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C. Neoformans and S In vivo imaging and analysis of host-pathogen interactions of intracellular pathogens By: Josie F Gibson A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Sheffield Department of Infection, Immunity & Cardiovascular disease Submission Date: September 2017 1 Abstract Cellular and extracellular host-pathogen interactions are important in the progression of infection. Extracellular survival and growth can be significant for pathogen dissemination. Equally, intracellular pathways, such as autophagy, can be employed in host cell defence. Autophagy is a cellular self-degradation process that recycles cellular components through lysosomal degradation. Primarily for regulating starvation and housekeeping pathways, autophagy is also important for degradation of invading intracellular pathogens. Selective autophagy receptors can also target pathogens for autophagic degradation. However, some intracellular pathogens are able to subvert or block host cell autophagy. Cryptococcus neoformans and Staphylococcus aureus represent fungal and bacterial pathogens which can reside either intracellularly or extracellularly. The role of host cell autophagy in C. neoformans and S. aureus infection is unclear. Infection of zebrafish with C. neoformans or S. aureus enabled in vivo imaging of host-pathogen interactions, to examine infection growth dynamics and dissemination, in addition to cellular level imaging of pathogen interactions with host cell autophagy components. Cryptococcal infection can cause cryptococcal meningitis, frequently associated with cerebral infarcts. Analysis of cryptococcal proliferation during infection suggested that formation of intravascular cryptococcal masses precedes invasion of surrounding tissue. Vessel integrity analysis highlighted cryptococcal-mediated vascular damage, potentially providing a route for tissue dissemination. Vasculature damage may explain the origin of cortical infarcts in disease. Autophagy mutant zebrafish were generated to analyse host autophagy in pathogen infection. Characterisation of mutant larvae revealed a clear survival and growth defect, indicating autophagy is required for larval development. Autophagy mutants were subsequently used to analyse the role of autophagy during infection through analysis of pathogenic burden and pathogen association with autophagosome marker LC3-II. LC3- II recruitment to pathogens was reduced in autophagy mutant neutrophils. Additionally, selective autophagy receptor p62 was recruited to S. aureus and C. neoformans within neutrophils, highlighting the involvement of host cell autophagy during infection. 2 Acknowledgements I would like to thank my supervisors, Dr Simon Johnston, Prof Stephen Renshaw and Prof Philip Ingham, for their advice, encouragement and support throughout my PhD, in many aspects of my PhD including scientific ideas and technique, training, critical thinking, presentation and scientific writing. I would like to thank my personal tutor Dr Heather Wilson for her advice and support. Special thanks to Adam Pyrah, my family and friends for their support and encouragement over the last four years. I would like to thank the entire D31 lab, the Johnston, Renshaw and Elks groups for their support, advice and friendship. Thanks to Aleksandra Bojarczuk for her support and for training in cryptococcal zebrafish infection and to Justyna Serba and Nelly Wagner for training in staphylococcus zebrafish infection. I am thankful to Ingham and Carney groups for their welcoming into the lab, support and advice. Many thanks to Abraha Gebregziabher, Raymond Lee, and Jia Jia Ma, for advice, scientific chats and friendship. Additionally I would like to thank many new friends I met after moving to Singapore for all the fun times and for their help in integrating into a new place. I would like to thank the aquarium staff, both at The University of Sheffield aquarium and the Institute of Molecular and Cell Biology fish facility (Singapore), particularly for their help in sending fish overseas, in use of the quarantine facility and for fish welfare. I would like to thank Darren Robinson and Felix Ellett for training and help with imaging. I am very thankful to the University of Sheffield and A*Star graduate academy for funding my project, enabling me to conduct research and complete my PhD. 3 TABLE OF CONTENTS Abstract ....................................................................................................................... 2 Acknowledgements .................................................................................................... 3 Abbreviations ............................................................................................................ 10 List of tables ............................................................................................................. 12 List of figures ............................................................................................................ 12 Chapter 1: Introduction ............................................................................................ 15 1.1 Innate immune system ..................................................................................................... 16 1.1.1 Role of the innate immune system in infection ......................................................... 16 1.1.2 Components of the innate immune system .............................................................. 16 1.1.3 Innate immune system links to the adaptive immune system .................................. 18 1.1.4 Phagocytic cells: macrophages and neutrophils ........................................................ 19 1.2 Cryptococcus neoformans ................................................................................................ 21 1.2.1 C. neoformans microbiology ...................................................................................... 21 1.2.2 Route of C. neoformans infection .............................................................................. 22 1.2.2 Cryptococcus gattii ..................................................................................................... 23 1.2.3 Pathology of cryptococcosis ...................................................................................... 23 1.2.4 Host defence interactions with C. neoformans ......................................................... 25 1.2.5 C. neoformans virulence factors and pathogenesis ................................................... 32 1.3 Staphylococcus aureus ...................................................................................................... 35 1.3.1 Microbiology of S. aureus .......................................................................................... 35 1.2.2 S. aureus route of infection ........................................................................................ 35 1.2.3 S. aureus disease and significance ............................................................................. 36 1.3.4 S. aureus host-pathogen interactions ........................................................................ 37 1.3.5 S. aureus virulence factors and pathogenesis ............................................................ 42 1.4 Autophagy ......................................................................................................................... 45 1.4.1 The role of macroautophagy ...................................................................................... 47 1.4.2 Microautophagy and chaperone mediated autophagy ............................................. 48 1.4.3 The macroautophagy pathway .................................................................................. 49 1.4.4 Selective autophagy ................................................................................................... 54 1.4.5 Canonical and non-canonical autophagy ................................................................... 55 1.4.6 Host-pathogen interactions in macroautophagy ....................................................... 56 1.4.7 Autophagy in S. aureus infection ............................................................................... 57 1.4.8 Autophagy in C. neoformans infection ...................................................................... 59 1.5 Zebrafish ........................................................................................................................... 60 1.5.1 Zebrafish as a model organism .................................................................................. 60 1.5.2 Zebrafish transgenic reporter lines ............................................................................ 60 4 1.5.3 Genetic manipulation of Zebrafish ............................................................................. 61 1.5.4 Drug treatment in zebrafish ....................................................................................... 62 1.5.5 The zebrafish innate immune system ........................................................................ 62 1.5.6 Study of autophagy in zebrafish ................................................................................. 63 1.5.8 Using the zebrafish model to study infection ............................................................. 65 1.5.9 Zebrafish as an infection model for S. aureus ............................................................ 66 1.5.10 Zebrafish as an infection model for C. neoformans ................................................
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