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Strukturní Charakterizace Fágové Infekce V Bakteriálním Biofilmu

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Strukturní Charakterizace Fágové Infekce V Bakteriálním Biofilmu PŘÍRODOVĚDECKÁ FAKULTA Strukturní charakterizace fágové infekce v bakteriálním biofilmu Diplomová práce YULIIA MIRONOVA Vedoucí práce: Ing. Zuzana Cieniková PhD Ústav experimentální biologie Obor Molekulární biologie a genetika Brno 2021 STRUKTURNÍ CHARAKTERIZACE FÁGOVÉ INFEKCE V BAKTERIÁLNÍM BIOFILMU STRUKTURNÍ CHARAKTERIZACE FÁGOVÉ INFEKCE V BAKTERIÁLNÍM BIOFILMU Bibliografický záznam Autor: Yuliia Mironova Přírodovědecká fakulta Masarykova univerzita Ústav experimentální biologie Název práce: Strukturní charakterizace fágové infekce v bakteriálním biofilmu Studijní program: Molekulární biologie a genetika Studijní obor: Molekulární biologie a genetika Vedoucí práce: Ing. Zuzana Cieniková PhD Rok: 2021 Počet stran: 75 Klíčová slova: kryo-EM, kryo-ET, bakteriofágy, fágová terapie, fág T7, Kmvviry, Pseudomonas aeruginosa, biofilm STRUKTURNÍ CHARAKTERIZACE FÁGOVÉ INFEKCE V BAKTERIÁLNÍM BIOFILMU Bibliographic record Author: Yuliia Mironova Faculty of Science Masaryk University Department of Experimental Biology Title of Thesis: Structural characterisation of phage infection in bacterial biofilm Degree Programme: Molecular biology and genetics Field of Study: Molecular biology and genetics Supervisor: Ing. Zuzana Cieniková PhD Year: 2021 Number of Pages: 75 Keywords: cryo-EM, cryo-ET, bacteriophages, phage therapy, phage T7, Phikmvviruses, Pseudomonas aeruginosa, biofilm STRUKTURNÍ CHARAKTERIZACE FÁGOVÉ INFEKCE V BAKTERIÁLNÍM BIOFILMU Anotace Bakteriofágy rodu Phikmvvirus jsou slibnými kandidáty pro fágovou terapií cílenou na Pseudomonas aeruginosa. Tato diplomová práce se zabývá strukturní charakteri- zací fága z rodu Phikmvvirus a procesu jeho infekce v buňkách P. aeruginosa. Jednot- livé stadia infekce byli analyzovány a struktura zralých virionů naplněných geno- mem byla vyřešena s pomocí kombinace kryo-elektronové mikroskopie a tomogra- fie. Nakonec byl předložen protokol fluorescenčního značení fága pro sledování in- fekce v bakteriálním biofilmu za použití “light-sheet” fluorescenční mikroskopie. STRUKTURNÍ CHARAKTERIZACE FÁGOVÉ INFEKCE V BAKTERIÁLNÍM BIOFILMU Abstract Phikmvviruses are promising candidates for phage therapy targeting Pseudomonas aeruginosa. This diploma thesis focuses on the structural characterization of a phage belonging to the Phikmvvirus genus and its infection in P. aeruginosa cells. Stages of the phage infection were investigated, and the structure of the mature virion filled with genome was determined using cryo-electron microscopy. Lastly, a protocol for phage fluorescent labelling, designed to study the phage infection in biofilm using light-sheet fluorescent microscopy, was developed. STRUKTURNÍ CHARAKTERIZACE FÁGOVÉ INFEKCE V BAKTERIÁLNÍM BIOFILMU STRUKTURNÍ CHARAKTERIZACE FÁGOVÉ INFEKCE V BAKTERIÁLNÍM BIOFILMU Declaration Prohlašuji, že jsem svoji práci vypracovala samostatně pod vedením vedoucího práce s využitím informačních zdrojů, které jsou v práci citovány. Brno June 15, 2021 ....................................... Yuliia Mironova STRUKTURNÍ CHARAKTERIZACE FÁGOVÉ INFEKCE V BAKTERIÁLNÍM BIOFILMU Acknowledgements I want to thank Ing. Zuzana Cieniková, PhD. and doc. Pavel Plevka, PhD for the possi- bility to join the research group, for the help and supervision during the experiments and writing of this thesis. I would also like to thank Mgr. Markéta Londýnová and Mgr. Marta Šiborová for the help with the phage propagation method, Ing. Ti- bor Füzik, Ph.D for the much appreciated help with reconstructions and dealing with computational problems, Mgr. Dominik Hrebík for providing the initial model of the phage, Mgr. Pavol Bardý Ph.D for the help with bioinformatics and the rest of Struc- tural virology group for all the help, support and friendliness they provided during the last two years. Core Facility Cryo-electron Microscopy and Tomography of CEITEC Masaryk University and CEITEC Proteomics Core Facility are gratefully acknowledged for the obtaining of the scientific data presented in this diploma thesis STRUKTURNÍ CHARAKTERIZACE FÁGOVÉ INFEKCE V BAKTERIÁLNÍM BIOFILMU Table of Contents Abbreviations 12 1 Theoretical introduction 13 1.1 Pseudomonas aeruginosa .......................................................................................... 13 1.1.1 Biofilm formation ............................................................................................. 13 1.1.2 Antibiotic resistance of P. aeruginosa .............................................................. 14 1.2 Phage therapy ............................................................................................................ 