity for various S. aureus strains with their certain DNA sequence features allowed us to demonstrate the avoidance of restriction sites for staphylococcal type I restriction-modification systems as a main basis of the wide strain specificity of these phages. The analysis of predicted proteomes of kayviruses and enterobacterial phage P1 of a large genome, followed by functional and mutational analyses of redundant lytic functions of these phages, allowed us to demonstrate their lytic system complexity and the contribution of this complexity in P1 to the wide host range. The initiation of productive phage infection is associated with the redirection of bacterial metabolism and intracellular stress. Phage-encoded mechanisms that prevent the stress-induced apoptosis of infected cells are essential for infection spread and contribute to the phage host range. Putative components of anti-phage defense avoidance systems that were predicted by us in the proteomes of analyzed phages will be studied in detail in the nearest future in parallel with studies on relevant bacterial stress-response mechanisms that are a part of anti-phage defense.

Methodology

Our strategy to elucidate functions of bacteriophage genes is based on reverse genetics, comparative genomics, and the results of comparative proteomic and physiological studies of related phages as well as on the phenotypic analyses of infected bacterial cells or cells that express selected phage genes. The contribution of particular phage genes to phage host specificity determination is studied using diversified bacterial strain collections in collaboration with the National Institute of Medicine. Phage mutants that lack particular genes or exhibit changes in particular gene sequences are acquired by random or site-directed mutagenesis and recombineering and directly selected or isolated using CRISPR-Cas-based counterselection procedures. Monoclonal phage preparations for analysis are acquired using prophage and plasmid-free bacterial strains that have been obtained in our laboratory. Certain phage properties and phage-bacteria interactions are studied under conditions that mimic the natural conditions of host bacteria growth, including artificial sputum, bodies of infected organisms, and surfaces that promote biofilm formation. Our methodology includes basic microbiological and molecular biology techniques, gene cloning and expression, genomic, transcriptomic, and proteomic analyses, random and site-directed mutagenesis, recombineering, the CRISPR-Cas-mediated selection of desired mutants, fluorescent, confocal, and electron microscopy, protein purification, enzymatic assays, and comparative genomic studies. Interactions between pathogenic bacteria and their phages are studied in the biosafety level 2 laboratory, which is equipped with a Bioscreen C Automated Microbiology Growth Curve Analysis System for parallel measurements of 96 bacterial culture growth or lysis kinetics. Studies of the outcome of pathogenic bacteria interactions with their phages in vivo, in the bodies of infected organisms, are performed using a simple model system that was developed in our laboratory. In this model, the nematode Caenorhabditis elegans serves as a natural environment to study these interactions. The vulnerability of C. elegans to several human bacterial pathogens allows the use of this model to evaluate and compare the therapeutic efficacy of various phages and their mutants in curing bacterial infections.

Selected Publications

  • A Kayvirus distant homolog of staphylococcal virulence determinants and VISA biomarker is a phage lytic enzyme. Głowacka-Rutkowska A, Ulatowska M, Empel J, Kowalczyk M, Boreczek J, Łobocka M. Viruses. 2020. doi: 10.3390/v12030292.
  • The ability of lytic staphylococcal podovirus vB_SauP_phiAGO1.3 to coexist in an equilibrium with its host facilitates the selection of host mutants of attenuated virulence but does not preclude the phage antistaphylococcal activity in a nematode infection model. Głowacka-Rutkowska A, Gozdek A, Empel J, Gawor J, Żuchniewicz K, Kozińska A, Dębski J, Gromadka R, Łobocka M. Frontiers Microbiol. doi: 10.3389/fmicb.2018.03227.
  • Protein AMPylation by an Evolutionarily Conserved Pseudokinase. Sreelatha A, Yee SS, Lopez VA, Park BC, Kinch LN, Pilch S, Servage KA, Zhang J, Jiou J, Karasiewicz-Urbańska M, Łobocka M, Grishin NV, Orth K, Kucharczyk R, Pawłowski K, Tomchick DR, Tagliabracci VS. Cell. 2018. doi: 10.1016/j.cell.2018.08.046.
  • Genomics of staphylococcal Twort-like phages – potential therapeutics of the post-antibiotic era. Łobocka M, Hejnowicz MS, Dąbrowski K, Gozdek A, Kosakowski J, Witkowska M, Ulatowska MI, Weber-Dąbrowska B, Kwiatek M, Parasion S, Gawor J, Kosowska H, Głowacka A.Adv. Virus. Res. 2012. doi: 10.1016/B978-0-12-394438-2.00005-0.

Collaborations

  • Evelien Adriaenssens, Quadram Institute, Norwich, United Kingdom, https://quadram.ac.uk/people/evelien-adriaenssens/
  • Joana Azeredo, Centre of Biological Engineering, University of Minho, Braga, Portugal, www.ceb.uminho.pt/People/Details/6008668A-8F77-4D3D-9A98-1C0F7161AD2D
  • Joanna Empel, Department of Epidemiology and Clinical Microbiology, National Medicines Institute, Warsaw, Poland.
  • Marek Gniadkowski, Department of Molecular Microbiology, National Medicines Institute, Warsaw, Poland.
  • Andrzej Górski, Laboratory of Bacteriophages, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland, www.iitd.pan.wroc.pl/pl/SLBF/
  • Roman Gryko, Biological Threat Identification and Countermeasure Centre of the Military Institute of Hygiene and Epidemiology, Puławy, Poland
  • Petar Knezevic, Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Novi Sad, Republik of Serbia, wwwold.dbe.pmf.uns.ac.rs/o_departmanu/imenik/petar_knezevic
  • Andrew Kropinski, Departments of Food Science and Pathobiology, University of Guelph, Guelph, Canada; https://ovc.uoguelph.ca/pathobiology/people/faculty/Andrew-Kropinski
  • Hugo Oliveira, Centre of Biological Engineering, University of Minho, Braga, Portugal, www.ceb.uminho.pt/People/Details/0f372f0a-487f-490d-9e4c-847eec964324
  • Hanna Rekosz-Burlaga, Department of Biochemistry and Microbiology, Faculty of Agriculture and Biology, Warsaw University of Life Sciences, Warsaw, Poland.
  • Vincent Tagliabracci, Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, USA, www.utsouthwestern.edu/labs/tagliabracci/

Publications

Patents

  • A method of evaluating the therapeutic efficacy of bacteriophages. Łobocka M, Głowacka A, Dąbrowski K, Hejnowicz MS, Gozdek A, Weber-Dąbrowska B, Górski A, Empel J, Hryniewicz W, Kwiatek M, Parasion S, Gryko R. WO/2014/012872 A1, 2014. Polish Patent PL 219654 B1, European Patent EP 2872156 B1, USA Patent US 9,678,063 B2.