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, 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, , 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 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 – 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 of antibiotics, antibiotic resistance began to emerge in differ- ent bacterial species (Willmann et al., 2013; Zainab et al., 2020). P. aeruginosa has multiple intrinsic and acquired mechanisms of antibiotic resistance, such as efflux pumps, ß-lactamase production, and lower membrane permeability (Hancock, 1997; Hancock and Speert, 2000; Nakae et al., 1999; Nikaido, 1994). A major accelerator of the resistance spread is antibiotic misuse in health-care sphere and agriculture (Manyi-Loh et al., 2018; Pachori et al., 2019). The ěé+í Annual report by European Antimicrobial Resistance Surveillance Network showed 14 THEORETICAL INTRODUCTION that more than 30% of clinical isolates of P. aeruginosa were resistant to multiple groups of antibiotics (European Centre for Disease Prevention and Control, 2019). According to the World Health Organisation, antibiotic resistance crisis poses a major threat to human health and the worldwide economy (WHO, 2017). Therefore, the development of new treatment methods against antibiotic resistant bacteria became the focus of intense research.

1.2 Phage therapy

Bacteriophages are infecting prokaryotic organisms, specifically bacteria. Phage therapy is an alternative to antibiotics treatment. It utilizes phages as natural antimicrobials against multi-drug resistant and not resistant strains of pathogenic bacteria (Romero-Calle et al., 2017; Sulakvelidze et al., 2001; Wittebole et al., 2014). Mixtures of bacteriophages were used experimentally to treat and prevent infections caused by P. aeruginosa in zebrafish and mouse models and in vitro lung-like environment (Cafora et al., 2019; Furusawa et al., 2016; Waters et al., 2017). Experimental phage therapy was also used to treat severe pseudomonal infections in human patients (Maddocks et al., 2019).

1.2.1 Candidate phages for the phage therapy

P. aeruginosa is infected by a structurally and genetically diverse group of phages be- longing to the orders , Levivirales, Tubulavirales and Mindivirales (Secor et al., 2020; Sepúlveda-Robles et al., 2012; Tars et al., 2000; Yang et al., 2016). Caudovirales phages have a dsDNA genome packaged into an icosahedral or oblate protein capsid with a tail. Mor- phologically, tailed phages are divided into three groups: long tailed non-contractile phages (Fig. 2A), long tailed contractile phages (Fig. 2B), and short tailed non-contractile phages (Fig. 2C). Levivirales phages are small icosahedral viruses with a positive-strand RNA genome (Fig. 2D). Mindivirales phages are icosahedral or spherical viruses enveloped by a membrane (Fig. 2E). Their genome is segmented into three linear double-stranded RNA molecules. Lastly, Tubulavirales are filamentous phages with a single-stranded DNA genome (Fig. 2F).

15 THEORETICAL INTRODUCTION

Figure 2. Morphological types of phages A. Caudovirales phage with long non-contractile tail; B. Caudovirales phage with long con- tractile tail; C. Caudovirales phage with short non-contractile tail; D. Levivirales phage; E. Mindivirales enveloped phage; F. Tubulavirales filamentous phage.

The suitability of phages for phage therapy depends on several criteria. Phage life cycle is either lysogenic or lytic. Lysogenic phages integrate their DNA into the host chromosome, not killing the bacterium (Howard-Varona et al., 2017). These phages can transduce bacterial DNA and promote the spread of resistance in bacterial populations (Keen et al., 2017). Lytic phages replicate inside the host cell and lyse it at the end of the cycle. After lysis, non-degraded bacterial DNA can also promote horizontal gene transfer among bacterial communities. Thus, only lytic phages that degrade host DNA are appropriate for the purposes of phage therapy. Other necessary requirements are a wide host range, easy propagation, and stability of the phage particles (Fernández et al, 2019).

1.2.2 Pseudomonas phages from Phikmvvirus genus

Phikmvvirus phages are Caudovirales phages infecting P. aeruginosa. They emerged as the most promising phage therapy candidates. The genus contains lytic phages with a wide range of host-strains among clinical P. aeruginosa isolates. Phikmvviruses are closely related to the coliphage T7 and have the typical morphology: a small icosahedral head with a diameter of 60 nm and a short tail (Fig. 3.A). Their are linear terminally re- dundant dsDNA molecules approximately 41 kb long, with characteristic localised single-

16 THEORETICAL INTRODUCTION

B

Figure 3. Phikmvvirus phage and phage T7 structure A. Electron micrograph of negatively stained phage LUZ19 (Ceyssens, 2009; edited); B. Schematic representation of phage T7 structure. The light blue colour on the capsid indicates pentamers and the green colour indicates hexamers. The tail is shown in dark green and the tail fibres in yellow. The dark orange colour indicates the portal and the inner core is shown in blue and purple. strand breaks. Phage are organised in three spatio-temporal groups: early, middle, and late genes. The early genes are transcribed by the host immediately after the infection and are involved in host take-over and host DNA degradation. The middle genes are responsible for phage genome replication and host RNA polymerase inhibition. Phikmvviruses encode single- subunit RNA-polymerase which facilitates transcription of the late genes which code for struc- tural proteins (Adriaenssens et al., 2020; Ceyssens, 2009; Kulakov et al., 2009; Lammens et al., 2009; Lavigne et al., 2013).

1.3 Phage T7 structure and life cycle

Phage T7 is the closest relative to Phikmvviruses with a known structure (Fig. 3.B) and a described infection process (Fig. 4). The capsid of T7 exhibits T=7 quasi-symmetry, meaning that each asymmetric unit of the capsid shell is made of seven subunits of the major capsid protein (Steven et al., 1983). The capsid with the triangulation number T=7 contains 12 pen- tamers located at five-fold axes and 60 hexamers making up the faces of the icosahedron. The mature capsid of phage T7 consists of 415 copies of the major capsid protein (MCP) (gp10A and gp10B in 9:1 ratio) possessing the HK97 fold (Appendix A). One five-fold axis vertex is modified and carries the portal complex. The portal complex (gp8) has a ring-like structure and a 12-fold symmetry, resulting in a symmetry mismatch with the five-fold symmetry of the capsid shell. The portal monomer consists of four sub-domains: crown, wing, stem and clip (Appendix A). The phage T7 inner core is located inside the capsid on top of the portal and has 17 THEORETICAL INTRODUCTION a four-fold symmetry. It consists of three types of proteins (gp14, gp15 and gp16) and facili- tates genome release during the infection (Cuervo et al., 2019; Duda and Teschke, 2019; Guo et al., 2014; Pérez-Ruiz et al., 2021). The tail complex has a six-fold symmetry and comprises the adaptor (gp11), the nozzle (gp12) and trimeric protein fibres (gp17). The adaptor interacts with the portal through the embracing helix and with the nozzle through the helix bundle. The interface between the adaptor and the nozzle serves for the attachment of tail fibres through their fibre dock domains. The nozzle is formed by six copies of a 700 residues long protein. It creates a platform domain interacting with the adaptor, a fibre dock that also facilitates fibre attachment to the tail, and a ß-propeller and a nozzle-tip that make up the main tail body (Ap- pendix A). The core, the portal, and the tail together form a tunnel through which DNA escapes from the capsid during the genome release process (Chen et al., 2020; Cuervo et al., 2019, 2013). The life cycle of the phage T7 starts with adsorption to a primary receptor. Next, phage attaches to the cell surface and injects its genome using the tail machinery (González-García

Figure 4. Life cycle of phage T7 I. Attachment to a primary receptor; II. Attachment to the cell surface and genome ejection; III. Early and middle gene transcription by host RNA-polymerase, host take-over; IV. Late gene transcription by viral RNA-polymerase, prohead assembly; V. capsid expansion, genome pack- ing, mature virion formation; VI. Host cell lysis and progeny release.

