STIMULATION OF INNATE IMMUNITY LEADS TO CLEARANCE OF C. NEOFORMANS INFECTION IN ZEBRAFISH ALFRED ALINAFE KAMUYANGO A thesis submitted to the University of Sheffield for fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Infection, Immunity and Cardiovascular Diseases December 2017 Abstract Cryptococcus neoformans is an opportunistic pathogen and a leading cause of life‑threatening fungal infections in the immunocompromised. Individuals that are at greater risk are those with defective T-cell mediated immunity such as those with HIV/AIDS. Despite treatment with anti-fungal drugs, mortality remains excessively high and patients experience serious side effects hence the need to explore new therapeutic strategies. Activation of macrophages is essential for the control of cryptococcal infection. However, in the absence of T-cell mediated immunity, activation of macrophages is disrupted and clearance of cryptococcal infection is abrogated. Herein I first report a zebrafish-C. neoformans model of infection and show that zebrafish can clear, control or fail to control cryptococcal infection. I then go on to test whether cytokines or PRR ligands are capable of stimulating innate immune resistance to C. neoformans in zebrafish. I demonstrate that concomitant injection of IFNγ with C. neoformans results in reduced fungal burden and increased fungal clearance. IFNγ increases the recruitment of phagocytes to the site of infection and enhances phagocytosis by macrophages. Macrophage deficient larvae fail to clear or suppress cryptococcal infection despite treatment with IFNγ. In addition, infected macrophages display low lysosomal pH and elevated expression of IL-1β in IFNγ-treated larvae. Although neutrophils take up the fungus, their depletion does not alter cryptococcal burden. Secondly, I demonstrate that S. aureus CWP is a potent inducer of innate defences against C. neoformans. Using chemically digested S. aureus CWP to remove wall teichoic acid or mutants that do not produce wall teichoic acid (tarO) or lipoproteins (lgt), I establish that protective effects of S. aureus CWP require wall teichoic acid but not lipoproteins. Protection by S. aureus CWP are associated with increased recruitment of macrophages but not enhanced phagocytosis or TNFα expression. ABSTRACT i This work provides new insights into cellular responses and effector molecules in the context of cryptococcal disease progression while identifying potential immunomodulatory targets. ABSTRACT ii Acknowledgements Not just one or two, but many people have made a positive contribution to this piece of work and I express my sincere appreciation. Firstly, I would like to express my heartfelt appreciation to my supervisor Simon Johnston. Simon, you have provided extraordinary support and guidance throughout my PhD. Your help was beyond expectation. You were so dedicated as if I was the only student you had. You have been very encouraging, for example, whenever I went to your office with my results I thought were not very good, I would come out smiling and feeling encouraged. I could not ask for a better supervisor, your approach is thrilling and I have learned a lot from you. You have been a blessing. I am forever thankful to Jenny Wright for proofreading my thesis. I also thank Josie Gibson who selflessly provided a graph for my thesis. I would also like to thank Josh Hooker, a technician in Simon Foster’s laboratory for making all the S. aureus cell wall preparations used in my project. My thanks also go to Victoriano Mulero at Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, Spain for providing Vibrio anguillarum DNA. I would also like to express my appreciation to my lab colleagues in the Johnston lab. Our socialising and laughter were comforts in my stressful moments, and your hard work and encouragement kept me focused on my work in those tough times. Words cannot articulate how grateful I am. Special thanks go to Robert Evans, Aleksandra Bojarczuk and Kate Pline. I am very grateful to the Wellcome for funding my PhD. Last, with love, extra special thanks go to all my family and friends both in Malawi and in the UK. Many thanks for the conversations, for texts and words of encouragement. I am eternally grateful. Truly, I am thankful to and humbled by all those who have contributed to my success. My prayers are with you always. Above all, I thank God for bringing each and every individual mentioned here to support me. ACKNOWLEDGEMENTS iii Table of Contents Table of Figures Table of Tables CHAPTER 1 INTRODUCTION ....................................................................... 1 1.1 Cryptococcus and cryptococcosis ................................................... 2 1.2 Pathogenic Cryptococcus species ................................................... 2 1.2.1 Cryptococcus neoformans ............................................................. 3 1.2.2 Cryptococcus gattii ........................................................................ 4 1.3 Life cycle of Cryptococcus ................................................................ 8 1.3.1 Infective forms of C. neoformans ................................................. 10 1.3.2 C. neoformans genome ............................................................... 10 1.4 Cryptococcosis ................................................................................. 11 1.4.1 Cause, transmission and affected organs, life cycle .................... 11 1.4.2 Epidemiology ............................................................................... 13 1.4.2.1 In HIV/AIDS patients ............................................................ 13 1.4.2.2 In HIV naïve patients ............................................................ 15 1.4.3 Clinical manifestations ................................................................. 17 1.4.3.1 CNS cryptococcosis ............................................................. 17 1.4.3.2 Pulmonary cryotococcosis .................................................... 17 1.4.3.3 Cryptococcosis in other sites ................................................ 18 1.4.4 Diagnosis ..................................................................................... 19 1.4.4.1 Cryptococcal meningitis ....................................................... 19 1.4.4.2 Pulmonary cryptococcosis .................................................... 20 1.4.5 Management of cryptococcal disease ......................................... 21 1.4.5.1 Management of meningeal cryptococcosis .......................... 21 1.4.5.2 Management of cryptococcosis complications ..................... 22 1.4.5.3 Management of non-meningeal cryptococcosis ................... 23 1.4.6 C. neoformans virulence factors .................................................. 24 1.4.6.1 The Capsule ......................................................................... 24 1.4.6.2 Melanin ................................................................................. 28 1.4.6.3 Anti-phagocytic protein 1 ...................................................... 28 CONTENTS iv 1.4.6.4 Urease .................................................................................. 29 1.4.6.5 Thermotolerance .................................................................. 29 1.4.6.6 Phospholipase ...................................................................... 30 1.4.6.7 Other virulence factors ......................................................... 30 1.4.7 Pathogenesis of C. neoformans .................................................. 30 1.4.7.1 Dissemination to the CNS .................................................... 33 1.5 Host immunity to Cryptococcus neoformans ................................ 35 1.5.1 Innate immune and cellular responses against C. neoformans ... 35 1.5.2 Phagocytes .................................................................................. 39 1.5.2.1 Neutrophils ........................................................................... 39 1.5.2.2 Dendritic cells ....................................................................... 41 1.5.2.3 Macrophages ........................................................................ 44 1.6 Experimental models for cryptococcosis ....................................... 48 1.6.1 In vitro cell models ....................................................................... 48 1.7 Invertebrate models .......................................................................... 49 1.7.1 Galleria mellonella ....................................................................... 49 1.7.2 Drosophila melanogaster and Drosophila S2 cells ...................... 50 1.7.3 Caenorhabditis elegans ............................................................... 50 1.7.4 Dictyostelium discoideum (Social amoebae) ............................... 51 1.7.5 Acanthamoeba castellanii ............................................................ 51 1.8 Vertebrate models ............................................................................ 52 1.8.1 Mouse (Mus musculus) ............................................................... 52 1.8.2 Rat (Rattus rattus) ....................................................................... 52 1.8.3 Rabbit (Oryctolagus cuniculus) .................................................... 53 1.8.4 Guinea pigs (Cavia porcellus) ..................................................... 53 1.8.5 The zebrafish ..............................................................................