15 1.2.1 Candidate phages for the phage therapy ........................................................... 15 1.2.2 Pseudomonas phages from Phikmvvirus genus ................................................ 16 1.3 Phage T7 structure and life cycle .............................................................................. 17 1.4 Characterization of the phage structure and host-pathogen interactions ................... 19 1.4.1 Cryo-EM ........................................................................................................... 19 1.4.2 Fluorescent microscopy .................................................................................... 22 1.4.3 Fluorescent labelling of the phage and bacterium ............................................ 23 2 Aims and objectives 25 Appendix A Phage T7 protein structure 36 ABBREVIATIONS Abbreviations cryoEM – cryo-electron microscopy cryo-ET – cryo-electron tomography CTF – contrast transfer function EPS – extracellular matrix gpXX – gene product XX LC-MS/MS – Liquid Chromatography - Tandem Mass Spectrometry LSFM – light-sheet fluorescent microscopy MCP – major capsid protein MOI – multiplicity of infection PI – post infection TEM – transmission electron microscopy SNR – signal to noise ration SPA – single particle analysis STA – subtomogram averaging THEORETICAL INTRODUCTION 1 Theoretical introduction 1.1 Pseudomonas aeruginosa Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen. It is com- monly associated with nosocomial diseases such as acute and chronic pneumonia, as well as surgical site, bloodstream, and urinary infections (Barbier et al., 2013; Weinstein et al., 2005). P. aeruginosa infections affect primarily immunocompromised individuals and are a leading cause of mortality in patients With cystic fibrosis (Lyczak et al., 2002; Sadikot et al., 2005). The spread of P. aeruginosa in hospitals is difficult to control due to its multi-drug resistance and the ability to form biofilms (Kerr and Snelling, 2009; Nemec et al., 2010; Soares et al., 2020). 1.1.1 Biofilm formation A biofilm is an aggregate of bacterial cells Which adhered to a surface and each other and are embedded in extracellular polymeric substances (EPS) (Vert et al., 2012). Its formation starts With the reversible adhesion of a planktonic cell to a surface (Fig. 1.I.). This attachment and later the sWitch to biofilm cell state is induced and regulated by quorum sensing (Kirisits and Parsek, 2006; Yan and Wu, 2019). In the right environmental conditions and due to meta- bolic changes, the attachment becomes irreversible, and the biofilm begins to grow and produce EPS (Fig 1.II.). In mature biofilm (Fig 1.III), EPS consists of eDNA, proteins and exopolysac- charides. The biofilm matrix provides structural integrity to the biofilm and mediates interac- tions betWeen the clustered cells. As the last stage of the development (Fig 1.IV) the biofilm disperses through release of planktonic cells (Flemming and Wingender, 2010; Harmsen et al., 2010; Maurice et al., 2018; Mulcahy et al., 2014; Rasamiravaka et al., 2015). The gene expression profile of P. aeruginosa biofilm cells differs from that of planktonic cells. Some metabolic changes in biofilm cells are associated With EPS components produc- tion, most notably alginate, Psl and Pel polysaccharides (Colvin et al., 2012; Flemming and Wingender, 2010; Vital-Lopez et al., 2015). Apart from that, the biofilm contains concentra- tion gradients of metabolites and oxygen, resulting in a metabolically heterogenous cell 13 THEORETICAL INTRODUCTION population (Wessel et al., 2014). Metabolically inactive persister cells that are resistant to an- tibiotics are located deep inside the biofilm While surface cells are metabolically active (Wood et al., 2013). Depending on available energy sources, the biofilm can form different morphol- ogies. “Mushroom”-like biofilm forms in the presence of glucose. Non-motile P. aeruginosa cells form the “mushroom” stack While motile cells are localized on the surface of the cap. In contrast, flat non-motile biofilm forms in the presence of citrate. Type IV pili and flagellum play a role in P. aeruginosa attachment and increase the structural integrity of the biofilm (Harmsen et al., 2010). Figure 1. Stages of biofilm development. I. – reversible attachment; II. – irreversible attachment; III. – biofilm maturation; IV. – biofilm dis- persal. Biofilm consists of heterogenous cell population: light green colour indicates planktonic cells, lime-green colour indicates cap-forming cells, red and pink colour indicates stack-forming cells, blue indicates persister cells. 1.1.2 Antibiotic resistance of P. aeruginosa Not long after the discovery
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