18 THEORETICAL INTRODUCTION et al., 2015). After the host take-over and phage gene transcription, phage particles are assem- bled. First, capsid proteins assemble around the portal with the help of the scaffold protein, resulting in a prohead. Then, the proheads become expanded empty particles (Agirrezabala et al., 2007, 2005; Guo et al., 2014, 2013). Two terminase subunits fill the expanded particles with the genome, resulting in mature phage virions (Serwer, 2011, 2010; White and Richardson, 1987). The final step of the phage infection is the cell lysis and the release of the progeny (Abdelrahman et al., 2021; Cahill and Young, 2019). Although phage T7 infects Escherichia coli, it can provide a reference point in studying the structure and the infection cycle of Pseudomonas phages from the Phikmvvirus genus. No- table differences in genome organisation are observed particularly in the early and middle gene segments. Structural genes of Phikmvvirus phages are arranged in the same order as in phage T7, except for some additional structural genes at the 3’ end of Phikmvvirus genomes (Ceyssens, 2009). Another crucial difference is that unlike phage T7, some of the Phikmvvirus phages depend on Type IV pili during the infection process (Chibeu et al., 2009).

1.4 Characterization of the phage structure and host-pathogen interactions

1.4.1 Cryo-EM

Cryogenic electron microscopy or simply cryo-EM is a structural biology technique that enables the determination of 3D structures of macromolecular assemblies from their 2D pro- jections. Advances in cryogenics, freezing methods, data collection and image analysis have led to a revolution in structural biology and made cryo-EM the method of choice to study large heterogeneous macromolecules such as tailed phages (Kühlbrandt, 2014; Nogales and Scheres, 2015). Samples for high resolution cryo-EM should be preferably homogenous, stable and of a high concentration. Phage samples are usually purified by ultracentrifugation methods (sac- charose or CsCl gradients), followed by concentration steps (Bachrach Uriel and Friedmann Adam, 1971; Bourdin et al., 2014; Luong et al., 2020). The sample is then applied onto a cryo- EM grid and vitrified in liquid ethane (Fig. 5.A). Grids for cryo-EM are thin copper or golden

19 THEORETICAL INTRODUCTION

B A

Figure 5. CryoEM method A. Process of sample vitrification in liquid ethane. Bottom left image shows vitrified sample in a grid hole (Murata and Wolf, 2018); B. Scheme of the transmission electron microscope (from https://myscope.training/#/TEM) discs covered by a carbon coating that vary in mesh and hole sizes. Rapid freezing prevents crystalline ice formation, which could deform or damage the sample. As a result, biomolecules are embedded in a thin vitreous ice layer in a grid hole. Cryo-EM method uses transmission electron microscope (TEM) (Fig. 5.B) to image sam- ples at low temperatures (-172 to -196 C°). Electrons are released from the electron gun at the top of the microscope and then travel through the column, where their path is shaped by a series of electromagnetic lenses. TEM image formation occurs in two stages. First, electron beam is scattered by the sample and focused by the objective lens to form the primary image. Next, the primary image is magnified with additional lenses to form the final image. The final image is recorded by a direct electron detector (Murata and Wolf, 2018). The image contrast in cryo-EM arises from electron wave amplitude and phase changes as a result of scattering. Atoms making up the organic matter generate a low amplitude contrast on their own. Negative staining by heavy metal derivates is one of the techniques to increase

20 THEORETICAL INTRODUCTION

Figure 6. Differences in data acquisition procedures for SPA and STA A. In the SPA method the stage remains stationary; it is assumed that the particles adopt different orientations in the sample; B. In tomography, particle orientations are collected through the stage rotation (Murata and Wolf, 2018) the contrast, however it degrades the resolution of the final structure (De Carlo and Harris, 2011; Scarff et al., 2018). Therefore, images in cryo-EM are collected with high defocus for increased phase contrast. The resulting image is strongly modulated with contrast transfer func- tion (CTF). CTF is estimated as a part of image pre-processing and later used for deconvolution (Wade, 1992; Zhang, 2016). Fourier slice theorem is the main principle behind structure determination by cryo-EM. It describes the relationship between a 3D object and 2D projections of that object. According to this theorem Fourier transform of a 2D projection represents a slice through the Fourier trans- form of the 3D object (Singer, 2018). There are two main approaches to structure determination by cryo-EM: single-particle analysis (SPA) and sub-tomogram averaging (STA). Thus, by obtaining a large number of 2D projections and determining their relative orientations, a full 3D volume of an object can be reconstructed. (Jonić et al., 2008; Murata and Wolf, 2018). SPA operates on the assumption that particles are differently oriented in the vitreous ice (Fig. 7.A). Thousands or even hundreds of thousands of particles are required to obtain a high-resolution structure of a macromolecule, requiring automated data collection (Cheng et al., 2016; Mastronarde, 2005; Schorb et al.,

21 THEORETICAL INTRODUCTION

2019; Tan et al., 2016)). In STA (Fig. 7.B), instead of obtaining one image of a particle, a series of images (tomographic tilt series) of the same particle is collected. The microscope stage is incrementally tilted, usually in a range of ±60° or ±70°. The aligned tilt series are then used to calculate a tomographic reconstruction (Briggs, 2013; Doerr, 2016; Mastronarde and Held, 2017). Particles of interest are identified in electron micrographs and picked either manually or with the use of correlation-based or neural network-based software (Bell et al., 2016; Wagner et al., 2019; Woolford et al., 2007). Then, several rounds of 2D- or 3D classifications are per- formed to address sample heterogeneity and to determine particle orientations and symmetries. Selected particle classes are then subjected to numerous rounds of 3D refinement of particle translational and angular alignments resulting in an averaged electron density volume (Scheres, 2013, 2012). Finally, an atomic model of the protein is built into the electron density volume, either de novo or based on homology modelling (Pfab et al., 2021; Wang et al., 2015; Zhu et al., 2010).

