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Staphylococcus Infection Dynamics Staphylococcus infection dynamics. By Rebecca Hodges, BSc. A thesis submitted for the degree of Doctor of Philosophy Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN Summary Staphylococcus aureus is a clinically significant human pathogen which poses an increasing healthcare threat due to the spread of antibiotic resistance. To better understand the process of S. aureus pathogenesis, a vertebrate model for infection, using zebrafish embryos, was previously pioneered at The University of Sheffield. In this study I have utilised this systemic embryonic model of S. aureus infection in combination with a recently developed fluorescence microscopy technique – light sheet fluorescence microscopy, in order to investigate the real-time dynamics of S. aureus infection within a living host. The first aim of this project was to develop methodology that enables the imaging of infected, living transgenic embryos, over extended time scales. Having established mounting and imaging parameters, infection progression was followed using fluorescent S. aureus reporter strains and fluorescently labelled host phagocytes. The 4D imaging of these interactions identified macrophages as the host-niche in which bacterial expansion, followed by phagocyte escape, occurs. Furthermore, by using bacterial population studies it was confirmed that depletion of macrophages abolishes the immune bottleneck which proceeds clonal, population expansion of S. aureus. When imaging embryos in the terminal stages of infection it was apparent that the large bacterial aggregates which form within the host, have biofilm-like characteristics. As such, the role of staphylococcal proteins involved in biofilm formation, during infection progression was investigated using fluorescent reporters for gene expression. It was determined that S. aureus nuclease is produced both inside of host phagocytes and later by bacteria associated with large aggregates. Nuclease was also identified as a novel virulence factor in the zebrafish embryo model of S. aureus infection. Light sheet fluorescence microscopy has proven a useful tool to gain further insight into the temporal and spatial dynamics of S. aureus pathogenesis and to dissect real-time host- pathogen interactions on a cellular level. Acknowledgements Firstly, I would like to the Professor Simon Foster for the wonderful opportunity to carry out my PhD and all the support and guidance he has provided me with along this journey, and the encouragement to pursue the more bizaar ideas. For this I am forever grateful. It was a pleasure to be welcomed into F18 by many, many lovely members of the lab, past and present, who have not only provided me with technical assistance but support, friendship and hilarity. Thank you for all the after work drinking that lubricated the more frustrating times and for listening to my moaning in the coffee room. I am particularly grateful to Tomasz Prajsnar and Emma Boldock for the help with learning the zebrafish model and Robert Turner for his everyday brilliance and occupying the desk next to me as I think out loud. Some of this work was performed with Logan Bulock, from the University of Nebraska Medical Centre and I am grateful for his valuable insight. Thank you to my absent friend Mark Cooke, who first had the hairbrained idea of using smURFP – it worked! I am sorry you didn’t get to see it fluoresce in its bright pink glory, you are dearly missed. I have met some incredible scientists who I am lucky to have made lifelong friends with, these women have set the bar very high, I hope I can pursue research with the same rigour and integrity. Thank you to these ‘F-floor girlies’, Victoria, Milena, Nicola, Kasia and Mina for wonderful adventures and accompanying me in consuming yet more gin. Hannah, thank you for the music, coffee and memorable nights. Outside of research, the past few years have been some of the most difficult times of my life and I absolutely would not have been able to weather the storm and finish this work without the support of brilliant friends. Thank you to Hayley who can always sense my anxieties and endeavours to ease them. Thanks to Anne, Sally, Leanne and Fionnuala for always being on the end of the phone, you are the best friends anyone could wish for. Special thanks to my lovely boyfriend James, who was my rock through the months of illness and kept me sane. I could not have got to this point without his love and support. I am especially grateful for all the times he patiently waited when my ‘ten minutes’ in the lab would routinely take hours. He has made me (and washed up) what must be a thousand cups of tea whilst I have been writing this thesis - I promise we will finally get a holiday. Finally, thank