1.4.2 Fluorescent microscopy

As was described above, cryo-EM is the method of choice to study phages and host-path- ogen interactions on a single cell level. Nevertheless, when it comes to the biofilm level, this technique provides a limited structural information because of the thickness of such a sample (Lučič et al., 2013). Fluorescent microscopy is a suitable technique to study phage infection on a biofilm level (Aulner et al., 2018; Baldvinsson et al., 2014; Bogachev et al., 2018; Rudilla et al., 2018; Szymczak et al., 2019). Fluorescent microscopy techniques allow to study phage infection dynamics in space and time. However, there are some limitations to time-lapsed con- focal microscopy connected to phototoxicity and fluorophore bleaching due to extensive illu- mination (Pawley, 2006). Recently, light sheet fluorescent microscopy (LSFM) emerged as a new technique that evades these limitations. LSFM uses a thin sheet of light to illuminate one slice of the sample at a time. It allows to study samples in volume and in time and may become a useful technique to study phage infection in biofilm (Power and Huisken, 2017).

22 THEORETICAL INTRODUCTION

1.4.3 Fluorescent labelling of the phage and bacterium

In order to observe biofilm samples using LSFM, both the bacterium and the phage need to be labelled by a fluorescent tag. Viability fluorescent staining using SYTO9 and propidium iodide is a conventional method for bacterial cell labelling that allows to differentiate between live and dead cells. However, this method has several drawbacks, most crucially a low speci- ficity and introduction of a high fluorescent background (Stiefel et al., 2015). Green fluorescent protein (GFP) and its derivatives are widely used for tagging of both live cells and individual proteins. In comparison to staining, this method is more precise and doesn’t introduce a high fluorescent background. The gene coding for the fluorescent protein can be introduced into bacteria either via a plasmid or directly to the chromosome using mini- transposons or CRIPR/Cas9 editing system (Barnes et al., 2008; Chen et al., 2018; Iino et al., 2001). Phage tagging with protein labels is complicated by the limited capacity of the capsid, a low percentage of non-coding sequences in the genome and the essentiality of majority of the phage genes (Hatfull and Hendrix, 2011; Jensen et al., 2020; Kiljunen et al., 2005). In phage T7, several non-essential sequences were identified among early and late genes, with the pro- duced deletion mutants exhibiting a four-fold decrease in progeny (Kemp et al., 2005; Kim and Chung, 1996; Tran et al., 2008).

A B

Figure 7. Methods of fluorescent labelling A. P. aeruginosa biofilm imaged using confocal microscopy. Live cells are stained green by SYTO9 and dead cells are stained red by propidium iodide (Rudilla et al., 2018; edited); B. Schematic rep- resentation of the splitGFP labelling system (FP = fluorescent protein) (Kamiyama et al., 2016). FP indicates fluorescent protein

23 THEORETICAL INTRODUCTION

One approach that can solve the problem of the limited capsid capacity is the use of splitGFP system. The GFP molecule has a ß-barrel structure with self-complementation prop- erties (Cabantous et al., 2005). SplitGFP is made of two fragments (GFP1-10 and GFP11) that can be used to localize proteins or protein interaction inside the cells (Kamiyama et al., 2016). This technique was used on viral proteins and has a potential in phage labelling (Hyun et al., 2015).

24 AIMS AND OBJECTIVES

2 Aims and objectives

Phage therapy has a potential in treating P. aeruginosa infections. However, P. aeruginosa phages are not well studied. This thesis focuses on the phage ……. from the genus Phikmv- . The aim is to characterise the phage …….. structure and its life cycle in P. aeruginosa cells and in biofilm using cryo-EM and fluorescent microscopy. To achieve these aims, the objectives were set as follows:

1. Determine the structure of the phage …….. mature virions using cryo-EM

2. Identify and build atomic models of the structural proteins of the mature virion

3. Study different stages of phage infection in P. aeruginosa cells using cryo-EM

4. Propose a protocol for fluorescent labelling of the phage …….

25 BIBLIOGRAPHY

Bibliography

Abdelrahman, F., Easwaran, M., Daramola, O.I., Ragab, S., Lynch, S., Oduselu, T.J., Khan, F.M., Ayobami, A., Adnan, F., Torrents, E., Sanmukh, S., El-Shibiny, A., 2021. Phage-Encoded Endolysins. Antibiotics 10, 124. https://doi.org/10.3390/antibiotics10020124 Adriaenssens, E.M., Sullivan, M.B., Knezevic, P., van Zyl, L.J., Sarkar, B.L., Dutilh, B.E., Alfenas-Zerbini, P., \Lobocka, M., Tong, Y., Brister, J.R., Moreno Switt, A.I., Klumpp, J., Aziz, R.K., Barylski, J., Uchiyama, J., Edwards, R.A., Kropin- ski, A.M., Petty, N.K., Clokie, M.R.J., Kushkina, A.I., Morozova, V.V., Duffy, S., Gillis, A., Rumnieks, J., Kurtböke, İ., Chanishvili, N., Goodridge, L., Witt- mann, J., Lavigne, R., Jang, H.B., Prangishvili, D., Enault, F., Turner, D., Po- ranen, M.M., Oksanen, H.M., Krupovic, M., 2020. Taxonomy of prokaryotic vi- ruses: 2018-2019 update from the ICTV Bacterial and Archaeal Viruses Subcom- mittee. Arch. Virol. 165, 1253–1260. https://doi.org/10.1007/s00705-020-04577- 8 Agirrezabala, X., Martín-Benito, J., Castón, J.R., Miranda, R., Valpuesta, J.M., Carras- cosa, J.L., 2005. Maturation of phage T7 involves structural modification of both shell and inner core components. EMBO J. 24, 3820–3829. https://doi.org/10.1038/sj.emboj.7600840 Agirrezabala, X., Velázquez-Muriel, J.A., Gómez-Puertas, P., Scheres, S.H.W., Carazo, J.M., Carrascosa, J.L., 2007. Quasi-Atomic Model of T7 Procap- sid Shell: Insights into the Structure and Evolution of a Basic Fold. Structure 15, 461–472. https://doi.org/10.1016/j.str.2007.03.004 Aulner, N., Danckaert, A., Fernandes, J., Nicola, M.A., Roux, P., Salles, A., Tinevez, J.Y., Shorte, S.L., 2018. Fluorescence imaging host pathogen interactions: fifteen years benefit of hindsight\ldots. Curr. Opin. Microbiol. 43, 193–198. https://doi.org/10.1016/j.mib.2018.03.001 Bachrach Uriel, Friedmann Adam, 1971. Practical Procedures for the Purification of Bacterial Viruses. Appl. Microbiol. 22, 706–715. https://doi.org/10.1128/am.22.4.706-715.1971 Bagdasarian, M.M., Amann, E., Lurz, R., Rückert, B., Bagdasarian, M., 1983. Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors. Gene 26, 273– 282. https://doi.org/10.1016/0378-1119(83)90197-x Baldvinsson, S.B., Sørensen, M.C.H., Vegge, C.S., Clokie, M.R.J., Brøndsted, L., 2014. Campylobacter jejuni Motility Is Required for Infection of the Flagellotropic Bacteriophage F341. Appl. Environ. Microbiol. 80, 7096–7106. https://doi.org/10.1128/AEM.02057-14 Barbier, F., Andremont, A., Wolff, M., Bouadma, L., 2013. Hospital-acquired pneumo- nia and ventilator-associated pneumonia: Recent advances in epidemiology and management. Curr. Opin. Pulm. Med. 19, 216–228. https://doi.org/10.1097/MCP.0b013e32835f27be

26 BIBLIOGRAPHY

Barnes, R.J., Leung, K.T., Schraft, H., Ulanova, M., 2008. Chromosomal gfp labelling of Pseudomonas aeruginosa using a mini-Tn7 transposon: application for studies of bacteria–host interactions. Can. J. Microbiol. 54, 48–57. https://doi.org/10.1139/W07-118 Bell, J.M., Chen, M., Baldwin, P.R., Ludtke, S.J., 2016. High resolution single particle refinement in EMAN2.1. Methods San Diego Calif 100, 25–34. https://doi.org/10.1016/j.ymeth.2016.02.018 Bogachev, M.I., Volkov, V.Y., Markelov, O.A., Trizna, E.Y., Baydamshina, D.R., Melnikov, V., Murtazina, R.R., Zelenikhin, P.V., Sharafutdinov, I.S., Kayumov, A.R., 2018. Fast and simple tool for the quantification of biofilm-embedded cells sub-populations from fluorescent microscopic images. PLOS ONE 13, e0193267. https://doi.org/10.1371/journal.pone.0193267 Bourdin, G., Schmitt, B., Marvin Guy, L., Germond, J.-E., Zuber, S., Michot, L., Reu- teler, G., Brüssow, H., 2014. Amplification and purification of T4-like esche- richia coli phages for phage therapy: from laboratory to pilot scale. Appl. Envi- ron. Microbiol. 80, 1469–1476. https://doi.org/10.1128/AEM.03357-13 Briggs, J.A., 2013. Structural biology in situ—the potential of subtomogram averaging. Curr. Opin. Struct. Biol. 23, 261–267. https://doi.org/10.1016/j.sbi.2013.02.003 Cabantous, S., Terwilliger, T.C., Waldo, G.S., 2005. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat. Biotech- nol. 23, 102–107. https://doi.org/10.1038/nbt1044 Cafora, M., Deflorian, G., Forti, F., Ferrari, L., Binelli, G., Briani, F., Ghisotti, D., Pis- tocchi, A., 2019. Phage therapy against Pseudomonas aeruginosa infections in a cystic fibrosis zebrafish model. Sci. Rep. 9, 1–10. https://doi.org/10.1038/s41598-018-37636-x Cahill, J., Young, R., 2019. Phage Lysis: Multiple Genes for Multiple Barriers. Adv. Vi- rus Res. 103, 33–70. https://doi.org/10.1016/bs.aivir.2018.09.003 Ceyssens, P.-J., 2009. Isolation and Characterization of lytic bacteriophages infecting Pseudomonas aerugionsa. Chen, W., Xiao, H., Wang, X., Song, S., Han, Z., Li, X., Yang, F., Wang, L., Song, J., Liu, H., Cheng, L., 2020. Structural changes of a bacteriophage upon DNA pack- aging and maturation. Protein Cell. https://doi.org/10.1007/s13238-020-00715-9 Chen, W., Zhang, Ya, Zhang, Yifei, Pi, Y., Gu, T., Song, L., Wang, Y., Ji, Q., 2018. CRISPR/Cas9-based Genome Editing in Pseudomonas aeruginosa and Cytidine Deaminase-Mediated Base Editing in Pseudomonas Species. iScience 6, 222– 231. https://doi.org/10.1016/j.isci.2018.07.024 Cheng, A., Tan, Y.Z., Dandey, V.P., Potter, C.S., Carragher, B., 2016. Strategies for Au- tomated CryoEM Data Collection Using Direct Detectors, 1st ed, Methods in En- zymology. Elsevier Inc. https://doi.org/10.1016/bs.mie.2016.04.008 Chibeu, A., Ceyssens, P.J., Hertveldt, K., Volckaert, G., Cornelis, P., Matthijs, S., Lavigne, R., 2009. The adsorption of Pseudomonas aeruginosa bacteriophage ϕkMV is dependent on expression regulation of type IV pili genes. FEMS Micro- biol. Lett. 296, 210–218. https://doi.org/10.1111/j.1574-6968.2009.01640.x Colvin, K.M., Irie, Y., Tart, C.S., Urbano, R., Whitney, J.C., Ryder, C., Howell, P.L., Wozniak, D.J., Parsek, M.R., 2012. The Pel and Psl polysaccharides provide

27 BIBLIOGRAPHY

Pseudomonas aeruginosa structural redundancy within the biofilm matrix: Poly- saccharides of the P. aeruginosa biofilm matrix. Environ. Microbiol. 14, 1913– 1928. https://doi.org/10.1111/j.1462-2920.2011.02657.x Coot: model-building tools for molecular graphics, n.d. Cuervo, A., Fàbrega-Ferrer, M., Machón, C., Conesa, J.J., Fernández, F.J., Pérez-Luque, R., Pérez-Ruiz, M., Pous, J., Vega, M.C., Carrascosa, J.L., Coll, M., 2019. Struc- tures of T7 bacteriophage portal and tail suggest a viral DNA retention and ejec- tion mechanism. Nat. Commun. 10, 3746. https://doi.org/10.1038/s41467-019- 11705-9 Cuervo, A., Pulido-Cid, M., Chagoyen, M., Arranz, R., González-García, V.A., Garcia- Doval, C., Castón, J.R., Valpuesta, J.M., Van Raaij, M.J., Martín-Benito, J., Car- rascosa, J.L., 2013. Structural characterization of the bacteriophage T7 tail ma- chinery. J. Biol. Chem. 288, 26290–26299. https://doi.org/10.1074/jbc.M113.491209 De Carlo, S., Harris, J.R., 2011. Negative staining and cryo-negative staining of macro- molecules and viruses for TEM. Micron Oxf. Engl. 1993 42, 117–131. https://doi.org/10.1016/j.micron.2010.06.003 Doerr, A., 2016. Cryo-electron tomography. Nat. Methods 14, 34. https://doi.org/10.1038/nmeth.4115 Duda, R.L., Teschke, C.M., 2019. The amazing HK97 fold: versatile results of modest differences. Curr. Opin. Virol. 36, 9–16. https://doi.org/10.1016/j.coviro.2019.02.001 Edgar, R., Rokney, A., Feeney, M., Semsey, S., Kessel, M., Goldberg, M.B., Adhya, S., Oppenheim, A.B., 2008. Bacteriophage infection is targeted to cellular poles. Mol. Microbiol. 68, 1107–1116. https://doi.org/10.1111/j.1365- 2958.2008.06205.x Emsley, P., Cowtan, K., 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr Biol Crystallogr 60, 2126–2132. https://doi.org/10.1107/S0907444904019158 European Centre for Disease Prevention and Control, 2019. Antimicrobial resistance in the EU/EEA (EARS-Net) - Annual Epidemiological Report 2019. Flemming, H.C., Wingender, J., 2010. The biofilm matrix. Nat. Rev. Microbiol. 8, 623– 633. https://doi.org/10.1038/nrmicro2415 Furusawa, T., Iwano, H., Hiyashimizu, Y., Matsubara, K., Higuchi, H., Nagahata, H., Niwa, H., Katayama, Y., Kinoshita, Y., Hagiwara, K., Iwasaki, T., Tanji, Y., Yo- kota, H., Tamura, Y., 2016. Phage therapy is effective in a mouse model of bac- terial equine keratitis. Appl. Environ. Microbiol. 82, 5332–5339. https://doi.org/10.1128/AEM.01166-16 González-García, V.A., Bocanegra, R., Pulido-Cid, M., Martín-Benito, J., Cuervo, A., Carrascosa, J.L., 2015. Characterization of the initial steps in the T7 DNA ejec- tion process. Bacteriophage 5, e1056904. https://doi.org/10.1080/21597081.2015.1056904 Guo, F., Liu, Z., Fang, P.A., Zhang, Q., Wright, E.T., Wu, W., Zhang, C., Vago, F., Ren, Y., Jakana, J., Chiu, W., Serwer, P., Jiang, W., 2014. Capsid expansion mecha- nism of bacteriophage T7 revealed by multistate atomic models derived from