you to my family, for your patience and understanding of my absence. My parents have provided me with endless love and support. I would not have got here without their encouragement and belief in my ability. They also funded my last four months in the lab when I did not receive sick pay and paid for my mortgage and food whilst writing up. This thesis literally would not exist if it wasn’t for them. They did whatever it took to make sure I got to this point and I love them to pieces. This work was funded by The University of Sheffield Abbreviations 3D Three dimensional 4D Four dimensional Ab antibody Agr accessory gene regulator AIP auto-inducing peptide AMPs antimicrobial peptides ATP Adenosine tri-phosphate bp base pairs BP band pass Bbp bone sialo-binding protein BiSAb bi-specific antibody C5aR1 C5a receptor 1 C centigrade CA community associated cas CRISPR associated protein CF cystic fibrosis CFU clony forming units CHIPs chemotaxis inhibitory protein of staphylococcus ClfA clumping factor A ClfB clumping factor B Cna collagen-binding protein Coa Coagulase protein CP5 capsular polysaccharide 5 CP8 capsular polysaccharide 8 CRISP clustered regularly interspersed short palindromic repeats CWA cell wall anchored adhesin DNA deoxyribonucleic acid dpf days post fertilisation Eap extracellular adherence protein ECM extracellular matrix eDNA extracellular DNA Efb extracellular fibrinogen-binding protein Ery erythromycin Fab antigen binding fragment Fc constant region FDA Federal Drug Administration FEP fluorinated ethylene propylene Fg fibrinogen fMLF N-formylated tripeptide Nformylmethionyl-leucyl-phenylalanine FnBPA fibronectin binding protein A FnBPB fibronectin binding protein B FOV field of view FPR1 formyl-peptide receptor GB gigabyte GFP Green Fluorescent Protein GSK Glaxo Smith Kline h hours H2O2 hydrogen peroxide HK histidine kinase Hla α-toxin HlgAB γ-haemolysin AB HlgCD γ-haemolysin CD HNP human neutrolphil peptide HNP1 human neutrophil peptide 1 HNP2 human neutrophil peptide 2 HNP3 human neutrophil peptide 3 HOCl hypochlorous acid hpf hours post-fertilisation hpi hours post-infection IAF-1 lymphocyte-function-associated antigen 1 ICAM-1 intracellular adhesion molecule-1 IE infective endocarditis IM intra-muscular IL interleukin IsdA iron-regulated surface determinant protein A IsdB iron-regulated surface determinant protein B L Litre LLO lysteriolisin O LMP low melting point LP long pass LPS lipopolysaccharide LSFM light sheet flourescence microscopy LSM Laser scanning microscopy LukAB leukocidin AB LukED leukocidin ED LWT London wild type M molar mAb monoclonal antibody MaxIP maximum intensity projection MIC minimum inhibitory concentration mg milligrams min minutes ml milliliters mm millimeters MntC manganese transporter MO morpholino antisense oligomers MPO myeloperoxidase MRSA methicillin resistant S. aureus MSCRAMMs microbial surface components recognising adhesive matrix molecules MSSA methicillin sensitive S. aureus M-CSF macrophage colony stimulating factor NA numerical aperture NE neutrophil elastase NEAT near iron transporter NET neutrophil extracellular traps NK Natural Killer NLRs NOD-like receptors nm nanometer NSG non-obese diabetic/ severe combined immunodeficient mouse with null mutation in Il2R common gamma chain NSPs neutrophil serine proteases Nuc nuclease OatA O-acetyltransferase OD outer diameter OD600 optical density at 600 nm PAMPs pathogen associated molecular patterns PBP2a penicillin binding protein PCR polymerase chain reaction PGRPs peptidoglycan recognition proteins PRRs pattern recognition receptors PSF point spread function PSMs phenyl soluble modulins PVL Panton-Valentine leukocidin RAM random access memory RAID redundant array of independent discs ROI Region of interest ROS reactive oxygen species RT room temperature sae S. aureus exoprotein expression SAK staphylococcal kinase SCIN staphylococcal complement inhibitor ScpA staphopain A sCMOS scientific complementary metal-oxide-semiconductor SdrC serine-aspartate repeat protein C SdrD serine-aspartate repeat protein D SEB staphylococcal enterotoxin B SELX staphylococcal enterotoxin-like toxin X sGFP superfolder GFP smURFP small ultra red fluorescent protein SNR signal-to-noise ratio SP short pass SpA protein A SPIM selective plane illumination microscopy SPIN staphylococcal peroxide inhibitor SSL13 staphylococcal superantigen like protein 13 SSL3 staphylococcal superantigen like protein 3 SSL5 staphylococcal superantigen like protein 5 SSS scalded skin syndrome SSTIs skin and soft tissue infections STRIVE Staphylococcus aureus surgical inpatient vaccine efficacy T3SS type III secretion system TB terabyte TCS two-component regulatory systems Tet tetracyline TLRs Toll-like receptors TNF- α tumour necrosis
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