28 BIBLIOGRAPHY

cryo-EM reconstructions. Proc. Natl. Acad. Sci. U. S. A. 111, E4606–E4614. https://doi.org/10.1073/pnas.1407020111 Guo, F., Liu, Z., Vago, F., Ren, Y., Wu, W., Wright, E.T., Serwer, P., Jiang, W., 2013. Visualization of uncorrelated, tandem symmetry mismatches in the internal ge- nome packaging apparatus of bacteriophage T7. Proc. Natl. Acad. Sci. 110, 6811–6816. https://doi.org/10.1073/pnas.1215563110 Hancock, R., 1997. The bacterial outer membrane as a drug barrier. Trends Microbiol. 5, 37–42. https://doi.org/10.1016/S0966-842X(97)81773-8 Hancock, R.E.W., Speert, D.P., 2000. Antibiotic resistance in Pseudomonas aeruginosa: Mechanisms and impact on treatment. Drug Resist. Updat. 3, 247–255. https://doi.org/10.1054/drup.2000.0152 Harmsen, M., Yang, L., Pamp, S.J., Tolker-Nielsen, T., 2010. An update on Pseudomo- nas aeruginosa biofilm formation, tolerance, and dispersal. FEMS Immunol. Med. Microbiol. 59, 253–268. https://doi.org/10.1111/j.1574-695X.2010.00690.x Hatfull, G.F., Hendrix, R.W., 2011. Bacteriophages and their genomes. Curr. Opin. Vi- rol. 1, 298–303. https://doi.org/10.1016/j.coviro.2011.06.009 Himes, B.A., Zhang, P., 2018. emClarity: software for high-resolution cryo-electron to- mography and subtomogram averaging. Nat. Methods 15, 955–961. https://doi.org/10.1038/s41592-018-0167-z Howard-Varona, C., Hargreaves, K.R., Abedon, S.T., Sullivan, M.B., 2017. Lysogeny in nature: mechanisms, impact and ecology of temperate phages. ISME J. 11, 1511– 1520. https://doi.org/10.1038/ismej.2017.16 Hrebík, D., Štveráková, D., Škubník, K., Füzik, T., Pantůček, R., Plevka, P., 2019. Structure and genome ejection mechanism of Staphylococcus aureus phage P68. Sci. Adv. 5, eaaw7414. https://doi.org/10.1126/sciadv.aaw7414 Hu, B., Margolin, W., Molineux, I.J., Liu, J., 2013. The Bacteriophage T7 Virion Under- goes Extensive Structural Remodeling During Infection. Science 339, 576–579. https://doi.org/10.1126/science.1231887 Hyun, S.I., Maruri-Avidal, L., Moss, B., 2015. Topology of Endoplasmic Reticulum-As- sociated Cellular and Viral Proteins Determined with Split-GFP. Traffic 16, 787– 795. https://doi.org/10.1111/tra.12281 Iino, R., Koyama, I., Kusumi, A., 2001. Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface. Bio- phys. J. 80, 2667–2677. https://doi.org/10.1016/S0006-3495(01)76236-4 Jansons, I., Touchie, G., Sharp, R., Almquist, K., Farinha, M.A., Lam, J.S., Kropinski, A.M., 1994. Deletion and transposon mutagenesis and sequence analysis of the pRO1600 OriR region found in the broad-host-range plasmids of the pQF series. Plasmid 31, 265–274. Jensen, J.D., Parks, A.R., Adhya, S., Rattray, A.J., Court, D.L., 2020. λ Recombineering Used to Engineer the Genome of Phage T7. Antibiotics 9, 805. https://doi.org/10.3390/antibiotics9110805 Jonić, S., Sorzano, C.O.S., Boisset, N., 2008. Comparison of single-particle analysis and electron tomography approaches: An overview. J. Microsc. 232, 562–579. https://doi.org/10.1111/j.1365-2818.2008.02119.x

29 BIBLIOGRAPHY

Källberg, M., Wang, H., Wang, S., Peng, J., Wang, Z., Lu, H., Xu, J., 2012. Template- based protein structure modeling using the RaptorX web server. Nat. Protoc. 7, 1511–1522. https://doi.org/10.1038/nprot.2012.085 Kamiyama, D., Sekine, S., Barsi-Rhyne, B., Hu, J., Chen, B., Gilbert, L.A., Ishikawa, H., Leonetti, M.D., Marshall, W.F., Weissman, J.S., Huang, B., 2016. Versatile protein tagging in cells with split fluorescent protein. Nat. Commun. 7, 1–9. https://doi.org/10.1038/ncomms11046 Keen, E.C., Bliskovsky, V.V., Malagon, F., Baker, J.D., Prince, J.S., Klaus, J.S., Adhya, S.L., 2017. crossm Transformation. mBio 8, 1–12. Kemp, P., Garcia, L.R., Molineux, I.J., 2005. Changes in bacteriophage T7 virion struc- ture at the initiation of infection. Virology 340, 307–317. https://doi.org/10.1016/j.virol.2005.06.039 Kerr, K.G., Snelling, A.M., 2009. Pseudomonas aeruginosa: a formidable and ever-pre- sent adversary. J. Hosp. Infect. 73, 338–344. https://doi.org/10.1016/j.jhin.2009.04.020 Kiljunen, S., Vilen, H., Pajunen, M., Savilahti, H., Skurnik, M., 2005. Nonessential genes of phage phiYeO3-12 include genes involved in adaptation to growth on Yersinia enterocolitica serotype O:3. J. Bacteriol. 187, 1405–1414. https://doi.org/10.1128/JB.187.4.1405-1414.2005 Kim, S.-H., Chung, Y.-B., 1996. Isolation of a Mutant Bacteriophage T7 Deleted in Nonessential Genetic Elements, Gene19.5andm. Virology 216, 20–25. https://doi.org/10.1006/viro.1996.0030 Kirisits, M.J., Parsek, M.R., 2006. Does Pseudomonas aeruginosa use intercellular sig- nalling to build biofilm communities? Cell. Microbiol. 8, 1841–1849. https://doi.org/10.1111/j.1462-5822.2006.00817.x Klimuk, E., Akulenko, N., Makarova, K.S., Ceyssens, P.-J., Volchenkov, I., Lavigne, R., Severinov, K., 2013. Host RNA polymerase inhibitors encoded by ϕKMV-like phages of pseudomonas. Virology 436, 67–74. https://doi.org/10.1016/j.vi- rol.2012.10.021 Kühlbrandt, W., 2014. The resolution revolution. Science 343, 1443–1444. https://doi.org/10.1126/science.1251652 Kulakov, L.A., Ksenzenko, V.N., Shlyapnikov, M.G., Kochetkov, V.V., Del Casale, A., Allen, C.C.R., Larkin, M.J., Ceyssens, P.J., Lavigne, R., 2009. Genomes of “phiKMV-like viruses” of Pseudomonas aeruginosa contain localized single- strand interruptions. Virology 391, 1–4. https://doi.org/10.1016/j.vi- rol.2009.06.024 Laloux, G., Jacobs-Wagner, C., 2014. How do bacteria localize proteins to the cell pole? J. Cell Sci. 127, 11–19. https://doi.org/10.1242/jcs.138628 Lammens, E., Ceyssens, P.J., Voet, M., Hertveldt, K., Lavigne, R., Volckaert, G., 2009. Representational Difference Analysis (RDA) of bacteriophage genomes. J. Mi- crobiol. Methods 77, 207–213. https://doi.org/10.1016/j.mimet.2009.02.006 Lavigne, R., Lecoutere, E., Wagemans, J., Cenens, W., Aertsen, A., Schoofs, L., Landuyt, B., Paeshuyse, J., Scheer, M., Schobert, M., Ceyssens, P.-J., 2013. A Multifaceted Study of Pseudomonas aeruginosa Shutdown by Virulent Podovirus LUZ19. mBio 4. https://doi.org/10.1128/mBio.00061-13

30 BIBLIOGRAPHY

Lučič, V., Rigort, A., Baumeister, W., 2013. Cryo-electron tomography: the challenge of doing structural biology in situ. J. Cell Biol. 202, 407–419. https://doi.org/10.1083/jcb.201304193 Luong, T., Salabarria, A.-C., Edwards, R.A., Roach, D.R., 2020. Standardized bacterio- phage purification for personalized phage therapy. Nat. Protoc. 15, 2867–2890. https://doi.org/10.1038/s41596-020-0346-0 Lyczak, J.B., Cannon, C.L., Pier, G.B., 2002. Lung infections associated with cystic fi- brosis. Clin. Microbiol. Rev. 15, 194–222. https://doi.org/10.1128/CMR.15.2.194-222.2002 Maddocks, S., Fabijan, A.P., Ho, J., Lin, R.C.Y., Ben Zakour, N.L., Dugan, C., Kliman, I., Branston, S., Morales, S., Iredell, J.R., 2019. Bacteriophage therapy of ventila- tor-associated pneumonia and empyema caused by pseudomonas aeruginosa. Am. J. Respir. Crit. Care Med. 200, 1179–1181. https://doi.org/10.1164/rccm.201904-0839LE Manyi-Loh, C., Mamphweli, S., Meyer, E., Okoh, A., 2018. Antibiotic Use in Agricul- ture and Its Consequential Resistance in Environmental Sources: Potential Public Health Implications. Molecules 23, 795. https://doi.org/10.3390/mole- cules23040795 Mastronarde, D.N., 2005. Automated electron microscope tomography using robust pre- diction of specimen movements. J. Struct. Biol. 152, 36–51. https://doi.org/10.1016/j.jsb.2005.07.007 Mastronarde, D.N., Held, S.R., 2017. Automated tilt series alignment and tomographic reconstruction in IMOD. J. Struct. Biol. 197, 102–113. https://doi.org/10.1016/j.jsb.2016.07.011 Maurice, N.M., Bedi, B., Sadikot, R.T., 2018. Pseudomonas aeruginosa biofilms: Host response and clinical implications in lung infections. Am. J. Respir. Cell Mol. Biol. 58, 428–439. https://doi.org/10.1165/rcmb.2017-0321TR Meng, R., Jiang, M., Cui, Z., Chang, J.-Y., Yang, K., Jakana, J., Yu, X., Wang, Z., Hu, B., Zhang, J., 2019. Structural basis for the adsorption of a single-stranded RNA bacteriophage. Nat. Commun. 10, 3130. https://doi.org/10.1038/s41467-019- 11126-8 Mulcahy, L.R., Isabella, V.M., Lewis, K., 2014. Pseudomonas aeruginosa Biofilms in Disease. Microb. Ecol. 68, 1–12. https://doi.org/10.1007/s00248-013-0297-x Murata, K., Wolf, M., 2018. Cryo-electron microscopy for structural analysis of dy- namic biological macromolecules. Biochim. Biophys. Acta - Gen. Subj. 1862, 324–334. https://doi.org/10.1016/j.bbagen.2017.07.020 Nakae, T., Nakajima, A., Ono, T., Saito, K., Yoneyama, H., 1999. Resistance to ␤-Lac- tam Antibiotics in Pseudomonas aeruginosa Due to Interplay between the MexAB-OprM Efflux Pump and ␤-Lactamase 3. Nayeemul Bari, S.M., Hatoum-Aslan, A., 2019. CRISPR-Cas10 assisted editing of viru- lent staphylococcal phages. Methods Enzymol. 616, 385–409. https://doi.org/10.1016/bs.mie.2018.10.023 Nemec, A., Krizova, L., Maixnerova, M., Musilek, M., 2010. Multidrug-resistant epi- demic clones among bloodstream isolates of Pseudomonas aeruginosa in the

31 BIBLIOGRAPHY

Czech Republic. Res. Microbiol. 161, 234–242. https://doi.org/10.1016/j.resmic.2010.02.002 Nikaido, H., 1994. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264, 382–388. https://doi.org/10.1126/sci- ence.8153625 Nogales, E., Scheres, S.H.W., 2015. Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity. Mol. Cell 58, 677–689. https://doi.org/10.1016/j.molcel.2015.02.019 Pachori, P., Gothalwal, R., Gandhi, P., 2019. Emergence of antibiotic resistance Pseudo- monas aeruginosa in intensive care unit; a critical review. Genes Dis. 6, 109–119. https://doi.org/10.1016/j.gendis.2019.04.001 Pawley, J.B., 2006. Fundamental limits in confocal microscopy. Handb. Biol. Confocal Microsc. Third Ed. 20–42. https://doi.org/10.1007/978-0-387-45524-2_2 Pérez-Ruiz, M., Pulido-Cid, M., Luque-ortega, J.R., Valpuesta, J.M., Cuervo, A., Carras- cosa, J.L., 2021. Assisted assembly of bacteriophage T7 core components for ge- nome translocation across the bacterial envelope 1–28. Persat, A., Inclan, Y.F., Engel, J.N., Stone, H.A., Gitai, Z., 2015. Type IV pili mechano- chemically regulate virulence factors in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. 112, 7563–7568. https://doi.org/10.1073/pnas.1502025112 Pettersen, E., Goddard, T., Huang, C., Couch, G., Greenblatt, D., Meng, E., Ferrin, T., n.d. UCSF Chimera--a visualization system for exploratory research and analy- sis. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. Pfab, J., Phan, N.M., Si, D., 2021. DeepTracer for fast de novo cryo-EM protein struc- ture modeling and special studies on CoV-related complexes. Proc. Natl. Acad. Sci. 118. https://doi.org/10.1073/pnas.2017525118 Power, R.M., Huisken, J., 2017. A guide to light-sheet fluorescence microscopy for mul- tiscale imaging. Nat. Methods 14, 360–373. https://doi.org/10.1038/nmeth.4224 Rasamiravaka, T., Labtani, Q., Duez, P., El Jaziri, M., 2015. The Formation of Biofilms by Pseudomonas aeruginosa : A Review of the Natural and Synthetic Com- pounds Interfering with Control Mechanisms. BioMed Res. Int. 2015, 1–17. https://doi.org/10.1155/2015/759348 Romero-Calle, D., Benevides, R.G., Góes-Neto, A., Billington, C., 2017. Bacteriophages as alternatives to antibiotics. Antibiotics 1–5. Rudilla, H., Merlos, A., Sans-Serramitjana, E., Fuste, E., M. Sierra, J., Zalacain, A., Vi- nuesa, T., Vinas, M., 1 Department of Pathology & Experimental therapeu- tics, Faculty of Medicine & Health Sciences, University of Barcelona, Feixa Llarga s/n 08907 Hospitalet, Barcelona, Spain, 2 Department of Clinical Sci- ences, Faculty of Medicine & Health Sciences, University of Barcelona, Feixa Llarga s/n 08907 Hospitalet, Barcelona, Spain, 2018. New and old tools to evaluate new antimicrobial peptides. AIMS Microbiol. 4, 522–540. https://doi.org/10.3934/microbiol.2018.3.522 Sadikot, R.T., Blackwell, T.S., Christman, J.W., Prince, A.S., 2005. Pathogen-host inter- actions in pseudomonas aeruginosa pneumonia. Am. J. Respir. Crit. Care Med. 171, 1209–1223. https://doi.org/10.1164/rccm.200408-1044SO

32 BIBLIOGRAPHY

Scarff, C.A., Fuller, M.J.G., Thompson, R.F., Iadaza, M.G., 2018. Variations on nega- tive stain electron microscopy methods: Tools for tackling challenging systems. J. Vis. Exp. 2018, 1–8. https://doi.org/10.3791/57199 Scheres, S.H.W., 2013. RELION-3.0-tutorial. Manuals 1–21. Scheres, S.H.W., 2012. RELION: Implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530. https://doi.org/10.1016/j.jsb.2012.09.006 Scheres, S.H.W., n.d. Single-particle processing in relion-3.1 72. Schorb, M., Haberbosch, I., Hagen, W.J.H., Schwab, Y., Mastronarde, D.N., 2019. Soft- ware tools for automated transmission electron microscopy. Nat. Methods 16, 471–477. https://doi.org/10.1038/s41592-019-0396-9 Secor, P.R., Burgener, E.B., Kinnersley, M., Jennings, L.K., Roman-Cruz, V., Popescu, M., Van Belleghem, J.D., Haddock, N., Copeland, C., Michaels, L.A., de Vries, C.R., Chen, Q., Pourtois, J., Wheeler, T.J., Milla, C.E., Bollyky, P.L., 2020. Pf Bacteriophage and Their Impact on Pseudomonas Virulence, Mammalian Im- munity, and Chronic Infections. Front. Immunol. 11, 1–18. https://doi.org/10.3389/fimmu.2020.00244 Sepúlveda-Robles, O., Kameyama, L., Guarneros, G., 2012. High diversity and novel species of Pseudomonas aeruginosa bacteriophages. Appl. Environ. Microbiol. 78, 4510–4515. https://doi.org/10.1128/AEM.00065-12 Serwer, P., 2011. Proposed Ancestors of Phage Nucleic Acid Packaging Motors (and Cells). Viruses 3, 1249–1280. https://doi.org/10.3390/v3071249 Serwer, P., 2010. A hypothesis for bacteriophage DNA packaging motors. Viruses 2, 1821–1843. https://doi.org/10.3390/v2091821 Singer, A., 2018. Mathematics for cryo-electron microscopy. ArXiv180306714 Phys. Soares, A., Alexandre, K., Etienne, M., 2020. Tolerance and Persistence of Pseudomo- nas aeruginosa in Biofilms Exposed to Antibiotics: Molecular Mechanisms, Anti- biotic Strategies and Therapeutic Perspectives. Front. Microbiol. 11, 1–11. https://doi.org/10.3389/fmicb.2020.02057 Steven, A.C., Serwer, P., Bisher, M.E., Trus, B.L., 1983. Molecular architecture of bac- teriophage T7 capsid. Virology 124, 109–120. Stiefel, P., Schmidt-Emrich, S., Maniura-Weber, K., Ren, Q., 2015. Critical aspects of using bacterial cell viability assays with the fluorophores SYTO9 and propidium iodide. BMC Microbiol. 15, 36. https://doi.org/10.1186/s12866-015-0376-x Sulakvelidze, A., Alavidze, Z., Morris, J.G., Jr, 2001. Bacteriophage therapy. Antimi- crob. Agents Chemother. 45, 649–659. https://doi.org/10.1128/AAC.45.3.649- 659.2001 Sullivan, M.J., Petty, N.K., Beatson, S.A., 2011. Easyfig: a genome comparison visual- izer. Bioinforma. Oxf. Engl. 27, 1009–1010. https://doi.org/10.1093/bioinformat- ics/btr039 Szymczak, P., Rau, M.H., Monteiro, J.M., Pinho, M.G., Filipe, S.R., Vogensen, F.K., Zeidan, A.A., Janzen, T., 2019. A comparative genomics approach for identify- ing host-range determinants in Streptococcus thermophilus bacteriophages. Sci. Rep. 9, 1–15. https://doi.org/10.1038/s41598-019-44481-z

33 BIBLIOGRAPHY

Tan, Y.Z., Cheng, A., Potter, C.S., Carragher, B., 2016. Automated data collection in single particle electron microscopy. Microsc. Oxf. Engl. 65, 43–56. https://doi.org/10.1093/jmicro/dfv369 Tars, K., Fridborg, K., Bundule, M., Liljas, L., 2000. Structure determination of bacteri- ophage PP7 from Pseudomonas aeruginosa: From poor data to a good map. Acta Crystallogr. D Biol. Crystallogr. 56, 398–405. https://doi.org/10.1107/S0907444900001232 Tran, N.Q., Rezende, L.F., Qimron, U., Richardson, C.C., Tabor, S., 2008. Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine. Proc. Natl. Acad. Sci. 105, 9373–9378. https://doi.org/10.1073/pnas.0804164105 Turoňová, B., Schur, F.K.M., Wan, W., Briggs, J.A.G., 2017. Efficient 3D-CTF correc- tion for cryo-electron tomography using NovaCTF improves subtomogram aver- aging resolution to 3.4Å. J. Struct. Biol. 199, 187–195. https://doi.org/10.1016/j.jsb.2017.07.007 Vert, M., Doi, Y., Hellwich, K.-H., Hess, M., Hodge, P., Kubisa, P., Rinaudo, M., Schué, F., 2012. Terminology for biorelated polymers and applications (IUPAC Recommendations 2012). Pure Appl. Chem. 84, 377–410. https://doi.org/10.1351/PAC-REC-10-12-04 Vital-Lopez, F.G., Reifman, J., Wallqvist, A., 2015. Biofilm Formation Mechanisms of Pseudomonas aeruginosa Predicted via Genome-Scale Kinetic Models of Bacte- rial Metabolism. PLoS Comput. Biol. 11, 1–24. https://doi.org/10.1371/jour- nal.pcbi.1004452 Wade, R., 1992. A brief look at imaging and contrast transfer. Ultramicroscopy 46, 145– 156. Wagner, T., Merino, F., Stabrin, M., Moriya, T., Antoni, C., Apelbaum, A., Hagel, P., Sitsel, O., Raisch, T., Prumbaum, D., Quentin, D., Roderer, D., Tacke, S., Siebolds, B., Schubert, E., Shaikh, T.R., Lill, P., Gatsogiannis, C., Raunser, S., 2019. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun. Biol. 2, 218. https://doi.org/10.1038/s42003-019-0437-z Wang, R.Y.-R., Kudryashev, M., Li, X., Egelman, E.H., Basler, M., Cheng, Y., Baker, D., DiMaio, F., 2015. De novo protein structure determination from near-atomic- resolution cryo-EM maps. Nat. Methods 12, 335–338. https://doi.org/10.1038/nmeth.3287 Waters, E.M., Neill, D.R., Kaman, B., Sahota, J.S., Clokie, M.R.J., Winstanley, C., Kadioglu, A., 2017. Phage therapy is highly effective against chronic lung infec- tions with Pseudomonas aeruginosa. Thorax 72, 666. https://doi.org/10.1136/thoraxjnl-2016-209265 Weinstein, R.A., Gaynes, R., Edwards, J.R., National Nosocomial Infections Surveil- lance System, 2005. Overview of Nosocomial Infections Caused by Gram-Nega- tive Bacilli. Clin. Infect. Dis. 41, 848–854. https://doi.org/10.1086/432803 Wessel, A.K., Arshad, T.A., Fitzpatrick, M., Connell, J.L., Bonnecaze, R.T., Shear, J.B., Whiteley, M., 2014. Oxygen Limitation within a Bacterial Aggregate. mBio 5, e00992-14. https://doi.org/10.1128/mBio.00992-14 White, J., Richardson, C., 1987. Processing of concatemers of bacteriophage T7 DNA in vitro. J. Biol. Chem. 262, 8851—8860.

34 BIBLIOGRAPHY

WHO, 2017. Global action plan on antimicrobial resistance. World Health Organ. 1–28. Willmann, M., Marschal, M., Hölzl, F., Schröppel, K., Autenrieth, I.B., Peter, S., 2013. Time series analysis as a tool to predict the impact of antimicrobial restriction in antibiotic stewardship programs using the example of multidrug-resistant Pseu- domonas aeruginosa. Antimicrob. Agents Chemother. 57, 1797–1803. https://doi.org/10.1128/AAC.02142-12 Wittebole, X., De Roock, S., Opal, S.M., 2014. A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens. Virulence 5, 226–235. https://doi.org/10.4161/viru.25991 Wood, T.K., Knabel, S.J., Kwan, B.W., 2013. Bacterial persister cell formation and dor- mancy. Appl. Environ. Microbiol. 79, 7116–7121. https://doi.org/10.1128/AEM.02636-13 Woolford, D., Ericksson, G., Rothnagel, R., Muller, D., Landsberg, M.J., Pantelic, R.S., McDowall, A., Pailthorpe, B., Young, P.R., Hankamer, B., Banks, J., 2007. SwarmPS: rapid, semi-automated single particle selection software. J. Struct. Biol. 157, 174–188. https://doi.org/10.1016/j.jsb.2006.04.006 Xu, J., Gui, M., Wang, D., Xiang, Y., 2016. The bacteriophage ϕ29 tail possesses a pore- forming loop for cell membrane penetration. Nature 534, 544–547. https://doi.org/10.1038/nature18017 Yan, S., Wu, G., 2019. Can Biofilm Be Reversed Through Quorum Sensing in Pseudo- monas aeruginosa? Front. Microbiol. 10, 1–9. https://doi.org/10.3389/fmicb.2019.01582 Yang, Y., Lu, S., Shen, W., Zhao, X., Shen, M., Tan, Y., Li, G., Li, M., Wang, J., Hu, F., Le, S., 2016. Characterization of the first double-stranded RNA bacteriophage in- fecting Pseudomonas aeruginosa. Sci. Rep. 6, 1–9. https://doi.org/10.1038/srep38795 Zainab, S.M., Junaid, M., Xu, N., Malik, R.N., 2020. Antibiotics and antibiotic resistant genes (ARGs) in groundwater: A global review on dissemination, sources, inter- actions, environmental and human health risks. Water Res. 187, 116455. https://doi.org/10.1016/j.watres.2020.116455 Zhang, K., 2016. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12. https://doi.org/10.1016/j.jsb.2015.11.003 Zhu, J., Cheng, L., Fang, Q., Zhou, Z.H., Honig, B., 2010. Building and refining protein models within cryo-electron microscopy density maps based on homology mod- eling and multiscale structure refinement. J. Mol. Biol. 397, 835–851. https://doi.org/10.1016/j.jmb.2010.01.041 Zivanov, J., Nakane, T., Forsberg, B.O., Kimanius, D., Hagen, W.J., Lindahl, E., Scheres, S.H., 2018. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166. https://doi.org/10.7554/eLife.42166

35 BIBLIOGRAPHY

Appendix A Phage T7 protein structure

A B

N- terminal arm A-domain

E-loop

Main helix

P-domain

C D

Figure A. Atomic models of phage T7 proteins A. Major capsid protein (Guo et al., 2014); B. portal protein monomer; C. tail nozzle protein monomer; D. adaptor protein monomer (Cuervo et al., 2019; edited)

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